Microorganisms For Enhancing The Availability Of Reducing Equivalents In The

Presence Of Methanol,

And For Producing 3-Hydroxyisobutyrate

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Serial No. 61/766,660 filed February 19, 2013, and U.S. Serial No. 61/722,657 filed November 5, 2012, each of which is incorporated herein by reference in its entirety.

1. SUMMARY

[0001] Provided herein are methods generally relating to metabolic and biosynthetic processes and microbial organisms capable of producing organic compounds. Specifically, provided herein is a non-naturally occurring microbial organism (NNOMO) having a methanol metabolic pathway (MMP) that can enhance the availability of reducing equivalents in the presence of methanol and/or convert methanol to formaldehyde. Such NNOMOs and reducing equivalents can be used to increase the product yield of organic compounds produced by the microbial organism, such as 3-hydroxyisobutyrate (3-HIB) and/or methacrylic acid (MAA). Also provided herein are NNOMOs and methods thereof to produce optimal yields of 3-HIB and/or MAA.

[0002] In a first aspect, provided herein is a NNOMO having a MMP, wherein said organism comprises at least one exogenous nucleic acid encoding a MMP enzyme (MMPE) expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol and/or convert methanol to formaldehyde. In certain embodiments, the MMP comprises one or more enzymes selected from the group consisting of a methanol methyltransferase (EMI); a methylenetetrahydrofolate reductase (EM2); a methylenetetrahydrofolate dehydrogenase (EM3); a methenyltetrahydrofolate cyclohydrolase (EM4); a formyltetrahydrofolate deformylase (EM5); a formyltetrahydrofolate synthetase (EM6); a formate hydrogen lyase (EM 15); a hydrogenase (EM 16); a formate dehydrogenase (EM8); a methanol dehydrogenase (EM9); a formaldehyde activating enzyme (EM 10); a formaldehyde dehydrogenase (EMI 1); a S- (hydroxymethyl)glutathione synthase (EM 12); a glutathione-dependent formaldehyde dehydrogenase (EM13); and an S-formylglutathione hydrolase (EM14). Such organisms, in certain embodiments, advantageously allow for the production of reducing equivalents, which can then be used by the organism for the production of 3-HIB or MAA using any one of the 3- HIBPs or MAAPs provided herein.

[0003] In one embodiment, the MMP comprises an EM9. In another embodiment, the MMP comprises an EM9 and an EM10. In other embodiments, the MMP comprises an EMI and an EM2. In one embodiment, the MMP comprises an EM9, an EM3, an EM4 and an EM5. In another embodiment, the MMP comprises an EM9, an EM3, an EM4 and an EM6. In other embodiments, the MMP comprises an EM9 and an EMI 1. In another embodiment, the MMP comprises an EM9, an EM12, and EM13 and an EM14. In other embodiments, the MMP comprises an EM9, an EM13 and an EM14. In an embodiment, the MMP comprises an EM9, an EM 10, an EM3, an EM4 and an EM5. In another embodiment, the MMP comprises an EM9, an EM10, an EM3, an EM4 and an EM6. In other embodiments, the MMP comprises an EMI, an EM2, an EM3, and EM4, and EM5. In one embodiment, the MMP comprises an EMI, an EM2, an EM3, an EM4 and EM6. In certain of the above embodiments, the MMP further comprises an EM8. In other of the above embodiments, the MMP further comprises and EMI 5. In yet other of the above embodiments, the MMP further comprises an EM 16. In certain embodiments, the organism comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a MMPE.

[0004] In a second aspect, provided herein is a NNOMO having (1) a MMP, wherein said organism comprises at least one exogenous nucleic acid encoding a MMPE expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol; and (2) a 3-HIB pathway (3-HIBP), wherein said organism comprises at least one exogenous nucleic acid encoding a 3-HIBP enzyme (3-HIBPE) expressed in a sufficient amount to produce 3-HIB. In certain embodiments, the 3-HIBPE is selected from the group consisting of a succinyl-CoA transferase (EMA1A), succinyl-CoA ligase (EMA1B), or succinyl-CoA synthetase (EMA1C); methylmalonyl-CoA mutase (EMA2); methylmalonyl-CoA epimerase

(EMA3); methylmalonyl-CoA reductase (aldehyde forming) (EMA4); methylmalonate semialdehyde reductase (EMA5); and methylmalonyl-CoA reductase (alcohol forming) (EMA7). [0005] In a third aspect, provided herein is a NNOMO having (1) a MMP, wherein said organism comprises at least one exogenous nucleic acid encoding a MMPE expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol; and (2) a MAA pathway (MAAP), wherein said organism comprises at least one exogenous nucleic acid encoding a MAAP enzyme (MAAPE) expressed in a sufficient amount to produce MAA. In certain embodiments, the MAAPE is selected from the group consisting of an EMA1A, EMA1B, or EMA1C; EMA2; EM A3; EMA4; EMA5; 3-HIB dehydratase (EMA6); and EMA7.

[0006] In other embodiments, the organism having a MMP, either alone or in combination with a 3-HIBP or MAAP, as provided herein, further comprises a formaldehyde assimilation pathway (FAP) that utilizes formaldehyde, e.g., obtained from the oxidation of methanol, in the formation of intermediates of certain central metabolic pathways that can be used, for example, in the formation of biomass. In certain embodiments, the organism further comprises a FAP, wherein said organism comprises at least one exogenous nucleic acid encoding a formaldehyde assimilation pathway enzyme (FAPE) expressed in a sufficient amount to produce an

intermediate of glycolysis and/or a metabolic pathway that can be used in the formation of biomass. In one embodiment, the FAPE is expressed in a sufficient amount to produce an intermediate of glycolysis. In another embodiment, the FAPE is expressed in a sufficient amount to produce an intermediate of a metabolic pathway that can be used in the formation of biomass. In some of the embodiments, the FAP comprises a hexulose-6-phosphate (H6P) synthase (EFl), a 6-phospho-3-hexuloisomerase (EF2), a dihydroxyacetone (DHA) synthase (EF3) or an EF4 (EF4). In one embodiment, the FAP comprises an EFl and an EF2. In one embodiment, the intermediate is a H6P, a fructose-6-phosphate (F6P), or a combination thereof. In other embodiments, the FAP comprises an EF3 or an EF4. In one embodiment, the intermediate is a DHA, a DHA phosphate (DHAP), or a combination thereof. In certain embodiments, the organism comprises two exogenous nucleic acids, each encoding a FAPE.

[0007] In certain embodiments, provided herein is a NNOMO having a MMP, wherein said organism comprises at least one exogenous nucleic acid encoding an EM9 expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol and/or expressed in a sufficient amount to convert methanol to formaldehyde. In some embodiments, the organism comprises at least one exogenous nucleic acid encoding an EM9 expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol. In other embodiments, the organism comprises at least one exogenous nucleic acid encoding an EM9 expressed in a sufficient amount to convert methanol to formaldehyde. In some embodiments, the microbial organism further comprises a FAP. In certain embodiments, the organism further comprises at least one exogenous nucleic acid encoding a FAPE expressed in a sufficient amount to produce an intermediate of glycolysis. In certain embodiments, the FAPE is selected from the group consisting of an EFl, an EF2, an EF3 and an EF4.

[0008] In some embodiments, provided herein is a NNOMO having a MMP, either alone or in combination with a 3-HIBP, MAAP and/or a FAP as provided herein, wherein said organism further comprises a formaldehyde reutilization pathway (FRP). In certain embodiments the organism comprises at least one exogenous nucleic acid encoding a FRP enzyme (FRPE) expressed in a sufficient amount to produce formaldehyde, pyruvate or acetyl-CoA. In some embodiments, the FRP comprises: (1) a formate reductase (EFR1); (2) (i) a formate ligase

(EFR2A), a formate transferase (EFR2B), or a formate synthetase (EFR2C), and (ii) a formyl-

CoA reductase (EFR3); (3) (i) a formyltetrahydrofolate synthetase (EFR4), (ii) a

methenyltetrahydrofolate cyclohydrolase (EFR5), (iii) a methylenetetrahydrofolate

dehydrogenase (EFR6) and (iv) a formaldehyde-forming enzyme (EFR7) or spontaneous; (6) (i) an EFR4, (ii) an EFR5, (iii) an EFR6, (iv) a glycine cleavage system (EFR8), (v) a serine hydroxymethyltransferase (EFR9), and (vi) a serine deaminase (EFR10); (7) (i) an EFR1, (ii) an

EFR4, (iii) an EFR5, (iv) an EFR6, (v) an EFR8, (vi) an EFR9, and (vii) an EFR10; (8) (i) an

EFR2A, an EFR2B or an EFR2C, (ii) an EFR3, (iii) an EFR4, (iv) an EFR5, (v) an EFR6, (vi) an

EFR8, (vii) an EFR9, and (viii) an EFR10; (9) (i) an EFR7 or spontaneous, (ii) an EFR4, (iii) an

EFR5, (iv) an EFR6, (v) an EFR8, (vi) an EFR9, and (vii) an EFR10; and (10) (i) an EFR4, (ii) an EFR5, (iii) an EFR6, (iv) a methylenetetrahydrofolate reductase (EFR11), and (v) an acetyl-

CoA synthase (EFR12). In some embodiments, the organism comprises two, three, four, five, six, seven or eight exogenous nucleic acids, each encoding a FRPE. In other embodiments, the at least one exogenous nucleic acid encoding a FRPE is a heterologous nucleic acid. In some embodiments, the FRP further comprises (i) a pyruvate formate lyase (EFR13); (ii) a pyruvate dehydrogenase (EFR14A), a pyruvate ferredoxin oxidoreductase (EFR14B), or a pyruvate :NADP+ oxidoreductase (EFR14C); (iii) a formate dehydrogenase (EFR15); or (iv) an EFR14A, EFR14B, or EFR14C; and an EFR15.

[0009] In certain embodiments, at least one exogenous nucleic acid is a heterologous nucleic acid. In some embodiments, the organism is in a substantially anaerobic culture medium. In some embodiments, the microbial organism is a species of bacteria, yeast, or fungus.

[0010] In some embodiments, the organism further comprises one or more gene disruptions, occurring in one or more endogenous genes encoding protein(s) or enzyme(s) involved in native production of ethanol, glycerol, acetate, lactate, formate, C0 ₂ , and/or amino acids by said microbial organism, wherein said one or more gene disruptions confer increased production of 3- HIB or MAA in said microbial organism. In some embodiments, one or more endogenous enzymes involved in native production of ethanol, glycerol, acetate, lactate, formate, C0 ₂ and/or amino acids by the microbial organism, has attenuated enzyme activity or expression levels. In certain embodiments, the organism comprises from one to twenty- five gene disruptions. In other embodiments, the organism comprises from one to twenty gene disruptions. In some

embodiments, the organism comprises from one to fifteen gene disruptions. In other

embodiments, the organism comprises from one to ten gene disruptions. In some embodiments, the organism comprises from one to five gene disruptions. In certain embodiments, the organism comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 gene disruptions or more.

[0011] In another aspect, provided herein is a method of producing formaldehyde, comprising culturing a NNOMO provided herein under conditions and for a sufficient period of time to produce formaldehyde. In certain embodiment, the NNOMO comprises an exogenous nucleic acid encoding an EM9. In certain embodiments, the formaldehyde is consumed to provide a reducing equivalent. In other embodiments, the formaldehyde is consumed to incorporate into 3- HIB or MAA or another target product.

[0012] In another aspect, provided herein is a method of producing an intermediate of glycolysis and/or a metabolic pathway that can be used in the formation of biomass, comprising culturing a NNOMO provided herein under conditions and for a sufficient period of time to produce the intermediate. In certain embodiment, the NNOMO comprises an exogenous nucleic acid encoding an EM9. In certain embodiments, the formaldehyde is consumed to provide a reducing equivalent. In other embodiments, the formaldehyde is consumed to incorporate into 3- HIB or MAA or another target product.

[0013] In another aspect, provided herein are methods for producing 3-HIB, comprising culturing any one of the NNOMOs comprising a MMP and a 3-HIBP provided herein under conditions and for a sufficient period of time to produce 3-HIB. In certain embodiments, the organism is cultured in a substantially anaerobic culture medium. In some embodiments, the NNOMO further comprises a FAP, FRP or a combination thereof, as provided herein.

[0014] In yet another aspect, provided herein are methods for producing MAA, comprising culturing any one of the NNOMOs comprising a MMP and a MAAP provided herein under conditions and for a sufficient period of time to produce MAA. In certain embodiments, the organism is cultured in a substantially anaerobic culture medium. In some embodiments, the NNOMO further comprises a FAP, FRP or a combination thereof, as provided herein.

2. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 shows exemplary metabolic pathways enabling the extraction of reducing equivalents from methanol. The enzymatic transformations shown are carried out by the following enzymes: 1A) a methanol methyltransferase (EMI), IB) a methylenetetrahydro folate reductase (EM2), 1C) a methylenetetrahydro folate dehydrogenase (EM3), ID) a

methenyltetrahydro folate cyclohydrolase (EM4), IE) a formyltetrahydro folate deformylase (EM5), IF) a formyltetrahydrofolate synthetase (EM6), 1G) a formate hydrogen lyase (EM15), 1H) a hydrogenase (EM 16), II) a formate dehydrogenase (EM8), 1 J) a methanol dehydrogenase (EM9), IK) a formaldehyde activating enzyme (EM 10), 1L) a formaldehyde dehydrogenase (EMU), 1M) a S-(hydroxymethyl)glutathione synthase (EM 12), IN) a glutathione-dependent formaldehyde dehydrogenase (EM13), and 10) a S-formyl glutathione hydrolase (EM14). In certain embodiments, steps K and/or M are spontaneous.

[0016] FIG. 2 shows exemplary 3-HIBPs and MAAPs, which can be used to increase 3-HIB or MAA yields from carbohydrates when reducing equivalents produced by a MMP provided herein are available. The enzymatic transformations shown are carried out by the following enzymes: 2A) a succinyl-CoA transferase (EMA1A), succinyl-CoA ligase (EMA1B), or succinyl-CoA synthetase (EMA1C); 2B) methylmalonyl-CoA mutase (EMA2); 2C) methylmalonyl-CoA epimerase (EMA3); 2D) methylmalonyl-CoA reductase (aldehyde forming) (EMA4); 2E) methylmalonate semialdehyde reductase (EMA5); 2F) 3-HIB dehydratase

(EMA6); and 2G) methylmalonyl-CoA reductase (alcohol forming) (EMA7). 3-HIB production can be carried out by 2A, 2B, 2C, 2D and 2E; 2A, 2B, 2D and 2E; or 2A, 2B and 2G. MAA production can be carried out by 2A, 2B, 2C, 2D, 2E and 2F; 2A, 2B, 2D, 2E and 2F; or 2A, 2B, 2G and 2F.

[0017] FIG. 3 shows an exemplary FAP. The enzymatic transformations are carried out by the following enzymes: 3A) a H6P synthase (EFl), and 3B) a 6-phospho-3-hexuloisomerase (EF2).

[0018] FIG. 4 shows an exemplary FAP. The enzymatic transformations are carried out by the following enzymes: 4A) a DHA synthase (EF3), and 4B) a DHA kinase (EF4).

[0019] FIG. 5 shows exemplary metabolic pathways enabling the conversion of C0 ₂ , formate, formaldehyde, MeOH, glycerol, and glucose to 2-hydroxyisobutyric acid, 3-HIB or MAA. The enzymatic transformations shown are carried out by the following enzymes: A) methanol dehydrogenase (EM9) (see also FIG. 1, step J); B) 3-hexulose-6-phosphate synthase (EFl) (see also FIG.3, step A); C) 6-phospho-3-hexuloisomerase (EF2) (see also FIG. 3, step B); D) DHA synthase (EF3) (see also FIG. 4, step A); E) a formate reductase (EFR1); F) a formate ligase (EFR2A), formate transferase (EFR2B), or formate synthetase (EFR2C); G) a formyl-CoA reductase (EFR3); H) a formyltetrahydrofolate synthetase (EFR4); I) a methenyltetrahydrofolate cyclohydrolase (EFR5); J) a methylenetetrahydrofolate dehydrogenase (EFR6); K) a

formaldehyde-forming enzyme (EFR7); L) a glycine cleavage system (EFR8); M) a serine hydroxymethyltransferase (EFR9); N) a serine deaminase (EFR10); O) a

methylenetetrahydrofolate reductase (EFR11); P) an acetyl-CoA synthase (EFR12); Q) a pyruvate formate lyase (EFR13); R) a pyruvate dehydrogenase (EFR14A), pyruvate ferredoxin oxidoreductase (EFR14B), or pyruvate :NADP+ oxidoreductase (EFR14C); S) a formate dehydrogenase (EFR15); T) a PEP carboxylase (EFR16A) or PEP carboxykinase (EFR16B), U) a pyruvate carboxylase (EFR17); V) a malate dehydrogenase (EFR18); W) a malic enzyme (EFR19); X) a fumarase (EFR20A), fumarate reductase (EFR20B), succinyl-CoA synthetase (EFR20C), succinyl-CoA ligase (EFR20D), or succinyl-CoA transferase (EFR20E); and Y) a citrate synthase (EFR21A), aconitase (EFR21B), or alpha-ketoglutarate dehydrogenase

(EFR21C). In some embodiments, step K is spontaneous. Exemplary pathways for the conversion of sucinyl-CoA to 3-HIB or MAA can be found in FIG. 2.

3. DETAILED DESCRIPTION

3.1 Definitions

[0020] As used herein, the term "non-naturally occurring" when used in reference to a microbial organism or microorganism provided herein is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes or proteins within a 3- HIB or MAA biosynthetic pathway.

[0021] As used herein, "3-hydroxyisobutyric acid" (IUPAC name 3-hydroxy-2- methylpropanoic acid), is the acid form of 3-HIB, and it is understood that 3-HIB and 3- hydroxyisobutyric acid can be used interchangeably throughout to refer to the compound in any of its neutral or ionized forms, including any salt forms thereof. It is understood by those skilled understand that the specific form will depend on the pH. The chemical structure of 3- hydroxyisobutyric acid, acid is shown below:

[0022] As used herein, "methacrylic acid" (MAA) having the chemical formula

CH ₂ =C(CH ₃ )C0 ₂ (RJPAC name 2-methyl-2-propenoic acid), is the acid form of methacrylate, and it is understood that MAA and methacrylate can be used interchangeably throughout to refer to the compound in any of its neutral or ionized forms, including any salt forms thereof. It is understood by those skilled understand that the specific form will depend on the pH. The chemical structure of MAA is shown below:

[0023] A metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, NNOMOs can have genetic modifications to nucleic acids encoding metabolic polypeptides, or functional fragments thereof. Exemplary metabolic modifications are disclosed herein.

[0024] As used herein, the term "isolated" when used in reference to a microbial organism is intended to mean an organism that is substantially free of at least one component as the referenced microbial organism is found in nature. The term includes a microbial organism that is removed from some or all components as it is found in its natural environment. The term also includes a microbial organism that is removed from some or all components as the microbial organism is found in non-naturally occurring environments. Therefore, an isolated microbial organism is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments. Specific examples of isolated microbial organisms include partially pure microbes, substantially pure microbes and microbes cultured in a medium that is non-naturally occurring.

[0025] As used herein, the terms "microbial," "microbial organism" or "microorganism" are intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical. [0026] As used herein, the term "CoA" or "coenzyme A" is intended to mean an organic cofactor or prosthetic group (nonprotein portion of an enzyme) whose presence is required for the activity of many enzymes (the apoenzyme) to form an active enzyme system. Coenzyme A functions in certain condensing enzymes, acts in acetyl or other acyl group transfer and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation.

[0027] As used herein, the term "substantially anaerobic" when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media. The term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen.

[0028] As used herein, the term "gene disruption," or grammatical equivalents thereof, is intended to mean a genetic alteration that renders the encoded gene product inactive or attenuated. The genetic alteration can be, for example, deletion of the entire gene, deletion of a regulatory sequence required for transcription or translation, deletion of a portion of the gene which results in a truncated gene product, or by any of various mutation strategies that inactivate or attenuate the encoded gene product. One particularly useful method of gene disruption is complete gene deletion because it reduces or eliminates the occurrence of genetic reversions in the NNOMOs provided herein. A gene disruption also includes a null mutation, which refers to a mutation within a gene or a region containing a gene that results in the gene not being transcribed into RNA and/or translated into a functional gene product. Such a null mutation can arise from many types of mutations including, for example, inactivating point mutations, deletion of a portion of a gene, entire gene deletions, or deletion of chromosolmal segments. The phenotypic effect of a gene disruption can be a null mutation, which can arise from many types of mutations including inactivating point mutations, entire gene deletions, and deletions of chromosomal segments or entire chromosomes. Specific antisense nucleic acid compounds and enzyme inhibitors, such as antibiotics, can also produce null mutant phenotype, therefore being equivalent to gene disruption.

[0029] As used herein, the term "growth-coupled" when used in reference to the production of a biochemical product is intended to mean that the biosynthesis of the referenced biochemical product is produced during the growth phase of a microorganism. In a particular embodiment, the growth-coupled production can be obligatory, meaning that the biosynthesis of the referenced biochemical is an obligatory product produced during the growth phase of a microorganism. The term "growth-coupled" when used in reference to the consumption of a biochemical is intended to mean that the referenced biochemical is consumed during the growth phase of a

microorganism.

[0030] As used herein, the term "attenuate," or grammatical equivalents thereof, is intended to mean to weaken, reduce or diminish the activity or amount of an enzyme or protein.

Attenuation of the activity or amount of an enzyme or protein can mimic complete disruption if the attenuation causes the activity or amount to fall below a critical level required for a given pathway to function. However, the attenuation of the activity or amount of an enzyme or protein that mimics complete disruption for one pathway, can still be sufficient for a separate pathway to continue to function. For example, attenuation of an endogenous enzyme or protein can be sufficient to mimic the complete disruption of the same enzyme or protein for production of a fatty alcohol, fatty aldehyde or fatty acid product provided herein, but the remaining activity or amount of enzyme or protein can still be sufficient to maintain other pathways, such as a pathway that is critical for the host microbial organism to survive, reproduce or grow.

Attenuation of an enzyme or protein can also be weakening, reducing or diminishing the activity or amount of the enzyme or protein in an amount that is sufficient to increase yield of a fatty alcohol, fatty aldehyde or fatty acid product provided herein, but does not necessarily mimic complete disruption of the enzyme or protein.

[0031] "Exogenous" as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term "endogenous" refers to a referenced molecule or activity that is present in the host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term "heterologous" refers to a molecule or activity derived from a source other than the referenced species whereas

"homologous" refers to a molecule or activity derived from the host microbial organism.

Accordingly, exogenous expression of an encoding nucleic acid provided herein can utilize either or both a heterologous or homologous encoding nucleic acid.

[0032] It is understood that when more than one exogenous nucleic acid is included in a microbial organism that the more than one exogenous nucleic acids refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above. It is further understood, as disclosed herein, that such more than one exogenous nucleic acids can be introduced into the host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one exogenous nucleic acid. For example, as disclosed herein a microbial organism can be engineered to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein. In the case where two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids. Similarly, it is understood that more than two exogenous nucleic acids can be introduced into a host organism in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids, for example three exogenous nucleic acids. Thus, the number of referenced exogenous nucleic acids or biosynthetic activities refers to the number of encoding nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism.

[0033] The NNOMOs provided herein can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.

[0034] Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.

[0035] An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor. [0036] Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the NNOMO. An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species. A specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase. A second example is the separation of mycoplasma 5 '-3' exonuclease and Drosophila DNA polymerase III activity. The DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa.

[0037] In contrast, paralogs are homologs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions. Paralogs can originate or derive from, for example, the same species or from a different species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent two distinct enzymes, co- evolved from a common ancestor, that catalyze distinct reactions and have distinct functions in the same species. Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor. Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others.

[0038] A nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species. Although generally, a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene.

[0039] Therefore, in identifying and constructing the NNOMOs provided herein having 3- HIB or MAA biosynthetic capability, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and/or nonorthologous gene displacements are present in the referenced

microorganism that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes.

[0040] Orthologs, paralogs and nonorthologous gene displacements can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to indicate the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity. Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related. Additional statistical analysis to determine the significance of such matches given the size of the data set can be carried out to determine the relevance of these sequences.

[0041] Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sept-16-1998) and the following parameters: Match: 1; mismatch: -2; gap open: 5; gap extension: 2; x dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.

[0042] Feedstock refers to a substance used as a raw material for the growth of an organism, including an industrial growth process. When used in reference to a culture of microbial organisms such as a fermentation process with cells, the term refers to the raw material used to supply a carbon or other energy source for the cells. A "renewable" feedstock refers to a renewable energy source such as material derived from living organisms or their metabolic byproducts including material derived from biomass, often consisting of underutilized components like chaff. Agricultural products specifically grown for use as renewable feedstocks include, for example, corn, soybeans and cotton, primarily in the United States; flaxseed and rapeseed, primarily in Europe; sugar cane in Brazil and palm oil in South-East Asia. Therefore, the term includes the array of carbohydrates, fats and proteins derived from agricultural or animal products across the planet.

[0043] Biomass refers to any plant-derived organic matter. In the context of post- fermentation processing, biomass can be used to refer to the microbial cell mass produced during fermentation. Biomass available for energy on a sustainable basis includes herbaceous and woody energy crops, agricultural food and feed crops, agricultural crop wastes and residues, wood wastes and residues, aquatic plants, and other waste materials including some municipal wastes. Biomass feedstock compositions, uses, analytical procedures and theoretical yields are readily available from the U.S. Department of Energy and can be found described, for example, at the URL 1. eere.energy.gov/biomass/information_resources.html, which includes a database describing more than 150 exemplary kinds of biomass sources. Exemplary types of biomasses that can be used as feedstocks in the methods provided herein include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, galactose, mannose, fructose, starch and the like.

[0044] The following is a list of abbreviations and their corresponding compound or composition names used herein. These abbreviations, which are used throughout the disclosure and the figures. It is understood that one of ordinary skill in the art can readily identify these compounds/compositions by such nomenclature. MeOH or MEOH = methanol; Fald = formaldehyde; GLC = glucose; G6P = glucose-6-phosphate; H6P = hexulose-6-phosphate; F6P = fructose-6-phosphate; FDP =fructose diphosphate or fructose- 1 ,6-diphosphate; DHA = dihydroxyacetone; DHAP = dihydroxyacetone phosphate; G3P = and glyceraldehyde-3- phosphate; PYR = pyruvate; Sugar 3 = arabinose; ACCOA = acetyl-CoA; AACOA = acetoacetyl-CoA; FTHF = formyltetrahydrofolate; THF = tetrahydrofolate; E4P = erythrose-4- phosphate: Xu5P = xyulose-5 -phosphate; Ru5P = ribulose-5 -phosphate; S7P = sedoheptulose-7- phosphate: R5P = ribose-5 -phosphate; OAA = oxaloacetate; MAL = malate

[0045] It is also understood that association of multiple steps in a pathway can be indicated by linking their step identifiers with or without spaces or punctuation; for example, the following are equivalent to describe the 4-step pathway comprising Step W, Step X, Step Y and Step Z: steps WXYZ or W,X,Y,Z or W;X;Y;Z or W-X-Y-Z.

3.2 Microbial Organisms that Utilize Reducing Equivalents Produced by the Metabolism of Methanol

[0046] Provided herein are NNOMOs and MMPs engineered to improve the availability of reducing equivalents, which can be used for the production of product molecules. Exemplary product molecules include, without limitation, 3-HIB and MAA, although given the teachings and guidance provided herein, it will be recognized by one skilled in the art that any product molecule that utilizes reducing equivalents in its production can exhibit enhanced production through the biosynthetic pathways provided herein.

[0047] The present invention relates generally to biosynthetic processes, and more specifically to organisms having 3-HIB and/or MAA biosynthetic capabilities.

[0048] Methyl methacrylate (MMA) is an organic compound with the formula

CH ₂ =C(CH ₃ )C0 ₂ CH ₃ . This colorless liquid is the methyl ester of MMA and is the monomer for the production of the transparent plastic polymethyl methacrylate (PMMA).

[0049] The principal application of methyl methacrylate is the production of polymethyl methacrylate acrylic plastics. Also, methyl methacrylate is used for the production of the copolymer methyl methacrylate-butadiene-styrene (MBS), used as a modifier for PVC. Methyl methacrylate polymers and co-polymers are used for waterborne coatings, such as latex paint. Uses are also found in adhesive formulations. Contemporary applications include the use in plates that keep light spread evenly across liquid crystal display (LCD) computer and TV screens. Methyl methacrylate is also used to prepare corrosion casts of anatomical organs, such as coronary arteries of the heart.

[0050] MAA, or 2-methyl-2-propenoic acid, is a low molecular weight carboxylic acid that occurs naturally in small amounts in the oil of Roman chamomile. It is a corrosive liquid with an acrid unpleasant odor. It is soluble in warm water and miscible with most organic solvents. MAA polymerizes readily upon heating or treatment with a catalytic amount of strong acid, such as HC1. The resulting polymer is a ceramic-looking plastic. MAA is used industrially in the preparation of its esters, known collectively as methacrylates, such as methyl methacrylate. The methacrylates have numerous uses, most notably in the manufacture of polymers.

[0051] Most commercial producers apply an acetone cyanohydrin (ACH) route to produce MAA, with acetone and hydrogen cyanide as raw materials. The intermediate cyanohydrin is converted with sulfuric acid to a sulfate ester of the methacrylamide, hydrolysis of which gives ammonium bisulfate and MAA. Some producers start with an isobutylene or, equivalently, tert- butanol, which is oxidized to methacrolein, and again oxidized to MAA. MAA is then esterified with methanol to MMA. [0052] The conventional production process, using the acetone cyanohydrin route, involves the conversion of hydrogen cyanide (HCN) and acetone to acetone cyanohydrin, which then undergoes acid assisted hydrolysis and esterification with methanol to give MMA. Difficulties in handling potentially deadly HCN along with the high costs of byproduct disposal (1.2 tons of ammonium bisulfate are formed per ton of MMA) have sparked a great deal of research aimed at cleaner and more economical processes. A number of new processes have been commercialized over the last two decades and many more are close to commercialization. The Asahi "Direct Metha" route, which involves the oxidation of isobutylene to methacrolein, which is then mixed with methanol, oxidized with air, and esterified to MMA, has been described as an economical process.

[0053] Other than MMA polymers, the other major product of this industry is crude MAA, which accounts for about 20 percent of the total production of MMA. Crude MAA is processed into butyl methacrylates and/or "glacial" MAA, which is highly purified crude MAA. Glacial MAA can be used directly as a comonomer in various polymers and is also used to make a variety of small volume methacrylates. On the other hand, MAA can also be converted into MMA via esterification with methanol.

[0054] There exists a need for the development of methods for effectively producing commercial quantities of compounds, such as 3-HIB and MAA.

[0055] Accordingly, provided herein is bioderived 3-HIB produced according to the methods described herein and biobased products comprising or obtained using the bioderived 3-HIB. The biobased product can comprise at least 5%, at least 10%, at least 20%>, at least 30%>, at least 40%> or at least 50%> bioderived 3-HIB. The biobased product can comprises a portion of said bioderived 3-HIB as a repeating unit. The biobased product can be a molded product obtained by molding the biobased product.

[0056] Also provided herein is bioderived MAA produced according to the methods described herein and biobased products comprising or obtained using the bioderived MAA. The biobased product can comprise at least 5%, at least 10%>, at least 20%>, at least 30%>, at least 40%> or at least 50%) bioderived MAA. The biobased product can comprises a portion of said bioderived MAA as a repeating unit. The biobased product can be a molded product obtained by molding the biobased product.

[0057] Methanol is a relatively inexpensive organic feedstock that can be derived from synthesis gas components, CO and H ₂ , via catalysis. Methanol can be used as a source of reducing equivalents to increase the molar yield of product molecules from carbohydrates.

[0058] Methanol can be used as a redox, energy, and carbon source for the production of chemicals such as 3-HIB and MAA, and their intermediates, by employing one or more methanol metabolic enzymes as described herein, for example as shown in FIGS. 1-5. Methanol can enter central metabolism in most production hosts by employing methanol dehydrogenase (FIG. 5, step A (see also FIG. 1, step J)) along with a pathway for formaldehyde assimilation. One exemplary FAP that can utilize formaldehyde produced from the oxidation of methanol is shown in FIG. 5, which involves condensation of formaldehyde and D-ribulose-5 -phosphate to form

2_ _| _

H6P by H6P synthase (FIG. 5, step B (see also, FIG. 3, step A)). The enzyme can use Mg or

2_ _| _

Mn for maximal activity, although other metal ions are useful, and even non-metal-ion- dependent mechanisms are contemplated. H6P is converted into F6P by 6-phospho-3- hexuloisomerase (FIG. 5, step C (see also FIG. 3, step B)). Another exemplary pathway that involves the detoxification and assimilation of formaldehyde produced from the oxidation of methanol proceeds through DHA. DHA synthase (FIG. 5, step D (see also FIG. 4, step A)) is a transketolase that first transfers a glycoaldehyde group from xylulose-5 -phosphate to

formaldehyde, resulting in the formation of DHA and glyceraldehyde-3 -phosphate (G3P), which is an intermediate in glycolysis. The DHA obtained from DHA synthase can be then further phosphorylated to form DHAP by a DHA kinase. DHAP can be assimilated into glycolysis, e.g., via isomerization to G3P, and several other pathways. Alternatively, DHA and G3P can be converted by F6P aldolase to form F6P.

[0059] By combining the pathways for methanol oxidation (FIG. 5, step A (see also FIG. 1, step J)) and formaldehyde assimilation (also called formaldehyde fixation herein) (FIG. 5, steps B and C (see also FIG. 3, steps A and B) or FIG. 5, step D (see also FIG. 4, step A)) improved molar yields of product/mol methanol can be achieved for 3-HIB or MAA and their

intermediates. [0060] The yield on several substrates, including methanol, can be further increased by capturing some of the carbon lost from the conversion of pathway intermediates, e.g., pyruvate to acetyl-CoA, using one of the formate reutilization (also called formate assimilation herein) pathways shown in FIG. 5. For example, the C0 ₂ generated by conversion of pyruvate to acetyl- CoA (FIG. 5, step R) can be converted to formate via formate dehydrogenase (FIG. 5, step S). Alternatively, pyruvate formate lyase, which forms formate directly instead of C0 ₂ , can be used to convert pyruvate to acetyl-CoA (FIG. 5, step Q). Formate can be converted to formaldehyde by using: 1) formate reductase (FIG. 5, step E), 2) a formyl-CoA synthetase, transferase, or ligase along with formyl-CoA reductase (FIG. 5, steps F-G), or 3) formyltetrahydro folate synthetase, methenyltetrahydrofolate cyclohydrolase, methylenetetrahydrofolate dehydrogenase, and formaldehyde-forming enzyme (FIG. 5, steps H-I-J-K). Conversion of methylene-THF to formaldehyde alternatively will occur spontaneously. Alternatively, formate can be reutilized by converting it to pyruvate or acetyl-CoA using FIG. 5, steps H-I-J-L-M-N or FIG. 5, steps H-I-J- O-P, respectively. Formate reutilization is also useful when formate is an external carbon source. For example, formate can be obtained from organocatalytic, electrochemical, or photoelectrochemical conversion of C0 ₂ to formate. An alternative source of methanol for use in the present methods is organocatalytic, electrochemical, or photoelectrochemical conversion of C0 ₂ to methanol. By combining the pathways for methanol oxidation (FIG. 5, step A), formaldehyde assimilation (FIG. 5, Steps B and C or Step D), and formate reutilization, even higher molar yields mol product/mol methanol can be achieved for 3-HIB or MAA. By combining pathways for formaldehyde assimilation and formate reutilization, yield increases on additional substrates are also available including but not limited to glucose, glycerol, sucrose, fructose, xylose, arabinose and galactose.

[0061] In numerous engineered pathways, realization of maximum product yields based on carbohydrate feedstock is hampered by insufficient reducing equivalents or by loss of reducing equivalents to byproducts. Methanol is a relatively inexpensive organic feedstock that can be used to generate reducing equivalents by employing one or more methanol metabolic enzymes as shown in FIG. 1. Reducing equivalents can also be extracted from hydrogen and carbon monoxide by employing hydrogenase and carbon monoxide dehydrogenase enzymes, respectively. The reducing equivalents are then passed to acceptors such as oxidized ferredoxins, oxidized quinones, oxidized cytochromes, NAD(P)+, water, or hydrogen peroxide to form reduced ferredoxin, reduced quinones, reduced cytochromes, NAD(P)H, H ₂ , or water, respectively. Reduced ferredoxin, reduced quinones and NAD(P)H are particularly useful as they can serve as redox carriers for various Wood-Ljungdahl pathway, reductive TCA cycle, or product pathway enzymes. The reducing equivalents produced by the metabolism of methanol, hydrogen, and carbon monoxide can be used to power several 3-HIB and MAA pathway. In some embodiments, the reducing equivalents produced by the metabolism of methanol by one or more of the MMPs can then be used to power the glucose to 3-HIB or MAA production pathways, for example, as shown in FIG. 2.

[0062] The product yields per C-mol of substrate of microbial cells synthesizing reduced fermentation products such as 3-HIB and MAA are limited by insufficient reducing equivalents in the carbohydrate feedstock. Reducing equivalents, or electrons, can be extracted from methanol using one or more of the enzymes described in FIG. 1. The reducing equivalents are then passed to acceptors such as oxidized ferredoxins, oxidized quinones, oxidized cytochromes, NAD(P)+, water, or hydrogen peroxide to form reduced ferredoxin, reduced quinones, reduced cytochromes, NAD(P)H, H ₂ , or water, respectively. Reduced ferredoxin, reduced quinones and NAD(P)H are particularly useful as they can serve as redox carriers for various Wood-Ljungdahl pathway, reductive TCA cycle, or product pathway enzymes.

[0063] Specific examples of how additional redox availability from methanol can improve the yield of reduced products such as 3-HIB or MAA are shown.

[0064] The maximum theoretical yield of 3-HIB or MAA via the pathway shown in FIG. 2 supplemented with the reactions of the oxidative TCA cycle (e.g. , citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase) is 1.09 mol/mol.

[0065] 1 C ₆ Hi ₂ 0 ₆ → 1.09 C ₄ Hi ₀ O ₂ + 1.64 C0 ₂ + 0.55 H ₂ 0

[0066] When both feedstocks of sugar and methanol are available, the methanol can be utilized to generate reducing equivalents by employing one or more of the enzymes shown in FIG. 1. The reducing equivalents generated from methanol can be utilized to power the glucose to 3-HIB or MAA production pathways, e.g., as shown in FIG. 2. Theoretically, all carbons in glucose will be conserved, thus resulting in a maximal theoretical yield to produce 3-HIB from glucose at 2 mol 3-HIB per mol of glucose under either aerobic or anaerobic conditions as shown in FIG. 2:

[0067] 10 CH ₃ OH + 3 C ₆ Hi ₂ 0 ₆ = 6 C ₄ Hio0 ₂ + 8 H ₂ 0 + 4 C0 ₂

[0068] In a similar manner, the maximum theoretical yields of MAA can reach 2 mol/mol glucose using the reactions shown in FIGS. 1 and 2.

[0069] C ₆ Hi ₂ 0 ₆ + 0.667 CH ₃ OH + 1.333 C0 ₂ → 2 C ₄ H ₆ 0 ₄ + 1.333 H ₂ 0

[0070] C ₆ Hi ₂ 0 ₆ + 2 CH ₃ OH→ 2 C ₄ H ₈ 0 ₃ + 2 H ₂ 0

[0071] Exemplary flux distributions can demonstrate how the maximum theoretical yield of 3-HIB or MAA from glucose and glycerol can be increased by enabling assimilation of formaldehyde, formate reutilization, and extraction of reducing equivalents from an external source such as hydrogen. By combining pathways for formaldehyde assimilation, formate reutilization, reducing equivalent extraction, and product synthesis, maximum theoretical yield stoichiometries for 3-HIB or MAA on glucose and glycerol are made possible. In certain embodiments, achieving such maximum yield stoichiometries may require some oxidation of reducing equivalents (e.g., H ₂ + ½ 0 ₂ -> H ₂ 0, CO + ½ 0 ₂ -> C0 ₂ , CH ₄ 0 + 1.5 0 ₂ -> C0 ₂ + 2 H ₂ 0, C ₆ Hi ₂ 06 + 6 0 ₂ -> 6 C0 ₂ + 6 H ₂ 0) to provide sufficient energy for the substrate to product pathways to operate. Nevertheless, if sufficient reducing equivalents are available, enabling pathways for assimilation of formaldehyde, formate reutilization, extraction of reducing equivalents, and product synthesis can even lead to production of 3-HIB or MAA and their intermediates, directly from C0 ₂ .

[0072] Pathways provided herein, and particularly pathways exemplified in specific combinations presented herein, are superior over other pathways based in part on the applicant's ranking of pathways based on attributes including maximum theoretical BDO yield, maximal carbon flux, maximal production of reducing equivalents, minimal production of C0 ₂ , pathway length, number of non-native steps, thermodynamic feasibility, number of enzymes active on pathway substrates or structurally similar substrates, and having steps with currently

characterized enzymes, and furthermore, the latter pathways are even more favored by having in addition at least the fewest number of non-native steps required, the most enzymes known active on pathway substrates or structurally similar substrates, and the fewest total number of steps from central metabolism.

[0073] In a first aspect, provided herein is a NNOMO having a MMP, wherein said organism comprises at least one exogenous nucleic acid encoding a MMPE expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol. In other embodiments, the MMPE is expressed in a sufficient amount to convert methanol to

formaldehyde. In certain embodiments, the MMP comprises one or more enzymes selected from the group consisting of an EMI; an EM2; an EM3; an EM4; an EM5; an EM6; an EMI 5; an EM16; an EM8; an EM9; an EMlO; an EMU; an EM12; an EM13; and an EM14. Such organisms, in certain embodiments, advantageously allow for the production of reducing equivalents, which can then be used by the organism for the production of 3-HIB or MAA using any one of the 3-HIBPs or MAAPs provided herein.

[0074] In certain embodiments, the MMP comprises 1A, IB, 1C, ID, IE, IF, 1G, 1H, II, 1J, IK, 1L, 1M, IN, or 10 or any combination of 1 A, IB, 1C, ID, IE, IF, 1G, 1H, II, 1J, IK, 1L, 1M, IN, and 10, thereof, wherein 1A is an EMI; IB is an EM2; 1C is an EM3; ID is an EM4; IE is an EM5; IF is an EM6; 1G is an EM15; 1H is an EM16, II is an EM8; 1J is an EM9; IK is an EMlO; 1L is an EMU; lM is an EM12; lN is EM13; and 10 is EM14. In some

embodiments, IK is spontaneous. In other embodiments, IK is an EM 10. In some

embodiments, 1M is spontaneous. In other embodiments, 1M is an EM 12.

[0075] In one embodiment, the MMP comprises 1 A. In another embodiment, the MMP comprises IB. In another embodiment, the MMP comprises 1C. In yet another embodiment, the MMP comprises ID. In one embodiment, the MMP comprises IE. In another embodiment, the MMP comprises IF. In another embodiment, the MMP comprises 1G. In yet another embodiment, the MMP comprises 1H. In one embodiment, the MMP comprises II. In another embodiment, the MMP comprises 1 J. In another embodiment, the MMP comprises IK. In yet another embodiment, the MMP comprises 1L. In yet another embodiment, the MMP comprises 1M. In another embodiment, the MMP comprises IN. In yet another embodiment, the MMP comprises 10. Any combination of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen MMPEs 1A, IB, IC, ID, IE, IF, 1G, 1H, II, IJ, IK, IL, IM, IN, and 10 is also contemplated.

[0076] In some embodiments, the MMP is a MMP depicted in FIG. 1.

[0077] In one aspect, provided herein is a NNOMO having a MMP, wherein said organism comprises at least one exogenous nucleic acid encoding a MMPE expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol, wherein said MMP comprises: (i) 1A and IB, (ii) 1 J; or (iii) IJ and IK. In one embodiment, the MMP comprises 1A and IB. In another embodiment, the MMP comprises 1 J. In one embodiment, the MMP comprises IJ and IK. In certain embodiments, the MMP comprises 1A, IB, IC, ID, and IE. In some embodiments, the MMP comprises 1A, IB, IC, ID and IF. In some embodiments, the MMP comprises IJ, IC, ID and IE. In one embodiment, the MMP comprises IJ, IC, ID and IF. In another embodiment, the MMP comprises IJ and IL. In yet another embodiment, the MMP comprises IJ, IM, IN and 10. In certain embodiments, the MMP comprises IJ, IN and 10. In some embodiments, the MMP comprises IJ, IK, IC, ID and IE. In one embodiment, the MMP comprises 1 J, IK, IC, ID and IF. In some embodiments, IK is spontaneous. In other embodiments, IK is an EM10. In some embodiments, IM is spontaneous. In other

embodiments, IM is an EM 12.

[0078] In certain embodiments, the MMP comprises II. In certain embodiments, the MMP comprises 1A, IB, IC, ID, IE and II. In some embodiments, the MMP comprises 1A, IB, IC, ID, IF and II. In some embodiments, the MMP comprises 1 J, IC, ID, IE and II. In one embodiment, the MMP comprises IJ, IC, ID, IF and II. In another embodiment, the MMP comprises 1 J, IL and II. In yet another embodiment, the MMP comprises 1 J, IM, IN, 10 and II. In certain embodiments, the MMP comprises IJ, IN, 10 and II. In some embodiments, the MMP comprises 1 J, IK, IC, ID, IE and II. In one embodiment, the MMP comprises 1 J, IK,

IC, ID, IF and II. In some embodiments, IK is spontaneous. In other embodiments, IK is an EM10. In some embodiments, IM is spontaneous. In other embodiments, IM is an EM12.

[0079] In certain embodiments, the MMP comprises 1G. In certain embodiments, the MMP comprises 1A, IB, IC, ID, IE and 1G. In some embodiments, the MMP comprises 1A, IB, IC,

ID, IF and 1G. In some embodiments, the MMP comprises 1 J, IC, ID, IE and 1G. In one embodiment, the MMP comprises IJ, 1C, ID, IF and IG. In another embodiment, the MMP comprises IJ, 1L and IG. In yet another embodiment, the MMP comprises IJ, 1M, IN, 10 and IG. In certain embodiments, the MMP comprises IJ, IN, 10 and IG. In some embodiments, the MMP comprises 1 J, IK, 1C, ID, IE and IG. In one embodiment, the MMP comprises 1 J, IK, 1C, ID, IF and IG. In some embodiments, IK is spontaneous. In other embodiments, IK is an EM10. In some embodiments, 1M is spontaneous. In other embodiments, 1M is an EM12.

[0080] In certain embodiments, the MMP comprises IG and 1H. In certain embodiments, the MMP comprises 1A, IB, 1C, ID, IE, IG and 1H. In some embodiments, the MMP comprises 1A, IB, 1C, ID, IF, IG and 1H. In some embodiments, the MMP comprises IJ, 1C, ID, IE, IG and 1H. In one embodiment, the MMP comprises IJ, 1C, ID, IF, IG and 1H. In another embodiment, the MMP comprises 1 J, 1L, IG and 1H. In yet another embodiment, the MMP comprises IJ, 1M, IN, 10, IG and 1H. In certain embodiments, the MMP comprises IJ, IN, 10, IG and 1H. In some embodiments, the MMP comprises 1 J, IK, 1C, ID, IE, IG and 1H. In one embodiment, the MMP comprises 1 J, IK, 1C, ID, IF, IG and 1H. In some embodiments, IK is spontaneous. In other embodiments, IK is an EM10. In some embodiments, 1M is spontaneous. In other embodiments, 1M is an EM 12.

[0081] In certain embodiments, the formation of 5-hydroxymethylglutathione from

formaldehyde is spontaneous (see, e.g., FIG. 1, step M). In some embodiments, the formation of 5-hydroxymethylglutathione from formaldehyde is catalyzed by an EM12 (see, e.g., FIG. 1, step M). In certain embodiments, the formation of methylene-THF from formaldehyde is

spontaneous (see, e.g., FIG. 1, step K). In certain embodiments, the formation of methylene- THF from formaldehyde is catalyzed by an EM 10 (see, e.g., FIG. 1, step K).

[0082] In certain embodiments, the organism comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a MMPE. In certain embodiments, the organism comprises two exogenous nucleic acids, each encoding a MMPE. In certain embodiments, the organism comprises three exogenous nucleic acids, each encoding a MMPE. In certain embodiments, the organism comprises four exogenous nucleic acids, each encoding a MMPE. In certain embodiments, the organism comprises five exogenous nucleic acids, each encoding a MMPE. In certain embodiments, the organism comprises six exogenous nucleic acids, each encoding a MMPE. In certain embodiments, the organism comprises seven exogenous nucleic acids, each encoding a MMPE.

[0083] Any NNOMO comprising a MMP and engineered to comprise a MMPE, such as those provided herein, can be engineered to further comprise one or more 3-HIBPEs or MAAPEs. Such organisms can further be engineered to comprise a FAP, a FRP or both a FAP and a FRP as provided herein.

[0084] In one embodiment, the NNOMO further comprises a 3-HIBP, wherein said organism comprises at least one exogenous nucleic acid encoding a 3-HIBPE expressed in a sufficient amount to produce 3-HIB. In certain embodiments, the 3-HIBPE is selected from the group consisting of an EMA1A, EMA1B, or EMA1C; EMA2; EMA3; EMA4; EMA5; and EMA7.

[0085] In another embodiment, the NNOMO further comprises a MAAP, wherein said organism comprises at least one exogenous nucleic acid encoding a MAAPE expressed in a sufficient amount to produce MAA. In certain embodiments, the MAAPE is selected from the group consisting of an EMA1A, EMA1B, or EMA1C; EMA2; EM A3; EMA4; EMA5; EMA6; and EMA7.

[0086] In some embodiments, the NNOMOs having a 3-HIBP include a set of 3-HIBPEs. In other embodiments, the NNOMOs having a MAAP include a set of MAAPEs.

[0087] Enzymes, genes and methods for engineering pathways from succinate or succinyl- CoA to various products, such as 3-HIB or MAA, into a microorganism, are now known in the art, as are enzymes for the conversion of glucose to phosphoenolpyruvate (PEP),

phosphoenolpyruvate to oxaloacetate, oxaloacetate to malate, malate to fumarate, and fumarate to succinate (see, e.g., U.S. Publ. No. 2011/0201089 and WO 2012/135789, each of which is herein incorporated by reference in its entirety). A set of 3-HIBPEs or MAAPEs represents a group of enzymes that can convert succinate to 3-HIB or MAA, respectively, as shown in FIG. 2. The additional reducing equivalents obtained from the MMPs, as disclosed herein, improve the yields of all these products when utilizing carbohydrate-based feedstock. [0088] Exemplary enzymes for the conversion of succinate to 3-HIB include EMA1 A, EMA1B, or EMA1C (FIG. 2, step A); EMA2 (FIG. 2, step B); EMA3 (FIG. 2, step C); EMA4 (FIG. 2, step D); EMA5 (FIG. 2, step E); and EMA7 (FIG. 2, step G).

[0089] In one aspect, provided herein is a NNOMO, comprising (1) a MMP, wherein said organism comprises at least one exogenous nucleic acid encoding a MMPE in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol; and (2) a 3-HIBP, wherein said organism comprises at least one exogenous nucleic acid encoding a 3- HIBPE expressed in a sufficient amount to produce 3-HIB. In one embodiment, the at least one exogenous nucleic acid encoding the MMPE enhances the availability of reducing equivalents in the presence of methanol in a sufficient amount to increase the amount of 3-HIB produced by the non-naturally microbial organism. In some embodiments, the MMP comprises any of the various combinations of MMPEs described above or elsewhere herein.

[0090] In certain embodiments, (1) the MMP comprises: 1A, IB, 1C, ID, IE, IF, 1G, 1H, II, 1J, IK, 1L, 1M, IN, or 10 or any combination of 1A, IB, 1C, ID, IE, IF, 1G, 1H, II, 1J, IK, 1L, 1M, IN, or 10, thereof, wherein 1A is an EMI; IB is an EM2; 1C is an EM3; ID is an EM4; IE is an EM5; IF is an EM6; 1G is an EM15; 1H is an EM16, II is an EM8; 1J is an EM9; IK is spontaneous or EM 10; 1L is an EMI 1; 1M is spontaneous or an EM 12; IN is EM 13 and 10 is EM14; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D, 2E or 2G, or any combination thereof, wherein 2A is an EMA1A, EMA1B, or EMA1C; 2B is an EMA2; 2C is an EM A3; 2D is an EMA4; 2E is an EMA5; and 2G is an EMA7. In some embodiments, IK is spontaneous. In other embodiments, IK is an EM10. In some embodiments, 1M is spontaneous. In other embodiments, 1M is an EM12. In some embodiments, 2A is an EMA1A. In other

embodiments, 2 A is an EMA1B. In certain embodiments, 2 A is an EMA1C.

[0091] In one embodiment, the 3-HIBP comprises 2 A. In another embodiment, the 3-HIBP comprises 2B. In an embodiment, the 3-HIBP comprises 2C. In another embodiment, the 3- HIBP comprises 2D. In another embodiment, the 3-HIBP comprises 2E. In another

embodiment, the 3-HIBP comprises 2G. Any combination of two, three, four, five or six 3- HIBPEs 2A, 2B, 2C, 2D, 2E and 2G is also contemplated. [0092] In some embodiments, the MMP is a MMP depicted in FIG. 1 , and the 3-HIBP is a 3- HIBP depicted in FIG. 2.

[0093] Exemplary sets of 3-HIBPEs to convert succinate to 3-HIB, according to FIG. 2, include (i) 2A, 2B, 2C, 2D and 2E; (ii) 2A, 2B, 2D and 2E; and (iii) 2A, 2B and 2G.

[0094] In one embodiment, (1) the MMP comprises 1A and IB; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In another embodiment, (1) the MMP comprises 1 J; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the MMP comprises 1J and IK; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the MMP comprises 1A, IB, IC, ID, and IE; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the MMP comprises 1A, IB, IC, ID and IF; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the MMP comprises 1J, IC, ID and IE; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the MMP comprises 1 J, IC, ID and IF; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In another embodiment, (1) the MMP comprises 1J and 1L; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In yet another embodiment, (1) the MMP comprises 1 J, 1M, IN and 10; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the MMP comprises 1J, IN and 10; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the MMP comprises 1 J, IK, IC, ID and IE; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the MMP comprises 1 J, IK, IC, ID and IF; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the MMP comprises II; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the MMP comprises 1A, IB, IC, ID, IE and II; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the MMP comprises 1A, IB, IC, ID, IF and II; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the MMP comprises 1 J, IC, ID, IE and II; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the MMP comprises 1J, IC, ID, IF and II; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In another embodiment, (1) the MMP comprises 1J, 1L and II; and (2) the 3-HIBP comprises 2 A, 2B, 2C, 2D and 2E. In yet another embodiment, (1) the MMP comprises 1 J, 1M, IN, 10 and II; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the MMP comprises 1J, IN, 10 and II; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the MMP comprises IJ, IK, 1C, ID, IE and II; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the MMP comprises 1 J, IK, 1C, ID, IF and II; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the MMP comprises IG; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IE and IG; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IF and IG; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the MMP comprises IJ, 1C, ID, IE and IG; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the MMP comprises IJ, 1C, ID, IF and IG; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In another embodiment, (1) the MMP comprises 1 J, 1L and IG; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In yet another embodiment, (1) the MMP comprises IJ, 1M, IN, 10 and IG; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the MMP comprises IJ, IN, 10 and IG; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the MMP comprises 1 J, IK, 1C, ID, IE and IG; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the MMP comprises 1 J, IK, 1C, ID, IF and IG; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the MMP comprises IG and IH; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IE, IG and IH; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IF, IG and IH; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the MMP comprises IJ, 1C, ID, IE, IG and IH; and (2) the 3- HIBP comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the MMP comprises 1 J, 1C, ID, IF, IG and IH; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In another embodiment, (1) the MMP comprises IJ, 1L, IG and IH; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In yet another embodiment, (1) the MMP comprises IJ, 1M, IN, 10, IG and IH; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the MMP comprises IJ, IN, 10, IG and IH; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the MMP comprises IJ, IK, 1C, ID, IE, IG and IH; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the MMP comprises IJ, IK, 1C, ID, IF, IG and IH; and (2) the 3-HIBP comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, 1M is spontaneous. In other embodiments, 1M is an EM12. In some embodiments, 2A is an EMA1A. In other embodiments, 2 A is an EMA1B. In certain embodiments, 2 A is an EMA1C.

[0095] In one embodiment, (1) the MMP comprises 1A and IB; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In another embodiment, (1) the MMP comprises 1 J; and (2) the 3- HIBP comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the MMP comprises IJ and IK; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the MMP comprises 1A, IB, 1C, ID, and IE; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the MMP comprises 1A, IB, 1C, ID and IF; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the MMP comprises 1 J, 1C, ID and IE; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the MMP comprises 1 J, 1C, ID and IF; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In another embodiment, (1) the MMP comprises IJ and 1L; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In yet another embodiment, (1) the MMP comprises IJ, 1M, IN and 10; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the MMP comprises IJ, IN and 10; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the MMP comprises IJ, IK, 1C, ID and IE; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the MMP comprises 1 J, IK, 1C, ID and IF; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the MMP comprises II; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IE and II; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IF and II; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the MMP comprises IJ, 1C, ID, IE and II; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the MMP comprises IJ, 1C, ID, IF and II; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In another embodiment, (1) the MMP comprises 1 J, 1L and II; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In yet another embodiment, (1) the MMP comprises 1 J, 1M, IN, 10 and II; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the MMP comprises 1 J, IN, 10 and II; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the MMP comprises IJ, IK, 1C, ID, IE and II; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the MMP comprises 1 J, IK, 1C, ID, IF and II; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the MMP comprises 1G; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IE and IG; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IF and IG; and (2) the 3- HIBP comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the MMP comprises 1J, 1C, ID, IE and IG; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the MMP comprises 1 J, 1C, ID, IF and IG; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In another embodiment, (1) the MMP comprises 1J, 1L and IG; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In yet another embodiment, (1) the MMP comprises 1 J, 1M, IN, 10 and IG; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the MMP comprises 1 J, IN, 10 and IG; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the MMP comprises 1 J, IK, 1C, ID, IE and IG; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the MMP comprises 1J, IK, 1C, ID, IF and IG; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the MMP comprises IG and 1H; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IE, IG and 1H; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IF, IG and 1H; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the MMP comprises 1J, 1C, ID, IE, IG and 1H; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the MMP comprises 1J, 1C, ID, IF, IG and 1H; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In another embodiment, (1) the MMP comprises 1J, 1L, IG and 1H; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In yet another embodiment, (1) the MMP comprises 1 J, 1M, IN, 10, IG and 1H; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the MMP comprises 1J, IN, 10, IG and 1H; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the MMP comprises 1J, IK, 1C, ID, IE, IG and 1H; and (2) the 3- HIBP comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the MMP comprises 1 J, IK, 1C, ID, IF, IG and 1H; and (2) the 3-HIBP comprises 2A, 2B, 2D and 2E. In some embodiments, 1M is spontaneous. In other embodiments, 1M is an EM12. In some embodiments, 2A is an EMA1A. In other embodiments, 2 A is an EMA1B. In certain embodiments, 2 A is an EMA1C.

[0096] In one embodiment, (1) the MMP comprises 1A and IB; and (2) the 3-HIBP comprises 2A, 2B and 2G. In another embodiment, (1) the MMP comprises 1 J; and (2) the 3-

HIBP comprises 2A, 2B and 2G. In one embodiment, (1) the MMP comprises 1J and IK; and

(2) the 3-HIBP comprises 2A, 2B and 2G. In certain embodiments, (1) the MMP comprises 1A, IB, IC, ID, and IE; and (2) the 3-HIBP comprises 2A, 2B and 2G. In some embodiments, (1) the MMP comprises 1A, IB, IC, ID and IF; and (2) the 3-HIBP comprises 2A, 2B and 2G. In some embodiments, (1) the MMP comprises 1 J, IC, ID and IE; and (2) the 3-HIBP comprises 2A, 2B and 2G. In one embodiment, (1) the MMP comprises IJ, IC, ID and IF; and (2) the 3- HIBP comprises 2A, 2B and 2G. In another embodiment, (1) the MMP comprises IJ and 1L; and (2) the 3-HIBP comprises 2A, 2B and 2G. In yet another embodiment, (1) the MMP comprises 1 J, 1M, IN and 10; and (2) the 3-HIBP comprises 2A, 2B and 2G. In certain embodiments, (1) the MMP comprises IJ, IN and 10; and (2) the 3-HIBP comprises 2A, 2B and 2G. In some embodiments, (1) the MMP comprises IJ, IK, IC, ID and IE; and (2) the 3-HIBP comprises 2A, 2B and 2G. In one embodiment, (1) the MMP comprises 1 J, IK, IC, ID and IF; and (2) the 3-HIBP comprises 2A, 2B and 2G. In certain embodiments, (1) the MMP comprises II; and (2) the 3-HIBP comprises 2A, 2B and 2G. In certain embodiments, (1) the MMP comprises 1A, IB, IC, ID, IE and II; and (2) the 3-HIBP comprises 2A, 2B and 2G. In some embodiments, (1) the MMP comprises 1A, IB, IC, ID, IF and II; and (2) the 3-HIBP comprises 2A, 2B and 2G. In some embodiments, (1) the MMP comprises IJ, IC, ID, IE and II; and (2) the 3-HIBP comprises 2A, 2B and 2G. In one embodiment, (1) the MMP comprises IJ, IC, ID, IF and II; and (2) the 3-HIBP comprises 2A, 2B and 2G. In another embodiment, (1) the MMP comprises 1 J, 1L and II; and (2) the 3-HIBP comprises 2A, 2B and 2G. In yet another embodiment, (1) the MMP comprises IJ, 1M, IN, 10 and II; and (2) the 3-HIBP comprises 2A, 2B and 2G. In certain embodiments, (1) the MMP comprises IJ, IN, 10 and II; and (2) the 3- HIBP comprises 2A, 2B and 2G. In some embodiments, (1) the MMP comprises IJ, IK, IC, ID, IE and II; and (2) the 3-HIBP comprises 2A, 2B and 2G. In one embodiment, (1) the MMP comprises IJ, IK, IC, ID, IF and II; and (2) the 3-HIBP comprises 2A, 2B and 2G. In certain embodiments, (1) the MMP comprises 1G; and (2) the 3-HIBP comprises 2A, 2B and 2G. In certain embodiments, (1) the MMP comprises 1A, IB, IC, ID, IE and 1G; and (2) the 3-HIBP comprises 2A, 2B and 2G. In some embodiments, (1) the MMP comprises 1A, IB, IC, ID, IF and 1G; and (2) the 3-HIBP comprises 2A, 2B and 2G. In some embodiments, (1) the MMP comprises 1 J, IC, ID, IE and 1G; and (2) the 3-HIBP comprises 2A, 2B and 2G. In one embodiment, (1) the MMP comprises IJ, IC, ID, IF and 1G; and (2) the 3-HIBP comprises 2A, 2B and 2G. In another embodiment, (1) the MMP comprises IJ, 1L and 1G; and (2) the 3-HIBP comprises 2A, 2B and 2G. In yet another embodiment, (1) the MMP comprises 1 J, 1M, IN, 10 and IG; and (2) the 3-HIBP comprises 2A, 2B and 2G. In certain embodiments, (1) the MMP comprises IJ, IN, 10 and IG; and (2) the 3-HIBP comprises 2A, 2B and 2G. In some embodiments, (1) the MMP comprises IJ, IK, IC, ID, IE and IG; and (2) the 3-HIBP comprises 2A, 2B and 2G. In one embodiment, (1) the MMP comprises IJ, IK, IC, ID, IF and IG; and (2) the 3-HIBP comprises 2A, 2B and 2G. In certain embodiments, (1) the MMP comprises IG and IH; and (2) the 3-HIBP comprises 2A, 2B and 2G. In certain embodiments, (1) the MMP comprises 1A, IB, IC, ID, IE, IG and IH; and (2) the 3-HIBP comprises 2A, 2B and 2G. In some embodiments, (1) the MMP comprises 1A, IB, IC, ID, IF, IG and IH; and (2) the 3-HIBP comprises 2A, 2B and 2G. In some embodiments, (1) the MMP comprises 1 J, IC, ID, IE, IG and IH; and (2) the 3-HIBP comprises 2A, 2B and 2G. In one embodiment, (1) the MMP comprises 1 J, IC, ID, IF, IG and IH; and (2) the 3-HIBP comprises 2A, 2B and 2G. In another embodiment, (1) the MMP comprises IJ, 1L, IG and IH; and (2) the 3-HIBP comprises 2 A, 2B and 2G. In yet another embodiment, (1) the MMP comprises 1 J, 1M, IN, 10, IG and IH; and (2) the 3-HIBP comprises 2A, 2B and 2G. In certain embodiments, (1) the MMP comprises 1 J, IN, 10, IG and IH; and (2) the 3-HIBP comprises 2A, 2B and 2G. In some embodiments, (1) the MMP comprises IJ, IK, IC, ID, IE, IG and IH; and (2) the 3-HIBP comprises 2A, 2B and 2G. In one embodiment, (1) the MMP comprises IJ, IK, IC, ID, IF, IG and IH; and (2) the 3-HIBP comprises 2A, 2B and 2G. In some embodiments, 1M is spontaneous. In other embodiments, 1M is an EM12. In some embodiments, 2A is an EMA1A. In other embodiments, 2A is an EMA1B. In certain embodiments, 2 A is an EMA1C.

[0097] In one embodiment, the NNOMO comprises (1) a MMP comprising 1A and IB; 1 J; IJ and IK; 1A, IB, IC, ID, and IE; 1A, IB, IC, ID and IF; IJ, IC, ID and IE; IJ, IC, ID and IF; IJ and 1L; IJ, 1M, IN and 10; IJ, IN and 10; IJ, IK, IC, ID and IE; IJ, IK, IC, ID and IF; II; 1A, IB, IC, ID, IE and II; 1A, IB, IC, ID, IF and II; IJ, IC, ID, IE and II; IJ, IC, ID, IF and II; IJ, 1L and II; IJ, 1M, IN, 10 and II; IJ, IN, 10 and II; IJ, IK, IC, ID, IE and II; IJ, IK, IC, ID, IF and II; IG; 1A, IB, IC, ID, IE and IG; 1A, IB, IC, ID, IF and IG; IJ, IC, ID, IE and IG; IJ, IC, ID, IF and IG; IJ, 1L and IG; IJ, 1M, IN, 10 and IG; IJ, IN, 10 and IG; IJ, IK, IC, ID, IE and IG; IJ, IK, IC, ID, IF and IG; IG and IH; 1A, IB, IC, ID, IE, IG and IH; 1A, IB, IC, ID, IF, IG and IH; IJ, IC, ID, IE, IG and IH; IJ, IC, ID, IF, IG and IH; IJ, 1L, IG and IH; IJ, 1M, IN, 10, IG and IH; IJ, IN, 10, IG and IH; IJ, IK, IC, ID, IE, IG and IH; or 1 J, IK, IC, ID, IF, IG and IH; and (2) a 3-HIBP. In some embodiments, IK is spontaneous. In other embodiments, IK is an EM10. In some embodiments, 1M is spontaneous. In other embodiments, 1M is an EM 12.

[0098] Any MMP provided herein can be combined with any 3-HIBP provided herein.

[0099] In certain embodiments, the 3-HIBP further comprises enzymes depicted in FIG. 5. In one embodiment, the 3-HIBP further comprises 5T, 5U, 5V, 5W, 5X and/or 5Y, wherein 5T is a PEP carboxylase (EFR16A) or PEP carboxykinase (EFR16B); 5U is a pyruvate carboxylase (EFR17); 5V is a malate dehydrogenase (EFR18); 5W is a malic enzyme (EFR19); 5X is a fumarase (EFR20A), fumarate reductase (EFR20B), succinyl-CoA synthetase (EFR20C), succinyl-CoA ligase (EFR20D), or succinyl-CoA transferase (EFR20E); and 5Y is a citrate synthase (EFR21A), aconitase (EFR21B), or alpha-ketoglutarate dehydrogenase (EFR21C). In one embodiment, the 3-HIBP comprises 5T. In another embodiment, the 3-HIBP comprises 5U. In another embodiment, the 3-HIBP comprises 5V. In other embodiment, the 3-HIBP comprises 5W. In another embodiment, the 3-HIBP comprises 5X. In another embodiment, the 3-HIBP comprises 5Y. In some embodiments, the 3-HIBP comprises 5T, 5V and 5X. In another embodiment, the 3-HIBP comprises 5U, 5V and 5X. In another embodiment, the 3-HIBP comprises 5W and 5X. In one embodiment, 5T is EFR16A. In other embodiments, 5T is EFR16B. In some embodiments, 5X is EFR20A. In other embodiments, 5X is EFR20B. In other embodiments, 5X is EFR20C. In one embodiment, 5X is EFR20D. In another

embodiment, 5X is EFR20E. In one embodiment, 5Y is EFR21A. In an embodiment, 5Y is EFR21B. In another embodiment, 5Y is EFR21C.

[0100] Exemplary enzymes for the conversion of succinate to MAA include EMA1 A, EMA1B, or EMA1C (FIG. 2, step A); EMA2 (FIG. 2, step B); EMA3 (FIG. 2, step C); EMA4 (FIG. 2, step D); EMA5 (FIG. 2, step E); EMA6 (FIG. 2, step F); and EMA7 (FIG. 2, step G).

[0101] In one aspect, provided herein is a NNOMO, comprising (1) a MMP, wherein said organism comprises at least one exogenous nucleic acid encoding a MMPE in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol; and (2) a MAAP, wherein said organism comprises at least one exogenous nucleic acid encoding a MAAPE expressed in a sufficient amount to produce MAA. In one embodiment, the at least one exogenous nucleic acid encoding the MMPE enhances the availability of reducing equivalents in the presence of methanol in a sufficient amount to increase the amount of MAA produced by the non-naturally microbial organism. In some embodiments, the MMP comprises any of the various combinations of MMPEs described above or elsewhere herein.

[0102] In certain embodiments, (1) the MMP comprises: 1A, IB, 1C, ID, IE, IF, 1G, 1H, II, 1J, IK, 1L, 1M, IN, or 10 or any combination of 1A, IB, 1C, ID, IE, IF, 1G, 1H, II, 1J, IK, 1L, 1M, IN, or 10, thereof, wherein 1A is an EMI; IB is an EM2; 1C is an EM3; ID is an EM4; IE is an EM5; IF is an EM6; 1G is an EM15; 1H is an EM16, II is an EM8; 1J is an EM9; IK is spontaneous or EM 10; 1L is an EMI 1; 1M is spontaneous or an EM 12; IN is EM 13 and 10 is EM14; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E, 2F or 2G, or any combination thereof, wherein 2A is an EMA1A, EMA1B, or EMA1C; 2B is an EMA2; 2C is an EM A3; 2D is an EMA4; 2E is an EMA5; 2F is an EMA6; and 2G is an EMA7. In some embodiments, IK is spontaneous. In other embodiments, IK is an EM10. In some embodiments, 1M is spontaneous. In other embodiments, 1M is an EM 12. In some embodiments, 2 A is an EMA1A. In other embodiments, 2 A is an EMA1B. In certain embodiments, 2 A is an EMA1C.

[0103] In one embodiment, the MAAP comprises 2A. In another embodiment, the MAAP comprises 2B. In an embodiment, the MAAP comprises 2C. In another embodiment, the MAAP comprises 2D. In another embodiment, the MAAP comprises 2E. In another

embodiment, the MAAP comprises 2F. In another embodiment, the MAAP comprises 2G. Any combination of two, three, four, five, six or seven MAAPEs 2A, 2B, 2C, 2D, 2E, 2F and 2G is also contemplated.

[0104] In some embodiments, the MMP is a MMP depicted in FIG. 1, and the MAAP is a MAAP depicted in FIG. 2.

[0105] Exemplary sets of MAAPEs to convert succinate to MAA, according to FIG. 2, include (i) 2A, 2B, 2C, 2D, 2E and 2F; (ii) 2A, 2B, 2D, 2E and 2F; and (iii) 2A, 2B, 2G and 2F.

[0106] In one embodiment, (1) the MMP comprises 1A and IB; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In another embodiment, (1) the MMP comprises 1 J; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises 1J and IK; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises 1A, IB, IC, ID, and IE; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1A, IB, IC, ID and IF; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1 J, IC, ID and IE; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises 1J, IC, ID and IF; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In another embodiment, (1) the MMP comprises 1J and 1L; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In yet another embodiment, (1) the MMP comprises 1 J, 1M, IN and 10; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises 1J, IN and 10; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1J, IK, IC, ID and IE; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises 1 J, IK, IC, ID and IF; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises II; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises 1A, IB, IC, ID, IE and II; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1A, IB, IC, ID, IF and II; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1J, IC, ID, IE and II; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises 1 J, IC, ID, IF and II; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In another embodiment, (1) the MMP comprises 1 J, 1L and II; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In yet another embodiment, (1) the MMP comprises 1J, 1M, IN, 10 and II; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises 1 J, IN, 10 and II; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1J, IK, IC, ID, IE and II; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises 1 J, IK, IC, ID, IF and II; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises 1G; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises 1A, IB, IC, ID, IE and 1G; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1A, IB, IC, ID, IF and 1G; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1 J, IC, ID, IE and 1G; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises IJ, 1C, ID, IF and IG; and (2) the MAAP comprises 2 A, 2B, 2C, 2D, 2E and 2F. In another embodiment, (1) the MMP comprises IJ, IL and IG; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In yet another embodiment, (1) the MMP comprises 1 J, 1M, IN, 10 and IG; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises IJ, IN, 10 and IG; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises IJ, IK, 1C, ID, IE and IG; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises IJ, IK, 1C, ID, IF and IG; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises IG and 1H; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises 1A, IB,

IC, ID, IE, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IF, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1 J, 1C,

ID, IE, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises IJ, 1C, ID, IF, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In another embodiment, (1) the MMP comprises IJ, IL, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In yet another embodiment, (1) the MMP comprises IJ, 1M, IN, 10, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises IJ, IN, 10, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises IJ, IK, 1C, ID, IE, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises IJ, IK, 1C, ID, IF, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, 1M is spontaneous. In other embodiments, 1M is an EM12. In some embodiments, 2A is an EMA1A. In other

embodiments, 2 A is an EMA1B. In certain embodiments, 2 A is an EMA1C.

[0107] In one embodiment, (1) the MMP comprises 1A and IB; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In another embodiment, (1) the MMP comprises 1 J; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises IJ and IK; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises 1A, IB, 1C, ID, and IE; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F.

In some embodiments, (1) the MMP comprises 1A, IB, 1C, ID and IF; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1 J, 1C, ID and IE; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises 1 J, 1C, ID and IF; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In another embodiment, (1) the MMP comprises 1J and 1L; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In yet another embodiment, (1) the MMP comprises 1J, 1M, IN and 10; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises 1 J, IN and 10; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1 J, IK, 1C, ID and IE; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises 1J, IK, 1C, ID and IF; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises II; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IE and II; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IF and II; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1J, 1C, ID, IE and II; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises 1 J, 1C, ID, IF and II; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In another embodiment, (1) the MMP comprises 1J, 1L and II; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In yet another embodiment, (1) the MMP comprises 1 J, 1M, IN, 10 and II; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises 1 J, IN, 10 and II; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1J, IK, 1C, ID, IE and II; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises 1J, IK, 1C, ID, IF and II; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises 1G; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IE and 1G; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IF and 1G; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1 J, 1C, ID, IE and 1G; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises 1J, 1C, ID, IF and 1G; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In another embodiment, (1) the MMP comprises 1J, 1L and 1G; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In yet another embodiment, (1) the MMP comprises 1J, 1M, IN, 10 and IG; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises 1J, IN, 10 and IG; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1 J, IK, 1C, ID, IE and IG; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises 1J, IK, 1C, ID, IF and IG; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises IG and 1H; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IE, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IF, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1J, 1C, ID, IE, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises 1 J, 1C, ID, IF, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In another

embodiment, (1) the MMP comprises 1J, 1L, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In yet another embodiment, (1) the MMP comprises 1 J, 1M, IN, 10, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the MMP comprises 1 J, IN, 10, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the MMP comprises 1J, IK, 1C, ID, IE, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the MMP comprises 1 J, IK, 1C, ID, IF, IG and 1H; and (2) the MAAP comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, 1M is spontaneous. In other embodiments, 1M is an EM12. In some embodiments, 2A is an EMA1A. In other embodiments, 2 A is an EMA1B. In certain embodiments, 2 A is an EMA1C.

[0108] In one embodiment, (1) the MMP comprises 1A and IB; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In another embodiment, (1) the MMP comprises 1 J; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the MMP comprises 1J and IK; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the MMP comprises 1A, IB, 1C, ID, and IE; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the MMP comprises 1A, IB, 1C, ID and IF; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the MMP comprises 1J, 1C, ID and IE; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the MMP comprises 1 J, 1C, ID and IF; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In another embodiment, (1) the MMP comprises 1J and 1L; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In yet another embodiment, (1) the MMP comprises IJ, 1M, IN and 10; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the MMP comprises IJ, IN and 10; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the MMP comprises IJ, IK, 1C, ID and IE; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the MMP comprises 1 J, IK, 1C, ID and IF; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the MMP comprises II; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IE and II; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IF and II; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the MMP comprises 1 J, 1C, ID, IE and II; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the MMP comprises IJ, 1C, ID, IF and II; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In another embodiment, (1) the MMP comprises 1 J, 1L and II; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In yet another embodiment, (1) the MMP comprises 1 J, 1M, IN, 10 and II; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In certain embodiments,

(1) the MMP comprises 1 J, IN, 10 and II; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the MMP comprises IJ, IK, 1C, ID, IE and II; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the MMP comprises IJ, IK, 1C, ID, IF and II; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the MMP comprises IG; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IE and IG; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the MMP comprises 1A, IB, 1C, ID, IF and IG; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the MMP comprises 1 J, 1C, ID, IE and IG; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the MMP comprises 1 J, 1C, ID, IF and IG; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In another embodiment, (1) the MMP comprises IJ, 1L and IG; and (2) the MAAP comprises 2 A, 2B, 2G and 2F. In yet another embodiment, (1) the MMP comprises IJ, 1M, IN, 10 and IG; and

(2) the MAAP comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the MMP comprises

1 J, IN, 10 and IG; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the MMP comprises IJ, IK, 1C, ID, IE and IG; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the MMP comprises IJ, IK, 1C, ID, IF and IG; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the MMP comprises IG and 1H; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the MMP comprises 1A, IB, IC, ID, IE, IG and IH; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the MMP comprises 1A, IB, IC, ID, IF, IG and IH; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the MMP comprises IJ, IC, ID, IE, IG and IH; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the MMP comprises 1 J, IC, ID, IF, IG and IH; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In another embodiment, (1) the MMP comprises IJ, 1L, IG and IH; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In yet another embodiment, (1) the MMP comprises 1 J, 1M, IN, 10, IG and IH; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the MMP comprises IJ, IN, 10, IG and IH; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the MMP comprises IJ, IK, IC, ID, IE, IG and IH; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the MMP comprises 1 J, IK, IC, ID, IF, IG and IH; and (2) the MAAP comprises 2A, 2B, 2G and 2F. In some embodiments, 1M is spontaneous. In other embodiments, 1M is an EM12. In some embodiments, 2A is an EMA1A. In other embodiments, 2A is an EMA1B. In certain embodiments, 2A is an EMA1C.

[0109] In one embodiment, the NNOMO comprises (1) a MMP comprising 1A and IB; 1 J; IJ and IK; 1A, IB, IC, ID, and IE; 1A, IB, IC, ID and IF; IJ, IC, ID and IE; IJ, IC, ID and IF; IJ and 1L; IJ, 1M, IN and 10; IJ, IN and 10; IJ, IK, IC, ID and IE; IJ, IK, IC, ID and IF; II; 1A, IB, IC, ID, IE and II; 1A, IB, IC, ID, IF and II; IJ, IC, ID, IE and II; IJ, IC, ID, IF and II; IJ, 1L and II; IJ, 1M, IN, 10 and II; 1 J, IN, 10 and II; IJ, IK, IC, ID, IE and II; IJ, IK, IC, ID, IF and II; IG; 1A, IB, IC, ID, IE and IG; 1A, IB, IC, ID, IF and IG; IJ, IC, ID, IE and IG; IJ, IC, ID, IF and IG; IJ, 1L and IG; IJ, 1M, IN, 10 and IG; IJ, IN, 10 and IG; IJ, IK, IC, ID, IE and IG; IJ, IK, IC, ID, IF and IG; IG and IH; 1A, IB, IC, ID, IE, IG and IH; 1A, IB, IC, ID, IF, IG and IH; IJ, IC, ID, IE, IG and IH; IJ, IC, ID, IF, IG and IH; IJ, 1L, IG and IH; IJ, 1M, IN, 10, IG and IH; IJ, IN, 10, IG and IH; IJ, IK, IC, ID, IE, IG and IH; or IJ, IK, IC, ID, IF, IG and IH; and (2) a MAAP. In some embodiments, IK is spontaneous. In other embodiments, IK is an EM10. In some embodiments, 1M is spontaneous. In other embodiments, 1M is an EM 12.

[0110] Any MMP provided herein can be combined with any MAAP provided herein. [0111] In certain embodiments, the MAAP further comprises enzymes depicted in FIG. 5. In one embodiment, the MAAP further comprises 5T, 5U, 5V, 5W, 5X and/or 5Y. In one embodiment, the MAAP comprises 5T. In another embodiment, the MAAP comprises 5U. In another embodiment, the MAAP comprises 5V. In other embodiment, the MAAP comprises 5W. In another embodiment, the MAAP comprises 5X. In another embodiment, the MAAP comprises 5Y. In some embodiments, the MAAP comprises 5T, 5V and 5X. In another embodiment, the MAAP comprises 5U, 5V and 5X. In another embodiment, the MAAP comprises 5W and 5X. In one embodiment, 5T is EFR16A. In other embodiments, 5T is EFR16B. In some embodiments, 5X is EFR20A. In other embodiments, 5X is EFR20B. In other embodiments, 5X is EFR20C. In one embodiment, 5X is EFR20D. In another

embodiment, 5X is EFR20E. In one embodiment, 5Y is EFR21A. In an embodiment, 5Y is EFR21B. In another embodiment, 5Y is EFR21C.

[0112] Also provided herein are exemplary pathways, which utilize formaldehyde produced from the oxidation of methanol (e.g., as provided in FIG. 1 , step J) in the formation of intermediates of certain central metabolic pathways that can be used for the formation of biomass. One exemplary FAP that can utilize formaldehyde produced from the oxidation of methanol (e.g., as provided in FIG. 1) is shown in FIG. 3, which involves condensation of formaldehyde and D-ribulose-5-phosphate to form H6P by EF1 (FIG. 3, step A). The enzyme can use Mg2+ or Mn2+ for maximal activity, although other metal ions are useful, and even non- metal-ion-dependent mechanisms are contemplated. H6P is converted into F6P by EF2 (FIG. 3, step B). Another exemplary pathway that involves the detoxification and assimilation of formaldehyde produced from the oxidation of methanol (e.g., as provided in FIG. 1) is shown in FIG. 4 and proceeds through DHA. EF3 is a special transketolase that first transfers a

glycoaldehyde group from xylulose-5 -phosphate to formaldehyde, resulting in the formation of DHA and glyceraldehyde-3 -phosphate (G3P), which is an intermediate in glycolysis (FIG. 4, step A). The DHA obtained from DHA synthase is then further phosphorylated to form DHAP by an EF4 (FIG. 4, step B). DHAP can be assimilated into glycolysis and several other pathways. Rather than converting formaldehyde to formate and on to C02 off-gassed, the pathways provided in FIGS 3 and 4 show that carbon is assimilated, going into the final product. [0113] Thus, in one embodiment, an organism having a MMP, either alone or in combination with a 3-HIBP or MAAP, as provided herein, further comprises a FAP that utilizes

formaldehyde, e.g. , obtained from the oxidation of methanol, in the formation of intermediates of certain central metabolic pathways that can be used, for example, in the formation of biomass. In some of embodiments, the FAP comprises 3 A or 3B, wherein 3 A is an EFl and 3B is an EF2 In other embodiments, the FAP comprises 4A or 4B, wherein 4A is an EF3 and 4B is a EF4.

[0114] In certain embodiments, provided herein is a NNOMO having a MMP, wherein said organism comprises at least one exogenous nucleic acid encoding an EM9 (1 J) expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol and/or expressed in a sufficient amount to convert methanol to formaldehyde. In some embodiments, the organism comprises at least one exogenous nucleic acid encoding an EM9 expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol. In other embodiments, the organism comprises at least one exogenous nucleic acid encoding an EM9 expressed in a sufficient amount to convert methanol to formaldehyde. In some embodiments, the microbial organism further comprises a FAP. In certain embodiments, the organism further comprises at least one exogenous nucleic acid encoding a FAPE expressed in a sufficient amount to produce an intermediate of glycolysis and/or a metabolic pathway that can be used, for example, in the formation of biomass. In certain embodiments, the FAPE is selected from the group consisting of an EFl (3 A), EF2 (3B), EF3 (4A) and EF4 (4B). In certain embodiments, the NNOMO further comprises a 3-HIBP or MAAP. In some embodiments, the NNOMO further comprises a FRP.

[0115] In some embodiments, the exogenous nucleic acid encoding an EM9 is expressed in a sufficient amount to produce an amount of formaldehyde greater than or equal to 1 μΜ, 10 μΜ, 20 μΜ, or 50 μΜ, or a range thereof, in culture medium or intracellularly. In other

embodiments, the exogenous nucleic acid encoding an EM9 is capable of producing an amount of formaldehyde greater than or equal to 1 μΜ, 10 μΜ, 20 μΜ, or 50 μΜ, or a range thereof, in culture medium or intracellularly. In some embodiments, the range is from 1 μΜ to 50 μΜ or greater. In other embodiments, the range is from 10 μΜ to 50 μΜ or greater. In other embodiments, the range is from 20 μΜ to 50 μΜ or greater. In other embodiments, the amount of formaldehyde production is 50 μΜ or greater, for example, 55 mM, 60 μΜ, 65 mM, 70 μΜ, 75 μΜ, 80 μΜ, 85 μΜ, 90 μΜ, 95 μΜ or 100 μΜ. In specific embodiments, the amount of formaldehyde production is in excess of, or as compared to, that of a negative control, e.g., the same species of organism that does not comprise the exogenous nucleic acid, such as a wild-type microbial organism or a control microbial organism thereof. In certain embodiments, the EM9 is selected from those provided herein, e.g., as exemplified in Example I (see FIG. 1, step J). In certain embodiments, the amount of formaldehyde production is determined by a whole cell assay, such as that provided in Example I (see FIG. 1, step J), or by another assay provided herein or otherwise known in the art. In certain embodiments, formaldehyde utilization activity is absent in the whole cell.

[0116] In certain embodiments, the exogenous nucleic acid encoding an EM9 is expressed in a sufficient amount to produce at least IX, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 15X, 20X, 3 OX, 40X, 5 OX, 100X or more formaldehyde in culture medium or intracellularly. In other embodiments, the exogenous nucleic acid encoding an EM9 is capable of producing an amount of formaldehyde at least IX, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 15X, 20X, 30X, 40X, 50X, 100X, or a range thereof, in culture medium or intracellularly. In some embodiments, the range is from IX to 100X. In other embodiments, the range is from 2X to 100X. In other

embodiments, the range is from 5X to 100X. In other embodiments, the range is from 10X to 100X. In other embodiments, the range is from 50X to 100X. In some embodiments, the amount of formaldehyde production is at least 20X. In other embodiments, the amount of formaldehyde production is at least 5 OX. In specific embodiments, the amount of formaldehyde production is in excess of, or as compared to, that of a negative control, e.g., the same species of organism that does not comprise the exogenous nucleic acid, such as a wild-type microbial organism or a control microbial organism thereof. In certain embodiments, the EM9 is selected from those provided herein, e.g., as exemplified in Example I (see FIG. 1, step J). In certain embodiments, the amount of formaldehyde production is determined by a whole cell assay, such as that provided in Example I (see FIG. 1, step J), or by another assay provided herein or otherwise known in the art. In certain embodiments, formaldehyde utilization activity is absent in the whole cell.

[0117] In one aspect, provided herein is a NNOMO, comprising (1) a MMP, wherein said organism comprises at least one exogenous nucleic acid encoding a MMPE in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol and/or expressed in a sufficient amount to convert methanol to formaldehyde; and (2) a FAP, wherein said organism comprises at least one exogenous nucleic acid encoding a FAPE expressed in a sufficient amount to produce an intermediate of glycolysis and/or a metabolic pathway that can be used, for example, in the formation of biomass. In some embodiments, the organism comprises at least one exogenous nucleic acid encoding an EM9 expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol. In other embodiments, the organism comprises at least one exogenous nucleic acid encoding an EM9 expressed in a sufficient amount to convert methanol to formaldehyde. In specific embodiments, the MMP comprises an EM9 (1 J). In certain embodiments, the FAPE is 3 A, and the

intermediate is a H6P, a F6P, or a combination thereof. In other embodiments, the FAPE is 3B, and the intermediate is a H6P, a F6P, or a combination thereof. In yet other embodiments, the FAPE is 3A and 3B, and the intermediate is a H6P, a F6P, or a combination thereof. In some embodiments, the FAPE is 4A, and the intermediate is a DHA, a DHAP, or a combination thereof. In other embodiments, the FAPE is 4B, and the intermediate is a DHA, a DHAP, or a combination thereof. In yet other embodiments, the FAPE is 4A and 4B, and the intermediate is a DHA, a DHAP, or a combination thereof. In one embodiment, the at least one exogenous nucleic acid encoding the MMPE, in the presence of methanol, sufficiently enhances the availability of reducing equivalents and sufficiently increases formaldehyde assimilation to increase the production of 3-HIB, MAA or other products described herein by the non-naturally microbial organism. In some embodiments, the MMP comprises any of the various

combinations of MMPEs described above or elsewhere herein.

[0118] In certain embodiments, (1) the MMP comprises: 1A, IB, 1C, ID, IE, IF, 1G, 1H, II, 1J, IK, 1L, 1M, IN, or 10 or any combination of 1A, IB, 1C, ID, IE, IF, 1G, 1H, II, 1J, IK, 1L, 1M, IN, or 10, thereof, wherein 1A is an EMI; IB is an EM2; 1C is an EM3; ID is an EM4; IE is an EM5; IF is an EM6; 1G is an EM15; 1H is an EM16, II is an EM8; 1J is an EM9; IK is spontaneous or EMIO; 1L is an EMI 1; 1M is spontaneous or an EM 12; IN is EM 13 and 10 is EM 14; and (2) the FAP comprises 3 A, 3B or a combination thereof, wherein 3 A is an EF1, and 3B is an EF2. In some embodiments, IK is spontaneous. In other embodiments, IK is an EMIO. In some embodiments, 1M is spontaneous. In other embodiments, 1M is an EM12. In some embodiments, the intermediate is a H6P. In other embodiments, the intermediate is a F6P. In yet other embodiments, the intermediate is a H6P and a F6P.

[0119] In one embodiment, the FAP comprises 3 A. In another embodiment, the FAP comprises 3B. In one embodiment, the FAP comprises 3A and 3B.

[0120] In some embodiments, the MMP is a MMP depicted in FIG. 1, and a FAP depicted in FIG. 3. An exemplary set of FAPEs to convert D-ribulose-5-phosphate and formaldehyde to F6P (via H6P) according to FIG. 3 include 3A and 3B.

[0121] In a specific embodiment, (1) the MMP comprises 1 J; and (2) the FAP comprises 3A and 3B. In other embodiments, (1) the MMP comprises IJ and IK; and (2) the FAP comprises 3A and 3B. In some embodiments, (1) the MMP comprises IJ, IC, ID and IE; and (2) the FAP comprises 3A and 3B. In one embodiment, (1) the MMP comprises IJ, IC, ID and IF; and (2) the FAP comprises 3A and 3B. In another embodiment, (1) the MMP comprises IJ and 1L; and (2) the FAP comprises 3A and 3B. In yet another embodiment, (1) the MMP comprises 1 J, 1M, IN and 10; and (2) the FAP comprises 3A and 3B. In certain embodiments, (1) the MMP comprises IJ, IN and 10; and (2) the FAP comprises 3A and 3B. In some embodiments, (1) the MMP comprises IJ, IK, IC, ID and IE; and (2) the FAP comprises 3A and 3B. In one embodiment, (1) the MMP comprises IJ, IK, IC, ID and IF; and (2) the FAP comprises 3A and 3B. In some embodiments, (1) the MMP comprises IJ, IC, ID, IE and II; and (2) the FAP comprises 3A and 3B. In one embodiment, (1) the MMP comprises IJ, IC, ID, IF and II; and (2) the FAP comprises 3A and 3B. In another embodiment, (1) the MMP comprises 1 J, 1L and II; and (2) the FAP comprises 3 A and 3B. In yet another embodiment, (1) the MMP comprises IJ, 1M, IN, 10 and II; and (2) the FAP comprises 3A and 3B. In certain embodiments, (1) the MMP comprises IJ, IN, 10 and II; and (2) the FAP comprises 3 A and 3B. In some

embodiments, (1) the MMP comprises IJ, IK, IC, ID, IE and II; and (2) the FAP comprises 3 A and 3B. In one embodiment, (1) the MMP comprises IJ, IK, IC, ID, IF and II; and (2) the FAP comprises 3A and 3B. In some embodiments, (1) the MMP comprises IJ, IC, ID, IE and 1G; and (2) the FAP comprises 3A and 3B. In one embodiment, (1) the MMP comprises 1 J, IC, ID, IF and 1G; and (2) the FAP comprises 3A and 3B. In another embodiment, (1) the MMP comprises IJ, 1L and 1G; and (2) the FAP comprises 3 A and 3B. In yet another embodiment, (1) the MMP comprises 1J, 1M, IN, 10 and 1G; and (2) the FAP comprises 3A and 3B. In certain embodiments, (1) the MMP comprises 1J, IN, 10 and 1G; and (2) the FAP comprises 3 A and 3B. In some embodiments, (1) the MMP comprises 1J, IK, IC, ID, IE and 1G; and (2) the FAP comprises 3A and 3B. In one embodiment, (1) the MMP comprises 1J, IK, IC, ID, IF and 1G; and (2) the FAP comprises 3A and 3B. In some embodiments, (1) the MMP comprises 1J, IC, ID, IE, 1G and 1H; and (2) the FAP comprises 3A and 3B. In one embodiment, (1) the MMP comprises 1J, IC, ID, IF, 1G and 1H; and (2) the FAP comprises 3A and 3B. In another embodiment, (1) the MMP comprises 1J, 1L, 1G and 1H; and (2) the FAP comprises 3A and 3B. In yet another embodiment, (1) the MMP comprises 1J, 1M, IN, 10, 1G and 1H; and (2) the FAP comprises 3A and 3B. In certain embodiments, (1) the MMP comprises 1J, IN, 10, 1G and 1H; and (2) the FAP comprises 3A and 3B. In some embodiments, (1) the MMP comprises 1 J, IK, IC, ID, IE, 1G and 1H; and (2) the FAP comprises 3A and 3B. In one embodiment, (1) the MMP comprises 1J, IK, IC, ID, IF, 1G and 1H; and (2) the FAP comprises 3A and 3B. In some embodiments, IK is spontaneous. In other embodiments, IK is an EMIO. In some embodiments, 1M is spontaneous. In some embodiments, the intermediate is a H6P. In other embodiments, the intermediate is a F6P. In yet other embodiments, the intermediate is a H6P and a F6P.

[0122] In certain embodiments, (1) the MMP comprises: 1A, IB, IC, ID, IE, IF, 1G, 1H, II, 1J, IK, 1L, 1M, IN, or 10 or any combination of 1A, IB, IC, ID, IE, IF, 1G, 1H, II, 1J, IK, 1L, 1M, IN, or 10, thereof, wherein 1A is an EMI; IB is an EM2; IC is an EM3; ID is an EM4; IE is an EM5; IF is an EM6; 1G is an EM15; 1H is an EM16, II is an EM8; 1J is an EM9; IK is spontaneous or EMIO; 1L is an EMI 1; 1M is spontaneous or an EM 12; IN is EM 13 and 10 is EM 14; and (2) the FAP comprises 4A, 4B or a combination thereof, wherein 4A is an EF3 and 4B is an EF4. In some embodiments, IK is spontaneous. In other embodiments, IK is an EMIO. In some embodiments, 1M is spontaneous. In other embodiments, 1M is an EM12. In some embodiments, the intermediate is a DHA. In other embodiments, the intermediate is a DHAP. In yet other embodiments, the intermediate is a DHA and a DHAP.

[0123] In one embodiment, the FAP comprises 4A. In another embodiment, the FAP comprises 4B. In one embodiment, the FAP comprises 4 A and 4B. [0124] In some embodiments, the MMP is a MMP depicted in FIG. 1, and a FAP depicted in FIG. 4. An exemplary set of FAPEs to convert xyulose-5 -phosphate and formaldehyde to DHAP (via DHA) according to FIG. 4 include 4A and 4B.

[0125] In a specific embodiment, (1) the MMP comprises 1 J; and (2) the FAP comprises 4A and 4B. In other embodiments, (1) the MMP comprises IJ and IK; and (2) the FAP comprises 4A and 4B. In some embodiments, (1) the MMP comprises IJ, IC, ID and IE; and (2) the FAP comprises 4A and 4B. In one embodiment, (1) the MMP comprises IJ, IC, ID and IF; and (2) the FAP comprises 4 A and 4B. In another embodiment, (1) the MMP comprises IJ and 1L; and (2) the FAP comprises 4 A and 4B. In yet another embodiment, (1) the MMP comprises 1 J, 1M, IN and 10; and (2) the FAP comprises 4A and 4B. In certain embodiments, (1) the MMP comprises IJ, IN and 10; and (2) the FAP comprises 4A and 4B. In some embodiments, (1) the MMP comprises 1 J, IK, IC, ID and IE; and (2) the FAP comprises 4A and 4B. In one embodiment, (1) the MMP comprises IJ, IK, IC, ID and IF; and (2) the FAP comprises 4A and 4B. In some embodiments, (1) the MMP comprises IJ, IC, ID, IE and II; and (2) the FAP comprises 4A and 4B. In one embodiment, (1) the MMP comprises IJ, IC, ID, IF and II; and (2) the FAP comprises 4 A and 4B. In another embodiment, (1) the MMP comprises 1 J, 1L and II; and (2) the FAP comprises 4 A and 4B. In yet another embodiment, (1) the MMP comprises IJ, 1M, IN, 10 and II; and (2) the FAP comprises 4A and 4B. In certain embodiments, (1) the MMP comprises IJ, IN, 10 and II; and (2) the FAP comprises 4A and 4B. In some

embodiments, (1) the MMP comprises IJ, IK, IC, ID, IE and II; and (2) the FAP comprises 4A and 4B. In one embodiment, (1) the MMP comprises IJ, IK, IC, ID, IF and II; and (2) the FAP comprises 4A and 4B. In some embodiments, (1) the MMP comprises IJ, IC, ID, IE and 1G; and (2) the FAP comprises 4A and 4B. In one embodiment, (1) the MMP comprises 1 J, IC, ID, IF and 1G; and (2) the FAP comprises 4 A and 4B. In another embodiment, (1) the MMP comprises 1 J, 1L and 1G; and (2) the FAP comprises 4 A and 4B. In yet another embodiment, (1) the MMP comprises 1 J, 1M, IN, 10 and 1G; and (2) the FAP comprises 4A and 4B. In certain embodiments, (1) the MMP comprises IJ, IN, 10 and 1G; and (2) the FAP comprises 4A and 4B. In some embodiments, (1) the MMP comprises IJ, IK, IC, ID, IE and 1G; and (2) the FAP comprises 4A and 4B. In one embodiment, (1) the MMP comprises IJ, IK, IC, ID, IF and 1G; and (2) the FAP comprises 4A and 4B. In some embodiments, (1) the MMP comprises 1 J, IC, ID, IE, 1G and 1H; and (2) the FAP comprises 4A and 4B. In one embodiment, (1) the MMP comprises 1 J, 1C, ID, IF, 1G and 1H; and (2) the FAP comprises 4A and 4B. In another embodiment, (1) the MMP comprises 1 J, 1L, 1G and 1H; and (2) the FAP comprises 4 A and 4B. In yet another embodiment, (1) the MMP comprises 1J, 1M, IN, 10, 1G and 1H; and (2) the FAP comprises 4A and 4B. In certain embodiments, (1) the MMP comprises 1 J, IN, 10, 1G and 1H; and (2) the FAP comprises 4A and 4B. In some embodiments, (1) the MMP comprises 1 J, IK, 1C, ID, IE, 1G and 1H; and (2) the FAP comprises 4A and 4B. In one embodiment, (1) the MMP comprises 1 J, IK, 1C, ID, IF, 1G and 1H; and (2) the FAP comprises 4A and 4B. In some embodiments, IK is spontaneous. In other embodiments, IK is an EM 10. In some

embodiments, 1M is spontaneous. In some embodiments, the intermediate is a DHA. In other embodiments, the intermediate is a DHAP. In yet other embodiments, the intermediate is a DHA and a DHAP.

[0126] Any MMP provided herein can be combined with any FAP provided herein. In addition, any MMP provided herein can be combined with any 3-HIBP or MAAP and any FAP provided herein. In other embodiments, these pathways can be further combined with any FRP provided herein.

[0127] Also provided herein are methods of producing formaldehyde comprising culturing a NNOMO having a MMP provided herein. In some embodiments, the MMP comprises 1 J. In certain embodiments, the organism is cultured in a substantially anaerobic culture medium. In specific embodiments, the formaldehyde is an intermediate that is consumed (assimilated) in the production of 3-HIB, MAA and other products described herein.

[0128] Also provided herein are methods of producing an intermediate of glycolysis and/or a metabolic pathway that can be used, for example, in the formation of biomass, comprising culturing a NNOMO having a MMP and a FAP, as provided herein, under conditions and for a sufficient period of time to produce the intermediate. In some embodiments, the intermediate is a H6P. In other embodiments, the intermediate is a F6P. In yet other embodiments, the intermediate is a H6P and a F6P. In some embodiments, the intermediate is a DHA. In other embodiments, the intermediate is a DHAP. In yet other embodiments, the intermediate is a DHA and a DHAP. In some embodiments, the MMP comprises 1 J. In certain embodiments, the organism is cultured in a substantially anaerobic culture medium. Such biomass can also be used in methods of producing any of the products, such as the biobased products, provided elsewhere herein.

[0129] In certain embodiments, the organism comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a 3-HIBPE or MAAPE. In some embodiments, the organism comprises two exogenous nucleic acids, each encoding a 3-HIBPE or MAAPE. In some embodiments, the organism comprises three exogenous nucleic acids, each encoding a 3- HIBPE or MAAPE. In some embodiments, the organism comprises four exogenous nucleic acids, each encoding a 3-HIBPE or MAAPE. In other embodiments, the organism comprises five exogenous nucleic acids, each encoding a 3-HIBPE or MAAPE. In some embodiments, the organism comprises six exogenous nucleic acids, each encoding a 3-HIBPE or MAAPE. In some embodiments, the organism comprises seven exogenous nucleic acids, each encoding a 3-HIBPE or MAAPE. In certain embodiments, the organism comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a 3-HIBPE or MAAPE; and the organism further comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a MMPE. In certain embodiments, the organism further comprises two exogenous nucleic acids, each encoding a MMPE. In certain embodiments, the organism further comprises three exogenous nucleic acids, each encoding a MMPE. In certain embodiments, the organism comprises further four exogenous nucleic acids, each encoding a MMPE. In certain embodiments, the organism further comprises five exogenous nucleic acids, each encoding a MMPE. In certain

embodiments, the organism further comprises six exogenous nucleic acids, each encoding a MMPE. In certain embodiments, the organism further comprises seven exogenous nucleic acids, each encoding a MMPE.

[0130] In some embodiments, the organism comprises two or more exogenous nucleic acids, each encoding a FAPE. In some embodiments, the organism comprises two exogenous nucleic acids, each encoding a FAPE. In certain embodiments, the organism comprises two exogenous nucleic acids, each encoding a FAPE; and the organism further comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a MMPE. In certain embodiments, the organism further comprises two exogenous nucleic acids, each encoding a MMPE. In certain embodiments, the organism further comprises three exogenous nucleic acids, each encoding a MMPE. In certain embodiments, the organism comprises further four exogenous nucleic acids, each encoding a MMPE. In certain embodiments, the organism further comprises five exogenous nucleic acids, each encoding a MMPE. In certain embodiments, the organism further comprises six exogenous nucleic acids, each encoding a MMPE. In certain embodiments, the organism further comprises seven exogenous nucleic acids, each encoding a MMPE.

[0131] In some embodiments, the at least one exogenous nucleic acid encoding a MMPE is a heterologous nucleic acid. In other embodiments, the at least one exogenous nucleic acid encoding a FAPE is a heterologous nucleic acid. In other embodiments, the at least one exogenous nucleic acid encoding a 3-HIBPE or MAAPE is a heterologous nucleic acid. In certain embodiments, the at least one exogenous nucleic acid encoding a MMPE is a

heterologous nucleic acid, and the at least one exogenous nucleic acid encoding a 3-HIBPE or MAAPE is a heterologous nucleic acid. In other embodiments, the at least one exogenous nucleic acid encoding a MMPE is a heterologous nucleic acid, and the at least one exogenous nucleic acid encoding a FAPE is a heterologous nucleic acid.

[0132] In certain embodiments, the organism is in a substantially anaerobic culture medium.

[0133] In some embodiments, provided herein is a NNOMO comprising a MMP. In certain embodiments, provided herein is a NNOMO comprising a FAP. In other embodiments, provided herein is a FRP. In some embodiments, provided herein is a NNOMO comprising a 3-HIBP. In other embodiments, provided herein is a NNOMO comprising a MAAP. A NNOMO comprising any combination of one, two, three, four or five of the various FAPs, FRPs, MMPs, 3-HIBPs and MAAPs provided herein are also contemplated. In one embodiment, a NNOMO comprises a MMP and a 3-HIBP provided herein. In another embodiment, a NNOMO comprises a MMP, a FAP and a 3-HIBP pathway provided herein. In other embodiments, a NNOMO comprises a MMP, a FAP, a FRP and a 3-HIBP provided herein. In one embodiment, a NNOMO comprises a MMP and a MAAP provided herein. In another embodiment, a NNOMO comprises a MMP, a FAP and a MAAP pathway provided herein. In other embodiments, a NNOMO comprises a MMP, a FAP, a FRP and a MAAP provided herein. Exemplary MMP, FAP, 1, 3-HIBP, MAAP pathways are provided in FIGS. 1-5 and elsewhere herein.

[0134] In certain embodiments, the NNOMOs provided herein comprises at least one exogenous nucleic acid encoding a MMP, a FAP, a FRP, a 3-HIBP and/or a MAAP enzyme or protein. In some embodiments, the NNOMO comprises an exogenous nucleic acid encoding a MMP enzyme or protein. In some embodiments, the NNOMO comprises an exogenous nucleic acid encoding a FAP enzyme or protein. In some embodiments, the NNOMO comprises an exogenous nucleic acid encoding a FRP enzyme or protein. In some embodiments, the NNOMO comprises an exogenous nucleic acid encoding a 3-HIBP enzyme or protein. In some

embodiments, the NNOMO comprises an exogenous nucleic acid encoding a MAAP enzyme or protein. In certain embodiments, the exogenous nucleic acid is a heterologous nucleic acid.

[0135] In certain embodiments, provided herein is a NNOMO having a FAP and a FRP. In certain embodiments, the organism comprises (i) at least one exogenous nucleic acid encoding a FAPE, wherein said FAP comprises 3A (see also 5B), 3B (see also 5C), or 4A (see also 5D) or any combination thereof, wherein 3 A is a 3-hexulose-6-phosphate synthase (EF1), wherein 3B is a 6-phospho-3-hexuloisomerase (EF2), wherein 4A is a DHA synthase (EF3). In certain embodiments, the FAPE is expressed in a sufficient amount to produce pyruvate. In certain embodiments, the NNOMO further comprises a MMP provided herein. In other embodiments, the NNOMO further comprises a 3-HIBP or MAAP provided herein. In some embodiments, the NNOMO further comprises a MMP and a 3-HIBP or MAAP provided herein.

[0136] In certain embodiments, the organism comprises at least one exogenous nucleic acid encoding a FRP enzyme (FRPE), wherein said FRP comprises 5E, 5F,5G, 5H, 51, 5J, 5K, 5L, 5M, 5N, 50, or 5P or any combination thereof, wherein 5E is a formate reductase (EFR1), 5F is a formate ligase (EFR2A), a formate transferase (EFR2B), or a formate synthetase (EFR2C), wherein 5G is a formyl-CoA reductase (EFR3), wherein 5H is a formyltetrahydrofolate synthetase (EFR4), wherein 51 is a methenyltetrahydrofolate cyclohydrolase (EFR5), wherein 5J is a methylenetetrahydrofolate dehydrogenase (EFR6), wherein 5K is a formaldehyde-forming enzyme (EFR7) or spontaneous, wherein 5L is a glycine cleavage system (EFR8), wherein 5M is a serine hydroxymethyltransferase (EFR9), wherein 5N is a serine deaminase (EFR10), wherein 50 is a methylenetetrahydrofolate reductase (EFR11), wherein 5P is an acetyl-CoA synthase (EFR12). In certain embodiments, the FRPE is expressed in a sufficient amount to produce formaldehyde. In certain embodiments, the FRPE is expressed in a sufficient amount to produce pyruvate. In certain embodiments, the FRPE is expressed in a sufficient amount to produce acetyl-CoA. In some embodiments, 5K is spontaneous. In some embodiments, 5F is an EFR2A. In other embodiments, 5F is an EFR2B. In other embodiments, 5F is an EFR2C. In certain embodiments, the NNOMO further comprises a MMP provided herein. In other embodiments, the NNOMO further comprises a 3-HIBP or MAAP provided herein. In some embodiments, the NNOMO further comprises a MMP and a 3-HIBP or MAAP provided herein.

[0137] In one embodiment, the FAP comprises 3A. In one embodiment, the FAP comprises 3B. In one embodiment, the FAP comprises 4A. In one embodiment, the FRP comprises 5E. In one embodiment, the FRP comprises 5F. In some embodiments, 5F is an EFR2A. In other embodiments, 5F is an EFR2B. In other embodiments, 5F is an EFR2C. In one embodiment, the FRP comprises 5G. In one embodiment, the FRP comprises 5H. In one embodiment, the FRP comprises 51. In one embodiment, the FRP comprises 5 J. In one embodiment, the FRP comprises 5K. In some embodiments, 5K is spontaneous. In one embodiment, the FRP comprises 5L. In one embodiment, the FRP comprises 5M. In one embodiment, the FRP comprises 5N. In one embodiment, the FRP comprises 50. In one embodiment, the FRP comprises 5P. Any combination of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen pathway enzymes of 3A, 3B, 4A, 5E, 5F,5G, 5H, 51, 5 J, 5K, 5L, 5M, 5N, 50, or 5P is also contemplated.

[0138] In one aspect, provided herein is a NNOMO having a FAP and a FRP, wherein said organism comprises (i) at least one exogenous nucleic acid encoding a FAPE, wherein said FAP comprises: (5) 3A and 3B; or (2) 4A; and (ii) at least one exogenous nucleic acid encoding a FRPE, wherein said FRP comprises a pathway selected from: (3) 5E; (4) 5F, and 5G; (5) 5H, 51, 5J, and 5K; (6) 5H, 51, 5J, 5L, 5M, and 5N; (7) 5E, 5H, 51, 5J, 5L, 5M, and 5N; (8) 5F, 5G, 5H, 51, 5 J, 5L, 5M, and 5N; (9) 5K, 5H, 51, 5 J, 5L, 5M, and 5N; and (10) 5H, 51, 5 J, 50, and 5P. In certain embodiments, the FAPE is expressed in a sufficient amount to produce pyruvate. In some embodiments, the FRPE is expressed in a sufficient amount to produce formaldehyde. In other embodiments, the FRPE is expressed in a sufficient amount to produce pyruvate. In certain embodiments, the FRPE is expressed in a sufficient amount to produce acetyl-CoA. In some embodiments, 5K is spontaneous. In some embodiments, 5F is an EFR2A. In other

embodiments, 5F is an EFR2B. In other embodiments, 5F is an EFR2C. In certain

embodiments, the NNOMO further comprises a MMP provided herein. In other embodiments, the NNOMO further comprises a 3-HIBP or MAAP provided herein. In some embodiments, the NNOMO further comprises a MMP and a 3-HIBP or MAAP provided herein.

[0139] In certain embodiments, the FAP comprises 3A and 3B. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5E. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5F and 5G. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5H, 51, 5 J, and 5K. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5E, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5F, 5G, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5K, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5H, 51, 5J, 50, and 5P. In some embodiments, 5K is spontaneous. In some embodiments, 5F is an EFR2A. In other embodiments, 5F is an EFR2B. In other embodiments, 5F is an EFR2C.

[0140] In certain embodiments, the FAP comprises 4A. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5E. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5F, and 5G. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5H, 51, 5 J, and 5K. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5E, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5F, 5G, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5K, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5H, 51, 5J, 50, and 5P. In some embodiments, 5K is spontaneous. In some embodiments, 5F is an EFR2A. In other embodiments, 5F is an EFR2B. In other embodiments, 5F is an EFR2C.

[0141] In certain embodiments, the FRP further comprises 5Q, 5R, or 5S or any combination thereof, wherein 5Q is a pyruvate formate lyase (EFR13); 5R is a pyruvate dehydrogenase (EFR14A), a pyruvate ferredoxin oxidoreductase (EFR14B), or a pyruvate :NADP+

oxidoreductase (EFR14C); and 5S is a formate dehydrogenase (EFR15). Thus, in certain embodiments the FRP comprises 5Q. In certain embodiments the FRP comprises 5R. In certain embodiments the FRP comprises 5S. In certain embodiments, the FRP comprises 5R and 5S. In some embodiments, 5R is an EFR14A. In other embodiments, 5R is an EFR14B. In other embodiments, 5R is an EFR14C.

[0142] In certain embodiments, FRP comprises 5Q, or 5R and 5S, and the FAP comprises 3A and 3B. In certain embodiments, FRP comprises 5Q, or 5R and 5S, and the FAP comprises 4A. In certain embodiments the FAP comprises 3A and 3B, and the FRP comprises 5Q, and 5E. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5Q, 5F, and 5G. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5Q, 5H, 51, 5J, and 5K. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5Q, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5Q, 5E, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5Q, 5F, 5G, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5Q, 5K, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5Q, 5H, 51, 5J, 50, and 5P. In certain embodiments the FAP comprises 4A, and the FRP comprises 5Q, and 5E. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5Q, 5F, and 5G. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5Q, 5H, 51, 5J, and 5K. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5Q, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5Q, 5E, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5Q, 5F, 5G, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5Q, 5K, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5Q, 5H, 51, 5J, 50, and 5P. In certain embodiments the FAP comprises 3A and 3B, and the FRP comprises 5R, 5S, and 5E. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5R, 5S, 5F, and 5G. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5R, 5S, 5H, 51, 5 J, and 5K. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5R, 5S, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5R, 5S, 5E, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5R, 5S, 5F, 5G, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5R, 5S, 5K, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 3A and 3B, and the FRP comprises 5R, 5S, 5H, 51, 5J, 50, and 5P. In certain embodiments the FAP comprises 4A, and the FRP comprises 5R, 5S, and 5E. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5R, 5S, 5F, and 5G. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5R, 5S, 5H, 51, 5J, and 5K. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5R, 5S, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5R, 5S, 5E, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5R, 5S, 5F, 5G, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5R, 5S, 5K, 5H, 51, 5J, 5L, 5M, and 5N. In certain embodiments, the FAP comprises 4A, and the FRP comprises 5R, 5S, 5H, 51, 5J, 50, and 5P. In some embodiments, 5K is spontaneous. In some embodiments, 5F is an EFR2A. In other embodiments, 5F is an

EFR2B. In other embodiments, 5F is an EFR2C. In some embodiments, 5R is an EFR14A. In other embodiments, 5R is an EFR14B. In other embodiments, 5R is an EFR14C.

[0143] In certain embodiments, the FAP is a pathway depicted in FIG. 3. In certain embodiments, the FAP is a pathway depicted in FIG. 4. In certain embodiments, the FAP is a pathway depicted in FIG. 5. In certain embodiments, the FRP is a pathway depicted in FIG. 5. In certain embodiments, the FAP and the FRP is a pathway depicted in FIG. 5.

[0144] In certain embodiments, provided herein is a NNOMO having a FAP, a FRP and a MMP. In some embodiments, the organism comprises (i) at least one exogenous nucleic acid encoding a FAPE, wherein said FAP comprises: (1) 3A and 3B; or (2) 4A; (ii) at least one exogenous nucleic acid encoding a FRPE, wherein said FRP comprises a pathway selected from: (3) 5E; (4) 5F, and 5G; (5) 5H, 51, 5J, and 5K; (6) 5H, 51, 5J, 5L, 5M, and 5N; (7) 5E, 5H, 51, 5J, 5L, 5M, and 5N; (8) 5F, 5G, 5H, 51, 5J, 5L, 5M, and 5N; (9) 5K, 5H, 51, 5J, 5L, 5M, and 5N; and (10) 5H, 51, 5J, 50, and 5P, and (iii) at least one exogenous nucleic acid encoding a MMPE, wherein said MMP comprises a pathway selected from: (1) 1 J; (2) 1A and IB; (3) 1A, IB and IC; (4) IJ, IK and IC; (5) IJ, 1M, and IN; (6) IJ and 1L; (7) 1A, IB, IC, ID, and IE; (8) 1A, IB, IC, ID, and IF; (9) IJ, IK, IC, ID, and IE; (10) IJ, IK, IC, ID, and IF; (11) IJ, 1M, IN, and 10; (12) 1A, IB, IC, ID, IE, and IG; (13) 1A, IB, IC, ID, IF, and IG; (14) IJ, IK, IC, ID, IE, and IG; (15) IJ, IK, IC, ID, IF, and IG; (16) IJ, 1M, IN, 10, and IG; (17) 1A, IB, IC, ID, IE, and II; (18) 1A, IB, 1C, ID, IF, and II; (19) 1J, IK, 1C, ID, IE, and II; (20) 1J, IK,

IC, ID, IF, and II; and (21) 1J, 1M, IN, 10, and II. In certain embodiments, the FAPE is expressed in a sufficient amount to produce pyruvate. In some embodiments, the FRPE is expressed in a sufficient amount to produce formaldehyde. In other embodiments, the FRPE is expressed in a sufficient amount to produce pyruvate. In certain embodiments, the FRPE is expressed in a sufficient amount to produce acetyl-CoA. In some embodiments, the MMP enzyme expressed in a sufficient amount to produce formaldehyde and/or produce or enhance the availability of reducing equivalents in the presence of methanol. In some embodiments, 5K is spontaneous. In some embodiments, 5F is an EFR2A. In other embodiments, 5F is an EFR2B. In other embodiments, 5 F is an EFR2C. In some embodiments, IK is spontaneous. In other embodiments, IK is an EM10. In some embodiments, 1M is spontaneous. In other

embodiments, 1M is an EM12. In certain embodiments, NNOMO further comprises a 3-HIBP or MAAP provided herein.

[0145] In certain embodiments, the MMP comprises 1 A. In certain embodiments, the MMP comprises IB. In certain embodiments, the MMP comprises 1C. In certain embodiments, the MMP comprises ID. In certain embodiments, the MMP comprises IE. In certain embodiments, the MMP comprises IF. In certain embodiments, the MMP comprises 1G. In certain

embodiments, the MMP comprises 1H. In certain embodiments, the MMP comprises II. In certain embodiments, the MMP comprises 1 J. In certain embodiments, the MMP comprises IK. In certain embodiments, the MMP comprises 1L. In certain embodiments, the MMP comprises 1M. In certain embodiments, the MMP comprises IN. In certain embodiments, the MMP comprises 10. In some embodiments, IK is spontaneous. In other embodiments, IK is an EM10. In some embodiments, 1M is spontaneous. In other embodiments, 1M is an EM12. In certain embodiments, the MMP comprises 1 J. In certain embodiments, the MMP comprises 1A and IB. In certain embodiments, the MMP comprises 1A, IB and 1C. In certain embodiments, the MMP comprises 1J, IK and 1C. In certain embodiments, the MMP comprises 1J, 1M, and IN. In certain embodiments, the MMP comprises 1 J and 1L. In certain embodiments, the MMP comprises 1A, IB, 1C, ID, and IE. In certain embodiments, the MMP comprises 1A, IB, 1C,

ID, and IF. In certain embodiments, the MMP comprises 1J, IK, 1C, ID, and IE. In certain embodiments, the MMP comprises 1J, IK, 1C, ID, and IF. In certain embodiments, the MMP comprises 1J, 1M, IN, and 10. In certain embodiments, the MMP comprises 1A, IB, 1C, ID, IE, and 1G. In certain embodiments, the MMP comprises 1A, IB, 1C, ID, IF, and 1G. In certain embodiments, the MMP comprises 1J, 1K, 1C, ID, IE, and 1G. In certain embodiments, the MMP comprises 1J, 1K, 1C, ID, IF, and 1G. In certain embodiments, the MMP comprises 1J, 1M, IN, 10, and 1G. In certain embodiments, the MMP comprises 1A, IB, 1C, ID, IE, and 11. In certain embodiments, the MMP comprises 1A, IB, 1C, ID, IF, and 11. In certain embodiments, the MMP comprises 1J, 1K, 1C, ID, IE, and 11. In certain embodiments, the MMP comprises 1J, 1K, 1C, ID, IF, and 11. In certain embodiments, the MMP comprises 1J, 1M, IN, 10, and 11. In some embodiments, 1K is spontaneous. In other embodiments, 1K is an EM10. In some embodiments, 1M is spontaneous. In other embodiments, 1M is an EM12.

[0146] In certain embodiments, provided herein is a NNOMO having a FAP, a FRP and a MMP. In some embodiments, the organism comprises (i) at least one exogenous nucleic acid encoding a FAPE, wherein said FAP comprises: (1) 3A and 3B; or (2) 4A, (ii) at least one exogenous nucleic acid encoding a FRPE, wherein said FRP comprises a pathway selected from: (3) 5E; (4) 5F, and 5G; (5) 5H, 51, 5J, and 5K; (6) 5H, 51, 5J, 5L, 5M, and 5N; (7) 5E, 5H, 51, 5J, 5L, 5M, and 5N; (8) 5F, 5G, 5H, 51, 5J, 5L, 5M, and 5N; (9) 5K, 5H, 51, 5J, 5L, 5M, and 5N; and (10) 5H, 51, 5J, 50, and 5P, and (iii) at least one exogenous nucleic acid encoding a MMPE (e.g., a methanol oxidation pathway enzyme) expressed in a sufficient amount to produce

formaldehyde in the presence of methanol, wherein said MMP comprises 1J (see also 5 A). In certain embodiments, the FAPE is expressed in a sufficient amount to produce pyruvate. In some embodiments, the FRPE is expressed in a sufficient amount to produce formaldehyde. In other embodiments, the FRPE is expressed in a sufficient amount to produce pyruvate. In certain embodiments, the FRPE is expressed in a sufficient amount to produce acetyl-CoA. In some embodiments, 5K is spontaneous. In some embodiments, 5F is an EFR2A. In other

embodiments, 5F is an EFR2B. In other embodiments, 5F is an EFR2C. In certain

embodiments, NNOMO further comprises a 3-HIBP or MAAP provided herein.

[0147] In certain embodiments, provided herein is a NNOMO having a FAP and a MMP. In some embodiments, the organism comprises (i) at least one exogenous nucleic acid encoding a FAPE, wherein said FAP comprises: (1) 3A and 3B; or (2) 4A; and (ii) at least one exogenous nucleic acid encoding a MMPE (e.g., a methanol oxidation pathway enzyme) expressed in a sufficient amount to produce formaldehyde in the presence of methanol, wherein said MMP comprises 1 J. In certain embodiments, the FAPE is expressed in a sufficient amount to produce pyruvate. In certain embodiments, NNOMO further comprises a 3-HIBP or MAAP provided herein.

[0148] In certain embodiments, provided herein is a NNOMO having a FAP, a FRP, and a MMP. In certain embodiments, the organism further comprises 1H or IP, wherein 1H is a hydrogenase (EM16) and IP a carbon monoxide dehydrogenase that converts CO to C0 ₂ . In some embodiments, the organism comprises (i) at least one exogenous nucleic acid encoding a FAPE, wherein said FAP comprises: (1) 3A and 3B; or (2) 4A, (ii) at least one exogenous nucleic acid encoding a FRPE, wherein said FRP comprises a pathway selected from: (3) 5E; (4) 5F, and 5G; (5) 5H, 51, 5J, and 5K; (6) 5H, 51, 5J, 5L, 5M, and 5N; (7) 5E, 5H, 51, 5J, 5L, 5M, and 5N; (8) 5F, 5G, 5H, 51, 5 J, 5L, 5M, and 5N; (9) 5K, 5H, 51, 5 J, 5L, 5M, and 5N; and (10) 5H, 51, 5J, 50, and 5P; and (iii) at least one exogenous nucleic acid encoding a MMP enzyme, wherein said MMP comprises a pathway selected from: (1) 1 J; (2) 1A and IB; (3) 1A, IB and IC; (4) IJ, IK and IC; (5) IJ, IM, and IN; (6) IJ and 1L; (7) 1A, IB, IC, ID, and IE; (8) 1A,

IB, IC, ID, and IF; (9) IJ, IK, IC, ID, and IE; (10) IJ, IK, IC, ID, and IF; (11) IJ, IM, IN, and 10; (12) 1A, IB, IC, ID, IE, and IG; (13) 1A, IB, IC, ID, IF, and IG; (14) IJ, IK, IC, ID, IE, and IG; (15) IJ, IK, IC, ID, IF, and IG; (16) IJ, IM, IN, 10, and IG; (17) 1A, IB, IC, ID, IE, and II; (18) 1A, IB, IC, ID, IF, and II; (19) IJ, IK, IC, ID, IE, and II; (20) IJ, IK,

IC, ID, IF, and II; and (21) IJ, IM, IN, 10, and II. In certain embodiments, the FAPE is expressed in a sufficient amount to produce pyruvate. In some embodiments, the FRPE is expressed in a sufficient amount to produce formaldehyde. In other embodiments, the FRPE is expressed in a sufficient amount to produce pyruvate. In certain embodiments, the FRPE is expressed in a sufficient amount to produce acetyl-CoA. In some embodiments, the MMP enzyme expressed in a sufficient amount to produce formaldehyde and/or produce or enhance the availability of reducing equivalents in the presence of methanol. In some embodiments, 5K is spontaneous. In some embodiments, 5F is an EFR2A. In other embodiments, 5F is an EFR2B. In other embodiments, 5F is an EFR2C. In some embodiments, IK is spontaneous. In other embodiments, IK is an EM10. In some embodiments, IM is spontaneous. In other

embodiments, IM is an EM12.In certain embodiments, NNOMO further comprises a 3-HIBP or MAAP provided herein. [0149] In certain embodiments, provided herein is a NNOMO having a FAP, a FRP, and a MMP. In certain embodiments, the organism further comprises 1H or IP, wherein 1H is a hydrogenase (EM16) and IP a carbon monoxide dehydrogenase that converts CO to C0 ₂ . In some embodiments, the organism comprises (i) at least one exogenous nucleic acid encoding a FAPE, wherein said FAP comprises: (1) 3A and 3B; or (2) 4A, wherein 3A is a 3-hexulose-6- phosphate synthase, wherein 3B is a 6-phospho-3-hexuloisomerase, wherein 4A is a DHA synthase, (ii) at least one exogenous nucleic acid encoding a FRPE, wherein said FRP comprises a pathway selected from: (3) 5E; (4) 5F, and 5G; (5) 5H, 51, 5J, and 5K; (6) 5H, 51, 5J, 5L, 5M, and 5N; (7) 5E, 5H, 51, 5J, 5L, 5M, and 5N; (8) 5F, 5G, 5H, 51, 5J, 5L, 5M, and 5N; (9) 5K, 5H, 51, 5 J, 5L, 5M, and 5N; and (10) 5H, 51, 5 J, 50, and 5P, and (iii) at least one exogenous nucleic acid encoding a MMPE (e.g., a methanol oxidation pathway enzyme) expressed in a sufficient amount to produce formaldehyde in the presence of methanol, wherein said MMP comprises 1 J. In certain embodiments, the FAPE is expressed in a sufficient amount to produce pyruvate. In some embodiments, the FRPE is expressed in a sufficient amount to produce formaldehyde. In other embodiments, the FRPE is expressed in a sufficient amount to produce pyruvate. In certain embodiments, the FRPE is expressed in a sufficient amount to produce acetyl-CoA. In some embodiments, 5K is spontaneous. In some embodiments, 5F is an EFR2A. In other embodiments, 5F is an EFR2B. In other embodiments, 5F is an EFR2C. In certain

embodiments, NNOMO further comprises a 3 -HIBP or MAAP provided herein.

[0150] In certain embodiments, provided herein is a NNOMO having a FAP, a FRP, a MMP (e.g., a methanol oxidation pathway, comprising 1 J), a hydrogenase, a carbon monoxide dehydrogenase or any combination described above, wherein the organism further comprises a 3- HIBP. In other embodiments, provided herein is a NNOMO having a FAP, a FRP, a MMP (e.g., a methanol oxidation pathway, comprising 1 J), a hydrogenase, a carbon monoxide

dehydrogenase or any combination described above, wherein the organism further comprises a MAAP.

[0151] In some embodiments, formaldehyde produced from EM9 (FIG. 1 , step J) in certain of the NNOMO provided herein is used for generating energy, redox and/or formation of biomass. Two such pathways are shown in FIG. 3. Additionally, several organisms use an alternative pathway called the "serine cycle" for formaldehyde assimilation. These organisms include the methylotroph, Methylobacterium extorquens AMI, and another, Methylobacterium organophilum. The net balance of this cycle is the fixation of two mols of formaldehyde and 1 mol of C0 ₂ into 1 mol of 3-phosphoglycerate, which is used for biosynthesis, at the expense of 2 mols ATP and the oxidation of 2 mols of NAD(P)H.

[0152] In the first reaction of the serine pathway, formaldehyde reacts with glycine to form serine. The reaction is catalyzed by serine hydroxymethyltransferase (SHMT), an enzyme that uses tetrahydro folate (THF) as a cofactor. This leads to the formation of 5,10- methylenetetrahydro folate. During the reaction, formaldehyde is transferred from 5,10- methylenetetrahydrofolate to the glycine, forming L-serine. In the next step, serine is transaminated with glyoxylate as the amino group acceptor by the enzyme serine-glyoxylate aminotransferase, to produce hydroxypyruvate and glycine. Hydroxypyruvate is reduced to glycerate by hydroxypyruvate reductase. Glycerate 2-kinase catalyzes the addition of a phosphate group from ATP to produce 2-phosphoglycerate.

[0153] Some of the 2-phosphoglycerate is converted by phosphoglycerate mutase to 3- phosphoglycerate, which is an intermediate of the central metabolic pathways and used for biosynthesis. The rest of the 2-phosphoglycerate is converted by an enolase to

phosphoenolpyruvate (PEP). PEP carboxylase then catalyzes the fixation of carbon dioxide coupled to the conversion of PEP to oxaloacetate, which is reduced to malate by malate dehydrogenase, an NAD-linked enzyme. In some embodiments, the exogenous malate dehydrogenase genes are Rhizopus delemar malate dehydrogenase genes encoding the amino acid sequence disclosed in WO2013112939 as SEQ ID NO: 167 or its variants. Malate is activated to malyl coenzyme A by malate thiokinase and is cleaved by malyl coenzyme A lyase into acetyl coA and glyoxylate. These two enzymes (malate thiokinase and malyl coenzyme A lyase), as well as hydroxypyruvate reductase and glycerate -2 -kinase, are uniquely present in methylotrophs that contain the serine pathway.

[0154] In organisms that possess isocitrate lyase, a key enzyme of the glyoxylate cycle, acetyl CoA is converted to glyoxylate by the glyoxylate cycle. However, if the enzyme is missing, it is converted by another unknown pathway (deVries et al, FEMS Microbiol Rev, 6 (1): 57-101 (1990)). The resulting glyoxylate can serve as substrate for serine-glyoxylate aminotransferase, regenerating glycine and closing the circle.

[0155] It is understood that any of the pathways disclosed herein, as described in the

Examples and exemplified in the figures, including the pathways of FIGS. 1, 2, 3, 4 and 5, can be utilized to generate a NNOMO that produces any pathway intermediate or product, as desired. Non- limiting examples of such intermediate or products are 3-HIB or MAA. As disclosed herein, such a microbial organism that produces an intermediate can be used in combination with another microbial organism expressing downstream pathway enzymes to produce a desired product. However, it is understood that a NNOMO that produces a 3-HIBP or MAAP intermediate can be utilized to produce the intermediate as a desired product.

[0156] In certain embodiments, a NNOMO comprising a MMP and a 3-HIBP or MAAP provided herein, either alone or in combination with a FAP and/or a FRP provided herein, further comprises one or more gene disruptions. In certain embodiments, the one or more gene disruptions confer increased production of 3-HIB or MAA in the organism. In other

embodiments, a NNOMO comprising a MMP, FAP and/or FRP provided herein, further comprises one or more gene disruptions. In some embodiments, the gene disruption is in an endogenous gene encoding a protein and/or enzyme involved in native production of ethanol, glycerol, acetate, lactate, formate, C0 ₂ , amino acids, or any combination thereof, by said microbial organism. In one embodiment, the gene disruption is in an endogenous gene encoding a protein and/or enzyme involved in native production of ethanol. In another embodiment, the gene disruption is in an endogenous gene encoding a protein and/or enzyme involved in native production of glycerol. In other embodiments, the gene disruption is in an endogenous gene encoding a protein and/or enzyme involved in native production of acetate. In another embodiment, the gene disruption is in an endogenous gene encoding a protein and/or enzyme involved in native production of lactate. In one embodiment, the gene disruption is in an endogenous gene encoding a protein and/or enzyme involved in native production of formate. In another embodiment, the gene disruption is in an endogenous gene encoding a protein and/or enzyme involved in native production of C0 ₂ . In other embodiments, the gene disruption is in an endogenous gene encoding a protein and/or enzyme involved in native production of amino acids by said microbial organism. In some embodiments, the protein or enzyme is a pyruvate decarboxylase, an ethanol dehydrogenase, a glycerol dehydrogenase, a glycerol-3 -phosphatase, a glycerol-3 -phosphate dehydrogenase, a lactate dehydrogenase, an acetate kinase, a

phosphotransacetylase, a pyruvate oxidase, a pyruvate :quinone oxidoreductase, a pyruvate formate lyase, an alcohol dehydrogenase, a lactate dehydrogenase, a pyruvate dehydrogenase, a pyruvate formate-lyase-2-ketobutyrate formate-lyase, a pyruvate transporter, a monocarboxylate transporter, a NADH dehydrogenase, a cytochrome oxidase, a pyruvate kinase, or any

combination thereof. In certain embodiments, the one or more gene disruptions confer increased production of formaldehyde in the organism. In another embodiment, the gene disruption is in an endogenous gene encoding a protein and/or enzyme involved in a native formaldehyde utilization pathway. In certain embodiments, the organism comprises from one to twenty- five gene disruptions. In other embodiments, the organism comprises from one to twenty gene disruptions. In some embodiments, the organism comprises from one to fifteen gene disruptions. In other embodiments, the organism comprises from one to ten gene disruptions. In some embodiments, the organism comprises from one to five gene disruptions. In certain

embodiments, the organism comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 gene disruptions or more.

[0157] In other embodiments, a NNOMO comprising a MMP and a 3-HIBP or MAAP provided herein, either alone or in combination with a FAP and/or a FRP provided herein, further comprises one or more endogenous proteins or enzymes involved in native production of ethanol, glycerol, acetate, lactate, formate, C0 ₂ and/or amino acids by said microbial organism, wherein said one or more endogenous proteins or enzymes has attenuated protein or enzyme activity and/or expression levels. In some embodiments, a NNOMO comprising a MMP and a FAP provided herein, either alone or in combination with a FAP and/or a FRP provided herein, further comprises one or more endogenous proteins or enzymes involved in native production of ethanol, glycerol, acetate, lactate, formate, C02 and/or amino acids by said microbial organism, wherein said one or more endogenous proteins or enzymes has attenuated protein or enzyme activity and/or expression levels. In one embodiment the endogenous protein or enzyme is a pyruvate decarboxylase, an ethanol dehydrogenase, a glycerol dehydrogenase, a glycerol-3- phosphatase, a glycerol-3 -phosphate dehydrogenase, a lactate dehydrogenase, an acetate kinase, a phosphotransacetylase, a pyruvate oxidase, a pyruvate :quinone oxidoreductase, a pyruvate formate lyase, an alcohol dehydrogenase, a lactate dehydrogenase, a pyruvate dehydrogenase, a pyruvate formate-lyase-2-ketobutyrate formate-lyase, a pyruvate transporter, a monocarboxylate transporter, a NADH dehydrogenase, a cytochrome oxidase, a pyruvate kinase, or any combination thereof.

[0158] Each of the non-naturally occurring alterations provided herein result in increased production and an enhanced level of 3-HIB or MAA, for example, during the exponential growth phase of the microbial organism, compared to a strain that does not contain such metabolic alterations, under appropriate culture conditions. Appropriate conditions include, for example, those disclosed herein, including conditions such as particular carbon sources or reactant availabilities and/or adaptive evolution.

[0159] In certain embodiments, provided herein are NNOMO having genetic alterations such as gene disruptions that increase production of, for example, 3-HIB or MAA, for example, growth-coupled production of 3-HIB or MAA. Product production can be, for example, obligatorily linked to the exponential growth phase of the microorganism by genetically altering the metabolic pathways of the cell, as disclosed herein. The genetic alterations can increase the production of the desired product or even make the desired product an obligatory product during the growth phase. Appropriate conditions include, for example, those disclosed herein, including conditions such as particular carbon sources or reactant availabilities and/or adaptive evolution.

[0160] Given the teachings and guidance provided herein, those skilled in the art will understand that to introduce a metabolic alteration, such as attenuation of an enzyme, it can be necessary to disrupt the catalytic activity of the one or more enzymes involved in the reaction. Alternatively, a metabolic alteration can include disrupting expression of a regulatory protein or cofactor necessary for enzyme activity or maximal activity. Furthermore, genetic loss of a cofactor necessary for an enzymatic reaction can also have the same effect as a disruption of the gene encoding the enzyme. Disruption can occur by a variety of methods including, for example, deletion of an encoding gene or incorporation of a genetic alteration in one or more of the encoding gene sequences. The encoding genes targeted for disruption can be one, some, or all of the genes encoding enzymes involved in the catalytic activity. For example, where a single enzyme is involved in a targeted catalytic activity, disruption can occur by a genetic alteration that reduces or eliminates the catalytic activity of the encoded gene product. Similarly, where the single enzyme is multimeric, including heteromeric, disruption can occur by a genetic alteration that reduces or destroys the function of one or all subunits of the encoded gene products. Destruction of activity can be accomplished by loss of the binding activity of one or more subunits required to form an active complex, by destruction of the catalytic subunit of the multimeric complex or by both. Other functions of multimeric protein association and activity also can be targeted in order to disrupt a metabolic reaction provided herein. Such other functions are well known to those skilled in the art. Similarly, a target enzyme activity can be reduced or eliminated by disrupting expression of a protein or enzyme that modifies and/or activates the target enzyme, for example, a molecule required to convert an apoenzyme to a holoenzyme. Further, some or all of the functions of a single polypeptide or multimeric complex can be disrupted according to the invention in order to reduce or abolish the catalytic activity of one or more enzymes involved in a reaction or metabolic modification provided herein.

Similarly, some or all of enzymes involved in a reaction or metabolic modification provided herein can be disrupted so long as the targeted reaction is reduced or eliminated.

[0161] Given the teachings and guidance provided herein, those skilled in the art also will understand that an enzymatic reaction can be disrupted by reducing or eliminating reactions encoded by a common gene and/or by one or more orthologs of that gene exhibiting similar or substantially the same activity. Reduction of both the common gene and all orthologs can lead to complete abolishment of any catalytic activity of a targeted reaction. However, disruption of either the common gene or one or more orthologs can lead to a reduction in the catalytic activity of the targeted reaction sufficient to promote coupling of growth to product biosynthesis.

Exemplified herein are both the common genes encoding catalytic activities for a variety of metabolic modifications as well as their orthologs. Those skilled in the art will understand that disruption of some or all of the genes encoding a enzyme of a targeted metabolic reaction can be practiced in the methods provided herein and incorporated into the NNOMOs provided herein in order to achieve the increased production of 3-HIB or MAA or growth-coupled product production.

[0162] Given the teachings and guidance provided herein, those skilled in the art also will understand that enzymatic activity or expression can be attenuated using well known methods. Reduction of the activity or amount of an enzyme can mimic complete disruption of a gene if the reduction causes activity of the enzyme to fall below a critical level that is normally required for a pathway to function. Reduction of enzymatic activity by various techniques rather than use of a gene disruption can be important for an organism's viability. Methods of reducing enzymatic activity that result in similar or identical effects of a gene disruption include, but are not limited to: reducing gene transcription or translation; destabilizing mRNA, protein or catalytic RNA; and mutating a gene that affects enzyme activity or kinetics (See, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999). Natural or imposed regulatory controls can also accomplish enzyme attenuation including: promoter replacement (See, Wang et al., Mol. Biotechnol. 52(2):300-308 (2012)); loss or alteration of transcription factors (Dietrick et al., Annu. Rev. Biochem. 79:563-590 (2010); and Simicevic et al., Mol. Biosyst. 6(3):462-468 (2010)); introduction of inhibitory RNAs or peptides such as siRNA, antisense RNA, RNA or peptide/small-molecule binding aptamers, ribozymes, aptazymes and riboswitches (Wieland et al., Methods 56(3):351-357 (2012); O'Sullivan, Anal. Bioanal. Chem. 372(l):44-48 (2002); and Lee et al, Curr. Opin. Biotechnol. 14(5):505-511 (2003)); and addition of drugs or other chemicals that reduce or disrupt enzymatic activity such as an enzyme inhibitor, an antibiotic or a target-specific drug.

[0163] One skilled in the art will also understand and recognize that attenuation of an enzyme can be done at various levels. For example, at the gene level, a mutation causing a partial or complete null phenotype, such as a gene disruption, or a mutation causing epistatic genetic effects that mask the activity of a gene product (Miko, Nature Education 1(1) (2008)), can be used to attenuate an enzyme. At the gene expression level, methods for attenuation include: coupling transcription to an endogenous or exogenous inducer, such as isopropylthio-β- galactoside (IPTG), then adding low amounts of inducer or no inducer during the production phase (Donovan et al., J. Ind. Microbiol. 16(3): 145-154 (1996); and Hansen et al., Curr.

Microbiol. 36(6):341-347 (1998)); introducing or modifying a positive or a negative regulator of a gene; modify histone acetyl ation/deacetylation in a eukaryotic chromosomal region where a gene is integrated (Yang et al., Curr. Opin. Genet. Dev. 13(2): 143-153 (2003) and Kurdistani et al., Nat. Rev. Mol. Cell Biol. 4(4):276-284 (2003)); introducing a transposition to disrupt a promoter or a regulatory gene (Bleykasten-Brosshans et al., C. R. Biol. 33(8-9):679-686 (2011); and McCue et al., PLoS Genet. 8(2):el002474 (2012)); flipping the orientation of a transposable element or promoter region so as to modulate gene expression of an adjacent gene (Wang et al., Genetics 120(4):875-885 (1988); Hayes, Annu. Rev. Genet. 37:3-29 (2003); in a diploid organism, deleting one allele resulting in loss of heterozygosity (Daigaku et al., Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 600(1-2)177-183 (2006)); introducing nucleic acids that increase RNA degradation (Houseley et al., Cell, 136(4):763-776 (2009); or in bacteria, for example, introduction of a transfer-messenger RNA (tmRNA) tag, which can lead to RNA degradation and ribosomal stalling (Sunohara et al., RNA 10(3):378-386 (2004); and Sunohara et al, J. Biol. Chem. 279: 15368-15375 (2004)). At the translational level, attenuation can include: introducing rare codons to limit translation (Angov, Biotechnol. J.

6(6):650-659 (2011)); introducing RNA interference molecules that block translation (Castel et al., Nat. Rev. Genet. 14(2): 100-112 (2013); and Kawasaki et al., Curr. Opin. Mol. Ther.

7(2): 125-131 (2005); modifying regions outside the coding sequence, such as introducing secondary structure into an untranslated region (UTR) to block translation or reduce efficiency of translation (Ringner et al., PLoS Comput. Biol. I(7):e72 (2005)); adding RNAase sites for rapid transcript degradation (Pasquinelli, Nat. Rev. Genet. 13(4):271-282 (2012); and Arraiano et al., FEMS Microbiol. Rev. 34(5):883-932 (2010); introducing antisense RNA oligomers or antisense transcripts (Nashizawa et al., Front. Biosci. 17:938-958 (2012)); introducing RNA or peptide aptamers, ribozymes, aptazymes, riboswitches (Wieland et al., Methods 56(3):351-357 (2012); O'Sullivan, Anal. Bioanal. Chem. 372(l):44-48 (2002); and Lee et al., Curr. Opin. Biotechnol. 14(5):505-511 (2003)); or introducing translational regulatory elements involving RNA structure that can prevent or reduce translation that can be controlled by the presence or absence of small molecules (Araujo et al., Comparative and Functional Genomics, Article ID 475731, 8 pages (2012)). At the level of enzyme localization and/or longevity, enzyme attenuation can include: adding a degradation tag for faster protein turnover (Hochstrasser, Annual Rev. Genet. 30:405- 439 (1996); and Yuan et al, PLoS One 8(4):e62529 (2013)); or adding a localization tag that results in the enzyme being secreted or localized to a subcellular compartment in a eukaryotic cell, where the enzyme would not be able to react with its normal substrate (Nakai et al.

Genomics 14(4):897-911 (1992); and Russell et al., J. Bact. 189(21)7581-7585 (2007)). At the level of post-translational regulation, enzyme attenuation can include: increasing intracellular concentration of known inhibitors; or modifying post-translational modified sites (Mann et al., Nature Biotech. 21 :255-261 (2003)). At the level of enzyme activity, enzyme attenuation can include: adding an endogenous or an exogenous inhibitor, such as an enzyme inhibitor, an antibiotic or a target-specific drug, to reduce enzyme activity; limiting availability of essential cofactors, such as vitamin B12, for an enzyme that requires the co factor; chelating a metal ion that is required for enzyme activity; or introducing a dominant negative mutation. The applicability of a technique for attenuation described above can depend upon whether a given host microbial organism is prokaryotic or eukaryotic, and it is understand that a determination of what is the appropriate technique for a given host can be readily made by one skilled in the art.

[0164] In some embodiments, microaerobic designs can be used based on the growth- coupled formation of the desired product. To examine this, production cones can be constructed for each strategy by first maximizing and, subsequently minimizing the product yields at different rates of biomass formation feasible in the network. If the rightmost boundary of all possible phenotypes of the mutant network is a single point, it implies that there is a unique optimum yield of the product at the maximum biomass formation rate possible in the network. In other cases, the rightmost boundary of the feasible phenotypes is a vertical line, indicating that at the point of maximum biomass the network can make any amount of the product in the calculated range, including the lowest amount at the bottommost point of the vertical line. Such designs are given a low priority.

[0165] The 3-HIB or MAA-production strategies identified by the methods disclosed herein such as the OptKnock framework are generally ranked on the basis of their (i) theoretical yields, and (ii) growth-coupled 3-HIB or MAA formation characteristics.

[0166] The 3-HIB- or MAA-production strategies provided herein can be disrupted to increase production of 3-HIB or MAA. Accordingly, also provided herein is a NNOMO having metabolic modifications coupling 3-HIB or MAA production to growth of the organism, where the metabolic modifications includes disruption of one or more genes selected from the genes encoding proteins and/or enzymes provided herein.

[0167] Each of the strains can be supplemented with additional deletions if it is determined that the strain designs do not sufficiently increase the production of 3-HIB or MAA and/or couple the formation of the product with biomass formation. Alternatively, some other enzymes not known to possess significant activity under the growth conditions can become active due to adaptive evolution or random mutagenesis. Such activities can also be knocked out. However, gene deletions provided herein allow the construction of strains exhibiting high-yield production of 3-HIB or MAA, including growth-coupled production of 3-HIB or MAA.

[0168] In another aspect, provided herein is a method for producing 3-HIB or MAA, comprising culturing any one of the NNOMOs comprising a MMP and an 3-HIBP or MAAP provided herein under conditions and for a sufficient period of time to produce 3-HIB or MAA. In certain embodiments, the organism is cultured in a substantially anaerobic culture medium.

[0169] In one embodiment, provided herein are methods for producing 3-HIB, comprising culturing an organism provided herein (e.g., a NNOMOs comprising a MMP and an 3-HIBP either alone or in combination with a FAP and/or a FRP provided herein) under conditions and for a sufficient period of time to produce 3-HIB. In some embodiments, the method comprises culturing, for a sufficient period of time to produce 3-HIB, a NNOMO, comprising (1) a MMP, wherein said organism comprises at least one exogenous nucleic acid encoding a MMPE in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol; and (2) an 3-HIBP, comprising at least one exogenous nucleic acid encoding an 3- HIBPE expressed in a sufficient amount to produce 3-HIB. In certain embodiments, the NNOMO further comprises a FAP, comprising at least one exogenous nucleic acid encoding a FAPE as provided herein; and/or a FRP, comprising at least one exogenous nucleic acid encoding a RFPE as provided herein.

[0170] In another embodiment, provided herein are methods for producing MAA, comprising culturing an organism provided herein (e.g., a NNOMOs comprising a MMP and an MAAP) under conditions and for a sufficient period of time to produce MAA. In some embodiments, the method comprises culturing, for a sufficient period of time to produce MAA, a NNOMO, comprising (1) a MMP, wherein said organism comprises at least one exogenous nucleic acid encoding a MMPE in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol; and (2) an MAAP, comprising at least one exogenous nucleic acid encoding an MAAPE expressed in a sufficient amount to produce MAA. In certain embodiments, the NNOMO further comprises a FAP, comprising at least one exogenous nucleic acid encoding a FAPE as provided herein; and/or a FRP, comprising at least one exogenous nucleic acid encoding a RFPE as provided herein.

[0171] In certain embodiments of the methods provided herein, the organism further comprises at least one nucleic acid encoding a 3-HIBPE or MAAPE expressed in a sufficient amount to produce 3-HIB or MAA. In some embodiments, the nucleic acid is an exogenous nucleic acid. In other embodiments, the nucleic acid is an endogenous nucleic acid. In some embodiments, the organism further comprises one or more gene disruptions provided herein that confer increased production of 3-HIB or MAA in the organism. In certain embodiments, the one or more gene disruptions occurs in an endogenous gene encoding a protein or enzyme involved in native production of ethanol, glycerol, acetate, lactate, formate, C0 ₂ and/or amino acids by said microbial organism. In other embodiments, the organism further comprises one or more endogenous proteins or enzymes involved in native production of ethanol, glycerol, acetate, lactate, formate, C0 ₂ and/or amino acids by said microbial organism, wherein said one or more endogenous proteins or enzymes has attenuated protein or enzyme activity and/or expression levels. In certain embodiments, the organism is a Crabtree positive, eukaryotic organism, and the organism is cultured in a culture medium comprising glucose. In certain embodiments, the organism comprises from one to twenty-five gene disruptions. In other embodiments, the organism comprises from one to twenty gene disruptions. In some embodiments, the organism comprises from one to fifteen gene disruptions. In other embodiments, the organism comprises from one to ten gene disruptions. In some embodiments, the organism comprises from one to five gene disruptions. In certain embodiments, the organism comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 gene disruptions or more. In certain embodiments, the NNOMO further comprises a FAP, comprising at least one exogenous nucleic acid encoding a FAPE as provided herein; and/or a FRP, comprising at least one exogenous nucleic acid encoding a RFPE as provided herein.

[0172] In an additional embodiment, provided is a NNOMO having a 3-HIBP or MAAP, formaldehyde assimilation and/or MMP, wherein the NNOMO comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product. By way of example, in FIG. 1, the substrate of 1J is methanol, and the product is formaldehyde; the substrate of 1L is formaldehyde, and the product is formate; and so forth. One skilled in the art will understand that these are merely exemplary and that any of the substrate-product pairs disclosed herein suitable to produce a desired product and for which an appropriate activity is available for the conversion of the substrate to the product can be readily determined by one skilled in the art based on the teachings herein. Thus, provided herein are NNOMOs containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of a MMP, such as that shown in FIG. 1; a 3-HIBP or MAAP, such as that shown in FIG. 2; a FAP, such as that shown in FIGS. 3-5, and/or a FRP, such as that shown in FIG. 5.

[0173] While generally described herein as a microbial organism that contains a 3-HIBP or MAAP, FAP, FRP, and/or a MMP, it is understood that provided herein are also NNOMO comprising at least one exogenous nucleic acid encoding a 3-HIBP or MAAP, FAP, FRP, and/or a MMPE expressed in a sufficient amount to produce an intermediate of a 3-HIBP or MAAP, FAP, FRP, and/or a MMP intermediate. For example, as disclosed herein, a 3-HIBP or MAAP is exemplified in FIG. 2. Therefore, in addition to a microbial organism containing a 3-HIBP or MAAP that produces 3-HIB or MAA, also provided herein is a NNOMO comprising at least one exogenous nucleic acid encoding a 3-HIBPE or MAAPE, where the microbial organism produces a 3-HIBP or MAAP intermediate.

[0174] In some embodiments, the carbon feedstock and other cellular uptake sources such as phosphate, ammonia, sulfate, chloride and other halogens can be chosen to alter the isotopic distribution of the atoms present in 3-HIB or MAA or any 3-HIBP or MAAP intermediate. The various carbon feedstock and other uptake sources enumerated above will be referred to herein, collectively, as "uptake sources." Uptake sources can provide isotopic enrichment for any atom present in the product 3-HIB or MAA and/or a 3-HIBP or MAAP intermediate, or for side products generated in reactions diverging away from a 3-HIBP or MAAP. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens. The same holds true for the MMPs, FAPs, FRPs, as well as intermediates thereof, provided herein.

[0175] In some embodiments, the uptake sources can be selected to alter the carbon-12, carbon-13, and carbon- 14 ratios. In some embodiments, the uptake sources can be selected to alter the oxygen-16, oxygen-17, and oxygen-18 ratios. In some embodiments, the uptake sources can be selected to alter the hydrogen, deuterium, and tritium ratios. In some embodiments, the uptake sources can selected to alter the nitrogen- 14 and nitrogen- 15 ratios. In some

embodiments, the uptake sources can be selected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35 ratios. In some embodiments, the uptake sources can be selected to alter the phosphorus-31 , phosphorus-32, and phosphorus-33 ratios. In some embodiments, the uptake sources can be selected to alter the chlorine-35, chlorine-36, and chlorine-37 ratios.

[0176] In some embodiments, the isotopic ratio of a target atom can be varied to a desired ratio by selecting one or more uptake sources. An uptake source can be derived from a natural source, as found in nature, or from a man-made source, and one skilled in the art can select a natural source, a man-made source, or a combination thereof, to achieve a desired isotopic ratio of a target atom. An example of a man-made uptake source includes, for example, an uptake source that is at least partially derived from a chemical synthetic reaction. Such isotopically enriched uptake sources can be purchased commercially or prepared in the laboratory and/or optionally mixed with a natural source of the uptake source to achieve a desired isotopic ratio. In some embodiments, a target isotopic ratio of an uptake source can be obtained by selecting a desired origin of the uptake source as found in nature For example, as discussed herein, a natural source can be a biobased derived from or synthesized by a biological organism or a source such as petroleum-based products or the atmosphere. In some such embodiments, a source of carbon, for example, can be selected from a fossil fuel-derived carbon source, which can be relatively depleted of carbon- 14, or an environmental or atmospheric carbon source, such as C0 ₂ , which can possess a larger amount of carbon- 14 than its petroleum-derived counterpart.

[0177] Isotopic enrichment is readily assessed by mass spectrometry using techniques known in the art such as Stable Isotope Ratio Mass Spectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques can be integrated with separation techniques such as liquid chromatography (LC) and/or high performance liquid chromatography (HPLC).

[0178] The unstable carbon isotope carbon- 14 or radiocarbon makes up for roughly 1 in 10 12 carbon atoms in the earth's atmosphere and has a half-life of about 5700 years. The stock of carbon is replenished in the upper atmosphere by a nuclear reaction involving cosmic rays and ordinary nitrogen ( ¹⁴ N). Fossil fuels contain no carbon-14, as it decayed long ago. Burning of fossil fuels lowers the atmospheric carbon-14 fraction, the so-called "Suess effect".

[0179] Methods of determining the isotopic ratios of atoms in a compound are well known to those skilled in the art. Isotopic enrichment is readily assessed by mass spectrometry using techniques known in the art such as accelerated mass spectrometry (AMS), Stable Isotope Ratio Mass Spectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques can be integrated with separation techniques such as liquid chromatography (LC), high performance liquid

chromatography (HPLC) and/or gas chromatography, and the like.

[0180] In the case of carbon, ASTM D6866 was developed in the United States as a standardized analytical method for determining the biobased content of solid, liquid, and gaseous samples using radiocarbon dating by the American Society for Testing and Materials (ASTM) International. The standard is based on the use of radiocarbon dating for the determination of a product's biobased content. ASTM D6866 was first published in 2004, and the current active version of the standard is ASTM D6866-11 (effective April 1, 2011). Radiocarbon dating techniques are well known to those skilled in the art, including those described herein.

[0181] The biobased content of a compound is estimated by the ratio of carbon-14 ( ¹⁴ C) to carbon- 12 ( 12 C). Specifically, the Fraction Modern (Fm) is computed from the expression: Fm = (S-B)/(M-B), where B, S and M represent the ¹⁴ C/ ¹² C ratios of the blank, the sample and the modern reference, respectively. Fraction Modern is a measurement of the deviation of the ¹⁴ C/ ¹² C ratio of a sample from "Modern." Modern is defined as 95% of the radiocarbon concentration (in AD 1950) of National Bureau of Standards (NBS) Oxalic Acid I (i.e., standard

13

reference materials (SRM) 4990b) normalized to δ CV _PDB =-19 per mil (Olsson, The use of Oxalic acid as a Standard, in, Radiocarbon Variations and Absolute Chronology, Nobel Symposium, 12th Proc, John Wiley & Sons, New York (1970)). Mass spectrometry results, for example, measured by ASM, are calculated using the internationally agreed upon definition of 0.95 times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalized to 5 ¹³ C _VPDB =-19 per mil. This is equivalent to an absolute (AD 1950) ¹⁴ C/ ¹² C ratio of 1.176 ± 0.010 x 10 ^~12 (Karlen et al., Arkiv Geofysik, 4:465-471 (1968)). The standard calculations take into account the differential uptake of one isotope with respect to another, for example, the preferential uptake in biological systems of C ¹² over C ¹³ over C ¹⁴ , and these corrections are reflected as a Fm

13

corrected for δ .

[0182] An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of 1955 sugar beet. Although there were 1000 lbs made, this oxalic acid standard is no longer commercially available. The Oxalic Acid II standard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of 1977 French beet molasses. In the early 1980's, a group of 12 laboratories measured the ratios of the two standards. The ratio of the activity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). The isotopic ratio of HOx II is -17.8 per mille. ASTM D6866-11 suggests use of the available Oxalic Acid II standard SRM 4990 C (Hox2) for the modern standard (see discussion of original vs. currently available oxalic acid standards in Mann, Radiocarbon, 25(2):519-527 (1983)). A Fm = 0% represents the entire lack of carbon-14 atoms in a material, thus indicating a fossil (for example, petroleum based) carbon source. A Fm = 100%, after correction for the post- 1950 injection of carbon-14 into the atmosphere from nuclear bomb testing, indicates an entirely modern carbon source. As described herein, such a "modern" source includes biobased sources.

[0183] As described in ASTM D6866, the percent modern carbon (pMC) can be greater than 100% because of the continuing but diminishing effects of the 1950s nuclear testing programs, which resulted in a considerable enrichment of carbon-14 in the atmosphere as described in ASTM D6866-11. Because all sample carbon-14 activities are referenced to a "pre -bomb" standard, and because nearly all new biobased products are produced in a post-bomb

environment, all pMC values (after correction for isotopic fraction) must be multiplied by 0.95 (as of 2010) to better reflect the true biobased content of the sample. A biobased content that is greater than 103% suggests that either an analytical error has occurred, or that the source of biobased carbon is more than several years old.

[0184] ASTM D6866 quantifies the biobased content relative to the material's total organic content and does not consider the inorganic carbon and other non-carbon containing substances present. For example, a product that is 50%> starch-based material and 50%> water would be considered to have a Biobased Content = 100% (50% organic content that is 100% biobased) based on ASTM D6866. In another example, a product that is 50% starch-based material, 25% petroleum-based, and 25% water would have a Biobased Content = 66.7% (75% organic content but only 50% of the product is biobased). In another example, a product that is 50% organic carbon and is a petroleum-based product would be considered to have a Biobased Content = 0% (50%) organic carbon but from fossil sources). Thus, based on the well known methods and known standards for determining the biobased content of a compound or material, one skilled in the art can readily determine the biobased content and/or prepared downstream products that utilize provided herein having a desired biobased content.

[0185] Applications of carbon- 14 dating techniques to quantify bio-based content of materials are known in the art (Currie et al., Nuclear Instruments and Methods in Physics Research B, 172:281-287 (2000)). For example, carbon- 14 dating has been used to quantify biobased content in terephthalate-containing materials (Colonna et al., Green Chemistry, 13:2543- 2548 (2011)). Notably, polypropylene terephthalate (PPT) polymers derived from renewable 1,3-propanediol and petroleum-derived terephthalic acid resulted in Fm values near 30% {i.e., since 3/11 of the polymeric carbon derives from renewable 1,3-propanediol and 8/11 from the fossil end member terephthalic acid) (Currie et al., supra, 2000). In contrast, polybutylene terephthalate polymer derived from both renewable BDO and renewable terephthalic acid resulted in bio-based content exceeding 90% (Colonna et al., supra, 2011).

[0186] Accordingly, in some embodiments, the present invention provides 3-HIB or MAA, or a 3-HIBP or MAAP intermediate that has a carbon-12, carbon-13, and carbon-14 ratio that reflects an atmospheric carbon, also referred to as environmental carbon, uptake source. For example, in some aspects, the 3-HIB or MAA, or a 3-HIB or MAA intermediate thereof can have an Fm value of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or as much as 100%). In some such embodiments, the uptake source is C0 ₂ . In some embodiments, the present invention provides 3-HIB or MAA, or a 3-HIB or MAA intermediate thereof, that has a carbon- 12, carbon-13, and carbon-14 ratio that reflects petroleum-based carbon uptake source. In this aspect, the a 3-HIB or MAA, or a 3-HIB or MAA intermediate can have an Fm value of less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%>, less than 55%, less than 50%>, less than 45%, less than 40%, less than 35%, less than 30%), less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2% or less than 1%. In some embodiments, the present invention provides a 3-HIB or MAA, or a 3-HIB or MAA intermediate thereof, that has a carbon-12, carbon-13, and carbon-14 ratio that is obtained by a combination of an atmospheric carbon uptake source with a petroleum-based uptake source. Using such a combination of uptake sources is one way by which the carbon-12, carbon-13, and carbon-14 ratio can be varied, and the respective ratios would reflect the proportions of the uptake sources.

[0187] Further, the present invention relates, in part, to biologically produced 3-HIB or MAA, or a 3-HIB or MAA intermediate thereof, as disclosed herein, and to the products derived therefrom, wherein the a 3-HIB or MAA, or an intermediate thereof, has a carbon-12, carbon-13, and carbon-14 isotope ratio of about the same value as the C0 ₂ that occurs in the environment. For example, in some aspects provided is bioderived 3-HIB or MAA, or an intermediate thereof, having a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the C0 ₂ that occurs in the environment, or any of the other ratios disclosed herein. It is understood, as disclosed herein, that a product can have a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the C0 ₂ that occurs in the environment, or any of the ratios disclosed herein, wherein the product is generated from bioderived 3-HIB or MAA, or an intermediate thereof, as disclosed herein, wherein the bioderived product is chemically modified to generate a final product. Methods of chemically modifying a bioderived product of 3-HIB or MAA, or an intermediate thereof, to generate a desired product are well known to those skilled in the art, as described herein. The invention further provides polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like, having a carbon- 12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the C0 ₂ that occurs in the environment, wherein the polymers, co-polymers, plastics, methacrylates (e.g. , a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like, are generated directly from or in combination with bioderived 3-HIB or MAA or a bioderived intermediate thereof, as disclosed herein. [0188] 3-HIB and MAA, as well as intermediates thereof, are chemicals used in commercial and industrial applications. Non-limiting examples of such applications include production of polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like. Moreover, 3-HIB and MAA are also used as a raw material in the production of a wide range of products including polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like. Accordingly, in some embodiments, provided is biobased polymers, co-polymers, plastics, methacrylates (e.g. , a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like, comprising one or more of bioderived 3-HIB or MAA, or a bioderived intermediate thereof, produced by a NNOMO provided herein or produced using a method disclosed herein.

[0189] As used herein, the term "bioderived" means derived from or synthesized by a biological organism and can be considered a renewable resource since it can be generated by a biological organism. Such a biological organism, in particular the microbial organisms disclosed herein, can utilize feedstock or biomass, such as, sugars or carbohydrates obtained from an agricultural, plant, bacterial, or animal source. Alternatively, the biological organism can utilize atmospheric carbon. As used herein, the term "biobased" means a product as described above that is composed, in whole or in part, of a bioderived compound provided herein. A biobased or bioderived product is in contrast to a petroleum derived product, wherein such a product is derived from or synthesized from petroleum or a petrochemical feedstock.

[0190] In some embodiments, provided is polymers, co-polymers, plastics, methacrylates (e.g. , a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like, comprising bioderived 3-HIB or MAA, or a bioderived intermediate thereof, wherein the bioderived 3-HIB or MAA, or bioderived intermediate thereof, includes all or part of the a 3-HIB or MAA, or an intermediate thereof, used in the production of polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like,. Thus, in some aspects, provided is a biobased polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like, comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%o or 100% bioderived 3-HIB or MAA, or a bioderived 3-HIB or MAA intermediate, as disclosed herein. Additionally, in some aspects, provided is biobased polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like, wherein the a 3-HIB or MAA, or a 3-HIB or MAA intermediate, used in its production is a combination of bioderived and petroleum derived 3-HIB or MAA, or a 3-HIB or MAA intermediate thereof. For example, biobased polymers, co-polymers, plastics, methacrylates (e.g. , a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like, can be produced using 50% bioderived 3-HIB or MAA and 50% petroleum derived 3-HIB or MAA or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%,

40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA aids, food and oral medicinal coatings/products, and the like, using the bioderived 3-HIB or MAA, or a bioderived 3-HIB or MAA intermediate thereof, provided herein are well known in the art.

[0191] In one embodiment, the product is a polymer. In one embodiment, the product is a polymer. . In one embodiment, the product is a co-polymer. In one embodiment, the product is a plastic. In one embodiment, the product is a methacrylate. In one embodiment, the product is a methyl methacrylate. In one embodiment, the product is a butyl methacrylate. In one

embodiment, the product is a glacial MAA.

[0192] In some embodiments, provided herein is a culture medium comprising bioderived 3- HIB. In some embodiments, the bioderived 3-HIB is produced by culturing a NNOMO having a MMP and 3-HIBP, as provided herein. In certain embodiments, the bioderived 3-HIB has a carbon-12, carbon-13 and carbon- 14 isotope ratio that reflects an atmospheric carbon dioxide uptake source. In one embodiment, the culture medium is separated from a NNOMO having a MMP and 3-HIBP.

[0193] In other embodiments, provided herein is a bioderived 3-HIB. In some embodiments, the bioderived 3-HIB is produced by culturing a NNOMO having a MMP and 3-HIBP, as provided herein. In certain embodiments, the bioderived 3-HIB has a carbon-12, carbon-13 and carbon- 14 isotope ratio that reflects an atmospheric carbon dioxide uptake source. In some embodiments, the bioderived 3-HIB has an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%. In certain embodiments, the bioderived 3-HIB is a component of culture medium.

[0194] In certain embodiments, provided herein is a composition comprising a bioderived 3- HIB provided herein, for example, a bioderived 3-HIB produced by culturing a NNOMO having a MMP and 3-HIBP, as provided herein. In some embodiments, the composition further comprises a compound other than said bioderived 3-HIB. In certain embodiments, the compound other than said bioderived 3-HIB is a trace amount of a cellular portion of a NNOMO having a MMP and a 3-HIBP, as provided herein.

[0195] In some embodiments, provided herein is a biobased product comprising a

bioderived 3-HIB provided herein. In certain embodiments, the biobased product is a polymer, co-polymer, plastic, methacrylate, methyl methacrylate, butyl methacrylate, or glacial MAA. In certain embodiments, the biobased product comprises at least 5% bioderived 3-HIB. In certain embodiments, the biobased product comprises at least 10%> bioderived 3-HIB. In some embodiments, the biobased product comprises at least 20%> bioderived 3-HIB. In other embodiments, the biobased product comprises at least 30%> bioderived 3-HIB. In some embodiments, the biobased product comprises at least 40%> bioderived 3-HIB. In other embodiments, the biobased product comprises at least 50%> bioderived 3-HIB. In one

embodiment, the biobased product comprises a portion of said bioderived 3-HIB as a repeating unit. In another embodiment, provided herein is a molded product obtained by molding the biobased product provided herein. In other embodiments, provided herein is a process for producing a biobased product provided herein, comprising chemically reacting said bioderived 3- HIB with itself or another compound in a reaction that produces said biobased product. In certain embodiments, provided herein is a polymer comprising or obtained by converting the bioderived 3-HIB. In other embodiments, provided herein is a method for producing a polymer, comprising chemically of enzymatically converting the bioderived 3-HIB to the polymer. In yet other embodiments, provided herein is a composition comprising the bioderived 3-HIB, or a cell lysate or culture supernatant thereof. [0196] In some embodiments, provided herein is a culture medium comprising bioderived MAA. In some embodiments, the bioderived MAA is produced by culturing a NNOMO having a MMP and MAAP, as provided herein. In certain embodiments, the bioderived MAA has a carbon- 12, carbon- 13 and carbon- 14 isotope ratio that reflects an atmospheric carbon dioxide uptake source. In one embodiment, the culture medium is separated from a NNOMO having a MMP and MAAP.

[0197] In other embodiments, provided herein is a bioderived MAA. In some embodiments, the bioderived MAA is produced by culturing a NNOMO having a MMP and MAAP, as provided herein. In certain embodiments, the bioderived MAA has a carbon- 12, carbon- 13 and carbon- 14 isotope ratio that reflects an atmospheric carbon dioxide uptake source. In some embodiments, the bioderived MAA has an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%. In certain embodiments, the bioderived MAA is a component of culture medium.

[0198] In certain embodiments, provided herein is a composition comprising a bioderived MAA provided herein, for example, a bioderived MAA produced by culturing a NNOMO having a MMP and MAAP, as provided herein. In some embodiments, the composition further comprises a compound other than said bioderived MAA. In certain embodiments, the compound other than said bioderived MAA is a trace amount of a cellular portion of a NNOMO having a MMP and a MAAP, as provided herein.

[0199] In some embodiments, provided herein is a biobased product comprising a bioderived MAA provided herein. In certain embodiments, the biobased product is a polymer, co-polymer, plastic, methacrylate, methyl methacrylate, butyl methacrylate, or glacial MAA. In certain embodiments, the biobased product comprises at least 5% bioderived MAA. In certain embodiments, the biobased product comprises at least 10%> bioderived MAA. In some embodiments, the biobased product comprises at least 20%> bioderived MAA. In other embodiments, the biobased product comprises at least 30%> bioderived MAA. In some embodiments, the biobased product comprises at least 40%> bioderived MAA. In other embodiments, the biobased product comprises at least 50%> bioderived MAA. In one

embodiment, the biobased product comprises a portion of said bioderived MAA as a repeating unit. In another embodiment, provided herein is a molded product obtained by molding the biobased product provided herein. In other embodiments, provided herein is a process for producing a biobased product provided herein, comprising chemically reacting said bioderived - MAA with itself or another compound in a reaction that produces said biobased product. In certain embodiments, provided herein is a polymer comprising or obtained by converting the bioderived MAA. In other embodiments, provided herein is a method for producing a polymer, comprising chemically of enzymatically converting the bioderived MAA to the polymer. In yet other embodiments, provided herein is a composition comprising the bioderived MAA, or a cell lysate or culture supernatant thereof.

[0200] Also provided herein is a method of producing formaldehyde, comprising culturing a NNOMO provided herein (e.g., comprising an exogenous nucleic acid encoding an EM9 (1 J)) under conditions and for a sufficient period of time to produce formaldehyde. In certain embodiments, the formaldehyde is consumed to provide a reducing equivalent. In other embodiments, the formaldehyde is consumed to incorporate into 3-HIB or MAA. In yet other embodiments, the formaldehyde is consumed to incorporate into another target product.

[0201] Also provided herein is a method of producing an intermediate of glycolysis and/or an intermediate of a metabolic pathway that can be used in the formation of biomass, comprising culturing a NNOMO provided herein (e.g., comprising an exogenous nucleic acid encoding an EM9 (1 J)) under conditions and for a sufficient period of time to produce the intermediate. In one embodiment, the method is a method of producing an intermediate of glycolysis. In other embodiments, the method is a method of producing an intermediate of a metabolic pathway that can be used in the formation of biomass. In certain embodiments, the intermediate is consumed to provide a reducing equivalent. In other embodiment, the intermediate is consumed to incorporate into 3-HIB or MAA. In yet other embodiments, the formaldehyde is consumed to incorporate into another target product.

[0202] A reducing equivalent can be readily obtained from the glycolysis intermediate by any of several central metabolic reactions including glyceraldehyde-3 -phosphate dehydrogenase, pyruvate dehydrogenase, pyruvate formate lyase and NAD(P)-dependant formate

dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase. Additionally, reducing equivalents can be generated from glucose 6-phosphate-l -dehydrogenase and 6-phosphogluconate dehydrogenase of the pentose phosphate pathway. Overall, at most twelve reducing equivalents can be obtained from a C6 glycolysis intermediate (e.g., glucose-6-phosphate, F6P, fructose- 1 ,6-diphosphate) and at most six reducing equivalents can be generated from a C3 glycolysis intermediate (e.g., DHAP, glyceraldehyde-3-phosphate).

[0203] The invention is described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction and that reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze, or proteins involved in, the referenced reaction, reactant or product. Likewise, given the well known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes, or a protein associated with the reaction, as well as the reactants and products of the reaction.

[0204] Microbial organisms generally lack the capacity to synthesize 3-HIB or MAA, and therefore any of the compounds disclosed herein to be within the 3-HIB or MAA family of compounds, or otherwise known by those in the art to be within the 3-HIB or MAA family of compounds. Moreover, organisms having all of the requisite metabolic enzymatic capabilities are not known to produce 3-HIB or MAA from the enzymes described and biochemical pathways exemplified herein. In contrast, the NNOMOs provided herein can generate 3-HIB or MAA as a product, as well as intermediates thereof. The biosynthesis of 3-HIB or MAA, as well as intermediates thereof, is particularly useful in chemical synthesis of 3-HIB or MAA family of compounds, it also allows for the further biosynthesis of 3-HIB or MAA family compounds and avoids altogether chemical synthesis procedures.

[0205] The NNOMOs provided herein that can produce 3-HIB or MAA are produced by ensuring that a host microbial organism includes functional capabilities for the complete biochemical synthesis of at least one 3-HIB or MAA biosynthetic pathway provided herein. Ensuring at least one requisite 3-HIB or MAA biosynthetic pathway confers 3-HIB or MAA biosynthesis capability onto the host microbial organism.

[0206] The organisms and methods are described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze or proteins involved in the referenced reaction, reactant or product. Likewise, given the well known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes or a protein associated with the reaction as well as the reactants and products of the reaction.

[0207] The NNOMOs described herein can be produced by introducing expressible nucleic acids encoding one or more of the enzymes or proteins participating in one or more methanol metabolic, formaldehyde assimilation, formate reutilization (assimilation), and/or 3-HIB or MAA biosynthetic pathways. Depending on the host microbial organism chosen for

biosynthesis, nucleic acids for some or all of a particular methanol metabolic, formaldehyde assimilation, formate reutilization (formate assimilation) and/or 3-HIB or MAA biosynthetic pathway can be expressed. For example, if a chosen host is deficient in one or more enzymes or proteins for a desired metabolic, assimilation, or biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous expression. Alternatively, if the chosen host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is needed for the deficient enzyme(s) or protein(s) to achieve 3-HIB or MAA biosynthesis and/or methanol metabolism. Thus, a NNOMO described herein can be produced by introducing exogenous enzyme or protein activities to obtain a desired metabolic pathway and/or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme or protein activities that, together with one or more endogenous enzymes or proteins, produces a desired product, such as 3-HIB or MAA.

[0208] Host microbial organisms can be selected from, and the NNOMOs generated in, for example, bacteria, yeast, fungus or any of a variety of other microorganisms applicable or suitable to fermentation processes. Exemplary bacteria include any species selected from the order Enter obacteriales, family Enter obacteriaceae, including the generan Escherichia and Klebsiella; the order Aeromonadales, family Succinivibrionaceae, including the genus

Anaerobiospirillum; the order Pasteur ellales, family Pasteur ellaceae, including the genera Actinobacillus and Mannheimia; the order Rhizobiales, family Bradyrhizobiaceae, including the genus Rhizobium; the order Bacillales, family Bacillaceae, including the genus Bacillus; the order Actinomycetales, families Corynebacteriaceae and Streptomycetaceae, including the genus Corynebacterium and the genus Streptomyces, respectively; order Rhodospirillales, family Acetobacteraceae, including the genus Gluconobacter; the order Sphingomonadales, family Sphingomonadaceae, including the genus Zymomonas; the order Lactobacillales, families Lactobacillaceae and Streptococcaceae, including the genus Lactobacillus and the genus Lactococcus, respectively; the order Clostridiales, family Clostridiaceae, genus Clostridium; and the order Pseudomonadales , family Pseudomonadaceae, including the genus Pseudomonas. Non- limiting species of host bacteria include Escherichia coli, Klebsiella oxytoca,

Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia

succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum,

Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and

Pseudomonas putida.

[0209] Similarly, exemplary species of yeast or fungi species include any species selected from the order Saccharomycetales, family Saccaromycetaceae, including the genera

Saccharomyces, Kluyveromyces and Pichia; the order Saccharomycetales, family

Dipodascaceae, including the genus Yarrowia; the order Schizosaccharomycetales, family Schizosaccaromycetaceae, including the genus Schizosaccharomyces; the order Eurotiales, family Trichocomaceae, including the genus Aspergillus; and the order Mucorales, family Mucoraceae, including the genus Rhizopus. Non-limiting species of host yeast or fungi include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, Yarrowia lipolytica, and the like. E. coli is a particularly useful host organism since it is a well characterized microbial organism suitable for genetic engineering. Other particularly useful host organisms include yeast such as Saccharomyces cerevisiae. It is understood that any suitable microbial host organism can be used to introduce metabolic and/or genetic modifications to produce a desired product.

[0210] In some embodiments, the host microbial organism can be a recombinant microbial organism having increased succinate (succinic acid) production as compared to the wild-type microbial organism. Increased succinate production can be generated by introduction of one or more gene disruptions of a host microbial organism gene and/or an exogenous nucleic acid. Methods of increasing succinate production in a microbial organism are well known in the art. For example, the host microbial organism can be a recombinant bacteria, such as a rumen bacteria, that includes a gene disruption in one or more genes selected from a lactate

dehydrogenase gene (IdhA), a pyruvate formate-lyase gene (pfl), a phosphotransacetylase gene (pta), and an acetate kinase gene (ackA) as described in U.S. Publication 2007-0054387, published March 8, 2007, now U.S. Patent 7,470,530, and U.S. Publication 2009-0203095, published Aug. 13, 2009. For example, in one aspect, the host microbial organism can include a gene disruption in a gene encoding IdhA, pta, and ackA, without disrupting a gene encoding pfl. Accordingly, in some aspects, the bacteria that can be used as a host microbial organism include, but are not limited to, a Mannheimia species (e.g. , Mannheimia sp. LPK, Mannheimia sp. LPK4, Mannheimia sp. LPK7, Mannheimia sp. LPK (KCTC 10558BP), Mannheimia

succiniciproducens MBEL55E (KCTC 0769BP), Mannheimia succiniciproducens PALK (KCTC10973BP), Mannheimia succiniciproducens ALK, or Mannheimia succiniciproducens ALKt), an Actinobacillus species (e.g. , Actinobacillus succinogenes), a Bacteroides species, a Succinimonas species, a Succinivibrio species, or an Anaerobio 'spirillum species (e.g.,

Anaerobiospirillum succiniciproducens) .

[0211] Additional methods for producing a host microbial organism having increased succinate production are also well known in the art. For example, the host microbial organism can have genes disruptions in genes encoding IdhA, pfl and a phosphopyruvate carboxylase (ppc), or alternatively/additionally gene disruptions in genes encoding a glucose

phosphotransferase (ptsG) and a pyruvate kinase (pykA and pykF), or alternatively/additionally gene disruptions in a gene encoding a succinic semialdehyde dehydrogenase (GabD), or alternatively/additionally introduction or amplification of a nucleic acid encoding a C4- dicarboxylate transport protein (DctA), which is associated with transport of succinate, as described in U.S. Publication 2010-0330634, published Dec. 30, 2010. Accordingly, a host microbial organism can include a Lumen bacteria, a Cory b acterium species, a Brevibacterium species or an Escherichia species {e.g., Escherichia coli, in particular strain W3110GFA, as disclosed in U.S. Publication 2009-0075352, published March 19, 2009). As yet another example, a host microbial organism having increased succinate production can be generated by introducing an exogenous nucleic acid encoding an enzyme or protein that increases production of succinate are described in U.S. Publication 2007-0042476, published Feb. 22, 2007, U.S. Publication 2007-0042477, published Feb. 22, 2007, and U.S. Publication 2008-0020436, published Jan. 24, 2008, which disclose introduction of a nucleic acid encoding a malic enzyme B (maeB), a fumarate hydratase C (fumC), a formate dehydrogenase D (fdhD) or a formate dehydrogenase E (fdhE). Additional useful host microbial organisms include, but are not limited to, a microbial organism that can produce succinate using glycerol as a carbon source, as disclosed in WO 2009/048202, or an organism that simultaneously use sucrose and glycerol as carbon sources to produce succinate by weakening a catabolic inhibition mechanism of the glycerol by sucrose as described in EP 2612905.

[0212] Additional microbes having high succinate production suitable for use as a host microbial organism for the pathways and methods described herein include those bacterial strains described in International Publications WO 2010/092155 and WO 2009/024294, and U.S.

Publication 2010-0159542, published June 24, 2010 and those yeast strains described in

International Publication WO 2013/112939, published August 1, 2013. For example, bacterial strains of the genus Pasteurella, which are gram negative, facultative anaerobes, motile, pleimorphic and often catalase-and oxidase-positive, specifically Pasteurella strain DDI and its variants, are suitable host microbial organisms. Pasteurella strain DDI is the bacterial strain deposited under the Budapest Treaty with DSMZ (Deutsche Sammlungvon Mikroorganismen und Zellkulturen, GmbH), Germany, having deposit number DSM 18541, and was originally isolated from the rumen of a cow of German origin. Improved variants of DDI, are described in WO 2010/092155, are also suitable host microbial organisms, and include, but are not limited to, LU15348 (DDI with deletion oipfl gene); LU15050 (DDI deletion oildh gene); and LU15224 (DDI with deletion of both pfl and Idh genes). Additional host bacteria include succinate - producers isolated from bovine rumen belonging to the genus Mannheimia, specifically the species Mannheimia succiniciproducens, and strain Mannheimia succiniciproducens MBEL55E and its variants.

[0213] Exemplary host yeast strains, as described in WO 2013/112939, can be genetically modified yeast cells that include modifications to enhance succinate production and/or export, and, in some aspects, selected for succinate tolerance. Accordingly, in some embodiments, the high succinate producing host cell can be a yeast cell comprising a genetic modification to enhance succinate production and/or export, and in some aspects be tolerant of increased intracellular and/or extracellular succinate concentrations. In some embodiments, the genetically modified yeast cell belongs to a genus selected from the group consisting of

Issatchenkia, Candida, Pichia, Zygosaccharomyces, Kluyveromyces, Saccharomyces,

Debaryomyces, and Saccharomycopsis. Thus, in some embodiments, the genetically modified yeast cell is a species selected from the group consisting of Issatchenkia orientalis, Candida lambica, Candida sorboxylosa, Candida zemplinina, Candida geochares, Pichia

membranifaciens, Zygosaccharomyces kombuchaensis, Candida sorbosivorans, Kluyveromyces marxianus, Candida vanderwaltii, Candida sorbophila, Zygosaccharomyces bisporus,

Zygosaccharomyces lentus, Saccharomyces bayanus, Saccharomyces bulderi, Debaryomyces castellii, Candida boidinii, Candida etchellsii, Kluyveromyces lactis, Pichia jadinii, Pichia anomala, Saccharomycopsis crataegensis, and Pichia jadinii. In some embodiments, the genetically modified yeast cell is from the Pichia fermentans/lssatchenkia orientalis clade.

[0214] Depending on the 3-HIB or MAA biosynthetic, methanol metabolic, FAP and/or FRP constituents of a selected host microbial organism, the NNOMOs provided herein will include at least one exogenously expressed 3-HIB or MAA, formaldehyde assimilation, formate reutilization and/or MMP-encoding nucleic acid and up to all encoding nucleic acids for one or more 3-HIB or MAA biosynthetic pathways, FAPs, FRPs and/or MMPs. For example, 3-HIB or MAA biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid. In a host deficient in all enzymes or proteins of a 3-HIBP or MAAP, exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins. For example, exogenous expression of all enzymes or proteins in a pathway for production of 3-HIB or MAA can be included. The same holds true for the MMPs, FAPs and FRPs provided herein.

[0215] Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding nucleic acids to introduce in an expressible form will, at least, parallel the 3-HIBP or MAAP, FAP, FRP, and MMP deficiencies of the selected host microbial organism. Therefore, a NNOMO provided herein can have one, two, three, four, five, six, seven, eight, nine, or up to all nucleic acids encoding the enzymes or proteins constituting a MMP, FAP, FRP, and/or 3-HIB or MAA biosynthetic pathway disclosed herein. In some embodiments, the NNOMOs also can include other genetic modifications that facilitate or optimize 3-HIB or MAA biosynthesis, formaldehyde assimilation, formate reutilization and/or methanol metabolism or that confer other useful functions onto the host microbial organism. One such other functionality can include, for example, augmentation of the synthesis of one or more of the 3-HIBP or MAAP precursors.

[0216] Generally, a host microbial organism is selected such that it produces the precursor of a 3-HIBP or MAAP, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced by the host microbial organism. A host organism can be engineered to increase production of a precursor, as disclosed herein. In addition, a microbial organism that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of a 3-HIBP or MAAP, either alone or in combination with a MMP, FAP and/or FRP.

[0217] In some embodiments, a NNOMO provided herein is generated from a host that contains the enzymatic capability to synthesize 3-HIB or MAA, assimilate formaldehyde, reutilize formate and/or metabolize methanol. In this specific embodiment it can be useful to increase the synthesis or accumulation of a 3-HIBP or MAAP product, FAP product, FRP product and/or MMP product {e.g., reducing equivalents and/or formaldehyde) to, for example, drive 3-HIBP or MAAP reactions toward 3-HIB or MAA production. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described 3-HIB or MAA, formaldehyde assimilation, formate reutilization, and/or MMP enzymes or proteins. Over expression the enzyme(s) and/or protein(s) of the 3-HIBP or MAAP, formaldehyde assimilation, formate reutilization and/or MMP can occur, for example, through exogenous expression of the endogenous gene(s), or through exogenous expression of the heterologous gene(s). Therefore, naturally occurring organisms can be readily generated to be NNOMOs, for example, producing 3-HIB or MAA through overexpression of one, two, three, four, five, six , seven, eight, up to all nucleic acids encoding 3- HIB or MAA biosynthetic pathway, formaldehyde assimilation, formate reutilization, and/or MMP enzymes or proteins. Naturally occurring organisms can also be readily generated to be NNOMOs, for example, assimilating formaldehyde and/or reutilizing formate, through overexpression of one, two, three, four, five, six , seven, eight, up to all nucleic acids encoding FAP, FRP, and/or MMP enzymes or proteins. In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the 3-HIB or MAA, formaldehyde assimilation, formate reutilization, and/or MMP biosynthetic pathway.

[0218] In particularly useful embodiments, exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user. However, endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene's promoter when linked to an inducible promoter or other regulatory element. Thus, an

endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a NNOMO.

[0219] It is understood that, in methods provided herein, any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a NNOMO provided herein. The nucleic acids can be introduced so as to confer, for example, a 3-HIB or MAA biosynthetic, formaldehyde assimilation, formate reutilization and/or MMP onto the microbial organism. Alternatively, encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer 3-HIB or MAA biosynthetic, formaldehyde assimilation, formate reutilization, and/or methanol metabolic capability. For example, a NNOMO having a 3-HIB or MAA biosynthetic pathway, FAP, FRP and/or MMP can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins. Thus, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway, FAP, FRP, and/or metabolic pathway can be included in a NNOMO provided herein. Similarly, it is understood that any combination of three or more enzymes or proteins of a biosynthetic pathway, FAP, FRP, and/or metabolic pathway can be included in a NNOMO provided herein, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway, FAP, FRP, and/or metabolic pathway results in production of the corresponding desired product. Similarly, any combination of four or more enzymes or proteins of a biosynthetic pathway, FAP, FRP, and/or MMP as disclosed herein can be included in a NNOMO provided herein, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic, assimilation, reutilization and/or metabolic pathway results in production of the corresponding desired product. In specific embodiments, the biosynthetic pathway is a 3-HIB or MAA biosynthetic pathway.

[0220] In addition to the metabolism of methanol, assimilation of formaldehyde, and biosynthesis of 3-HIB or MAA, as described herein, the NNOMOs and methods provided also can be utilized in various combinations with each other and with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes. For example, one alternative to produce 3-HIB or MAA, other than use of the 3-HIB or MAA producers is through addition of another microbial organism capable of converting a 3-HIBP or MAAP intermediate to 3-HIB or MAA. One such procedure includes, for example, the fermentation of a microbial organism that produces a 3-HIBP or MAAP intermediate. The 3-HIBP or MAAP intermediate can then be used as a substrate for a second microbial organism that converts the 3-HIBP or MAAP intermediate to 3-HIB or MAA. The 3-HIBP or MAAP intermediate can be added directly to another culture of the second organism or the original culture of the 3-HIBP or MAAP intermediate producers can be depleted of these microbial organisms by, for example, cell separation, and then subsequent addition of the second organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps. The same holds true for the MMPs, FAPs and FRPs provided herein.

[0221] In other embodiments, the NNOMOs and methods provided herein can be assembled in a wide variety of subpathways to achieve biosynthesis of, for example, 3-HIB or MAA. In these embodiments, biosynthetic pathways for a desired product can be segregated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product. In such a biosynthetic scheme, the product of one microbial organism is the substrate for a second microbial organism until the final product is synthesized. For example, the biosynthesis of 3-HIB or MAA can be accomplished by constructing a microbial organism that contains biosynthetic pathways for conversion of one pathway intermediate to another pathway intermediate or the product. Alternatively, 3-HIB or MAA also can be biosynthetically produced from microbial organisms through co-culture or co-fermentation using two organisms in the same vessel, where the first microbial organism produces a 3-HIB or MAA intermediate and the second microbial organism converts the intermediate to 3-HIB or MAA. The same holds true for the MMPs, FAPs and FRPs provided herein.

[0222] Given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of combinations and permutations exist for the NNOMOs and methods together with other microbial organisms, with the co-culture of other NNOMOs having subpathways and with combinations of other chemical and/or biochemical procedures well known in the art to produce 3-HIB or MAA and/or metabolize methanol.

[0223] Sources of encoding nucleic acids for a 3-HIB or MAA, formaldehyde assimilation, or MMP enzyme or protein can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction. Such species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human. Exemplary species for such sources include, for example, Escherichia coli, Saccharomyces cerevisiae, Saccharomyces kluyveri, Candida boidinii, Clostridium Jduyveri, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium difficile, Clostridium botulinum, Clostridium tyrobutyricum, Clostridium tetanomorphum, Clostridium tetani, Clostridium propionicum, Clostridium aminobutyricum, Clostridium subterminale, Clostridium sticklandii, Ralstonia eutropha, Mycobacterium bovis, Mycobacterium tuberculosis, Porphyromonas gingivalis, Arabidopsis thaliana, Thermus thermophilus, Pseudomonas species, including Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas fluorescens, Homo sapiens,

Oryctolagus cuniculus, Rhodobacter spaeroides, Thermoanaerobacter brockii, Metallosphaera sedula, Leuconostoc mesenteroides, Chloroflexus aurantiacus, Roseiflexus castenholzii,

Erythrobacter, Simmondsia chinensis, Acinetobacter species, including Acinetobacter calcoaceticus and Acinetobacter baylyi, Porphyromonas gingivalis, Sulfolobus tokodaii, Sulfolobus solfataricus, Sulfolobus acidocaldarius, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus brevis, Bacillus pumilus, Rattus norvegicus, Klebsiella pneumonia, Klebsiella oxytoca, Euglena gracilis, Treponema denticola, Moorella thermoacetica,

Thermotoga maritima, Halobacterium salinarum, Geobacillus stearothermophilus, Aeropyrum pernix, Sus scrofa, Caenorhabditis elegans, Corynebacterium glutamicum, Acidaminococcus fermentans, Lactococcus lactis, Lactobacillus plantarum, Streptococcus thermophilus,

Enterobacter aerogenes, Candida, Aspergillus terreus, Pedicoccus pentosaceus, Zymomonas mobilus, Acetobacter pasteurians, Kluyveromyces lactis, Eubacterium barkeri, Bacteroides capillosus, Anaerotruncus colihominis, Natranaerobius thermophilusm, Campylobacter jejuni, Haemophilus influenzae, Serratia marcescens, Citrobacter amalonaticus, Myxococcus xanthus, Fusobacterium nuleatum, Penicillium chrysogenum, marine gamma proteobacterium, butyrate- producing bacterium, Nocardia iowensis, Nocardia farcinica, Streptomyces griseus,

Schizosaccharomyces pombe, Geobacillus thermoglucosidasius, Salmonella typhimurium, Vibrio cholera, Heliobacter pylori, Nicotiana tabacum, Oryza sativa, Haloferax mediterranei,

Agrobacterium tumefaciens, Achromobacter denitriflcans, Fusobacterium nucleatum,

Streptomyces clavuligenus, Acinetobacter baumanii, Mus musculus, Lachancea kluyveri, Trichomonas vaginalis, Trypanosoma brucei, Pseudomonas stutzeri, Bradyrhizobium japonicum, Mesorhizobium loti, Bos taurus, Nicotiana glutinosa, Vibrio vulnificus, Selenomonas

ruminantium, Vibrio parahaemolyticus, Archaeoglobus fulgidus, Haloarcula marismortui, Pyrobaculum aerophilum, Mycobacterium smegmatis MC2 155, Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium marinum M, Tsukamurella paurometabola DSM 20162, Cyanobium PCC7001, Dictyostelium discoideum AX4, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes.

[0224] In certain embodiments, sources of encoding nucleic acids for a 3-HIBPE or MAAPE include Acetobacter aceti, Acinetobacter baylyi, Acinetobacter calcoaceticus, Acinetobacter sp. Strain M-1, Anaerotruncus colihominis, Arabidopsis thaliana, Aromatoleum aromaticum EbNl, Azoarcus sp. T Bacillus cereus ATCC 14579, Bacillus subtilis, Bacteroides capillosus,

Burkholderia xenovorans, Escherichia coli C, Caenorhabditis elegans, Campylobacter jejuni, Chloroflexus aurantiacus, Citrobacter youngae ATCC 29220, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium kluyveri, Clostridium saccharoperbutylacetonicum, Comamonas sp. CNB-1, Corynebacterium glutamicum, Erythrobacter sp. NAP 1, Escherichia coli, Escherichia coli K12, Escherichia coli str. K-12 substr. MG1655, Escherichia coli W, Eubacterium barkeri, Geobacter metallireducens GS-15, Helicobacter pylori, Homo sapiens, Klebsiella pneumonia, Leuconostoc mesenteroides, marine gamma proteobacterium HTCC2080, Metallosphaera sedula, Methanocaldococcus jannaschii, Methylibium petroleiphilum PM1, Methylobacterium extorquens, Natranaerobius thermophilus, Oryctolagus cuniculus,

Pelotomaculum thermopropionicum, Porphyromonas gingivalis, Propionibacterium acnes, Propionibacterium fredenreichii sp. Shermanii, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas sp, Ralstonia eutropha HI 6, Rattus norvegicus, Roseiflexus castenholzii,

Saccharomyces cerevisiae, Salmonella enteric, Salmonella enteric, Salmonella enterica subsp. arizonae serovar, Salmonella typhimurium, Shigella flexneri, Streptomyces avermitilis,

Streptomyces cinnamonensis, Streptomyces coelicolor A3 (2), Sulfolobus acidocaldarius, Sulfolobus solfataricus, Sulfolobus tokodaii, Thauera aromatic, Thermoproteus neutrophilus, Thermus thermophilus, Trichomonas vaginalis G3, Trypanosoma brucei, Yersinia frederiksenii, and Yersinia intermedia ATCC 29909, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes.

[0225] In certain embodiments, sources of encoding nucleic acids for a 3-HIBPE or MAAPE include Acetobacter aceti, Actinobacillus succinogenes, Anaerobio spirillum succiniciproducens, Ascaris suum, Aspergillus niger CBS 513.88, Bacillus subtilis, Campylobacter jejuni,

Clostridium beijerinckii, Clostridium kluyveri, Corynebacterium glutamicum, Escherichia coli, Escherichia coli K-12 MG1655, Haemophilus influenza, Helicobacter pylori, Homo sapiens, Mannheimia succiniciproducens, Methylobacterium extorquens, Mycobacterium smegmatis, Pelotomaculum thermopropionicum, Pseudomonas putida, Pyrococcus furiosus, Ralstonia eutropha, Rattus norvegicus, Rhodococcus ruber, Saccharomyces cerevisiae, Saccharomyces cerevisiae S288c, Salmonella typhimurium, Thermoanaerobacter brockii, Thermus thermophilus, Trichomonas vaginalis G3, Trypanosoma brucei, and Yarrowia lipolytica, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes.

[0226] In certain embodiments, sources of encoding nucleic acids for a MMPE include,

Acinetobacter baumannii Naval-82, Actinobacillus succinogenes 130Z, AUochromatium vinosum DSM 180, Azotobacter vinelandii DJ, Bacillus alcalophilus ATCC 27647, Bacillus azotoformans LMG 9581, Bacillus coagulans 36D1, Bacillus methanolicus MGA3, Bacillus methanolicus PBl, Bacillus methanolicus PB-1, Bacillus smithii, Bacillus subtilis, Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia multivorans, Burkholderia pyrrocinia, Burkholderia stabilis, Burkholderia thailandensis E264, Burkholderiales bacterium Joshi OOl, Campylobacter jejuni, Candida boidinii, Candida methylica, Carboxydothermus hydrogenoformans,

Carboxydothermus hydrogenoformans Z-2901, Caulobacter sp. AP07, Clostridium

acetobutylicum ATCC 824, Clostridium acidurici, Clostridium carboxidivorans P7, Clostridium cellulovorans 743B, Clostridium kluyveri, Clostridium kluyveri DSM 555, Clostridium ljungdahlii, Clostridium ljungdahlii DSM 13528, Clostridium pasteurianum, Clostridium pasteurianum DSM 525, Clostridium perfringens, Clostridium perfringens ATCC 13124, Clostridium perfringens str. 13, Clostridium phytofermentans ISDg, Corynebacterium

glutamicum ATCC 14067, Corynebacterium glutamicum R, Corynebacterium sp. U-96,

Corynebacterium variabile, Cupriavidus necator N-l, Desulfitobacterium hafniense,

Desulfitobacterium metallireducens DSM 15288, Desulfotomaculum reducens MI-1,

Desulfovibrio africanus str. Walvis Bay, Desulfovibrio fructosovorans JJ, Desulfovibrio vulgaris str. Hildenborough, Desulfovibrio vulgaris str. Miyazaki F', Escherichia coli, Escherichia coli K-12, Escherichia coli K-12 MG1655, Flavobacterium frigoris, Geobacillus sp. Y4.1MC1, Geobacillus themodenitrificans NG80-2, Geobacter bemidjiensis Bern, Geobacter

sulfurreducens, Geobacter sulfurreducens PCA, Helicobacter pylori, Homo sapiens, human gut metagenome, Hydrogenobacter thermophilus, Hyphomicrobium denitrificans ATCC 51888, Hyphomicrobium zavarzinii, Klebsiella pneumoniae subsp. pneumoniae MGH 78578,

Lysinibacillus fusiformis, Lysinibacillus sphaericus, Mesorhizobium loti MAFF303099, Methanosarcina acetivorans, Methanosarcina acetivorans C2A, Methanosarcina barkeri, Methanosarcina mazei TucOl, Methylobacter marinus, Methylobacterium extorquens, ,

Methylobacterium extorquens AMI, Methylococcus capsulatis, Moorella thermoacetica, Mycobacterium smegmatis, Nitrosopumilus salaria BD31, Nitrososphaera gargensis Ga9.2, Nostoc sp. PCC 7120, Paenibacillus peoriae KCTC 3763, Paracoccus denitrificans,

Photobacterium profundum 3TCK, Pichia pastoris, Picrophilus torridus DSM9790,

Pseudomonas aeruginosa PA01, Pseudomonas putida, Pseudomonas syringae pv. syringae B728a, Ralstonia eutropha, Ralstonia eutropha HI 6, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodobacter sphaeroides ATCC 17025, Rhodopseudomonas palustris,

Rhodopseudomonas palustris CGA009, Rhodopseudomonas palustris DX-1, Rhodospirillum rubrum, Saccharomyces cerevisiae, Saccharomyces cerevisiae S288c, Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, Sebaldella termitidis ATCC 33386, Shewanella oneidensis MR-1, Sinorhizobium meliloti 1021, Sulfolobus acidocalarius, Sulfolobus solfataricus P-2, Synechocystis str. PCC 6803, Syntrophobacter fumaroxidans, Thauera aromatica,

Thermoanaerobacter sp. X514, Thermococcus litoralis, Thermoplasma acidophilum, Thiocapsa roseopersicina, Vibrio harveyi ATCC BAA-1116, Xanthobacter autotrophicus Py2, and Zea mays, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes.

[0227] In certain embodiments, sources of encoding nucleic acids for a FAPE include

Aminomonas aminovorus, Bacillus methanolicus MGA3, Bacillus methanolicus PBl, Bacillus subtilis, Candida boidinii, Citrobacter freundii, Escherichia coli, Geobacillus sp. GHH01, Geobacillus sp. M10EXG, Geobacillus sp. Y4.1MC1, Klebsiella pneumonia, Methylobacillus flagellates, Methylobacillus flagellatus KT, Methylococcus capsulatas, Methylomicrobium album BG8, Methylomonas aminofaciens, Methylovorus glucosetrophus SIP3-4, Methylovorus sp. MP688, Mycobacter sp. strain JC1 DSM 3803, Mycobacterium gastri, Ogataea angusta, Ogataea parapolymorpha DL-1 (Hansenula polymorpha DL-1), Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii OT3, Saccharomyces cerevisiae S288c, and Thermococcus kodakaraensis, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes. [0228] In certain embodiments, sources of encoding nucleic acids for a FRPE include

Acinetobacter baylyi, Acinetobacter calcoaceticus, Acinetobacter sp. Strain M-1, Archaeglubus fulgidus, Archaeoglobus fulgidus DSM 4304, Arthrobacter globiformis, Bacillus methanolicus MGA3, Bacillus methanolicus PB1, Bacillus selenitireducens MLS10, Bacillus subtilis,

Burkholderia stabilis, Campylobacter jejuni, Candida albicans, Candida boidinii, Candida methylica, Carboxydothermus hydrogenoformans, Chlamydomonas reinhardtii, Citrobacter koseri ATCC BAA-895, Clostridium acetobutylicum, Clostridium acetobutylicum ATCC 824, Clostridium acidurici, Clostridium beijerinckii, Clostridium carboxidivoransP7, Clostridium cellulovorans 743B, Clostridium kluyveri, Clostridium kluyveri DSM 555, Clostridium ljungdahlii DSM, Clostridium ljungdahlii DSM 13528, Clostridium pasteurianum, Clostridium perfringens, Clostridium phytofermentans ISDg, Clostridium saccharoperbutylacetonicum, Corynebacterium glutamicum, Corynebacterium sp., Cryptosporidium parvum Iowa II,

Cyanobium PCC7001, Desulfatibacillum alkenivorans AK-01, Desulfitobacterium hafniense, Desulfovibrio africanus, Desulfovibrio fructosovorans JJ, Dictyostelium discoideum AX4, Escherichia coli, Euglena gracilis, Fusobacterium nucleatum, Geobacter sulfurreducens PCA, Haloarcula marismortui ATCC 43049, Helicobacter pylori, Homo sapiens, Klebsiella pneumoniae, Lactobacillus acidophilus, Lactobacillus brevis ATCC 367, Lactococcus lactis, Leuconostoc mesenteroides, Metallosphaera sedula, Metarhizium acridum CQMa 102,

Methanosarcina acetivorans, Methanothermobacter thermautotrophicus, Methylobacterium extorquens, Moorella thermoacetica, Mus musculus, Mycobacterium avium subsp.

paratuberculosis K-10, Mycobacterium bovis BCG, Mycobacterium marinum M, Mycobacterium smegmatis MC2 155, Nocardia farcinica IFM 10152, Nocardia iowensis (sp. NRRL 5646), Oxalobacter formigenes, Penicillium chrysogenum, Perkinsus marinus ATCC 50983,

Porphyromonas gingivalis, Porphyromonas gingivalis W83, Pseudomonas putida, Pseudomonas sp, Pyrobaculum aerophilum str. IM2, Ralstonia eutropha, Ralstonia eutropha HI 6, Rattus norvegicus, Rhizopus oryzae, Rhodococcus opacus B4, Saccharomyces cerevisiae,

Saccharomyces cerevisiae S288c, Salmonella enterica, Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, Salmonella enterica Typhimurium, Salmonella typhimurium, Schizosaccharomyces pombe, Streptococcus mutans, Streptomyces griseus subsp. griseus NBRC 13350, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Sulfolobus tokodaii, Syntrophobacter fumaroxidans, Thermoanaerobacter tengcongensis MB4, Trichomonas vaginalis G3, Trypanosoma brucei, and Tsukamurella paurometabola DSM 20162 , as well as other exemplary species disclosed herein or available as source organisms for corresponding genes.

[0229] However, with the complete genome sequence available for now more than 550 species (with more than half of these available on public databases such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes, the identification of genes encoding the requisite 3-HIB or MAA biosynthetic pathway, methanol metabolic and/or formaldehyde assimilation activity for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations allowing biosynthesis of 3-HIB or MAA, metabolism of methanol and/or assimilation of formaldehyde described herein with reference to a particular organism such as E. coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.

[0230] In some instances, such as when an alternative 3-HIB or MAA biosynthetic, formaldehyde assimilation and/or MMP exists in an unrelated species, 3-HIB or MAA

biosynthesis, formaldehyde assimilation and/or methanol metabolism can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the teachings and methods provided herein can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will synthesize 3-HIB or MAA, assimilate formaldehyde, and/or metabolize methanol.

[0231] A nucleic acid molecule encoding a 3-HIBP or MAAP enzyme or protein can also include a nucleic acid molecule that hybridizes to a nucleic acid disclosed herein by SEQ ID NO, GenBank and/or GI number or a nucleic acid molecule that hybridizes to a nucleic acid molecule that encodes an amino acid sequence disclosed herein by SEQ ID NO, GenBank and/or GI number. Hybridization conditions can include highly stringent, moderately stringent, or low stringency hybridization conditions that are well known to one of skill in the art such as those described herein. Similarly, a nucleic acid molecule that can be used in the invention can be described as having a certain percent sequence identity to a nucleic acid disclosed herein by SEQ ID NO, GenBank and/or GI number or a nucleic acid molecule that hybridizes to a nucleic acid molecule that encodes an amino acid sequence disclosed herein by SEQ ID NO, GenBank and/or GI number. For example, the nucleic acid molecule can have at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a nucleic acid described herein.

[0232] Stringent hybridization refers to conditions under which hybridized polynucleotides are stable. As known to those of skill in the art, the stability of hybridized polynucleotides is reflected in the melting temperature (Tm) of the hybrids. In general, the stability of hybridized polynucleotides is a function of the salt concentration, for example, the sodium ion concentration and temperature. A hybridization reaction can be performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Reference to hybridization stringency relates to such washing conditions. Highly stringent hybridization includes conditions that permit hybridization of only those nucleic acid sequences that form stable hybridized polynucleotides in 0.018M NaCl at 65°C, for example, if a hybrid is not stable in 0.018M NaCl at 65°C, it will not be stable under high stringency conditions, as contemplated herein. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.1X SSPE, and 0.1% SDS at 65°C. Hybridization conditions other than highly stringent hybridization conditions can also be used to describe the nucleic acid sequences disclosed herein. For example, the phrase moderately stringent hybridization refers to conditions equivalent to hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.2X SSPE, 0.2%) SDS, at 42°C. The phrase low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5X Denhart's solution, 6X SSPE, 0.2% SDS at 22°C, followed by washing in IX SSPE, 0.2% SDS, at 37°C. Denhart's solution contains 1%

Ficoll, 1%) polyvinylpyrolidone, and 1% bovine serum albumin (BSA). 20X SSPE (sodium chloride, sodium phosphate, ethylene diamide tetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M (EDTA). Other suitable low, moderate and high stringency hybridization buffers and conditions are well known to those of skill in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).

[0233] A nucleic acid molecule encoding a 3-HIBP or MAAP enzyme or protein can have at least a certain sequence identity to a nucleotide sequence disclosed herein. According, in some aspects of the invention, a nucleic acid molecule encoding a 3-HIBP or MAAP enzyme or protein has a nucleotide sequence of at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96%) identity, at least 97% identity, at least 98% identity, or at least 99% identity to a nucleic acid disclosed herein by SEQ ID NO, GenBank and/or GI number or a nucleic acid molecule that hybridizes to a nucleic acid molecule that encodes an amino acid sequence disclosed herein by SEQ ID NO, GenBank and/or GI number.

[0234] Sequence identity (also known as homology or similarity) refers to sequence similarity between two nucleic acid molecules or between two polypeptides. Identity can be determined by comparing a position in each sequence, which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of identity between sequences is a function of the number of matching or homologous positions shared by the sequences. The alignment of two sequences to determine their percent sequence identity can be done using software programs known in the art, such as, for example, those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).

Preferably, default parameters are used for the alignment. One alignment program well known in the art that can be used is BLAST set to default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the National Center for Biotechnology Information.

[0235] Methods for constructing and testing the expression levels of a non-naturally occurring 3-HIB- or MAA-producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).

[0236] Exogenous nucleic acid sequences involved in a pathway for metabolism of methanol, assimilation of formaldehyde and/or production of 3-HIB or MAA can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. For exogenous expression in E. or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.

[0237] An expression vector or vectors can be constructed to include one or more 3-HIB or MAA biosynthetic, formaldehyde assimilation and/or MMP encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the microbial host organisms provided include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media.

Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.

[0238] Suitable purification and/or assays to test, e.g., for the production of 3-HIB or MAA can be performed using well known methods. Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid

Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid

Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art. Exemplary assays for the activity of methanol dehydrogenase (FIG. 1, step J) are provided in the Example I.

[0239] The 3-HIB or MAA can be separated from other components in the culture using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.

[0240] Any of the NNOMOs described herein can be cultured to produce and/or secrete the biosynthetic products, or intermediates thereof. For example, the 3-HIB or MAA producers can be cultured for the biosynthetic production of 3-HIB or MAA. Accordingly, in some

embodiments, provided is culture medium having a 3-HIB or MAA, formaldehyde assimilation and/or MMP intermediate described herein. In some aspects, the culture medium can also be separated from the NNOMOs provided herein that produced the 3-HIB or MAA, formaldehyde assimilation and/or MMP intermediate. Methods for separating a microbial organism from culture medium are well known in the art. Exemplary methods include filtration, flocculation, precipitation, centrifugation, sedimentation, and the like.

[0241] In certain embodiments, for example, for the production of the production of 3-HIB or MAA, the recombinant strains are cultured in a medium with carbon source and other essential nutrients. It is sometimes desirable and can be highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap. For strains where growth is not observed anaerobically, microaerobic or substantially anaerobic conditions can be applied by perforating the septum with a small hole for limited aeration. Exemplary anaerobic conditions have been described previously and are well-known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in U.S. Publ. No. 2009/0047719. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein. Fermentations can also be conducted in two phases, if desired. The first phase can be aerobic to allow for high growth and therefore high productivity, followed by an anaerobic phase of high 3-HIB or MAA yields.

[0242] If desired, the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH. The growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time.

[0243] The growth medium, can include, for example, any carbohydrate source which can supply a source of carbon to the NNOMO. Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch; or glycerol, alone as the sole source of carbon or in combination with other carbon sources described herein or known in the art. In one embodiment, the carbon source is a sugar. In one embodiment, the carbon source is a sugar-containing biomass. In some embodiments, the sugar is glucose. In one embodiment, the sugar is xylose. In another embodiment, the sugar is arabinose. In one embodiment, the sugar is galactose. In another embodiment, the sugar is fructose. In other embodiments, the sugar is sucrose. In one embodiment, the sugar is starch. In certain

embodiments, the carbon source is glycerol. In some embodiments, the carbon source is crude glycerol. In one embodiment, the carbon source is crude glycerol without treatment. In other embodiments, the carbon source is glycerol and glucose. In another embodiment, the carbon source is methanol and glycerol. In one embodiment, the carbon source is carbon dioxide. In one embodiment, the carbon source is formate. In one embodiment, the carbon source is methane. In one embodiment, the carbon source is methanol In certain embodiments, methanol is used alone as the sole source of carbon or in combination with other carbon sources described herein or known in the art. In a specific embodiment, the methanol is the only (sole) carbon source. In one embodiment, the carbon source is chemoelectro-generated carbon (see, e.g. , Liao et al.

(2012) Science 335: 1596). In one embodiment, the chemoelectro-generated carbon is methanol. In one embodiment, the chemoelectro-generated carbon is formate. In one embodiment, the chemoelectro-generated carbon is formate and methanol. In one embodiment, the carbon source is a carbohydrate and methanol. In one embodiment, the carbon source is a sugar and methanol. In another embodiment, the carbon source is a sugar and glycerol. In other embodiments, the carbon source is a sugar and crude glycerol. In yet other embodiments, the carbon source is a sugar and crude glycerol without treatment. In one embodiment, the carbon source is a sugar- containing biomass and methanol. In another embodiment, the carbon source is a sugar- containing biomass and glycerol. In other embodiments, the carbon source is a sugar-containing biomass and crude glycerol. In yet other embodiments, the carbon source is a sugar-containing biomass and crude glycerol without treatment. In some embodiments, the carbon source is a sugar-containing biomass, methanol and a carbohydrate. Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods provided herein include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms provided herein for the production of 3-HIB or MAA, and other pathway intermediates.

[0244] In one embodiment, the carbon source is glycerol. In certain embodiments, the glycerol carbon source is crude glycerol or crude glycerol without further treatment. In a further embodiment, the carbon source comprises glycerol or crude glycerol, and also sugar or a sugar- containing biomass, such as glucose. In a specific embodiment, the concentration of glycerol in the fermentation broth is maintained by feeding crude glycerol, or a mixture of crude glycerol and sugar (e.g., glucose). In certain embodiments, sugar is provided for sufficient strain growth. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of from 200: 1 to 1 :200. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of from 100: 1 to 1 : 100. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of from 100: 1 to 5 : 1. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of from 50: 1 to 5 : 1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 100: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 90: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 80: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 70: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 60: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 50: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 40: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 30: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 20: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 10: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 5 : 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 2: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1 : 1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1 : 100. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1 :90. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1 :80. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1 :70. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1 :60. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1 :50. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1 :40. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1 :30. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1 :20. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1 : 10. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1 :5. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1 :2. In certain embodiments of the ratios provided above, the sugar is a sugar-containing biomass. In certain other embodiments of the ratios provided above, the glycerol is a crude glycerol or a crude glycerol without further treatment. In other embodiments of the ratios provided above, the sugar is a sugar-containing biomass, and the glycerol is a crude glycerol or a crude glycerol without further treatment.

[0245] Crude glycerol can be a by-product produced in the production of biodiesel, and can be used for fermentation without any further treatment. Biodiesel production methods include

(1) a chemical method wherein the glycerol-group of vegetable oils or animal oils is substituted by low-carbon alcohols such as methanol or ethanol to produce a corresponding fatty acid methyl esters or fatty acid ethyl esters by transesterification in the presence of acidic or basic catalysts;

(2) a biological method where biological enzymes or cells are used to catalyze transesterification reaction and the corresponding fatty acid methyl esters or fatty acid ethyl esters are produced; and (3) a supercritical method, wherein transesterification reaction is carried out in a supercritical solvent system without any catalysts. The chemical composition of crude glycerol can vary with the process used to produce biodiesel, the transesterification efficiency, recovery efficiency of the biodiesel, other impurities in the feedstock, and whether methanol and catalysts were recovered. For example, the chemical compositions of eleven crude glycerol collected from seven Australian biodiesel producers reported that glycerol content ranged between 38% and 96%, with some samples including more than 14% methanol and 29% ash. In certain

embodiments, the crude glycerol comprises from 5% to 99% glycerol. In some embodiments, the crude glycerol comprises from 10%> to 90%> glycerol. In some embodiments, the crude glycerol comprises from 10%> to 80%> glycerol. In some embodiments, the crude glycerol comprises from 10%) to 70%) glycerol. In some embodiments, the crude glycerol comprises from 10%> to 60%> glycerol. In some embodiments, the crude glycerol comprises from 10%> to 50%> glycerol. In some embodiments, the crude glycerol comprises from 10%> to 40%> glycerol. In some embodiments, the crude glycerol comprises from 10%> to 30%> glycerol. In some embodiments, the crude glycerol comprises from 10%> to 20%> glycerol. In some embodiments, the crude glycerol comprises from 80%> to 90%> glycerol. In some embodiments, the crude glycerol comprises from 70%> to 90%> glycerol. In some embodiments, the crude glycerol comprises from 60%) to 90%) glycerol. In some embodiments, the crude glycerol comprises from 50%> to 90%> glycerol. In some embodiments, the crude glycerol comprises from 40%> to 90%> glycerol. In some embodiments, the crude glycerol comprises from 30%> to 90%> glycerol. In some embodiments, the crude glycerol comprises from 20% to 90%> glycerol. In some embodiments, the crude glycerol comprises from 20%> to 40%> glycerol. In some embodiments, the crude glycerol comprises from 40%> to 60%> glycerol. In some embodiments, the crude glycerol comprises from 60%> to 80%> glycerol. In some embodiments, the crude glycerol comprises from 50%o to 70%) glycerol. In one embodiment, the glycerol comprises 5% glycerol. In one embodiment, the glycerol comprises 10% glycerol. In one embodiment, the glycerol comprises 15% glycerol. In one embodiment, the glycerol comprises 20% glycerol. In one embodiment, the glycerol comprises 25% glycerol. In one embodiment, the glycerol comprises 30%> glycerol. In one embodiment, the glycerol comprises 35% glycerol. In one embodiment, the glycerol comprises 40% glycerol. In one embodiment, the glycerol comprises 45% glycerol. In one embodiment, the glycerol comprises 50%> glycerol. In one embodiment, the glycerol comprises 55%o glycerol. In one embodiment, the glycerol comprises 60%> glycerol. In one embodiment, the glycerol comprises 65%> glycerol. In one embodiment, the glycerol comprises 70%> glycerol. In one embodiment, the glycerol comprises 75% glycerol. In one embodiment, the glycerol comprises 80%> glycerol. In one embodiment, the glycerol comprises 85% glycerol. In one embodiment, the glycerol comprises 90%> glycerol. In one embodiment, the glycerol comprises 95%o glycerol. In one embodiment, the glycerol comprises 99% glycerol.

[0246] In one embodiment, the carbon source is methanol or formate. In certain

embodiments, methanol is used as a carbon source in the FAPs provided herein. In one embodiment, the carbon source is methanol or formate. In other embodiments, formate is used as a carbon source in the FAPs provided herein. In specific embodiments, methanol is used as a carbon source in the MMPs provided herein, either alone or in combination with the product pathways provided herein. In one embodiment, the carbon source is methanol. In another embodiment, the carbon source is formate.

[0247] In one embodiment, the carbon source comprises methanol, and sugar (e.g., glucose) or a sugar-containing biomass. In another embodiment, the carbon source comprises formate, and sugar (e.g., glucose) or a sugar-containing biomass. In one embodiment, the carbon source comprises methanol, formate, and sugar (e.g., glucose) or a sugar-containing biomass. In specific embodiments, the methanol or formate, or both, in the fermentation feed is provided as a mixture with sugar (e.g., glucose) or sugar-comprising biomass. In certain embodiments, sugar is provided for sufficient strain growth.

[0248] In certain embodiments, the carbon source comprises methanol and a sugar (e.g., glucose). In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of from 200: 1 to 1 :200. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of from 100: 1 to 1 : 100. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of from 100: 1 to 5 : 1. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of from 50: 1 to 5 : 1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 100: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 90: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 80: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 70: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 60: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 50: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 40: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 30: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 20: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 10: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 5 : 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 2: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 : 1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar

concentration ratio of methanol to sugar of 1 : 100. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :90. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :80. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :70. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :60. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :50. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :40. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :30. In one embodiment, the sugar (e.g., glucose) is provided at a molar

concentration ratio of methanol to sugar of 1 :20. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 : 10. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :5. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :2. In certain embodiments of the ratios provided above, the sugar is a sugar- containing biomass.

[0249] In certain embodiments, the carbon source comprises formate and a sugar (e.g., glucose). In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of from 200: 1 to 1 :200. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of from 100: 1 to 1 : 100. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of from 100: 1 to 5 : 1. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of from 50: 1 to 5 : 1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 100: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 90: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 80: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 70: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 60: 1. In one

embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 50: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 40: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 30: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 20: 1. In one

embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 10: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 5 : 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 2: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1 : 1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1 : 100. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1 :90. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1 :80. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1 :70. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1 : 60. In one

embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1 :50. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1 :40. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1 :30. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1 : 20. In one

embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1 : 10. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1 :5. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1 :2. In certain embodiments of the ratios provided above, the sugar is a sugar-containing biomass.

[0250] In certain embodiments, the carbon source comprises a mixture of methanol and formate, and a sugar (e.g., glucose). In certain embodiments, sugar is provided for sufficient strain growth. In some embodiments, the sugar (e.g., glucose) is provided at a molar

concentration ratio of methanol and formate to sugar of from 200: 1 to 1 :200. In some

embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of from 100: 1 to 1 : 100. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of from 100: 1 to 5 : 1. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of from 50: 1 to 5 : 1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 100: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 90: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar

- I l l - concentration ratio of methanol and formate to sugar of 80: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 70: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 60: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 50: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 40: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 30: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 20: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 10: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 5 : 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 2: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 : 1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 : 100. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 : 90. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :80. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :70. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :60. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :50. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :40. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :30. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :20. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 : 10. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :5. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :2. In certain embodiments of the ratios provided above, the sugar is a sugar-containing biomass.

[0251] Given the teachings and guidance provided herein, those skilled in the art will understand that a NNOMO can be produced that secretes the biosynthesized compounds when grown on a carbon source such as a carbohydrate. Such compounds include, for example, 3-HIB or MAA and any of the intermediate metabolites in the 3-HIBP or MAAP. All that is required is to engineer in one or more of the required enzyme or protein activities to achieve biosynthesis of the desired compound or intermediate including, for example, inclusion of some or all of the 3- HIB or MAA biosynthetic pathways. Accordingly, provided herein is a NNOMO that produces and/or secretes 3-HIB or MAA when grown on a carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the 3-HIBP or MAAP when grown on a carbohydrate or other carbon source. The 3-HIB or MAA-producing microbial organisms provided herein can initiate synthesis from an intermediate. The same holds true for intermediates in the formaldehyde assimilation and MMPs.

[0252] The NNOMOs provided herein are constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding a 3-HIB or MAA biosynthetic pathway and/or MMP enzyme or protein in sufficient amounts to produce 3- HIB or MAA. It is understood that the microbial organisms are cultured under conditions sufficient to produce 3-HIB or MAA. Following the teachings and guidance provided herein, the NNOMOs can achieve biosynthesis of 3-HIB or MAA, resulting in intracellular concentrations between about 0.1-500 mM or more. Generally, the intracellular concentration of 3-HIB or MAA is between about 3-150 mM, particularly between about 5-125 mM and more particularly between about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, or more.

Intracellular concentrations between and above each of these exemplary ranges also can be achieved from the NNOMOs provided herein.

[0253] In some embodiments, culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been described previously and are well known in the art. Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S. Publ. No. 2009/0047719. Any of these conditions can be employed with the NNOMOs as well as other anaerobic conditions well known in the art. Under such anaerobic or substantially anaerobic conditions, the 3-HIB or MAA producers can synthesize 3-HIB or MAA at intracellular concentrations of 5- 100 mM or more as well as all other concentrations exemplified herein. It is understood that, even though the above description refers to intracellular concentrations, 3-HIB or MAA can produce 3-HIB or MAA intracellularly and/or secrete the product into the culture medium.

[0254] Exemplary fermentation processes include, but are not limited to, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation; and continuous fermentation and continuous separation. In an exemplary batch fermentation protocol, the production organism is grown in a suitably sized bioreactor sparged with an appropriate gas. Under anaerobic conditions, the culture is sparged with an inert gas or combination of gases, for example, nitrogen, N2/C02 mixture, argon, helium, and the like. As the cells grow and utilize the carbon source, additional carbon source(s) and/or other nutrients are fed into the bioreactor at a rate approximately balancing consumption of the carbon source and/or nutrients. The temperature of the bioreactor is maintained at a desired temperature, generally in the range of 22-37 degrees C, but the temperature can be maintained at a higher or lower temperature depending on the growth characteristics of the production organism and/or desired conditions for the fermentation process. Growth continues for a desired period of time to achieve desired characteristics of the culture in the fermenter, for example, cell density, product concentration, and the like. In a batch fermentation process, the time period for the fermentation is generally in the range of several hours to several days, for example, 8 to 24 hours, or 1, 2, 3, 4 or 5 days, or up to a week, depending on the desired culture conditions. The pH can be controlled or not, as desired, in which case a culture in which pH is not controlled will typically decrease to pH 3-6 by the end of the run. Upon completion of the cultivation period, the fermenter contents can be passed through a cell separation unit, for example, a centrifuge, filtration unit, and the like, to remove cells and cell debris. In the case where the desired product is expressed intracellularly, the cells can be lysed or disrupted enzymatically or chemically prior to or after separation of cells from the fermentation broth, as desired, in order to release additional product. The fermentation broth can be transferred to a product separations unit.

Isolation of product occurs by standard separations procedures employed in the art to separate a desired product from dilute aqueous solutions. Such methods include, but are not limited to, liquid- liquid extraction using a water immiscible organic solvent (e.g. , toluene or other suitable solvents, including but not limited to diethyl ether, ethyl acetate, tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane, hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE), dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and the like) to provide an organic solution of the product, if appropriate, standard distillation methods, and the like, depending on the chemical characteristics of the product of the

fermenation process.

[0255] In an exemplary fully continuous fermentation protocol, the production organism is generally first grown up in batch mode in order to achieve a desired cell density. When the carbon source and/or other nutrients are exhausted, feed medium of the same composition is supplied continuously at a desired rate, and fermentation liquid is withdrawn at the same rate. Under such conditions, the product concentration in the bioreactor generally remains constant, as well as the cell density. The temperature of the fermenter is maintained at a desired temperature, as discussed above. During the continuous fermentation phase, it is generally desirable to maintain a suitable pH range for optimized production. The pH can be monitored and maintained using routine methods, including the addition of suitable acids or bases to maintain a desired pH range. The bioreactor is operated continuously for extended periods of time, generally at least one week to several weeks and up to one month, or longer, as appropriate and desired. The fermentation liquid and/or culture is monitored periodically, including sampling up to every day, as desired, to assure consistency of product concentration and/or cell density. In continuous mode, fermenter contents are constantly removed as new feed medium is supplied. The exit stream, containing cells, medium, and product, are generally subjected to a continuous product separations procedure, with or without removing cells and cell debris, as desired.

Continuous separations methods employed in the art can be used to separate the product from dilute aqueous solutions, including but not limited to continuous liquid-liquid extraction using a water immiscible organic solvent (e.g., toluene or other suitable solvents, including but not limited to diethyl ether, ethyl acetate, tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane, hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE), dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and the like), standard continuous distillation methods, and the like, or other methods well known in the art. [0256] In addition to the culturing and fermentation conditions disclosed herein, growth condition for achieving biosynthesis of 3 -HIB or MAA can include the addition of an

osmoprotectant to the culturing conditions. In certain embodiments, the NNOMOs provided herein can be sustained, cultured or fermented as described herein in the presence of an osmoprotectant. Briefly, an osmoprotectant refers to a compound that acts as an osmolyte and helps a microbial organism as described herein survive osmotic stress. Osmoprotectants include, but are not limited to, betaines, amino acids, and the sugar trehalose. Non-limiting examples of such are glycine betaine, praline betaine, dimethylthetin, dimethylslfonioproprionate, 3- dimethylsulfonio-2-methylproprionate, pipecolic acid, dimethylsulfonioacetate, choline, L- carnitine and ectoine. In one aspect, the osmoprotectant is glycine betaine. It is understood to one of ordinary skill in the art that the amount and type of osmoprotectant suitable for protecting a microbial organism described herein from osmotic stress will depend on the microbial organism used. The amount of osmoprotectant in the culturing conditions can be, for example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM, no more than about lOmM, no more than about 50mM, no more than about lOOmM or no more than about 500mM.

[0257] The culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described herein, particularly useful yields of the biosynthetic products provided herein can be obtained under anaerobic or substantially anaerobic culture conditions.

[0258] As described herein, one exemplary growth condition for achieving biosynthesis of 3- HIB or MAA, as well as other pathway intermediates, includes anaerobic culture or fermentation conditions. In certain embodiments, the NNOMOs provided can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions. Briefly, anaerobic conditions refer to an environment devoid of oxygen. Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation. Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1% oxygen. The percent of oxygen can be maintained by, for example, sparging the culture with an N ₂ /CO ₂ mixture or other suitable non-oxygen gas or gases.

[0259] The culture conditions described herein can be scaled up and grown continuously for manufacturing of 3-HIB or MAA. Exemplary growth procedures include, for example, fed- batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of 3-HIB or MAA. Generally, and as with non-continuous culture procedures, the continuous and/or near-continuous production of 3-HIB or MAA will include culturing a non-naturally occurring 3-HIB or MAA producing organism provided herein in sufficient nutrients and medium to sustain and/or nearly sustain growth in an exponential phase. Continuous culture under such conditions can be included, for example, growth or culturing for 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include longer time periods of 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, organisms provided can be cultured for hours, if suitable for a particular application. It is to be understood that the continuous and/or near-continuous culture conditions also can include all time intervals in between these exemplary periods. It is further understood that the time of culturing the microbial organism provided herein is for a sufficient period of time to produce a sufficient amount of product for a desired purpose.

[0260] Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of 3-HIB or MAA can be utilized in, for example, fed-batch

fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.

[0261] In addition to the above fermentation procedures using the 3-HIB or MAA producers for continuous production of substantial quantities of 3-HIB or MAA, the 3-HIB or MAA producers also can be, for example, simultaneously subjected to chemical synthesis procedures to convert the product to other compounds or the product can be separated from the fermentation culture and sequentially subjected to chemical and/or enzymatic conversion to convert the product to other compounds, if desired.

[0262] To generate better producers, metabolic modeling can be utilized to optimize growth conditions. Modeling can also be used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. Publ. Nos. 2002/0012939, 2003/0224363, 2004/0029149, 2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466, and U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of 3-HIB or MAA.

[0263] One computational method for identifying and designing metabolic alterations favoring biosynthesis of a desired product is the OptKnock computational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)). OptKnock is a metabolic modeling and simulation program that suggests gene deletion or disruption strategies that result in genetically stable microorganisms which overproduce the target product. Specifically, the framework examines the complete metabolic and/or biochemical network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become an obligatory byproduct of cell growth. By coupling biochemical production with cell growth through strategically placed gene deletions or other functional gene disruption, the growth selection pressures imposed on the engineered strains after long periods of time in a bioreactor lead to improvements in performance as a result of the compulsory growth-coupled biochemical production. Lastly, when gene deletions are constructed there is a negligible possibility of the designed strains reverting to their wild-type states because the genes selected by OptKnock are to be completely removed from the genome. Therefore, this computational methodology can be used to either identify alternative pathways that lead to biosynthesis of a desired product or used in connection with the NNOMOs for further optimization of biosynthesis of a desired product.

[0264] Briefly, OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism. The OptKnock program relates to a framework of models and methods that incorporate particular constraints into flux balance analysis (FBA) models. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data. OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the performance limits of metabolic networks in the presence of gene additions or deletions. OptKnock computational framework allows the construction of model formulations that allow an effective query of the performance limits of metabolic networks and provides methods for solving the resulting mixed-integer linear programming problems. The metabolic modeling and simulation methods referred to herein as OptKnock are described in, for example, U.S. Publ. No. 2002/0168654, International Patent Application No. PCT/US02/00660, and U.S. Publ. No. 2009/0047719.

[0265] Another computational method for identifying and designing metabolic alterations favoring biosynthetic production of a product is a metabolic modeling and simulation system termed SimPheny®. This computational method and system is described in, for example, U.S. Publ. No. 2003/0233218, and International Patent Application No. PCT/US03/18838.

SimPheny® is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system. This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components.

[0266] These computational approaches are consistent with biological realities because biological systems are flexible and can reach the same result in many different ways. Biological systems are designed through evolutionary mechanisms that have been restricted by fundamental constraints that all living systems must face. Therefore, constraints-based modeling strategy embraces these general realities. Further, the ability to continuously impose further restrictions on a network model via the tightening of constraints results in a reduction in the size of the solution space, thereby enhancing the precision with which physiological performance or phenotype can be predicted. [0267] Given the teachings and guidance provided herein, those skilled in the art will be able to apply various computational frameworks for metabolic modeling and simulation to design and implement biosynthesis of a desired compound in host microbial organisms. Such metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny® and OptKnock. For illustration, some methods are described herein with reference to the OptKnock computation framework for modeling and simulation. Those skilled in the art will know how to apply the identification, design and implementation of the metabolic alterations using OptKnock to any of such other metabolic modeling and simulation

computational frameworks and methods well known in the art.

[0268] The methods described above will provide one set of metabolic reactions to disrupt. Elimination of each reaction within the set or metabolic modification can result in a desired product as an obligatory product during the growth phase of the organism. Because the reactions are known, a solution to the bilevel OptKnock problem also will provide the associated gene or genes encoding one or more enzymes that catalyze each reaction within the set of reactions. Identification of a set of reactions and their corresponding genes encoding the enzymes participating in each reaction is generally an automated process, accomplished through correlation of the reactions with a reaction database having a relationship between enzymes and encoding genes.

[0269] Once identified, the set of reactions that are to be disrupted in order to achieve production of a desired product are implemented in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction within the set. One particularly useful means to achieve functional disruption of the reaction set is by deletion of each encoding gene. However, in some instances, it can be beneficial to disrupt the reaction by other genetic aberrations including, for example, mutation, deletion of regulatory regions such as promoters or cis binding sites for regulatory factors, or by truncation of the coding sequence at any of a number of locations. These latter aberrations, resulting in less than total deletion of the gene set can be useful, for example, when rapid assessments of the coupling of a product are desired or when genetic reversion is less likely to occur. [0270] To identify additional productive solutions to the above described bilevel OptKnock problem which lead to further sets of reactions to disrupt or metabolic modifications that can result in the biosynthesis, including growth-coupled biosynthesis of a desired product, an optimization method, termed integer cuts, can be implemented. This method proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration. Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous iteration that obligatorily couples product biosynthesis to growth. For example, if a previously identified growth-coupled metabolic modification specifies reactions 1 , 2, and 3 for disruption, then the following constraint prevents the same reactions from being simultaneously considered in subsequent solutions. The integer cut method is well known in the art and can be found described in, for example, Burgard et al., Biotechnol. Prog. 17:791-797 (2001). As with all methods described herein with reference to their use in combination with the OptKnock computational framework for metabolic modeling and simulation, the integer cut method of reducing redundancy in iterative computational analysis also can be applied with other computational frameworks well known in the art including, for example, SimPheny®.

[0271] The methods exemplified herein allow the construction of cells and organisms that biosynthetically produce a desired product, including the obligatory coupling of production of a target biochemical product to growth of the cell or organism engineered to harbor the identified genetic alterations. Therefore, the computational methods described herein allow the

identification and implementation of metabolic modifications that are identified by an in silico method selected from OptKnock or SimPheny®. The set of metabolic modifications can include, for example, addition of one or more biosynthetic pathway enzymes and/or functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion.

[0272] As discussed above, the OptKnock methodology was developed on the premise that mutant microbial networks can be evolved towards their computationally predicted maximum- growth phenotypes when subjected to long periods of growth selection. In other words, the approach leverages an organism's ability to self-optimize under selective pressures. The

OptKnock framework allows for the exhaustive enumeration of gene deletion combinations that force a coupling between biochemical production and cell growth based on network stoichiometry. The identification of optimal gene/reaction knockouts requires the solution of a bilevel optimization problem that chooses the set of active reactions such that an optimal growth solution for the resulting network overproduces the biochemical of interest (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)).

[0273] An in silico stoichiometric model of E. coli metabolism can be employed to identify essential genes for metabolic pathways as exemplified previously and described in, for example, U.S. Publ. Nos. 2002/0012939, 2003/0224363, 2004/0029149, 2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466, and in U.S. Patent No. 7,127,379. As disclosed herein, the OptKnock mathematical framework can be applied to pinpoint gene deletions leading to the growth-coupled production of a desired product. Further, the solution of the bilevel OptKnock problem provides only one set of deletions. To enumerate all meaningful solutions, that is, all sets of knockouts leading to growth-coupled production formation, an optimization technique, termed integer cuts, can be implemented. This entails iteratively solving the OptKnock problem with the incorporation of an additional constraint referred to as an integer cut at each iteration, as discussed above.

[0274] As disclosed herein, a nucleic acid encoding a desired activity of a 3-HIBP or MAAP, FAP, FRP, and/or MMP can be introduced into a host organism. In some cases, it can be desirable to modify an activity of a 3-HIBP or MAAP, FAP, FRP, or MMP enzyme or protein to increase production of 3-HIB or MAA; formaldehyde, and/or reducing equivalents. For example, known mutations that increase the activity of a protein or enzyme can be introduced into an encoding nucleic acid molecule. Additionally, optimization methods can be applied to increase the activity of an enzyme or protein and/or decrease an inhibitory activity, for example, decrease the activity of a negative regulator.

[0275] One such optimization method is directed evolution. Directed evolution is a powerful approach that involves the introduction of mutations targeted to a specific gene in order to improve and/or alter the properties of an enzyme. Improved and/or altered enzymes can be identified through the development and implementation of sensitive high-throughput screening assays that allow the automated screening of many enzyme variants (for example, >10 ⁴ ). Iterative rounds of mutagenesis and screening typically are performed to afford an enzyme with optimized properties. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened. Numerous directed evolution technologies have been developed (for reviews, see Hibbert et al., Biomol. Eng. 22: 11-19 (2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax. Biomol. Eng. 22: 1-9 (2005).; and Sen et al., Appl Biochem.Biotechnol 143:212-223 (2007)) to be effective at creating diverse variant libraries, and these methods have been successfully applied to the improvement of a wide range of properties across many enzyme classes. Enzyme characteristics that have been improved and/or altered by directed evolution technologies include, for example: selectivity/specificity, for conversion of non-natural substrates; temperature stability, for robust high temperature processing; pH stability, for bioprocessing under lower or higher pH conditions; substrate or product tolerance, so that high product titers can be achieved; binding (K _m ), including broadening substrate binding to include non-natural substrates; inhibition (K;), to remove inhibition by products, substrates, or key intermediates; activity (kcat), to increases enzymatic reaction rates to achieve desired flux; expression levels, to increase protein yields and overall pathway flux; oxygen stability, for operation of air sensitive enzymes under aerobic conditions; and anaerobic activity, for operation of an aerobic enzyme in the absence of oxygen.

[0276] A number of exemplary methods have been developed for the mutagenesis and diversification of genes to target desired properties of specific enzymes. Such methods are well known to those skilled in the art. Any of these can be used to alter and/or optimize the activity of a 3-HIBP or MAAP and/or a methanol metabolic enzyme or protein. Such methods include, but are not limited to EpPCR, which introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions (Pritchard et al., J. Theor. Biol. 234:497-509 (2005)); Error-prone Rolling Circle Amplification (epRCA), which is similar to epPCR except a whole circular plasmid is used as the template and random 6-mers with exonuclease resistant thiophosphate linkages on the last 2 nucleotides are used to amplify the plasmid followed by transformation into cells in which the plasmid is re-circularized at tandem repeats (Fujii et al., Nucleic Acids Res. 32:el45 (2004); and Fujii et al., Nat. Protocols 1 :2493-2497 (2006)); DNA or Family Shuffling, which typically involves digestion of two or more variant genes with nucleases such as Dnase I or EndoV to generate a pool of random fragments that are reassembled by cycles of annealing and extension in the presence of DNA polymerase to create a library of chimeric genes (Stemmer, Proc. Natl. Acad. Sci. U.S.A. 91 : 10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994)); Staggered Extension (StEP), which entails template priming followed by repeated cycles of 2 step PCR with denaturation and very short duration of annealing/extension (as short as 5 sec) (Zhao et al., Nat. Biotechnol. 16:258-261 (1998)); Random Priming

Recombination (RPR), in which random sequence primers are used to generate many short DNA fragments complementary to different segments of the template (Shao et al., Nucleic Acids Res. 26:681-683 (1998)).

[0277] Additional methods include Heteroduplex Recombination, in which linearized plasmid DNA is used to form heteroduplexes that are repaired by mismatch repair (Volkov et al,

Nucleic Acids Res. 27:el 8 (1999); and Volkov et al, Methods Enzymol. 328:456-463 (2000));

Random Chimeragenesis on Transient Templates (RACHITT), which employs Dnase I fragmentation and size fractionation of single stranded DNA (ssDNA) (Coco et al., Nat.

Biotechnol. 19:354-359 (2001)); Recombined Extension on Truncated templates (RETT), which entails template switching of unidirectionally growing strands from primers in the presence of unidirectional ssDNA fragments used as a pool of templates (Lee et al., J. Molec. Catalysis

26: 119-129 (2003)); Degenerate Oligonucleotide Gene Shuffling (DOGS), in which degenerate primers are used to control recombination between molecules; (Bergquist and Gibbs, Methods

Mol. Biol. 352: 191-204 (2007); Bergquist et al, Biomol. Eng. 22:63-72 (2005); Gibbs et al.,

Gene 271 : 13-20 (2001)); Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY), which creates a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest (Ostermeier et al., Proc. Natl. Acad. Sci. U.S.A. 96:3562-3567 (1999); and Ostermeier et al., Nat. Biotechnol. 17: 1205-1209 (1999)); Thio-Incremental Truncation for the Creation of

Hybrid Enzymes (THIO-ITCHY), which is similar to ITCHY except that phosphothioate dNTPs are used to generate truncations (Lutz et al., Nucleic Acids Res. 29:E16 (2001)); SCRATCHY, which combines two methods for recombining genes, ITCHY and DNA shuffling (Lutz et al.,

Proc. Natl. Acad. Sci. U.S.A. 98: 11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in which mutations made via epPCR are followed by screening/selection for those retaining usable activity (Bergquist et al., Biomol. Eng. 22:63-72 (2005)); Sequence Saturation Mutagenesis

(SeSaM), a random mutagenesis method that generates a pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage, which is used as a template to extend in the presence of "universal" bases such as inosine, and replication of an inosine- containing complement gives random base incorporation and, consequently, mutagenesis (Wong et al, Biotechnol. J. 3:74-82 (2008); Wong et al, Nucleic Acids Res. 32:e26 (2004); and Wong et αΙ, ΑηαΙ Biochem. 341 : 187-189 (2005)); Synthetic Shuffling, which uses overlapping oligonucleotides designed to encode "all genetic diversity in targets" and allows a very high diversity for the shuffled progeny (Ness et al, Nat. Biotechnol 20: 1251-1255 (2002));

Nucleotide Exchange and Excision Technology NexT, which exploits a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine to perform endpoint DNA fragmentation (Muller et al, Nucleic Acids Res. 33:el 17 (2005)).

[0278] Further methods include Sequence Homology- Independent Protein Recombination (SHIPREC), in which a linker is used to facilitate fusion between two distantly related or unrelated genes, and a range of chimeras is generated between the two genes, resulting in libraries of single-crossover hybrids (Sieber et al, Nat. Biotechnol. 19:456-460 (2001)); Gene Site Saturation Mutagenesis™ (GSSM™), in which the starting materials include a supercoiled double stranded DNA (dsDNA) plasmid containing an insert and two primers which are degenerate at the desired site of mutations (Kretz et al, Methods Enzymol. 388:3-11 (2004)); Combinatorial Cassette Mutagenesis (CCM), which involves the use of short oligonucleotide cassettes to replace limited regions with a large number of possible amino acid sequence alterations (Reidhaar-Olson et al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al. Science 241 :53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis (CMCM), which is essentially similar to CCM and uses epPCR at high mutation rate to identify hot spots and hot regions and then extension by CMCM to cover a defined region of protein sequence space (Reetz et al, Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); the Mutator Strains technique, in which conditional ts mutator plasmids, utilizing the mutD5 gene, which encodes a mutant subunit of DNA polymerase III, to allow increases of 20 to 4000-X in random and natural mutation frequency during selection and block accumulation of deleterious mutations when selection is not required (Selifonova et al, Appl. Environ. Microbiol. 67:3645-3649 (2001)); Low et al, J. Mol. Biol. 260:359-3680 (1996)). [0279] Additional exemplary methods include Look- Through Mutagenesis (LTM), which is a multidimensional mutagenesis method that assesses and optimizes combinatorial mutations of selected amino acids (Rajpal et al, Proc. Natl. Acad. Sci. U.S.A. 102:8466-8471 (2005)); Gene Reassembly, which is a DNA shuffling method that can be applied to multiple genes at one time or to create a large library of chimeras (multiple mutations) of a single gene (Tunable

GeneReassembly™ (TGR™) Technology supplied by Verenium Corporation), in Silico Protein Design Automation (PDA), which is an optimization algorithm that anchors the structurally defined protein backbone possessing a particular fold, and searches sequence space for amino acid substitutions that can stabilize the fold and overall protein energetics, and generally works most effectively on proteins with known three-dimensional structures (Hayes et al., Proc. Natl. Acad. Sci. U.S.A. 99: 15926-15931 (2002)); and Iterative Saturation Mutagenesis (ISM), which involves using knowledge of structure/function to choose a likely site for enzyme improvement, performing saturation mutagenesis at chosen site using a mutagenesis method such as Stratagene QuikChange (Stratagene; San Diego CA), screening/selecting for desired properties, and, using improved clone(s), starting over at another site and continue repeating until a desired activity is achieved (Reetz et al., Nat. Protocols 2:891-903 (2007); and Reetz et al., Angew. Chem. Int. Ed Engl. 45:7745-7751 (2006)).

[0280] Any of the aforementioned methods for mutagenesis can be used alone or in any combination. Additionally, any one or combination of the directed evolution methods can be used in conjunction with adaptive evolution techniques, as described herein.

[0281] 3-HIB or MAA can be harvested or isolated at any time point during the culturing of the microbial organism, for example, in a continuous and/or near-continuous culture period, as disclosed herein. Generally, the longer the microorganisms are maintained in a continuous and/or near-continuous growth phase, the proportionally greater amount of 3-HIB or MAA can be produced.

[0282] Therefore, additionally provided is a method for producing 3-HIB or MAA that includes culturing a non-naturally occurring microbial organism having one or more gene disruptions, as disclosed herein. The disruptions can occur in one or more genes encoding an enzyme that increases production of 3-HIB or MAA, including optionally coupling 3-HIB or MAA production to growth of the microorganism when the gene disruption reduces or eliminates an activity of the enzyme. For example, the disruptions can confer stable growth-coupled production of 3-HIB or MAA onto the non-naturally microbial organism.

[0283] In some embodiments, the gene disruption can include a complete gene deletion. In some embodiments other methods to disrupt a gene include, for example, frameshifting by omission or addition of oligonucleotides or by mutations that render the gene inoperable. One skilled in the art will recognize the advantages of gene deletions, however, because of the stability it confers to the non-naturally occurring organism from reverting to a parental phenotype in which the gene disruption has not occurred. In particular, the gene disruptions are selected from the gene sets as disclosed herein.

[0284] Once computational predictions are made of gene sets for disruption to increase production of 3-HIB or MAA, the strains can be constructed, evolved, and tested. Gene disruptions, including gene deletions, are introduced into host organism by methods well known in the art. A particularly useful method for gene disruption is by homologous recombination, as disclosed herein.

[0285] The engineered strains can be characterized by measuring the growth rate, the substrate uptake rate, and/or the product/byproduct secretion rate. Cultures can be grown and used as inoculum for a fresh batch culture for which measurements are taken during exponential growth. The growth rate can be determined by measuring optical density using a

spectrophotometer (A600). Concentrations of glucose and other organic acid byproducts in the culture supernatant can be determined by well known methods such as HPLC, GC-MS or other well known analytical methods suitable for the analysis of the desired product, as disclosed herein, and used to calculate uptake and secretion rates.

[0286] Strains containing gene disruptions can exhibit suboptimal growth rates until their metabolic networks have adjusted to their missing functionalities. To assist in this adjustment, the strains can be adaptively evolved. By subjecting the strains to adaptive evolution, cellular growth rate becomes the primary selection pressure and the mutant cells are compelled to reallocate their metabolic fluxes in order to enhance their rates of growth. This reprogramming of metabolism has been recently demonstrated for several E. coli mutants that had been adaptively evolved on various substrates to reach the growth rates predicted a priori by an in silico model (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004)). The growth improvements brought about by adaptive evolution can be accompanied by enhanced rates of 3-HIB or MAA production. The strains are generally adaptively evolved in replicate, running in parallel, to account for differences in the evolutionary patterns that can be exhibited by a host organism (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004); Fong et al, J. Bacteriol. 185:6400-6408 (2003); Ibarra et al., Nature 420: 186-189 (2002)) that could potentially result in one strain having superior production qualities over the others. Evolutions can be run for a period of time, typically 2-6 weeks, depending upon the rate of growth improvement attained. In general, evolutions are stopped once a stable phenotype is obtained.

[0287] Following the adaptive evolution process, the new strains are characterized again by measuring the growth rate, the substrate uptake rate, and the product/byproduct secretion rate. These results are compared to the theoretical predictions by plotting actual growth and production yields alongside the production envelopes from metabolic modeling. The most successful design/evolution combinations are chosen to pursue further, and are characterized in lab-scale batch and continuous fermentations. The growth-coupled biochemical production concept behind the methods disclosed herein such as OptKnock approach should also result in the generation of genetically stable overproducers. Thus, the cultures are maintained in continuous mode for an extended period of time, for example, one month or more, to evaluate long-term stability. Periodic samples can be taken to ensure that yield and productivity are maintained.

[0288] Adaptive evolution is a powerful technique that can be used to increase growth rates of mutant or engineered microbial strains, or of wild-type strains growing under unnatural environmental conditions. It is especially useful for strains designed via methods such as OptKnock, which results in growth-coupled product formation. Therefore, evolution toward optimal growing strains will indirectly optimize production as well. Unique strains of E. coli K- 12 MG1655 were created through gene knockouts and adaptive evolution. (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004)). In this work, all adaptive evolutionary cultures were maintained in prolonged exponential growth by serial passage of batch cultures into fresh medium before the stationary phase was reached, thus rendering growth rate as the primary selection pressure. Knockout strains were constructed and evolved on minimal medium supplemented with different carbon substrates (four for each knockout strain). Evolution cultures were carried out in duplicate or triplicate, giving a total of 50 evolution knockout strains. The evolution cultures were maintained in exponential growth until a stable growth rate was reached. The computational predictions were accurate (within 10%) at predicting the post- evolution growth rate of the knockout strains in 38 out of the 50 cases examined. Furthermore, a combination of OptKnock design with adaptive evolution has led to improved lactic acid production strains. (Fong et al., Biotechnol. Bioeng. 91 :643-648 (2005)). Similar methods can be applied to the strains disclosed herein and applied to various host strains.

[0289] There are a number of developed technologies for carrying out adaptive evolution. Exemplary methods are disclosed herein. In some embodiments, optimization of a NNOMOs provided herein includes utilizing adaptive evolution techniques to increase 3-HIB or MAA production and/or stability of the producing strain.

[0290] Serial culture involves repetitive transfer of a small volume of grown culture to a much larger vessel containing fresh growth medium. When the cultured organisms have grown to saturation in the new vessel, the process is repeated. This method has been used to achieve the longest demonstrations of sustained culture in the literature (Lenski and Travisano, Proc. Natl. Acad. Sci. USA 91 :6808-6814 (1994)) in experiments which clearly demonstrated consistent improvement in reproductive rate over a period of years. Typically, transfer of cultures is usually performed during exponential phase, so each day the transfer volume is precisely calculated to maintain exponential growth through the next 24 hour period. Manual serial dilution is inexpensive and easy to parallelize.

[0291] In continuous culture the growth of cells in a chemostat represents an extreme case of dilution in which a very high fraction of the cell population remains. As a culture grows and becomes saturated, a small proportion of the grown culture is replaced with fresh media, allowing the culture to continually grow at close to its maximum population size. Chemostats have been used to demonstrate short periods of rapid improvement in reproductive rate

(Dykhuizen, Methods Enzymol. 613-631 (1993)). The potential usefulness of these devices was recognized, but traditional chemostats were unable to sustain long periods of selection for increased reproduction rate, due to the unintended selection of dilution-resistant (static) variants. These variants are able to resist dilution by adhering to the surface of the chemostat, and by doing so, outcompete less adherent individuals, including those that have higher reproductive rates, thus obviating the intended purpose of the device (Chao and Ramsdell, J. Gen. Microbiol 20: 132-138 (1985)). One possible way to overcome this drawback is the implementation of a device with two growth chambers, which periodically undergo transient phases of sterilization, as described previously (Marliere and Mutzel, U.S. Patent No. 6,686,194).

[0292] Evolugator™ is a continuous culture device developed by Evolugate, LLC

(Gainesville, FL) and exhibits significant time and effort savings over traditional evolution techniques (de Crecy et al.,. Appl. Microbiol. Biotechnol. 77:489-496 (2007)). The cells are maintained in prolonged exponential growth by the serial passage of batch cultures into fresh medium before the stationary phase is attained. By automating optical density measurement and liquid handling, the Evolugator™ can perform serial transfer at high rates using large culture volumes, thus approaching the efficiency of a chemostat in evolution of cell fitness. For example, a mutant of Acinetobacter sp ADP1 deficient in a component of the translation apparatus, and having severely hampered growth, was evolved in 200 generations to 80% of the wild-type growth rate. However, in contrast to the chemostat which maintains cells in a single vessel, the machine operates by moving from one "reactor" to the next in subdivided regions of a spool of tubing, thus eliminating any selection for wall-growth. The transfer volume is adjustable, and normally set to about 50%. A drawback to this device is that it is large and costly, thus running large numbers of evolutions in parallel is not practical. Furthermore, gas addition is not well regulated, and strict anaerobic conditions are not maintained with the current device configuration. Nevertheless, this is an alternative method to adaptively evolve a production strain.

[0293] In one aspect, provided herein is a NNOMO comprising: (A) a methanol metabolic pathway (MMP), wherein said organism comprises at least one exogenous nucleic acid encoding a MMP enzyme (MMPE) expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol, wherein said MMP comprises: (i) a methanol dehydrogenase (EM9); (ii) an EM9 and a formaldehyde activating enzyme (EM 10); or (iii) a methanol methyltransferase (EMI) and a methylenetetrahydro folate reductase (EM2); and (B)a 3-hydroxyisobutyrate (3-HIB) pathway (3-HIBP). In one embodiment, the organism comprises at least one exogenous nucleic acid encoding a 3-HIBP enzyme (3-HIBPE) expressed in a sufficient amount to produce 3-HIB, wherein said 3-HIBP comprises (1) (i) a succinyl-CoA transferase (EMA1A), ligase (EMA1B), or synthetase (EMA1C); (ii) a methylmalonyl-CoA mutase (EMA2); (iii) a methylmalonyl-CoA epimerase (EMA3); (iv) a methylmalonyl-CoA reductase (aldehyde forming) (EMA4); and (v) a methylmalonate semialdehyde reductase (EMA5); (2) (i) an EMA1A, EMA1B, or EMA1C; (ii) an EMA2; (iii) an EMA4; and (iv) an EMA5; or (3) (i) an EMA1A, EMA1B, or EMA1C; (ii) an EMA2; and (iii) a methylmalonyl- CoA reductase (alcohol forming) (EMA7). In one embodiment, the organism of claim 1 or 2, wherein the 3-HIBP comprises (i) an EMA1A, EMA1B, or EMA1C; (ii) an EMA2; (iii) an EMA3; (iv) an EMA4; and (v) an EMA5. In another embodiment, the 3-HIBP comprises (i) an EMA1A, EMA1B, or EMA1C; (ii) an EMA2; (iii) an EMA4; and (iv) an EMA5. In other embodiments, the 3-HIBP comprises (i) an EMA1A, EMA1B, or EMA1C; (ii) an EMA2; and

(iii) an EMA7. In some embodiments, the 3-HIBP comprises a succinyl-CoA transferase. In other embodiments, the 3-HIBP comprises a succinyl-CoA ligase. In another embodiment, the 3-HIBP comprises a succinyl-CoA synthetase. In certain embodiments, the organism comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a 3-HIBPE. In some embodiments, the at least one exogenous nucleic acid encoding a 3-HIBPE is a heterologous nucleic acid.

[0294] In another aspect, provided herein is a NNOMO comprising: (A) a MMP, wherein said organism comprises at least one exogenous nucleic acid encoding a MMPE expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol, wherein said MMP comprises: (i) an EM9; (ii) an EM9 and an EM10; or (iii) an EMI and an EM2; and (B) a methacryic acid (MAA) pathway (MAAP). In one embodiment, the organism comprises at least one exogenous nucleic acid encoding a MAAP enzyme (MAAPE) expressed in a sufficient amount to produce MAA, wherein said MAAP comprises (1) (i) an EMA1 A, EMA1B, or EMA1C; (ii) an EMA2; (iii) an EM A3; (iv) an EMA4; (v) an EMA5; and (vi) a 3- HIB dehydratase (EMA6); (2) (i) an EMA1A, EMA1B, or EMA1C; (ii) an EMA2; (iii) an EMA4; (iv) an EMA5; and (v) an EMA6; or (3) (i) an EMA1A, EMA1B, or EMA1C; (ii) an EMA2; (iii) an EMA7; and (iv) an EMA6. In one embodiment, the MAAP comprises (i) an EMA1A, EMA1B, or EMA1C; (ii) an EMA2; (iii) an EMA3; (iv) an EMA4; (v) an EMA5; and (vi) an EMA6. In another embodiment, the MAAP comprises (i) an EMA1A, EMA1B, or EMA1C; (ii) an EMA2; (iii) an EMA4; (iv) an EMA5; and (v) an EMA6. In one embodiment, the MAAP comprises (i) an EMA1A, EMA1B, or EMA1C; (ii) an EMA2; (iii) an EMA7; and

(iv) an EMA6. In some embodiments, the MAAP comprises an EMA1 A. In other embodiments, the MAAP comprises an EMA1B. In yet other embodiments, the MAAP comprises an EMA1C. In some embodiments, the organism comprises two, three, four, five or six exogenous nucleic acids, each encoding a MAAPE. In certain embodiments, the at least one exogenous nucleic acid encoding a MAAPE is a heterologous nucleic acid.

[0295] In certain embodiments of the NNOMOs provided herein, the MMP comprises an EMI and an EM2. In other embodiments, the MMP comprises an EM9. In some embodiments, the MMP comprises an EM9 and an EM10. In certain embodiments, the MMP comprises an EMI, an EM2, a methylenetetrahydrofolate dehydrogenase (EM3), a methenyltetrahydrofolate cyclohydrolase (EM4), and a formyltetrahydrofolate deformylase (EM5). In some embodiments, the MMP comprises an EMI, an EM2, an EM3, an EM4 and a formyltetrahydrofolate synthetase (EM6). In one embodiment, the MMP comprises an EM9, an EM3, an EM4 and an EM5. In certain embodiments, the MMP comprises an EM9, an EM3, an EM4 and an EM6. In some embodiments, the MMP comprises an EM9 and a formaldehyde dehydrogenase (EMI 1). In other embodiments, the MMP comprises an EM9, an EM 12, a glutathione-dependent formaldehyde dehydrogenase (EM 13) and a S-formylglutathione hydrolase (EM 14). In some embodiments, the MMP comprises an EM9, an EM13 and an EM14. In an embodiment, the MMP comprises an EM9, an EM10, an EM3, an EM4 and an EM5. In other embodiments, the MMP comprises an EM9, an EM10, an EM3, an EM4 and an EM6. In certain embodiments, the MMP further comprises a formate dehydrogenase (EM8). In some embodiments, the MMP further comprises a formate hydrogen lyase (EM 15). In yet other embodiments, the MMP further comprises a hydrogenase (EM16). In certain embodiments, the organism comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a MMPE. In some embodiments, the at least one exogenous nucleic acid encoding a MMPE is a heterologous nucleic acid.

[0296] In some embodiments of the NNOMO provided herein, the organism further comprises one or more gene disruptions, wherein said one or more gene disruptions occur in one or more endogenous genes encoding protein(s) or enzyme(s) involved in native production of ethanol, glycerol, acetate, lactate, formate, C0 ₂ , and/or amino acids, by said microbial organism, and wherein said one or more gene disruptions confers increased production of 3-HIB or MAA in said microbial organism. [0297] In certain embodiments of the NNOMO provided herein, the one or more endogenous enzymes involved in: native production of ethanol, glycerol, acetate, lactate, formate, C0 ₂ and/or amino acids by said microbial organism, has attenuated enzyme activity or expression levels.

[0298] In other embodiments of the NNOMO provided herein, the organism further comprises a formaldehyde assimilation pathway (FAP), wherein said organism comprises at least one exogenous nucleic acid encoding a FAPE expressed in a sufficient amount to produce an intermediate of glycolysis and/or a metabolic pathway that can be used in the formation of biomass, and wherein said FAP comprises a hexulose-6-phosphate (H6P) synthase (EF1) and a 6-phospho-3-hexuloisomerase (EF2). In one embodiment, the intermediate is a H6P, a fructose- 6-phosphate (F6P), or a combination thereof. In other embodiments of the NNOMO provided herein, the organism further comprises a FAP, wherein said organism comprises at least one exogenous nucleic acid encoding a FAPE expressed in a sufficient amount to produce an intermediate of glycolysis and/or a metabolic pathway that can be used in the formation of biomass, and wherein said FAP comprises a dihydroxyacetone (DHA) synthase (EF3) and a DHA kinase (EF4). In some embodiments, the intermediate is a DHA, a DHAP, or a

combination thereof. In some embodiments, the organism comprises two exogenous nucleic acids, each encoding a FAPE. In other embodiments, the at least one exogenous nucleic acid is a heterologous nucleic acid.

[0299] In some embodiments, the organism is in a substantially anaerobic culture medium. In certain embodiments, the microbial organism is a species of bacteria, yeast, or fungus.

[0300] In some embodiments, also provided herein is a method for producing 3-HIB, comprising culturing a NNOMO having a 3-HIBP provided herein under conditions and for a sufficient period of time to produce 3-HIB. Also provided herein is a bioderived or biobased product comprising 3-HIB, or an intermediate thereof, produced according to the method. In certain embodiments, the bioderived or biobased product is selected from the group consisting of a polymer, a co-polymer, a plastic, a methacrylate, a methyl methacrylate, a butyl methacrylate, glacial MAA, and combinations thereof. Also provided is a bioderived 3-HIB produced according to the method. Also provided is a culture medium comprising said bioderived 3-HIB. In certain embodiments, of the culture medium, said bioderived 3-HIB or has a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source. In other embodiments, the culture medium is separated from the NNOMO having the 3-HIBP. In certain embodiments, the bioderived 3-HIB has a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source. In some embodiments, the bioderived 3-HIB has an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%). Also provided is a composition comprising said bioderived 3-HIB, and a compound other than said bioderived 3-HIB. In one embodiment, the compound other than said bioderived 3-HIB is a trace amount of a cellular portion of a NNOMO having a 3-HIBP. Also provided is a biobased product comprising said bioderived 3-HIB, wherein said biobased product is a polymer, a co-polymer, a plastic, a methacrylate, a methyl methacrylate, a butyl methacrylate, glacial MAA, or combination thereof. In certain embodiments, at least 5%, at least 10%, at least 20%, at least 30%>, at least 40%> or at least 50%> bioderived 3-HIB. In some embodiments, the biobased product comprises a portion of said bioderived 3-HIB as a repeating unit. In certain

embodiments, provided herein is a molded product obtained by molding the biobased product. Also provided herein is a process for producing the biobased product provided herein, comprising chemically reacting said 3-HIB with itself or another compound in a reaction that produces said biobased product. Also provided herein is a polymer comprising or obtained by converting the bioderived 3-HIB provided herein. Also provided is a method for producing a polymer, comprising chemically of enzymatically converting the bioderived 3-HIB to the polymer. Also provided is a composition comprising the bioderived 3-HIB, or a cell lysate or culture supernatant thereof.

[0301] In other embodiments, also provided herein is a method for producing MAA, comprising culturing a NNOMO having a MAAP provided herein under conditions and for a sufficient period of time to produce MAA. Also provided herein is a bioderived or biobased product comprising MAA, or an intermediate thereof, produced according to the method. In certain embodiments, the bioderived or biobased product is selected from the group consisting of a polymer, a co-polymer, a plastic, a methacrylate, a methyl methacrylate, a butyl methacrylate, glacial MAA, and combinations thereof. Also provided is a bioderived MAA produced according to the method. Also provided is a culture medium comprising said bioderived MAA.

In certain embodiments, of the culture medium, said bioderived MAA or has a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source. In other embodiments, the culture medium is separated from the NNOMO having the MAAP. In certain embodiments, the bioderived MAA has a carbon- 12, carbon- 13 and carbon- 14 isotope ratio that reflects an atmospheric carbon dioxide uptake source. In some embodiments, the bioderived MAA has an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%). Also provided is a composition comprising said bioderived MAA, and a compound other than said bioderived MAA. In one embodiment, the compound other than said bioderived MAA is a trace amount of a cellular portion of a NNOMO having a MAAP. Also provided is a biobased product comprising said bioderived MAA, wherein said biobased product is a polymer, a co-polymer, a plastic, a methacrylate, a methyl methacrylate, a butyl methacrylate, glacial MAA, or combination thereof. In certain embodiments, at least 5%, at least 10%, at least 20%, at least 30%, at least 40% or at least 50% bioderived MAA. In some embodiments, the biobased product comprises a portion of said bioderived MAA as a repeating unit. In certain

embodiments, provided herein is a molded product obtained by molding the biobased product. Also provided herein is a process for producing the biobased product provided herein, comprising chemically reacting said MAA with itself or another compound in a reaction that produces said biobased product. Also provided herein is a polymer comprising or obtained by converting the bioderived MAA provided herein. Also provided is a method for producing a polymer, comprising chemically of enzymatically converting the bioderived MAA to the polymer. Also provided is a composition comprising the bioderived MAA, or a cell lysate or culture supernatant thereof.

[0302] Also provided herein is a method of producing formaldehyde, comprising culturing a NNOMO provided herein under conditions and for a sufficient period of time to produce formaldehyde, and optionally wherein the formaldehyde is consumed to provide a reducing equivalent or to incorporate into 3-HIB, MAA, or target product.

[0303] Also provided herein is a method of producing an intermediate of glycolysis and/or an intermediate of a metabolic pathway that can be used in the formation of biomass, comprising culturing a NNOMO provided herein under conditions and for a sufficient period of time to produce the intermediate, and optionally wherein the intermediate is consumed to provide a reducing equivalent or to incorporate into 3-HIB, MAA, or target product. [0304] In certain embodiments, the organism is cultured in a medium comprising biomass, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, starch, glycerol, methanol, carbon dioxide, formate, methane, or any combination thereof as a carbon source.

[0305] Throughout this application various publications have been referenced. The disclosures of these publications in their entireties, including GenBank and GI number publications, are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.

[0306] It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also included within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.

4. EXAMPLES

4.1 Example I - Production of reducing equivalents via a MMP

Exemplary MMPs are provided in FIG.1. FIG. 1, Step A - Methanol Methyltransferase (EMI)

[0307] A complex of 3 -methyltransferase proteins, denoted MtaA, MtaB, and MtaC, perform the desired EMI activity (Sauer et al., Eur. J. Biochem. 243:670-677 (1997); Naidu and

Ragsdale, J. Bacteriol. 183:3276-3281 (2001); Tallant and Krzycki, J. Biol. Chem. 276:4485- 4493 (2001); Tallant and Krzycki, J. Bacteriol. 179:6902-6911 (1997); Tallant and Krzycki, J. Bacteriol. 178: 1295-1301 (1996); Ragsdale, S.W., Crit. Rev. Biochem. Mol. Biol. 39: 165-195 (2004)).

[0308] MtaB is a zinc protein that can catalyze the transfer of a methyl group from methanol to MtaC, a corrinoid protein. Exemplary genes encoding MtaB and MtaC can be found in methanogenic archaea such as Methanosarcina barkeri (Maeder et al., J. Bacteriol. 188:7922- 7931 (2006) and Methanosarcina acetivorans (Galagan et al., Genome Res. 12:532-542 (2002), as well as the acetogen, Moorella thermoacetica (Das et al., Proteins 67: 167-176 (2007). In general, the MtaB and MtaC genes are adjacent to one another on the chromosome as their activities are tightly interdependent. The protein sequences of various MtaB and MtaC encoding genes in M. barkeri, M. acetivorans, and M. thermoaceticum can be identified by their following GenBank accession numbers.

[0309] The MtaB 1 and MtaC 1 genes, YP 304299 and YP 304298, from M. barkeri were cloned into E. coli and sequenced ( Sauer et al., Eur. J. Biochem. 243:670-677 (1997)). The crystal structure of this methanol-cobalamin methyltransferase complex is also available (Hagemeier et al, Proc. Natl. Acad. Sci. U.S.A. 103: 18917-18922 (2006)). The MtaB genes, YP 307082 and YP 304612, in barkeri were identified by sequence homology to

YP 304299. In general, homology searches are an effective means of identifying EM Is because MtaB encoding genes show little or no similarity to methyltransferases that act on alternative substrates such as trimethylamine, dimethylamine, monomethylamine, or dimethylsulfide. The MtaC genes, YP 307081 and YP 304611 were identified based on their proximity to the MtaB genes and also their homology to YP 304298. The three sets of MtaB and MtaC genes from acetivorans have been genetically, physiologically, and biochemically characterized (Pritchett and Metcalf, Mol. Microbiol. 56: 1183-1194 (2005)). Mutant strains lacking two of the sets were able to grow on methanol, whereas a strain lacking all three sets of MtaB and MtaC genes sets could not grow on methanol. This suggests that each set of genes plays a role in methanol utilization. The M. thermoacetica MtaB gene was identified based on homology to the methanogenic MtaB genes and also by its adjacent chromosomal proximity to the methanol- induced corrinoid protein, MtaC, which has been crystallized (Zhou et al., Acta Crystallogr. Sect. F. Struct. Biol. Cyrst. Commun. 61 :537-540 (2005) and further characterized by Northern hybridization and Western Blotting ((Das et al., Proteins 67: 167-176 (2007)).

[0310] MtaA is zinc protein that catalyzes the transfer of the methyl group from MtaC to either Coenzyme M in methanogens or methyltetrahydrofolate in acetogens. MtaA can also utilize methylcobalamin as the methyl donor. Exemplary genes encoding MtaA can be found in methanogenic archaea such as Methanosarcina barkeri (Maeder et al., J. Bacteriol. 188:7922- 7931 (2006) and Methanosarcina acetivorans (Galagan et al., Genome Res. 12:532-542 (2002), as well as the acetogen, Moorella thermoacetica ((Das et al., Proteins 67: 167-176 (2007)). In general, MtaA proteins that catalyze the transfer of the methyl group from CH ₃ -MtaC are difficult to identify bioinformatically as they share similarity to other corrinoid protein methyltransferases and are not oriented adjacent to the MtaB and MtaC genes on the

chromosomes. Nevertheless, a number of MtaA encoding genes have been characterized. The protein sequences of these genes in M. barkeri and M. acetivorans can be identified by the following GenBank accession numbers.

[0311] The MtaA gene, YP 304602, from M. barkeri was cloned, sequenced, and functionally overexpressed in E. coli (Harms and Thauer, Eur. J. Biochem. 235:653-659 (1996)). In M. acetivorans, MtaAl is required for growth on methanol, whereas MtaA2 is dispensable even though methane production from methanol is reduced in MtaA2 mutants (Bose et al., J. Bacteriol. 190:4017-4026 (2008)). There are multiple additional MtaA homologs in M barkeri and M. acetivorans that are as yet uncharacterized, but may also catalyze corrinoid protein methyltransferase activity.

[0312] Putative MtaA encoding genes in M. thermoacetica were identified by their sequence similarity to the characterized methanogenic MtaA genes. Specifically, three M. thermoacetica genes show high homology (>30% sequence identity) to YP 304602 from M. barkeri. Unlike methanogenic MtaA proteins that naturally catalyze the transfer of the methyl group from C¾- MtaC to Coenzyme M, an M. thermoacetica MtaA is likely to transfer the methyl group to methyltetrahydrofolate given the similar roles of methyltetrahydrofolate and Coenzyme M in methanogens and acetogens, respectively. The protein sequences of putative MtaA encoding genes from M. thermoacetica can be identified by the following GenBank accession numbers.

FIG. 1, Step B - Methylenetetrahydrofolate Reductase (EM2)

[0313] The conversion of methyl-THF to methylenetetrahydrofolate is catalyzed by EM2. In M. thermoacetica, this enzyme is oxygen-sensitive and contains an iron-sulfur cluster (Clark and Ljungdahl, J. Biol. Chem. 259: 10845-10849 (1984). This enzyme is encoded by metF in E. coli (Sheppard et al., J. Bacteriol. 181 :718-725 (1999) and CHY_1233 in C. hydrogenoformans (Wu et al., PLoS Genet. I :e65 (2005). The thermoacetica genes, and its C. hydrogenoformans counterpart, are located near the CODH/ACS gene cluster, separated by putative EM 16 and heterodisulfide reductase genes. Some additional gene candidates found bioinformatically are listed below. In Acetobacterium woodii metF is coupled to the Rnf complex through RnfC2 (Poehlein et al, PLoS One. 7:e33439). Homologs of RnfC are found in other organisms by blast search. The Rnf complex is known to be a reversible complex (Fuchs (201 1) Annu. Rev.

Microbiol. 65:631-658). Protein GenBank ID GI number Organism

Moth_1191 YP 430048.1 83590039 Moorella thermoacetica

Moth_1192 YP 430049.1 83590040 Moorella thermoacetica metF NP_418376.1 16131779 Escherichia coli

CHY 1233 YP 360071.1 78044792 Carboxydothermus

hydrogenoformans

CLJU_c37610 YP 003781889.1 300856905 Clostridium ljungdahlii DSM

13528

DesfrDRAFT_3717 ZP_07335241.1 303248996 Desulfovibrio fructosovorans JJ

CcarbDRAFT_2950 ZP 05392950.1 255526026 Clostridium carboxidivorans

P7

Ccel74_010100023124 ZP 07633513.1 307691067 Clostridium cellulovorans

743B

Cphy_3110 YP 001560205.1 160881237 Clostridium

phytofermentans ISDg

FIG. 1, Steps C and D - Methylenetetrahydrofolate Dehydrogenase (EM3),

Methenyltetrahydrofolate Cyclohydrolase (EM4)

[0314] In thermoacetica, E. coli, and C. hydrogenoformans, EM4 and EM3 are carried out by the bi-functional gene products of Moth_1516,y¾/D, and CHY 1878, respectively (Pierce et al, Environ. Microbiol. 10:2550-2573 (2008); Wu et al, PLoS Genet. I :e65 (2005); D'Ari and Rabinowitz, J. Biol. Chem. 266:23953-23958 (1991)). A homolog exists in C. carboxidivorans P7. Several other organisms also encode for this bifunctional protein as tabulated below.

folD NP_348702.1 15895353 Clostridium

acetobutylicum ATCC 824 folD YP 696506.1 110800457 Clostridium perfringens

MGA3 09460 EIJ83438.1 387591119 Bacillus methanolicus MGA3

PB1 14689 ZP 10132349.1 387929672 Bacillus methanolicus PB1

FIG. 1, Step E - Formyltetrahydrofolate Deformylase (EMS)

[0315] This enzyme catalyzes the hydrolysis of 10-formyltetrahydro folate (formyl-THF) to THF and formate. In E. coli, this enzyme is encoded by purl! and has been overproduced, purified, and characterized (Nagy, et al., J. Bacteriol. 3: 1292-1298 (1995)). Homologs exist in Corynebacterium sp. U-96 (Suzuki, et al., Biosci. Biotechnol. Biochem. 69(5):952-956 (2005)), Corynebacterium glutamicum ATCC 14067, Salmonella enterica, and several additional organisms.

FIG. 1, Step F - Formyltetrahydrofolate Synthetase (EM6)

[0316] EM6 ligates formate to tetrahydro folate at the expense of one ATP. This reaction is catalyzed by the gene product of Moth_0109 in M. thermoacetica (O'brien et al., Experientia Suppl. 26:249-262 (1976); Lovell et al, Arch. Microbiol. 149:280-285 (1988); Lovell et al, Biochemistry 29:5687-5694 (1990)), FHS in Clostridium acidurici (Whitehead and Rabinowitz, J. Bacteriol. 167:203-209 (1986); Whitehead and Rabinowitz, J. Bacteriol. 170:3255-3261 (1988), and CHY_2385 in C. hydrogenoformans (Wu et al, PLoS Genet. I :e65 (2005).

Homologs exist in C. carboxidivorans P7. This enzyme is found in several other organisms as listed below. Protein GenBank ID GI number Organism

Moth_0109 YP 428991.1 83588982 Moorella thermoacetica

CHY 2385 YP 361 182.1 78045024 Carboxydothermus

hydrogenoformans

FHS P13419.1 120562 Clostridium acidurici

CcarbDRAFT l 913 ZP 05391913.1 255524966 Clostridium carboxidivorans P7

CcarbDRAFT_2946 ZP 05392946.1 255526022 Clostridium carboxidivorans P7

Dhaf_0555 ACL18622.1 219536883 Desulfitobacterium hafniense

Fhs YP 001393842.1 153953077 Clostridium kluyveri DSM 555

Fhs YP 003781893.1 300856909 Clostridium ljungdahlii DSM

13528

MGA3 08300 EIJ83208.1 387590889 Bacillus methanolicus MGA3

PB1 13509 ZP 101321 13.1 387929436 Bacillus methanolicus PB1

FIG. 1, Step G - Formate Hydrogen Lyase (EM 15)

[0317] A EM 15 enzyme can be employed to convert formate to carbon dioxide and hydrogen. An exemplary EM 15 enzyme can be found in Escherichia coli. The E. coli EM 15 consists of hydrogenase 3 and formate dehydrogenase-H (Maeda et al., Appl Microbiol

Biotechnol 77:879-890 (2007)). It is activated by the gene product of fhlA. (Maeda et al., Appl Microbiol Biotechnol 77:879-890 (2007)). The addition of the trace elements, selenium, nickel and molybdenum, to a fermentation broth has been shown to enhance EM 15 activity (Soini et al., Microb. Cell Fact. 7:26 (2008)). Various hydrogenase 3, EM8 and transcriptional activator genes are shown below.

hycl NP_417197 16130624 Escherichia coli K-12 MG1655 fdhF NP_418503 16131905 Escherichia coli K-12 MG1655 fhlA NP_417211 16130638 Escherichia coli K-12 MG1655

[0318] A EM 15 enzyme also exists in the hyperthermophilic archaeon, Thermococcus litoralis (Takacs et al, BMC.Microbiol 8:88 (2008)).

[0319] Additional EM15 systems have been found in Salmonella typhimurium, Klebsiella pneumoniae, Rhodospirillum rubrum, Methanobacterium formicicum (Vardar-Schara et al., Microbial Biotechnology 1 : 107-125 (2008)).

FIG. 1, Step H - Hydrogenase (EM16)

[0320] Hydrogenase enzymes can convert hydrogen gas to protons and transfer electrons to acceptors such as ferredoxins, NAD+, or NADP+. Ralstonia eutropha HI 6 uses hydrogen as an energy source with oxygen as a terminal electron acceptor. Its membrane-bound uptake [NiFe]- hydrogenase is an "02-tolerant" hydrogenase (Cracknell, et al. Proc Nat Acad Sci, 106(49) 20681-20686 (2009)) that is periplasmically-oriented and connected to the respiratory chain via a b-type cytochrome (Schink and Schlegel, Biochim. Biophys. Acta, 567, 315-324 (1979);

Bernhard et al., Eur. J. Biochem. 248, 179-186 (1997)). R. eutropha also contains an 0 ₂ -tolerant soluble EM 16 encoded by the Hox operon which is cytoplasmic and directly reduces NAD+ at the expense of hydrogen (Schneider and Schlegel, Biochim. Biophys. Acta 452, 66-80 (1976); Burgdorf, J. Bact. 187(9) 3122-3132(2005)). Soluble EM16 enzymes are additionally present in several other organisms including Geobacter sulfurreducens (Coppi, Microbiology 151, 1239— 1254 (2005)), Synechocystis str. PCC 6803 (Germer, J. Biol. Chem., 284(52), 36462-36472 (2009)), and Thiocapsa roseopersicina (Rakhely, Appl. Environ. Microbiol. 70(2) 722-728 (2004)). The Synechocystis enzyme is capable of generating NADPH from hydrogen.

Overexpression of both the Hox operon from Synechocystis str. PCC 6803 and the accessory genes encoded by the Hyp operon from Nostoc sp. PCC 7120 led to increased EM16 activity compared to expression of the Hox genes alone (Germer, J. Biol. Chem. 284(52), 36462-36472 (2009)).

HoxlH AAP50523.1 37787355 Thiocapsa roseopersicina

[0321] The genomes of E. coli and other enteric bacteria encode up to four EM 16 enzymes (Sawers, G., Antonie Van Leeuwenhoek 66:57-88 (1994); Sawers et al., J Bacteriol. 164: 1324- 1331 (1985); Sawers and Boxer, EurJ Biochem. 156:265-275 (1986); Sawers et al., J Bacteriol. 168:398-404 (1986)). Given the multiplicity of enzyme activities E. coli or another host organism can provide sufficient EM 16 activity to split incoming molecular hydrogen and reduce the corresponding acceptor. Endogenous hydrogen-lyase enzymes of E. coli include

hydrogenase 3, a membrane -bound enzyme complex using ferredoxin as an acceptor, and hydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3 and 4 are encoded by the hyc and hyf gene clusters, respectively. EM 16 activity in E. coli is also dependent upon the expression of the hyp genes whose corresponding proteins are involved in the assembly of the EM16 complexes (Jacobi et al., Arch.Microbiol 158:444-451 (1992); Rangarajan et al., J Bacteriol. 190: 1447-1458 (2008)). The M. thermoacetica and Clostridium ljungdahli EM 16s are suitable for a host that lacks sufficient endogenous EM 16 activity. M. thermoacetica and C. ljungdahli can grow with C0 ₂ as the exclusive carbon source indicating that reducing equivalents are extracted from H ₂ to enable acetyl-CoA synthesis via the Wood-Ljungdahl pathway (Drake, H. L., J Bacteriol. 150:702-709 (1982); Drake and Daniel, Res Microbiol 155:869-883 (2004); Kellum and Drake, J Bacteriol. 160:466-469 (1984)). M. thermoacetica has homologs to several hyp, hyc, and hyf genes from E. coli. These protein sequences encoded for by these genes are identified by the following GenBank accession numbers. In addition, several gene clusters encoding EM 16 functionality are present in M thermoacetica and C. ljungdahli (see for example US 2012/0003652).

Protein GenBank ID GI Number Organism

HypA NP 417206 16130633 Escherichia coli

HypB NP 417207 16130634 Escherichia coli

HypC NP 417208 16130635 Escherichia coli

HypD NP 417209 16130636 Escherichia coli

HypE NP 417210 226524740 Escherichia coli

HypF NP 417192 16130619 Escherichia coli

HycA NP 417205 16130632 Escherichia coli

HycB NP 417204 16130631 Escherichia coli

HycC NP 417203 16130630 Escherichia coli

HycD NP 417202 16130629 Escherichia coli HycE NP 417201 16130628 Escherichia coli

HycF NP 417200 16130627 Escherichia coli

HycG NP 417199 16130626 Escherichia coli

HycH NP 417198 16130625 Escherichia coli

Hycl NP 417197 16130624 Escherichia coli

HyfA NP 416976 90111444 Escherichia coli

HyfB NP 416977 16130407 Escherichia coli

HyfC NP 416978 90111445 Escherichia coli

HyfD NP 416979 16130409 Escherichia coli

Hyffi NP 416980 16130410 Escherichia coli

HyfF NP 416981 16130411 Escherichia coli

HyfG NP 416982 16130412 Escherichia coli

HyfH NP 416983 16130413 Escherichia coli

Hyfl NP 416984 16130414 Escherichia coli

HyfJ NP 416985 90111446 Escherichia coli

HyfR NP 416986 90111447 Escherichia coli

[0322] Proteins in M. thermoacetica whose genes are homologous to the E. coli EM 16 genes are shown below.

Moth 0810 YP 429671 83589662 Moorella thermoacetica

Moth 0811 YP 429672 83589663 Moorella thermoacetica

Moth 0812 YP 429673 83589664 Moorella thermoacetica

Moth 0814 YP 429674 83589665 Moorella thermoacetica

Moth 0815 YP 429675 83589666 Moorella thermoacetica

Moth 0816 YP 429676 83589667 Moorella thermoacetica

Moth 1193 YP 430050 83590041 Moorella thermoacetica

Moth 1194 YP 430051 83590042 Moorella thermoacetica

Moth 1195 YP 430052 83590043 Moorella thermoacetica

Moth 1196 YP 430053 83590044 Moorella thermoacetica

Moth 1717 YP 430562 83590553 Moorella thermoacetica

Moth 1718 YP 430563 83590554 Moorella thermoacetica

Moth 1719 YP 430564 83590555 Moorella thermoacetica

Moth 1883 YP 430726 83590717 Moorella thermoacetica

Moth 1884 YP 430727 83590718 Moorella thermoacetica

Moth 1885 YP 430728 83590719 Moorella thermoacetica

Moth 1886 YP 430729 83590720 Moorella thermoacetica

Moth 1887 YP 430730 83590721 Moorella thermoacetica

Moth 1888 YP 430731 83590722 Moorella thermoacetica

Moth 1452 YP 430305 83590296 Moorella thermoacetica

Moth 1453 YP 430306 83590297 Moorella thermoacetica

Moth 1454 YP 430307 83590298 Moorella thermoacetica

[0323] Genes encoding EM 16 enzymes from C ljungdahli are shown below.

[0324] In some cases, EM 16 encoding genes are located adjacent to a CODH. In Rhodospirillum rubrum, the encoded CODH/hydrogenase proteins form a membrane -bound enzyme complex that has been indicated to be a site where energy, in the form of a proton gradient, is generated from the conversion of CO and H ₂ 0 to C0 ₂ and H ₂ (Fox et al., J Bacteriol. 178:6200-6208 (1996)). The CODH-I of C. hydrogenoformans and its adjacent genes have been proposed to catalyze a similar functional role based on their similarity to the R. rubrum

CODH/hydrogenase gene cluster (Wu et al., PLoS Genet. 1 :e65 (2005)). The C.

hydrogenoformans CODH-I was also shown to exhibit intense CO oxidation and C0 ₂ reduction activities when linked to an electrode (Parkin et al., J Am.Chem.Soc. 129: 10328-10329 (2007)).

[0325] Some EM 16 and CODH enzymes transfer electrons to ferredoxins. Ferredoxins are small acidic proteins containing one or more iron-sulfur clusters that function as intracellular electron carriers with a low reduction potential. Reduced ferredoxins donate electrons to Fe- dependent enzymes such as ferredoxin-NADP ⁺ oxidoreductase, pyruvate :ferredoxin

oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (OFOR). The H.

thermophilus gene fdxl encodes a [4Fe-4S]-type ferredoxin that is required for the reversible carboxylation of 2-oxoglutarate and pyruvate by OFOR and PFOR, respectively (Yamamoto et al., Extremophiles 14:79-85 (2010)). The ferredoxin associated with the Sulfolobus solfataricus 2-oxoacid:ferredoxin reductase is a monomeric dicluster [3Fe-4S][4Fe-4S] type ferredoxin (Park et al. 2006). While the gene associated with this protein has not been fully sequenced, the N- terminal domain shares 93% homology with the zfx ferredoxin from S. acidocaldarius. The E. coli genome encodes a soluble ferredoxin of unknown physiological function, fdx. Some evidence indicates that this protein can function in iron-sulfur cluster assembly (Takahashi and Nakamura, 1999). Additional ferredoxin proteins have been characterized in Helicobacter pylori (Mukhopadhyay et al. 2003) and Campylobacter jejuni (van Vliet et al. 2001). A 2Fe-2S ferredoxin from Clostridium pasteurianum has been cloned and expressed in E. coli (Fujinaga and Meyer, Biochemical and Biophysical Research Communications, 192(3): (1993)).

Acetogenic bacteria such as Moorella thermoacetica, Clostridium carboxidivorans P7,

Clostridium ljungdahli and Rhodospirillum rubrum are predicted to encode several ferredoxins, listed below.

Fdx YP 002801146.1 226946073 Azotobacter vinelandii DJ

CKL 3790 YP 001397146.1 153956381 Clostridium kluyveri DSM 555 ferl NP 949965.1 39937689 Rhodopseudomonas palustris CGA009

Fdx CAA12251.1 3724172 Thauera aromatica

CHY 2405 YP 361202.1 78044690 Carboxydothermus hydrogenoformans

Fer YP 359966.1 78045103 Carboxydothermus hydrogenoformans

Fer AAC83945.1 1146198 Bacillus subtilis

fdxl NP 249053.1 15595559 Pseudomonas aeruginosa PA01 fhL AP 003148.1 89109368 Escherichia coli K-12

CLJU c00930 ADK13195.1 300433428 Clostridium ljungdahli

CLJU cOOOlO ADK13115.1 300433348 Clostridium ljungdahli

CLJU c01820 ADK13272.1 300433505 Clostridium ljungdahli

CLJU cl 7980 ADK14861.1 300435094 Clostridium ljungdahli

CLJU c 17970 ADK14860.1 300435093 Clostridium ljungdahli

CLJU c22510 ADK15311.1 300435544 Clostridium ljungdahli

CLJU c26680 ADK15726.1 300435959 Clostridium ljungdahli

CLJU c29400 ADK15988.1 300436221 Clostridium ljungdahli

[0326] Ferredoxin oxidoreductase enzymes transfer electrons from ferredoxins or flavodoxins to NAD(P)H. Two enzymes catalyzing the reversible transfer of electrons from reduced ferredoxins to NAD(P)+ are ferredoxin :NAD+ oxidoreductase (EC 1.18.1.3) and ferredoxin :NADP+ oxidoreductase (FNR, EC 1.18.1.2). Ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) has a noncovalently bound FAD cofactor that facilitates the reversible transfer of electrons from NADPH to low-potential acceptors such as ferredoxins or flavodoxins (Blaschkowski et al., Eur. J. Biochem. 123:563-569 (1982); Fujii et al., 1977). The

Helicobacter pylori FNR, encoded by HP 1164 (fqrB), is coupled to the activity of

pyruvate: ferredoxin oxidoreductase (PFOR) resulting in the pyruvate-dependent production of NADPH (St et al. 2007). An analogous enzyme is found in Campylobacter jejuni (St Maurice et al., J. Bacteriol. 189:4764-4773 (2007)). A ferredoxin :NADP+ oxidoreductase enzyme is encoded in the E. coli genome by fpr (Bianchi et al. 1993). Ferredoxin :NAD+ oxidoreductase utilizes reduced ferredoxin to generate NADH from NAD+. In several organisms, including E. coli, this enzyme is a component of multifunctional dioxygenase enzyme complexes. The ferredoxin :NAD+ oxidoreductase of E. coli, encoded by hcaD, is a component of the 3- phenylproppionate dioxygenase system involved in involved in aromatic acid utilization (Diaz et al. 1998). NADH: ferredoxin reductase activity was detected in cell extracts of

Hydrogenobacter thermophilus, although a gene with this activity has not yet been indicated (Yoon et al. 2006). Additional ferredoxin:NAD(P)+ oxidoreductases have been annotated in Clostridium carboxydivorans P7. The NADH-dependent reduced ferredoxin: NADP oxidoreductase of C. kluyveri, encoded by nfnAB, catalyzes the concomitant reduction of ferredoxin and NAD+ with two equivalents of NADPH (Wang et al, J Bacteriol 192: 5115-5123 (2010)). Finally, the energy-conserving membrane-associated Rnf-type proteins (Seedorf et al, PNAS 105:2128-2133 (2008); and Herrmann, J. Bacteriol 190:784-791 (2008)) provide a means to generate NADH or NADPH from reduced ferredoxin.

CLJU_c37220 (NfnAB) YP 003781850.1 300856866 Clostridium ljungdahlii

FIG. 1, Step I - Formate Dehydrogenase (EM8)

[0327] Formate dehydrogenase (FDH; EM8) catalyzes the reversible transfer of electrons from formate to an acceptor. Enzymes with FDH activity utilize various electron carriers such as, for example, NADH (EC 1.2.1.2), NADPH (EC 1.2.1.43), quinols (EC 1.1.5.6), cytochromes (EC 1.2.2.3) and EM16s (EC 1.1.99.33). FDH enzymes have been characterized from Moorella thermoacetica (Andreesen and Ljungdahl, J Bacteriol 116:867-873 (1973); Li et al., J Bacteriol 92:405-412 (1966); Yamamoto et al., JBiol Chem. 258: 1826-1832 (1983). The loci,

Moth_2312 is responsible for encoding the alpha subunit of EM8 while the beta subunit is encoded by Moth_2314 (Pierce et al., Environ Microbiol (2008)). Another set of genes encoding EM8 activity with a propensity for C0 ₂ reduction is encoded by Sfum_2703 through Sfum_2706 in Syntrophobacter fumaroxidans (de Bok et al., Eur J Biochem. 270:2476-2485 (2003)); Reda et al., PNAS 105: 10654-10658 (2008)). A similar set of genes presumed to carry out the same function are encoded by CHY 0731, CHY 0732, and CHY 0733 in C. hydrogenoformans (Wu et al., PLoS Genet 1 :e65 (2005)). EM8s are also found many additional organisms including C. carboxidivorans P7, Bacillus methanolicus, Burkholderia stabilis, Moorella thermoacetica ATCC 39073, Candida boidinii, Candida methylica, and Saccharomyces cerevisiae S288c. The soluble EM8 from Ralstonia eutropha reduces NAD ⁺ (fdsG, -B, -A, -C, -D) (Oh and Bowien, 1998).

[0328] Several EM8 enzymes have been identified that have higher specificity for NADP as the cofactor as compared to NAD. This enzyme has been deemed as the NADP-dependent formate dehydrogenase and has been reported from 5 species of the Burkholderia cepacia complex. It was tested and verified in multiple strains of Burkholderia multivorans, Burkholderia stabilis, Burkholderia pyrrocinia, and Burkholderia cenocepacia (Hatrongjit et ah, Enzyme and Microbial Tech., 46: 557-561 (2010)). The enzyme from Burkholderia stabilis has been characterized and the apparent K _m of the enzyme were reported to be 55.5 mM, 0.16 mM and 1.43 mM for formate, NADP, and NAD respectively. More gene candidates can be identified using sequence homology of proteins deposited in Public databases such as NCBI, JGI and the metagenomic databases. Sfum_2703 YP 846816.1 116750129 Syntrophobacter fumaroxidans

Sfum 2704 YP 846817.1 116750130 Syntrophobacter fumaroxidans

Sfum 2705 YP 846818.1 116750131 Syntrophobacter fumaroxidans

Sfum 2706 YP 846819.1 116750132 Syntrophobacter fumaroxidans

CHY 0731 YP 359585.1 78044572 Carboxydothermus hydrogenoformans

CHY 0732 YP 359586.1 78044500 Carboxydothermus hydrogenoformans

CHY 0733 YP 359587.1 78044647 Carboxydothermus hydrogenoformans

CcarbDRAFT 0901 ZP 05390901.1 255523938 Clostridium carboxidivorans P7

CcarbDRAFT 4380 ZP 05394380.1 255527512 Clostridium carboxidivorans P7 fdhA, EIJ82879.1 387590560 Bacillus methanolicus MGA3

MGA3 06625

fdhA, PB1 11719 ZP 10131761.1 387929084 Bacillus methanolicus PB1

fdhD, EIJ82880.1 387590561 Bacillus methanolicus MGA3

MGA3 06630

fdhD, PB1 11724 ZP 10131762.1 387929085 Bacillus methanolicus PB1

fdh ACF35003.1 194220249 Burkholderia stabilis

fdh ACF35004.1 194220251 Burkholderia pyrrocinia

fdh ACF35002.1 194220247 Burkholderia cenocepacia

fdh ACF35001.1 194220245 Burkholderia multivorans

fdh ACF35000.1 194220243 Burkholderia cepacia

FDH1 AAC49766.1 2276465 Candida boidinii

fdh CAA57036.1 1181204 Candida methylica

FDH2 P0CF35.1 294956522 Saccharomyces cerevisiae S288c

FDH1 NP O 15033.1 6324964 Saccharomyces cerevisiae S288c fdsG YP_725156.1 113866667 Ralstonia eutropha

fdsB YP_725157.1 113866668 Ralstonia eutropha

fdsA YP_725158.1 113866669 Ralstonia eutropha

fdsC YP_725159.1 113866670 Ralstonia eutropha

fdsD YP 725160.1 113866671 Ralstonia eutropha

FIG. 1, Step J - Methanol Dehydrogenase (EM9)

[0329] NAD+ dependent EM9 enzymes (EC 1.1.1.244) catalyze the conversion of methanol and NAD+ to formaldehyde and NADH. An enzyme with this activity was first characterized in Bacillus methanolicus (Heggeset et al, Applied and Environmental Microbiology, 78(15):5170— 5181 (2012)). This enzyme is zinc and magnesium dependent, and activity of the enzyme is enhanced by the activating enzyme encoded by act (Kloosterman et al, J Biol Chem 277:34785- 92 (2002)). They act is a Nudix hydrolase. Several of these candidates have been identified and shown to have activity on methanol. Additional NAD(P)+ dependent enzymes can be identified by sequence homology. EM9 enzymes utilizing different electron acceptors are also known in the art. Examples include cytochrome dependent enzymes such as mxalF of the methylotroph Methylobacterium extorquens (Nunn et al, Nucl Acid Res 16:7722 (1988)). EM9 enzymes of methanotrophs such as Methylococcus capsulatis function in a complex with methane

monooxygenase (MMO) (Myronova et al., Biochem 45: 11905-14 (2006)). Methanol can also be oxidized to formaldehyde by alcohol oxidase enzymes such as methanol oxidase (EC 1.1.3.13) of Candida boidinii (Sakai et al., Gene 114: 67-73 (1992)).

Protein ( ,cn Bank ID GI Number Organism

mdh, MGA3J7392 EIJ77596.1 387585261 Bacillus methanolicus MGA3 mdh2, MGA3 07340 EIJ83020.1 387590701 Bacillus methanolicus MGA3 mdh3, MGA3 10725 EIJ80770.1 387588449 Bacillus methanolicus MGA3 act, MGA3 09170 EIJ83380.1 387591061 Bacillus methanolicus MGA3 mdh, PBl 17533 ZP 10132907.1 387930234 Bacillus methanolicus PB1 mdhl. PBl 14569 ZP 10132325.1 387929648 Bacillus methanolicus PB1 mdh2, PB1 12584 ZP 10131932.1 387929255 Bacillus methanolicus PB1 act, PB1 14394 ZP 10132290.1 387929613 Bacillus methanolicus PB1

BFZC1 05383 ZP 07048751.1 299535429 Lysinibacillus fusiformis

BFZC1 20163 ZP 07051637.1 299538354 Lysinibacillus fusiformis

Bsph_4187 YP 001699778.1 169829620 Lysinibacillus sphaericus

Bsph_1706 YP 001697432.1 169827274 Lysinibacillus sphaericus mdh2 YP 004681552.1 339322658 Cupriavidus necator N-l nudFl YP 004684845.1 339325152 Cupriavidus necator N-l

BthaA O 10200007655 ZP_05587334.1 Burkholderia thailandensis

257139072 E264

BTH 11076 YP 441629.1 Burkholderia thailandensis (MutT/NUDIX NTP E264

pyrophosphatase) 83721454

BalcAV_11743 ZP l 0819291.1 Bacillus alcalophilus ATCC

402299711 27647

BalcAV_05251 ZP l 0818002.1 Bacillus alcalophilus ATCC

402298299 27647

alcohol dehydrogenase YP 725376.1 113866887 Ralstonia eutropha HI 6

Vibrio harveyi A TCC BAA- alcohol dehydrogenase YP 001447544 156976638 1116

Photobacterium profundum

P3TCK 27679 ZP 01220157.1 90412151 3TCK

Clostridium perfringens alcohol dehydrogenase YP 694908 110799824 ATCC 13124 Protein ( ,cn Bank ID GI Number Organism

adhB NP 717107 24373064 Shewanella oneidensis MR-1

Pseudomonas syringae pv. alcohol dehydrogenase YP 237055 66047214 syringae B728a

Carboxydothermus alcohol dehydrogenase YP 359772 78043360 hydrogenoformans Z-2901 alcohol dehydrogenase YP 003990729 312112413 Geobacillus sp. Y4.1MC1

Paenibacillus peoriae KCTC

PpeoK3 010100018471 ZP 10241531.1 390456003 3763

OBE 12016 EKC54576 406526935 human gut metagenome

Sebaldella termitidis ATCC alcohol dehydrogenase YP 003310546 269122369 33386

Actinobacillus succinogenes alcohol dehydrogenase YP 001343716 152978087 130Z

Clostridium pasteurianum dhaT AAC45651 2393887 DSM 525

Clostridium perfringens str. alcohol dehydrogenase NP 561852 18309918 13

Bacillus azotoformans LMG

BAZO 10081 ZP 11313277.1 410459529 9581

Methanosarcina mazei alcohol dehydrogenase YP 007491369 452211255 TucOl

alcohol dehydrogenase YP 004860127 347752562 Bacillus coagulans 36D1 alcohol dehydrogenase YP 002138168 197117741 Geobacter bemidjiensis Bern

Desulfitobacterium

DesmeDRAFT 1354 ZP 08977641.1 354558386 metallireducens DSM 15288

Klebsiella pneumoniae subsp. pneumoniae MGH alcohol dehydrogenase YP 001337153 152972007 78578

Desulfotomaculum reducens alcohol dehydrogenase YP 001113612 134300116 MI-1

Thermoanaerobacter sp. alcohol dehydrogenase YP 001663549 167040564 X514

Acinetobacter baumannii

ACINNAV82 2382 ZP 16224338.1 421788018 Naval-82

Desulfovibrio vulgaris str.

DVU2405 YP 011618 46580810 Hildenborough

Desulfovibrio africanus str. alcohol dehydrogenase YP 005052855 374301216 Walvis Bay

Desulfovibrio vulgaris str. alcohol dehydrogenase YP 002434746 218885425 Miyazaki F'

alcohol dehydrogenase AGF87161 451936849 uncultured organism

Desulfovibrio fructosovorans

DesfrDRAFT 3929 ZP 07335453.1 303249216 JJ Protein ( ,cn Bank ID GI Number Organism

Methanosarcina acetivorans alcohol dehydrogenase NP 617528 20091453 C2A

alcohol dehydrogenase NP 343875.1 15899270 Sulfolobus solfataricus P-2

Nitrososphaera gargensis

YP 006863258

adh4 408405275 Ga9.2

Nitrosopumilus salaria

ZP 10117398.1

BD31 10957 386875211 BD31

Rhodopseudomonas palustris

YP 004108045.1

alcohol dehydrogenase 316933063 DX-1

Ta0841 NP 394301.1 16081897 Thermoplasma acidophilum

Picrophilus torridus

YP 023929.1

PT01151 48478223 DSM9790

alcohol dehydrogenase ZP 10129817.1 387927138 Bacillus methanolicus PB-1

Corymb acterium

cgR 2695 YP 001139613.1 145296792 glutamicum R

Corymb acterium variabile alcohol dehydrogenase YP 004758576.1 340793113

HMPREF1015 01790 ZP 09352758.1 365156443 Bacillus smithii

ADH1 NP 014555.1 6324486 Saccharomyces cerevisiae

NADH-dependent

Geobacillus

butanol dehydrogenase YP 001126968.1 138896515

themodenitrificans NG80-2 A

alcohol dehydrogenase WP 007139094.1 494231392 Flavobacterium frigoris methanol

WP 003897664.1 489994607 Mycobacterium smegmatis dehydrogenase

ADH1B NP 000659.2 34577061 Homo sapiens

PMI01 01199 ZP 10750164.1 399072070 Caulobacter sp. AP07

Burkholderiales bacterium

BurJl DRAFT 3901 ZP 09753449.1 375107188 Joshi 001

YiaY YP 026233.1 49176377 Escherichia coli

MCA0299 YP 112833.1 53802410 Methylococcus capsulatis

MCA0782 YP 113284.1 53804880 Methylococcus capsulatis mxal YP 002965443.1 Methylob acterium

240140963 extorquens

mxaF YP 002965446.1 Methylob acterium

240140966 extorquens

AOD1 AAA34321.1 170820 Candida boidinii

[0330] An in vivo assay was developed to determine the activity of methanol

dehydrogenases. This assay relies on the detection of formaldehyde (HCHO), thus measuring the forward activity of the enzyme (oxidation of methanol). To this end, a strain comprising a

BDOP and lacking frmA, frmB, frmR was created using Lamba Red recombinase technology

(Datsenko and Wanner, Proc. Natl. Acad. Sci. USA, 6 97(12): 6640-5 (2000). Plasmids expressing methanol dehydrogenases were transformed into the strain, then grown to saturation in LB medium + antibiotic at 37° C with shaking. Transformation of the strain with an empty vector served as a negative control. Cultures were adjusted by O.D. and then diluted 1 : 10 into M9 medium + 0.5% glucose + antibiotic and cultured at 37° C with shaking for 6-8 hours until late log phase. Methanol was added to 2% v/v and the cultures were further incubated for 30 min. with shaking at 37° C. Cultures were spun down and the supernatant was assayed for formaldehyde produced using DETECTX Formaldehyde Detection kit (Arbor Assays; Ann Arbor, MI) according to manufacturer's instructions. The frmA, frmB, frmR deletions resulted in the native formaldehyde utilization pathway to be deleted, which enables the formation of formaldehyde that can be used to detect methanol dehydrogenase activity in the NNOMO.

[0331] The activity of several enzymes was measured using the assay described above. The results of four independent experiments are provided in Table 1 below.

Table 1: Results of in vivo assays showing formaldehyde (HCHO) production by various

NNOMO comprising a plasmid expressing a methanol dehydrogenase.

Empty vector 0.11

FIG. 1, Step K - Spontaneous or Formaldehyde Activating Enzyme (EM10)

[0332] The conversion of formaldehyde and THF to methylenetetrahydrofolate can occur spontaneously. It is also possible that the rate of this reaction can be enhanced by an EM 10. A formaldehyde activating enzyme (Fae) has been identified in Methylobacterium extorquens AMI which catalyzes the condensation of formaldehyde and tetrahydromethanopterm to methylene tetrahydromethanopterm (Vorholt, et al., J. BacterioL, 182(23), 6645-6650 (2000)). It is possible that a similar enzyme exists or can be engineered to catalyze the condensation of formaldehyde and tetrahydrofolate to methylenetetrahydrofolate. Homologs exist in several organisms including Xanthobacter autotrophicus Py2 and Hyphomicrobium denitrificans ATCC 51888.

FIG. 1, Step L - Formaldehyde Dehydrogenase (EMU)

[0333] Oxidation of formaldehyde to formate is catalyzed by EMI 1. An NAD+ dependent EMI 1 enzyme is encoded by fidhA of Pseudomonas putida (Ito et al, J Bacteriol 176: 2483-2491 (1994)). Additional EMI 1 enzymes include the NAD+ and glutathione independent EMI 1 from Hyphomicrobium zavarzinii (Jerome et al, Appl Microbiol Biotechnol 77:779-88 (2007)), the glutathione dependent EMI 1 of Pichia pastoris (Sunga et al, Gene 330:39-47 (2004)) and the NAD(P)+ dependent EMI 1 of Methylobacter marinus (Speer et al, FEMS Microbiol Lett, 121(3):349-55 (1994)). Protein ( ,cn Bank ID GI Number Organism

fdhA P46154.3 1169603 Pseudomonas putida fiaoA CAC85637.1 19912992 Hyphomicrobium zavarzinii

Fldl CCA39112.1 328352714 Pichia pastoris

fidh P47734.2 221222447 Methylobacter marinus

[0334] In addition to the EMI 1 enzymes listed above, alternate enzymes and pathways for converting formaldehyde to formate are known in the art. For example, many organisms employ glutathione-dependent formaldehyde oxidation pathways, in which formaldehyde is converted to formate in three steps via the intermediates S-hydroxymethylglutathione and S- formylglutathione (Vorholt et al, J Bacteriol 182:6645-50 (2000)). The enzymes of this pathway are EM12 (EC 4.4.1.22), glutathione-dependent formaldehyde dehydrogenase (EC 1.1.1.284) and S -formylglutathione hydrolase (EC 3.1.2.12).

FIG. 1, Step M - Spontaneous or S-(hydroxymethyl)glutathione Synthase (EM12)

[0335] While conversion of formaldehyde to S-hydroxymethylglutathione can occur spontaneously in the presence of glutathione, it has been shown by Goenrich et al (Goenrich, et al., J Biol Chem 277(5);3069-72 (2002)) that an enzyme from Paracoccus denitrificans can accelerate this spontaneous condensation reaction. The enzyme catalyzing the conversion of formaldehyde and glutathione was purified and named glutathione-dependent formaldehyde- activating enzyme (Gfa). The gene encoding it, which was named gfa, is located directly upstream of the gene for glutathione-dependent formaldehyde dehydrogenase, which catalyzes the subsequent oxidation of S-hydroxymethylglutathione. Putative proteins with sequence identity to Gfa from P. denitrificans are present also in Rhodobacter sphaeroides, Sinorhizobium meliloti, and Mesorhizobium loti.

FIG. 1, Step N - Glutathione-Dependent Formaldehyde Dehydrogenase (EM13)

[0336] Glutathione-dependent formaldehyde dehydrogenase (GS-FDH) belongs to the family of class III alcohol dehydrogenases. Glutathione and formaldehyde combine non-enzymatically to form hydroxymethylglutathione, the true substrate of the GS-FDH catalyzed reaction. The product, S-formylglutathione, is further metabolized to formic acid.

FIG. 1, Step O - S-Formylglutathione Hydrolase (EM14)

[0337] EM14 is a glutathione thiol esterase found in bacteria, plants and animals. It catalyzes conversion of S-formylglutathione to formate and glutathione. T efghA gene of P. denitrificans is located in the same operon with gfa and flhA, two genes involved in the oxidation of formaldehyde to formate in this organism. In E. coli, FrmB is encoded in an operon with FrmR and FrmA, which are proteins involved in the oxidation of formaldehyde. YeiG of E. coli is a promiscuous serine hydrolase; its highest specific activity is with the substrate S- formylglutathione.

4.2 Example II - Enhanced Yield of 3-HIB or MAA from Carbohydrates using Methanol

[0338] Exemplary MMPs for enhancing the availability of reducing equivalents are provided in FIG.1.

[0339] 3-HIB and/or MAA production can be achieved in a recombinant organism by the pathway shown in FIG. 2. For example, MAA and/or 3-HIB can be produced from succinate via a methylmalonyl-CoA intermediate as shown in FIG. 2. Exemplary enzymes for the conversion of succinate to MAA or 3-hydroxyisobutyric acid by this route include EMA1 A, EMA1B, or EMA1C; EMA2; EMA3; EMA4; EMA5; EMA6; and EMA7.

[0340] In this pathway, central metabolic intermediates are first channeled into succinate. For formation of succinate, phosphoenolpyruvate (PEP) is converted into oxaloacetate either via PEP carboxykinase or PEP carboxylase. Alternatively, PEP is converted first to pyruvate by pyruvate kinase and then to oxaloacetate by methylmalonyl-CoA carboxytransferase or pyruvate carboxylase. Oxaloacetate is then converted to succinate by means of the reductive TCA cycle.

[0341] Succinate is then activated to succinyl-CoA by an EMA1 A or synthetase. EMA2 then forms methylmalonyl-CoA from succinyl-CoA. Methylmalonyl-CoA is then reduced to methylmalonate semialdehyde. Further reduction of methylmalonate semialdehyde yields 3- hydroxyisobutyric acid, which can be secreted as a product or further transformed to MAA via dehydration.

[0342] Further enzymes that can be used in conjunction with the 3-HIBP and MAAPs provided in FIG. 2 are provided in FIG. 5 (e.g., FIG. 5, steps T-Y).

[0343] Exemplary enzyme candidates for the transformations shown in FIGS. 2 and 5 are described below.

FIG. 2, Step A - Succinyl-CoA transferase (EMA1A), ligase (EMA1B), or synthetase (EMA1C)

[0344] The ATP-dependent acylation of succinate to succinyl-CoA is catalyzed by EMA1C (EC 6.2.1.5). The product of the LSC1 and LSC2 genes of S. cerevisiae and the sucC and sucD genes of E. coli naturally form an EMA1C complex that catalyzes the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP, a reaction which is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)). These proteins are identified below:

Protein ( ,cn Bank ID GI Number Organism sucD AAC73823.1 1786949 Escherichia coli

[0345] EMA1 A catalyzes the conversion of succinyl-CoA to succinate while transferring the CoA moiety to a CoA acceptor molecule. Many transferases have broad specificity and can utilize CoA acceptors as diverse as acetate, succinate, propionate, butyrate, 2- methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate, valerate, crotonate, 3- mercaptopropionate, propionate, vinylacetate, and butyrate, among others.

[0346] The conversion of succinate to succinyl-CoA can be carried by a transferase which does not require the direct consumption of an ATP or GTP. This type of reaction is common in a number of organisms. The conversion of succinate to succinyl-CoA can also be catalyzed by succinyl-CoA:Acetyl-CoA transferase. The gene product of catl of Clostridium kluyveri has been shown to exhibit succinyl-CoA: acetyl-CoA transferase activity (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)). In addition, the activity is present in Trichomonas vaginalis (van Grinsven et al., J Biol Chem. 283: 1411-1418 (2008)) and Trypanosoma brucei (Riviere et al, J. Biol. Chem. 279(44):45337-45346 (2004)). The succinyl-Co A: acetate CoA-transferase from Acetobacter aceti, encoded by aarC, replaces EMAIC in a variant TCA cycle (Mullins et al, J. Bacteriol. 190(14):4933-4940 (2008)). Similar EMA1A activities are also present in Trichomonas vaginalis (van Grinsven et al, supra, 2008), Trypanosoma brucei (Riviere et al, supra, 2004) and Clostridium kluyveri (Sohling and Gottschalk, supra, 1996). The beta- ketoadipate:EMAl A encoded by peal and pcaJ in Pseudomonas putida is yet another candidate (Kaschabek et al, J. Bacteriol. 184(1):207-215 (2002)). The aforementioned proteins are identified below.

[0347] An additional exemplary transferase that converts succinate to succinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid is succinyl-CoA:3:ketoacid-CoA transferase (EC 2.8.3.5). Exemplary succinyl-CoA:3:ketoacid-CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al., J. Biol. Chem. 272(41):25659-25667 (1997)), Bacillus subtilis, and Homo sapiens (Fukao et al., Genomics 68(2): 144-151 (2000); Tanaka et al., Mol. Hum. Reprod. 8(1): 16-23 (2002)). The aforementioned proteins are identified below.

[0348] Converting succinate to succinyl-CoA by succinyl-CoA:3 :ketoacid-CoA transferase requires the simultaneous conversion of a 3-ketoacyl-CoA such as acetoacetyl-CoA to a 3- ketoacid such as acetoacetate. Conversion of a 3-ketoacid back to a 3-ketoacyl-CoA can be catalyzed by an acetoacetyl-CoA:acetate:CoA transferase. Acetoacetyl-CoA: acetate: Co A transferase converts acetoacetyl-CoA and acetate to acetoacetate and acetyl-CoA, or vice versa. Exemplary enzymes include the gene products of atoAD from E. coli (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007), ctfAB from C. acetobutylicum (Jojima et al., Appl Microbiol Biotechnol 77: 1219-1224 (2008), and ctfAB from Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem. 71 :58-68 (2007)) are shown below.

[0349] Yet another possible CoA acceptor is benzylsuccinate. Succinyl-CoA:(R)- Benzylsuccinate CoA-Transferase functions as part of an anaerobic degradation pathway for toluene in organisms such as Thauera aromatica (Leutwein and Heider, J. Bact. 183(14) 4288- 4295 (2001)). Homologs can be found m Azoarcus sp. T, Aromatoleum aromaticum EbNl, and Geobacter metallireducens GS-15. The aforementioned proteins are identified below.

[0350] Additionally, ygfH encodes a propionyl CoA:succinate CoA transferase in E. coli (Haller et al., Biochemistry, 39(16) 4622-4629). Close homologs can be found in, for example, Citrobacter youngae ATCC 29220, Salmonella enterica subsp. arizonae serovar, and Yersinia intermedia ATCC 29909. The aforementioned proteins are identified below.

FIG. 2, Step B - Methylmalonyl-CoA mutase (EMA2)

[0351] Methylmalonyl-CoA mutase (MCM) (EMA2) (EC 5.4.99.2) is a cobalamin- dependent enzyme that converts succinyl-CoA to methylmalonyl-CoA. In E. coli, the reversible adenosylcobalamin-dependant mutase participates in a three-step pathway leading to the conversion of succinate to propionate (Haller et al., Biochemistry 39:4622-4629 (2000)).

Overexpression of the EMA2 gene candidate along with the deletion of YgfG can be used to prevent the decarboxylation of methylmalonyl-CoA to propionyl-CoA and to maximize the methylmalonyl-CoA available for MAA synthesis. EMA2 is encoded by genes scpA in Escherichia coli (Bobik and Rasche, Anal. Bioanal. Chem. 375:344-349 (2003); Haller et al., Biochemistry 39:4622-4629 (2000)) and mutA in Homo sapiens (Padovani and Banerjee, Biochemistry 45:9300-9306 (2006)). In several other organisms EMA2 contains alpha and beta subunits and is encoded by two genes. Exemplary gene candidates encoding the two-subunit protein are Propionibacterium fredenreichii sp. shermani mutA and mutB (Korotkova and Lidstrom, J. Biol. Chem. 279: 13652-13658 (2004)), Methylobacterium extorquens mcmA and mcmB (Korotkova and Lidstrom, supra, 2004), and Ralstonia eutropha sbml and sbm2

(Peplinski et al., Appl. Microbiol. Biotech. 88: 1145-59 (2010)). Additional enzyme candidates identified based on high homology to the E. coli spcA gene product are also listed below.

[0352] These sequences can be used to identify homologue proteins in GenBank or other databases through sequence similarity searches (for example, BLASTp). The resulting homologue proteins and their corresponding gene sequences provide additional exogenous DNA sequences for transformation into E. coli or other suitable host microorganisms to generate production hosts. Additional gene candidates include the following, which were identified based on high homology to the E. coli spcA gene product.

[0353] There further exists evidence that genes adjacent to the EMA2 catalytic genes contribute to maximum activity. For example, it has been demonstrated that the meaB gene from M. extorquens forms a complex with EMA2, stimulates iin vitro mutase activity, and possibly protects it from irreversible inactivation (Korotkova and Lidstrom, J. Biol. Chem. 279: 13652- 13658 (2004)). The M. extorquens meaB gene product is highly similar to the product of the E. coli argK gene (BLASTp: 45% identity, e-value: 4e-67), which is adjacent to scpA on the chromosome. No sequence for a meaB homolog in P. freudenreichii is catalogued in GenBank. However, the Propionibacterium acnes KPA171202 gene product, YP_055310.1, is 51% identical to the M. extorquens meaB protein and its gene is also adjacent to the EMA2 gene on the chromosome. A similar gene is encoded by H16 B1839 of Ralstonia eutropha H16.

[0354] E. coli can synthesize adenosylcobalamin, a necessary cofactor for this reaction, only when supplied with the intermediates cobinamide or cobalamin (Lawrence and Roth. J.

Bacteriol. 177:6371-6380 (1995); Lawrence and Roth, Genetics 142: 11-24 (1996)).

Alternatively, the ability to synthesize cobalamins de novo has been conferred upon E. coli following the expression of heterologous genes (Raux et al., J. Bacteriol. 178:753-767 (1996)).

[0355] Alternatively, isobutyryl-CoA mutase (ICM) (EC 5.4.99.13) could catalyze the proposed transformation shown in FIG., step B. ICM is a cobalamin-dependent methylmutase in the EMA2 family that reversibly rearranges the carbon backbone of butyryl-CoA into isobutyryl- CoA (RatnatiUeke et al., J. Biol. Chem. 274:31679-31685 (1999)). A recent study of a novel ICM in Methylibium petroleiphilum, along with previous work, provides evidence that changing a single amino acid near the active site alters the substrate specificity of the enzyme (RatnatiUeke et al, J. Biol. Chem. 274:31679-31685 (1999); Rohwerder et al, Appl. Environ. Microbiol. 72:4128-4135. (2006)). This indicates that, if a native enzyme is unable to catalyze or exhibits low activity for the conversion of 4HB-CoA to 3HIB-CoA, the enzyme can be rationally engineered to increase this activity. Exemplary ICM genes encoding homodimeric enzymes include icmA in Streptomyces coelicolor A3 (Alhapel et al., Proc. Natl. Acad. Sci. USA

103: 12341-12346 (2006)) and Mpe_B0541 in Methylibium petroleiphilum PM1 (RatnatiUeke et al, J. Biol. Chem. 274:31679-31685 (1999); Rohwerder et al, Appl. Environ. Microbiol.

72:4128-4135 (2006)). Genes encoding heterodimeric enzymes include icm and icmB in

Streptomyces cinnamonensis (Ratnatilleke et al, J. Biol. Chem. 274:31679-31685 (1999);

Vrijbloed et al, J. Bacteriol. 181 :5600-5605. (1999); Zerbe-Burkhardt et al, J. Biol. Chem. 273:6508-6517 (1998)). Enzymes encoded by icmA and icmB genes in Streptomyces avermitilis MA-4680 show high sequence similarity to known ICMs. These genes/proteins are identified below.

FIG. 2, Step C - Methylmalonyl-CoA epimerase (EMA3)

[0356] Methylmalonyl-CoA epimerase (MMCE) (EMA3) is the enzyme that interconverts (R)-methylmalonyl-CoA and (S)-methylmalonyl-CoA. EMA3 is an essential enzyme in the breakdown of odd-numbered fatty acids and of the amino acids valine, isoleucine, and methionine. EMA3 activity is not believed to be encoded in the E. coli genome (Boynton et al, J. Bacteriol.178:3015-3024 (1996)), but is present in other organisms such as Homo sapiens {YqjC) (Fuller and Leadlay,. Biochem. J. 213:643-650 (1983)), Rattus norvegicus (Mcee) (Bobik and Rasche, J. Biol. Chem. 276:37194-37198 (2001)), Propionibacterium shermanii (AF454511) (Fuller, and Leadlay, Biochem. J. 213:643-650 (1983); Haller et al, Biochemistry 39:4622-4629 (2000); McCarthy et al.,. Structure 9:637-646.2001)) and Caenorhabditis elegans (mmce) (Kuhnl et al, FEBSJ. 272: 1465-1477 (2005)). An additional gene candidate, AE016877 in Bacillus cereus, has high sequence homology to other characterized enzymes. This enzymatic step may or may not be necessary depending upon the stereospecificity of the enzyme or enzymes used for the conversion of methylmalonyl-CoA to 3-HIB (steps 3-4 in FIG. 3). These genes/proteins are described below. Gene ( ,cn Bank ID GI Number Organism

YqjC NP_390273 255767522 Bacillus subtilis

MCEE Q96PE7.1 50401130 Homo sapiens

Mcee _predicted NP OO 1099811.1 157821869 Rattus norvegicus

AF454511 AAL57846.1 18042135 Propionibacterium fredenreichii sp.

shermanii

mmce AAT92095.1 51011368 Caenorhabditis elegans

AEO 16877 AAP08811.1 29895524 Bacillus cereus ATCC 14579

FIG. 2, Step D - Methylmalonyl-CoA reductase (aldehyde forming) (EMA4)

[0357] The reduction of methylmalonyl-CoA to its corresponding aldehyde, methylmalonate semialdehyde, is catalyzed by a CoA-dependent aldehyde dehydrogenase (EC 1.2.1.-).

Conversion of methylmalonyl-CoA to methylmalonic semialdehyde, is accomplished by a CoA- dependent aldehyde dehydrogenase. An enzyme encoded by a malonyl-CoA reductase gene from Sulfolobus tokodaii (Alber et. al, J. Bacteriol. 188(24):8551-8559 (2006)), has been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde

(WO2007141208). A similar enzyme exists in Metallosphaera sedula (Alber et. al, J. Bacteriol. 188(24):8551-8559 (2006)). Several additional CoA dehydrogenases are capable also of reducing an acyl-CoA to its corresponding aldehyde. The reduction of methylmalonyl-CoA to its corresponding aldehyde, methylmalonate semialdehyde, is catalyzed by a CoA-dependent aldehyde dehydrogenase. Exemplary enzymes include fatty acyl-CoA reductase, succinyl-CoA reductase (EC 1.2.1.76), acetyl-CoA reductase and butyryl-CoA reductase. Exemplary fatty acyl-CoA reductase enzymes are encoded by acrl of Acinetobacter calcoaceticus (Reiser and Somerville. J. Bacteriol. 179:2969-2975 (1997)), and Acinetobacter sp. M-l fatty acyl-CoA reductase (Ishige et al., Appl. Environ. Microbiol. 68: 1192-1195 (2002)). Also known is a CoA- and NADP- dependent succinate semialdehyde dehydrogenase (also referred to as succinyl-CoA reductase) encoded by the sucD gene in Clostridium kluyverx (Sohling and Gottschalk, J.

Bacteriol. 178:871-880 (1996); Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)) and sucD of P. gingivalis (Takahashi, J. Bacteriol 182:4704-4710 (2000)). Additional succinyl-CoA reductase enzymes participate in the 3-hydroxypropionate/4-hydroxybutyrate cycle of thermophilic archaea including Metallosphaera sedula (Berg et al., Science 318: 1782-1786 (2007)) and Thermoproteus neutrophilus (Ramos-Vera et al., J Bacteriol, 191 :4286-4297 (2009)). The M. sedula enzyme, encoded by Msed_0709, is strictly NADPH-dependent and also has malonyl-CoA reductase activity. The T. neutrophilus enzyme is active with both NADPH and NADH. The enzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is also a good candidate as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, formaldehyde and the branched-chain compound

isobutyraldehyde (Powlowski et al., J. Bacteriol. 175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mes enter oides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya, J. Gen. Appl. Microbiol. 18:43-55 (1972); and Koo et al, Biotechnol Lett. 27:505-510 (2005)). Butyraldehyde dehydrogenase catalyzes a similar reaction, conversion of butyryl-CoA to butyraldehyde, in solventogenic organisms such as Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci Biotechnol Biochem., 71 :58-68 (2007)).

malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archaeal bacteria (Berg, Science 318: 1782-1786 (2007); and Thauer, Science 318: 1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus sp. (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Hugler, J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed_0709 in

Metallosphaera sedula (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Berg, Science 318: 1782-1786 (2007)). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli (Alber et al., J. Bacteriol 188:8551-8559 (2006). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WO2007141208 (2007)). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate

semialdehyde. Additional gene candidates can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius and have been listed below. Yet another candidate for CoA-acylating aldehyde dehydrogenase is the aid gene from Clostridium beijerinckii (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE that encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999).

[0359] A bifunctional enzyme with acyl-CoA reductase and alcohol dehydrogenase activity can directly convert methylmalonyl-CoA to 3-HIB. Exemplary bifunctional oxidoreductases that convert an acyl-CoA to alcohol include those that transform substrates such as acetyl-CoA to ethanol (for example, adhE from E. coli (Kessler et al., FEBS.Lett. 281 :59-63 (1991))) and butyryl-CoA to butanol (for example, adhE 2 from C. acetobutylicum (Fontaine et al.,

J. Bacteriol. 184:821-830 (2002)). The C. acetobutylicum enzymes encoded by bdh I and bdh II (Walter, et al, J. Bacteriol. 174:7149-7158 (1992)), reduce acetyl-CoA and butyryl-CoA to ethanol and butanol, respectively. In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxide the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al, J.Gen.Appl.Microbiol. 18:43- 55 (1972); Koo et al., Biotechnol Lett, 27:505-510 (2005)). Another exemplary enzyme can convert malonyl-CoA to 3 -HP. An NADPH-dependent enzyme with this activity has

characterized in Chloroflexus aurantiacus where it participates in the 3-hydroxypropionate cycle (Hugler et al, J Bacteriol, 184:2404-2410 (2002); Strauss et al, Eur J Biochem, 215:633-643 (1993)). This enzyme, with a mass of 300 kDa, is highly substrate-specific and shows little sequence similarity to other known oxidoreductases (Hugler et al, supra). No enzymes in other organisms have been shown to catalyze this specific reaction; however there is bioinformatic evidence that other organisms may have similar pathways (Klatt et al, Env Microbiol, 9:2067- 2078 (2007)). Enzyme candidates in other organisms including Roseiflexus castenholzii, Erythrobacter sp. NAP1 and marine gamma proteobacterium HTCC2080 can be inferred by sequence similarity.

FIG. 2, Step E - Methylmalonate semialdehyde reductase (EMA5)

[0360] The reduction of methylmalonate semialdehyde to 3-HIB is catalyzed by EMA5 or 3- HIB dehydrogenase. This enzyme participates in valine, leucine and isoleucine degradation and has been identified in bacteria, eukaryotes, and mammals. The enzyme encoded by P84067 from Thermus thermophilus HB8 has been structurally characterized (Lokanath et al, J. Mol. Biol. 352:905-917 (2005)). The reversibility of the human 3-HIB dehydrogenase was demonstrated using isotopically-labeled substrate (Manning and Pollitt, Biochem. J. 231 :481-484 (1985)). Additional genes encoding this enzyme include 3hidh in Homo sapiens (Hawes et al, Methods Enzymol. 324:218-228 (2000)) and Oryctolagus cuniculus (Chowdhury et al, Biosci. Biotechnol. Biochem. 60:2043-2047 (1996); Hawes et al., Methods Enzymol. 324:218-228 (2000)), mmsb in Pseudomonas aeruginosa, and dhat in Pseudomonas putida (Aberhart and Hsu J

Chem.Soc.fPerkin 1] 6: 1404-1406 (1979); Chowdhury et al., Biosci. Biotechnol. Biochem.

67:438-441 (2003); Chowdhury et al, Biosci. Biotechnol. Biochem. 60:2043-2047 (1996)). Several 3-HIB dehydrogenase enzymes have been characterized in the reductive direction, including mmsB from Pseudomonas aeruginosa (Gokarn et al., US Patent 7,393,676 (2008)) and mmsB from Pseudomonas putida.

FIG. 2, Step F - 3-HIB dehydratase (EMA6)

[0361] The dehydration of 3-HIB to MAA is catalyzed by an enzyme with EMA6 activity (EC 4.2.1.-). The final step involves the dehydration of 3-HIB to MAA The dehydration of 3- HIB to MAA is catalyzed by an enzyme with EMA6 activity. Although no direct evidence for this specific enzymatic transformation has been identified, most dehydratases catalyze the α,β- elimination of water, which involves activation of the a-hydrogen by an electron-withdrawing carbonyl, carboxylate, or CoA-thiol ester group and removal of the hydroxyl group from the β- position (Buckel and Barker, J Bacteriol. 117: 1248-1260 (1974); Martins et al, Proc. Natl. Acad. Sci. USA 101 : 15645-15649 (2004)). This is the exact type of transformation proposed for the final step in the methacrylate pathway. In addition, the proposed transformation is highly similar to the 2-(hydroxymethyl)glutarate dehydratase of Eubacterium barkeri, which can catalyze the conversion of 2-hydroxymethyl glutarate to 2-methylene glutarate. This enzyme has been studied in the context of nicotinate catabolism and is encoded by hmd (Alhapel et al., Proc. Natl. Acad. Sci. USA 103: 12341-12346 (2006)). Similar enzymes with high sequence homology are found in Bacteroides capillosus, Anaerotruncus colihominis, and Natranaerobius thermophilics. Several enzymes are known to catalyze the alpha, beta elimination of hydroxyl groups. Exemplary enzymes include 2-(hydroxymethyl)glutarate dehydratase (EC 4.2.1.-), fumarase (EC 4.2.1.2), 2-keto-4-pentenoate dehydratase (EC 4.2.1.80), citramalate hydrolyase and

dimethylmaleate hydratase.

[0362] 2-(Hydroxymethyl)glutarate dehydratase is a [4Fe-4S]-containing enzyme that dehydrates 2-(hydroxymethyl)glutarate to 2-methylene-glutarate, studied for its role in nicontinate catabolism in Eubacterium barkeri (formerly Clostridium barkeri) (Alhapel et al., Proc Natl Acad Sci USA 103: 12341-12346 (2006)). Similar enzymes with high sequence homology are found in Bacteroides capillosus, Anaerotruncus colihominis, and Natranaerobius thermophilius . These enzymes are also homologous to the a- and β-subunits of [4Fe-4S]- containing bacterial serine dehydratases, for example, E. coli enzymes encoded by tdcG, sdhB, and sdaA). An enzyme with similar functionality in E. barkeri is dimethylmaleate hydratase, a reversible Fe2+-dependent and oxygen-sensitive enzyme in the aconitase family that hydrates dimethylmaeate to form (2R,3S)-2,3-dimethylmalate. This enzyme is encoded by dmdAB (Alhapel et al, Proc Natl Acad Sci USA 103: 12341-6 (2006); Kollmann-Koch et al., Hoppe Seylers. Z.Physiol Chem. 365:847-857 (1984)).

[0363] Fumarate hydratase enzymes, which naturally catalyze the reversible hydration of fumarate to malate. Although the ability of fumarate hydratase to react on branched substrates with 3-oxobutanol as a substrate has not been described, a wealth of structural information is available for this enzyme and other researchers have successfully engineered the enzyme to alter activity, inhibition and localization (Weaver, Acta Crystallogr. D Biol. Crystallogr. 61 : 1395- 1401 (2005)). E. coli has three fumarases: FumA, FumB, and FumC that are regulated by growth conditions. FumB is oxygen sensitive and only active under anaerobic conditions.

FumA is active under microanaerobic conditions, and FumC is the only active enzyme in aerobic growth (Tseng et al., J. Bacteriol. 183:461-467 (2001); Woods et al., Biochem. Biophys. Acta 954: 14-26 (1988); Guest et al, J Gen Microbiol 131 :2971-2984 (1985)). Exemplary enzyme candidates include those encoded by fumC from Escherichia coli (Estevez et al., Protein Sci. 11 : 1552-1557 (2002); Hong and Lee, Biotechnol. Bioprocess Eng. 9:252-255 (2004); Rose and Weaver, Proc. Natl. Acad. Sci. USA 101 :3393-3397 (2004)), and enzymes found in

Campylobacter jejuni (Smith et al., Int. J. Biochem.Cell Biol. 31 :961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch. Biochem. Biophys. 355:49-55 (1998)), and Rattus norvegicus (Kobayashi et al., J. Biochem. 89: 1923-1931 (1981)). Similar enzymes with high sequence homology include fuml from Arabidopsis thaliana and fumC from Corynebacterium glutamicum. The MmcBC fumarase from Pelotomaculum thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., FEMS Microbiol Lett. 270:207-213 (2007)).

[0364] Dehydration of 4 -hydroxy-2-oxo valerate to 2-oxopentenoate is catalyzed by 4- hydroxy-2-oxovalerate hydratase (EC 4.2.1.80). This enzyme participates in aromatic degradation pathways and is typically co-transcribed with a gene encoding an enzyme with 4- hydroxy-2-oxovalerate aldolase activity. Exemplary gene products are encoded by mhpD of E. coli (Ferrandez et al., J Bacteriol. 179:2573-2581 (1997); Pollard et al., Eur J Biochem. 251 :98- 106 (1998)), todG and cmtF of Pseudomonas putida (Lau et al., Gene 146:7-13 (1994); Eaton, J Bacteriol. 178: 1351-1362 (1996)), cnbE of Comamonas sp. CNB-1 (Ma et al., Appl Environ Microbiol 73:4477-4483 (2007)) and mhpD of Burkholderia xenovorans (Wang et al., FEBSJ 272:966-974 (2005)). A closely related enzyme, 2-oxohepta-4-ene-l,7-dioate hydratase, participates in 4-hydroxyphenylacetic acid degradation, where it converts 2-oxo-hept-4-ene-l,7- dioate (OHED) to 2-oxo-4-hydroxy-hepta-l,7-dioate using magnesium as a cofactor (Burks et al., J.Am.Chem.Soc. 120: (1998)). OHED hydratase enzyme candidates have been identified and characterized in E. coli C (Roper et al., Gene 156:47-51 (1995); Izumi et al., J Mol.Biol.

370:899-911 (2007)) and E. coli ^(Prieto et al., J Bacteriol. 178: 111-120 (1996)). Sequence comparison reveals homologs in a wide range of bacteria, plants and animals. Enzymes with highly similar sequences are contained in Klebsiella pneumonia (91% identity, eval = 2e-138) and Salmonella enterica (91% identity, eval = 4e-138), among others.

[0365] Another enzyme candidate is citramalate hydrolyase (EC 4.2.1.34), an enzyme that naturally dehydrates 2-methylmalate to mesaconate. This enzyme has been studied in

Methanocaldococcus jannaschii in the context of the pyruvate pathway to 2-oxobutanoate, where it has been shown to have a broad substrate specificity (Drevland et al., J Bacteriol. 189:4391- 4400 (2007)). This enzyme activity was also detected in Clostridium tetanomorphum,

Morganella morganii, Citrobacter amalonaticus where it is thought to participate in glutamate degradation (Kato et al., Arch. Microbiol 168:457-463 (1997)). The M. jannaschii protein sequence does not bear significant homology to genes in these organisms.

2_ _| _

[0366] Dimethylmaleate hydratase (EC 4.2.1.85) is a reversible Fe -dependent and oxygen- sensitive enzyme in the aconitase family that hydrates dimethylmaeate to form (2R,3S)-2,3- dimethylmalate. This enzyme is encoded by dmdAB in Eubacterium barkeri (Alhapel et al., supra; Kollmann-Koch et al., Hoppe Seylers. Z.Physiol Chem. 365:847-857 (1984)).

FIG. 2, Step G - Methylmalonyl-CoA reductase (alcohol forming) (EMA7)

[0367] Referring to FIG. 2, step G can involve a combined Alcohol/ Aldehyde dehydrogenase (EC 1.2.1.-). Methylmalonyl-CoA can be reduced to 3-HIB in one step by a multifunctional enzyme with dual acyl-CoA reductase and alcohol dehydrogenase activity. Although the direct conversion of methylmalonyl-CoA to 3-HIB has not been reported, this reaction is similar to the common conversions such as acetyl-CoA to ethanol and butyryl-CoA to butanol, which are catalyzed by CoA-dependant enzymes with both alcohol and aldehyde dehydrogenase activities. Gene candidates include the E. coli adhE (Kessler et al., FEB S Lett. 281 :59-63 (1991)) and C. acetobutylicum bdh I and bdh II (Walter, et al., J. Bacteriol. 174:7149-7158 (1992)), which can reduce acetyl-CoA and butyryl-CoA to ethanol and butanol, respectively. In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mes enter oides has been shown to oxide the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al, J. Gen. Appl. Microbiol. 18:43-55 (1972); Koo et al., Biotechnol. Lett. 27:505-510 (2005)). An additional candidate enzyme for converting methylmalonyl-CoA directly to 3-HIB is encoded by a malonyl-CoA reductase from Chloroflexus aurantiacus (Hiigler, et al., J. Bacteriol. 184(9):2404-2410 (2002).

FIG. 5, Step T - PEP Carboxylase (EFR16A) or PEP Carboxykinase (EFR16B)

[0368] Carboxylation of phosphoenolpyruvate to oxaloacetate is catalyzed by

phosphoenolpyruvate carboxylase. Exemplary PEP carboxylase enzymes are encoded by ppc in E. coli (Kai et al., Arch. Biochem. Biophys. 414: 170-179 (2003), ppcA in Methylobacterium extorquens AMI (Arps et al., J. Bacteriol. 175:3776-3783 (1993), and ppc in Corynebacterium glutamicum (Eikmanns et αΙ., ΜοΙ. Gen. Genet. 218:330-339 (1989).

[0369] An alternative enzyme for converting phosphoenolpyruvate to oxaloacetate is PEP carboxykinase, which simultaneously forms an ATP while carboxylating PEP. In most organisms PEP carboxykinase serves a gluconeogenic function and converts oxaloacetate to PEP at the expense of one ATP. S. cerevisiae is one such organism whose native PEP carboxykinase, PCKI, serves a gluconeogenic role (Valdes-Hevia et al., FEBS Lett. 258:313-316 (1989). E. coli is another such organism, as the role of PEP carboxykinase in producing oxaloacetate is believed to be minor when compared to PEP carboxylase, which does not form ATP, possibly due to the higher K _m for bicarbonate of PEP carboxykinase (Kim et al., Appl. Environ. Microbiol. 70: 1238- 1241 (2004)). Nevertheless, activity of the native E. coli PEP carboxykinase from PEP towards oxaloacetate has been recently demonstrated in ppc mutants of E. coli K-12 (Kwon et al., J. Microbiol. Biotechnol. 16: 1448-1452 (2006)). These strains exhibited no growth defects and had increased succinate production at high NaHC0 ₃ concentrations. Mutant strains of E. coli can adopt Pck as the dominant C02-fixing enzyme following adaptive evolution (Zhang et al.

2009). In some organisms, particularly rumen bacteria, PEP carboxykinase is quite efficient in producing oxaloacetate from PEP and generating ATP. Examples of PEP carboxykinase genes that have been cloned into E. coli include those from Mannheimia succiniciproducens (Lee et al., Biotechnol. Bioprocess Eng. 7:95-99 (2002)), Anaerobiospirillum succiniciproducens

(Laivenieks et αΙ., ΑρρΙ. Environ. Microbiol. 63:2273-2280 (1997), and Actinobacillus

- Ill - succinogenes (Kim et al. supra). The PEP carboxykinase enzyme encoded by Haemophilus influenza is effective at forming oxaloacetate from PEP.

FIG. 5, Step U - Pyruvate Carboxylase (EFR17)

[0370] Pyruvate carboxylase (EC 6.4.1.1) directly converts pyruvate to oxaloacetate at the cost of one ATP. Pyruvate carboxylase enzymes are encoded by PYCl (Walker et al., Biochem. Biophys. Res. Commun. 176: 1210-1217 (1991) and PYC2 (Walker et al., supra) in

Saccharomyces cerevisiae, and pyc in Mycobacterium smegmatis (Mukhopadhyay and

Purwantini, Biochim. Biophys. Acta 1475: 191-206 (2000)).

FIG. 5, Step V - Malate Dehydrogenase (EFR18)

[0371] Malate dehydrogenase converts oxaloacetate to malate. Exemplary enzymes are found in several organisms including E. coli, S. cerevisiae, Bacillus subtilis, and Rhizopus oryzae. MDHl, MDH2, and MDH3 from S. cerevisiae are known to localize to the

mitochondrion, cytosol, and peroxisome, respectively. Protein ( ,cn Bank ID GI number Organism

mdh AAC76268.1 1789632 Escherichia coli

MDH1 NP_012838.1 6322765 Saccharomyces cerevisiae

MDH2 NP_014515.2 116006499 Saccharomyces cerevisiae

MDH3 NP O 10205.1 6320125 Saccharomyces cerevisiae

mdh NP 390790.1 16079964 Bacillus subtilis

MDH ADG65261.1 296011196 Rhizopus oryzae

FIG. 5, Step W - Malic enzyme (EFR19)

[0372] Malic enzyme can be applied to convert C0 ₂ and pyruvate to malate at the expense of one reducing equivalent. Malic enzymes for this purpose can include, without limitation, malic enzyme (NAD-dependent) and malic enzyme (NADP-dependent). For example, one of the E. coli malic enzymes (Takeo, J. Biochem. 66:379-387 (1969)) or a similar enzyme with higher activity can be expressed to enable the conversion of pyruvate and C0 ₂ to malate. By fixing carbon to pyruvate as opposed to PEP, malic enzyme allows the high-energy phosphate bond from PEP to be conserved by pyruvate kinase whereby ATP is generated in the formation of pyruvate or by the phosphotransferase system for glucose transport. Although malic enzyme is typically assumed to operate in the direction of pyruvate formation from malate, overexpression of the NAD-dependent enzyme, encoded by maeA, has been demonstrated to increase succinate production in E. coli while restoring the lethal Apfl-AldfiA phenotype under anaerobic conditions by operating in the carbon- fixing direction (Stols and Donnelly, Appl. Environ. Microbiol. 63(7) 2695-2701 (1997)). A similar observation was made upon overexpressing the malic enzyme from Ascaris suum in E. coli (Stols et al., Appl. Biochem. Biotechnol. 63-65(1), 153-158 (1997)). The second E. coli malic enzyme, encoded by maeB, is NADP-dependent and also

decarboxylates oxaloacetate and other alpha-keto acids (Iwakura et al., J. Biochem. 85(5): 1355- 65 (1979)).

maeB NP_416958 16130388 Escherichia coli

NAD-ME P27443 126732 Ascaris suum

FIG. 5, Step X - Fumarase (EFR20A), Fumarate Reductase (EFR20B), Succinyl-CoA Synthetase (EFR20C), Succinyl-CoA Ligase (EFR20D), Succinyl-CoA Transferase

(EFR20E)

[0373] Fumarate hydratase (EC 4.2.1.2) catalyzes the reversible hydration of fumarate to malate. The three fumarases of E. coli, encoded by fumA,fumB and fumC, are regulated under different conditions of oxygen availability. FumB is oxygen sensitive and is active under anaerobic conditions. FumA is active under microanaerobic conditions, and FumC is active under aerobic growth conditions (Tseng et al., J. Bacteriol. 183:461-467 (2001);Woods et al., Biochim. Biophys. Acta 954: 14-26 (1988); Guest et al., J. Gen. Microbiol. 131 :2971-2984 (1985)). S. cerevisiae contains one copy of a fumarase-encoding gene, FUM1, whose product localizes to both the cytosol and mitochondrion (Sass et al., J. Biol. Chem. 278:45109-45116 (2003)). Additional fumarase enzymes are found in Campylobacter jejuni (Smith et al., Int. J. Biochem. Cell. Biol. 31 :961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch.

Biochem. Biophys. 355:49-55 (1998)) and Rattus norvegicus (Kobayashi et al., J. Biochem. 89: 1923-1931 (1981)). Similar enzymes with high sequence homology include fuml from Arabidopsis thaliana, FUM1 from Rhizopus oryzae, and fumC from Cory neb acterium

glutamicum. The MmcBC fumarase from Pelotomaculum thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., FEMS Microbiol. Lett. 270:207-213 (2007)).

Protein ( ,cn Bank ID GI Number Organism fumH P14408.1 120605 Rattus norvegicus

MmcB YP OO 1211906 147677691 Pelotomaculum thermopropionicum

MmcC YP OO 1211907 147677692 Pelotomaculum thermopropionicum

FUM1 ADG65260.1 296011194 Rhizopus oryzae

[0374] Fumarate reductase catalyzes the reduction of fumarate to succinate. The fumarate reductase of E. coli, composed of four subunits encoded by frdABCD, is membrane -bound and active under anaerobic conditions. The electron donor for this reaction is menaquinone and the two protons produced in this reaction do not contribute to the proton gradient (Iverson et al., Science 284: 1961-1966 (1999)). The yeast genome encodes two soluble fumarate reductase isozymes encoded by FRDS1 (Enomoto et al., DNA Res. 3:263-267 (1996)) and FRDS2 (Muratsubaki et al., Arch. Biochem. Biophys. 352: 175-181 (1998)), which localize to the cytosol and promitochondrion, respectively, and are used for anaerobic growth on glucose (Arikawa et al., FEMS Microbiol. Lett. 165: 111-116 (1998)).

[0375] The ATP-dependent acylation of succinate to succinyl-CoA is catalyzed by succinyl- CoA synthetase (EC 6.2.1.5). The product of the LSCl and LSC2 genes of S. cerevisiae and the sucC and sucD genes of E. coli naturally form a succinyl-CoA synthetase complex that catalyzes the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP, a reaction which is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)). These proteins are identified below: Protein ( ,cn Bank ID GI Number Organism

LSC1 NP_014785 6324716 Saccharomyces cerevisiae

LSC2 NP O 11760 6321683 Saccharomyces cerevisiae sucC NP_415256.1 16128703 Escherichia coli

sucD AAC73823.1 1786949 Escherichia coli

[0376] Succinyl-CoA transferase catalyzes the conversion of succinyl-CoA to succinate while transferring the CoA moiety to a CoA acceptor molecule. Many transferases have broad specificity and can utilize CoA acceptors as diverse as acetate, succinate, propionate, butyrate, 2- methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate, valerate, crotonate, 3- mercaptopropionate, propionate, vinylacetate, and butyrate, among others.

[0377] The conversion of succinate to succinyl-CoA can be carried by a transferase which does not require the direct consumption of an ATP or GTP. This type of reaction is common in a number of organisms. The conversion of succinate to succinyl-CoA can also be catalyzed by succinyl-CoA:Acetyl-CoA transferase. The gene product of catl of Clostridium kluyveri has been shown to exhibit succinyl-CoA: acetyl-CoA transferase activity (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)). In addition, the activity is present in Trichomonas vaginalis (van Grinsven et al. 2008) and Trypanosoma brucei (Riviere et al. 2004). The succinyl- CoA:acetate CoA-transferase from Acetobacter aceti, encoded by aarC, replaces succinyl-CoA synthetase in a variant TCA cycle (Mullins et al. 2008). Similar succinyl-CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al. 2008), Trypanosoma brucei (Riviere et al. 2004) and Clostridium kluyveri (Sohling and Gottschalk, 1996c). The beta-ketoadipate:succinyl-CoA transferase encoded by peal and pcaJ in Pseudomonas putida is yet another candidate (Kaschabek et al. 2002). The aforementioned proteins are identified below.

Protein ( ,cn Bank ID GI Number Organism

catl P38946.1 729048 Clostridium kluyveri

TVAG 95550 XP 001330176 123975034 Trichomonas vaginalis G3

Tbl 1.02.0290 XP_828352 71754875 Trypanosoma brucei peal AAN69545.1 24985644 Pseudomonas putida pcaJ NP_746082.1 26990657 Pseudomonas putida aarC ACD85596.1 189233555 Acetobacter aceti

[0378] An additional exemplary transferase that converts succinate to succinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid is succinyl-CoA:3:ketoacid-CoA transferase (EC 2.8.3.5). Exemplary succinyl-CoA:3:ketoacid-CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al. 1997), Bacillus subtilis, and Homo sapiens (Fukao et al. 2000; Tanaka et al. 2002). The aforementioned proteins are identified below.

FIG. 5, Step Y - Citrate Synthase (EFR21A), Aconitase (EFR21B), Alpha-ketoglutarate Dehydrogenase (EFR21C)

[0379] The conversion of ACCOA and OAA to succinyl-CoA can be catalyzed by a citrate synthase. Exemplary genes are provided below.

ANI_1_1226134 XP 001396731.1 145250435 Aspergillus niger CBS 513.88 gltA NP_415248.1 16128695 Escherichia coli K-12 MG1655

[0380] Aconitase (EC 4.2.1.3) is an iron-sulfur-containing protein catalyzing the reversible isomerization of citrate and iso-citrate via the intermediate czs-aconitate. Two aconitase enzymes are encoded in the E. coli genome by acnA and acnB. AcnB is the main catabolic enzyme, while AcnA is more stable and appears to be active under conditions of oxidative or acid stress (Cunningham et al., Microbiology 143 (Pt 12):3795-3805 (1997)). Two isozymes of aconitase in Salmonella typhimurium are encoded by acnA and acnB (Horswill and Escalante-Semerena, Biochemistry 40:4703-4713 (2001)). The S. cerevisiae aconitase, encoded by ACOl, is localized to the mitochondria where it participates in the TCA cycle (Gangloff et al., Mol. Cell. Biol. 10:3551-3561 (1990)) and the cytosol where it participates in the glyoxylate shunt (Regev- Rudzki et al., Mol. Biol. Cell. 16:4163-4171 (2005)).

[0381] Alpha-ketoglutarate dehydrogenase (AKGD) converts alpha-ketoglutarate to succinyl-CoA. Exemplary enzymes are found in E. coli, B. subtilis, and S. cerevisiae. Alpha- ketoglutarate dehydrogenase (AKGD) converts alpha-ketoglutarate to succinyl-CoA and is the primary site of control of metabolic flux through the TCA cycle (Hansford, 1980, Curr. Top. Bioenerg. 10:217-278). Encoded by genes sucA, sucB and Ipd in E. coli, AKGD gene expression is downregulated under anaerobic conditions and during growth on glucose (Park et al, 1995, Mol. Microbiol, 15:473-482). The Bacillus subtilis AKGD, encoded by odhAB (El and E2) and pdhD (E3, shared domain), is regulated at the transcriptional level and is dependent on the carbon source and growth phase of the organism (Resnekov et al, 1992, Mol. Gen. Genet, 234:285-296). In yeast, the LPD1 gene encoding the E3 component is regulated at the transcriptional level by glucose (Roy and Dawes, 1987, J. Gen. Microbiol, 133:925-933). The El component, encoded by KGDI, is also regulated by glucose and activated by the products of HAP2 and HAP3 (Repetto and Tzagoloff, 1989, Mol. Cell Biol. 9:2695-2705).

4.3 Example III - Methods of using formaldehyde produced from the oxidation of methanol in the formation of intermediates of central metabolic pathways for the formation of biomass

[0382] Provided herein are exemplary pathways, which utilize formaldehyde produced from the oxidation of methanol (see, e.g., FIG. 1, step J) in the formation of intermediates of certain central metabolic pathways that can be used for the formation of biomass. Exemplary MMPs for enhancing the availability of reducing equivalents, as well as the producing formaldehyde from methanol (step J), are provided in FIG.1.

[0383] One exemplary pathway that can utilize formaldehyde produced from the oxidation of methanol {e.g., as provided in FIG. 1) is shown in FIG. 3, which involves condensation of formaldehyde and D-ribulose-5-phosphate to form H6P by EF1 (FIG. 3, step A). The enzyme can use Mg 2+ or Mn 2+ for maximal activity, although other metal ions are useful, and even non- metal-ion-dependent mechanisms are contemplated. H6P is converted into F6P by EF2 (FIG. 3, step B).

[0384] Another exemplary pathway that involves the detoxification and assimilation of formaldehyde produced from the oxidation of methanol {e.g., as provided in FIG. 1) is shown in FIG. 4 and proceeds through DHA. EF3 is a special transketolase that first transfers a

glycoaldehyde group from xylulose-5 -phosphate to formaldehyde, resulting in the formation of DHA and glyceraldehyde-3 -phosphate (G3P), which is an intermediate in glycolysis (FIG. 4, step A). The DHA obtained from DHA synthase is then further phosphorylated to form DHAP by an EF4 (FIG. 4, step B). DHAP can be assimilated into glycolysis and several other pathways.

FIG. 3, Steps A and B - H6P synthase (EF1) (Step A) and 6-phospho-3-hexuloisomerase (EF2) (Step B)

[0385] Both of the EF1 and EF2 enzymes are found in several organisms, including methanotrops and methylotrophs where they have been purified (Kato et al., 2006, BioSci Biotechnol Biochem. 70(1): 10-21. In addition, these enzymes have been reported in heterotrophs such as Bacillus subtilis also where they are reported to be involved in formaldehyde

detoxification (Mitsui et al, 2003, AEM 69(10):6128-32, Yasueda et al. , 1999. J Bac

181(23):7154-60. Genes for these two enzymes from the methylotrophic bacterium

Mycobacterium gastri MB 19 have been fused and E. coli strains harboring the hps-phi constmct showed more efficient utilization of formaldehyde (Orita et al., 2007, Appl Microbiol

Biotechnol. 76:439 -445). In some organisms, these two enzymes naturally exist as a fused version that is bifunctional.

[0386] Exemplary candidate genes for hexulose-6-phopshate synthase are:

[0387] Exemplary gene candidates for EF2 are: Protein ( ,cn Bank ID GI number Organism

Phi AAR39393.1 40074228 Bacillus methanolicus MGA3

Phi EIJ81376.1 387589056 Bacillus methanolicus PB1

Phi BAA83098.1 5706383 Methylomonas aminofaciens

RmpB BAA90545.1 6899860 Mycobacterium gastri

Phi YP_545762.1 91776006 Methylobacillus flagellatus KT

Phi YP 003051269.1 253999206 Methylovorus glucosetrophus SIP3- 4

Phi YP 003990383.1 3121 12067 Geobacillus sp. Y4.1MC1

Phi YP 007402408.1 448238350 Geobacillus sp. GN OI

[0388] Candidates for enzymes where both of these functions have been fused into a single open reading frame include the following:

[0389] An experimental system was designed to test the ability of a methanol dehydrogenase (MeDH) in conjunction with the enzymes H6P synthase (HPS) and 6-phospho-3- hexuloisomerase (PHI) of the Ribulose Monophosphate (RuMP) pathway to assimilate methanol carbon into the glycolytic pathway and the TCA cycle. Escherichia coli strain ECh-7150 (AlacIA, ApflB, Aptsl, APpckA(pckA), APglk(glk), glk: :glfB, AhycE, AfrmR, AfrmA, AfrmB) was constructed to remove the glutathione-dependent formaldehyde detoxification capability encoded by the FrmA and FrmB enzyme. This strain was then transformed with plasmid pZA23S variants that either contained or lacked gene 2616A encoding a fusion of the HPS and PHI enzymes. These two transformed strains were then each transformed with pZS* 13S variants that contained gene 2315L (encoding an active MeDH), or gene 2315 RIP2 (encoding a catalytically inactive MeDH), or no gene insertion. Genes 2315 and 2616 are internal nomenclatures for NAD-dependent methanol dehydrogenase from Bacillus methanolicus MGA3 and 2616 is a fused phs-hpi constructs as described in Orita et al. (2007) Appl Microbiol

Biotechnol 76:439-45.

[0390] The six resulting strains were aerobically cultured in quadruplicate, in 5 ml minimal medium containing 1% arabinose and 0.6 M 13C-methanol as well as 100 ug/ml carbenicillin and 25 μg/ml kanamycin to maintain selection of the plasmids, and 1 mM IPTG to induce expression of the methanol dehydrogenase and HPS-PHI fusion enzymes. After 18 hours incubation at 37°C, the cell density was measured spectrophotometrically at 600 nM wavelength and a clarified sample of each culture medium was submitted for analysis to detect evidence of incorporation of the labeled methanol carbon into TCA-cycle derived metabolites. The label can be further enriched by deleting the gene araD that competes with ribulose-5-phosphate.

13

[0391] C carbon derived from labeled methanol provided in the experiment was found to be significantly enriched in the TCA-cycle derived amino acid glutamate, but only in the strain expressing both catalytically active MeDH 2315L and the HPS-PHI fusion 2616A together (data not shown). Moreover, this strain grew significantly better than the strain expressing

catalytically active MeDH but lacking expression of the HPS-PHI fusion (data not shown), suggesting that the HPS-PHI enzyme is capable of reducing growth inhibitory levels of formaldehyde that cannot be detoxified by other means in this strain background. These results show that co-expression of an active MeDH and the enzymes of the RuMP pathway can effectively assimilate methanol derived carbon and channel it into TCA-cycle derived products.

FIG. 4, Step A - DHA synthase (EF3)

[0392] Another exemplary pathway that involves the detoxification and assimilation of formaldehyde produced from the oxidation of methanol {e.g., as provided in FIG. 1) is shown in FIG. 4 and proceeds through DHA. EF3 is a special transketolase that first transfers a

glycoaldehyde group from xylulose-5 -phosphate to formaldehyde, resulting in the formation of DHA and glyceraldehyde-3 -phosphate (G3P), which is an intermediate in glycolysis (FIG. 4, step A). The DHA obtained from DHA synthase is then further phosphorylated to form DHAP by an EF4 (FIG. 4, step B). DHAP can be assimilated into glycolysis and several other pathways. 2_ _| _

[0393] The EF3 enzyme in Candida boidinii uses thiamine pyrophosphate and Mg as cofactors and is localized in the peroxisome. The enzyme from the methanol-growing carboxydobacterium, Mycobacter sp. strain JC1 DSM 3803, was also found to have DHA synthase and kinase activities (Ro et al., 1997, JBac 179(19):6041-7). DHA synthase from this organism also has similar cofactor requirements as the enzyme from C. boidinii. The K _m s for formaldehyde and xylulose 5-phosphate were reported to be 1.86 mM and 33.3 microM, respectively. Several other mycobacteria, excluding only Mycobacterium tuberculosis, can use methanol as the sole source of carbon and energy and are reported to use EF3 (Part et al., 2003, JBac 185(l): 142-7.

FIG. 4, Step B - DHA kinase (EF4)

[0394] DHA obtained from DHA synthase is further phosphorylated to form DHAP by an EF4. DHAP can be assimilated into glycolysis and several other pathways. EF4 has been purified from Ogataea angusta to homogeneity (Bystrkh, 1983, Biokhimiia, 48(10): 1611-6). The enzyme, which phosphorylates DHA and, to a lesser degree, glyceraldehyde, is a homodimeric protein of 139 kDa. ATP is the preferred phosphate group donor for the enzyme. When ITP, GTP, CTP and UTP are used, the activity drops to about 30%. In several organisms such as Klebsiella pneumoniae and Citrobacter fruendii (Daniel et al., 1995, JBac 177(15):4392-40), DHA is formed as a result of oxidation of glycerol and is converted into DHAP by the kinase EF4 of K. pneumoniae has been characterized (Jonathan et al, 1984, JBac 160(l):55-60). It is very specific for DHA, with a K _m of 4 μΜ, and has two apparent K _m values for ATP, one at 25 to 35 μΜ, and the other at 200 to 300 μΜ. DHA can also be phosphorylated by glycerol kinases but the EF4 from K. puemoniae is different from glycerol kinase in several respects. While both enzymes can phosphorylate DHA, EF4 does not phosphorylate glycerol, neither is it inhibited by fructose- 1,6-diphosphate. In Saccharomyces cerevisiae, EF4s (I and II) are involved in rescuing the cells from toxic effects of DHA (Molin et al., 2003, J Biol Chem. 17; 278(3): 1415-23).

[0395] In Escherichia coli, EF4 is composed of the three subunits DhaK, DhaL, and DhaM and it functions similarly to a phosphotransferase system (PTS) in that it utilizes

phosphoenolpyruvate as a phosphoryl donor (Gutknecht et al., 2001, EMBO J. 20(10):2480-6). It differs in not being involved in transport. The phosphorylation reaction requires the presence of the EI and HPr proteins of the PTS system. The DhaM subunit is phosphorylated at multiple sites. DhaK contains the substrate binding site (Garcia-Alles et al., 2004, 43(41): 13037-45;

Siebold et al., 2003, PNAS. 100(14):8188-92). The K _M for DHA for the E. coli enzyme has been reported to be 6 μΜ. The K subunit is similar to the N-terminal half of ATP-dependent EF4 of Citrobacter freundii and eukaryotes.

[0396] Exemplary EF4 gene candidates for this step are:

4.4 Example IV - Methods for Handling Anaerobic Cultures

[0397] This example describes methods used in handling anaerobic cultures.

[0398] A. Anaerobic chamber and conditions. Exemplary anaerobic chambers are available commercially (see, for example, Vacuum Atmospheres Company, Hawthorne CA; MBraun, Newburyport MA). Conditions included an 0 ₂ concentration of 1 ppm or less and 1 atm pure N ₂ . In one example, 3 oxygen scrubbers/catalyst regenerators were used, and the chamber included an 0 ₂ electrode (such as Teledyne; City of Industry CA). Nearly all items and reagents were cycled four times in the airlock of the chamber prior to opening the inner chamber door. Reagents with a volume >5mL were sparged with pure N ₂ prior to introduction into the chamber. Gloves are changed twice/yr and the catalyst containers were regenerated periodically when the chamber displays increasingly sluggish response to changes in oxygen levels. The chamber's pressure was controlled through one-way valves activated by solenoids. This feature allowed setting the chamber pressure at a level higher than the surroundings to allow transfer of very small tubes through the purge valve.

[0399] The anaerobic chambers achieved levels of 0 ₂ that were consistently very low and were needed for highly oxygen sensitive anaerobic conditions. However, growth and handling of cells does not usually require such precautions. In an alternative anaerobic chamber configuration, platinum or palladium can be used as a catalyst that requires some hydrogen gas in the mix. Instead of using solenoid valves, pressure release can be controlled by a bubbler.

Instead of using instrument-based 0 ₂ monitoring, test strips can be used instead.

[0400] B. Anaerobic microbiology. Serum or media bottles are fitted with thick rubber stoppers and aluminum crimps are employed to seal the bottle. Medium, such as Terrific Broth, is made in a conventional manner and dispensed to an appropriately sized serum bottle. The bottles are sparged with nitrogen for ~30 min of moderate bubbling. This removes most of the oxygen from the medium and, after this step, each bottle is capped with a rubber stopper (such as Bellco 20 mm septum stoppers; Bellco, Vineland, NJ) and crimp-sealed (Bellco 20 mm). Then the bottles of medium are autoclaved using a slow (liquid) exhaust cycle. At least sometimes a needle can be poked through the stopper to provide exhaust during autoclaving; the needle needs to be removed immediately upon removal from the autoclave. The sterile medium has the remaining medium components, for example buffer or antibiotics, added via syringe and needle. Prior to addition of reducing agents, the bottles are equilibrated for 30 - 60 minutes with nitrogen (or CO depending upon use). A reducing agent such as a 100 x 150 mM sodium sulfide, 200 mM cysteine -HC1 is added. This is made by weighing the sodium sulfide into a dry beaker and the cysteine into a serum bottle, bringing both into the anaerobic chamber, dissolving the sodium sulfide into anaerobic water, then adding this to the cysteine in the serum bottle. The bottle is stoppered immediately as the sodium sulfide solution generates hydrogen sulfide gas upon contact with the cysteine. When injecting into the culture, a syringe filter is used to sterilize the solution. Other components are added through syringe needles, such as B12 (10 μΜ

cyanocobalamin), nickel chloride (NiCl ₂ , 20 microM final concentration from a 40 mM stock made in anaerobic water in the chamber and sterilized by autoclaving or by using a syringe filter upon injection into the culture), and ferrous ammonium sulfate (final concentration needed is 100 μΜ— made as 100-lOOOx stock solution in anaerobic water in the chamber and sterilized by autoclaving or by using a syringe filter upon injection into the culture). To facilitate faster growth under anaerobic conditions, the 1 liter bottles were inoculated with 50 mL of a preculture grown anaerobically. Induction of the pAl-lacOl promoter in the vectors was performed by addition of isopropyl β-D-l-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM and was carried out for about 3 hrs.

[0401] Large cultures can be grown in larger bottles using continuous gas addition while bubbling. A rubber stopper with a metal bubbler is placed in the bottle after medium addition and sparged with nitrogen for 30 minutes or more prior to setting up the rest of the bottle. Each bottle is put together such that a sterile filter will sterilize the gas bubbled in and the hoses on the bottles are compressible with small C clamps. Medium and cells are stirred with magnetic stir bars. Once all medium components and cells are added, the bottles are incubated in an incubator in room air but with continuous nitrogen sparging into the bottles.

4.5 Example V - In vivo labeling assay for conversion of methanol to C0 ₂

[0402] This example describes a functional methanol pathway in a microbial organism.

[0403] Strains with functional reductive TCA branch and pyruvate formate lyase deletion were grown aerobically in LB medium overnight, followed by inoculation of M9 high-seed media containing IPTG and aerobic growth for 4 hrs. These strains had methanol

dehydrogenase/ ACT pairs in the presence and absence of formaldehyde dehydrogenase or formate dehydrogenase. ACT is an activator protein (a Nudix hydrolase). At this time, strains

13

were pelleted, resuspended in fresh M9 medium high-seed media containing 2% CH ₃ OH, and sealed in anaerobic vials. Head space was replaced with nitrogen and strains grown for 40 hours at 37°C. Following growth, headspace was analyzed for ¹³ C0 ₂ . Media was examined for residual methanol as well as BDO and byproducts. All constructs expressing methanol dehydrogenase (MeDH) mutants and MeDH/ ACT pairs grew to slightly lower ODs than strains containing empty vector controls. This is likely due to the high expression of these constructs (Data not shown). One construct (2315/2317) displayed significant accumulation of labeled C0 ₂ relative to controls in the presence of FalDH, FDH or no coexpressed protein. This shows a functional MeOH pathway in E. coli and that the endogenous glutathione-dependent formaldehyde detoxification genes (frmAB) are sufficient to carry flux generated by the current MeDH/ ACT constructs.

[0404] 2315 is internal laboratory designation for the MEDH from Bacillus methanolicus MGA3 (GenBank Accession number: EIJ77596.1; GI number: 387585261), and 2317 is internal laboratory designation for the activator protein from the same organism (locus tag:

MGA3 09170; GenBank Accession number:EIJ83380; GI number: 387591061).

[0405] Sequence analysis of the NADH-dependent methanol dehydrogenase from Bacillus methanolicus places the enzyme in the alcohol dehydrogenase family III. It does not contain any tryptophan residues, resulting in a low extinction coefficient (18,500 M ^"1 , cm ^"1 ) and should be detected on SDS gels by Coomassie staining.

[0406] The enzyme has been characterized as a multisubunit complex built from 43 kDa subunits containing one Zn and 1 -2 Mg atoms per subunit. Electron microscopy and

sedimentation studies determined it to be a decamer, in which two rings with five-fold symmetry are stacked on top of each other (Vonck et al., J. Biol. Chem. 266:3949-3954, 1991). It is described to contain a tightly but not covalently bound cofactor and requires exogenous NAD ⁺ as e ^" -acceptor to measure activity iin vitro. A strong increase (10-40-fold) of iin vitro activity was observed in the presence of an activator protein (ACT), which is a homodimer (21 kDa subunits) and contains one Zn and one Mg atom per subunit.

[0407] The mechanism of the activation was investigated by Kloosterman et al., J. Biol. Chem. 277:34785-34792, 2002, showing that ACT is a Nudix hydrolase and Hektor et al, J. Biol. Chem. 277:46966-46973, 2002, demonstrating that mutation of residue S97 to G or T in MeDH changes activation characteristics along with the affinity for the cofactor. While mutation of residues G15 and D88 had no significant impact, a role of residue G13 for stability as well as of residues G95, D100, and K103 for the activity is suggested. Both papers together propose a hypothesis in which ACT cleaves MeDH-bound NAD . MeDH retains AMP bound and enters an activated cycle with increased turnover.

[0408] The stoichiometric ratio between ACT and MeDH is not well defined in the literature. Kloosterman et al., supra determine the ratio of dimeric Act to decameric MeDH for full iin vitro activation to be 10: 1. In contrast, Arfman et al. J. Biol. Chem. 266:3955-3960, 1991 determined a ratio of 3 : 1 iin vitro for maximum and a 1 :6 ratio for significant activation, but observe a high sensitivity to dilution. Based on expression of both proteins in Bacillus, the authors estimate the ratio in vivo to be around 1 : 17.5.

[0409] However, our iin vitro experiments with purified activator protein (2317A) and methanol dehydrogenase (2315 A) showed the ratio of ACT to MeDH to be 10: 1. This iin vitro test was done with 5 M methanol, 2 mM NAD and 10 μΜ methanol dehydrogenase 2315 A at pH 7.4.

4.6 Example VI - Formate Assimilation Pathways

[0410] This example describes a functional methanol pathway in a microbial organism.

[0411] This example describes enzymatic pathways for converting pyruvate to

formaldehyde, and optionally in combination with producing acetyl-CoA and/or reproducing pyruvate.

FIG. 5, Step E - Formate Reductase (EFR1)

[0412] The conversion of formate to formaldehyde can be carried out by a formate reductase (step E, Figure 1). A suitable enzyme for these transformations is the aryl-aldehyde

dehydrogenase, or equivalently a carboxylic acid reductase, from Nocardia iowensis. Carboxylic acid reductase catalyzes the magnesium, ATP and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes (Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007)). This enzyme, encoded by car, was cloned and functionally expressed in E. coli

(Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007)). Expression of the npt gene product improved activity of the enzyme via post-transcriptional modification. The npt gene encodes a specific phosphopantetheine transferase (PPTase) that converts the inactive apo- enzyme to the active holo-enzyme. The natural substrate of this enzyme is vanillic acid, and the enzyme exhibits broad acceptance of aromatic and aliphatic substrates (Venkitasubramanian et al., in Biocatalysis in the Pharmaceutical and Biotechnology Industires, ed. R.N. Patel, Chapter 15, pp. 425-440, CRC Press LLC, Boca Raton, FL. (2006)). Information related to these proteins and genes is shown below.

[0413] Additional car and npt genes can be identified based on sequence homology.

Tsukamurella paurometabola

TpauDRAFT_33060 ZP 04027864.1 227980601

DSM 20162

Tsukamurella paurometabola

TpauDRAFT 20920 ZP 04026660.1 227979396

DSM 20162

CPCC7001J320 ZP 05045132.1 254431429 Cyanobium PCC7001

DDBDRAFT 01877 Dictyostelium discoideum

XP 636931.1 66806417

29 AX4

[0414] An additional enzyme candidate found in Streptomyces griseus is encoded by the griC and griD genes. This enzyme is believed to convert 3-amino-4-hydroxybenzoic acid to 3- amino-4-hydroxybenzaldehyde as deletion of either griC or griD led to accumulation of extracellular 3-acetylamino-4-hydroxybenzoic acid, a shunt product of 3-amino-4- hydroxybenzoic acid metabolism (Suzuki, et al., J. Antibiot. 60(6):380-387 (2007)). Co- expression of griC and griD with SGR 665, an enzyme similar in sequence to the Nocardia iowensis npt, can be beneficial. Information related to these proteins and genes is shown below.

[0415] An enzyme with similar characteristics, alpha-aminoadipate reductase (AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in some fungal species. This enzyme naturally reduces alpha-aminoadipate to alpha-aminoadipate semialdehyde. The carboxyl group is first activated through the ATP-dependent formation of an adenylate that is then reduced by NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizes magnesium and requires activation by a PPTase. Enzyme candidates for AAR and its corresponding PPTase are found in Saccharomyces cerevisiae (Morris et al., Gene 98: 141-145 (1991)), Candida albicans (Guo et al., Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe (Ford et al., Curr. Genet. 28: 131-137 (1995)). The AAR from S. pombe exhibited significant activity when expressed in E. coli (Guo et al., Yeast 21 : 1279-1288 (2004)). The AAR from Penicillium chrysogenum accepts S-carboxymethyl-L-cysteine as an alternate substrate, but did not react with adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J. Biol. Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenum PPTase has not been identified to date.

Information related to these proteins and genes is shown below.

[0416] Tani et al (Agric Biol Chem, 1978, 42: 63-68; Agric Biol Chem, 1974, 38: 2057- 2058) showed that purified enzymes from Escherichia coli strain B could reduce the sodium salts of different organic acids (e.g. formate, glycolate, acetate, etc.) to their respective aldehydes (e.g. formaldehyde, glycoaldehyde, acetaldehyde, etc.). Of three purified enzymes examined by Tani et al (1978), only the "A" isozyme was shown to reduce formate to formaldehyde. Collectively, this group of enzymes was originally termed glycoaldehyde dehydrogenase; however, their novel reductase activity led the authors to propose the name glycolate reductase as being more appropriate (Morita et al, Agric Biol Chem, 1979, 43: 185-186). Morita et al (Agric Biol Chem, 1979, 43: 185-186) subsequently showed that glycolate reductase activity is relatively

widespread among microorganisms, being found for example in: Pseudomonas, Agrobacterium, Escherichia, Flavobacterium, Micrococcus, Staphylococcus, Bacillus, and others. Without wishing to be bound by any particular theory, it is believed that some of these glycolate reductase enzymes are able to reduce formate to formaldehyde.

[0417] Any of these CAR or CAR- like enzymes can exhibit formate reductase activity or can be engineered to do so.

FIG. 5, Step F - Formate Ligase (EFR2A), Formate Transferase (EFR2B), Formate Synthetase (EFR2C)

[0418] The acylation of formate to formyl-CoA is catalyzed by enzymes with formate transferase, synthetase, or ligase activity (Step F, Figure 1). Formate transferase enzymes have been identified in several organisms including Escherichia coli (Toyota, et al., J Bacteriol. 2008 Apr;190(7):2556-64), Oxalobacter formigenes (Toyota, et al., J Bacteriol. 2008

Apr;190(7):2556-64; Baetz et al., J Bacteriol. 1990 Jul; 172(7) :3537-40 ; Ricagno, et al., EMBO J. 2003 Jul l;22(13):3210-9)), and Lactobacillus acidophilus (Azcarate-Peril, et αΙ., ΑρρΙ.

Environ. Microbiol. 2006 72(3) 1891-1899). Homologs exist in several other organisms.

Enzymes acting on the CoA-donor for formate transferase may also be expressed to ensure efficient regeneration of the CoA-donor. For example, if oxalyl-CoA is the CoA donor substrate for formate transferase, an additional transferase, synthetase, or ligase may be required to enable efficient regeneration of oxalyl-CoA from oxalate. Similarly, if succinyl-CoA or acetyl-CoA is the CoA donor substrate for formate transferase, an additional transferase, synthetase, or ligase may be required to enable efficient regeneration of succinyl-CoA from succinate or acetyl-CoA from acetate, respectively.

[0419] Suitable CoA-donor regeneration or formate transferase enzymes are encoded by the gene products of catl, cat2, and cat3 of Clostridium kluyveri. These enzymes have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA acetyltransferase activity, respectively (Seedorf et al., Proc. Natl. Acad. Sci. USA 105:2128-2133 (2008); Sohling and Gottschalk, J Bacteriol 178:871-880 (1996)). Similar CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al., J. Biol. Chem. 283: 1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., J. Biol. Chem. 279:45337-45346 (2004)). Yet another transferase capable of the desired conversions is butyryl-CoA:acetoacetate CoA-transferase. Exemplary enzymes can be found in Fusobacterium nucleatum (Barker et al., J. Bacteriol. 152(l):201-7 (1982)), Clostridium SB4 (Barker et al., J. Biol. Chem. 253(4): 1219-25 (1978)), and Clostridium acetobutylicum (Wiesenborn et al., Appl. Environ. Microbiol. 55(2):323-9 (1989)). Although specific gene sequences were not provided for butyryl-CoA:acetoacetate CoA-transferase in these references, the genes FN0272 and FN0273 have been annotated as a butyrate-acetoacetate CoA-transferase (Kapatral et al., J. Bact. 184(7) 2005-2018 (2002)). Homologs in Fusobacterium nudeatum such as FN1857 and FN1856 also likely have the desired acetoacetyl-CoA transferase activity. FN1857 and FN1856 are located adjacent to many other genes involved in lysine fermentation and are thus very likely to encode an acetoacetate:butyrate CoA transferase (Kreimeyer, et al, J. Biol. Chem. 282 (10) 7191-7197 (2007)). Additional candidates from Porphyrmonas gingivalis and Thermoanaerobacter tengcongensis can be identified in a similar fashion (Kreimeyer, et al., J. Biol. Chem. 282 (10) 7191-7197 (2007)). Information related to these proteins and genes is shown below.

[0420] Additional transferase enzymes of interest include the gene products of atoAD from E. coli (Hanai et al, Appl Environ Microbiol 73:7814-7818 (2007)), ctfAB from C.

acetobutylicum (Jojima et al., Appl Microbiol Biotechnol 77: 1219-1224 (2008)), and ctfAB from Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci.Biotechnol Biochem. 71 :58-68 (2007)). Information related to these proteins and genes is shown below. AtoD P76458.1 2492990 Escherichia coli

CtfA NPJ49326.1 15004866 Clostridium acetobutylicum

OJB NPJ49327.1 15004867 Clostridium acetobutylicum

CtfA AAP42564.1 31075384 Clostridium saccharoperbutylacetonicum

CtJB AAP42565.1 31075385 Clostridium saccharoperbutylacetonicum

[0421] Succinyl-CoA:3-ketoacid-CoA transferase naturally converts succinate to succinyl- CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid. Exemplary succinyl-CoA:3:ketoacid- CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al., J.Biol. Chem.

272:25659-25667 (1997)), Bacillus subtilis (Stols et al, Protein.Expr.Purif. 53:396-403 (2007)), and Homo sapiens (Fukao et al., Genomics 68: 144-151 (2000); Tanaka et al., Mol.Hum.Reprod. 8: 16-23 (2002)). Information related to these proteins and genes is shown below.

[0422] Two additional enzymes that catalyze the activation of formate to formyl-CoA reaction are AMP-forming formyl-CoA synthetase and ADP-forming formyl-CoA synthetase. Exemplary enzymes, known to function on acetate, are found in E. coli (Brown et al., J. Gen. Microbiol. 102:327-336 (1977)), Ralstonia eutropha (Priefert and Steinbuchel, J. Bacteriol. 174:6590-6599 (1992)), Methanothermobacter thermautotrophicus (Ingram-Smith and Smith, Archaea 2:95-107 (2007)), Salmonella enterica (Gulick et al., Biochemistry 42:2866-2873 (2003)) and Saccharomyces cerevisiae (Jogl and Tong, Biochemistry 43: 1425-1431 (2004)). Such enzymes may also acylate formate naturally or can be engineered to do so.

acsl ABC87079.1 86169671 Methanothermobacter thermautotrophicus acsl AAL23099.1 16422835 Salmonella enterica

ACS1 Q01574.2 257050994 Saccharomyces cerevisiae

[0423] ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is another candidate enzyme that couples the conversion of acyl-CoA esters to their corresponding acids with the concurrent synthesis of ATP. Several enzymes with broad substrate specificities have been described in the literature. ACD I from Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain substrates including acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate, butyrate, isobutyryate, isovalerate, succinate, fumarate, phenylacetate, indoleacetate (Musfeldt et al., J. Bacteriol. 184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al., Arch. Microbiol.

182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al., supra (2004)). The enzymes from A. fulgidus, H. marismortui and . aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Musfeldt et al., supra; Brasen et al., supra (2004)). Additional candidates include the succinyl-CoA synthetase encoded by sucCD in E. coli (Buck et al., Biochemistry 24:6245-6252 (1985)) and the acyl-CoA ligase from Pseudomonas putida (Fernandez- Valverde et al., Appl. Environ. Microbiol. 59: 1149-1154 (1993)). Such enzymes may also acylate formate naturally or can be engineered to do so.

Information related to these proteins and genes is shown below.

paaF AAC24333.2 22711873 Pseudomonas putida

[0424] An alternative method for adding the CoA moiety to formate is to apply a pair of enzymes such as a phosphate-transferring acyltransferase and a kinase. These activities enable the net formation of formyl-CoA with the simultaneous consumption of ATP. An exemplary phosphate -transferring acyltransferase is phosphotransacetylase, encoded by pta. The pta gene from E. coli encodes an enzyme that can convert acetyl-CoA into acetyl-phosphate, and vice versa (Suzuki, T. Biochim.Biophys.Acta 191 :559-569 (1969)). This enzyme can also utilize propionyl-CoA instead of acetyl-CoA forming propionate in the process (Hesslinger et al.

Mol.Microbiol 27:477-492 (1998)). Homologs exist in several other organisms including Salmonella enterica and Chlamydomonas reinhardtii. Such enzymes may also phosphorylate formate naturally or can be engineered to do so.

[0425] An exemplary acetate kinase is the E. coli acetate kinase, encoded by ackA (Skarstedt and Silverstein J.Biol. Chem. 251 :6775-6783 (1976)). Homologs exist in several other organisms including Salmonella enterica and Chlamydomonas reinhardtii. It is likely that such enzymes naturally possess formate kinase activity or can be engineered to have this activity. Information related to these proteins and genes is shown below:

[0426] The acylation of formate to formyl-CoA can also be carried out by a formate ligase. For example, the product of the LSCl and LSC2 genes of S. cerevisiae and the sucC and sucD genes of E. coli naturally form a succinyl-CoA ligase complex that catalyzes the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP, a reaction which is reversible in vivo (Gruys et al, US Patent No. 5,958,745, filed September 28, 1999). Such enzymes may also acylate formate naturally or can be engineered to do so. Information related to these proteins and genes is shown below.

[0427] Additional exemplary CoA-ligases include the rat dicarboxylate-CoA ligase for which the sequence is yet uncharacterized (Vamecq et al., Biochemical J. 230:683-693 (1985)), either of the two characterized phenylacetate-CoA ligases from P. chrysogenum (Lamas- Maceiras et al., Biochem. J. 395: 147-155 (2005); Wang et al., Biochem Biophy Res Commun 360(2):453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonas putida (Martinez- Bianco et al., J. Biol. Chem. 265:7084-7090 (1990)), and the 6-carboxyhexanoate-CoA ligase from Bacilis subtilis (Boweret al., J. Bacteriol. 178(14):4122-4130 (1996)). Additional candidate enzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa et al., Biochim. Biophys. Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al., Biochem. Pharmacol. 65:989-994 (2003)), which naturally catalyze the ATP-dependant conversion of acetoacetate into acetoacetyl-CoA. 4-Hydroxybutyryl-CoA synthetase activity has been demonstrated in Metallosphaera sedula (Berg et al., Science 318: 1782-1786 (2007)). This function has been tentatively assigned to the Msed_1422 gene. Such enzymes may also acylate formate naturally or can be engineered to do so. Information related to these proteins and genes is shown below. PhlB ABS19624.1 152002983 Penicillium chrysogenum

PaaF AAC24333.2 22711873 Pseudomonas putida

BioW NP 390902.2 50812281 Bacillus subtilis

AACS NP_084486.1 21313520 Mus musculus

AACS NP_076417.2 31982927 Homo sapiens

Msed_1422 YP 001191504 146304188 Metallosphaera sedula

FIG. 5, Step G - Formyl-CoA reductase (EFR3)

[0428] Several acyl-CoA dehydrogenases are capable of reducing an acyl-CoA {e.g., formyl- CoA) to its corresponding aldehyde {e.g., formaldehyde) (Steps F, Figure 1). Exemplary genes that encode such enzymes include the Acinetobacter calcoaceticus acrl encoding a fatty acyl- CoA reductase (Reiser and Somerville, J. Bacteriol. 179:2969-2975 (1997), the Acinetobacter sp. M-l fatty acyl-CoA reductase (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195 (2002), and a CoA- and NADP- dependent succinate semialdehyde dehydrogenase encoded by the sucD gene in Clostridium kluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996); Sohling and Gottschalk, J. Bacteriol. 1778:871-880 (1996)). SucD of P. gingivalis is another succinate semialdehyde dehydrogenase (Takahashi et al., J. Bacteriol. 182:4704-4710 (2000). The enzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is yet another candidate as it has been demonstrated to oxidize and acylate acetaldehyde,

propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski et al., J.

Bacteriol. 175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J. Gen. Appl. Microbiol. 18:45- 55 (1972); Koo et al., Biotechnol. Lett. 27:505-510 (2005)). Butyraldehyde dehydrogenase catalyzes a similar reaction, conversion of butyryl-CoA to butyraldehyde, in solventogenic organisms such as Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol. Biochem. 71 :58-68 (2007)). Additional aldehyde dehydrogenase enzyme candidates are found in Desulfatibacillum alkenivorans, Citrobacter koseri, Salmonella enterica, Lactobacillus brevis and Bacillus selenitireducens. Such enzymes may be capable of naturally converting formyl- CoA to formaldehyde or can be engineered to do so. Protein ( ,cn Bank ID GI number Organism

acrl YP 047869.1 50086355 Acinetobacter calcoaceticus

acrl AAC45217 1684886 Acinetobacter baylyi

acrl BAB85476.1 18857901 Acinetobacter sp. Strain M-l

sucD P38947.1 172046062 Clostridium kluyveri

sucD NP 904963.1 34540484 Porphyromonas gingivalis

bphG BAA03892.1 425213 Pseudomonas sp

adhE AAV66076.1 55818563 Leuconostoc mesenteroides

Bid AAP42563.1 31075383 Clostridium

saccharoperbutylacetonicum

Aid ACL06658.1 218764192 Desulfatibacillum alkenivorans AK-01

Aid YP 001452373 157145054 Citrobacter koseri ATCC BAA-895 pduP NP 460996.1 16765381 Salmonella enterica Typhimurium pduP ABJ64680.1 116099531 Lactobacillus brevis ATCC 367

BselDRA ZP 02169447 163762382 Bacillus selenitireducens MLS 10

FT 1651

[0429] An additional enzyme type that converts an acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archaeal bacteria (Berg et al., Science 318: 1782-1786 (2007); Thauer, Science 318: 1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus spp (Alber et al., J. Bacteriol. 188:8551-8559 (2006); Hugler et al., J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed_0709 in

Metallosphaera sedula (Alber et al., supra (2006); Berg et al., Science 318: 1782-1786 (2007)). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and

heterologously expressed in E. coli (Alber et al., J. Bacteriol. 188:8551-8559 (2006)). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its

corresponding aldehyde (WO 2007/141208 (2007)). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional gene candidates can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius and have been listed below. Yet another candidate for CoA-acylating aldehyde dehydrogenase is the aid gene from Clostridium beijerinckii (Toth et al., Appl. Environ. Microbiol. 65:4973-4980 (1999). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE that encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth et al., supra). Such enzymes may be capable of naturally converting formyl-CoA to formaldehyde or can be engineered to do so.

FIG. 5, Step H - Formyltetrahydrofolate synthetase (EFR4)

[0430] Formyltetrahydrofolate synthetase ligates formate to tetrahydrofolate at the expense of one ATP. This reaction is catalyzed by the gene product of Moth_0109 in M. thermoacetica (O'brien et al, Experientia Suppl. 26:249-262 (1976); Lovell et al., Arch. Microbiol. 149:280- 285 (1988); Lovell et al, Biochemistry 29:5687-5694 (1990)), FHS va. Clostridium acidurici (Whitehead and Rabinowitz, J. Bacteriol. 167:203-209 (1986); Whitehead and Rabinowitz, J. Bacteriol. 170:3255-3261 (1988), and CHY_2385 in C. hydrogenoformans (Wu et al., PLoS Genet. I :e65 (2005). Homologs exist in C. carboxidivorans P7. This enzyme is found in several other organisms as listed below.

hydrogenoformans

FHS P13419.1 120562 Clostridium acidurici

CcarbDRAFT_ 1913 ZP 05391913.1 255524966 Clostridium carboxidivorans

P7

CcarbDRAFT_2946 ZP 05392946.1 255526022 Clostridium carboxidivorans

P7

Dhaf_0555 ACL18622.1 219536883 Desulfitobacterium hafniense fhs YP 001393842.1 153953077 Clostridium kluyveri DSM 555 fhs YP 003781893.1 300856909 Clostridium ljungdahlii DSM

13528

MGA3 08300 EIJ83208.1 387590889 Bacillus methanolicus MGA3

PB1 13509 ZP 101321 13.1 387929436 Bacillus methanolicus PB1

FIG. 5, Steps I and J - Formyltetrahydrofolate synthetase (EFR5) and

Methylenetetrahydrofolate dehydrogenase (EFR6)

[0431] In thermoacetica, E. coli, and C. hydrogenoformans, methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolate dehydrogenase are carried out by the bi-functional gene products of Moth l 516, folD, and CHY_1878, respectively (Pierce et al., Environ.

Microbiol. 10:2550-2573 (2008); Wu et al., PLoS Genet. I :e65 (2005); DAri and Rabinowitz, J. Biol. Chem. 266:23953-23958 (1991)). A homolog exists in C. carboxidivorans P7. Several other organisms also encode for this bifunctional protein as tabulated below.

MGA3 09460 EIJ83438.1 387591119 Bacillus methanolicus MGA3

PB1 14689 ZP 10132349.1 387929672 Bacillus methanolicus PB1

FIG. 5, Step K - Formaldehyde-forming enzyme (EFR7) or Spontaneous

[0432] Methylene-THF, or active formaldehyde, will spontaneously decompose to formaldehyde and THF (Thorndike and Beck, Cancer Res. 1977, 37(4) 1125-32; Ordonez and Caraballo, Psychopharmacol Commun. 1975 1(3) 253-60; Kallen and Jencks, 1966, J Biol Chem 241(24) 5851-63). To achieve higher rates, a formaldehyde-forming enzyme can be applied. Such an activity can be obtained by engineering an enzyme that reversibly forms methylene-THF from THF and a formaldehyde donor, to release free formaldehyde. Such enzymes include glycine cleavage system enzymes which naturally transfer a formaldehyde group from methylene-THF to glycine (see Step L, Figure 1 for candidate enzymes). Additional enzymes include serine hydroxymethyltransferase (see Step M, Figure 1 for candidate enzymes), dimethylglycine dehydrogenase (Porter, et al., Arch Biochem Biophys. 1985, 243(2) 396-407; Brizio et al., 2004, (37) 2, 434-442), sarcosine dehydrogenase (Porter, et al., Arch Biochem Biophys. 1985, 243(2) 396-407), and dimethylglycine oxidase (Leys, et al., 2003, The EMBO Journal 22(16) 4038-4048).

FIG. 5, Step L - Glycine cleavage system (EFR8)

[0433] The reversible NAD(P)H-dependent conversion of 5 , 10-methylenetetrahydro folate and C0 ₂ to glycine is catalyzed by the glycine cleavage complex, also called glycine cleavage system, composed of four protein components; P, H, T and L. The glycine cleavage complex is involved in glycine catabolism in organisms such as E. coli and glycine biosynthesis in eukaryotes (Kikuchi et al, Proc Jpn Acad Ser 84:246 (2008)). The glycine cleavage system of E. coli is encoded by four genes: gcvPHT and IpdA (Okamura et al, Eur J Biochem 216:539-48 (1993);Heil et al, Microbiol 148:2203-14 (2002)). Activity of the glycine cleavage system in the direction of glycine biosynthesis has been demonstrated in vivo in Saccharomyces cerevisiae (Maaheimo et al, Eur J Biochem 268:2464-79 (2001)). The yeast GCV is encoded by GCV1, GCV2, GCV3 and LPDl .

FIG. 5, Step M - Serine hydroxymethyltransferase (EFR9)

[0434] Conversion of glycine to serine is catalyzed by serine hydroxymethyltransferase, also called glycine hydroxymethyltranferase. This enzyme reversibly converts glycine and 5,10- methylenetetrahydrofolate to serine and THF. Serine methyltransferase has several side reactions including the reversible cleavage of 3-hydroxyacids to glycine and an aldehyde, and the hydrolysis of 5,10-methenyl-THF to 5-formyl-THF. This enzyme is encoded by glyA of E. coli (Plamann et al, Gene 22:9-18 (1983)). Serine hydroxymethyltranferase enzymes of S. cerevisiae include SHM1 (mitochondrial) and SHM2 (cytosolic) (McNeil et al, J Biol Chem 269:9155-65 (1994)). Similar enzymes have been studied in Corynebacterium glutamicum and

Methylobacterium extorquens (Chistoserdova et al, J Bacteriol 176:6759-62 (1994); Schweitzer et al, JBiotechnol 139:214-21 (2009)).

SHM2 NP 013159.1 6323087 Saccharomyces cerevisiae glyA AAA64456.1 496116 Methylobacterium extorquens glyA AAK60516.1 14334055 Corynebacterium glutamicum

FIG. 5, Step N - Serine deaminase (EFR10)

[0435] Serine can be deaminated to pyruvate by serine deaminase. Serine deaminase enzymes are present in several organisms including Clostridium acidurici (Carter, et al., 1972, J Bacteriol., 109(2) 757-763), Escherichia coli (Cicchillo et al., 2004, J Biol Chem., 279(31) 32418-25), and Corneybacterium sp. (Netzer et al., Appl Environ Microbiol. 2004

Dec;70(12):7148-55).

FIG. 5, Step O - Methylenetetrahydrofolate reductase (EFR11)

[0436] In M. thermoacetica, this enzyme is oxygen-sensitive and contains an iron-sulfur cluster (Clark and Ljungdahl, J. Biol. Chem. 259: 10845-10849 (1984). This enzyme is encoded by metF in E. coli (Sheppard et al, J. Bacteriol. 181 :718-725 (1999) and CHY_1233 in C. hydrogenoformans (Wu et al., PLoS Genet. 1 :e65 (2005). The M. thermoacetica genes, and its C. hydrogenoformans counterpart, are located near the CODH/ACS gene cluster, separated by putative hydrogenase and heterodisulfide reductase genes. Some additional gene candidates found bioinformatically are listed below. In Acetobacterium woodii metF is coupled to the Rnf complex through RnfC2 (Poehlein et al, PLoS One. 7:e33439). Homologs of RnfC are found in other organisms by blast search. The Rnf complex is known to be a reversible complex (Fuchs (2011) Annu. Rev. Microbiol. 65:631-658). Moth_1 192 YP 430049.1 83590040 Moorella thermoacetica metF NP_418376.1 16131779 Escherichia coli

CHY 1233 YP 360071.1 78044792 Carboxydothermus

hydrogenoformans

CLJU_c37610 YP 003781889.1 300856905 Clostridium ljungdahlii

DSM 13528

DesfrDRAFT_3717 ZP_07335241.1 303248996 Desulfovibrio

fructosovorans JJ

CcarbDRAFT_2950 ZP 05392950.1 255526026 Clostridium

carboxidivoransP 7

Ccel74_010100023124 ZP 07633513.1 307691067 Clostridium cellulovorans

743B

Cphy_31 10 YP 001560205.1 160881237 Clostridium

phytofermentans ISDg

FIG. 5, Step P - Acetyl-CoA synthase (EFR12)

[0437] Acetyl-CoA synthase is the central enzyme of the carbonyl branch of the Wood- Ljungdahl pathway. It catalyzes the synthesis of acetyl-CoA from carbon monoxide, coenzyme A, and the methyl group from a methylated corrinoid-iron-sulfur protein. The corrinoid-iron- sulfur-protein is methylated by methyltetrahydrofolate via a methyltransferase. Expression in a foreign host entails introducing one or more of the following proteins and their corresponding activities: Methyltetrahydrofolate orrinoid protein methyltransferase (AcsE), Corrinoid iron- sulfur protein (AcsD), Nickel-protein assembly protein (AcsF), Ferredoxin (Orf7), Acetyl-CoA synthase (AcsB and AcsC), Carbon monoxide dehydrogenase (AcsA), and Nickel-protein assembly protein {CooC).

[0438] The genes used for carbon-monoxide dehydrogenase/acetyl-CoA synthase activity typically reside in a limited region of the native genome that can be an extended operon

(Ragsdale, S.W., Crit. Rev. Biochem. Mol. Biol. 39: 165-195 (2004); Morton et al., J. Biol. Chem. 266:23824-23828 (1991); Roberts et al, Proc. Natl. Acad. Sci. U.S.A. 86:32-36 (1989). Each of the genes in this operon from the acetogen, M. thermoacetica, has already been cloned and expressed actively in E. coli (Morton et al. supra; Roberts et al. supra; Lu et al., J. Biol. Chem. 268:5605-5614 (1993). The protein sequences of these genes can be identified by the following GenBank accession numbers. Protein ( ,cn Bank ID GI number Organism

AcsE YP 430054 83590045 Moorella thermoacetica

AcsD YP 430055 83590046 Moorella thermoacetica

AcsF YP 430056 83590047 Moorella thermoacetica

Orf7 YP 430057 83590048 Moorella thermoacetica

AcsC YP 430058 83590049 Moorella thermoacetica

AcsB YP 430059 83590050 Moorella thermoacetica

AcsA YP 430060 83590051 Moorella thermoacetica

CooC YP 430061 83590052 Moorella thermoacetica

[0439] The hydrogenic bacterium, Carboxydothermus hydrogenoformans, can utilize carbon monoxide as a growth substrate by means of acetyl-CoA synthase (Wu et al., PLoS Genet. 1 :e65 (2005)). In strain Z-2901, the acetyl-CoA synthase enzyme complex lacks carbon monoxide dehydrogenase due to a frameshift mutation (Wu et al. supra (2005)) , whereas in strain DSM 6008, a functional unframeshifted full-length version of this protein has been purified

(Svetlitchnyi et al, Proc. Natl. Acad. Sci. U.S.A. 101 :446-451 (2004)). The protein sequences of the C. hydrogenoformans genes from strain Z-2901 can be identified by the following GenBank accession numbers.

[0440] Homologous ACS/CODH genes can also be found in the draft genome assembly of Clostridium carboxidivorans P7. CooC ZP 05392945.1 255526021 Clostridium carboxidivorans P7

AcsF ZP 05392952.1 255526028 Clostridium carboxidivorans P7

AcsD ZP 05392953.1 255526029 Clostridium carboxidivorans P7

AcsC ZP 05392954.1 255526030 Clostridium carboxidivorans P7

AcsE ZP 05392955.1 255526031 Clostridium carboxidivorans P7

AcsB ZP 05392956.1 255526032 Clostridium carboxidivorans P7

Orf7 ZP 05392958.1 255526034 Clostridium carboxidivorans P7

[0441] The methanogenic archaeon, Methanosarcina acetivorans, can also grow on carbon monoxide, exhibits acetyl-CoA synthase/carbon monoxide dehydrogenase activity, and produces both acetate and formate (Lessner et al., Proc. Natl. Acad. Sci. U.S.A. 103: 17921-17926 (2006)). This organism contains two sets of genes that encode ACS/CODH activity (Rother and Metcalf, Proc. Natl. Acad. Sci. U.S.A. 101 : 16929-16934 (2004)). The protein sequences of both sets of M. acetivorans genes are identified by the following GenBank accession numbers.

[0442] The AcsC, AcsD, AcsB, AcsEps, and AcsA proteins are commonly referred to as the gamma, delta, beta, epsilon, and alpha subunits of the methanogenic CODH/ACS. Homo logs to the epsilon encoding genes are not present in acetogens such as M. thermoacetica or

hydrogenogenic bacteria such as C. hydrogenoformans . Hypotheses for the existence of two active CODH/ACS operons in acetivorans include catalytic properties {i.e., K _m , V _max , k _cat ) that favor carboxidotrophic or aceticlastic growth or differential gene regulation enabling various stimuli to induce CODH/ACS expression (Rother et al, Arch. Microbiol. 188:463-472 (2007)).

FIG. 5, Step Q - Pyruvate Formate Lyase (EFR13)

[0443] Pyruvate formate-lyase (PFL, EC 2.3.1.54), encoded by pflB in E. coli, can convert pyruvate into acetyl-CoA and formate. The activity of PFL can be enhanced by an activating enzyme encoded by pflA (Knappe et al, Proc.Natl.Acad.Sci U.S.A 81 : 1332-1335 (1984); Wong et al, Biochemistry 32: 14102-14110 (1993)). Keto-acid formate-lyase (EC 2.3.1.-), also known as 2-ketobutyrate formate-lyase (KFL) and pyruvate formate-lyase 4, is the gene product of tdcE in E. coli. This enzyme catalyzes the conversion of 2-ketobutyrate to propionyl-CoA and formate during anaerobic threonine degradation, and can also substitute for pyruvate formate- lyase in anaerobic catabolism (Simanshu et al, JBiosci. 32: 1195-1206 (2007)). The enzyme is oxygen-sensitive and, like PflB, can require post-translational modification by PFL-AE to activate a glycyl radical in the active site (Hesslinger et al, Mol.Microbiol 27:477-492 (1998)). A pyruvate formate-lyase from Archaeglubus fulgidus encoded by pflD has been cloned, expressed in E. coli and characterized (Lehtio et al, Protein Eng Des Sel 17:545-552 (2004)). The crystal structures of the A.fulgidus and E. coli enzymes have been resolved (Lehtio et al, J Mol.Biol. 357:221-235 (2006); Leppanen et al, Structure. 7:733-744 (1999)). Additional PFL and PFL-AE candidates are found in Lactococcus lactis (Melchiorsen et al, Appl Microbiol Biotechnol 58:338-344 (2002)), and Streptococcus mutans (Takahashi-Abbe et al,

Oral. Microbiol Immunol. 18:293-297 (2003)), Chlamydomonas reinhardtii (Hemschemeier et al, Eukaryot.Cell 7:518-526 (2008b); Atteia et al, J.Biol.Chem. 281 :9909-9918 (2006)) and Clostridium pasteurianum (Weidner et al, J Bacteriol. 178:2440-2444 (1996)). pflA NP_415422.1 16128869 Escherichia coli

tdcE AAT48170.1 48994926 Escherichia coli

pflD NP_070278.1 11499044 Archaeglubus fulgidus

Pfl CAA03993 2407931 Lactococcus lactis

Pfl BAA09085 1129082 Streptococcus mutans

PFL1 XP OO 1689719.1 159462978 Chlamydomonas reinhardtii

pflAl XP 001700657.1 159485246 Chlamydomonas reinhardtii

Pfl Q46266.1 2500058 Clostridium pasteurianum

Act CAA63749.1 1072362 Clostridium pasteurianum

FIG. 5, Step R - Pyruvate Dehydrogenase (EFR14A), Pyruvate Ferredoxin Oxidoreductase (EFR14B), Pyruvate:NADP+ Oxidoreductase (EFR14C)

[0444] The pyruvate dehydrogenase (PDH) complex catalyzes the conversion of pyruvate to acetyl-CoA (Figure 2H). The E. coli PDH complex is encoded by the genes aceEF and IpdA.

Enzyme engineering efforts have improved the E. coli PDH enzyme activity under anaerobic conditions (Kim et al., J.Bacteriol. 190:3851-3858 (2008); Kim et al., Appl.Environ.Microbiol.

73: 1766-1771 (2007); Zhou et al., Biotechnol.Lett. 30:335-342 (2008)). In contrast to the E. coli

PDH, the B. subtilis complex is active and required for growth under anaerobic conditions

(Nakano et al., 179:6749-6755 (1997)). The Klebsiella pneumoniae PDH, characterized during growth on glycerol, is also active under anaerobic conditions (Menzel et al., 56: 135-142

(1997)). Crystal structures of the enzyme complex from bovine kidney (Zhou et al., 98: 14802-

14807 (2001)) and the E2 catalytic domain from Azotobacter vinelandii are available (Mattevi et al., Science. 255: 1544-1550 (1992)). Some mammalian PDH enzymes complexes can react on alternate substrates such as 2-oxobutanoate. Comparative kinetics of Rattus norvegicus PDH and BCKAD indicate that BCKAD has higher activity on 2-oxobutanoate as a substrate (Paxton et al., Biochem.J. 234:295-303 (1986)). The S. cerevisiae PDH complex canconsist of an E2

(LAT1) core that binds El (PDA1, PDB1), E3 (LPD1), and Protein X (PDX1) components

(Pronk et al., Yeast 12: 1607-1633 (1996)). The PDH complex of S. cerevisiae is regulated by phosphorylation of El involving PKP1 (PDH kinase I), PTC5 (PDH phosphatase I), PKP2 and

PTC6. Modification of these regulators may also enhance PDH activity. Coexpression of lipoyl ligase (LplA of E. coli and AIM22 in S. cerevisiae) with PDH in the cytosol may be necessary for activating the PDH enzyme complex. Increasing the supply of cytosolic lipoate, either by modifying a metabolic pathway or media supplementation with lipoate, may also improve PDH activity.

[0445] As an alternative to the large multienzyme PDH complexes described above, some organisms utilize enzymes in the 2-ketoacid oxidoreductase family (OFOR) to catalyze acylating oxidative decarboxylation of 2-keto-acids. Unlike the PDH complexes, PFOR enzymes contain iron-sulfur clusters, utilize different cofactors and use ferredoxin or flavodixin as electron acceptors in lieu of NAD(P)H. Pyruvate ferredoxin oxidoreductase (PFOR) can catalyze the oxidation of pyruvate to form acetyl-CoA (Figure 2H). The PFOR from Desulfovibrio africanus has been cloned and expressed in E. coli resulting in an active recombinant enzyme that was stable for several days in the presence of oxygen (Pieulle et al., J Bacteriol. 179:5684-5692 (1997)). Oxygen stability is relatively uncommon in PFORs and is believed to be conferred by a 60 residue extension in the polypeptide chain of the D. africanus enzyme. The M. thermoacetica PFOR is also well characterized (Menon et al., Biochemistry 36:8484-8494 (1997)) and was even shown to have high activity in the direction of pyruvate synthesis during autotrophic growth (Furdui et al., J Biol Chem. 275:28494-28499 (2000)). Further, E. coli possesses an

uncharacterized open reading frame, ydbK, that encodes a protein that is 51% identical to the M. thermoacetica PFOR. Evidence for pyruvate oxidoreductase activity in E. coli has been described (Blaschkowski et al., Eur. J Biochem. 123:563-569 (1982)). Several additional PFOR enzymes are described in Ragsdale, Chem. Rev. 103:2333-2346 (2003). Finally, flavodoxin reductases (e.g., fqrB from Helicobacter pylori or Campylobacter jejuni (St Maurice et al., J. Bacteriol. 189:4764-4773 (2007))) or Rnf-type proteins (Seedorf et al, Proc.Natl.Acad.Sci. U S.A. 105:2128-2133 (2008); Herrmann et al., J.Bacteriol. 190:784-791 (2008)) provide a means to generate NADH or NADPH from the reduced ferredoxin generated by PFOR. These proteins are identified below.

[0446] Pyruvate :NADP oxidoreductase (PNO) catalyzes the conversion of pyruvate to acetyl-CoA. This enzyme is encoded by a single gene and the active enzyme is a homodimer, in contrast to the multi-subunit PDH enzyme complexes described above. The enzyme from Euglena gracilis is stabilized by its cofactor, thiamin pyrophosphate (Nakazawa et al, Arch Biochem Biophys 411 : 183-8 (2003)). The mitochondrial targeting sequence of this enzyme should be removed for expression in the cytosol. The PNO protein of E. gracilis and other NADP-dependant pyruvate :NADP+ oxidoreductase enzymes are listed in the table below.

FIG. 5, Step S - Formate Dehydrogenase (EFR15)

[0447] Formate dehydrogenase (FDH) catalyzes the reversible transfer of electrons from formate to an acceptor. Enzymes with FDH activity utilize various electron carriers such as, for example, NADH (EC 1.2.1.2), NADPH (EC 1.2.1.43), quinols (EC 1.1.5.6), cytochromes (EC 1.2.2.3) and hydrogenases (EC 1.1.99.33). FDH enzymes have been characterized from

Moorella thermoacetica (Andreesen and Ljungdahl, J Bacteriol 116:867-873 (1973); Li et al., J Bacteriol 92:405-412 (1966); Yamamoto et al, J Biol Chem. 258: 1826-1832 (1983). The loci, Moth_2312 is responsible for encoding the alpha subunit of formate dehydrogenase while the beta subunit is encoded by Moth_2314 (Pierce et al., Environ Microbiol (2008)). Another set of genes encoding formate dehydrogenase activity with a propensity for C0 ₂ reduction is encoded by Sfum_2703 through Sfum_2706 in Syntrophobacter fumaroxidans (de Bok et al., Eur J Biochem. 270:2476-2485 (2003)); Reda et al., PNAS 105: 10654-10658 (2008)). A similar set of genes presumed to carry out the same function are encoded by CHY 0731, CHY 0732, and CHY_0733 in C. hydrogenoformans (Wu et al., PLoS Genet 1 :e65 (2005)). Formate dehydrogenases are also found many additional organisms including C. carboxidivorans P7, Bacillus methanolicus, Burkholderia stabilis, Moorella thermoacetica ATCC 39073, Candida boidinii, Candida methylica, and Saccharomyces cerevisiae S288c. The soluble formate dehydrogenase from Ralstonia eutropha reduces NAD ⁺ (fdsG, -B, -A, -C, -D) (Oh and Bowien, 1998) Protein GenBank ID GI Number Organism

Moth_2312 YP_431142 148283121 Moorella thermoacetica

Moth_2314 YP_431144 83591135 Moorella thermoacetica

Sfum_2703 YP 846816.1 116750129 Syntrophobacter fumaroxidans

Sfum_2704 YP_846817.1 116750130 Syntrophobacter fumaroxidans

Sfum_2705 YP_846818.1 116750131 Syntrophobacter fumaroxidans

Sfum_2706 YP 846819.1 116750132 Syntrophobacter fumaroxidans

CHY 0731 YP_359585.1 78044572 Carboxydothermus

hydrogenoformans

CHY 0732 YP 359586.1 78044500 Carboxydothermus

hydrogenoformans

CHY 0733 YP_359587.1 78044647 Carboxydothermus

hydrogenoformans

CcarbDRAFT 0901 ZP 05390901.1 255523938 Clostridium carboxidivorans P7

CcarbDRAFT 4380 ZP 05394380.1 255527512 Clostridium carboxidivorans P7 fdhA, EIJ82879.1 387590560 Bacillus methanolicus MGA3

MGA3 06625

fdhA, PB1 11719 ZP 10131761.1 387929084 Bacillus methanolicus PB1 fdhD, EIJ82880.1 387590561 Bacillus methanolicus MGA3

MGA3 06630

fdhD, PB 1 11724 ZP 10131762.1 387929085 Bacillus methanolicus PB1 fdh ACF35003. 194220249 Burkholderia stabilis

FDH1 AAC49766.1 2276465 Candida boidinii

Fdh CAA57036.1 1181204 Candida methylica

FDH2 P0CF35.1 294956522 Saccharomyces cerevisiae S288c

FDH1 NP O 15033.1 6324964 Saccharomyces cerevisiae S288c

[0448] Throughout this application various publications have been referenced. The disclosures of these publications in their entireties, including GenBank and GI number publications, are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples and embodiments provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.