COMPOSITIONS AND METHODS REGARDING DIRECT

NADH UTILIZATION TO PRODUCE 3- HYDROXYPROPIONIC ACID, DERIVED CHEMICALS AND FURTHER DERIVED PRODUCTS

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 61/536,464, filed

September 19, 2011, and U.S. Provisional Application No. 61/536,558, filed September 19, 2011, and U.S. Provisional Application No. 61/536,539, filed September 19, 2011, each of which is incorporated herein by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with Government support under DE-AR0000088 awarded by the United States Department of Energy. The Government has certain rights in this invention.

FIELD OF THE INVENTION

[0003] The present invention has to deal with the production of malonate semialdehyde, 3- hydroxypropionate, and products derived thereof using a combinations of malonyl CoA reductase domains and 3 -HP dehydrogenase domains with altered cofactor specificities for NADH and NADPH. The present invention also has to deal with the methods for engineering these proteins, the genetically engineered organisms used for production of products with these altered enzymes, the methods for producing such organisms. The present invention deals with the production of malonic acid. The present invention also deals with the methods for engineering the genetically engineered organisms used for production.

REFERENCE TO A SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 19, 2012, is named 34246761601, is a .txt file, and is 795 kilobytes in size.

BACKGROUND OF THE INVENTION

[0005] Efforts are increasing to develop microbial fermentation alternatives to production of industrial chemicals and fuels that currently are largely derived from petroleum. These efforts include the use of metabolic engineering approaches to improve performance of such fermentation alternatives.

[0006] As fermentation models are refined toward reaching economic viability on an at-cost replacement basis for petro^based chemicals, microbial performance, including production rate and efficiency, remains as a target for improvement. The performance based on any one improvement often requires coordination with other modifications. [0007] Notwithstanding advances in the field, there remains a need to further improve microbial performance particularly with regard to improving production of 3-hydroxypropionic acid or malonic acid, which can be converted to many useful monomers (including acrylic acid), industrial chemicals, including polymers, and consumer products.

SUMMARY OF THE INVENTION

[0008] The present invention regards microbial production of malonate semialdehyde, 3- hydroxypropionate, and products derived thereof, including using a combinations of malonyl CoA reductase domains and 3-HP dehydrogenase domains with altered cofactor specificities for NADH and NADPH. Embodiments of the present invention also include methods for engineering these proteins, including the polynucleotides that encode them, the genetically engineered microorganisms used for production with these altered enzymes, methods for producing such organisms, and methods of making compounds, downstream compounds, and downstream, products.

[0009] Various embodiments of the invention are directed to a method of making 3-hydroxypropionic acid comprising: combining in a vessel media, a carbon source, and any recombinant microorganism as described herein; maintaining the vessel under suitable conditions to obtain a detectable amount of 3- hydroxypionic acid or its salt in a fermentation broth; and recovering the 3-hydroxypropionic acid or its salt ("3-HP").

[0010] Various embodiments of the invention comprise an isolated or recombinant polynucleotide encoding a polypeptide that exhibits malonyl-CoA reductase activity, 3-HP dehydrogenase activity, or both, wherein the polypeptide so encoded is selected from the group consisting of:

any one of SEQ ID NOs:002 to 057;

any conservatively modified variants of SEQ ID NOs:002 to 057; and a polypeptide having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least

99% identity with a portion of any of SEQ ID NOs:002 to 057.

[0011] Further embodiments are directed to an isolated or recombinant polynucleotide comprising a nucleic acid sequence that hybridizes under stringent conditions to any one of the above described polynucleotides. More specific embodiments of the above-described isolated or recombinant polynucleotides are where the indicated portion is at least 200, at least 300, at least 400, at least 450, at least 500, or at least 550 contiguous amino acids, or wherein such portion is between about 200, 300, 400 and 500, 500 and 600, 600 and 700, or 700 and 1220 contiguous amino acids. More particularly, in various embodiments the portion is selected from the group consisting of SEQ. ID NOs.: 070, 071, 072, 076, 077, 078, 079, and conservatively modified variants thereof.

[0012] Also, in various embodiments the above-described isolated or recombinant polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 059, 059, 060, 061, and 062, and optionally SEQ ID NO: 065. Also, in various embodiments any of the above-described isolated or recombinant polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 066, 067, 068, and 069.

[0013] Embodiments variously comprise any of the above-described isolated or recombinant polynucleotides that encode a polypeptide that exhibits malonyl-CoA reductase activity, and/or that encodes a polypeptide that exhibits 3-HP dehydrogenase activity.

[0014] Embodiments of the invention comprise any of the following:

a mutant malonyl-CoA reductase encoded by any of the above polynucleotides, wherein the mutant malonyl-CoA reductase preferentially utilizes NADH rather than NADPH;

a mutant malonyl-CoA reductase encoded by any of the above polynucleotides, wherein the mutant malonyl-CoA reductase demonstrates a switch in cofactor preference fromNADPH to NADH as compared to a corresponding wild-type malonyl-CoA reductase;

a mutant 3-HP dehydrogenase encoded by any of the above polynucleotides, wherein the mutant 3-HP dehydrogenase preferentially utilizes NADH rather than NADPH;

a mutant 3-HP dehydrogenase encoded by any of the above polynucleotides, wherein the mutant 3-HP dehydrogenase demonstrates a switch in cofactor preference from NADPH to NADH as compared to a corresponding wild-type 3-HP dehydrogenase.

[0015] Embodiments of the invention also comprise an isolated or recombinant polypeptide encoded by any of the above-described polynucleotides.

[0016] Further, embodiments are directed to recombinant microorganisms each comprising at least one of the above-'describedpolynucleotides and/or at least one of the above-described polypeptides.

[0017] More particular embodiments include such recombinant microorganism wherein the

microorganism has one or more of: an increased NADH dependent malonyl-CoA reductase activity; an increased NADH dependent 3-HP dehydrogenase activity. Without limitation, polypeptides in such microorganisms may be selected from the group consisting of SEQ ID NOs: 082-089, 091-095, conservatively modified variants thereof, functional variants and functional homologs thereof.

[0018] Further, in various embodiments a polypeptide in any such microorganism comprises a fusion of any two or more of the polypeptides of any of the above claims.

[0019] Further, in various embodiments the recombinant microorganism is additionally engineered for production of 3- hydroxypropionic acid (3-HP), such as by any of the general or specific approaches described herein, including the figures, and also including in particular references that are incorporated by reference herein.

[0020] Any of the above recombinant microorganisms may have an increased utilization of NADH for 3- HP production, and more particularly in various embodiments the increased NADH utilization for 3-HP production is at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 percent greater than NADH utilization for 3-HP production in a microorganism lacking the polynucleotide(s). [0021] Also, any of the above-described recombinant microorganisms may comprise one or more of the following modifications:

a disruption of one or more of the enzymes of the fatty acid synthase system;

an increase in overall activity of acetyl-CoA carboxylase;

a disruption in a gene conferring lactic acid production;

a disruption in a gene conferring acetic acid production;

a disruption in a gene conferring ethanol production;

a disruption in a gene conferring methylglyoxal production;

a disruption in a gene conferring formate production;

a disruption in a gene conferring metabolism of 3 -HP to 3-hydroxypropionaldehyde a disruption in a gene conferring metabolism of 3-HP to malonate semialdehyde.

[0022] Also, any of the above-described recombinant microorganisms may further comprise a modification to increase expression of a transhydrogenase, or alternatively may have transhydrogenase function that remains unmodified or is decreased.

[0023] Also, any of the above-described recombinant microorganisms may additionally comprising further modification(s) to decrease activity of one or more of the activities in Table 10, in any

combination.

[0024] Also, any of the above-described recombinant microorganisms may comprise any one or more of the modifications of FIGs. 2A-G.

[0025] Any of the above-described recombinant microorganisms has or demonstrates NADH dependent malonyl-CoA reductase activity measured in cell lysate that is greater than 0.01 U/mg of total cell protein.

[0026] Any of the above-described recombinant microorganisms has or demonstrates NADH dependent 3-HP dehydrogenase activity measured in cell lysate is greater than 0.01 U/mg of total cell protein.

[0027] Any of the above-described recombinant microorganisms may further be additionally engineered for production of 3-hydroxypropionaldehyde, such as by modification to provide increased activity of 3- hydroxypropionaldehyde dehydrogenase, which may more particularly comprise providing increased activity of NADH dependent 3- hydroxypropionaldehyde dehydrogenase, such as (but not limited to) providing increased activity of NADH dependent 3-hydroxypropionaldehyde dehydrogenase with a polypeptide encoded by the puuC gene of E. coli or conservatively modified variants or functional variants thereof.

[0028] Any of the above-described recombinant microorganisms, which comprises at least one of the polynucleotides or polypeptides described above, may be additionally engineered for production of 1,3 propanediol. For example, a recombinant microorganism may comprise increased activity of 1,3 propanediol dehydrogenase, more particularly an NADH dependent 1,3 propanediol dehydrogenase, such as (but not limited to) providing increased activity of an NADH dependent 1,3 propanediol dehydrogenase with a polypeptide encoded by the dhaT gene of a Clostridia species or conservatively modified variants or functional variants thereof. [0029] The recombinant microorganisms of the invention may be or may be developed from any microorganism species, including but not limited to those described and/or listed herein.

[0030] In various embodiments of the methods described herein a malonyl-CoA reductase uses at least 0.25 molecule of NADH for each molecule of 3-HP that is produced, and/or a 3-HP dehydrogenase uses at least 0.25 molecule of NADH for each molecule of 3-HP that is produced, and/or 3-HP is produced at a yield of greater than 50 percent theoretical. In various embodiments any such method may additionally comprise any one or more of the steps of converting the 3-HP to acrylic acid, to an acrylic acid product, and to an acrylic acid-based consumer product (including those described in this specification).

[0031] Further embodiments of the invention are directed to culture system comprising a carbon source in an aqueous medium and a recombinant microorganism as described above, wherein the recombinant organism is present in an amount selected from greater than 0.05 gDCW/L, 0.1 gDCW/L, greater than 1 gDCW/L, greater than 5 gDCW/L, greater than 10 gDCW/L, greater than 15 gDCW/L or greater than 20 gDCW/L. In various culture system embodiments the volume of the aqueous medium is selected from greater than 5 mL, greater than 100 mL, greater than 0.5L, greater than 1L, greater than 2 L, greater than 10 L, greater than 250 L, greater than 1000L, greater than 10,000L, greater than 50,000 L, greater than 100,000 L or greater than 200,000 L. In various culture system embodiments the volume of the aqueous medium is greater than 250 L and is contained within a steel vessel. In any such culture systems, in various embodiments the carbon source is selected from dextrose, sucrose, a pentose, a polyol, a hexose, both a hexose and a pentose, and combinations thereof.

[0032] The invention also regards any of the above culture systems wherein the pH of the aqueous medium is less than 7.5. The invention also regards any of the above culture systems wherein the culture system is aerated, and in more particular embodiments wherein the culture system is aerated with an oxygen transfer rate selected from:

greater than 0 mmole/L-hr of oxygen and less than 100 mmole/L-hr oxygen;

greater than 0 mmole/L-hr of oxygen and less than 50 mmole/L-hr oxygen; greater than 0 mmole/L-hr of oxygen and less than 20 mmole/L-hr oxygen; and greater than 0 mmole/L-hr of oxygen and less than 10 mmole/L-hr oxygen.

[0033] Any of the above-described culture systems may provide an aqueous broth that comprises:

a concentration of 3-hydroxypropionate selected from greater than 5g/L, greater than lOg/L, greater than 15 g/L, greater than 20g/L, greater than 25g/L, greater than 30g/L, greater than 35g/L, greater than 40g/L, greater than 50g/L, greater than 60g/L, greater than 70g/L, greater than 80g/L, greater than 90g/L, or greater than lOOg/L 3-hydroxypropionate; and optionally

a concentration of 1,3-propanediol selected from less than 30g/L; less than 20g/L; less than lOg/L; less than 5g/L; less than 1 g/L; or less than 0.5 g/L.

[0034] Any such aqueous may comprise an amount of biomass selected from less than 20 gDCW/L biomass, less than 15 gDCW/L biomass, less than 10 gDCW/L biomass, less than 5 gDCW/L biomass or less than 1 gDCW/L biomass. [0035] In various embodiments variations of the six-polypeptide putative phosphate binding loop sequence may be employed. In some of such embodiments arginine (R) is not substituted into position 2, and/or aspartic acid (D) is substituted into position 1.

[0036] Also, in various embodiments the production of malonate semialdehyde, 3-HP, and/or other products of interest is not linked to microorganism growth, that is to say, there are non-growth coupled embodiments in which production rate is not linked metabolically to cellular growth. Products that may be made from 3-HP using the embodiments herein include derived chemicals and derived products, such as those disclosed in Table 12, incorporated into this section.

INCORPORATION BY REFERENCE

[0037] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The novel features of the invention are set forth with particularity in the claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0039] FIG. 1A depicts representative metabolic pathways to produce end products (chemical products) produced from 3-HP which include, but are not limited to, 3-hydroxypropionaldehyde, 1,3 propanediol, poly-3-hydroxypropionate. FIG. IB depicts representative metabolic pathways to produce end products (chemical products) produced from malonate semialdehyde.

[0040] FIGs. 2A to 2G depict various genetic modifications that may be made in a microorganism cell according to various embodiments of the present invention. In various figures gene names such as those of E. coli are shown at certain enzymatic steps. These are provided as an example and are not meant to be limiting.

[0041] FIG. 3 depicts reactions of a representative fatty acid synthase complex, other reactions directed to embodiments of the present invention, and includes indications of effects of various inhibitors. The gene names of E. coli are shown at various enzymatic steps. These are provided as an example and are not meant to be limiting. A line indicating feedback inhibition also is shown.

[0042] FIG. 4 A, B, and C show a schematic of an entire process of converting biomass to a finished product.

[0043] FIG. 5 A and B show a schematic of production of a diaper which may be followed using downstream product(s) of the present invention. FIGs. 4A-C and 5A, B are meant to be exemplary and not limiting. [0044] FIG. 6 comprises a schematic showing truncated constructs of Chloroflexus aurantiacus bi- functional malonyl-CoA reductase enzyme in relationship to the full length protein.

[0045] FIG. 7 provides results of malonyl-CoA reductase (Reaction 1) specific activity of N-terminal end MCR truncations assayed without YdfG.

[0046] FIG. 8 provides results of malonyl-CoA reductase (Reaction 1) specific activity of N-terminal end MCR truncations assayed with YdfG.

[0047] FIG. 9 provides results of assays 3-HP dehydrogenase activity (Reaction 2) including for specific

C-terminal truncations of the full length bifunctional MCR protein. These results are of specific activities of whole cell lysates of cells expressing various 3-HP dehydrogenase domains.

[0048] FIG. 10 provides results of assays using plasmids able to express the putative NADH-specific loop variants (variant 1 thru 4) (SEQ ID NO: 046-049) of the malonyl CoA reductase domain.

[0049] FIG. 11 provides 3-HP GC-MS results regarding the assays described for FIG. 10.

[0050] FIG. 12 provides specific activity results for variants 5-8 which comprise the secondary mutation for malonyl-CoA reductase (Reaction 1). This secondary mutation was introduced to each of the mutations made to create variants 1 -4 for the same reaction.

[0051] FIG. 13 provides a sequence comparison of the cofactor binding regions of the malonyl-CoA reductase and 3-HP dehydrogenase domains of the Chloroflexus aurantiacus bi-functional malonyl-CoA reductase enzyme.

[0052] FIG. 14 provides results showing the NADH-specific activities and NADPH-specific activities of variants 9-12. FIG. 15 provides a calibration curve for 3-HP conducted with HPLC.

[0053] FIG. 16 provides a calibration curve for 3-HP conducted for GC/MS.

[0054] FIG. 17 provides a representative standard curve for the enzymatic assay for 3-HP.

[0055] FIG. 18 schematically depicts a first metabolic approach to production of malonate from malonyl-

CoA.

[0056] FIG. 19 schematically depicts a second metabolic approach to production of malonate from malonyl-CoA

[0057] FIG. 20 depicts reactions of a representative fatty acid synthase complex, other reactions directed to embodiments of the present invention, and includes indications of effects of various inhibitors. The gene names of E. coli are shown at various enzymatic steps. These are provided as an example and are not meant to be limiting. A line indicating feedback inhibition also is shown.

[0058] FIG. 21 depicts various genetic modifications that may be made in a microorganism cell according to various embodiments of the present invention. In various figures gene names such as those of E. coli are shown at certain enzymatic steps. These are provided as an example and are not meant to be limiting.

[0059] FIG. 22 depicts various genetic modifications that may be made in a microorganism cell according to various embodiments of the present invention. In various figures gene names such as those of E. coli are shown at certain enzymatic steps. These are provided as an example and are not meant to be limiting.

[0060] FIG. 23 depicts malonate and 3-HP production 20 hours after induction according to Example 6a.

DETAILED DESCRIPTION OF THE INVENTION

[0061] Abbreviations and Definitions

[0062] The meaning of abbreviations is as follows: "C" means Celsius or degrees Celsius, as is clear from its usage, DCW means dry cell weight, "s" means second(s), "min" means minute(s), "h," "hr," or "hrs" means hour(s), "psi" means pounds per square inch, "nm" means nanometers, "d" means day(s), "μΐ " or "uL" or "ul" means microliter(s), "mL" means milliliter(s), "L" means liter(s), "mm" means millimeter(s), "nm" means nanometers, "mM" means millimolar, "μΜ" or "uM" means micromolar, "M" means molar, "mmol" means millimole(s), "μιηοΐ" or "uMol" means micromole(s)", "g" means gram(s), "^g" or "ug" means microgram(s) and "ng" means nanogram(s), "PCRn" means polymerase chain reaction, "OD" means optical density, "OD 600" means the optical density measured at a photon wavelength of 600 nm, "kDa" means kilodaltons, "g" means the gravitation constant, "bp" means base pair(s), "kbp" means kilobase pair(s), "% w/v" means weight/volume percent, "% v/v" means

volume/volume percent, "IPTG" means isopropyl-d-D-thiogalactopyranoiside, "RBS" means ribosome binding site, "rpm" means revolutions per minute, "HPLC" means high performance liquid

chromatography, and "GC" means gas chromatography. As disclosed herein, "3-HP" and "3HP" means 3-hydroxypropionic acid. Also, 10"5 and the like are taken to mean 10 ⁵ and the like.

[0063] As used in the specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "expression vector" includes a single expression vector as well as a plurality of expression vectors, either the same (e.g., the same operon) or different; reference to "microorganism" includes a single microorganism as well as a plurality of microorganisms; and the like.

[0064] As used herein, dry cell weight (DCW) for E. coli strains is calculated as 0.41 times the measured OD ₆ oo value, based on baseline DCW to OD ₆ oo determinations.

[0065] Unless defined or used otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.

[0066] Any publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.

[0067] The term "microorganism" includes prokaryotic and eukaryotic microbial species from the Domains Archaea, Bacteria and Eukarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. The terms "microbial cells" and "microbes" are used interchangeably with the term microorganism.

[0068] Various primers may be used in the methods of making and using embodiments of the invention; such primers generally are synthetic polynucleotide, and more particularly synthetic oligonucleotide, constructs. Thus, it is noted that all primers disclosed herein are artificial sequences.

[0069] As used herein, "fatty acid synthase," whether followed by "pathway," "system," or "complex," is meant to refer to a metabolic pathway, often involving cyclic reactions to elongate to biosynthesize fatty acids in a host cell. It is noted that this may also be referred to as a "fatty acid synthesis," a "fatty acid biosynthesis," (or a "fatty acid synthetase") "pathway," "system," or "complex."

[0070] As used herein, "reduced enzymatic activity," "reducing enzymatic activity," and the like is meant to indicate that a microorganism cell's, or an isolated enzyme, exhibits a lower level of activity than that measured in a comparable cell of the same species or its native enzyme. That is, enzymatic conversion of the indicated substrate(s) to indicated product(s) under known standard conditions for that enzyme is at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 percent less than the enzymatic activity for the same biochemical conversion by a native (non-modified) enzyme under a standard specified condition. This term also can include elimination of that enzymatic activity. That is, the term "reduction" or "to reduce" when used in such phrases and their grammatical equivalents is intended to encompass a complete elimination of such conversion(s). A cell having reduced enzymatic activity of an enzyme can be identified using any method known in the art. For example, enzyme activity assays can be used to identify cells having reduced enzyme activity. See, for example, Enzyme Nomenclature, Academic Press, Inc., New York 2007.

[0071] The term "heterologous DNA," "heterologous polynucleotide," "heterologous nucleic acid sequence," and the like as used herein refers to a nucleic acid sequence wherein at least one of the following is true: (a) the sequence of nucleic acids is foreign to (i.e., not naturally found in) a given host microorganism; (b) the sequence may be naturally found in a given host microorganism, but in an unnatural (e.g., greater than expected) amount; or (c) the sequence of nucleic acids comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, regarding instance (c), a heterologous nucleic acid sequence that is recombinantly produced will have two or more sequences from unrelated genes arranged to make a new functional nucleic acid.

[0072] The term "heterologous" is intended to include the term "exogenous" as the latter term is generally used in the art. With reference to the host microorganism's genome prior to the introduction of a heterologous nucleic acid sequence, the nucleic acid sequence that codes for the enzyme is heterologous (whether or not the heterologous nucleic acid sequence is introduced into that genome).

[0073] By "increase production," "increase the production," and like terms is meant to increase the quantity of one or more of enzymes, the enzymatic activity, the enzymatic specificity, and/or the overall flux through an enzymatic conversion step, biosynthetic pathway, or portion of a biosynthetic pathway. A discussion of non-limiting genetic modification techniques is discussed, infra, which may be used either for increasing or decreasing a particular enzyme's quantity, activity, specificity, flux, etc.

[0074] As used herein, the terms "disrupt," "disruption," "gene disruption," or grammatical equivalents thereof (and including "to disrupt enzymatic function," "disruption of enzymatic function," and the like), are intended to mean a genetic modification to a microorganism that renders the encoded gene product as having a reduced polypeptide activity compared with polypeptide activity in or from a microorganism cell not so modified. The genetic modification can be, for example, deletion of the entire gene, deletion or other modification of a regulatory sequence required for transcription or translation, deletion of a portion of the gene which results in a truncated gene product (e.g., enzyme) or by any of various mutation strategies that reduces activity (including to no detectable activity level) the encoded gene product. A disruption may broadly include a deletion of all or part of the nucleic acid sequence encoding the enzyme, and also includes, but is not limited to other types of genetic modifications, e.g., introduction of stop codons, frame shift mutations, introduction or removal of portions of the gene, and introduction of a degradation signal, those genetic modifications affecting m NA transcription levels and/or stability, and altering the promoter or repressor upstream of the gene encoding the enzyme.

[0075] In various contexts, a gene disruption is taken to mean any genetic modification to the DNA, mRNA encoded from the DNA, and the corresponding amino acid sequence that results in reduced polypeptide activity. Many different methods can be used to make a cell having reduced polypeptide activity. For example, a cell can be engineered to have a disrupted regulatory sequence or polypeptide-encoding sequence using common mutagenesis or knock-out technology. See, e.g., Methods in Yeast Genetics (1997 edition), Adams et al., Cold Spring Harbor Press (1998). One particularly useful method of gene disruption is complete gene deletion because it reduces or eliminates the occurrence of genetic reversions in the genetically modified microorganisms of the invention. Accordingly, a disruption of a gene whose product is an enzyme thereby disrupts enzymatic function. Alternatively, antisense technology can be used to reduce the activity of a particular polypeptide. For example, a cell can be engineered to contain a cDNA that encodes an antisense molecule that prevents a polypeptide from being translated. Further, gene silencing can be used to reduce the activity of a particular polypeptide.

[0076] The term "antisense molecule" as used herein encompasses any nucleic acid molecule or nucleic acid analog (e.g., peptide nucleic acids) that contains a sequence that corresponds to the coding strand of an endogenous polypeptide. An antisense molecule also can have flanking sequences (e.g., regulatory sequences). Thus, antisense molecules can be ribozymes or antisense oligonucleotides.

[0077] As used herein, a ribozyme can have any general structure including, without limitation, hairpin, hammerhead, or axhead structures, provided the molecule cleaves RNA. [0078] Bio-production and other culture of microorganisms, as used herein, may be aerobic,

microaerobic, or anaerobic. As used herein, the language "sufficiently identical" refers to proteins or portions thereof that have amino acid sequences that include a minimum number of identical or equivalent amino acid residues when compared to an amino acid sequence of the amino acid sequences provided in this application (including the SEQ ID Nos./sequence listings) such that the protein or portion thereof is able to achieve the respective enzymatic reaction and/or other function. To determine whether a particular protein or portion thereof is sufficiently homologous may be determined by an assay of enzymatic activity, such as those commonly known in the art.

[0079] Descriptions and methods for sequence identity and homology are intended to be exemplary and it is recognized that these concepts are well-understood in the art. Further, it is appreciated that nucleic acid sequences may be varied and still encode an enzyme or other polypeptide exhibiting a desired functionality, and such variations are within the scope of the present invention. Also, it is intended that the phrase "equivalents thereof is mean to indicate functional equivalents of a referred to gene, enzyme or the like. Such an equivalent may be for the same species or another species, such as another microorganism species.

[0080] Further to nucleic acid sequences, a nucleic acid is "hybridizable" to another nucleic acid when a single stranded form of the nucleic acid can anneal to the other nucleic acid under appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook (1989) supra, (see in particular chapters 9 and 11), incorporated by reference to such teachings. Low stringency hybridization conditions correspond to a Tm of 55°C (for example 5xSSC, 0.1% SDS, 0.25 milk and no formamide or 5xSSC, 0.5% SDS and 30% formamide). Moderate stringency hybridization conditions correspond for example, to Tm of 60°C (for example 6xSSC, 0.1%> SDS, 0.05%> milk with or without formamide, and stringent hybridization conditions correspond for example, to a Tm of 65° C. and O. lxSSC and 0.1% SDS. For various embodiments of the invention a sequence of interest may be hybridizable under any such stringency condition -low, moderate or high. The term "identified enzymatic functional variant" means a polypeptide that is determined to possess an enzymatic activity and specificity of an enzyme of interest but which has an amino acid sequence different from such enzyme of interest. A corresponding "variant polynucleotide" (also, "variant nucleic acid sequence") may be constructed that is determined to encode such an identified enzymatic functional variant. These may be identified and/or developed from orthologs, paralogs, or nonorthologous gene displacements. Also, it is recognized that a subset within such functional variants comprises conservatively modified variants of sequences provided herein.

[0081] The use of the phrase "segment of interest" is meant to include both a gene and any other nucleic acid sequence segment of interest. One example of a method used to obtain a segment of interest is to acquire a culture of a microorganism, where that microorganism's genome includes the gene or nucleic acid sequence segment of interest. When the genetic modification of a gene product, i.e., an enzyme, is referred to herein, including the claims, it is understood that the genetic modification is of a nucleic acid sequence, such as or including the gene, that normally encodes the stated gene product, i.e., the enzyme.

[0082] The ability to genetically modify a host cell is essential for the production of any genetically modified (recombinant) microorganism. The mode of gene transfer technology may be by

electroporation, conjugation, transduction, or natural transformation. A broad range of host conjugative plasmids and drug resistance markers are available. The cloning vectors are tailored to the host organisms based on the nature of antibiotic resistance markers that can function in that host. Also, as disclosed herein, a genetically modified (recombinant) microorganism may comprise modifications other than via plasmid introduction, including modifications to its genomic DNA.

[0083] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature, such as, MOLECULAR CLONING: A LABORATORY MANUAL, second edition (Sambrook et al., 1989) Cold Spring Harbor Laboratory Press; CURRENT PROTOCOLS IN

MOLECULAR BIOLOGY (F. M. Ausubel et al., eds., 1987 and annual updates);

OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait, ed., 1984); PCR: THE POLYMERASE CHAIN REACTION, (Mullis et al., eds., 1994); MANUAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY, Second Edition (A. L. Demain, et al., eds. 1999); MANUAL OF METHODS FOR GENERAL BACTERIOLOGY (Phillip Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W.

Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds.), pp. 210-213 American Society for Microbiology, Washington, D.C. and BIOTECHNOLOGY: A TEXTBOOK OF INDUSTRIAL

MICROBIOLOGY, (Thomas D. Brock) Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass. These references are hereby incorporated by reference for their respective teachings of laboratory methods which may be used herein.

[0084] The practice of the invention may include cultivating or culturing (meant to be synonymous) cells, including in large-scale fermentations. Batch, fed-batch and other approaches to fermentation practices are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227, (1992), and

Biochemical Engineering Fundamentals, 2nd Ed. J. E. Bailey and D. F. 01 lis, McGraw Hill, New York, 1986, herein incorporated by reference for general instruction on biosynthesis of chemical products.

[0085] Also, the following published resources are incorporated by reference herein for their respective teachings to indicate the level of skill in these relevant arts, and as needed to support a disclosure that teaches how to make and use methods of industrial biosynthesis of 3 -HP, or other product(s) produced under the invention, from sugar sources, and also industrial systems that may be used to achieve such conversion with any of the recombinant microorganisms of the present invention (Biochemical

Engineering Fundamentals, 2nd Ed. J. E. Bailey and D. F. 01 lis, McGraw Hill, New York, 1986, entire book for purposes indicated and Chapter 9, pages 533-657 in particular for biological reactor design; Unit Operations of Chemical Engineering, 5th Ed., W. L. McCabe et al., McGraw Hill, New York 1993, entire book for purposes indicated, and particularly for process and separation technologies analyses;

Equilibrium Staged Separations, P. C. Wankat, Prentice Hall, Englewood Cliffs, NJ USA, 1988, entire book for separation technologies teachings). Generally, it is further appreciated, in view of the disclosure, that any of the above methods and systems may be used for production of chemical products other than 3- HP.

[0086] It is noted that embodiments of the invention may be practiced in large-scale fermentation vessels, such as steel vessels, for cost-effective commercial production of a selected chemical product. For example, a steel or other vessel may be greater than 250 L, greater than 1,000 L, greater than 10,000 L, greater than 50,000 L, greater than 100,000 L or greater than 200,000 L. The specific examples below are not intended to limit the scope of size of vessels in which the any embodiment of the invention may be practiced.

[0087] Description

[0088] Various embodiments of the present invention are directed to improved use of NADH by polypeptides that catalyze one or by both of the enzymatic functions of a bi-functional malonyl-CoA reductase. The constructs taught herein are, in further various embodiments, used in recombinant microorganisms to increase the biosynthetic production of the chemical malonate semialdehyde (MSA, CAS No. 926-61-4) and/or 3-hydroxypropionic acid (3-HP, CAS No. 503-66-2). These embodiments are believed to substantially improve the economic production of 3-HP and the various chemicals and products that can be made from MSA or 3-HP.

[0089] The bi-functional malonyl-CoA reductase of Chloroflexus aurantiacus functions to catalyze the following two reactions:

Malonyl-CoA+ NADPH + H ⁺ -*malonate semialdehyde + NADP ⁺ + coenzyme A (EC No. 1.2.1.75); and

Malonate semialdehyde + NADPH + H ⁺ 4 3-hydroxypropionate + NADP ⁺ (EC No. 1.1.1.298)

[0090] Reaction 1 is referred to herein as "malonyl-CoA reductase" reaction, activity, or the like, and reaction 2 is referred to as "3-HP dehydrogenase" reaction, activity, or the like, including in the claims. These terms may be used whether the cofactor is nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NADH), or some combination of the two.

[0091] When the native Chloroflexus aurantiacus bi-functional malonyl-CoA reductase enzyme is operative in the directions indicated so as to produce 3-HP, two molecules of NADPH are consumed per molecule of 3-HP produced from malonyl-CoA. This exerts a costly burden on microorganisms that are engineered to efficiently produce 3-HP. This is based in part on the additional metabolic burden of converting NADH, the product of many catabolic reactions, to NADP(H), which also is generally present at substantially lower cellular concentration than NAD(H). As to the reactions 1 and 2 to produce 3 -HP from malonyl-CoA, embodiments of the present invention decrease this burden, and thereby increase 3-HP production efficiency, by providing particular variants of polypeptides that are able to catalyze reactions 1 and 2 with increased use of NADH as the cofactor. A portion of the increased 3-HP production efficiency is notable as increased yield from a carbon source such as glucose, sucrose, and other sugars. In various embodiments one, or both, of reactions 1 and 2 utilize increased amounts and/or proportions of NADH. More particularly, based on analyses and evaluations a number of specific mutations were identified that increase utilization of NADH in both enzymatic domains of the bi-functional malonyl CoA reductase of Chloroflexus aurantiacus (SEQ ID NO: 001) The FASTA sequence is shown in SEQ ID NO: 001 (gil42561982IgbIAAS20429.1 1 malonyl-CoA reductase {Chloroflexus aurantiacus)). SEQ ID NOs: 002 to 045 present full-length mutant (variant) sequences based on SEQ ID NO: 001 however comprising replacements at one or more locations along a respective sequence ("replacement sequences," or "replacement sequence locations" or "replacement sequence regions"). Targets for the replacement sequences include putative phosphate binding sites that in the native sequence are expected to bind the phosphate of NADPH.

[0092] It is appreciated that various polynucleotide sequences may encode for a particular polypeptide sequence of SEQ ID NOs: 002 to 057 based on codon degeneracy. Any such

polynucleotides may be referred to as isolated polynucleotides, as may be applicable during construction, or recombinant polynucleotides, as may be applicable both during construction and also when incorporated into a recombinant microorganism.

[0093] Further, as described in greater detail herein it has been determined that enzymatic functionality is maintained even when the polypeptide sequence is truncated.

[0094] Accordingly, embodiments of the present invention may be directed to the full length variant sequences based on the full length Chloroflexus aurantiacus malonyl-CoA reductase (i.e., SEQ ID NOs: 002 to 045), or alternatively to truncated sequences (e.g., SEQ ID NOs: 070 to 081) that similarly may comprise one or more of the replacement sequence locations, such as are shown (not to be limiting) in SEQ ID NOs: 046 to 057.

[0095] Table 1 lists variant sequences when compared to the parent sequence (SEQ ID NO: 001), that have increase specificity and or activity with NADH compared to NADPH. Additionally, Table 1 describes how to construct these sequences by combining different wild type and mutant sequences into a total complete sequence with NADH dependent malonyl-CoA reductase activity or NADH dependent 3-HP dehydrogenase activity or both. [0096] Table 1

Table 1 Parent Protein Used to Make Malonyl CoA Reductase 3. IIP Dehydrogenase

Combinations Domain (Reaction 1) Domain (Reaction 2)

Sequenc Region Name of SEQ Region of SEQ Unmutat Putativ Sequ e Name of Unmutated ID No. full length ID No. ed e ence full Parent MCR of parent Phosph Nam length Sequence Unmut sequence ate e MCR ated Bindin

Parent g

Sequen Loop ce Sequen

ce

12

[0097] Table 2 provides a summary of sequence numbers and respective measured enzymatic activities for full length C. aurantiacus bi-functional malonyl-CoA reductase (SEQ ID NO: 001) and various truncated polypeptide sequences of this sequence. Each of the latter is named "MCR(x-y)" where x is the start of the truncated sequence, y is the end of the truncated sequence, based on the polypeptide sequence numbering of the native full length C. aurantiacus bifunctional malonyl-CoA reductase (SEQ ID NO: 001). "MCR" is an abbreviation for the latter gene or enzyme, "malonyl-CoA reductase," as the case may be, but does not necessarily refer to the enzymatic function of a truncated sequence.

[0098] Table 2

[0099] It is noted that not all truncations provide both, or any, enzymatic activities, and this is instructive regarding how well any truncation other than those indicated reasonably can be expected to perform. This is discussed in greater detail herein.

[00100] Considering Tables 1 and 2, and further considering SEQ ID NOs: 046 to 057, it is appreciated that various other constructs may be reasonably prepared, such as by using a truncated sequence other than MCR(177-1220) and inserting any of SEQ ID NOs: 059-062 and/or SEQ ID NO: 064 into a truncated sequence having malonyl-CoA reductase activity (Reaction 1), or any of SEQ ID NOs: 066-069 into a truncated sequence having 3-HP dehydrogenase activity (Reaction 2). The particular truncation lengths of Table 2 are not meant to be limiting, and from these teachings truncations of various N- and/or C-terminals (i.e., removing sequences from such terminals) may be made that employ the any of SEQ ID NOs: 059-062 and/or SEQ ID NO: 064 into any such truncated sequence that has malonyl-CoA reductase activity (Reaction 1), and/or any of SEQ ID NOs:066-069 into any such truncated sequence that has 3-HP dehydrogenase activity (Reaction 2).

[00101] In further embodiments, some substitutions may be made to SEQ ID NOs:059-062 and 066-069 and such newly substituted sequences may be employed as taught for the respective non-substituted sequences. Such substituted sequences, which may include conservatively modified variants, may be evaluated for performance as described herein. However, in particular subsets of these further embodiments, arginine (R) is not substituted into position 2, and/or aspartic acid (D) is substituted into position 1.

[00102] It is further appreciated that these replacement sequences may be inserted into sequences that are homologous to, and/or have a specified identity with, the respective portions of Chloroflexus aurantiacus malonyl CoA reductase. For example, and not to be limiting, a BLAST for homology with Chloroflexus aurantiacus malonyl CoA reductase provides the following 8 different sequences when searching over the entire protein. The portion of a CLUSTAL 2.0.11 multiple sequence alignment identifies these eight sequences with respective SEQ ID NOs: 001, and 082-087, as shown in the following table. These sequences represent non- limiting examples of homologues to Chloroflexus aurantiacus malonyl CoA reductase that may be modified with mutations listed in Table 1 , to confer NADH dependent activity.

[00103] Table 3

[00104] Also, it is noted that another malonyl-CoA reductase is known in Metallosphaera sedula (Msed_709, identified as malonyl-CoA reductase/succinyl-CoA reductase), and a malonyl-CoA reductase identified as mono- functional in Sulfolobus tokodaii (sequence provided below). [00105] By providing nucleic acid sequences that encode polypeptides having the above enzymatic activities with any combination of the indicated replacement sequences (see Table 1), so as to shift to greater utilization of NADH compared with utilization of NADPH, a genetically modified microorganism may comprise an effective 3-HP pathway to convert malonyl-CoA to 3-HP in accordance with the embodiments of the present invention. It is further appreciated that the advances taught herein for reactions 1 and 2 may be employed in microorganisms to produce 3-HP. This chemical can be further converted by use of various metabolic pathways, which may be engineered, introduced, and/or enhanced in a recombinant microorganism. Examples of commercially useful end products produced from such 3-HP include, but are not limited to, 3-hydroxypropionaldehyde, 1 ,3 propanediol, poly3-hydroxypropionate. Figure 1A provides representative pathways for such conversions.

[00106] It is further appreciated that the advances taught herein for reaction 1 , the malonyl-CoA reductase reaction, may be employed in microorganisms to produce malonate semialdehyde. This chemical can be further converted by use of various metabolic pathways, which may be engineered, introduced, and/or enhanced in a recombinant microorganism. Examples of commercially useful end products produced from such malonate semialdehyde include, but are not limited to, propenoate and 0-alanine. Figure IB provides representative pathways for such conversions. The conversions and noted genes/enzymes are not meant to be limiting. Embodiments also include additional

modifications to a microorganism for any one or more reasons.

[00107] In some embodiments genetic modifications are provided to delete or otherwise decrease activity of enzymes to reduce formation of undesired metabolites and chemical end products.

[00108] In some embodiments genetic modifications are made to increase overall enzymatic activity of certain enzymatic functions. For example, enzymatic activities as provided in Table 4 may be provided with any of the other modifications described herein, in any combination.

[00109] Table 4: Additional/Supplemental Modifications

[00110] Accordingly, in some embodiments the protein function for converting malonate semialdehyde to 3-HP is a native or mutated form of mmsB from Pseudomonas aeruginosa, or a functional equivalent thereof. Alternatively, or additionally, this protein function can be a native or mutated form of ydfG from E. coli , or a functional equivalent thereof. Alternatively, or additionally, this protein function can be a native or mutated form of nemA or rutE from E. coli, or a functional equivalent thereof. Alternatively, these may supplement such activity of a bi-functional malonyl-CoA reductase as described above, having increased NADH utilization, and/or of a mono-functional dehydrogenase having increased NADH utilization (in which latter case a polypeptide having reaction 1 function also may be provided).

[00111] FIGs. 2A to 2G also summarize various genetic modifications that may be made to a

microorganism cell, using E. coli gene names, underlining those to be, introduced according to the present teachings, increased and/or overexpressed, and providing Xs and dashed Xs to those steps/reactions to be eliminated (e.g., disrupted) or reduced (such as transiently), respectively. In these figures El is taken to refer to sequences providing Reaction 1 activity, and E2 is taken to refer to sequences providing Reaction 2 activity, noting that a single sequence can be introduced that provides both reaction functions.

[00112] The teachings of the following publications as to genetic modification combinations, culture methods, and other aspects to produce 3 -HP and other chemical products are hereby incorporated by reference into this application: Application number PCT/US2010/050436, published March 31, 2011 as WO/2011/038364; Application number PCT/US/057690, published May 26, 2011 as WO/2011/063363, and Application number PCT/US2011/022790 published August 4, 2011 as WO/2011/094457. The respective teachings therein regarding increasing tolerance to 3-HP also are incorporated by reference herein.

[00113] Also, particular note is made of the use of modifications to reduce activity of one or more fatty acid synthase system enzymatic functions. This is described in the incorporated Application numbers PCT/US2010/050436 and PCT/US2011/022790. Briefly, any one or more enzymes of a microorganism cell's fatty acid synthase (synthetase) system may be modified so reduce or eliminate its function, including by use of temperature-sensitive mutants and shifting culture temperature to a temperature (such as after a desired biomass is achieved) at which there is reduced activity. This leads to a shifting use of malonyl-CoA toward production of 3-HP (or alternatively other chemicals of interest). By use of such genetic modifications (and also corresponding culture conditions, though the latter are optional in various embodiments), improved production of a chemical product of interest, such as 3-HP, may be achieved in a manner that is not growth-coupled. While not meant to be limiting, in E. coli the polypeptides having such activities are encoded by genes such as fabl, fabB, fabF, fabD, and fabH. These genes are shown in FIG. 3 which depicts a representative fatty acid synthase complex.

[00114] As shown in the figures, two primary pathways to malonate from malonyl-CoA are proposed. The first relies on the reduction of malonyl-CoA to malonate semialdehyde (reaction 1 below), followed by the oxidation of malonate semialdehyde to malonate (reaction 2 below). The second pathway relies on the hydrolysis of malonyl-coA by a malonyl-coA thioesterase.

(1) Malonyl-CoA+ NADPH + H+ -*malonate semialdehyde + NADP+ + coenzyme A (EC No. 1.2.1.75)

(2) Malonate semialdehyde + NAD+ + 4 malonate + NADH + H+ (EC No. 1.2.1.X)

(3) Malonyl-CoA+ H20 + 4 malonate + coenzyme A (EC No. TBD)

[00115] Reaction 1 is referred to herein as "malonyl-CoA reductase" reaction, activity, or the like, and reaction 2 is referred to as "malonate semialdehyde dehydrogenase" reaction, activity, or the like, including in the claims. Reaction 3 is referred to as "malonyl-CoA thioesterase" reaction, activity, or the like. These terms for reactions 1 and 2 may be used whether the cofactor is nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NADH), or some combination of the two.

[00116] Several eznymes have been characterized to perform reaction 2. These include dehydrogenases that operate on malonate semialdehyde and use either NADH or NADPH as a reductant. Of particular note are the glycoaldehyde dehydrogenase , encoded by the aldA gene of E. coli,( Tani, Y.; Morita, H.; Nishise, H.; Ogata, K.; Agric. Biol. Chem. 42, 63-68 (1978)) and succinate semialdehyde dehydrogenase of Euglena gracilis (Tokunaga, M.; Nakano, Y.; Kitaoka, S.; Biochim. Biophys. Acta 429, 55-62 (1976)) which have been shown to have malonate dehydrogenase activity. It is further appreciated that these malonate dehydrogenases may be obtained from polypeptide or corresponding polynucleotide sequences that are homologous to, and/or have a specified identity with, the respective portions of aldA gene of E. coli. . For example, and not to be limiting, a BLAST for homology with aldA gene of E. coli. provides 18 different sequences when searching over the entire polynucleotide. These sequences represent non- limiting examples of homologues to the aldA gene of E. coli. Methods in the art can be used to improve the use of malonate semialdehyde as a substrate with any of these sequences, by mutation and screening.

[00117] Carbon Sources

[00118] In addition, fermentable sugars may be obtained from cellulosic and lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in U.S. Patent Publication No. 2007/0031918A1, which is herein incorporated by reference. Biomass refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein and/or lipid. Biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass could comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers and animal manure. Any such biomass may be used in a bio-production method or system to provide a carbon source. Various approaches to breaking down cellulosic biomass to mixtures of more available and utilizable carbon molecules, including sugars, include: heating in the presence of concentrated or dilute acid (e.g., < 1% sulfuric acid); treating with ammonia; treatment with ionic salts; enzymatic degradation; and combinations of these. These methods normally follow mechanical separation and milling, and are followed by appropriate separation processes. Bio-production media, which is used in the present invention with recombinant microorganisms having a biosynthetic pathway for 3 -HP or malonic acid, must contain suitable carbon sources or substrates for the intended metabolic pathways. Suitable substrates may include, but are not limited to, monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Additionally the carbon substrate may also be one-carbon substrates such as carbon dioxide, carbon monoxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated.

[00119] Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention as a carbon source, common carbon substrates used as carbon sources are glucose, fructose, and sucrose, as well as mixtures of any of these sugars. Other suitable substrates include xylose, arabinose, other cellulose-based C-5 sugars, high- fructose corn syrup, and various other sugars and sugar mixtures as are available commercially. Sucrose may be obtained from feedstocks such as sugar cane, sugar beets, cassava, bananas or other fruit, and sweet sorghum. Glucose and dextrose may be obtained through saccharification of starch based feedstocks including grains such as corn, wheat, rye, barley, and oats. Also, in some embodiments all or a portion of the carbon source may be glycerol. Alternatively, glycerol may be excluded as an added carbon source.

[00120] In one embodiment, the carbon source is selected from glucose, fructose, sucrose, dextrose, lactose, glycerol, and mixtures thereof. Variously, the amount of these components in the carbon source may be greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or more, up to 100% or essentially 100% of the carbon source.

[00121] In addition, methylotrophic organisms are known to utilize a number of other carbon containing compounds in addition to one and two carbon substrates, such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeasts are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth CI Compd. (Int. Symp.), 7th (1993), 415-32. Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Suiter et al., Arch. Microbiol. 153:485-489 (1990)). Hence it is contemplated that the source of carbon utilized in embodiments of the present invention may encompass a wide variety of carbon-containing substrates.

[00122] In various embodiments, any of a wide range of sugars, including, but not limited to sucrose, glucose, xylose, cellulose or hemicellulose, are provided to a microorganism, such as in an industrial system comprising a reactor vessel in which a defined media (such as a minimal salts media including but not limited to M9 minimal media, potassium sulfate minimal media, yeast synthetic minimal media and many others or variations of these), an inoculum of a microorganism providing one or more of the 3-HP or malonic acid biosynthetic pathway alternatives, and the a carbon source may be combined. The carbon source enters the cell and is catabolized by well-known and common metabolic pathways to yield common metabolic intermediates, including phosphoenolpyruvate (PEP). (See Molecular Biology of the Cell, 3rd Ed., B. Alberts et al. Garland Publishing, New York, 1994, pp. 42-45, 66-74, incorporated by reference for the teachings of basic metabolic catabolic pathways for sugars; Principles of Biochemistry, 3rd Ed., D. L. Nelson & M. M. Cox, Worth Publishers, New York, 2000, pp. 527-658, incorporated by reference for the teachings of major metabolic pathways; and Biochemistry, 4th Ed., L. Stryer, W. H. Freeman and Co., New York, 1995, pp. 463-650, also incorporated by reference for the teachings of major metabolic pathways.)

[00123] Bio-based carbon can be distinguished from petroleum-based carbon according to a variety of methods, including without limitation ASTM D6866, or various other techniques. For example, carbon- 14 and carbon- 12 ratios differ in bio-based carbon sources versus petroleum-based sources, where higher carbon- 14 ratios are found in bio-based carbon sources. In various embodiments, the carbon source is not petroleum-based, or is not predominantly petroleum based. In various embodiments, the carbon source is greater than about 50% non-petroleum based, greater than about 60%> non-petroleum based, greater than about 70%) non-petroleum based, greater than about 80% non-petroleum based, greater than about 90% non-petroleum based, or more. In various embodiments, the carbon source has a carbon- 14 to carbon- 12 ratio of about 1.0 x 10 ^"14 or greater.

[00124] Various components may be excluded from the carbon source. For example, in some embodiments, acrylic acid, 1 ,4-butanediol, and/or glycerol are excluded or essentially excluded from the carbon source. As such, the carbon source according to some embodiments of the invention may be less than about 50% glycerol, less than about 40% glycerol, less than about 30% glycerol, less than about 20% glycerol, less than about 10% glycerol, less than about 5% glycerol, less than about 1% glycerol, or less. For example, the carbon source may be essentially glycerol-free. By essentially glycerol- free is meant that any glycerol that may be present in a residual amount does not contribute substantially to the production of the target chemical compound.

[00125] Microorganisms

[00126] Features as described and claimed herein may be provided in a microorganism selected from the listing herein, or another suitable microorganism, that also comprises one or more natural, introduced, or enhanced chemical product biosynthesis pathway. Thus, in some embodiments the microorganism comprises an endogenous chemical product biosynthesis pathway (which may, in some such

embodiments, be enhanced), whereas in other embodiments the microorganism does not comprise an endogenous biosynthesis pathway for the selected chemical product (such as 3-HP or malonic acid).

[00127] Varieties of these genetically modified microorganisms may comprise genetic modifications and/or other system alterations as may be described in other patent applications of one or more of the present inventor(s) and/or subject to assignment or license to the owner of the present patent application.

[00128] The examples describe specific modifications and evaluations to certain bacterial and yeast microorganisms. The scope of the invention is not meant to be limited to such species, but to be generally applicable to a wide range of suitable microorganisms. Generally, a microorganism used for the present invention may be selected from bacteria, cyanobacteria, filamentous fungi and yeasts.

[00129] For some embodiments, microbial hosts initially selected for 3-HP or malonic acid or other chemical product biosynthesis should also utilize sugars including glucose at a high rate. Most microbes are capable of utilizing carbohydrates. However, certain environmental microbes cannot utilize carbohydrates to high efficiency, and therefore may not be suitable hosts for such

embodiments that are intended for glucose or other carbohydrates as the principal added carbon source.

[00130] As the genomes of various species become known, the present invention easily may be applied to an ever-increasing range of suitable microorganisms. Further, given the relatively low cost of genetic sequencing, the genetic sequence of a species of interest may readily be determined to make application of aspects of the present invention more readily obtainable (based on the ease of application of genetic modifications to an organism having a known genomic sequence). Public database sites, such as «www.metacyc.org», «www.ecocyc.org», «www.biocyc.org», and «www.ncbi.gov», « http:/www.nchi.nlm.nih.gov/» have various genetic and genomic information and associated tools to identify enzymes in various species that have desired function or that may be modified to achieve such desired function.

[00131] More particularly, based on the various criteria described herein, suitable microbial hosts for the biosynthesis of a chemical product generally may include, but are not limited to, any gram negative organisms, more particularly a member of the family Enterobacteriaceae, such as E. coli, or Oligotropha carboxidovorans, or Pseudomononas sp.; any gram positive microorganism, for example Bacillus subtilis, Lactobaccilus sp. or Lactococcus sp.; a yeast, for example Saccharomyces cerevisiae, Pichia pastoris or Pichia stipitis; and other groups or microbial species including those found in Actinomycetes (also referred to as Actinobacteria). More particularly, suitable microbial hosts for the biosynthesis of a chemical product generally include, but are not limited to, members of the genera Clostridium,

Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus,

Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces. Hosts that may be particularly of interest include: Oligotropha carboxidovorans (such as strain OMS), Escherichia coli, Alcaligenes eutrophus (Cupriavidus necator), Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis and Saccharomyces cerevisiae.

[00132] More particularly, suitable microbial hosts for the biosynthesis of 3-HP or malonic acid and other chemical products generally include, but are not limited to, members of the genera Clostridium,

Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus,

Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium,

Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces. Hosts that may be particularly of interest include: Oligotropha carboxidovorans (such as strain 0M5T), Escherichia coli, Alcaligenes eutrophus (Cupriavidus necator), Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtil is and Saccharomyces cerevisiae. Also, any of the known strains of these species may be utilized as a starting microorganism, as may any of the following species including respective strains thereof- Cupriavidus basilensis, Cupriavidus campinensis, Cupriavidus gilardi, Cupriavidus laharsis, Cupriavidus metallidurans, Cupriavidus oxalaticus, Cupriavidus pauculus, Cupriavidus pinatubonensis, Cupriavidus respiraculi, and Cupriavidus taiwanensis.

[00133] In some embodiments, the recombinant microorganism is a gram-negative bacterium. In some embodiments, the recombinant microorganisms are selected from the genera Zymomonas, Escherichia, Pseudomonas, Alcaligenes, and Klebsiella. In some embodiments, the recombinant microorganisms are selected from the species Escherichia coli, Cupriavidus necator, Oligotropha carboxidovorans, and Pseudomonas putida. In some embodiments, the recombinant microorganism is an E. coli strain.

[00134] In some embodiments, the recombinant microorganism is a gram-positive bacterium. In some embodiments, the recombinant microorganism is selected from the genera Clostridium, Salmonella, Rhodococcus, Bacillus, Lactobacillus, Enterococcus, Paenibacillus, Arthrobacter, Corynebacterium, and Brevibacterium. In some embodiments, the recombinant microorganism is selected from the species Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, and Bacillus subtilis. In particular embodiments, the recombinant microorganism is a B. subtilis is strain.

[00135] In some embodiments, the recombinant microorganism is a yeast. In some embodiments, the recombinant microorganism is selected from the genera Pichia, Candida, Hansenula and Saccharomyces. In particular embodiments, the recombinant microorganism is Saccharomyces cerevisiae.

[00136] It is noted that as to the microorganisms that may be used in the practice of various embodiments of the invention, these microorganisms include any species of microorganism, and more particularly, any species of any taxonomic group disclosed herein, and more particularly, any species disclosed herein.

[00137] The ability to genetically modify the host is essential for the production of any recombinant microorganism. The mode of gene transfer technology may be by electr op oration, conjugation, transduction or natural transformation. A broad range of host conjugative plasmids and drug resistance markers are available. The cloning vectors are tailored to the host organisms based on the nature of antibiotic resistance markers that can function in that host.

[00138] Media and Culture Conditions

[00139] In addition to an appropriate carbon source, such as selected from one of the herein-disclosed types, bio-production media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for 3 -HP or malonic acid production, or other products made under the present invention.

[00140] Another aspect of the invention regards media and culture conditions that comprise genetically modified microorganisms of the invention and optionally supplements.

[00141]Typically cells are grown at a temperature in the range of about 25° C to about 40° C in an appropriate medium, as well as up to 70° C for thermophilic microorganisms. Suitable growth media in the present invention are common commercially prepared media such as Luria Bertani (LB) broth, M9 minimal media, Sabouraud Dextrose (SD) broth, Yeast medium (YM) broth, (Ymin) yeast synthetic minimal media, and minimal media as may be described herein, such as M9 minimal media. Other defined or synthetic growth media may also be used, and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or bio-production science. In various embodiments a minimal media may be developed and used that does not comprise, or that has a low level of addition of various components, for example less than 10, 5, 2 or 1 g/L of a complex nitrogen source including but not limited to yeast extract, peptone, tryptone, soy flour, corn steep liquor, or casein. These minimal medias may also have limited supplementation of vitamin mixtures including biotin, vitamin B12 and derivatives of vitamin B12, thiamin, pantothenate and other vitamins. Minimal medias may also have limited simple inorganic nutrient sources containing less than 28, 17, or 2.5 mM phosphate, less than 25 or 4 mM sulfate, and less than 130 or 50mM total nitrogen.

[00142] Bio-production media, which is used in embodiments of the present invention with genetically modified microorganisms, must contain suitable carbon substrates for the intended metabolic pathways. As described hereinbefore, suitable carbon substrates include carbon monoxide, carbon dioxide, and various monomeric and oligomeric sugars.

[00143] Suitable pH ranges for the bio-production are between pH 3.0 to pH 10.0, where pH 6.0 to pH 8.0 is a typical pH range for the initial condition. However, the actual culture conditions for a particular embodiment are not meant to be limited by these pH ranges.

[00144]Bio-productions may be performed under aerobic, microaerobic, or anaerobic conditions, with or without agitation. The amount of 3 -HP or malonic acid or other product(s) produced in a bio- production media generally can be determined using a number of methods known in the art, for example, high performance liquid chromatography (HPLC), gas chromatography (GC), GC/Mass Spectroscopy (MS), or spectrometry.

[00145]Bio-production Reactors and Systems

[00146] Fermentation systems utilizing methods and/or compositions according to the invention are also within the scope of the invention.

[00147] Any of the recombinant microorganisms as described and/or referred to herein may be introduced into an industrial bio-production system where the microorganisms convert a carbon source into a selected chemical product, such as 3 -HP or malonic acid, in a commercially viable operation. The bio-production system includes the introduction of such a recombinant

microorganism into a bioreactor vessel, with a carbon source substrate and bio-production media suitable for growing the recombinant microorganism, and maintaining the bio-production system within a suitable temperature range (and dissolved oxygen concentration range if the reaction is aerobic or microaerobic) for a suitable time to obtain a desired conversion of a portion of the substrate molecules to 3 -HP or malonic acid. Industrial bioproduction systems and their operation are well-known to those skilled in the arts of chemical engineering and bioprocess engineering.

[00148]Bio-productions may be performed under aerobic, microaerobic, or anaerobic conditions, with or without agitation. The operation of cultures and populations of microorganisms to achieve aerobic, microaerobic and anaerobic conditions are known in the art, and dissolved oxygen levels of a liquid culture comprising a nutrient media and such microorganism populations may be monitored to maintain or confirm a desired aerobic, microaerobic or anaerobic condition. When syngas is used as a feedstock, aerobic, microaerobic, or anaerobic conditions may be utilized. When sugars are used, anaerobic, aerobic or microaerobic conditions can be implemented in various embodiments. Any of the recombinant microorganisms as described and/or referred to herein may be introduced into an industrial bio-production system where the microorganisms convert a carbon source into 3 -HP or malonic acid, and optionally in various embodiments also to one or more downstream compounds of 3 -HP or malonic acid in a commercially viable operation. The bioproduction system includes the introduction of such a recombinant microorganism into a bioreactor vessel, with a carbon source substrate and bio-production media suitable for growing the recombinant microorganism, and maintaining the bio-production system within a suitable temperature range (and dissolved oxygen concentration range if the reaction is aerobic or microaerobic) for a suitable time to obtain a desired conversion of a portion of the substrate molecules to 3 -HP or malonic acid.

[00149]In various embodiments, syngas components or sugars are provided to a microorganism, such as in an industrial system comprising a reactor vessel in which a defined media (such as a minimal salts media including but not limited to M9 minimal media, potassium sulfate minimal media, yeast synthetic minimal media and many others or variations of these), an inoculum of a microorganism providing an embodiment of the biosynthetic pathway(s) taught herein, and the carbon source may be combined. The carbon source enters the cell and is catabolized by well-known and common metabolic pathways to yield common metabolic intermediates, including phosphoenolpyruvate (PEP). (See Molecular Biology of the Cell, 3 ^rd Ed., B. Alberts et al. Garland Publishing, New York, 1994, pp. 42- 45, 66-74, incorporated by reference for the teachings of basic metabolic catabolic pathways for sugars; Principles of Biochemistry, 3 ' ^d Ed., D. L. Nelson & M. M. Cox, Worth Publishers, New York, 2000, pp. 527-658, incorporated by reference for the teachings of major metabolic pathways; and Biochemistry, 4 ^th Ed., L. Stryer, W. H. Freeman and Co., New York, 1995, pp. 463-650, also incorporated by reference for the teachings of major metabolic pathways.). Further to types of industrial bio-production, various embodiments of the present invention may employ a batch type of industrial bioreactor. A classical batch bioreactor system is considered "closed" meaning that the composition of the medium is established at the beginning of a respective bio-production event and not subject to artificial alterations and additions during the time period ending substantially with the end of the bio-production event. Thus, at the beginning of the bio-production event the medium is inoculated with the desired organism or organisms, and bio-production is permitted to occur without adding anything to the system. Typically, however, a "batch" type of bio-production event is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly up to the time the bio-production event is stopped. Within batch cultures cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of a desired end product or intermediate.

[00150] A variation on the standard batch system is the fed-batch system. Fed-batch bio-production processes are also suitable in the present invention and comprise a typical batch system with the exception that the nutrients, including the substrate, are added in increments as the bio-production progresses. Fed- Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual nutrient concentration in Fed-Batch systems may be measured directly, such as by sample analysis at different times, or estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO ₂ . Batch and fed-batch approaches are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227, (1992), and Biochemical Engineering Fundamentals, 2 ^d Ed. J. E. Bailey and D. F. 01 lis, McGraw Hill, New York, 1986, herein incorporated by reference for general instruction on bio-production.

[00151] Although embodiments of the present invention may be performed in batch mode, or in fed-batch mode, it is contemplated that the invention would be adaptable to continuous bio-production methods. Continuous bioproduction is considered an "open" system where a defined bio-production medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous bioproduction generally maintains the cultures within a controlled density range where cells are primarily in log phase growth. Two types of continuous bioreactor operation include a chemostat, wherein fresh media is fed to the vessel while simultaneously removing an equal rate of the vessel contents. The limitation of this approach is that cells are lost and high cell density generally is not achievable. In fact, typically one can obtain much higher cell density with a fed-batch process. Another continuous bioreactor utilizes perfusion culture, which is similar to the chemostat approach except that the stream that is removed from the vessel is subjected to a separation technique which recycles viable cells back to the vessel. This type of continuous bioreactor operation has been shown to yield significantly higher cell densities than fed-batch and can be operated continuously. Continuous bio- production is particularly advantageous for industrial operations because it has less down time associated with draining, cleaning and preparing the equipment for the next bio-production event. Furthermore, it is typically more economical to continuously operate downstream unit operations, such as distillation, than to run them in batch mode.

[00152] Continuous bio-production allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Methods of modulating nutrients and growth factors for continuous bio-production processes as well as general techniques for maximizing the rate of product formation are known in the art of industrial microbiology and a variety of such methods are detailed by Brock, supra.

[00153] It is contemplated that embodiments of the present invention may be practiced using either batch, fed-batch or continuous processes and that any known mode of bio-production would be suitable. It is contemplated that cells may be immobilized on an inert scaffold as whole cell catalysts and subjected to suitable bio-production conditions for 3 -HP or malonic acid production, or be cultured in liquid media in a vessel, such as a culture vessel. Thus, embodiments used in such processes, and in bio-production systems using these processes, include a population of genetically modified microorganisms of the present invention, a culture system comprising such population in a media comprising nutrients for the population, and methods of making 3 -HP or malonic acid and thereafter, a downstream product of 3 -HP or malonic acid. Embodiments of the invention include methods of making 3-HP or malonic acid in a bio-production system, some of which methods may include obtaining 3-HP or malonic acid after such bio-production event. For example, a method of making 3-HP or malonic acid may comprise: providing to a culture vessel a media comprising suitable nutrients; providing to the culture vessel an inoculum of a genetically modified microorganism comprising genetic modifications described herein such that the microorganism produces 3-HP or malonic acid from syngas and/or a sugar molecule; and maintaining the culture vessel under suitable conditions for the genetically modified microorganism to produce 3-HP or malonic acid.

[00154] It is within the scope of the present invention to produce, and to utilize in bio-production methods and systems, including industrial bio-production systems for production of 3-HP, a recombinant microorganism genetically engineered to modify one or more aspects effective to increase tolerance to 3-HP (and, in some embodiments, also 3-HP bio-production) by at least 20 percent over control microorganism lacking the one or more modifications. In various embodiments, the invention is directed to a system for bioproduction of acrylic acid as described herein, said system comprising: a fermentation tank suitable for microorganism cell culture; a line for discharging contents from the fermentation tank to an extraction and/or separation vessel; an extraction and/or separation vessel suitable for removal of 3-hydroxypropionic acid from cell culture waste; a line for transferring 3-hydroxypropionic acid to a dehydration vessel; and a dehydration vessel suitable for conversion of 3-hydroxypropionic acid to acrylic acid. In various embodiments, the system includes one or more pre-fermentation tanks, distillation columns, centrifuge vessels, back extraction columns, mixing vessels, or combinations thereof.

[00155] Also, it is within the scope of the present invention to produce, and to utilize in bio-production methods and systems, including industrial bio-production systems for production of a selected chemical product other than 3 -HP, a recombinant microorganism genetically engineered to modify one or more aspects effective to increase the selected chemical product's bio-production by at least 20 percent over control microorganism lacking the one or more modifications.

[00156] In various embodiments, the invention is directed to a system for bio-production of a chemical product as described herein, said system comprising: a fermentation tank suitable for microorganism cell culture; a line for discharging contents from the fermentation tank to an extraction and/or separation vessel; and an extraction and/or separation vessel suitable for removal of the chemical product from cell culture waste. In various embodiments, the system includes one or more pre-fermentation tanks, distillation columns, centrifuge vessels, back extraction columns, mixing vessels, or combinations thereof.

[00157] The following published resources are incorporated by reference herein for their respective teachings to indicate the level of skill in these relevant arts, and as needed to support a disclosure that teaches how to make and use methods of industrial bio-production of 3 -HP, or other product(s) produced under the invention, from sugar sources, and also industrial systems that may be used to achieve such conversion with any of the recombinant microorganisms of the present invention

(Biochemical Engineering Fundamentals, 2 ^nd Ed. J. E. Bailey and D. F. 01 lis, McGraw Hill, New York, 1986, entire book for purposes indicated and Chapter 9, pages 533-657 in particular for biological reactor design; Unit Operations of Chemical Engineering, 5 ^th Ed., W. L. McCabe et al., McGraw Hill, New York 1993, entire book for purposes indicated, and particularly for process and separation technologies analyses; Equilibrium Staged Separations, P. C. Wankat, Prentice Hall, Englewood Cliffs, NJ USA, 1988, entire book for separation technologies teachings). Generally, it is further appreciated, in view of the disclosure, that any of the above methods and systems may be used for production of chemical products other than 3-HP.

[00158] Genetic Modifications, Nucleotide Sequences, and Amino Acid Sequences

[00159] Embodiments of the present invention may result from introduction of an expression vector into a host microorganism, wherein the expression vector contains a nucleic acid sequence coding for an enzyme that is, or is not, normally found in a host microorganism. [00160] The ability to genetically modify a host cell is essential for the production of any genetically modified (recombinant) microorganism. The mode of gene transfer technology may be by

electroporation, conjugation, transduction, or natural transformation. A broad range of host conjugative plasmids and drug resistance markers are available. The cloning vectors are tailored to the host organisms based on the nature of antibiotic resistance markers that can function in that host. Also, as disclosed herein, a genetically modified (recombinant) microorganism may comprise modifications other than via plasmid introduction, including modifications to its genomic DNA.

[00161] Embodiments of the present invention may involve various nucleic acid sequences, such as heterologous nucleic acid sequences introduced into a cell's genome or may be episomal, and also encompasses antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.

[00162]More generally, nucleic acid constructs can be prepared comprising an isolated polynucleotide encoding a polypeptide having enzyme activity operably linked to one or more (several) control sequences that direct the expression of the coding sequence in a microorganism, such as E. coli, under conditions compatible with the control sequences. The isolated polynucleotide may be manipulated to provide for expression of the polypeptide. Manipulation of the polynucleotide's sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. As noted herein, the techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well established in the art.

[00163] More generally, nucleic acid constructs can be prepared comprising an isolated polynucleotide encoding a polypeptide having enzyme activity operably linked to one or more (several) control sequences that direct the expression of the coding sequence in a microorganism, such as E. coli, under conditions compatible with the control sequences. The isolated polynucleotide may be manipulated to provide for expression of the polypeptide. Manipulation of the polynucleotide's sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well established in the art.

[00164] The control sequence may be an appropriate promoter sequence, a nucleotide sequence that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any nucleotide sequence that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. Examples of suitable promoters for directing transcription of the nucleic acid constructs, especially in an is. coli host cell, are the lac promoter (Gronenborn, 1976, MoT Gen. Genet. 148: 243-250), tac promoter (DeBoer et al 1983, Proceedings of the National Academy of Sciences USA 80: 21 - 25), trc promoter (Brosius et al, 1985, J. Biol. Chem. 260: 3539-3541), T7 RNA polymerase promoter (Studier and Moffatt, 1986, J. Mol. Biol. 189: 113-130), phage promoter p _L (Elvin et al., 1990, Gene 87: 123- 126), tetA promoter (Skerra, 1994, Gene 151 : 131-135), araBAD promoter (Guzman et al., 1995, J. Bacteriol. 177: 4121-4130), and rhaP _BA D promoter (Haldimann et al., 1998, J. Bacteriol. 180: 1277- 1286). Other promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242: 74-94; and in Sambrook and Russell, "Molecular Cloning: A Laboratory

Manual," Third Edition 2001 (volumes 1-3), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[00165] The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in an E. coli cell may be used in the present invention. It may also be desirable to add regulatory sequences that allow regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems.

[00166] For various embodiments of the invention the genetic manipulations may be described to include various genetic manipulations, including those directed to change regulation of, and therefore ultimate activity of, an enzyme or enzymatic activity of an enzyme identified in any of the respective pathways. Such genetic modifications may be directed to transcriptional, translational, and post-translational modifications that result in a change of enzyme activity and/or selectivity under selected and/or identified culture conditions and/or to provision of additional nucleic acid sequences such as to increase copy number and/or mutants of an enzyme related to 3-HP production. Specific methodologies and approaches to achieve such genetic modification are well known to one skilled in the art, and include, but are not limited to: increasing expression of an endogenous genetic element; decreasing functionality of a repressor gene; introducing a heterologous genetic element; increasing copy number of a nucleic acid sequence encoding a polypeptide catalyzing an enzymatic conversion step to produce 3-HP; mutating a genetic element to provide a mutated protein to increase specific enzymatic activity; over-expressing; under-expressing; over-expressing a chaperone; knocking out a protease; altering or modifying feedback inhibition; providing an enzyme variant comprising one or more of an impaired binding site for a repressor and/or competitive inhibitor; knocking out a repressor gene; evolution, selection and/or other approaches to improve mRNA stability as well as use of plasmids having an effective copy number and promoters to achieve an effective level of improvement. Random mutagenesis may be practiced to provide genetic modifications that may fall into any of these or other stated approaches. The genetic modifications further broadly fall into additions (including insertions), deletions (such as by a mutation) and substitutions of one or more nucleic acids in a nucleic acid of interest. In various embodiments a genetic modification results in improved enzymatic specific activity and/or turnover number of an enzyme. Without being limited, changes may be measured by one or more of the following: K _M ; K^; and [00167] In various embodiments, to function more efficiently, a microorganism may comprise one or more gene deletions. For example, in E. coli, the genes encoding the lactate dehydrogenase (ldhA), phosphate acetyltransferase (pta), pyruvate oxidase (poxB), and pyruvate-formate lyase (pflB) may be disrupted, including deleted. Such gene disruptions, including deletions, are not meant to be limiting, and may be implemented in various combinations in various embodiments. Gene deletions may be accomplished by mutational gene deletion approaches, and/or starting with a mutant strain having reduced or no expression of one or more of these enzymes, and/or other methods known to those skilled in the art. Gene deletions may be effectuated by any of a number of known specific methodologies, including but not limited to the RED/ET methods using kits and other reagents sold by Gene Bridges (Gene Bridges GmbH, Dresden, Germany, «www.genebridges.com»).

[00168] More particularly as to the latter method, use of Red/ET recombination, is known to those of ordinary skill in the art and described in U.S. Patent Nos. 6,355,412 and 6,509,156, issued to Stewart et al. and incorporated by reference herein for its teachings of this method. Material and kits for such method are available from Gene Bridges (Gene Bridges GmbH, Dresden, Germany, «www.genebridges.com»), and the method may proceed by following the manufacturer's instructions. The method involves replacement of the target gene by a selectable marker via homologous recombination performed by the recombinase from X-phage. The host organism expressing k-red recombinase is transformed with a linear DNA product coding for a selectable marker flanked by the terminal regions (generally— 50 bp, and alternatively up to about— 300 bp) homologous with the target gene. The marker could then be removed by another recombination step performed by a plasmid vector carrying the FLP-recombinase, or another recombinase, such as Cre.

[00169] Targeted deletion of parts of microbial chromosomal DNA or the addition of foreign genetic material to microbial chromosomes may be practiced to alter a host cell's metabolism so as to reduce or eliminate production of undesired metabolic products. This may be used in combination with other genetic modifications such as described herein in this general example. In this detailed description, reference has been made to multiple embodiments and to the accompanying drawings in which is shown by way of illustration specific exemplary embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that modifications to the various disclosed embodiments may be made by a skilled artisan.

[00170] Further, for 3-HP production, such genetic modifications may be chosen and/or selected for to achieve a higher flux rate through certain enzymatic conversion steps within the respective 3-HP production pathway and so may affect general cellular metabolism in fundamental and/or major ways.

[00171] It will be appreciated that amino acid "homology" includes conservative substitutions, i.e. those that substitute a given amino acid in a polypeptide by another amino acid of similar

characteristics. Recognized conservative amino acid substitutions comprise (substitutable amino acids following each colon of a set): ala:ser; arg:lys; asn:gln or his; asp:glu; cys:ser; gln:asn; glu:asp; gly:pro; his:asn or gin; ile:leu or val; leu:ile or val; lys: arg or gin or glu; met:leu or ile; phe:met or leu or tyr; ser:thr; thr:ser; trp:tyr; tyr:trp or phe; val:ile or leu. Also generally recognized as conservative substitutions are the following replacements: replacements of an aliphatic amino acid such as Ala, Val, Leu and Ile with another aliphatic amino acid; replacement of a Ser with a Thr or vice versa; replacement of an acidic residue such as Asp or Glu with another acidic residue;

replacement of a residue bearing an amide group, such as Asn or Gin, with another residue bearing an amide group; exchange of a basic residue such as Lys or Arg with another basic residue; and replacement of an aromatic residue such as Phe or Tyr with another aromatic residue. For all polynucleotide (nucleic acid) and polypeptide (amino acid) sequences provided herein, it is appreciated that conservatively modified variants of these sequences are included, and are within the scope of the invention in its various embodiments. Conservatively modified variant include amino acid conservative substitutions such as those described in the previous paragraph as well as modified polynucleotide sequences such as based on codon degeneracy described in the following paragraph and table. Further, the following table also provides characteristics of amino acids that provide for additional conservative substitutions that may fall within the scope of conservatively modified variants, based on commonly shared properties of particular amino acids. Also, in view of the teachings provided herein, conservatively modified variants also include truncated sequences, of polynucleotides as well as polypeptides, that maintain one or both functions with regard to Reaction 1 and Reaction 2. The present disclosure teaches that many deletions at either the N- or C- terminals can provide a construct (truncation) that nonetheless provides a desired functionality. Also, in various embodiments deletions and/or substitutions at either end, or in other regions, of a

polynucleotide or polypeptide may be practiced for other sequences, based on the present teachings and knowledge of those skilled in the art, and remain within the scope of conservatively modified variants. Accordingly, functionally equivalent polynucleotides and polypeptides (functional variants), which may include conservatively modified variants as well as more extensively varied sequences, which are well within the skill of the person of ordinary skill in the art, and

microorganisms comprising these, also are within the scope of various embodiments of the invention, as are methods and systems comprising such sequences and/or microorganisms. In various embodiments, nucleic acid sequences encoding sufficiently homologous proteins or portions thereof are within the scope of the invention. More generally, nucleic acids sequences that encode a particular amino acid sequence employed in the invention may vary due to the degeneracy of the genetic code, and nonetheless fall within the scope of the invention. The following table provides a summary of similarities among amino acids, upon which conservative substitutions may be based, and also various codon redundancies that reflect this degeneracy. [00172] Table 5

Legend: side groups and other related properties: A=acidic; B=basic; Ali=aliphatic; Ami=amine; Aro= aromatic; N=nonpolar; PU=polar uncharged; NEG=negatively charged; POS=positively charged.

[00173] It is noted that codon preferences and codon usage tables for a particular species can be used to engineer isolated nucleic acid molecules that take advantage of the codon usage preferences of that particular species. For example, the isolated nucleic acid provided herein can be designed to have codons that are preferentially used by a particular organism of interest. Numerous software and sequencing services are available for such codon-optimizing of sequences. It also is noted that less conservative substitutions may be made and still provide a functional variant.

[00174] It has long been recognized in the art that some amino acids in amino acid sequences can be varied without significant effect on the structure or function of proteins. Variants included can constitute deletions, insertions, inversions, repeats, and type substitutions so long as the indicated enzyme activity is not significantly adversely affected. Guidance concerning which amino acid changes are likely to be phenotypically silent can be found, inter alia, in Bowie, J. U., et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990). This reference is incorporated by reference for such teachings, which are, however, also generally known to those skilled in the art. [00175] Those skilled in the art also will understand that the genetic alterations, including metabolic modifications exemplified herein are described with reference to a suitable source organism such as E. coli, yeast, or other organisms disclosed herein and their corresponding metabolic enzymatic reactions or a suitable source organism for desired genetic material such as genes encoding enzymes for their corresponding metabolic enzymatic reactions. 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

microorganisms. 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.

[00176] 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. 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%o 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.

[00177] 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. 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.

[00178] In contrast, paralogs are homologues 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. 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. In some cases, functional similarity requires at least some structural similarity in the active site or binding region of a nonorthologous gene compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene.

[00179] Therefore, in identifying and designing a genetically modified microorganism of the present invention, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of genetic modifications can include identification and inclusion or inactivation 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 evolutionarily related genes. 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 Wand 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% sequence identity 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.

[00180] 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. Through such comparisons and analyses, one skilled in the art may be able to obtain a desired polypeptide in a particular species that functions similarly to a polypeptide (enzyme) disclosed herein, and/or a functional variant that possesses a desired enzymatic activity. Also, in various embodiments polypeptides, such as enzymes, obtained by the expression of the any of the various polynucleotide molecules (i.e., nucleic acid sequences) of the present invention may have at least approximately 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to one or more amino acid sequences encoded by the genes and/or nucleic acid sequences described herein.

[00181] Also, variants and portions of particular nucleic acid sequences, and respective encoded amino acid sequences recited herein may be exhibit a desired functionality, e.g., enzymatic activity at a selected level, when such nucleic acid sequence variant and/or portion contains a 15 nucleotide sequence identical to any 15 nucleotide sequence set forth in the nucleic acid sequences recited herein including, without limitation, the sequence starting at nucleotide number 1 and ending at nucleotide number 15, the sequence starting at nucleotide number 2 and ending at nucleotide number 16, the sequence starting at nucleotide number 3 and ending at nucleotide number 17, and so forth. It will be appreciated that the invention also provides isolated nucleic acid that contains a nucleotide sequence that is greater than 15 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides) in length and identical to any portion of the sequence set forth in nucleic acid sequences recited herein. For example, the invention provides isolated nucleic acid that contains a 25 nucleotide sequence identical to any 25 nucleotide sequence set forth in any one or more (including any grouping of) nucleic acid sequences recited herein including, without limitation, the sequence starting at nucleotide number 1 and ending at nucleotide number 25, the sequence starting at nucleotide number 2 and ending at nucleotide number 26, the sequence starting at nucleotide number 3 and ending at nucleotide number 27, and so forth. Additional examples include, without limitation, isolated nucleic acids that contain a nucleotide sequence that is 50 or more nucleotides (e.g., 100, 150, 200, 250, 300, or more nucleotides) in length and identical to any portion of any of the sequences disclosed herein. Such isolated nucleic acids can include, without limitation, those isolated nucleic acids containing a nucleic acid sequence represented in any one section of discussion and/or examples, such as regarding 3 -HP production pathways, nucleic acid sequences encoding enzymes of the fatty acid synthase system, or 3 -HP tolerance. For example, the invention provides an isolated nucleic acid containing a nucleic acid sequence listed herein that contains a single insertion, a single deletion, a single substitution, multiple insertions, multiple deletions, multiple substitutions, or any combination thereof (e. g., single deletion together with multiple insertions). Such isolated nucleic acid molecules can share at least 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, or 99 percent sequence identity with a nucleic acid sequence listed herein (i.e., in the sequence listing).

[00182] Additional examples include, without limitation, isolated nucleic acids that contain a nucleic acid sequence that encodes an amino acid sequence that is 50 or more amino acid residues (e.g., 100, 150, 200, 250, 300, or more amino acid residues) in length and identical to any portion of an amino acid sequence listed or otherwise disclosed herein. In addition, the invention provides isolated polynucleotide (nucleic acid) that contains a nucleic acid sequence that encodes an polypeptide amino acid sequence having a variation of an amino acid sequence listed or otherwise disclosed herein. For example, the invention provides isolated polypeptide containing a nucleic acid sequence encoding an amino acid sequence listed or otherwise disclosed herein that contains a single insertion, a single deletion, a single substitution, multiple insertions, multiple deletions, multiple substitutions, or any combination thereof (e.g., single deletion together with multiple insertions). Such isolated nucleic acid molecules can contain a nucleic acid sequence encoding an amino acid sequence that shares at least 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, or 99 percent sequence identity with an amino acid sequence listed or otherwise disclosed herein.

[00183] Examples of properties that provide the bases for conservative and other amino acid substitutions are exemplified in Table 5. Accordingly, one skilled in the art may make numerous substitutions to obtain an amino acid sequence variant that exhibits a desired functionality. BLASTP, CLUSTALP, and other alignment and comparison tools may be used to assess highly conserved regions, to which fewer substitutions may be made (unless directed to alter activity to a selected level, which may require multiple substitutions). More substitutions may be made in regions recognized or believed to not be involved with an active site or other binding or structural motif. In accordance with Table 5, for example, substitutions may be made of one polar uncharged (PU) amino acid for a polar uncharged amino acid of a listed sequence, optionally considering size/molecular weight (i.e., substituting a serine for a threonine).

[00184] The invention provides polypeptides that contain the entire amino acid sequence of an amino acid sequence listed or otherwise disclosed herein. In addition, the invention provides polypeptides that contain a portion of an amino acid sequence listed or otherwise disclosed herein. For example, the invention provides polypeptides that contain a 15 amino acid sequence identical to any 15 amino acid sequence of an amino acid sequence listed or otherwise disclosed herein including, without limitation, the sequence starting at amino acid residue number 1 and ending at amino acid residue number 15, the sequence starting at amino acid residue number 2 and ending at amino acid residue number 16, the sequence starting at amino acid residue number 3 and ending at amino acid residue number 17, and so forth. It will be appreciated that the invention also provides polypeptides that contain an amino acid sequence that is greater than 15 amino acid residues (e. g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues) in length and identical to any portion of an amino acid sequence listed or otherwise disclosed herein For example, the invention provides polypeptides that contain a 25 amino acid sequence identical to any 25 amino acid sequence of an amino acid sequence listed or otherwise disclosed herein including, without limitation, the sequence starting at amino acid residue number 1 and ending at amino acid residue number 25, the sequence starting at amino acid residue number 2 and ending at amino acid residue number 26, the sequence starting at amino acid residue number 3 and ending at amino acid residue number 27, and so forth. Additional examples include, without limitation, polypeptides that contain an amino acid sequence that is 50 or more amino acid residues (e.g., 100, 150, 200, 250, 300 or more amino acid residues) in length and identical to any portion of an amino acid sequence listed or otherwise disclosed herein. Further, it is appreciated that, per above, a 15 nucleotide sequence will provide a 5 amino acid sequence, so that the latter, and higher-length amino acid sequences, may be defined by the above-described nucleotide sequence lengths having identity with a sequence provided herein.

[00185] In addition, the invention provides polypeptides that an amino acid sequence having a variation of the amino acid sequence set forth in an amino acid sequence listed or otherwise disclosed herein. For example, the invention provides polypeptides containing an amino acid sequence listed or otherwise disclosed herein that contains a single insertion, a single deletion, a single substitution, multiple insertions, multiple deletions, multiple substitutions, or any combination thereof (e.g., single deletion together with multiple insertions). Such polypeptides can contain an amino acid sequence that shares at least 60, 65, 70, 75, 80, 85, 90, 95, 97, 98 or 99 percent sequence identity with an amino acid sequence listed or otherwise disclosed herein. A particular variant amino acid sequence may comprise any number of variations as well as any combination of types of variations. Certain embodiments of the invention additionally comprise a genetic modification to increase the availability of the cofactor NADPH, which can increase the NADPH/NADP+ ratio as may be desired. Non-limiting examples for such genetic modification are pgi (E.C. 5.3.1.9, in a mutated form), pntAB (E.C. 1.6.1.2), overexpressed, gapA (E.C. 1.2.1.12):gapN (E.C. 1.2.1.9, from Streptococcus mutans) substitution/replacement, and disrupting or modifying a soluble transhydrogenase such as sthA (E.C. 1.6.1.2), and/or genetic modifications of one or more of zwf (E.C. 1.1.1.49), gnd (E.C. 1.1.1.44), and edd (E.C. 4.2.1.12). Sequences of these genes are available at www.metacyc.org. Also, the sequences for the genes and encoded proteins for the E. coli gene names shown in Tables 6A, 6B, and 7 are provided in U.S. Provisional Patent Application No. : 61/246, 141 (priority claimed to this in

PCT/US2010/050436, published March 31 , 201 1 as WO/201 1/038364), incorporated herein in its entirety and for such sequences, and also are available at www.ncbi.gov as well as www.metacyc.org or www.ecocyc.org.

[00186] Also, genetic modifications may be provided to add functionality for breakdown of more complex carbon sources, such as cellulosic biomass or products thereof, for uptake, and/or for utilization of such carbon sources. For example, numerous cellulases and cellulase-based cellulose degradation systems have been studied and characterized (see, for example, and incorporated by reference herein for such teachings, Beguin, P and Aubert, J-P (1994) FEMS Microbial. Rev. 13 : 25- 58; Ohima, K. et al. (1997) Biotechnol. Genet. Eng. Rev. 14: 365414).

[00187] Any of various genetic modifications described herein may be provided to species not having such functionality, or having a less than desired level of such functionality.

[00188]More generally, and depending on the particular metabolic pathways of a microorganism selected for genetic modification, any subgroup of genetic modifications may be made to decrease cellular production of fermentation product(s) selected from the group consisting of acetate, acetoin, acetone, acrylic, malate, fatty acid ethyl esters, isoprenoids, glycerol, ethylene glycol, ethylene, propylene, butylene, isobutylene, ethyl acetate, vinyl acetate, other acetates, 1 ,4-butanediol, 2,3- butanediol, butanol, isobutanol, sec-butanol, butyrate, isobutyrate, 2-OH-isobutryate, 30H-butyrate, ethanol, isopropanol, D-lactate, L-lactate, pyruvate, itaconate, levulinate, glucarate, glutarate, caprolactam, adipic acid, propanol, isopropanol, fusel alcohols, and 1,2-propanediol, 1,3 -propanediol, formate, fumaric acid, propionic acid, succinic acid, valeric acid, and maleic acid. Gene deletions may be made as disclosed generally herein, and other approaches may also be used to achieve a desired decreased cellular production of selected fermentation products.

[00189] Separation and Purification of the Chemical Product 3-HP or malonic acid

[00190] When 3-HP or malonic acid is the chemical product, the 3-HP or malonic acid may be separated and purified by the approaches described in the following paragraphs, taking into account that many methods of separation and purification are known in the art and the following disclosure is not meant to be limiting. Osmotic shock, sonication, homogenization, and/or a repeated freeze-thaw cycle followed by filtration and/or centrifugation, among other methods, such as pH adjustment and heat treatment, may be used to produce a cell-free extract from intact cells. Any one or more of these methods also may be employed to release 3-HP or malonic acid from cells as an extraction step.

[00191] Further as to general processing of a bio-production broth comprising 3-HP or malonic acid, various methods may be practiced to remove biomass and/or separate 3-HP or malonic acid from the culture broth and its components. Methods to separate and/or concentrate the 3-HP or malonic acid include centrifugation, filtration, extraction, chemical conversion such as esterification, distillation (which may result in chemical conversion, such as dehydration to acrylic acid, under some reactive- distillation conditions), crystallization, chromatography, and ion-exchange, in various forms. Additionally, cell rupture may be conducted as needed to release 3-HP or malonic acid from the cell mass, such as by sonication, homogenization, pH adjustment or heating. 3-HP or malonic acid may be further separated and/or purified by methods known in the art, including any combination of one or more of centrifugation, liquid-liquid separations, including extractions such as solvent extraction, reactive extraction, two-phase aqueous extraction and two- phase solvent extraction, membrane separation technologies, distillation, evaporation, ion-exchange chromatography, adsorption chromatography, reverse phase chromatography and crystallization. Any of the above methods may be applied to a portion of a bioproduction broth (i.e., a fermentation broth, whether made under aerobic, anaerobic, or microaerobic conditions), such as may be removed from a bio-production event gradually or periodically, or to the broth at termination of a bio- production event. Conversion of 3-HP or malonic acid to downstream products, such as described herein, may proceed after separation and purification, or, such as with distillation, thin- film evaporation, or wiped- film evaporation optionally also in part as a separation means.

[00192] For various of these approaches, one may apply a counter-current strategy, or a sequential or iterative strategy, such as multi-pass extractions. For example, a given aqueous solution comprising 3-HP or malonic acid may be repeatedly extracted with a non-polar phase comprising an amine to achieve multiple reactive extractions.

[00193] When a culture event (fermentation event) is at a point of completion, the spent broth may be transferred to a seperate tank, or remain in the culture vessel, and in either case the temperature may be elevated to at least 60°C for a minimum of one hour in order to kill the microorganisms. (Alternatively, as noted above other approaches to killing the microorganisms may be practiced, or centrifugation may occur prior to heating.) By spent broth is meant the final liquid volume comprising the initial nutrient media, cells grown from the microorganism inoculum (and possibly including some original cells of the inoculum), 3-HP or malonic acid, and optionally liquid additions made after providing the initial nutrient media, such as periodic additions to provide additional carbon source, etc. It is noted that the spent broth may comprise organic acids other than 3-HP or malonic acid, such as for example acetic acid and/or lactic acid.

[00194] A centrifugation step may then be practiced to filter out the biomass solids (e.g., microorganism cells). This may be achieved in a continuous or batch centrifuge, and solids removal may be at least about 80%, 85%o, 90%), or 95% in a single pass, or cumulatively after two or more serial centrifugations.

[00195] An optional step is to polish the centrifuged liquid through a filter, such as microfiltration or ultrafiltration, or may comprise a filter press or other filter device to which is added a filter aid such as diatomaceous earth. Alternative or supplemental approaches to this and the centrifugation may include removal of cells by a flocculent, where the cells floe and are allowed to settle, and the liquid is drawn off or otherwise removed. A flocculent may be added to a fermentation broth after which settling of material is allowed for a time, and then separations may be applied, including but not limited to centrifugation.

[00196] After such steps, a spent broth comprising 3-HP or malonic acid and substantially free of solids is obtained for further processing. By "substantially free of solids" is meant that greater than 98%, 99%, or 99.5%) of the solids have been removed.

[00197] In various embodiments this spent broth comprises various ions of salts, such as Na, CI, S04, and P04 . In some embodiments these ions may be removed by passing this spent broth through ion exchange columns, or otherwise contacting the spent broth with appropriate ion exchange material. Here and elsewhere in this document, "contacting" is taken to mean a contacting for the stated purpose by any way known to persons skilled in the art, such as, for example, in a column, under appropriate conditions that are well within the ability of persons of ordinary skill in the relevant art to determine. As but one example, these may comprise sequential contacting with anion and cation exchange materials (in any order), or with a mixed anion/cation material. This demineralization step should remove most such inorganic ions without removing the 3-HP or malonic acid. This may be achieved, for example, by lowering the pH sufficiently to protonate 3-HP or malonic acid and similar organic acids so that these acids are not bound to the anion exchange material, whereas anions, such as CI and S04, that remain charged at such pH are removed from the solution by binding to the resin. Likewise, positively charged ions are removed by contacting with cation exchange material. Such removal of ions may be assessed by a decrease in conductivity of the solution. Such ion exchange materials may be regenerated by methods known to those skilled in the art.

[00198] In some embodiments, the spent broth (such as but not necessarily after the previous

demineralization step) is subjected to a pH elevation, after which it is passed through an ion exchange column, or otherwise contacted with an ion exchange resin, that comprises anionic groups, such as amines, to which organic acids, ionic at this pH, associate. Other organics that do not so associate with amines at this pH (which may be over 6.5, over 7.5, over 8.5, over 9.5, over 10.5, or higher pH) may be separated from the organic acids at this stage, such as by flushing with an elevated pH rinse. Thereafter elution with a lower pH and/or elevated salt content rinse may remove the organic acids. Eluting with a gradient of decreasing pH and/or increasing salt content rinses may allow more distinct separation of 3- HP from other organic acids, thereafter simplifying further processing.

[00199] This latter step of anion-exchange resin retention of organic acids may be practiced before or after the demineralization step. However, the following two approaches are alternatives to the anion- exchange resin step. A first alternative approach comprises reactive extraction (a form of liquid- liquid extraction) as exemplified in this and the following paragraphs. The spent broth, which may be at a stage before or after the demineralization step above, is combined with a quantity of a tertiary amine such as Alamine336® (Cognis Corp., Cincinnati, OH USA) at low pH. Co-solvents for the Alamine336 or other tertiary amine may be added and include, but are not limited to benzene, carbon tetrachloride, chloroform, cyclohexane, di-isobutyl ketone, ethanol, #2 fuel oil, isopropanol, kerosene, nbutanol, isobutanol, octanol, and n-decanol that increase the partition coefficient when combined with the amine. After appropriate mixing a period of time for phase separation transpires, after which the non-polar phase, which comprises 3-HP associated with the Alamine336 or other tertiary amine, is separated from the aqueous phase.

[00200] When a co-solvent is used that has a lower boiling point than the 3-HP/tertiary amine, a distilling step may be used to remove the co-solvent, thereby leaving the 3 -HP -tertiary amine complex in the non-polar phase.

[00201] Whether or not there is such a distillation step, a stripping or recovery step may be used to separate the 3-HP from the tertiary amine. An inorganic salt, such as ammonium sulfate, sodium chloride, or sodium carbonate, or a base such as sodium hydroxide or ammonium hydroxide, is added to the 3- HP/tertiary amine to reverse the amine protonation reaction, and a second phase is provided by addition of an aqueous solution (which may be the vehicle for provision of the inorganic salt). After suitable mixing, two phases result and this allows for tertiary amine regeneration and re-use, and provides the 3-HP in an aqueous solution. Alternatively, hot water may also be used without a salt or base to recover the 3HP from the amine.

[00202] In the above approach the phase separation and extraction of 3-HP to the aqueous phase can serve to concentrate the 3-HP. It is noted that chromatographic separation of respective organic acids also can serve to concentrate such acids, such as 3-HP. In similar approaches other suitable, non-polar amines, which may include primary, secondary and quaternary amines, may be used instead of and/or in combination with a tertiary amine.

[00203] A second alternative approach is crystallization. For example, the spent broth (such as free of biomass solids) may be contacted with a strong base such as ammonium hydroxide, which results in formation of an ammonium salt of 3-HP. This may be concentrated, and then ammonium-3-HP crystals are formed and may be separated, such as by filtration, from the aqueous phase. Once collected, ammonium-3-HP crystals may be treated with an acid, such as sulfuric acid, so that ammonium sulfate is regenerated, so that 3-HP and ammonium sulfate result.

[00204] Also, various aqueous two-phase extraction methods may be utilized to separate and/or concentrate a desired chemical product from a fermentation broth or later- obtained solution. The addition of polymers, such as dextran and glycol polymers, such as polyethylene glycol (PEG) and polypropylene glycol (PPG) to an aqueous solution may result in formation of two aqueous phases. In such systems a desired chemical product may segregate to one phase while cells and other chemicals partition to the other phase, thus providing for a separation without use of organic solvents. This approach has been demonstrated for some chemical products, but challenges associated with chemical product recovery from a polymer solution and low selectivities are recognized (See "Extractive Recovery of Products from Fermentation Broths," Joong Kyun Kim et al., Biotechnol. Bioprocess Eng., 1999(4)1-11, incorporated by reference for all of its teachings of extractive recovery methods).

[00205] Various substitutions and combinations of the above steps and processes may be made to obtain a relatively purified 3-HP solution. Also, methods of separation and purification disclosed in US 6,534,679, issued March 18, 2003, and incorporated by reference herein for such methods disclosures, may be considered based on a particular processing scheme. Also, in some culture events periodic removal of a portion of the liquid volume may be made, and processing of such portion(s) may be made to recover the 3-HP, including by any combination of the approaches disclosed above.

[00206] As noted, solvent extraction is another alternative. This may use any of a number of and/or combinations of solvents, including alcohols, esters, ketones, and various organic solvents. Without being limiting, after phase separation a distillation step or a secondary extraction may be employed to separate 3-HP from the organic phase.

[00207]The following published resources are incorporated by reference herein for their respective teachings to indicate the level of skill in these relevant arts, and as needed to support a disclosure that teaches how to make and use methods of industrial bio-production of 3 -HP, and also industrial systems that may be used to achieve such conversion with any of the recombinant microorganisms of the present invention (Biochemical Engineering Fundamentals, 2nd Ed. J. E. Bailey and D. F. 01 lis, McGraw Hill, New York, 1986, entire book for purposes indicated and Chapter 9, pp. 533657 in particular for biological reactor design; Unit Operations of Chemical Engineering, 5th Ed., W. L. McCabe et al., McGraw Hill, New York 1993, entire book for purposes indicated, and particularly for process and separation technologies analyses; Equilibrium Staged Separations, P. C. Wankat, Prentice Hall, Englewood Cliffs, NJ USA, 1988, entire book for separation technologies teachings).

[00208] Conversion of 3-HP to Acrylic Acid and Downstream Products

[00209] As discussed herein, various embodiments described herein are related to production of a particular chemical product, 3-hydroxypropionic acid (3-HP). This organic acid, 3-HP, may be converted to various other products having industrial uses, such as but not limited to acrylic acid, esters of acrylic acid, and other chemicals obtained from 3-HP, referred to as "downstream products." Under some approaches the 3-HP may be converted to acrylic acid, acrylamide, and/or other downstream chemical products, in some instances the conversion being associated with the separation and/or purification steps. Many conversions to such downstream products are described herein. The methods of the invention include steps to produce downstream products of 3-HP.

[00210] Also, it is noted that acrylic acid, first converted from 3-HP by dehydration, may be esterified with appropriate compounds to form a number of commercially important acrylate-based esters, including but not limited to methyl acrylate, ethyl acrylate, methyl acrylate, 2-ethylhexyl acrylate, butyl acrylate, and lauryl acrylate. Alternatively, 3 HP may be esterified to form an ester of 3 HP and then dehydrated to form the acrylate ester.

[00211] As will be further described, 3-HP can be converted into derivatives starting (i) substantially as the protonated form of 3-hydroxypropionic acid; (ii) substantially as the deprotonated form, 3- hydroxypropionate; or (iii) as mixtures of the protonated and deprotonated forms. Generally, the fraction of 3- HP present as the acid versus the salt will depend on the pH, the presence of other ionic species in solution, temperature (which changes the equilibrium constant relating the acid and salt forms), and to some extent pressure. Many chemical conversions may be carried out from either of the 3-HP forms, and overall process economics will typically dictate the form of 3-HP for downstream conversion.

[00212] Also, as an example of a conversion during separation, 3-HP in an amine salt form, such as in the extraction step herein disclosed using Alamine 336 as the amine, may be converted to acrylic acid by contacting a solution comprising the 3-HP amine salt with a dehydration catalyst, such as aluminum oxide, at an elevated temperature, such as 170 to 180 C, or 180 to 190 C, or 190 to 200 C, and passing the collected vapor phase over a low temperature condenser. Operating conditions, including 3-HP concentration, organic amine, co-solvent (if any), temperature, flow rates, dehydration catalyst, and condenser temperature, are evaluated and improved for commercial purposes. Conversion of 3-HP to acrylic acid is expected to exceed at least 80 percent, or at least 90 percent, in a single conversion event. The amine may be re-used, optionally after clean-up. Other dehydration catalysts, as provided herein, may be evaluated. It is noted that U.S. Patent

No.7, 186,856 discloses data regarding this conversion approach, albeit as part of an extractive salt- splitting conversion that differs from the teachings herein. However, U.S. Patent No. 7, 186,856 is incorporated by reference for its methods, including extractive salt- splitting, the latter to further indicate the various ways 3-HP may be extracted from a microbial fermentation broth. Further as to embodiments in which the chemical product being synthesized by the microorganism host cell is 3- HP, made as provided herein and optionally purified to a selected purity prior to conversion, the methods of the present invention can also be used to produce "downstream" compounds derived from 3-HP, such as polymerized-3-HP (poly-3-HP), acrylic acid, polyacrylic acid (polymerized acrylic acid, in various forms), and methyl acrylate. Numerous approaches may be employed for such downstream conversions, generally falling into enzymatic, catalytic (chemical conversion process using a catalyst), thermal, and combinations thereof (including some wherein a desired pressure is applied to accelerate a reaction).

[00213] As noted, an important industrial chemical product that may be produced from 3-HP is acrylic acid. Chemically, one of the carbon-carbon single bonds in 3-HP must undergo a dehydration reaction, converting to a carbon-carbon double bond and rejecting a water molecule. Dehydration of 3-HP in principle can be carried out in the liquid phase or in the gas phase. In some embodiments, the dehydration takes place in the presence of a suitable homogeneous or heterogeneous catalyst. Suitable dehydration catalysts are both acid and alkaline catalysts. Following dehydration, an acrylic acid-containing phase is obtained and can be purified where appropriate by further purification steps, such as by distillation methods, extraction methods, or crystallization methods, or combinations thereof.

[00214] Making acrylic acid from 3-HP via a dehydration reaction may be achieved by a number of commercial methodologies including via a distillation process, which may be part of the separation regime and which may include an acid and/or a metal ion as catalyst. More broadly, incorporated herein for its teachings of conversion of 3-HP, and other B-hydroxy carbonyl compounds, to acrylic acid and other related downstream compounds, is U.S. Patent Publication No. 2007/0219390 Al, published September 20, 2007, now abandoned. This publication lists numerous catalysts and provides examples of conversions, which are specifically incorporated herein. Also among the various specific methods to dehydrate 3-HP to produce acrylic acid is an older method, described in U.S. Patent No. 2,469,701 (Redmon). This reference teaches a method for the preparation of acrylic acid by heating 3-HP to a temperature between 130 and 190°C, in the presence of a dehydration catalyst, such as sulfuric acid or phosphoric acid, under reduced pressure. U.S. Patent Publication No. 2005/0222458 Al (Craciun et al.) also provides a process for the preparation of acrylic acid by heating 3- HP or its derivatives. Vapor- phase dehydration of 3-HP occurs in the presence of dehydration catalysts, such as packed beds of silica, alumina, or titania. These patent publications are incorporated by reference for their methods relating to converting 3 -HP to acrylic acid.

[00215] The dehydration catalyst may comprise one or more metal oxides, such as Ab03, Si02, or Ti02. In some embodiments, the dehydration catalyst is a high surface area Ab03 or a high surface area silica wherein the silica is substantially Si02. High surface area for the purposes of the invention means a surface area of at least about 50, 75, 100 m ² /g, or more. In some embodiments, the dehydration catalyst may comprise an aluminosilicate, such as a zeolite.

[00216] For example, including as exemplified from such incorporated references, 3-HP may be dehydrated to acrylic acid via various specific methods, each often involving one or more dehydration catalysts. One catalyst of particular apparent value is titanium, such as in the form of titanium oxide, Ti0(2). A titanium dioxide catalyst may be provided in a dehydration system that distills an aqueous solution comprising 3-HP, wherein the 3-HP dehydrates, such as upon volatilization, converting to acrylic acid, and the acrylic acid is collected by condensation from the vapor phase.

[00217] As but one specific method, an aqueous solution of 3-HP is passed through a reactor column packed with a titanium oxide catalyst maintained at a temperature between 170 and 190 C and at ambient atmospheric pressure. Vapors leaving the reactor column are passed over a low temperature condenser, where acrylic acid is collected. The low temperature condenser may be cooled to 30 C or less, 2 C or less, or at any suitable temperature for efficient condensation based on the flow rate and design of the system. Also, the reactor column temperatures may be lower, for instance when operating at a pressure lower than ambient atmospheric pressure. It is noted that Example 1 of U.S. Patent Publication No. 2007/0219390, published September 20, 2007, now abandoned, provides specific parameters that employs the approach of this method. As noted, this publication is incorporated by reference for this teaching and also for its listing of catalysts that may be used in a 3-HP to acrylic acid dehydration reaction.

[00218] Further as to dehydration catalysts, the following table summarizes a number of catalysts (including chemical classes) that may be used in a dehydration reaction from 3-HP (or its esters) to acrylic acid (or acrylate esters). Such catalysts, some of which may be used in any of solid, liquid or gaseous forms, may be used individually or in any combination. This listing of catalysts is not intended to be limiting, and many specific catalysts not listed may be used for specific dehydration reactions. Further without being limiting, catalyst selection may depend on the solution pH and/or the form of 3-HP in a particular conversion, so that an acidic catalyst may be used when 3-HP is in acidic form, and a basic catalyst may be used when the ammonium salt of 3-HP is being converted to acrylic acid. Also, some catalysts may be in the form of ion exchange resins. [00219] Table 6: Dehydration Catalysts

[00220] As to another specific method using one of these catalysts, concentrated sulfuric acid and an aqueous solution comprising 3-HP are separately flowed into a reactor maintained at 150 to 165°C at a reduced pressure of 100 mm Hg. Flowing from the reactor is a solution comprising acrylic acid. A specific embodiment of this method, disclosed in Example 1 of US2009/0076297, incorporated by reference herein, indicates a yield of acrylic acid exceeding 95 percent.

[00221] Based on the wide range of possible catalysts and knowledge in the art of dehydration reactions of this type, numerous other specific dehydration methods may be evaluated and implemented for commercial production. The dehydration of 3-HP may also take place in the absence of a dehydration catalyst. For example, the reaction may be run in the vapor phase in the presence of a nominally inert packing such as glass, ceramic, a resin, porcelain, plastic, metallic or brick dust packing and still form acrylic acid in reasonable yields and purity. The catalyst particles can be sized and configured such that the chemistry is, in some embodiments, mass-transfer-limited or kinetically limited. The catalyst can take the form of powder, pellets, granules, beads, extrudates, and so on. When a catalyst support is optionally employed, the support may assume any physical form such as pellets, spheres, monolithic channels, etc. The supports may be co-precipitated with active metal species; or the support may be treated with the catalytic metal species and then used as is or formed into the aforementioned shapes; or the support may be formed into the aforementioned shapes and then treated with the catalytic species.

[00222] A reactor for dehydration of 3-HP may be engineered and operated in a wide variety of ways. The reactor operation can be continuous, semi-continuous, or batch. It is perceived that an operation that is substantially continuous and at steady state is advantageous from operations and economics perspectives. The flow pattern can be substantially plug flow, substantially well-mixed, or a flow pattern between these extremes. A "reactor" can actually be a series or network of several reactors in various arrangements. [00223]For example, without being limiting, acrylic acid may be made from 3-HP via a dehydration reaction, which may be achieved by a number of commercial methodologies including via a distillation process, which may be part of the separation regime and which may include an acid and/or a metal ion as catalyst. More broadly, incorporated herein for its teachings of conversion of 3-HP, and other B- hydroxy carbonyl compounds, to acrylic acid and other related downstream compounds, is U.S. Patent Publication No. 2007/0219390 Al, published September 20, 2007, now abandoned. This publication lists numerous catalysts and provides examples of conversions, which are specifically incorporated herein.

[00224] For example, including as exemplified from such incorporated references, 3-HP may be dehydrated to acrylic acid via various specific methods, each often involving one or more dehydration catalysts. One catalyst of particular apparent value is titanium, such as in the form of titanium oxide, Ti02. A titanium dioxide catalyst may be provided in a dehydration system that distills an aqueous solution comprising 3-HP, wherein the 3-HP dehydrates, such as upon volatilization, converting to acrylic acid, and the acrylic acid is collected by condensation from the vapor phase.

[00225] As but one specific method, an aqueous solution of 3-HP is passed through a reactor column packed with a titanium oxide catalyst maintained at a temperature between 170 and 190°C and at ambient atmospheric pressure. Vapors leaving the reactor column are passed over a low temperature condenser, where acrylic acid is collected. The low temperature condenser may be cooled to 30°C or less, 20°C or less, 2°C or less, or at any suitable temperature for efficient condensation based on the flow rate and design of the system. Also, the reactor column temperatures may be lower, for instance when operating at a pressure lower than ambient atmospheric pressure. It is noted that Example 1 of U.S. Patent Publication No. 2007/0219390, published September 20, 2007, now abandoned, provides specific parameters that employs the approach of this method. As noted, this publication is incorporated by reference for this teaching and also for its listing of catalysts that may be used in a 3- HP to acrylic acid dehydration reaction. Crystallization of the acrylic acid obtained by dehydration of 3-HP may be used as one of the final separation/purification steps. Various approaches to

crystallization are known in the art, including crystallization of esters.

[00226]As noted above, in some embodiments, a salt of 3-HP is converted to acrylic acid or an ester or salt thereof. For example, U.S. Patent No.7, 186,856 (Meng et al.) teaches a process for producing acrylic acid from the ammonium salt of 3-HP, which involves a first step of heating the ammonium salt of 3-HP in the presence of an organic amine or solvent that is immiscible with water, to form a two-phase solution and split the 3-HP salt into its respective ionic constituents under conditions which transfer 3-HP from the aqueous phase to the organic phase of the solution, leaving ammonia and ammonium cations in the aqueous phase. The organic phase is then back-extracted to separate the 3- HP, followed by a second step of heating the 3 -HP-containing solution in the presence of a dehydration catalyst to produce acrylic acid. U.S. Patent No. 7, 186,856 is incorporated by reference for its methods for producing acrylic acid from salts of 3-HP. Various alternatives to the particular approach disclosed in this patent may be developed for suitable extraction and conversion processes.

[00227] Methyl acrylate may be made from 3-HP via dehydration and esterification, the latter to add a methyl group (such as using methanol), acrylamide may be made from 3-HP via dehydration and amidation reactions, acrylonitrile may be made via a dehydration reaction and forming a nitrile moiety, propriolactone may be made from 3-HP via a ring-forming internal esterification reaction (eliminating a water molecule), ethyl-3-

[00228] HP may be made from 3-HP via esterification with ethanol, malonic acid may be made from 3-HP via an oxidation reaction, and 1,3-propanediol may be made from 3-HP via a reduction reaction.

[00229] Malonic acid may be produced from oxidation of 3-HP as produced herein. U.S. Patent No. 5,817,870 (Haas et al.) discloses catalytic oxidation of 3-HP by a precious metal selected from Ru, Rh, Pd, Os, Ir or Pt. These can be pure metal catalysts or supported catalysts. The catalytic oxidation can be carried out using a suspension catalyst in a suspension reactor or using a fixed-bed catalyst in a fixed-bed reactor. If the catalyst, such as a supported catalyst, is disposed in a fixed-bed reactor, the latter can be operated in a trickle- bed procedure as well as also in a liquid-phase procedure. In the trickle-bed procedure the aqueous phase comprising the 3-HP starting material, as well as the oxidation products of the same and means for the adjustment of pH, and oxygen or an oxygen-containing gas can be conducted in parallel flow or counter-flow. In the liquid-phase procedure the liquid phase and the gas phase are conveniently conducted in parallel flow.

[00230] In order to achieve a sufficiently short reaction time, the conversion is carried out at a pH equal or greater than 6, such as at least 7, and in particular between 7.5 and 9. According to a particular embodiment, during the oxidation reaction the pH is kept constant, such as at a pH in the range between 7.5 and 9, by adding a base, such as an alkaline or alkaline earth hydroxide solution. The oxidation is usefully carried out at a temperature of at least 10°C and maximally 70°C. The flow of oxygen is not limited. In the suspension method it is important that the liquid and the gaseous phase are brought into contact by stirring vigorously. Malonic acid can be obtained in nearly quantitative yields. U.S. Patent No. 5,817,870 is incorporated by reference herein for its methods to oxidize 3-HP to malonic acid.

[00231] Also, addition reactions may yield acrylic acid or acrylate derivatives having alkyl or aryl groups at the carbonyl hydroxyl group. Such additions may be catalyzed chemically, such as by hydrogen, hydrogen halides, hydrogen cyanide, or Michael additions under alkaline conditions optionally in the presence of basic catalysts. Alcohols, phenols, hydrogen sulfide, and thiols are known to add under basic conditions. Aromatic amines or amides, and aromatic hydrocarbons, may be added under acidic conditions. These and other reactions are described in Ulmann's Encyclopedia of Industrial Chemistry, Acrylic Acid and Derivatives, Wil ey VCH Verlag GmbH, Wienham (2005), incorporated by reference for its teachings of conversion reactions for acrylic acid and its derivatives.

[00232] Acrylic acid obtained from 3-HP made by the present invention may be further converted to various chemicals, including polymers, which are also considered downstream products in some embodiments. Acrylic acid esters may be formed from acrylic acid (or directly from 3 -HP) such as by condensation esterification reactions with an alcohol, releasing water. This chemistry described in Monomeric Acrylic Esters, E. H. Riddle, Reinhold, NY (1954), incorporated by reference for its esterification teachings. Among esters that are formed are methyl acrylate, ethyl acrylate, n-butyl aery late, hydroxypropyl acrylate, hydroxy ethyl acrylate, isobutyl acrylate, and 2-ethylhexyl acrylate, and these and/or other acrylic acid and/or other acrylate esters may be combined, including with other compounds, to form various known acrylic acid-based polymers. Although acrylamide is produced in chemical syntheses by hydration of acrylonitrile, herein a conversion may convert acrylic acid to acrylamide by amidation. Acrylic acid obtained from 3-HP made by the present invention may be further converted to various chemicals, including polymers, which are also considered downstream products in some embodiments. Acrylic acid esters may be formed from acrylic acid (or directly from 3-HP) such as by condensation esterification reactions with an alcohol, releasing water. This chemistry is described in Monomeric Acrylic Esters, E. H. Riddle, Reinhold, NY (1954), incorporated by reference for its esterification teachings. Among esters that are formed are methyl acrylate, ethyl acrylate, n-butyl acrylate, hydroxypropyl acrylate, hydroxy ethyl acrylate, isobutyl acrylate, and 2- ethylhexyl acrylate, and these and/or other acrylic acid and/or other acrylate esters may be combined, including with other compounds, to form various known acrylic acid-based polymers. Although acrylamide is produced in chemical syntheses by hydration of acrylonitrile, herein a conversion may convert acrylic acid to acrylamide by amidation. Direct esterification of acrylic acid can take place by esterification methods known to the person skilled in the art, by contacting the acrylic acid obtained from 3-HP dehydration with one or more alcohols, such as methanol, ethanol, 1- propanol, 2-propanol, n-butanol, tert-butanol or isobutanol, and heating to a temperature of at least 50, 75, 100, 125, or 150°C. The water formed during esterification may be removed from the reaction mixture, such as by azeotropic distillation through the addition of suitable separation aids, or by another means of separation. Conversions up to 95%, or more, may be realized.

[00233] Several suitable esterification catalysts are commercially available, such as from Dow Chemical (Midland, Michigan US). For example, AmberlystTM 131 Wet Monodisperse gel catalyst confers enhanced hydraulic and reactivity properties and is suitable for fixed bed reactors. AmberlystTM 39Wet is a macroreticular catalyst suitable particularly for stirred and slurry loop reactors. AmberlystTM 46 is a macroporous catalyst producing less ether byproducts than conventional catalyst (as described in U.S. Patent No. 5,426,199 to Rohm and Haas, which patent is incorporated by reference for its teachings of esterification catalyst compositions and selection considerations).

[00234] Acrylic acid, and any of its esters, may be further converted into various polymers.

[00235] Polymerization may proceed by any of heat, light, other radiation of sufficient energy, and free radical generating compounds, such as azo compounds or peroxides, to produce a desired polymer of acrylic acid or acrylic acid esters. As one example, an aqueous acrylic acid solution's temperature raised to a temperature known to start polymerization (in part based on the initial acrylic acid concentration), and the reaction proceeds, the process frequently involving heat removal given the high exothermicity of the reaction. Many other methods of polymerization are known in the art. Some are described in Ulmann's Encyclopedia of Industrial Chemistry, Polyacrylamides and

Poly( Acrylic Acids), Wil ey VCH Verlag GmbH, Wienham (2005), incorporated by reference for its teachings of polymerization reactions.

[00236] For example, the free-radical polymerization of acrylic acid takes place by various polymerization methods and can be carried out either in an emulsion or suspension in aqueous solution or another solvent. Initiators, such as but not limited to organic peroxides, often are added to aid in the polymerization.

Among the classes of organic peroxides that may be used as initiators are diacyls, peroxydicarbonates, monoperoxycarbonates, peroxyketals, peroxyesters, dialkyls, and hydroperoxides. Another class of initiators is azo initiators, which may be used for acrylate polyermization as well as co-polymerization with other monomers. U.S. Patent Nos. 5,470,928; 5,510,307; 6,709,919; and 7,678,869 teach various approaches to polymerization using a number of initiators, including organic peroxides, azo compounds, and other chemical types, and are incorporated by reference for such teachings as applicable to the polymers described herein.

[00237] Accordingly, it is further possible for co-monomers, such as crosslinkers, to be present during the polymerization. The free-radical polymerization of the acrylic acid obtained from dehydration of 3 -HP, as produced herein, in at least partly neutralized form and in the presence of crosslinkers is practiced in certain embodiments. This polymerization may result in hydrogels which can then be comminuted, ground and, where appropriate, surface-modified, by known techniques.

[00238] An important commercial use of polyacrylic acid is for superabsorbent polymers. This specification hereby incorporates by reference Modern Superabsorbent Polymer Technology, Buchholz and Graham (Editors), WileyVCH, 1997, in its entirety for its teachings regarding superabsorbent polymers components, manufacture, properties and uses. Superabsorbent polymers are primarily used as absorbents for water and aqueous solutions for diapers, adult incontinence products, feminine hygiene products, and similar consumer products. In such consumer products, superabsorbent materials can replace traditional absorbent materials such as cloth, cotton, paper wadding, and cellulose fiber.

Superabsorbent polymers absorb, and retain under a slight mechanical pressure, up to 25 times or their weight in liquid. The swollen gel holds the liquid in a solid, rubbery state and prevents the liquid from leaking. Superabsorbent polymer particles can be surface- modified to produce a shell structure with the shell being more highly crosslinked. This technique improves the balance of absorption, absorption under load, and resistance to gel-blocking. It is recognized that superabsorbent polymers have uses in fields other than consumer products, including agriculture, horticulture, and medicine.

[00239] Superabsorbent polymers are prepared from acrylic acid (such as acrylic acid derived from 3 -HP provided herein) and a crosslinker, by solution or suspension polymerization. Exemplary methods include U.S. Patent Nos. 5,145,906; 5,350,799; 5,342,899; 4,857,610; 4,985,518; 4,708, 997; 5,180,798;

4,666,983;4,734,478; and 5,331,059, each incorporated by reference for their teachings relating to superabsorbent polymers. Among consumer products, a diaper, a feminine hygiene product, and an adult incontinence product are made with superabsorbent polymer that itself is made substantially from acrylic acid converted from 3 -HP made in accordance with the present invention. Diapers and other personal hygiene products may be produced that incorporate superabsorbent polymer made from acrylic acid made from 3-HP which is bio-produced by the teachings of the present application. The following provides general guidance for making a diaper that incorporates such superabsorbent polymer. The superabsorbent polymer first is prepared into an absorbent pad that may be vacuum formed, and in which other materials, such as a fibrous material (e.g., wood pulp) are added. The absorbent pad then is assembled with sheet(s) of fabric, generally a nonwoven fabric (e.g., made from one or more of nylon, polyester, polyethylene, and polypropylene plastics) to form diapers.

[00240] More particularly, in one non- limiting process, above a conveyer belt multiple pressurized nozzles spray superabsorbent polymer particles (such as about 400 micron size or larger), fibrous material, and/or a combination of these onto the conveyer belt at designated spaces/intervals. The conveyor belt is perforated and under vacuum from below, so that the sprayed on materials are pulled toward the belt surface to form a flat pad. In various embodiments, fibrous material is applied first on the belt, followed by a mixture of fibrous material and the superabsorbent polymer particles, followed by fibrous material, so that the superabsorbent polymer is concentrated in the middle of the pad. A leveling roller may be used toward the end of the belt path to yield pads of uniform thickness. Each pad thereafter may be further processed, such as to cut it to a proper shape for the diaper, or the pad may be in the form of a long roll sufficient for multiple diapers. Thereafter, the pad is sandwiched between a top sheet and a bottom sheet of fabric (one generally being liquid pervious, the other liquid

impervious), such as on a conveyor belt, and these are attached together such as by gluing, heating or ultrasonic welding, and cut into diaper-sized units (if not previously so cut). Additional features may be provided, such as elastic components, strips of tape, etc., for fit and ease of wearing by a person. FIG. 4 A, B, and C and FIG. 5 A and B show a schematic of an entire process of converting biomass to a finished product such as a diaper. These are meant to be exemplary and not limiting.

[00241] The ratio of the fibrous material to polymer particles is known to effect performance

characteristics. In some embodiments, this ratio is between 75:25 and 90:10 (see U.S. Patent No.

4,685,915, incorporated by reference for its teachings of diaper manufacture). Other disposable absorbent articles may be constructed in a similar fashion, such as for adult incontinence, feminine hygiene (sanitary napkins), tampons, etc. (see, for example, U.S. Patent Nos. 5,009,653, 5,558,656, and 5,827,255 incorporated by reference for their teachings of sanitary napkin manufacture).

[00242] Low molecular-weight polyacrylic acid has uses for water treatment, flocculants, and thickeners for various applications including cosmetics and pharmaceutical preparations. For these applications, the polymer may be uncrosslinked or lightly crosslinked, depending on the specific application. The molecular weights are typically from about 200 to about 1,000,000 g/mol. Preparation of these low molecular-weight polyacrylic acid polymers is described in U.S. Patent Nos. 3,904,685; 4,301,266; 2,798,053; and 5,093,472, each of which is incorporated by reference for its teachings relating to methods to produce these polymers.

[00243] Acrylic acid may be co-polymerized with one or more other monomers selected from acrylamide, 2-acrylamido-2- methylpropanesulfonic acid, Ν,Ν-dimethylacrylamide, N-isopropylacrylamide, methacrylic acid, and methacrylamide, to name a few. The relative reactivities of the monomers affect the microstructure and thus the physical properties of the polymer. Co-monomers may be derived from 3-HP, or otherwise provided, to produce copolymers. Ulmann's Encyclopedia of Industrial Chemistry,

Polyacrylamides and Poly( Acrylic Acids), Wil eyVCH Verlag GmbH, Wienham (2005), is incorporated by reference herein for its teachings of polymer and co-polymer processing.

[00244]Acrylic acid can in principle be copolymerized with almost any free-radically polymerizable monomers including styrene, butadiene, acrylonitrile, acrylic esters, maleic acid, maleic anhydride, vinyl chloride, acrylamide, itaconic acid, and so on. End-use applications typically dictate the copolymer composition, which influences properties. Acrylic acid also may have a number of optional substitutions on it, and after such substitutions be used as a monomer for polymerization, or co- polymerization reactions. As a general rule, acrylic acid (or one of its copolymerization monomers) may be substituted by any substituent that does not interfere with the polymerization process, such as alkyl, alkoxy, aryl, heteroaryl, benzyl, vinyl, allyl, hydroxy, epoxy, amide, ethers, esters, ketones, maleimides, succinimides, sulfoxides, glycidyl and silyl (see U.S. Patent No. 7,678,869, incorporated by reference above, for further discussion). The following paragraphs provide a few non-limiting examples of copolymerization applications.

[00245] Paints that comprise polymers and copolymers of acrylic acid and its esters are in wide use as industrial and consumer products. Aspects of the technology for making such paints can be found in U.S. Patent Nos. 3,687,885 and 3,891,591, incorporated by reference for its teachings of such paint manufacture. Generally, acrylic acid and its esters may form homopolymers or copolymers among themselves or with other monomers, such as amides, methacrylates, acrylonitrile, vinyl, styrene and butadiene. A desired mixture of homopolymers and/or copolymers, referred to in the paint industry as 'vehicle' (or 'binder') are added to an aqueous solution and agitated sufficiently to form an aqueous dispersion that includes sub-micrometer sized polymer particles. The paint cures by coalescence of these 'vehicle' particles as the water and any other solvent evaporate. Other additives to the aqueous dispersion may include pigment, filler (e.g., calcium carbonate, aluminum silicate), solvent (e.g., acetone, benzol, alcohols, etc., although these are not found in certain no VOC paints), thickener, and additional additives depending on the conditions, applications, intended surfaces, etc. In many paints, the weight percent of the vehicle portion may range from about nine to about 26 percent, but for other paints the weight percent may vary beyond this range. Acrylic-based polymers are used for many coatings in addition to paints. For example, for paper coating latexes, acrylic acid is used from 0.1-5.0%, along with styrene and butadiene, to enhance binding to the paper and modify rheology, freeze-thaw stability and shear stability. In this context, U.S. Patent Nos. 3,875,101 and 3,872,037 are incorporated by reference for their teachings regarding such latexes. Acrylate- based polymers also are used in many inks, particularly UV curable printing inks. For water treatment, acrylamide and/or hydroxy ethyl acrylate are commonly co- polymerized with acrylic acid to produce low molecular-weight linear polymers. In this context, U.S. Patent Nos. 4,431,547 and 4,029,577 are incorporated by reference for their teachings of such polymers. Copolymers of acrylic acid with maleic acid or itaconic acid are also produced for water- treatment applications, as described in U.S. Patent No. 5, 135,677, incorporated by reference for that teaching. Sodium acrylate (the sodium salt of glacial acrylic acid) can be co-polymerized with acrylamide (which may be derived from acrylic acid via amidation chemistry) to make an anionic copolymer that is used as a flocculent in water treatment.

[00246] For thickening agents, a variety of co-monomers can be used, such as described in U.S. Patent Nos. 4,268,641 and 3,915,921, incorporated by reference for description of these co-monomers. U.S. Patent No. 5,135,677 describes a number of co-monomers that can be used with acrylic acid to produce water-soluble polymers, and is incorporated by reference for such description.

[00247] Also as noted, some conversions to downstream products may be made enzymatically. For example, 3-HP may be converted to 3-HP-CoA, which then may be converted into polymerized 3-HP with an enzyme having polyhydroxyacid synthase activity (EC 2.3.1.-). Also, 1,3-propanediol can be made using polypeptides having oxidoreductase activity or reductase activity (e.g. , enzymes in the EC 1.1.1.- class of enzymes). Alternatively, when creating 1,3-propanediol from 3HP, a combination of (1) a polypeptide having aldehyde dehydrogenase activity (e.g., an enzyme from the 1.1.1.34 class) and (2) a polypeptide having alcohol dehydrogenase activity (e.g., an enzyme from the 1.1.1.32 class) can be used. Polypeptides having lipase activity may be used to form esters. Enzymatic reactions such as these may be conducted in vitro, such as using cell-free extracts, or in vivo. Thus, various embodiments of the present invention, such as methods of making a chemical, include conversion steps to any such noted downstream products of microbially produced 3-HP, including but not limited to those chemicals described herein and in the incorporated references (the latter for jurisdictions allowing this). For example, one embodiment is making 3-HP molecules by the teachings herein and further converting the 3-HP molecules to

polymerized-3-HP (poly-3-HP) or acrylic acid, and such as from acrylic acid then producing from the 3- HP molecules any one of polyacrylic acid (polymerized acrylic acid, in various forms), methyl acrylate, acrylamide, acrylonitrile, propiolactone, ethyl 3-HP, malonic acid, 1,3-propanediol, ethyl acrylate, n-butyl acrylate, hydroxypropyl acrylate, hydroxyethyl acrylate, isobutyl acrylate, 2-ethylhexyl acrylate, and acrylic acid or an acrylic acid ester to which an alkyl or aryl addition is made, and/or to which halogens, aromatic amines or amides, and aromatic hydrocarbons are added.

[00248] Also as noted, some conversions to downstream products may be made enzymatically. For example, 3-HP may be converted to 3-HP-CoA, which then may be converted into polymerized 3-HP with an enzyme having polyhydroxyacid synthase activity (EC 2.3.1.-). Also, 1,3-propanediol can be made using polypeptides having oxidoreductase activity or reductase activity (e.g., enzymes in the EC 1.1.1.- class of enzymes). Alternatively, when creating 1,3-propanediol from 3HP, a combination of (1) a polypeptide having aldehyde dehydrogenase activity (e.g., an enzyme from the 1.1.1.34 class) and (2) a polypeptide having alcohol dehydrogenase activity (e.g., an enzyme from the 1.1.1.32 class) can be used. Polypeptides having lipase activity may be used to form esters. Enzymatic reactions such as these may be conducted in vitro, such as using cell-free extracts, or in vivo. Thus, various embodiments of the present invention, such as methods of making a chemical, include conversion steps to any such noted downstream products of microbially produced 3-HP, including but not limited to those chemicals described herein and in the incorporated references (the latter for jurisdictions allowing this). For example, one embodiment is making 3-HP molecules by the teachings herein and further converting the 3-HP molecules to polymerized-3-HP (poly-3-HP) or acrylic acid, and such as from acrylic acid then producing from the 3-HP molecules any one of polyacrylic acid (polymerized acrylic acid, in various forms), methyl acrylate, acrylamide, acrylonitrile, propiolactone, ethyl3-HP, malonic acid, 1,3 -propanediol, ethyl acrylate, n-butyl acrylate, hydroxypropyl acrylate, hydroxy ethyl acrylate, isobutyl acrylate, 2- ethylhexyl acrylate, and acrylic acid or an acrylic acid ester to which an alkyl or aryl addition is made, and/or to which halogens, aromatic amines or amides, and aromatic hydrocarbons are added.

[00249]Reactions that form downstream compounds such as acrylates or acrylamides can be conducted in conjunction with use of suitable stabilizing agents or inhibiting agents reducing likelihood of polymer formation. See, for example, U.S. Patent Publication No. 2007/0219390 Al. Stabilizing agents and/or inhibiting agents include, but are not limited to, e.g., phenolic compounds (e.g., dimethoxyphenol (DMP) or alkylated phenolic compounds such as di-tert-butyl phenol), quinones (e.g., t-butyl hydroquinone or the monomethyl ether of hydroquinone (MEHQ)), and/or metallic copper or copper salts (e.g., copper sulfate, copper chloride, or copper acetate). Inhibitors and/or stabilizers can be used individually or in combinations. Also, in various embodiments, the one or more downstream compounds is/are recovered at a molar yield of up to about 100 percent, or a molar yield in the range from about 70 percent to about 90 percent, or a molar yield in the range from about 80 percent to about 100 percent, or a molar yield in the range from about 90 percent to about 100 percent. Such yields may be the result of single- pass (batch or continuous) or iterative separation and purification steps in a particular process.

[00250] Acrylic acid and other downstream products are useful as commodities in manufacturing, such as in the manufacture of consumer goods, including diapers, textiles, carpets, paint, and adhesives.

[00251]As indicated herein, the scope of the invention includes isolated and recombinant

polynucleotides and polypeptides, microorganisms made by the methods described herein, which may include such isolated and recombinant polynucleotides and polypeptides, culture systems employing these microorganisms to produce 3-hydroxypropionic acid (3-HP), as well as final fermentation broth (aqueous broth) comprising 3-HP. The 3-HP, in methods of the present invention, may then be converted enzymatically, catalytically (chemical conversion), and/or with thermal treatment to any of a first range of chemicals, which may be referred to as "derived chemicals." The advances described herein as far as protein and metabolic engineering effectively may be applied to methods to produce any of such range of chemicals, as well as to further derived products made by further steps by converting any one or more of these chemicals. For example, Table 12 lists derived chemicals and various further derived products.

[00252] As discussed herein, various embodiments are related to microbial production of 3 -HP and subsequent conversions of the 3 -HP so produced. Efficient and cost-effective production of 3 -HP, essentially a C3 building block, affords multiple opportunities through various chemical conversions to make commercially important intermediates, industrial end products, and consumer products.

Accordingly, the scope of the invention also includes methods and products made by those methods in which the microbially produced 3 -HP thereafter is converted to various chemicals and products.

[00253] For example, the methods of making may include making 3-HP microbially and further making any of the following derived chemicals: polymerized-3-HP (poly-3-HP), acrylic acid (CAS No. 79-10- 7), polyacrylic acid, acrylamide (CAS No. 79-06-01), acrylonitrile, propiolactone, ethyl 3-HP, malonic acid, and 1,3— propanediol. In various embodiments many of these are converted to other monomer forms and/or polymers, and are used in a variety of industrial and consumer products. Any of the various monomers may be further processed by methods of the present invention to yield another product of interest (e.g., a "further derived product"). An expanded listing of such monomers is acrylic acid, acrylamide, acrylonitrile, propiolactone, ethyl 3-HP, malonic acid, 1,3 -propanediol, hydroxypropyl acrylate, hydroxy ethyl acrylate, isobutyl acrylate, n-butyl acrylate, hydroxypropyl aery late, hydroxyethyl acrylate, isobutyl acrylate, ethyl acrylate, methyl acrylate, ethyl ethoxy propionate, ethyl -3- hydroxypropionate (ethyl-3-HP), 2-ethylhexyl acrylate and other acrylates (e.g., acrylic acid salts and esters).

[00254] Also, in various embodiments a "further derived product" includes within its scope any such derived product and also includes more complex polymers and products of any such derived product. For example, acrylamide may be made from 3-HP made by the methods described herein, and thereafter may be converted chemically to any of a plurality of polyacrylamides, such as by methods known in the art. Similarly, acrylonitrile may be made from 3-HP made by the methods described herein, and thereafter may be converted chemically to form (or be a component of) any of a plurality of plastics including but not limited to polyacrylonitrile, styrene-acrylonitrile, acrylonitrile butadiene styrene, acrylonitrile styrene acrylate, acrylonitrile butadiene, and adiponitrile. A further derivedproduct of 1,3-propanediol is Polytrimethylene terephthalate, or PTT. The present invention is contemplated to include use of modified microorganisms as described herein to produce 3-HP which is then converted ultimately to any derived chemical and further derived product, such as listed in Table 12.

[00255] Several conversion examples follow. Methyl acrylate may be made from 3-HP via dehydration and esterification, the latter to add a methyl group (such as using methanol). Acrylamide may be made from 3-HP via dehydration and amidation reactions. Acrylonitrile may be made via a dehydration reaction and forming a nitrile moiety. Propriolactone may be made from 3-HP via a ring- forming internal esterification reaction (eliminating a water molecule). Ethyl-3-HP may be made from 3 -HP via esterification with ethanol. Malonic acid may be made from 3 -HP via an oxidation reaction, and 1,3 -propanediol may be made from 3 -HP via a reduction reaction. Also, acrylic acid, first converted from 3 -HP by dehydration, may be esterified with appropriate compounds to form a number of commercially important acrylate -based esters, including but not limited to methyl acrylate, ethyl acrylate, methyl acrylate, 2-ethylhexyl acrylate, butyl acrylate, and lauryl acrylate. Alternatively, 3HP may be esterified to form an ester of 3HP and then dehydrated to form the acrylate ester.

[00256] Additionally, 3-HP may be oligomerized or polymerized to form poly(3-hydroxypropionate) homopolymers, or copolymerized with one or more other monomers to form various co-polymers.

Because 3- HP has only a single stereoisomer, polymerization of 3-HP is not complicated by the stereo- specificity of monomers during chain growth. This is in contrast to (S)-2-Hydroxypropanoic acid (also known as lactic acid), which has two (D, L) stereoisomers that must be considered during its

polymerizations.

[00257] Table 12

Monomer or polymer Further derived monomers or Additional further derived chemical polymer products derived products

from 3- HP (reaction type(s)) (reaction type(s))

(reaction(s) from 3-HP)

3- 3-aminopropanamide (activation of

hydroxylpro anamide the hydroxyl followed by

and related N-alkyl displacement with amine

derivatives (amidation) nucleophiles)

[00258] While various embodiments of the present invention have been shown and described herein, it is emphasized that such embodiments are provided by way of example only. Numerous variations, changes and substitutions may be made without departing from the invention herein in its various embodiments. Specifically, and for whatever reason, for any grouping of compounds, nucleic acids (polynucleotide, oligonucleotides), polypeptides including specific proteins including functional enzymes, metabolic pathway enzymes or intermediates, elements, or other compositions, metabolic (including biosynthetic), pathways or portions thereof, or concentrations stated or otherwise presented herein in a list, table, or other grouping (such as metabolic pathway enzymes shown in a figure), unless clearly stated otherwise, it is intended that each such grouping provides the basis for and serves to identify various subset embodiments, the subset embodiments in their broadest scope comprising every subset of such grouping by exclusion of one or more members (or subsets) of the respective stated grouping. Moreover, when any range is described herein, unless clearly stated otherwise, that range includes all values therein and subranges therein.

EXAMPLES

[00259] The examples herein provide some examples, not meant to be limiting, of combinations of genetic modifications and supplement additions. The following examples include both actual examples and prophetic examples. Unless indicated otherwise, temperature is in degrees Celsius and pressure is at or near atmospheric pressure at approximately 5,340 feet (1,628 meters) above sea level. It is noted that work done at external analytical and synthetic facilities is not conducted at or near atmospheric pressure at approximately 5,340 feet (1,628 meters) above sea level. All reagents, unless otherwise indicated, are obtained commercially. Species and other phylogenic identifications are according to the classification known to a person skilled in the art of microbiology. The names and city addresses of major suppliers are provided herein. In addition, as to Qiagen products, the DNeasy® Blood and Tissue Kit, Cat. No. 69506, is used in the methods for genomic DNA preparation; the QIAprep® Spin ("mini prep"), Cat. No. 27106, is used for plasmid DNA purification, and the QIAquick® Gel Extraction Kit, Cat. No. 28706, is used for gel extractions as described herein.

[00260] Example 1 : Determining the region encoding the malonyl CoA reductase domain of the full length Chloroflexus aurantiacus and a region that provides increased specific activity. [00261] Multiple plasmids able to express various truncated versions of the bifunctional malonyl CoA reductase of Chloroflexus aurantiacus were constructed to determine the regions important to the enzymatic activities of this enzyme. This enzyme has two functions. The first enzymatic function is a malonyl CoA reductase activity that is responsible for converting malonyl CoA to malonate semialdehyde (MSA) and a free CoA molecule. The second enzymatic function is a dehydrogenase, referred to herein as a 3 -HP dehydrogenase, able to convert the malonate semialdehyde produced in the first enzymatic reaction to 3-hydroxypropionate (3-HP). These truncations fall into two groups; N-terminal truncations were created to shorten the bifunctional enzyme from the N-terminal end of the protein, and C-terminal truncations were created to shorten the bifunctional enzyme from the C-terminal end of the protein. Assessing each of these truncated versions of the full length enzyme it was possible to determine the regions of the full length protein responsible for each of the two enzymatic functions.

[00262]The plasmids for expressing the various truncated versions of the bifunctional malonyl CoA reductase of Chloroflexus aurantiacus were created as follows. Using pTRC-ptrc-mcr (1 -1220) SEQ ID NO: 096) as template, each mcr variant was amplified by PCR using the primers (IDT, Coralville, IA) described in Table 7. All of the amplified products were subcloned into the pTRC(amp)HisA (Invitrogen, Carlsbad, CA) using NcoI(New England Biolabs, Ipswich, MA) and Hindlll (New England Biolabs, Ipswich, MA) restriction sites. Plasmid constructs were confirmed by restriction mapping and DNA sequencing (Genewiz, South Plainfield, NJ).

[00263]Table 7

[00264] The resulting mcr variants resulted to the proteins with N-terminal truncations: MCR( 177- 1220) (SEQ ID NO:070), MCR(366-1220) (SEQ ID NO: 071), MCR(496-1220) (SEQ ID NO: 072), MCR(691- 1220) (SEQ ID NO: 073), MCR(845-1220) (SEQ ID NO: 074); MCR(1031- 1220) (SEQ ID NO: 075) and C-terminal truncations: MCR(1-1053) (SEQ ID NO: 076); MCR(l-884) (SEQ ID NO: 077); MCR(1- 720) (SEQ ID NO: 078); MCR(l-554) (SEQ ID NO: 079); MCR(l-387) (SEQ ID NO: 080); MCR(l-300) (SEQ ID NO: 081). A schematic showing these constructs in relationship to the full length protein are shown in FIG. 6. Lysates for assaying the specific activities were prepared from over expressed cultures and assessed with an assay to measure malonyl-CoA reductase enzyme activity as discussed in the methods section. Briefly, cell lines carrying plasmids able to over express malonyl CoA reductases were grown with antibiotic selection in LB media overnight as starter cultures. These overnight starter cultures were used to inoculate either 50 mL to 100 mL expression cultures grown with antibiotic selection in LB media supplemented with ImM Isopropyl O-D-l-thiogalactopyranoside (IPTG) to induce protein production. Cultures were grown 24 hr, after which the cells were collected by centrifugation. Cell pellets were lysed using a mixture of Bugbuster, benzonase nuclease, and rLysozyme (all from Novagen). Once lysed, the lysate mixture was centrifuged at 14000 RPM in a standard table top centrifuge. The resulting supernatant was removed to another tube. The clarified supernatant was measured for protein concentration using a Biorad Total Protein determination kit (BioRad). For each measurement, 20 uL of lysate was added to a reaction buffer filled well of the 96-well plate used to perform the assay. The assay was initiated by addition of malonyl CoA to a final concentration of 0.3 mM or ImM, which is well above the reported Km binding constant for these enzymes. Once the reaction time course was read and the slopes of each well were calculated, the specific activities were compared to a negative control to determine a background rate. All values reported are the average specific activities measured in triplicate.

[00265] For determining the region of the full length protein that encodes the malonyl CoA reductase domain of the bifunctional MCR enzymes, the specific activities of lysates in the presence of NADPH were determined as described above. The results of these experiments are shown in FIG. 7. These results show enzymatic activity for the malonyl CoA reductase domain is encoded in the region comprising amino acids 496 to 1220 of the full length protein sequence. This construct MCR(496-1220) retains activity to convert malonyl CoA to Malonate semialdehyde in the assay. Truncations with fewer amino acids than MCR(496-1220) have activities equivalent or less than the negative control made from a cell lysate with a control plasmid without an MCR gene (BW25113). These truncations with fewer amino acids include MCR(691-1120), MCR(845-1220), and MCR(1031-1220). Truncations other than

MCR(496-1220) also showed enzymatic activity with malonyl CoA as substrate. These truncations encode more amino acids of the full length bifunctional MCR protein. These truncations included MCR(366-1220) and MCR(177-1220). More activity was observed with increased numbers of amino acids of the full length bifunctional protein being included in the truncated versions. Since the MCR(177- 1220) truncation construct (also referred to as "truncate" or "truncation"), which undergoes only one reaction to create a final product of malonate semialdehyde per malonyl CoA molecule at the expense of 1 NADPH, has an equivalent or greater specific activity compared with the full length bifunctional MCR positive control, which undergoes both enzymatic reactions to create 1 molecule 3 -HP per malonyl CoA at the expense of 2 NADPH molecules, this suggests that the residues in the region of 366 to 177 provide increased function to the malonyl CoA reductase domain. This is further confirmed in FIG. 8 that shows the same assay performed in the presence of YdfG (SEQ ID NO: 091). YdfG is a dehydrogenase able to convert malonate semialdehyde to 3-HP at the expense of 1 NADPH molecule. As the MCR( 177- 1220) truncate has about twice the activity of the full length bifunctional MCR in this coupled assay, MCR(177- 1220) has a higher specific activity.

[00266] Example 2: Determining the region of Chloroflexus aurantiacus that is responsible for 3-HP dehydrogenase activity.

[00267] The results of this assay are shown in FIG. 9 for the C-terminal truncations of the full length bifunctional MCR protein. As a negative control, a cell line carrying just vector and no protein coding sequence was used to make a cell lysate. This lysate showed little to no specific activity in this assay. Several positive controls were also performed. These positive controls included lysates from cell lines over expressing MmsB (SEQ ID NO: 092), YdfG (SEQ ID NO: 091), and full length bifunctional MCR(SEQ ID NO: 001). These lysates showed specific activities in this assay of 3.7 U/mg, 5.9 U/mg, and 3.4 U/mg, respectively. All C-terminal truncates that are larger than and including the MCR(l-554) truncate show specific activity for converting malonate semialdehyde to 3-HP. As constructs MCR(1- 300) and MCR(l-387) show no activity in this assay, the region between amino acid 1 and amino acid 554 contains the dehydrogenase domain. The MCR(l-554) truncate has a specific activity of 4 U/mg in this assay, which is about equivalent to the full length MCR enzyme. Truncates carrying more or the full length MCR peptide sequence all show activity. The specific activities for MCR(l-720), MCR (1-884), and MCR(1-1053) show activities of 1.8 U/mg, 0.4 U/mg, and 0.5 U/mg. These reduced activities as compared to the full length MCR enzyme and MCR(l-554) suggest that addition of extra amino acids that do not encode the full CoA reductase domain may cause reduced function of the 3-HP dehydrogenase domain, and that a truncation at or about amino acid 554 in the full length sequence appears to provide optimal activity.

[00268] Example 3: Engineering the malonyl CoA reductase domain of Chloroflexus aurantiacus for increased NADH utilization.

[00269] With knowledge about the domain boundaries, work was performed to increase the cofactor specificity of the truncated malonyl CoA reductase polypeptides to utilize nicotinamide adenine dinucleotide (NADH) over nicotinamide adenine dinucleotide phosphate (NADPH). In order to perform the "cofactor switching' on these truncates, the following steps were performed: First, the sequence of the truncated regions was compared to enzymes with similar sequences, as judged by standard sequence similarity and sequence identity percentages, compared with other proteins in the NCBI protein sequence database and against determined structures in the Protein Database (PDB). From these similar sequences, information concerning the amino acid regions and corresponding folds within the determine protein structures was used to suggest regions and sites within the malonyl CoA reductase domain that are responsible for binding NADPH over NADH. NADPH differs chemically from NADH due to a phosphate moiety located attached to the 2' carbon of its adenine-ribose ring. A number of studies have suggested how proteins are able to bind each of these cofactors, as well as the regions and sites that are likely responsible for giving these proteins there corresponding specificities (Bocanegra, J. A. et al, 1993). However, there is no standard procedure for altering these specificities. From analysis of this information, a putative phosphate loop was located within the sequence. This putative phosphate binding loop was located between and including amino acids 604 to 609 in the full length bifunctional MCR sequence (SEQ ID NO:058). Using this information, a new search of the NCBI sequence database was performed to find protein sequences that had similar proximal amino acid sequences around the putative phosphate loop that included 10 to 20 amino acids before and after the putative phosphate binding loop region. This search was performed with a sequence derived with three specifications as follows; First, the phosphate binding loop region in the search sequence was replaced with the sequence being 'XXXXXX'.

Second, the first location in the putative phosphate binding loop was replaced with a D to represent an aspartic acid residue (corresponding to position 604) as many NADH specific phosphate binding loops have an aspartic acid at this position. Finally, the search results were filtered to remove any sequences that showed an arginine residue at the second position in in the putative phosphate binding loop (position 605 in the full length bifunctional MCR protein). These results were compiled and used to derive a new search sequence for the putative phosphate binding loop. The new sequence being 'DINKDR' was used in place of the "XXXXXX' sequence used in the first search. The second database search results were used to identify four proteins having putative loops that can be used to replace the putative NADPH-specific phosphate binding loop of the malonyl CoA reductase domain, and/ or including the phosphate binding loops in various truncates described here. Searching for sequences with this method attempts create a chimeric sequence that finds putative NADH-specific loops that best fit the polypeptide sequence context of the enzymes that is to undergo cofactor switching. Also during the initial search a set of secondary site mutations were suggested due to similarity of this malonyl CoA reductase domain to a protein with a similar protein fold (Scrutton N.S. et al., 1990). These secondary mutations are specific to this enzymatic domain. Once these desired mutations were determined, they were introduced into the truncated malonyl CoA reductase truncate to assess their effects. Using pTRC(kan)-ptrc-mcr(496-1220) (SEQ ID NO: 072) as a template and the primers listed in Table 8, site-directed mutations were made using QuikChange Site- Directed Mutagenesis Kit (Agilent, Santa Clara, CA) as directed by the manufacturer's protocol. [00270] Table 8

These created vectors able to express variants 1 through 8 (SEQ ID NO 046-053).

[00271] Plasmids able to express the putative NADH-specific loop variants (variants 1 thru 4) (SEQ ID NO: 046-049) of the malonyl CoA reductase domain were assayed for function as described in the methods section and other examples. The results for these assays are shown in FIG. 10. As a positive control a cell lysate from cells expressing MCR(496-1220) was used as a control to compare NADH- specific activity and NADPH-specific activity of the variant malonyl CoA reductase domains. The MCR(496-1220) control has an activity an NADH-specific activity of nearly 0.01 U/mg and an NADPH- specific activity of about 0.42 U/mg in this assay. Notably, all variants (1 through 4) (SEQ ID NO:046- 049) showed a significant reduction of NADPH-specific activity as compared to control and showed increased NADH-specific activity over the control MCR(496-1220) NADH-specific activity. The NADHspecific activity for variant 1 was 0.018 U/mg, variant 2 was 0.14 U/mg, variant 3 was 0.023 U/mg, and variant 4 was 0.14 U/mg. These NADH-specific activities are a significant increase over the MCR (496-1220) control that had activity of only 0.01 U/mg with NADH in this assay.

[00272] In order to confirm that reactions showing malonyl CoA reductase activities could perform a reaction able to convert malonyl CoA to malonate semialdehdye, a coupled assay was used. The coupled assay uses lysates overexpressing MmsB, a dehydrogenase able to convert malonate semialdehyde to 3- hydroxypropionate. The formation of 3- hydroxypropionate was assessed using gas chromatography— mass spectrometry (GC-MS). The reactions for these assays were performed as 750 uL reactions containing 20 uL of clarified whole cell lysates from cells expressing the truncated version of malonyl CoA reductase or variant, 20 uL of clarified whole cell lysates from cells expressing MmsB

dehydrogenase, and buffer. The buffer conditions consisted of 1 mM malonyl CoA, 2 mM NADH or 2mM NADH, 5 mM dithiothreitol, 3 mM magnesium chloride, 100 mM Trizma-HCl pH7.6 buffer. Lysates for these assays were prepared from over expressed cultures. Cell line carrying plasmids able to over express mmsB or the malonyl CoA reductases were grown with antibiotic selection in LB media overnight as starter cultures. These overnight starter cultures were used to inoculate either 50 mL to 100 mL expression cultures grown with antibiotic selection in LB media supplemented with lmM Isopropyl 0- D-l-thiogalactopyranoside (IPTG) to induce protein production. Cultures were grown 24 hr, after which the cells were collected by centrifugation. Cell pellets were lysed using a mixture of Bugbuster, benzonase nuclease, and rLysozyme (all from Novagen). Once lysed, the lysate mixture was centrifuged at 14000 RPM in a standard table top centrifuge. The resulting supernatant was removed to another tube. The mmsB lysate added to the reaction buffer followed by the malonyl CoA reductase lysate being tested. Reactions were incubated at 37 degrees Celsius for 12 to 15 hours. With each assay set, negative control samples for the mmsB clarified cell lysate and the malonyl CoA reductases or variants samples were included to make sure no lysate had the ability to form 3-hydroxypropionate with a combination of a CoA reductase domain and a dehydrogenase domain. After incubating, all samples were submitted for GS-MS analysis as described below. For MmsB, the protein sequence (SEQ ID NO: 092) for 3- hydroxyisobutyrate dehydrogenase gene (SEQ ID NO: 121) or MmsB from Pseudomonas aeruginosa was codon optimized for E. coli according to a service from DNA 2.0 (Menlo Park, CA USA), a commercial DNA gene synthesis provider. This gene construct was synthesized by DNA 2.0 and provided in a pJ251 vector backbone. This was designated pJ251_26386 (SEQ ID NO: 122). This synthesized plasmid was used as a template to amplify the mmsB gene by PCR with forward primer,

GGATAGTCCCATGGCCGACATTGCGTTTCTGGGTC (SEQ ID NO: 123) and reverse primer, GCTAATATGGATCCACCTCTTTAATCTAATCCTTACCGCGATACAG, (SEQ ID NO: 124). The amplified gene was digested with Ncol and BamHI and subcloned into the pTRC(amp)-HisA (Invitrogen, Carslbad, CA) to make pTRC-mmsB (SEQ ID NO: 125). For reactions using ydfG, the malonic semialdehyde reductase gene (SEQ ID NO: 126), ydfG, was amplified by PCR from the E coli genome using the forward primer, GGATAGTCCCATGGTCGTTTTAGTAACTGGAGCAAC (SEQ ID NO: 127), and reverse primer, GCTAATATGGATCCACCTCTTTAATTTACTGACGGTGGACATTCAG (SEQ ID NO: 128). The amplified gene was digested with Ncol and BamHI and subcloned into the same restriction sites in the pTRC(amp)-HisA (Invitrogen, Carslbad, CA) to make pTRC-ydfG (SEQ ID NO: 129).

[00273] The following method is used for GC-MS analysis of 3 -HP. Soluble monomeric 3-HP is quantified using GC-MS after a single exaction with ethyl acetate from a reaction sample. Once the 3-HP has been extracted into the ethyl acetate, the active hydrogens on the 3-HP are replaced with trimethylsilyl groups using N, 0-Bis-(Trimethylsilyl) trifluoroacetamide to make the compound volatile for GC analysis. A standard curve of known 3-HP concentrations is prepared at the beginning of the run and a known quantity of ketohexanoic acid (lg/L) is added to both the standards and the samples to act as an internal standard for Quantitation, with tropic acid as an additional internal standard. The 3-HP content of individual samples is then assayed by examining the ratio of the ketohexanoic acid ion (m/z = 247) to the 3-HP ion (219) and compared to the standard curve. 3-HP is quantified using a 3HP standard curve at the beginning of the run and the data are analyzed using HP Chemstation. The GC-MS system consists of a Hewlett Packard model 5890 GC and Hewlett Packard model 5972 MS. The column is Supelco SPB- 1 (60m X 0.32mm X 0.25um film thickness). The capillary coating is a non-polar methylsilicone. The carrier gas is helium at a flow rate of lmL/min. The 3-HP as derivatized is separated from other components in the ethyl acetate extract using either of two similar temperature regimes. In a first temperature gradient regime, the column temperature starts with 40°C for 1 minute, then is raised at a rate of 10°C/minute to 235°C, and then is raised at a rate of 50°C/minute to 300°C. In a second temperature regime, which was demonstrated to process samples more quickly, the column temperature starts with 70°C which is held for 1 min, followed by a ramp-up of 10 °C/minute to 235°C which is followed by a ramp-up of 50 ^" C/minute to 300°C. All values reported are the average specific activities measured in triplicate.

[00274] In order to confirm that 3-HP was produced by variant 1 thru 4, the GC-MS assay as described above was performed with lysates of these variants results in FIG. 11. Detectable amounts of 3-HP where observed over the negative control samples for all variants.

[00275] As variants 1 through 4 showed better NADH-specific activities over controls and had confirmed 3-HP production as shown by GC-MS, the secondary site mutations were added as described above. The plasmids were sequenced, and cell lines expressing each of these plasmids able to express the putative NADH-specific phosphate loop variants containing the secondary site mutations (variants 5 through 8 in Table 1) (SEQ ID NO: 050-053) of the malonyl CoA reductase domains were assayed for function as described above. The results for these assays are shown in FIG. 12. As a positive control a cell lysate from cells expressing MCR( 177- 1220) was used as a control to compare NADH-specific activity and NADPH-specific activity of the variant malonyl CoA reductase domains. The MCR( 177- 1220) control has an activity an NADH-specific activity of nearly 0.01 U/mg and an NADPH-specific activity of about 0.60 U/mg in this assay. The variants (5 thru 8), with the secondary site mutations, all showed increased activity as compared to their corresponding non-secondary site mutation variants (variant 1 thru 4) (SEQ ID NO: 046-049). With the addition of the secondary site mutations, about a doubling of activity or more was witnessed over their corresponding non-secondary site mutation variants (1 through 4). Variant 6 had an NADHspecific activity of 0.59 U/mg. This was the highest of those evaluated. This is a 59 times improvement over a malonyl CoA reductase domain with a wild type sequence.

[00276] Example 4: Engineering the dehydrogenase domain of Chloroflexus aurantiacus for increased NADH utilization.

[00277] A similar procedure to the one described in the examples above to switch the cofactor utilization of the dehydrogenase domain was employed to change the cofactor specificity from NADPH to NADH. A sequence comparison showed that the malonyl CoA reductase domain of the full length bifunctional Chloroflexus aurantiacus MCR is very similar to the dehydrogenase domain, especially in the cofactor binding region (See FIG. 13). As a result, four NADH-specific putative loops were found for the dehydrogenase domain. These new loops are shown in Table 9 and were used to replace the sequence located at amino acids between and including positions 39 thru 44 in the polypeptide chain. These mutations were introduced site-directed mutations. Using pTRC(kan)-ptrc-mcr(l-554) (SEQ ID NO: 079) as a template and the primers listed in Table 9, site-directed mutations were made using QuikChange Site- Directed Mutagenesis Kit (Agilent, Santa Clara, CA) as directed by the manufacturer's protocol. [00278] Table 9

These created vectors able to express variants 9-12 (SEQ ID NO: 054-057).

[00279] Once plasmids were created, these dehydrogenase variants were assayed using the coupled- dehydrogenase assay described above. Variants 9 through 12 (SEQ ID NO: 054-057) were overexpressed and cell lysates were assayed. The results showing the NADH-specific activities and NADPH-specific activities of these variants are shown in FIG.14. As a control, MCR (1-554) lysate was also tested. The control showed strong NADPH specific activity of 1.53 U/mg and only 0.13 U/mg NADH-specific activity. Conversely, each of the dehydrogenase variants showed a switch of cofactor specificity. Each variant had an NADPH-specific activity of less than 0.04 U/mg. However, the NADH-specific activities for variant 9, variant 10, variant 11, and variant 12 are 0.67 U/mg, 0.5 U/mg, 0.70 U/mg, and 1.2 U/mg, respectively. These results signify a 5.1 times, 3.8 time, 5.3 times, and a 9.2 times improvement in NADH specific activity over the MCR (1-554) truncate without an NADH-specific phosphate binding loop.

[00280] Example 4a: Modifications for improved NADH utilization in several malonyl-coA reductases

[00281] Several different divergent malonyl-coA reductase sequences are used as a starting point for additional mutations that increase the ability of the enzyme to utilize NADH as reductant.

[00282] Three of these sequences include the MCRs from C. auranticus (Ca MCR), O. trichoides (OTMCR) and C. aggregans (Caggregans MCR). Using the Ca MCR as the reference sequence the O. trichoides' MCR is 61% identical and the C. aggregans' MCR is 88% identical. An alignment of these protein sequences can be obtained by comparing the published sequences. In addition to these, other sequences may also be used as a starting sequence. Using the alignment it is reasonable to mutate the sequences in the homologous MCRs to mimic the changes made in other examples to the chlorflexus sequence which resulted in increased NADH utilization. See SEQ ID NO. 149, SEQ ID NO. 150, and SEQ ID NO. 151. These mutations are shown in Table 9a below.

[00283] Table 9a. Mutations to homologous MCRs to increase NADH utilization. The mutated sequence on the left is used to replace the original amino sequence in the middle or right column. The position of mutation is indicated by the amino acids number referring to the original sequence. C. aggregans (original

Mutated sequence 0. trichoides (original sequence) sequnce)

DINKDR 39-44 (GRNRDK) 40-45 (GRNSAK)

DVDTRK 605-610 (ARDERK) 603-608 (ARDPHK)

781-794

GGLFGRRARLILEN 782-795 (PGLFLRRGRLILEN) (PGLFARRARLILEN)

[00284] Example 5: Switching the enzyme cofactor utilization from nicotinamide adenine dinucleotide and nicotinamide phosphate (NADPH) to adenine dinucleotide phosphate (NADH).

[00285] The procedure described in the examples above for switching the cofactor specificity could be used for converting any number of NADPH utilizing enzymes to NADH specificity. Possible domains or proteins that could be converted include SEQ ID NO 090-092. This could be combined with additional secondary site mutations in order to obtain additional increases in NADH-specific function or decreases in NADPH-specific function as needed. These mutations could be site-substitutions, insertions, and/or deletions to the polypeptide sequence of the proteins. They could be introduced by site-saturation mutagenesis, directed mutation, random mutagenesis, or other methods known in the art. Function and improvements of these new variants could be tested directly such as in methods similar to those listed above or indirectly through cellular screens that impart a detectable phenotype on a strain carrying such variants.

[00286] Example 6: Combining malonyl-CoA reductase and 3-HP dehydrogenase activities.

[00287] 3-HP is produced from malonyl-CoA by the sequential effects of a malonyl-CoA reductase and a 3-HP dehydrogenase activity. Production of 3-HP can thus be achieved in a microorganism comprising NADH-dependent malonyl-CoA reductase activity and a 3-HP dehydrogenase activity encoded by the ydfG gene of E. coli, which is an NADPH-specific dehydrogenase, or in a microorganism comprising NADH-dependent malonyl-CoA reductase activity and a 3-HP dehydrogenase activity encoded by the mmsB gene of Pseudomonas aeruginosa (SEQ ID NO: 092), which is a dehydrogenase with NADH substrate preference, or in a microorganism comprising NADHdependent malonyl-CoA reductase activity and 3-HP dehydrogenase activities encoded by the ydfG and mmsB gene products. Production of 3-HP can also be achieved in a microorganism comprising NADH-dependent malonyl-CoA reductase activity and NADH-dependent 3-HP dehydrogenase activity, the latter encoded by variants of MCR with NADH- dependent 3-HP dehydrogenase activity. Respective non-limiting examples of such combinations include variants 1-8(SEQ ID NOs:046-053) as to the former function and variants 9 -12 (SEQ ID NOs:054-057) as to the latter function. Production of 3-HP from malonyl-CoA can in addition be achieved with a NADH-dependent malonyl-CoA reductase activity and an activity that converts malonyl semialdehyde to 3-HP using a biological reductant other than NADPH or NADH, such as the activity encoded by the rutE gene of E. coli or by the nemA gene of E. coli which are reductases that utilize a flavin derivative as the reductant and which further require the activity of a function such as the fre gene product encoding FMN reductase to regenerate the reductant. See, for example, Kim et al., 2010, J. Bacteriol. 192(16): 4089- 4102.

[00288] While production of 3-HP from malonyl-CoA by NADH-dependent enzymes may be more favorable relative to NADPH, the above combinations may be constructed using a NADPH-dependent malonyl-CoA reductase, such as the malonyl-CoA reductase encoded by amino acid residues 177-1220 of Mcr.

[00289] Combinations of a gene encoding malonyl-CoA reductase activity and a gene or genes encoding 3-HP dehydrogenase can be achieved by cloning the respective genes behind promoters such that the genes are operably expressed in the microorganism under conditions that induce expression. The genes may be cloned in plasmid vectors, such as plasmids based on the ColEl replication or the pl5A replication, or may be inserted into the chromosome of the microorganism, such as at a locus which encodes a dispensable function, for example the aldA locus of E. coli. Expression of these genes can be driven by regulated promoters, such as the lac promoter or derivatives thereof, or by the T7 bacteriophage promoter, or by the arabinose promoter, or other DNA sequences known or found to drive expression in E. coli. These examples of constructs and promoters are not meant to be limiting.

[00290]Example 6a:

[00291]Two malonyl-coA reductases were evaluated to produce malonate as well as one malonate semialdehyde dehydrogenase. The malonyl-coA reductases used were the monofunctional malonyl- coA reductase from Solfolobus todakii ( StMCR) and the monofunctional MCR from chloroflexus auranticus comprising the 177-1220 amino acids of the full length bifunctional malonyl-coA reductase ( 177MCR). The malonate semialdehyde dehydrogenase evaluated was that from gabD gene from E. coli which encodes for a succinate semialdehyde dehydrogenase. The results are provided in Figure 23. Strains A-D are controls, while Strains E-J are strains modified according to the invention showing malonate production has been achieved via the expression of E. coli gabD from malonyl-coA via malonate semialdehyde produced from two independent malonyl-coA reductases.

[00292]Example 7: Sequence of the fabl ^ts mutation.

[00293] The nature of the exact sequence change in the fabr allele carried by strains JP1111 was reconfirmed. Confirmation of this change allows targeted mutagenesis to generate alternative strains with different temperature sensitivities and mutants with stabilities intermediate between wild type and the fabI392 temperature-sensitive allele, allowing growth at a constant temperature higher than 30°C while providing the benefit of increased internal malonyl-CoA pools. To confirm the DNA sequence of this segment of the chromosome of a wild type (BW25113) and the JP1111 mutant E. coli, chromosomal DNA was prepared from these strains. These DNA were used as templates in a PCR reaction and subsequent sequencing reaction by standard methods.

[00294] A comparison of the DNA sequence obtained from the fabI392 and wild type strains reveals a single difference between the alleles of C at position 722 of the wild type gene to T leading to a protein change of Ser at codon 241 to Phe. These changes are identical to those found by Bergler, H., Hogenauer, G., and Turnowsky, F., J. Gen. Microbiol. 138:2093-2100 (1992). The identification of the affected residue at codon 241 indicates that targeted mutagenesis at this codon, for example to amino acid residues such as Trp, Tyr, His, He, or other amino acids other than Ser or Phe, may result in fah./ alleles with different properties than the fabI392 originally isolated in JP1111. Targeted mutagenesis at codons near to codon 241 may also be contemplated to obtain the desired fah./ mutants with altered properties. Strains bearing the discussed fabl alleles can be used in combination with expression of NADH dependent 3-HP production pathways from malonyl-CoA using the enzymes variants described within the above examples. It has been shown that mutations in a microorganism's fatty acid synthase system can be used to increase the production of malonyl-CoA dependent products. The teachings of the following publications as to genetic modification combinations, culture methods, and other aspects to produce 3-HP and other chemical products are hereby incorporated by reference into this application: Application number PCT/US2010/050436, published March 31, 2011 as WO/2011/038364; and Application number PCT/US/057690, published May 26, 2011 as WO/2011/063363, and Application number

PCT/US2011/022790 published August 4, 201 1 as WO/2011/094457. The respective teachings therein regarding increasing tolerance to 3-HP also are incorporated by reference herein.

[00295] Example 8: Construction of Additional Strains for Evaluation.

[00296] Part 1 : Gene Deletions

[00297] The homologous recombination method using Red/ET recombination, as described elsewhere herein, was employed for gene deletion in E. coli strains. This method is known to those of ordinary skill in the art and described in U.S. Patent Nos. 6,355,412 and 6,509,156, issued to Stewart et al. and incorporated by reference herein for its teachings of this method. Material and kits for such method are available from Gene Bridges (Gene Bridges GmbH, Heidelberg (formerly Dresden), Germany,

«www.genebridges.com»), and the method proceeded by following the manufacturer's instructions. The method replaces the target gene by a selectable marker via homologous recombination performed by the recombinase from X-phage. The host organism expressing k-red recombinase is transformed with a linear DNA product coding for a selectable marker flanked by the terminal regions (generally— 50 bp, and alternatively up to about— 300 bp) homologous with the target gene or promoter sequence. The marker is thereafter removed by another recombination step performed by a plasmid vector carrying the FLP -recombinase, or another recombinase, such as Cre.

[00298] Specific deletions can be constructed by amplification using PCR from the Keio strains carrying particular deletions using primers as specified below. The Keio collection was obtained from Open Biosystems (Huntsville, AL USA 35806). Individual clones may be purchased from the Yale Genetic Stock Center (New Haven, CT USA 06520). These strains each contain a kanamycin marker in place of the deleted gene. In cases where the desired deletion was not in a Keio strain, for example ackA-pta, the deletion are constructed by the above-noted recombination method using the kanamycin resistance marker to replace the deleted sequence, followed by selection of a kanamycin resistance clone having the deletion. The PCR products are introduced into targeted strains using the above-noted recombination method. Combinations of deletions are generated sequentially to obtain strains as described in the following parts of this example. Table 10 gives a list of E. coli genes that may be deleted individually or in combination to increase the yield to 3-HP.

[00299] Table 10

[00300] These deletions may be employed in various microbial strains in combinations such as but not limited to those shown in FIGs. 2A-G.

[00301] Part 2: Construction of E. coli_651 having a fabl mutation

[00302] The fad ^s mutation (Ser241— *Phe) in E. coli strain JP1 1 11 significantly increases the malonyl-CoA concentration when cells are grown at the nonpermissive temperature (37°C) and thus produces more 3-HP at this temperature. However, JP1 1 11 is not an ideal strain for transitioning into pilot and commercial scale, since it is the product of NTG mutagenesis and thus may harbor unknown mutations, carries mutations in the stringency regulatory factors relA and spoT, and has enhanced conjugation propensity due to the presence of an Hfr factor. Thus the fabf mutation can be moved to other strains for the production of 3-HP, these strains also expressing NADH dependent malonyl-CoA reductase and/or NADH dependent 3-HP dehydrogenase activities as discussed in the other examples. Moving of this mutation can be accomplished with several standard molecular biology techniques.

[00303] Part 3: Promoter Replacement for Selected Genes in Chromosome [00304] The homologous recombination method described elsewhere herein was employed to replace promoters of various genes. As noted, use of Red/ET recombination is known to those of ordinary skill in the art and described in U.S. Patent Nos. 6,355,412 and 6,509,156, issued to Stewart et al. and incorporated by reference herein for its teachings of this method. Material and kits for such method are available from Gene Bridges (Gene Bridges GmbH, Heidelberg, Germany, «www.genebridges.com»), and the method may proceed by following the manufacturer's instructions. The method involves replacement of the target gene (or, in this case, a promoter region) by a selectable marker via homologous recombination performed by the recombinase from X-phage. The host organism expressing k-red recombinase is transformed with a linear DNA product coding for a selectable marker flanked by the terminal regions (generally— 50 bp, and alternatively up to about— 300 bp) homologous with the target gene or promoter sequence. The marker can then be removed by another recombination step performed by a plasmid vector carrying the FLP -recombinase, or another recombinase, such as Cre.

[00305] Example 9: Preparing a Genetically Modified E. coli Host Cell Comprising an NADH dependent malonyl-CoAreductase (Mcr) and or NADH dependent 3-HP dehydrogenase in Combination with Other Genetic Modifications to Increase 3-HP Production Relative to a Control E. coli Cell (Prophetic).

[00306] Genetic modifications are made to introduce a vector comprising mmsB such as from

Pseudomonas aeruginosa, which further is codon-optimized for E. coli, alternatively, E. coli genes encoding, ydfG, rutE, nemA and fire may be overexpressed by standard methodology. Vectors comprising galP and a native or mutated ppc also may be introduced by methods known to those skilled in the art (see, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third Edition 2001 (volumes 1-3), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., "Sambrook and Russell, 2001"), additionally recognizing that mutations may be made by a method using the XL1 -Red mutator strain, using appropriate materials following a manufacturer's instructions (Stratagene QuikChange Mutagenesis Kit, Stratagene, La Jolla, CA USA) and selected for or screened under standard protocols. Also, genetic modifications are made to reduce or eliminate the enzymatic activities of E.coli genes as desired. These genetic modifications are achieved by using the RED/ET homologous recombination method with kits supplied by Gene Bridges (Gene Bridges GmbH, Dresden, Germany, www.genebridges.com) according to manufacturer's instructions.

[00307] Also, in some embodiments genetic modifications are made to increase the NADPH cellular pool. Non- limiting examples of some targets for genetic modification are provided herein. These are pgi (in a mutated form), pntAB, overexpressed, gapA:gapN substitution/replacement, and disrupting or modifying a soluble transhydrogenase such as sthA, and genetic modifications of one or more of zwf, gnd, and edd.

[00308] The so-genetically modified microorganism of any such engineered embodiment is evaluated and found to exhibit higher productivity of 3-HP compared with a control E. coli lacking said genetic modifications. Productivity is measured by standard metrics, such as volumetric productivity (grams of 3- HP/hour) under similar culture conditions. [00309] Example 9b: Preparing a Genetically Modified E. coli Host Cell Comprising a malonyl-CoA- thioesterase in Combination with Other Genetic Modifications to Increase malonic acid Production Relative to a Control E. coli Cell (Prophetic)

[00310] Genetic modifications are made to introduce a vector comprising malonyl-CoA thioesterase variants as described above such as the ybgC gene from E. coli. In addition, vectors comprising galP and a native or mutated ppc also may be introduced by methods known to those skilled in the art (see, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third Edition 2001 (volumes 1-3), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., "Sambrook and Russell, 2001 "). In addition, strains may be genetically modified to have mutations in the strains fatty acid synthase system, such as the temperature sensitive fabl allele as described above. Also, genetic modifications are made to reduce or eliminate the enzymatic activities of E.coli genes as desired. These genetic modifications are achieved by using the RED/ET homologous recombination method with kits supplied by Gene Bridges (Gene Bridges GmbH, Dresden, Germany, www.genebridges.com) according to manufacturer's instructions.

[00311] The so-genetically modified microorganism of any such engineered embodiment is evaluated and found to exhibit higher productivity of malonic acid compared with a control E. coli lacking said genetic modifications. Productivity is measured by standard metrics, such as volumetric productivity (grams of malonic acid/hour) under similar culture conditions.

[00312] Example 9c: Genetic modification/introduction of Malonyl-CoA Reductase and malonate semialdehyde dehydrogenase for 3-HP production in Bacillus subtilis (Prophetic)

[00313] An alternative approach that could be utilized to express MCR variants or combinations of MCR and 3malonate semialdehyde dehydrogenasees in Bacillus subtilis is expression from a plasmid. Shuttle vectors are well known in the art that can carry an inducible Pgrac IPTG-inducible promoter. The genetic element containing these enzymes can be cloned under the control of the this promoter using standard molecular biology techniques creating a plasmid containing MCR or malonate semialdehyde

dehydrogenase varaints. These plasmids could then be transformed into different bacillus strains

[00314] Based on the present disclosure, it is noted that, in addition to introducing a nucleic acid construct that comprises a sequence encoding for malonyl-CoA reductase and or malonate semialdehyde dehydrogenase activity in a bacillus cell, in some embodiments additional genetic modifications are made to decrease enoyl-CoA reductase activity and/or other fatty acid synthase activity.

[00315] Example 10: Evaluation of Strains for 3-HP Production (Prophetic).

[00316] 3-HP production in biocatalysts (strains), discussed in the other examples, can be evaluated at 100-mL scale in SM3 (minimal salts) media. SM3 used is described under the Common Methods Section, but is supplemented with 200 mM MOPS. Cultures are started from LB plates containing antibiotics by standard practice (Sambrook and Russell, 2001) into 50 mL of TB media plus the appropriate antibiotic as indicated and grown to stationary phase overnight at 30°C with rotation at 250 rpm. Five ml of this culture are transferred to 100 ml of SM3 media plus 30 g/L glucose, antibiotic, and 1 mM IPTG in triplicate 250-ml baffled flasks and incubated at 30°C, 250 rpm. Flasks are shifted to 37°C, 250 rpm after 4 hours. To monitor cell growth and 3-HP production by these cultures, samples (2 ml) are withdrawn at 24 hours for optical density measurements at 600nm (OD ₆ oo, 1 cm path length) and pelleted by centrifugation at 14000 rpm for 5 mM and the supernatant collected for analysis of 3-HP production as described under "Analysis of cultures for 3-HP production" in the Common Methods section. 3-HP titer and standard deviation is expressed as g/L. Dry cell weight (DCW) is calculated as 0.41 times the measured OD ₆ oo value, based on baseline DCW per OD ₆ oo determinations.

[00317] Example 1 1 : General example of genetic modification to a host cell (prophetic and non-specific).

[00318] In addition to the above specific examples, this example is meant to describe a non-limiting approach to genetic modification of a selected microorganism to introduce a nucleic acid sequence of interest. Alternatives and variations are provided within this general example. The methods of this example are conducted to achieve a combination of desired genetic modifications in a selected microorganism species, such as a combination of genetic modifications as described in sections herein, and their functional equivalents, such as in other bacterial and other microorganism species. Several groups of genetic modifications that may be evaluated are described in figures 2A-G.

[00319] A gene or other nucleic acid sequence segment of interest is identified in a particular species (such as E. coli as described herein) and a nucleic acid sequence comprising that gene or segment is obtained.

[00320] Based on the nucleic acid sequences at the ends of or adjacent the ends of the segment of interest, 5' and 3 ' nucleic acid primers are prepared. Each primer is designed to have a sufficient overlap section that hybridizes with such ends or adjacent regions. Such primers may include enzyme recognition sites for restriction digest of transposase insertion that could be used for subsequent vector incorporation or genomic insertion. These sites are typically designed to be outward of the hybridizing overlap sections. Numerous contract services are known that prepare primer sequences to order (e.g., Integrated DNA Technologies, Coralville, IA USA).

[00321] Once primers are designed and prepared, polymerase chain reaction (PCR) is conducted to specifically amplify the desired segment of interest. This method results in multiple copies of the region of interest separated from the microorganism's genome. The microorganism's DNA, the primers, and a thermophilic polymerase are combined in a buffer solution with potassium and divalent cations (e.g., Mg or Mn) and with sufficient quantities of deoxynucleoside triphosphate molecules. This mixture is exposed to a standard regimen of temperature increases and decreases. However, temperatures, components, concentrations, and cycle times may vary according to the reaction according to length of the sequence to be copied, annealing temperature approximations and other factors known or readily learned through routine experimentation by one skilled in the art. [00322] In an alternative embodiment the segment of interest may be synthesized, such as by a commercial vendor, and prepared via PCR, rather than obtaining from a microorganism or other natural source of DNA.

[00323] The nucleic acid sequences then are purified and separated, such as on an agarose gel via electrophoresis. Optionally, once the region is purified it can be validated by standard DNA sequencing methodology and may be introduced into a vector. Any of a number of vectors may be used, which generally comprise markers known to those skilled in the art, and standard methodologies are routinely employed for such introduction. Commonly used vector systems are pSMART (Lucigen, Middleton, WI), pET E. coli EXPRESSION SYSTEM (Stratagene, La Jolla, CA), pSC-B StrataClone Vector (Stratagene, La Jolla, CA), pRANGER-BTB vectors (Lucigen, Middleton, WI), and TOPO vector (Invitrogen Corp, Carlsbad, CA, USA). Similarly, the vector then is introduced into any of a number of host cells.

Commonly used host cells are E. cloni 100 (Lucigen, Middleton, WI), E. cloni 10GF' (Lucigen,

Middleton, WI), StrataClone Competent cells (Stratagene, La Jolla, CA), E. coli BL21, E. coli BW25113, and E. coli K12 MG1655. Some of these vectors possess promoters, such as inducible promoters, adjacent the region into which the sequence of interest is inserted (such as into a multiple cloning site), while other vectors, such as pSMART vectors (Lucigen, Middleton, WI), are provided without promoters and with dephosporylated blunt ends. The culturing of such plasmid-laden cells permits plasmid replication and thus replication of the segment of interest, which often corresponds to expression of the segment of interest.

[00324] Various vector systems comprise a selectable marker, such as an expressible gene encoding a protein needed for growth or survival under defined conditions. Common selectable markers contained on backbone vector sequences include genes that encode for one or more proteins required for antibiotic resistance as well as genes required to complement auxotrophic deficiencies or supply critical nutrients not present or available in a particular culture media. Vectors also comprise a replication system suitable for a host cell of interest.

[00325] The plasmids containing the segment of interest can then be isolated by routine methods and are available for introduction into other microorganism host cells of interest. Various methods of introduction are known in the art and can include vector introduction or genomic integration. In various alternative embodiments the DNA segment of interest may be separated from other plasmid DNA if the former will be introduced into a host cell of interest by means other than such plasmid.

[00326] While steps of the general prophetic example involve use of plasmids, other vectors known in the art may be used instead. These include cosmids, viruses (e.g., bacteriophage, animal viruses, plant viruses), and artificial chromosomes (e.g., yeast artificial chromosomes (YAC) and bacteria artificial chromosomes (BAC)).

[00327] Host cells into which the segment of interest is introduced may be evaluated for performance as to a particular enzymatic step, and/or tolerance or bio-production of a chemical compound of interest. Selections of better performing genetically modified host cells may be made, selecting for overall performance, tolerance, or production or accumulation of the chemical of interest.

[00328] It is noted that this procedure may incorporate a nucleic acid sequence for a single gene (or other nucleic acid sequence segment of interest), or multiple genes (under control of separate promoters or a single promoter), and the procedure may be repeated to create the desired heterologous nucleic acid sequences in expression vectors, which are then supplied to a selected microorganism so as to have, for example, a desired complement of enzymatic conversion step functionality for any of the herein-disclosed metabolic pathways. However, it is noted that although many approaches rely on expression via transcription of all or part of the sequence of interest, and then translation of the transcribed mRNA to yield a polypeptide such as an enzyme, certain sequences of interest may exert an effect by means other than such expression.

[00329] The specific laboratory methods used for these approaches are well-known in the art and may be found in various references known to those skilled in the art, such as Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third Edition 2001 (volumes 1-3), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (hereinafter, Sambrook and Russell, 2001).

[00330] As an alternative to the above, other genetic modifications may also be practiced, such as a deletion of a nucleic acid sequence of the host cell's genome. One non- limiting method to achieve this is by use of Red/ET recombination, known to those of ordinary skill in the art and described in U.S. Patent Nos. 6,355,412 and 6,509,156, issued to Stewart et al. and incorporated by reference herein for its teachings of this method. Material and kits for such method are available from Gene Bridges (Gene Bridges GmbH, Dresden, Germany, «www.genebridges.com»), and the method may proceed by following the manufacturer's instructions. Targeted deletion of genomic DNA may be practiced to alter a host cell's metabolism so as to reduce or eliminate production of undesired metabolic products. This may be used in combination with other genetic modifications such as described herein in this general example.

[00331] Example 12. Utilization of sucrose as the feedstock for production of 3-HP and other products (Partial Prophetic).

[00332] Common laboratory and industrial strains of E. coli, such as the strains described herein, are not capable of utilizing sucrose as the sole carbon source, although this property is found in a number of wild strains, including pathogenic E. coli strains. Sucrose, and sucrose-containing feedstocks such as molasses, are abundant and often used as feedstocks for the production by microbial fermentation of organic acids, amino acids, vitamins, and other products. Thus further derivatives of the 3 -HP -producing strains that are capable of utilizing sucrose would expand the range of feedstocks that can be utilized to produce 3-HP.

[00333] Various sucrose uptake and metabolism systems are known in the art (for example, U.S. Pat. No. 6,960,455), incorporated by reference for such teachings. Described herein is the construction of E. coli strains that harbor the CSC genes conferring the ability to utilize sucrose via a non-phosphotransferase system, wherein the CSC genes constitute cscA, encoding a sucrose hydrolase, cscB, encoding a sucrose permease, cscK, encoding a fructokinase, and cscR, encoding a repressor. The sequences of these genes are annotated in the NCBI database as accession No. X81461 AF473544. To allow efficient expression utilizing codons that are highly abundant in E. coli genes, an operon containing cscB, cscK, and cscA was designed and synthesized using the services of a commercial synthetic DNA provider (DNA 2.0, Menlo Park, CA). These genes may be isolated or synthesized as described above, incorporated on a plasmid, and transformed into a suitable host cell simultaneously with plasmids that may express NADH dependent malonyl-CoA reductase or NADH dependent 3-HP dehydrogenase genes as described in other examples. Transformants carrying both plasmids are grown and evaluated for 3-HP production in shake flasks using SM3 medium where glucose is replaced with an equal concentration of sucrose.

[00334] Genes that confer functions to enable utilization of sucrose by E. coli can also be obtained from the natural isolate pUR400 (Cowan, P. J., et al. J. Bacteriol. 173:7464-7470, 1991) which carries genes for the phosphoenolpyruvatedependent carbohydrate uptake phosphotransferase system (PTS). These genes consist of scrA, encoding the enzyme II component of the PTS transport complex, scrB, encoding sucrose-6 phosphate hydrolase, scrK, encoding fructokinase, and scrY, encoding a porin. These genes may be isolated or synthesized as described above, incorporated on a plasmid, and transformed into a suitable host cell simultaneously with plasmids that may express

[00335] NADH dependent malonyl-CoA reductase or NADH dependent 3-HP dehydrogenase genes as described in other examples. Transformants carrying both plasmids are grown and evaluated for 3-HP production in shake flasks using SM3 medium where glucose is replaced with an equal concentration of sucrose.

[00336] Example 13: Conversion of 3-HP to Specified Derived Chemicals and Products

[00337] 3-HP is obtained in a relatively pure state from a microbial bio-production event, and is converted to any one or more of propriolactone via a ring-forming internal esterification reaction (eliminating a water molecule), ethyl-3-HP via esterification with ethanol, malonic acid via an oxidation reaction, and 1,3-propanediol via a reduction reaction. These conversions proceed such as by organic synthesis reactions known to those skilled in the art. Any of these conversions of 3-HP may proceed via a chemical synthesis reaction under controlled conditions to attain a high conversion rate and yield with acceptably low by-product formation. Table 12, incorporated into this example, provides additional derived chemicals and products that may be made, such as using indicated reactions.

[00338] Example 14: Genetic modification/introduction of Malonyl-CoA Reductase for 3-HP production in Bacillus subtilis (Prophetic).

[00339] An alternative approach that could be utilized to express MCR variants or combinations of MCR and 3-HP dehydrogenases in Bacillus subtilis is expression from a plasmid. Shuttle vectors are known in the art that carry an inducible Pgrac IPTG-inducible promoter. The genetic element containing these enzymes can be cloned under the control of this promoter using standard molecular biology techniques creating a plasmid containing MCR or 3-HP dehydrogenase variants. A plasmid based MCR could then be transformed into different bacillus strains

[00340] Based on the present disclosure, it is noted that, in addition to introducing a nucleic acid construct that comprises a sequence encoding for malonyl-CoA reductase and or 3-HP dehydrogenase activity in a bacillus cell, in some embodiments additional genetic modifications are made to decrease enoyl-CoA reductase activity and/or other fatty acid synthase activity.

[00341] Example 15: Yeast aerobic pathway for 3HP production (Prophetic).

[00342] An alternative approach could be utilized to express MCR variants or combinations of MCR and 3-HP dehydrogenases in yeast by expression from a plasmid. The genetic elements containing these enzymes under the control of numerous promoters in numerous vectors are known in the art by use of standard methods. A vector based MCR could then be transformed into different yeast strains.

[00343] Based on the present disclosure, it is noted that, in addition to introducing a nucleic acid construct that comprises a sequence encoding for malonyl-CoA reductase and or 3-HP dehydrogenase activity in a yeast cell, in some embodiments additional genetic modifications are made to decrease enoyl-CoA reductase activity and/or other fatty acid synthase activity.

[00344] Example 16: Yeast Strain construction.

[00345] Yeast strains are constructed using standard yeast transformation and selected for by

complementation of auxotrophic markers. For general yeast transformation methods, see Gietz, R.D. and R.A. Woods. (2002) "Transformation of Yeast by the Liac/SS Carrier DNA/PEG Method." Methods in Enzymology 350: 87-96. The following are non-limiting general prophetic examples directed to practicing the present invention in other microorganism species. These are meant to be instructive of approaches to practice embodiments of the invention in these as well as other microorganism species.

[00346] General Prophetic Example 17: Improvement of 3-HP Bio-production in Rhodococcus erythropolis .

[00347] A series of E. coli-Rhodococcus shuttle vectors are available for expression in R. erythropolis, including, but not limited to, pRhBR17 and pDA71 (Kostichka et al., Appl. Microbiol. Biotechnol. 62:61- 68(2003)). Additionally, a series of promoters are available for heterologous gene expression in R.

erythropolis (see for example Nakashima et al., Appl. Environ. Microbiol. 70:5557-5568 (2004), and Tao et al., Appl. Microbiol. Biotechnol. 2005, DOI 10.1007/s00253-005-0064). Targeted gene disruption of chromosomal genes in R. erythropolis may be created using the method described by Tao et al., supra, and Brans et al. (Appl. Environ. Microbiol. 66: 2029-2036 (2000)). These published resources are incorporated by reference for their respective indicated teachings and compositions. The nucleic acid sequences required for providing an increase in 3-HP production, as described herein, are cloned initially in pDA71 or pRhBR71 and transformed into E. coli. The vectors are then transformed into R. erythropolis by electroporation, as described by Kostichka et al., supra. The recombinants are grown in synthetic medium containing glucose and the 3-HP bio-production is followed using methods known in the art and/or described herein.

[00348] General Prophetic Example 18: Improvement of 3-HP Bio-production in B. licheniformis .

[00349] Most of the plasmids and shuttle vectors that replicate in B. subtilis are used to transform B. licheniformis by either protoplast transformation or electroporation. The nucleic acid sequences required for improvement of 3-HP biosynthesis are isolated from various sources, codon optimized as appropriate, and cloned in plasmids pBE20 or pBE60 derivatives (Nagarajan et al., Gene 114:121-126 (1992)).

Methods to transform B. licheniformis are known in the art (for example see Fleming et al. Appl. Environ. Microbiol., 61(11):3775-3780 (1995)). These published resources are incorporated by reference for their respective indicated teachings and compositions.

[00350] The plasmids constructed for expression in B. subtilis are transformed into B. licheniformis to produce a recombinant microorganism that then demonstrates improved 3-HP Bio-production.

[00351] General Prophetic Example 19a: Improvement of 3-HP Bio-production in Paenibacillus macerans.

[00352] Plasmids are constructed as described herein for expression in B. subtilis and used to transform Paenibacillus macerans by protoplast transformation to produce a recombinant microorganism that demonstrates improved 3-HP Bio-production.

[00353] General Prophetic Example 19b: Improvement of 3-HP Bio-production in Alcaligenes (Ralstonia) eutrophus (currently referred to as Cupriavidus necator).

[00354] Methods for gene expression and creation of mutations in Alcaligenes eutrophus are known in the art (see for example Taghavi et al., Appl. Environ. Microbiol., 60(10):3585-3591 (1994)). This published resource is incorporated by reference for its indicated teachings and compositions. Any of the nucleic acid sequences identified to improve 3-HP biosynthesis are isolated from various sources, codon optimized as appropriate, and cloned in any of the broad host range vectors described herein, and electroporated to generate recombinant microorganisms that demonstrate improved 3-HP Bio-production. The poly(hydroxybutyrate) pathway in Alcaligenes has been described in detail, a variety of genetic techniques to modify the Alcaligenes eutrophus genome is known, and those tools can be applied for engineering a recombinant microorganism that produces 3-HP and/or polymers thereof.

[00355] General Prophetic Example 20: Improvement of 3-HP Bio-production in Pseudomonas putida.

[00356] Methods for gene expression in Pseudomonas putida are known in the art (see for example Ben- Bassat et al., U.S. Patent No. 6,586,229, which is incorporated herein by reference for these teachings). Any of the nucleic acid sequences identified to improve 3-HP biosynthesis are isolated from various sources, codon optimized as appropriate, and cloned in any of the broad host range vectors described herein, and electr op orated to generate recombinant microorganisms that demonstrate improved 3-HP biosynthetic production. For example, these nucleic acid sequences are inserted into pUCP18 and this ligated DNA are electr op orated into electrocompetent Pseudomonas putida KT2440 cells to generate recombinant P. putida microorganisms that exhibit increased 3-HP Bio-production, comprised at least in part of introduced nucleic acid sequences.

[00357] General Prophetic Example 21 : Improvement of 3-HP Bio-production in Lactobacillus plantarum.

[00358] The Lactobacillus genus belongs to the Lactobacillales family and many plasmids and vectors used in the transformation of Bacillus subtilis and Streptococcus are used for Lactobacillus. Non-limiting examples of suitable vectors include pAM.beta. l and derivatives thereof (Renault et al., Gene 183 :175- 182 (1996); and O'Sullivan et al., Gene 137:227-231 (1993)); pMBBl and pHW800, a derivative of pMBBl (Wyckoff et al. Appl. Environ. Microbiol 62:1481-1486 (1996)); pMGl, a conjugative plasmid (Tanimoto et al., J. Bacteriol. 184:5800-5804 (2002)); pNZ9520 (Kleerebezem et al., Appl. Environ. Microbiol. 63:4581-4584 (1997)); pAM401 (Fujimoto et al., Appl. Environ. Microbiol. 67:1262-1267 (2001)); and pAT392 (Arthur et al., Antimicrob. Agents Chemother. 38:18991903 (1994)). Several plasmids from Lactobacillus plantarum have also been reported (e.g., van Kranenburg R, Golic N, Bongers R, Leer R J, de Vos W M, Siezen R J, Kleerebezem M. Appl. Environ. Microbiol. 2005 March; 71(3): 1223-1230).

[00359] General Prophetic Example 22: Improvement of 3-HP Bio-production in Enterococcus faecium, Enterococcus gallinarium, and Enterococcus faecalis.

[00360] The Enterococcus genus belongs to the Lactobacillales family and many plasmids and vectors used in the transformation of Lactobacillus, Bacillus subtilis, and Streptococcus are used for

Enterococcus. Non- limiting examples of suitable vectors include pAM.beta. l and derivatives thereof (Renault et al., Gene 183:175-182 (1996); and O'Sullivan et al., Gene 137:227-231 (1993)); pMBBl and pHW800, a derivative of pMBBl (Wyckoff et al. Appl. Environ. Microbiol. 62:1481-1486 (1996));

pMGl, a conjugative plasmid (Tanimoto et al., J. Bacteriol. 184:5800-5804 (2002)); pNZ9520

(Kleerebezem et al., Appl. Environ. Microbiol. 63:4581-4584 (1997)); pAM401 (Fujimoto et al., Appl. Environ. Microbiol. 67:1262-1267 (2001)); and pAT392 (Arthur et al., Antimicrob. Agents Chemother. 38:1899-1903 (1994)). Expression vectors for E. faecalis using the nisA gene from Lactococcus may also be used (Eichenbaum et al., Appl. Environ. Microbiol. 64:2763-2769 (1998). Additionally, vectors for gene replacement in the E. faecium chromosome are used (Nallaapareddy et al., Appl. Environ. Microbiol. 72:334-345 (2006)). [00361] For each of the examples above, the following 3-HP bio-production comparison may be incorporated thereto: Using analytical methods for 3-HP such as are described in Subsection III of Common Methods Section, 3-HP is obtained in a measurable quantity at the conclusion of a respective bio-production event conducted with the respective recombinant microorganism (see types of bio- production events, incorporated by reference into each respective General Prophetic Example). That measurable quantity is substantially greater than a quantity of 3-HP produced in a control bio-production event using a suitable respective control microorganism lacking the functional 3-HP pathway so provided in the respective General Prophetic Example.

COMMON METHODS SECTION

[00362] All methods in this Section are provided for incorporation into the Examples where so referenced.

[00363] Subsection I. Microorganism Species and Strains, Cultures, and Growth Media.

[00364] Bacterial species, which may be utilized as needed, are as follows:

Acinetobacter calcoaceticus (DSMZ # 1139) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Brain Heart Infusion (BHI) Broth (RPI Corp, Mt. Prospect, IL, USA). Serial dilutions of the resuspended A. calcoaceticus culture are made into BHI and are allowed to grow for aerobically for 48 hours at 37°C at 250 rpm until saturated. Bacillus subtilis is a gift from the Gill lab (University of Colorado at Boulder) and is obtained as an actively growing culture. Serial dilutions of the actively growing B. subtilis culture are made into Luria Broth (RPI Corp, Mt. Prospect, IL, USA) and are allowed to grow for aerobically for 24 hours at 37°C at 250 rpm until saturated. Chlorobium limicola (DSMZ# 245) is obtained from the German Collection of Microorganisms and Cell Cultures

(Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended using Pfennig's Medium I and II (#28 and 29) as described per DSMZ instructions. C limicola is grown at 25°C under constant vortexing. Citrobacter braakii (DSMZ # 30040) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Brain Heart Infusion (BHI) Broth ( RPI Corp, Mt. Prospect, IL, USA). Serial dilutions of the resuspended C. braakii culture are made into BHI and are allowed to grow for aerobically for 48 hours at 30°C at 250 rpm until saturated.

Clostridium acetobutylicum (DSMZ # 792) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Clostridium acetobutylicum medium (#411) as described per DSMZ instructions. C. acetobutylicum is grown anaerobically at 37°C at 250 rpm until saturated.

Clostridium aminobutyricum (DSMZ # 2634) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Clostridium aminobutyricum medium (#286) as described per DSMZ instructions. C. aminobutyricum is grown anaerobically at 37°C at 250 rpm until saturated.

Clostridium kluyveri (DSMZ #555) is obtained from the German Collection of

Microorganisms and Cell Cultures (Braunschweig, Germany) as an actively growing culture. Serial dilutions of C. kluyveri culture are made into Clostridium kluyveri medium (#286) as described per DSMZ instructions. C kluyveri is grown anaerobically at 37°C at 250 rpm until saturated.

Cupriavidus metallidurans (DMSZ # 2839) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Brain Heart Infusion (BHI) Broth (RPI Corp, Mt. Prospect, IL, USA). Serial dilutions of the resuspended C. metallidurans culture are made into BHI and are allowed to grow for aerobically for 48 hours at 30°C at 250 rpm until saturated.

Cupriavidus necator (DSMZ # 428) is obtained from the German Collection of

Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Brain Heart Infusion (BHI) Broth (RPI Corp, Mt. Prospect, IL, USA). Serial dilutions of the resuspended C. necator culture are made into BHI and are allowed to grow for aerobically for 48 hours at 30°C at 250 rpm until saturated. As noted elsewhere, previous names for this species are Alcaligenes eutrophus and Ralstonia eutrophus.

Desulfovibrio fructosovorans (DSMZ # 3604) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Desulfovibrio fructosovorans medium (#63) as described per DSMZ instructions. D. fructosovorans is grown anaerobically at 37°C at 250 rpm until saturated.

Escherichia coli Crooks (DSMZ#1576) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Brain Heart Infusion (BHI) Broth (RPI Corp, Mt. Prospect, IL, USA). Serial dilutions of the resuspended E. coli Crook culture are made into BHI and are allowed to grow for aerobically for 48 hours at 37°C at 250 rpm until saturated.

Escherichia coli K12 is a gift from the Gill lab (University of Colorado at Boulder) and is obtained as an actively growing culture. Serial dilutions of the actively growing E. coli K12 culture are made into Luria Broth (RPI Corp, Mt. Prospect, IL, USA) and are allowed to grow for aerobically for 24 hours at 37°C at 250 rpm until saturated. Halobacterium salinarum (DSMZ# 1576) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Halobacterium medium (#97) as described per DSMZ instructions. H. salinarum is grown aerobically at 37°C at 250 rpm until saturated.

Lactobacillus delbrueckii (#4335) is obtained from WYEAST USA (Odell, OR, USA) as an actively growing culture. Serial dilutions of the actively growing L. delbrueckii culture are made into Brain Heart Infusion (BHI) broth (RPI Corp, Mt. Prospect, IL, USA) and are allowed to grow for aerobically for 24 hours at 30°C at 250 rpm until saturated. Metallosphaera sedula (DSMZ #5348) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as an actively growing culture. Serial dilutions of M. sedula culture are made into Metallosphaera medium (#485) as described per DSMZ instructions. M. sedula is grown aerobically at 65°C at 250 rpm until saturated.

Propionibacterium freudenreichii subsp. shermanii (DSMZ# 4902) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in PYG-medium (#104) as described per DSMZ instructions. P. freudenreichii subsp. shermanii is grown anaerobically at 30°C at 250 rpm until saturated.

Pseudomonas putida is a gift from the Gill lab (University of Colorado at Boulder) and is obtained as an actively growing culture. Serial dilutions of the actively growing P. putida culture are made into Luria Broth (RPI Corp, Mt. Prospect, IL, USA) and are allowed to grow for aerobically for 24 hours at 37°C at 250 rpm until saturated. Streptococcus mutans (DSMZ# 6178) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Luria Broth (RPI Corp, Mt. Prospect, IL, USA). S. mutans is grown aerobically at 37°C at 250 rpm until saturated.

[00365] The following non- limiting strains may also be used as starting strains in the Examples: DF40 Hfr(P02A), garBlO, fhuA22, ompF627(T2R), fadL701(T2R), relAl, pitAlO, spoTl, rrnB-2, pgi-2, mcrBl, creC510, BW25113 F-, A(araD-araB)567, AlacZ4787(::rrnB-3), &lambda , rph-1, A(rhaD- rhaB)568, hsdR514, JP111 Hfr(POl), galE45(GalS), &lambda Jab 1392 (ts), relAl, spoTJ, thi-1. These strains possess recognized genetic modifications, and are available from public culture sources such as the Yale Coli Genetic Stock Collection (New Haven, CT USA). Strains developed from these strains are described in the Examples, including in Examples in applications that are incorporated by reference herein, particularly Application number PCT/US2010/050436, published March 31, 2011 as

WO/2011/038364; Application number PCT/US/057690, published May 26, 2011 as WO/2011/063363, and Application number PCT/US2011/022790 published August 4, 2011 as WO/2011/094457.

[00366] Bacterial growth culture media and associated materials and conditions, are as follows:

Fed-batch medium contained (per liter): 10 g tryptone, 5 g yeast extract, 1.5 g NaCl, 2 g Na ₂ HP0 ₄ .7 H ₂ 0, 1 g KH ₂ P0 ₄ , and glucose as indicated

AM2 medium contained (per liter): 2.87 g K ₂ HP0 ₄ , 1.50g KH ₂ P0 ₄ , 3.13g (NH ₄ ) ₂ S0 ₄ , 0.15 g KC1, 1.5 mM MgS0 ₄ , 0.1M K ⁺ MOPS pH 7.2, 30 g glucose, and 1 ml trace Mineral Stock prepared as described in Martinez et al. Biotechnol Lett 29:397-404 (2007)

SM3 minimal medium for E. coli (Final phosphate concentration = 27.5 mM; Final N concentration = 47.4 mM NH ₄ +).

Components per liter: 700 mL DI water, 100 mL 10X SM3 Salts, 2 ml 1M MgS0 ₄ , 1 ml 1000X Trace Mineral Stock, 60 mL 500 g/L glucose, 100 mL 0.1 M MOPS (pH 7.4), 0.1 mL of 1 M CaCl ₂ , Q.S. with DI water to 1000 mL, and 0.2 urn filter sterilize.

[00367] Preparation of Stock Solutions: To make 10X SM3 Salts (1 L): 800 mL DI water, 28.7 g K ₂ HP0 ₄ , 15 g KH ₂ P0 ₄ , 31.3 g (NH ₄ ) ₂ 50 ₄ , 1.5 g KC1, 0.5 g Citric Acid (anhydrous), and Q.S. with DI water to 1000 mL.

To make 1000X Trace Mineral Stock (1L): save in 50-ml portions at room temp

Per liter in 0.12M HC1 (dilute 10 ml cone HC1 into 1 liter water):2.4 g FeCL ₃ .6H ₂ 0, 0.17 g CoCl ₂ .6H ₂ 0, 0.15 g CuCl ₂ .2H ₂ 0, 0.3 g ZnCl ₂ , 0.3 g NaMo0 ₄ .2H ₂ 0 (Molybdic acid, disodium salt, dihydrate), 0.07 g H ₃ B0 ₃ , and 0.5 g M n C 1 ₂ . 4 H ₂ O .

To make 1M MOPS:209.3 g MOPS, dissolve in 700 ml water. Take 70-ml portions and adjust to desired pH with 50% KOH, adjust to 100 mL final volume, and 0.2 jtm filter sterilize.

To make 1M Mg50 ₄ : 120.37 g dissolved in 1000 mL water.

To make 500 g/L (50%) glucose stock solution: 900 mL DI water, 500 g glucose, and Q.S. to 1000 mL.

[00368] To make 1L M9 minimal media:

M9 minimal media was made by combining 5X M9 salts, 1M MgS0 ₄ , 20% glucose, 1M CaCl ₂ and sterile deionized water. The 5X M9 salts are made by dissolving the following salts in deionized water to a final volume of 1L: 64g Na ₂ HP0 ₄ 7H ₂ 0, 15g KH ₂ P0 ₄ ,2.5g NaCl, 5.0g NH ₄ C1. The salt solution was divided into 200mL aliquots and sterilized by autoclaving for 15minutes at 15psi on the liquid cycle. A IM solution of MgS0 and 1M CaCl ₂ were made separately, then sterilized by autoclaving. The glucose was filter sterilized by passing it thought a 0.22jtm filter. All of the components are combined as follows to make 1L of M9: 750mL sterile water, 200mL 5X M9 salts, 2mL of 1M MgS0 ₄ , 20mL 20% glucose, O. lmL CaCl ₂ , Q.S. to a final volume of 1L.

[00369] To make EZ rich media:

All media components were obtained from TEKnova (Hollister CA USA) and combined in the following volumes. lOOmL 10X MOPS mixture, lOmL 0.132M K2 HP0 ₄ , lOOmL 10X ACGU, 200mL 5X Supplement EZ, lOmL 20% glucose, 580mL sterile water.

[00370] Subsection II: Gel Preparation, DNA Separation, Extraction, Ligation, and Transformation Methods:

[00371] Molecular biology grade agarose (RPI Corp, Mt. Prospect, IL, USA) is added to lx TAE to make a 1% Agarose in TAE. To obtain 50x TAE add the following to 900ml distilled H ₂ 0 : 242g Tris base (RPI Corp, Mt. Prospect, IL, USA), 57.1ml Glacial Acetic Acid (Sigma- Aldrich, St. Louis, MO, USA), 18.6 g EDTA (Fisher Scientific, Pittsburgh, PA USA), and adjust volume to IL with additional distilled water. To obtain lx TAE, add 20mL of 50x TAE to 980mL of distilled water. The agarose-TAE solution is then heated until boiling occurred and the agarose is fully dissolved. The solution is allowed to cool to 50°C before lOmg/mL ethidium bromide (Acros Organics, Morris Plains, NJ, USA) is added at a concentration of 5ul per lOOmL of 1% agarose solution. Once the ethidium bromide is added, the solution is briefly mixed and poured into a gel casting tray with the appropriate number of combs (Idea Scientific Co., Minneapolis, MN, USA) per sample analysis. DNA samples are then mixed accordingly with 5X TAE loading buffer. 5X TAE loading buffer consists of 5X TAE(diluted from 50X TAE as described herein), 20% glycerol (Acros Organics, Morris Plains, NJ, USA), 0.125% Bromophenol Blue (Alfa Aesar, Ward Hill, MA, USA), and adjust volume to 50mL with distilled water. Loaded gels are then run in gel rigs (Idea Scientific Co., Minneapolis, MN, USA) filled with IX TAE at a constant voltage of 125 volts for 25-30 minutes. At this point, the gels are removed from the gel boxes with voltage and visualized under a UV transilluminator (FOTODYNE Inc., Hartland, WI, USA).

[00372] The DNA isolated through gel extraction is then extracted using the QIAquick Gel Extraction Kit following manufacturer' s instructions (Qiagen (Valencia CA USA)). Similar methods are known to those skilled in the art. The thus-extracted DNA then may be ligated into pSMART (Lucigen Corp, Middleton, WI, USA), StrataClone (Stratagene, La Jolla, CA, USA) or pCR2.1-TOPO TA (Invitrogen Corp, Carlsbad, CA, USA) according to manufacturer' s instructions. These methods are described in the next subsection of Common Methods.

[00373] Ligation Methods:

[00374] For ligations into pSMART vectors:

[00375] Gel extracted DNA is blunted using PCRTerminator (Lucigen Corp, Middleton, WI, USA) according to manufacturer' s instructions. Then 50Ong of DNA is added to 2.5 uL 4x CloneSmart vector premix, lul CloneSmart DNA ligase (Lucigen Corp, Middleton, WI, USA) and distilled water is added for a total volume of lOul. The reaction is then allowed to sit at room temperature for 30 minutes and then heat inactivated at 70°C for 15 minutes and then placed on ice. E. cloni 10G Chemically Competent cells (Lucigen Corp, Middleton, WI, USA) are thawed for 20 minutes on ice. 40ul of chemically competent cells are placed into a microcentrifuge tube and 1 ul of heat inactivated CloneSmart Ligation is added to the tube. The whole reaction is stirred briefly with a pipette tip. The ligation and cells are incubated on ice for 30 minutes and then the cells are heat shocked for 45 seconds at 42°C and then put back onto ice for 2 minutes. 960 ul of room temperature Recovery media (Lucigen Corp, Middleton, WI, USA) and places into microcentrifuge tubes. Shake tubes at 250 rpm for 1 hour at 37°C. Plate lOOul of transformed cells on Luria Broth plates (RPI Corp, Mt. Prospect, IL, USA) plus appropriate antibiotics depending on the pSMART vector used. Incubate plates overnight at 37°C.

[00376] For Ligations into StrataClone:

[00377] Gel extracted DNA is blunted using PCRTerminator (Lucigen Corp, Middleton, WI, USA) according to manufacturer' s instructions. Then 2ul of DNA is added to 3ul StrataClone Blunt Cloning buffer and 1 ul StrataClone Blunt vector mix amp/kan (Stratagene, La Jolla, CA, USA) for a total of 6ul . Mix the reaction by gently pipeting up at down and incubate the reaction at room temperature for 30 minutes then place onto ice. Thaw a tube of StrataClone chemically competent cells (Stratagene, La Jolla, CA, USA) on ice for 20 minutes. Add lul of the cloning reaction to the tube of chemically competent cells and gently mix with a pipette tip and incubate on ice for 20 minutes. Heat shock the transformation at 42°C for 45 seconds then put on ice for 2 minutes. Add 250ul pre-warmed Luria Broth (RPI Corp, Mt. Prospect, IL, USA) and shake at 250 rpm for 37°C for 2 hour. Plate 100 ul of the transformation mixture onto Luria Broth plates (RPI Corp, Mt. Prospect, IL, USA) plus appropriate antibiotics. Incubate plates overnight at 37°C.

[00378] For Ligations into pCR2.1-TOPO TA:

[00379] Add lul TOPO vector, lul Salt Solution (Invitrogen Corp, Carlsbad, CA, USA) and 3 ul gel extracted DNA into a microcentrifuge tube. Allow the tube to incubate at room temperature for 30 minutes then place the reaction on ice. Thaw one tube of TOPIOF' chemically competent cells

(Invitrogen Corp, Carlsbad, CA, USA) per reaction. Add luL of reaction mixture into the thawed TOPIOF' cells and mix gently by swirling the cells with a pipette tip and incubate on ice for 20 minutes. Heat shock the transformation at 42°C for 45 seconds then put on ice for 2 minutes. Add 250ul pre- warmed SOC media (Invitrogen Corp, Carlsbad, CA, USA) and shake at 250 rpm for 37°C for 1 hour. Plate 100 ul of the transformation mixture onto Luria Broth plates (RPI Corp, Mt. Prospect, IL, USA) plus appropriate antibiotics. Incubate plates overnight at 37°C.

[00380] General Transformation and Related Culture Methodologies:

[00381] Chemically competent transformation protocols are carried out according to the manufacturer's instructions or according to the literature contained in Molecular Cloning (Sambrook and Russell, 2001). Generally, plasmid DNA or ligation products are chilled on ice for 5 to 30 min. in solution with chemically competent cells. Chemically competent cells are a widely used product in the field of biotechnology and are available from multiple vendors, such as those indicated in this Subsection.

Following the chilling period cells generally are heat-shocked for 30 seconds at 42°C without shaking, re- chilled and combined with 250 microliters of rich media, such as S.O.C. Cells are then incubated at 37°C while shaking at 250 rpm for 1 hour. Finally, the cells are screened for successful transformations by plating on media containing the appropriate antibiotics.

[00382] Alternatively, selected cells may be transformed by electroporation methods such as are known to those skilled in the art.

[00383] The choice of an E. coli host strain for plasmid transformation is determined by considering factors such as plasmid stability, plasmid compatibility, plasmid screening methods and protein expression. Strain backgrounds can be changed by simply purifying plasmid DNA as described herein and transforming the plasmid into a desired or otherwise appropriate E. coli host strain such as determined by experimental necessities, such as any commonly used cloning strain (e.g., DH5a, Topi OF', E. cloni 10G, etc.).

[00384] Plasmid DNA was prepared using the commercial miniprep kit from Qiagen (Valencia, CA USA) according to manufacturer' s instructions.

[00385] Subsection Ma. 3 -HP Preparation [00386] A 3-HP stock solution was prepared as follows. A vial of 0-propriolactone (Sigma- Aldrich, St. Louis, MO, USA) was opened under a fume hood and the entire bottle contents was transferred to a new container sequentially using a 25-mL glass pipette. The vial was rinsed with 50 mL of HPLC grade water and this rinse was poured into the new container. Two additional rinses were performed and added to the new container. Additional HPLC grade water was added to the new container to reach a ratio of 50 mL water per 5 mL 0-propriolactone. The new container was capped tightly and allowed to remain in the fume hood at room temperature for 72 hours. After 72 hours the contents were transferred to centrifuge tubes and centrifuged for 10 minutes at 4,000 rpm. Then the solution was filtered to remove particulates and, as needed, concentrated by use of a rotary evaporator at room temperature. Assay for concentration was conducted, and dilution to make a standard concentration stock solution was made as needed.

[00387] Subsection Mb. HPLC, GC/MS and Other Analytical Methods for 3-HP Detection (Analysis of Cultures for 3-HP Production)

[00388] For HPLC analysis of 3-HP, the Waters chromatography system (Milford, MA) consisted of the following: 600S Controller, 616 Pump, 717 Plus Autosampler, 486 Tunable UV Detector, and an in-line mobile phase Degasser. In addition, an Eppendorf external column heater is used and the data are collected using an SRI (Torrance, CA) analog-to- digital converter linked to a standard desk top computer. Data are analyzed using the SRI Peak Simple software. A Coregel 64H ion exclusion column

(Transgenomic, Inc., San Jose, CA) is employed. The column resin is a sulfonated polystyrene divinyl benzene with a particle size of lOum and column dimensions are 300 x 7.8 mm. The mobile phase consisted of sulfuric acid (Fisher Scientific, Pittsburgh, PA USA) diluted with deionized (18 MSkm) water to a concentration of 0.02 N and vacuum filtered through a 0.2 μιη nylon filter. The flow rate of the mobile phase is 0.6 mL/min. The UV detector is operated at a wavelength of 210 nm and the column is heated to 60 °C. The same equipment and method as described herein is used for 3-HP analyses for relevant prophetic examples. A representative calibration curve using this HPLC method with a 3-HP standard (TCI America, Portland, OR) is provided in FIG. 15.

[00389] The following method is used for GC-MS analysis of 3-HP. Soluble monomeric 3-HP is quantified using GC-MS after a single extraction of the fermentation media with ethyl acetate. Once the 3-HP has been extracted into the ethyl acetate, the active hydrogens on the 3-HP are replaced with trimethylsilyl groups using N, 0-Bis(Trimethylsilyl) trifluoroacetamide to make the compound volatile for GC analysis. A standard curve of known 3-HP concentrations is prepared at the beginning of the run and a known quantity of ketohexanoic acid (lg/L) is added to both the standards and the samples to act as an internal standard for Quantitation, with tropic acid as an additional internal standard. The 3-HP content of individual samples is then assayed by examining the ratio of the ketohexanoic acid ion (m/z = 247) to the 3-HP ion (219) and compared to the standard curve. 3-HP is quantified using a 3HP standard curve at the beginning of the run and the data are analyzed using HP Chemstation. The GC-MS system consists of a Hewlett Packard model 5890 GC and Hewlett Packard model 5972 MS. The column is Supelco SPB-1 (60m X 0.32mm X 0.25um film thickness). The capillary coating is a non-polar methylsilicone. The carrier gas is helium at a flow rate of lmL/min. The 3 -HP as derivatized is separated from other components in the ethyl acetate extract using either of two similar temperature regimes. In a first temperature gradient regime, the column temperature starts with 40°C for 1 minute, then is raised at a rate of 10°C/minute to 235°C, and then is raised at a rate of 50°C/minute to 300°C. In a second temperature regime, which was demonstrated to process samples more quickly, the column temperature starts with 70°C which is held for 1 mM, followed by a ramp-up of 10 °C/minute to 235°C which is followed by a ramp-up of 50 C/minute to 300°C. A representative calibration curve is provided in FIG. 16.

[00390] A bioassay for detection of 3-HP also was used in various examples. This determination of 3-HP concentration was carried out based on the activity of the E. coli 3-HP dehydrogenase encoded by the ydfG gene (the YDFG protein). Reactions of 200-u.1 were carried out in 96-well microtiter plates, and contained 100 mM Tris-HCl, pH 8.8, 2.5 mM MgCl ₂ , 2.625 mM NADP ⁺ , 3 lig purified YDFG and 20 ul culture supernatant. Culture supernatants were prepared by centrifugation in a microfuge (14,000 rpm, 5 min) to remove cells. A standard curve of 3-HP (containing from 0.025 to 2 g/1) was used in parallel reactions to quantitate the amount of 3-HP in culture supernatants. Uninoculated medium was used as the reagent blank. Where necessary, the culture supernatant was diluted in medium to obtain a solution with 3-HP concentrations within that of the standard curve.

[00391] The reactions were incubated at 37°C for 1 hr, and 20 u. l of color developer containing 1.43 mM nitroblue tetrazolium, 0.143 phenazine methosulfate, and 2.4% bovine serum albumin were added to each reaction. Color development was allowed to proceed at 37°C for an additional hr, and the absorbance at 580nm was measured. 3-HP concentration in the culture supernatants was quantitated by comparison with the values obtained from the standard curve generated on the same microtiter plate. The results obtained with the enzymatic assay were verified to match those obtained by one of the analytical methods described above. FIG. 17 provides a representative standard curve.

[00392] A spectrophotometric assay was developed in order to biochemically measure malonyl CoA reductase activity of both full length and truncated version of malonyl CoA reductase. Activities were determined with either NADPH, NADH, or a mixture of NADPH and NADH as a cofactor and malonyl CoA as a substrate. Assays were performed as 200 microliter reactions in a 96-well plate format using a Molecular Dynamics SpectraMax 384 microplate reader with SoftmaxPro software (Molecular Dynamics, Sunnyvale CA) to quantitate the rate of change in the 340 nm absorbance. All assays were conducted at 37°C, and the instrument was allowed to mix the plate for 1 second prior to each measurement. The progress of each reaction was monitored for 30 minutes during which measurements were made every 20 seconds. The reaction conditions at the time of reaction consisted of 5 mM dithiothreitol, 3 mM magnesium chloride, 100 mM Trizma-HCl pH7.6 buffer. Unless otherwise noted, all chemicals were purchased from Sigma- Aldrich (St. Louis, MO). The nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) cofactors were added at lmM NADH, lmM NADPH, or a combination or 0.5 mM NADH (EMD Bioscience) and 0.5 mM NADPH (EMD

Bioscience) depending on the assay condition. In assays where purified YdfG (Molecular Throughput), was supplemented, the protein was added at a concentration of 0.075 mg/mL.

[00393] A spectrophotometric coupled assay was developed in order to biochemically measure the 3-HP dehydrogenase domain of malonyl CoA reductase to determine the specific activities of either the full length enzyme or truncated version thereof. This coupled assay allows for measurement of the dehydrogenase specific activity by providing malonate semialdehyde to the dehydrogenase. The malonate semialdehyde is produced using a purified beta-alanine aminotransaminase (BAAT). This enzyme is able to convert alpha-ketoglutarate and beta-alanine into glutamine and malonate semialdehyde. This malonate semialdehyde is they used as substrate for the dehydrogenase reaction that is monitor by recoding the decrease in 340 nm NADPH/NADH-specific signal with the spectrophotometer. Purified protein for this assay was provided by overexpressing the Saccharomyces kluyveri with a c-terminal SxHistidine purification tag. A plasmid encoding this codon optimized gene was synthesized using the services of Genscript, Inc. The sequence of the overexpressed protein is provided as SEQ ID NO:148. To purify the BAAT protein, a starter culture of the cell line carrying the BAAT overexpression plasmid was grown overnight in LB media with antibiotic selection. The next morning the overnight culture was used to inoculate 1 L culture of LB media with selection at 30 degrees Celsius. When the culture reached an optical density of 0.7, protein production was induced with 1 mM O-D-l-thiogalactopyranoside (IPTG). The culture was grown for an additional 7 hours at 30 degrees Celsius. After growth, cells containing the protein were pelleted by centrifugation. To purify the BAAT enzyme, the cell pellets were resuspended in 10 mL of 50 mM Tris pH 8.0, 500 mM sodium chloride, 10 mM imidazole, and 250 U/mL Benzonase (Novagen). All Chemicals were supplied by Sigma- Aldrich (St. Louis, MO) unless otherwise stated. The cells were mechanically lysed using a Mini-Beadb eater (Biospec Products, Bartelsville, OK). The cell lysate was clarified by centrifugation and 10000 G for 15 minutes. The his-tagged BAAT protein was then purified from the clarified cell lysate by mixing the lysate as a batch-purification with 3mL of Ni- NTA resin (Qiagen) at 4 degrees Celsius for 1 hour. The protein bound resin was then placed in an empty chromatography column (Biorad), and washed with a buffer consisting of 50 mM Tris pH 8.0, 500 mM sodium chloride, and 20 mM imidazole for 50 column volumes. The pure BBAT protein was eluted from the column using a buffer consisting of 50 mM Tris pH 8.0, 500 mM sodium chloride, 250 mM imidazole, and 0.1 mM pyridoxal 5' phosphate. Fractions were pooled and stored at -80 Celsius until the time of use.

[00394] Activities were determined with either NADPH, NADH, or a mixture of NADPH and NADH as a cofactor. Assays were performed as 200 microliter reactions in a 96-well plate format using a Molecular Dynamics SpectraMax 384 microplate reader with SoftmaxPro software (Molecular Dynamics, Sunnyvale CA) to quantitate the rate of change in the 340 nm absorbance. All assays were conducted at 37°C, and the instrument was allowed to mix the plate for 1 second prior to each measurement. The progress of each reaction was monitored for 30 minutes during which measurements were made every 20 seconds. The reaction conditions at the time of reaction consisted of 0.1 mM pyridoxal 5' phosphate, 10 mM alpha- ketoglutarate, 25 mM beta-alanine, 1 mM magnesium chloride, 50 mM Trizma-HCl pH8.0 buffer, and 0.006 mg of purified BAAT. Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO). The nicotinamide adenine dinucleotide and nicotinamide (NADH) adenine dinucleotide phosphate (NADPH) cofactors were added at lmM NADH, lmM NADPH. Reactions were initiated by the addition of 20 uL of diluted, clarified whole cell lysates of cells expressing the dehydrogenase being assessed to a reaction buffer filled well of the 96-well plate used to perform the assay . Once the reaction time course was read and the slopes of each well were calculated, the specific activities were compared to a negative control to determine a background rate. All values reported are the average specific activities measured in triplicate. The clarified supernatant was measure for protein concentration using a Biorad Total Protein determination kit (BioRad). For each dehydrogenase tested, a cell line containing the expression plasmid was grown as 50 mL cultures in LB with antibiotic selection. Expression cultures were started from overnight cultures grown overnight in LB at 30 degrees Celsius. After inoculation, cells were grown at 37 degrees Celsius for 1.5 hours and then protein production was induced with addition of 1 mM IPTG. Cultures were grown 5 hr, after which the cells were collected by centrifugation The cell pellets were resuspended in 50 mM Tris pH 8.0, 1 mM magnesium chloride, and 250 U/mL Benzonase (Novagen). All Chemicals were supplied by Sigma-Aldrich (St. Louis, MO) unless otherwise stated. The cells were mechanically lysed using a Mini-Beadb eater (Bioscpec Products, Bartelsville, OK). The resulting cell lysate was clarified by centrifugation and 10000 G for 15 minutes.

[00395] The embodiments, variations, sequences, and figures described herein should provide an indication of the utility and versatility of the present invention. Other embodiments that do not provide all of the features and advantages set forth herein may also be utilized without departing from the spirit and scope of the present invention. Such modifications and variations are considered to be within the scope of the invention.