ALCOHOL DEHYDROGENASE VARIANTS

Priority Claim

This application claims the benefit of U.S. Provisional Patent Application Serial Number 61/887,251 filed October 4, 2013, entitled ALCOHOL DEHYDROGENASE VARIANTS, the disclosure of which is incorporated herein by reference. Also, the entire contents of the ASCII text file entitled "GNO0003WO_Sequence_Listing_ST25.txt" created on October 3, 2014, having a size of 206 kilobytes is incorporated herein by reference.

Background

Alcohol dehydrogenases (ADHs; EC 1.1.1.1) promote the conversion of alcohols to and aldehydes or ketones, typically along with the reduction of nicotinamide adenine dinucleotide (NAD ⁺ to NADH). ADHs are instrumental in the generation of important compounds having aldehyde, ketone, and alcohol groups during biosynthesis of various metabolites.

One class of alcohol dehydrogenase is methanol dehydrogenases (MDHs). MDHs, converts methanol (MeOH) to formaldehyde (Fald), may be used in an enzymatic pathway engineered into a microbe to enable MeOH as a sole carbon source or as a co-carbon source with other feed stocks such as, for example, glucose, dextrose, plant biomass or syngas, to produce valuable products. Microorganisms have been reported that metabolize methanol, and in some instances do so via a methanol dehydrogenase, and in even fewer instances produce valuable products. Increasing MDH activity will enable improved use of MeOH, improving MeOH as a sole carbon source, decreasing production costs, decreasing amounts of any more expensive secondary or co-carbon source, e.g. glucose, increasing product yields, and providing faster rate of MeOH use.

Summary

Generally, presented herein are non-natural NAD ⁺ -dependent alcohol dehydrogenases (ADHs) (i.e. engineered enzymes and their encoding-polynucleotides) capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to its original or unmodified counterpart. Exemplary aspects describe non-natural NAD ⁺ -dependent methanol dehydrogenases (MDHs), in particular enzymes of the class EC 1.1.1.244.

The ADHs and MDHs have at least one amino acid substitution as compared to its corresponding natural or unmodified alcohol dehydrogenase. By unmodified alcohol

dehydrogenase is meant that the ADH or MDH may have been previously engineered (e.g., need not be naturally-occuring), prior to incorporating any modification described herein. Such alcohol dehydrogenases that are starting sequences for incorporating a modification described herein to generate the novel engineered enzyme may be alternatively referred to herein as wild-type, template, starting sequence, natural, naturally-occurring, unmodified, corresponding natural alcohol dehydrogenase, corresponding natural alcohol dehydrogenase without the amino acid substitution, corresponding alcohol dehydrogenase or corresponding alcohol dehydrogenase without the amino acid substitution. Experimental studies described herein demonstrate for the first time that a number of amino acid positions along the length of the amino acid sequence can be substituted to provide a non-natural dehydrogenase having increased substrate conversion. The studies also show that combinations of substitutions (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve, etc.) in an amino acid sequence can also provide even further increased substrate conversion. Provided herein therefore are single and combination variants of a starting or template or corresponding alcohol dehydrogenase, e.g., in particular enzymes of the class EC 1.1.1.244, having increased substrate conversion.

Embodiments of the disclosure provide a non-natural NAD ⁺ -dependent alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase, and capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, as measured relative to a corresponding alcohol dehydrogenase without amino acid substitution. Embodiments of the disclosure also provide a non-natural NAD ⁺ -dependent alcohol dehydrogenase comprising at least one amino acid substitution capable of at least two fold greater conversion of methanol or ethanol to_formaldehyde or acetaldehyde, respectively, as compared a second sequence that is an NAD ⁺ -dependent alcohol dehydrogenase, wherein the first and second sequences differ with regards to the at least one amino acid substitution. Embodiments are also directed to engineered cells expressing the non-natural NAD ⁺ - dependent alcohol dehydrogenase capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde as described.

Some embodiments of the current disclosure are directed to an engineered cell expressing a non-natural NAD ⁺ -dependent alcohol dehydrogenase comprising at least one amino acid substitution (including single and combination variants). The cells can be used to promote production of a target product or intermediate thereof. For example, the cell may provide either or both an increased amount of reducing equivalents, e.g. NADH, for an increase in a target product or may provide for increased fixation of carbon from formaldehyde and/or acetaldehyde into a target product. Exemplary products include (a) 1 ,4-butanediol and intermediates thereto, such as 4-hydroxybutanoic acid (4-hydroxybutanoate, 4-hydroxybutyrate, 4-HB), (b) butadiene and intermediates thereto, such as 1,4-butanediol, 1,3-butanediol, crotyl alcohol, 3-buten-2-ol (methyl vinyl carbinol) and 3-buten-l-ol, (c) 1,3-butanediol and intermediates thereto, such as 2,4- pentadienoate, crotyl alcohol or 3-buten-l-ol, (d) adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine and levulinic acid and their intermediates, e.g. 4-aminobutyryl-CoA, (e) methacrylic acid (2-methyl-2-propenoic acid) and its esters known collectively as methacrylates, such as methyl methacrylate, methyl methacrylate, 3-hydroxyisobutyrate and/or 2- hydroxyisobutyrate and their intermediates, (f) 1,2-propanediol (propylene glycol), n-propanol, 1,3-propanediol and glycerol, and their intermediates and (g) succinic acid and intermediates thereto.

Embodiments of the engineered cell may further optionally include one or more additional metabolic pathway transgene(s) to further promote production of the target product or intermediate thereof. In exemplary embodiments the cell further comprises one or more methanol metabolic pathway (MMP) transgene(s), such as a formaldehyde dehydrogenase transgene, allowing expression of the encoded pathway enzyme or accessory protein.

In exemplary embodiments the cell further comprises a product pathway comprising enzymes (and their endoding polynucleotides) for production of a target product, such as the enzymes described herein for production of 1 ,4-butanediol from glucose.

Other embodiments are directed to compositions including engineered cell, such as cell culture compositions, and also compositions including one or more product(s) produced from the engineered cell. For example, a composition can include a target product or intermediate thereof produced by the cells, where the composition has been purified to remove cells or other components useful for cell culturing. The composition may be treated to enrich or purify the target product or intermediate thereof.

Other embodiments of the disclosure are directed to products made from the target product obtained from methods using the engineered cell. Exemplary products include polymers made with target products, such as polymers made from diol target products combined with diacids, including target product succinic acid, such as polybutylene terephthalate (PBT) and polybutylene succinate (PBS) made from 1,4-butanediol polymerized with terephthalic acid or succinic acid respectively.

Other embodiments of the disclosure are directed to nucleic acids encoding the non-natural alcohol dehydrogenases with one or more variant amino acids, as well as expression constructs including the nucleic acids, and engineered cells comprising the nucleic acids or expression constructs.

In other embodiments the disclosure also provides methods for generating non-natural

NAD ⁺ -dependent alcohol dehydrogenases capable of at least two-fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to its unmodified (original; template) counterpart. In some embodiments, the method includes steps of (a) identifying a variant amino acid that provides increased conversion in a template sequence, (b) identifying corresponding amino acid position in a target sequence having identify to the template sequence, and (c) changing the amino acid at the corresponding amino acid position in a target sequence to the variant amino acid. The starting template for incorporation of modifications described herein can be a naturally-occurring enzyme sequence or a previously engineered enzyme sequence.

In other embodiments, the methods includes steps of (a) identifying an amino acid position in a non-natural NAD ⁺ -dependent alcohol dehydrogenases that is not a variant position, (b) providing, in a original template, a variation at an amino acid position that is a non-variant position, and (c) identifying variants from step (b) that provide increased conversion of the substrate. Description of the Drawings

Figures 1A- D are graphs listing amino acid positions of methanol dehydrogenase (2315 A) and the effect of substitution of those positions on enzyme activity.

Figure 2 illustrates a pathway using MDH to produce 1, 4-butanediol (BDO) in organism such as E. coli.

Figures 3A-D illustrate pathways using certain methanol metabolizing enzymes. Figure 3D illustrates using enzymes to fix carbon from methanol via formaldehyde assimilation into a product pathway of interest. The "Product Pathway" can be that of 1 ,4-butanediol as described herein or other product pathway.

Figure 4 is an amino acid sequence alignment of various Fe-dependent alcohol dehydrogenases with Bacillus MeDH.

Detailed Description

The embodiments of the description described herein are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed in the following detailed description. Rather, the embodiments are chosen and described so that others skilled in the art can appreciate and understand the principles and practices of the description.

All publications and patents mentioned herein are hereby incorporated by reference. The publications and patents disclosed herein are provided solely for their disclosure. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate any publication and/or patent, including any publication and/or patent cited herein.

Generally, the disclosure provides non-natural NAD ⁺ -dependent alcohol dehydrogenases (ADHs) capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to its unmodified counterpart. Nucleic acids encoding the non-natural alcohol dehydrogenases, as well as expression constructs including the nucleic acids, and engineered cells comprising the nucleic acids or expression constructs are described. Also described are engineered cells expressing a non-natural NAD -dependent alcohol dehydrogenase, optionally including one or more additional metabolic pathway transgene(s), methanol metabolic pathway genes, and/or target product pathway genes; cell culture compositions including the cells; methods for promoting production of the target product or intermediate thereof from the cells; compositions including the target product or intermediate; and products made from the target product or intermediate.

The term "non-naturally occurring", when used in reference to an organism (e.g., microbial) is intended to mean that the organism has at least one genetic alteration not normally found in a naturally occurring organism of the referenced species. Naturally-occurring organisms can be referred to as "wild-type" such as wild type strains of the referenced species. Likewise, a "non-natural" polypeptide or nucleic acid can include at least one genetic alteration not normally found in a naturally-occurring polypeptide or nucleic acid. Naturally-occurring organisms, nucleic acids, and polypeptides can be referred to as "wild-type" or "original" such as wild type strains of the referenced species. Likewise, amino acids found in the wild type organism can be referred to as "original" with regards to any amino acid position.

A genetic alteration that makes an organism non-natural can include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon.

An NAD(P)+-dependent methanol dehydrogenase from Bacillus methanolicus MGA3 (Genbank Accession number EIJ77596.1, GI number: 387585261; designated herein as MDH 2315, 382 amino acids long; SEQ ID NO: 1), was selected as a template for mutagenesis to identify variants with improved activity. This sequence was selected as it was surprisingly found to be very active on methanol, typically nearly twice as active as other alcohol dehydrogenases tested, and used NAD+ and thus able to regenerate NADH that can be useful to enzymes in target pathways. MDH 2315 is reported in the literature as an NAD(P)-dependent methanol dehydrogenase from Bacillus methanolicus MGA3 and its sequence was described in Brautaset et ah, "Plasmid-

Dependent Methylotrophy in Thermotolerant Bacillus methnolicus", Journal of Bacteriology, vol. 186, ppl229-1238 (2004). It is also referred to as MDH MGA3 in WO2013/110797 to Brautaset and MDH "M" in Krog et ah, "Methylotrophic Bacillus methanolicus Encodes Two Chromosomal and One Plasmid Born NAD+ Dependent Mathanol Dehydrogenase Paralogs with Different Catalytic and Biochemical Properties", PLOS ONE, pp. 1-11, (2013), which report addititional wild-type Bacillus MDHs. MDH 2315A was expressed in E. coli and a library of variants was generated by error prone PCR (the epPCR Library). Specifically, MDH 2315A was expressed in E. coli and subjected to saturation mutagenesis at 375 of 382 positions to generate a library of all single point substitutions of the common 20 amino acids (the NNK library).

In the primary screen, the NNK Library was screened by assaying individual colony cell extracts for MeOH to Fald conversion. The library was screened for variants that had greater than 2-fold activity over wild-type (defined as a positive). Such a library can contain variants with multiple mutations, deletions, etc. However, variants with single point substitutions were identified. In that primary screen about 150 positions gave colonies whose extracts had activity reliably 2-fold greater than wild-type. Of those, 21 positions gave colonies with activity reliably greater than 4-fold wild-type.

The primary screen of the NNK library identified about 10 positions where 90% or more of the colonies were inactive, suggesting that any amino acid other than wild-type at those positions led to enzyme inactivity (or rapid degradation). About 30 positions were identified in which less than 5% of the colonies were inactive. Many positions were identified in which no positives were observed but the total number of inactives was less than 90%. Many positions were identified in which the total number of positives and inactives were less than 95%.

Secondary in vitro assays in triplicate and sequencing were done on all positives greater than 4-fold, and many greater than 2-fold activity. At 40 positions, a total of 90 substitutions were determined to be responsible for the activity increase either greater than 2-fold or in many cases greater than 4-fold, and up to 10-fold. In many positions, only a single specific amino acid gave improvement, in other positions several amino acid substitutions gave improvement (e.g., at 213 only N213D, but at 121 both G121D, G121V, etc).

Secondary screening of the epPCR library revealed about 20 positions whose substitutions gave activity greater than 3-fold wild-type. Sequencing of these colonies revealed the specific substitutions at each position, for example, N213D (nomenclature: N is original or unmodified amino acid at position 213; D is the substitution). At these 20 positions, a total of 30 substitutions provided greater than 3 -fold wild-type activity of which about 15 variants provided greater than 4- fold activity.

Some of the colonies whose MDH variant sequence were identified were also assayed in vivo by measuring MeOH conversion to formaldehyde in a strain background in which genes for use of formaldehyde were inactivated, thus enabling accumulation of formaldehyde. Generally, all MDH variants that were positive (greater than wild-type) in in vitro screening also enabled greater than wild-type activity in vivo, although the in vitro to in vivo activities did not always correlate exactly. At some positions the frequency of a particular substitution in the total population of colonies at that position in a library was zero or less than statistically expected. Thus some substitutions at some positions may not have been present or present and not detected.

Table 1 lists amino acids mutations with respect to SEQ ID NO: 1, providing greater than two fold activity when present as single mutations. While not to be bound by theory, as depicted in Figure 1, functional features associated with amino acid positions of methanol dehydrogenase designated 2315A and corresponding positions in other methanol dehydrogenase described herein include: NADH cofactor binding correlating with positions 38D, 39A, 40F, 70D, 97G, 98S, 137T, 138T, 141T, 142G, 143S, 145T, 146T, 147S, 148L, 149A, 150V, 161P, 162V, 1631; activation site correlating with positions 95G, 97G, 98S; and substrate correlating with positions 145T, 146T, 147S, 148L, 149A, 150V, 161P, 162V, 1631, 253F, 258L, 266H, 359D, 360V, 361C.

As can be seen from Figure 1 depicting site saturation mutagenesis, numerous amino acid positons of methanol dehydrogenase designated 2315A are tolerant to substitution (indicated by a percentage of colonies having greater than 2-fold (designated "hits") or from 0.2 to less than 2-fold activity of wild-type enzyme) and others less tolerant (indicated by percentage of colonies where enzyme activity was less than 20% of wild-type enzyme activity (designated "dead"). For example, 168T and 270G are very tolerant to change, where substitutions at 168T generally had little effect on activity, whereas substitutions at 270G predominantly improved activity.

Art known methods can be used for the testing the enzymatic activity of alcohol dehydrogenases, and such methods can be used to test activity of alcohol dehydrogenase (ADH) variant enzymes as well. As a general matter, a reaction composition including the alcohol dehydrogenase (ADH) variant, an alcohol (substrate) and NAD (cofactor) can be converted to a dehydrogenated product. For example, conversion of ethanol is shown as follows:

Ethanol + β-NAD ^{Alcoho1 Dehvdj0¾enase} > Acetaldehyde + β-NADH

Reaction can be carried out at a desired temperature, such as 25° C, and pH, such as pH 7.

The ADH variant, can be defined in terms of its enzymatic activity with one unit of enzyme converting 1.0 μιηοΐε of alcohol to dehydrogenated product per minute at pH 8.8 @ 25° C. See, for example, Kagi, J.H.R. and Vallee, B.L. (1960) Journal of Biological Chemistry 235, 3188-3192

Of particular interest herein is conversion of methanol to formaldehyde to regenerate NADH. This conversion can be followed by either or both conversion to formate or fixation of the formaldehyde carbon into target product. The formate can be either or both converted to CO ₂ or have its carbon fixed into target product, such as by conversion back to formaldehyde. See the attached figures.

A representative in vivo assay was developed to determine the activity of methanol dehydrogenase variants in organisms is reported in U.S. Application Serial Number 13/975,678. This assay relies on the detection of formaldehyde (Palp), thus measuring the forward activity of the enzyme (oxidation of methanol). To this end, a strain comprising a BDOP and lacking frmA, frmB, frmR was created using Lamba Red recombinase technology (Datsenko and Wanner, Proc. Natl. Acad. Sci. USA, 6 97(12): 6640-5 (2000). Plasmids expressing methanol dehydrogenases were transformed into the strain, then grown to saturation in LB medium + antibiotic at 37°C with shaking. Transformation of the strain with an empty vector served as a negative control. Cultures were adjusted by O.D. and then diluted 1 : 10 into M9 medium + 0.5% glucose + antibiotic and cultured at 37°C with shaking for 6-8 hours until late log phase. Methanol was added to 2% v/v and the cultures were further incubated for 30 min. with shaking at 37°C. Cultures were spun down and the supernatant was assayed for formaldehyde produced using DETECTX Formaldehyde Detection kit (Arbor Assays; Ann Arbor, MI) according to manufacturer's instructions. The frmA, frmB, frmR deletions resulted in the native formaldehyde utilization pathway to be deleted, which enables the formation of formaldehyde that can be used to detect methanol dehydrogenase activity in the organism. These genes are deleted in this case solely to facilitate measurement of methanol conversion by preventing loss of the measured analyte, formaldehyde.

Enzymatic kinetic assays were done for 10 single point variants, and that 2-10 fold improvements in activity were reflected in 2-10 fold improvements in Km, Vmax or both. Co- factor binding nor substrate or product on-off rates were not measured.

Table 7 shows enzymology data from various wild type ADH proteins. Tables 8 and 9 show data for wild type and variant enzymes, with Table 8 showing activity using either methanol or 1,4-butanediol, and Table 9 showing 1 ,4-butanediol-dependent steady-state kinetic parameters for wild-type and variant methanol dehydrogenase.

Results of the mutagenesis procedures and rationale design and screening of the positives (by "positive" is meant a sequence modified as described herein having at least a two (2) fold increase in activity compared to the unmodified template sequence) revealed a number of amino acid variants along the MDH protein 2315A template for use in the invention. Positives showing greater than two fold increase in activity are shown in Table 1, and listed as follows:

SI IT, D38N, H42Q, E48D, N53I, E56K, D60E, V61A, I63F, P65Q, D70N, P71I, P71T, P71V, T74S, D81G, K84R, E86K, N87K, I94V, S99P, S99T, A103V, I106L, G107S, L108V, L108W, V109Y, Nl 12K, Nl 12R, Rl 15H, II 16F, Nl 17D, Nl 17Q, Nl 17Y, Q120H, Q120R, G121A, G121D, G121E, G121L, G121M, G121R, G121 S, G121T, G121V, G121W, G121Y, V122A,

V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, S124R, V125C, V125G, V125W, E126G, E126V, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T145M, T146N, S147R, L148A, L148F, L148G, L148I, L148T, L148V, L148W, A149L, A149M, A149T, A149V, V150A, V150I, T152M, A155V, K157N, V158E, V158H, V158K, V158W, P161A, P161G, P161Q, P161S, P161V, I163F, I163N, I163Q, I163T, D164G, D164N, E165G, K181R, A184T, L186M, T190A, T190S, I199V, Q217K, L226M, G256C, Q267H, G269S, G270M, G270S, G270Y, T296S, R298H, A300T, I302V,G312V, A316V, I323M, F333L, P336L, S337C, G343D, V344A, V344G, K345E, E350K, K354M, N355D, N355I, N355K, E358G, V360A, V360G, V360K, V360R, V360S, C361N, C361R, Q363K, and K379M. These changes, their positions in SEQ ID NO: 1, and their corresponding positions in other template sequences are described further in the tables and elsewhere herein.

Of more interest are positives showing greater than two fold increase in activity as single mutations shown in Table 1, and listed as follows: D38N, D60E, P71I, P71V, N87K, S99T, A103V, G107S, L108V, L108W, V109Y, Rl 15H, II 16F, Nl 17D, Nl 17Q, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, V125C, V125G, V125W, E126G, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T146N, A149L, A149M, A149T, A149V, V150A, K157N, V158E, V158H, V158K, V158W, I163Q, D164N, Q267H, G270M, G270S, G270Y, K345E, N355D, V360G, V360K, V360R, V360S, C361R. These changes, their positions in SEQ ID NO: 1, and their corresponding positions in other template sequences are described further in the tables and elsewhere herein.

Results of the rationale design mutagenesis procedures and the other library generation procedures described herein and screening of the positive revealed a number of combination amino acid variants along the MDH protein 2315A template. Positives showing greater than two fold increase in activity are shown in Tables 2 - 4, and listed as variations in the following sets: (a) D70N, L148G, P161G, V360A; (b) D70N, L148G, V360A, C361N; (c) D70N, L148V, V150I, P161A, V360G; (d) D70N, L148V, V360G; (e) D70N, P161A, V360A; (f) D70N, P161V, V360G, C361N; (g) D70N, V150I, P161A, V360A; (h) D70N, V150I, P161V, V360G, C361N; (i) E48D, L148V, P161A, V360A; (J) L148G, P161A, V360A, C361N; (k) L148G, P161A, V360G; (1) L148G, P161A, V360G, C361N; (m) L148G, P161G, V360A; (n) L148G, P161G, V360G, C361N; (o) L148G, V360A, C361N; (p) L148G, V360G, C361N; (q) L148I, P161G, V360G; (r) L148I, P161V, V360G; (s) L148T, V150I, V360A; (t) L148T, V360G; (u) L148V, P161A, V360A; (v) L148V, V150I, P161A, V360A; (w) L148V, V150I, P161A, V360A, C361N; (x) L148V, V150I, P161A, V360G; (y) L148V, V150I, P161A, V360G, C361N; (z) L148V, V150I, P161A, V360G, C361N; (aa) L148V, V150I, P161G, V360A; (ab) L148V, V150I, P161V, V360G, C361N; (ac) L148W, P161A, V360A, C361N; (ad) N112K , S147R, P161A,

V360A; (ae) P161A, Q217K, V360A, C361N; (af) P161A, V360A, C361N; (ag) P161A, V360G; (ah) P161V, E358G, V360G; (ai) P161V, V360A, C361N; (aj) L148W, P161A, V360A, C361N; (ak) N112K , S147R, P161A, V360A; (al) P161A, Q217K, V360A, C361N; (am) P161A, V360A, C361N; (an) P161A, V360G; (ao) P161V, E358G, V360G; (ap) P161V, V360A, C361N; (aq) P161V, V360G; (ar) P65Q, L148G, V150I, P161A, V360G, C361N; (as) S147R, L148A, V150I, P161A, V360G; (at) S147R, L148F, V150I, P161G, V360G; (au) S147R, L148V, P161G, V360A; (av) P161V, V360G; (aw) P65Q, L148G, V150I, P161A, V360G, C361N; (ax) S147R, L148A, V150I, P161A, V360G; (ay) S147R, L148F, V150I, P161G, V360G; (az) S147R, L148V, P161G, V360A; (aaa) S147R, L148V, P161V, V360G; (aab) S147R, L148V, V150I, P161A, C361N; (aac) S147R, L148V, V150I, P161G, V360G; (aad) S147R, P161A, V360A; (aae) S147R, P161A, V360A, C361N; (aaf) S147R, P161A, V360G; (aag) S147R, P161V, V360G; (aah) S147R, P161V, V360G, C361N; (aai) S147R, V150I, P161V, V360A; (aaj) S147R, V150I, V360A, C361N; (aak) T145M, L148I, V360G; (aal) V150I, I302V, V360G, C361N; (aam) V150I, P161A, C361N; (aan) V150I, P161G, V360A, C361N; (aao) V150I, P161G, V360G; (aap) V150I, P161G, V360G, C361N; (aaq) V150I, P161V, C361N; (aar) V150I, P161V, K354R, V360A, C361N; (aas) V150I, P161V, V360A, C361N; (aat) V150I,

P161V, V360G, C361N; (aau) V150I, V360A, C361N; (aav) V150I, V360G; (aaw) SI IT, T74S, G269S, V344A; (aax) K84R, I163T; (aay) V122A, I163N; (aaz) G107S, F333L; (aaaa) V129M, T152M, G343D; (aaab) I63F, N355K; (aaac) G107S, F333L; (aaad) E86K, S99T, A149V; (aaae) N53I, V158E; (aaaf) N355I, K379M; (aaag) H42Q, G107S; (aaah) Q120H, I163N; (aaai) A149V, I323M; (aaaj) G107S, F333L; (aaak) D164G, K181R; (aaal) A155V, R298H, N355D; (aaam) N123D, E165G; (aaan) I163F, L186M; (aaao) G121A, T296S; (aaap) I94V, S99P, N123I; (aaaq) E126V, V129M, V344G; (aaar) Q120R, S143T; (aaas) G256C, A316V; (aaat) P161Q, G312V; (aaau) L226M, A300T, V360A; (aaav) S337C, E350K, N355D, Q363K; (aaaw) D81G, V158E; (aaax) I106L, N117Y, E126V; (aaay) G107S, G121D; (aaaz) V61A, V158E; (aaaaa) N53I, V158E; (aaaab) N117Y, T190S; (aaaac) S124R, I199V; (aaaad) K354M, C361R; (aaaae) A184T, C361R; (aaaag) E56K, Q267H; (aaaag) S124R, E126G; (aaaah) T190A, N355K; (aaaai) P71T, F333L; (aaaaj) G107S, F333L; and (aaaak) N123I, P336L, (aaaal) D38D/A149V, (aaaam) D38N/V163V, (aaaan) D73D/L108V, (aaaao) G121R/P161 S, and (aaaap) Nl 12R/P161 S.

Also identified were positions in SEQ ID NO:l that are generally intolerant to substitution, including P8, V132, E177, A207, T208, Q246, 1264, P285, E308, Y339, A340, A353, T362, R367, P369, D373, and 1377; where original amino acids are of particular interest at these positions in SEQ ID NO: l and their corresponding positions in other templates as described herein.

Also identified were positions in SEQ ID NO:l that are generally very tolerant to substitution, including E56, E78, E86, T2, N4, E237, E240, E327, E341, E347, P9, V18, R24, T44, L46, K52, D60, A62, F64, A67, P71, A72, D73, T74, V80, K84, Q85, 1106, V109, Rl 15, II 16, Nl 17, V122, K127, 1135, T136, S154, A155, R156, P161 , V162, 1163, P169, T170, V171, V174, L178, M179, A184, G185, L186, A189, T190, A194, A198, Y202, V203, T211, F214, 1216, Q217, K220, L221, N223, Y225, A229, Y244, A245, M248, A252, N254, H266, G270, Y272, 1278, M284, H286, V287, N291, 1293, A294, R298, H301, 1302, L305, N309, A311, G312, T315, A317, R321, V324, 1329, S332, S337, M342, K345, K354, N355, A356, Y357, T370, A375, and 1378 in SEQ ID NO: l and their corresponding positions in other templates; where it was observed that typically all amino acids, except for occasionally 1 or 2, were neutral, beneficial, or less than an 80% decrease to activity at these positions. Of these, D60, 1378, L46, A62, A67, Nl 17, K52, 1135, Y202, 1216, M248, N291, M342, T2, E240, T44, II 16, V203, S332, and thus their corresponding positions in other templates, were tolerant to all changes.

Positions for additional interest for substitution to increase activity include 23T, 8 ID,

141T, 174V, 189A, 332S, 372Q and 379K and their corresponding positions in other templates.

Embodiments of the disclosure are also directed to the preparation and use of other alcohol dehydrogenase (ADH) variant enzymes including methanol dehydrogenases variant enzymes, based on the single and combination amino acid variants indentified in and with respect to the MDH protein 2315A template. In these embodiments generation of non-natural NAD ⁺ -dependent alcohol dehydrogenases (that are not 2315 A) capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to its original unmodified counterpart can be based on information of those MDH 2315A variants that provided increased activity.

In some embodiments, the method for preparing other ADH variants includes steps of (a) identifying a variant amino acid that provides increased conversion when present in the 2315A template sequence, (b) identifying a corresponding amino acid position in a target sequence (i.e., other ADH or MDH sequence) having identity to the template sequence, and (c) changing the amino acid at the corresponding amino acid position in the target sequence to the variant amino acid. For example, the D at position 38 of 2315A when substituted with N provides greater than two fold increase in activity compared to the unmodified 2315A sequence. A position corresponding to D38 is identified in the new target ADH or MDH template sequence, e.g., it may be D38 or D39 appropriate, and replaced with N to generate the new non-naturally-occurring ADH or MDH.

In some cases an "ortholog" of the NAD(P)-dependent methanol dehydrogenase from

Bacillus methanolicus MGA3 (2315A), SEQ ID NO: 1, is first identified. 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 45% to 100% amino acid sequence identity, and more preferably about 60% to 100% amino acid sequence identity. Genes sharing a desired amount of identify (e.g., 45%, 50%, 55%, or 60% or greater) to the NAD(P)-dependent methanol dehydrogenase from Bacillus methanolicus MGA3 (2315 A), including orthologs and paralogs, 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.

Computational approaches to sequence alignment and generation of sequence identity include global alignments and local alignments. Global alignment uses global optimization to forces alignment to span the entire length of all query sequences. Local alignments, by contrast, identify regions of similarity within long sequences that are often widely divergent overall. For understanding the indentity of a target sequence to the Bacillus methanolicus MGA3 (2315 A) template a global alignment can be used. Optionally, amino terminal and/or carboxy-terminal sequences of the target sequence that share little or no identify with the template sequence can be excluded for a global alignment and generation of an identify score.

Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity. Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 45% 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%).

Pairwise global sequence alignment was carried out for each of the template polypeptides with SEQ ID No.1 (2315 A) as the reference. The alignment was performed using the Needleman- Wunsch algorithm (Needleman, S. & Wunsch, C. A general method applicable to the search for similarities in the amino acid sequence of two proteins J. Mol. Biol, 1970, 48, 443-453) implemented through the BALIGN tool (http://balign.sourceforge.net/). Default parameters were used for the alignment and BLOSUM62 was used as the scoring matrix.

Table 10 provides target polypeptides details and alignment to SEQ ID NO.l (2315 A). These sequences represent target sequences in which one or more amino acid variations, based on the variant amino acids in the Bacillus methanolicus MGA3 (2315A) variants showing increased conversion, can be made. For example, as a general matter, this process can involve steps of aligning the template 2315A sequence to a target sequence, such as any sequence listed in Table 10. Next a position of the amino acid substitution/variant (or set of substitutions) in the template 2315A sequence providing the increased conversion of methanol or ethanol is identified. The amino acid alignment at the substitution/variant position is inspected to identify what amino acid position in the target sequence corresponds to that of the template 2315A sequence. Preferred target sequences for substitution with the amino acid variants based on the 2315A variants are highlighted.

In some cases the original amino acid and its position on the template 2315A sequence will precisely correlate with the original amino acid and position on the target. In other cases the original amino acid and its position on the template 2315A sequence will correlate with the original amino acid, but its position on the target will not be in the corresponding template position.

However, the corresponding amino acid on the target can be a predetermined distance from the position on the template, such as within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid positions from the template position. In other cases the original amino acid on the template 2315A sequence will not precisely correlate with the original amino acid on the target. However one can understand what the corresponding amino acid on the target sequence is based on the general location of the amino acid on the template and the sequence of amino acids in the vicinity of the target amino acid. For example, amino acids in the vicinity of the target amino acids may be viewed as a "sequence motif having a certain amount of identity or similarity to between the template and target sequences.

In some cases, it can be useful to use the Basic Local Alignment Search Tool (BLAST) algorithm to understand the sequence identity between an amino acid motif in a template sequence and a target sequence. Therefore, in preferred modes of practice, BLAST is used to identify or understand the identity of a shorter stretch of amino acids (e.g. a sequence motif) between a template and a target protein. BLAST finds similar sequences using a heuristic method that approximates the Smith- Waterman algorithm by locating short matches between the two sequences. The (BLAST) algorithm can identify library sequences that resemble the query sequence above a certain threshold. 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.

In view of and following the teachings herein, using methods known in the art such as sequence alignment and 3D modeling, the "corresponding positions and amino acids" for substitution in template polypeptides other than SEQ ID NO: 1 are readily determined. Table 11 indicates for each template the corresponding positions for substitution to improve polypeptide activity, from which each original amino acid, its location and substitution is specifically contemplated as if expressly listed. For example, using the 385 amino acid template polypeptide of ELT83020.1,GI:387590701 (SEQ ID NO:7), from Bacillus methanolicus MGA3, having 61% global identity and 79% similarity to SEQ ID NO: 1, the formula R ^J XR ² is directly and

unambiguously derived, is evident, and is contemplated, as if expressly listed herein and is from which the group of positions for R ^J XR ² are readily envisioned as: D41, E63, P74, N91, S102, G106, A110, Ll l l, V112, K118, 1119, H120, G124, V125, D126, V127, S128, K129, E130, P131, M132, V134, S146, T149, T152, 1153, K160, V161, V166, D167, Q270, G273, K348, N358, A363, C364. This is readily derived as evident in the following that depicts the amino acid position for each amino acid for substitution (corresponding to those of SEQ ID NO: 1 and accepting the corresponding substitution). The above approach applies to obtain a resulting R ^J XR ² formula for each template polypeptide herein, as well as for polypeptides sharing identity thereto as described herein.

GI : MTNTQSAFFMPSVNLFGAGSVNEVGTRLADLGVKKALLVTD41AGLHGLGLSEKISSIIR AAG

387590701 VE63VSIFPKAEPNP74TDKNVAEGLEAYNAEN91CDSIVTLGGGSS102HDAG106KAI A11

0L111V112AANGGK118I119H120DYEG124V125D126V127S128K129E130P13 1M1 32V134PLIAINTTAGTGS146ELT149KFT152I153ITDTERK160V161KMAIV166 D16

7KHVTPTLSINDPELMVGMPPSLTAATGLDALTHAIEAYVSTGATPITDALAIQAIK IISKYL PRAVANGKDIEAREQMAFAQS LAGMAFNNAGLGYVHAIAHQ270 LGG273FYNFPHGVCNAVL LPYVCRFNLISKVERYAEIAAFLGENVDGLSTYDAAEKAIKAIERMAKDLNIPKGFKELG AK3 48EEDIETLAKN358AMKDA363C364ALTNPRKPKLEEVIQI IKNAM In another example, the amino acids and positions for substitution R ^J XR ² in 387 amino acid template polypeptide YP 002138168.1 GI: 197117741 (SEQ ID NO: 17) from Geobacter bemidjiensis Bern with 52% global identity and 71% similarity to SEQ ID NO: 1 are: D43, S65, P76, G92, S104, A108, G112, Ml 13, V114, H120, 1121, R122, G126, V127, N128, K129, T130, T131, K132, P133, M134, P135, S148, T151, C154, 1155, H162, V163, V168, D169, Q272, G275, K350, N360, A365, C366. The following, readily derivable from the tables herein, indicates these positions.

GI : MALGEQTYGFYIPTVSLMGIGSAKETGGQIKALGASKALIVTD43KGLSAMGVADKIKSQ VEE

197117741 AGVS65AVIFDGAEPNP76TDINVHDGVKVYQDNG92CDAI ISLGGGSS104HDCA108KGIG 112M113V114IGNGGH120I121R122DLEG126V127N128K129T130T131K132P 133 M134P135AFVAINTTAGTAS148EMT151RFC154I155ITNTDTH162V163KMAIV1 68D

169WRCTPNVAINDPLLMVGKPAALTAATGMDALTHAVEAYVSTIATPITDACAIKA IELIAE FLSKAVANGEDLEARDKMAYAEYLAGMAFNNAS LGYVHSMAHQ272LGG275FYNLPHGVCNA ILLPAVSQYNLIACPKRFADIAKALGENIDGLSVTEAGQKAIDRIRTLSASIGIPTGLKA LNV

K350EADLTIMAEN360AKKDA365C366QFTNPRKATLEQWQIFKDAM

Table 11 provides amino acid sequences of target polypeptides, having underlined target amino acids for substitution with the variant amino acids generated in the Bacillus methanolicus MGA3 (2315A) variants. It is understood that upon replacement of amino acid in the target sequence (with a variant amino acid from the corresponding location in the 2315A variant), the substituted target sequence can be considered a "template sequence," useful in some embodiments for the further screening of polypeptides sequences for substitution.

Table 11 also illustrates a consensus of the templates of 60% or better identity to SEQ ID NO: 1 with positions for substitution indicated by underlining. Non-underlined positions are not required for substitution and, in embodiments, remain constant (identical across all templates). These positions can be tolerant to change by selection from at least amongst the wild-type alternatives indicated at a specific position, and tolerant sites for substitution with the substitutions at the variant amino acid positions.

Site-directed mutagenesis or sequence alteration (e.g., site-specific mutagenesis or oligonucleotide-directed)can be used to make specific changes to a target alcohol dehydrogenase DNA sequence to provide a variant DNA sequence encoding alcohol dehydrogenase with the desired amino acid substitution. As a general matter, an oligonucleotide having a sequence that provides a codon encoding the variant amino acid is used. Alternatively, artificial gene sequence of the entire coding region of the variant alcohol dehydrogenase DNA sequence can be performed as preferred alcohol dehydrogenases targeted for substitution are generally less than 400 amino acids long.

Exemplary techniques using mutagenic oligonucleotides for generation of a variant ADH sequence include the Kunkel method which may utilize an ADH gene sequence placed into a phagemid. The phagemid in E. coli produces ADH ssDNA which is template for mutagenesis using an oligonucleotide which is primer extended on the template.

Depending on the restriction enzyme sites flanking a location of interest in the ADH DNA, cassette mutagenesis may be used to create a variant sequence of interest. For cassette mutagenesis, a DNA fragment is synthesized inserted into a plasmid, cleaved with a restriction enzyme, and and then subsequently ligated to a pair of complementary oligonucleotides containing the ADH variant mutation The restriction fragments of the plasmid and oligonucleotide can be ligated to one another. Another technique that can be used to generate the variant ADH sequence is PCR site directed mutagensis. Mutageneic oligonucleotide primers are used to introduce the desired mutation and to provide a PCR fragment carrying the mutated sequence. Additional

oligonucleotides may be used to extend the ends of the mutated fragment to provide restriction sites suitable for restriction enzyme digestion and insertion into the gene.

Commercial kits for site-directed mutagenesis techniques are also available. For example, the Quikchange™ kit uses complementary mutagenic primers to PCR amplify a gene region using a high-fidelity non-strand-displacing DNA polymerase such as pfu polymerase. The reaction generates a nicked, circular DNA which is relaxed. The template DNA is eliminated by enzymatic digestion with a restriction enzyme such as Dpnl which is specific for methylated DNA.

An expression vector or vectors can be constructed to include one or more variant ADH encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the microbial host organisms provided include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.

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

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

Exogenous variant ADH-encoding nucleic acid sequences can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. Optionally, for exogenous expression in E. co\i or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329- 4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties.

Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.

The terms "microbial," "microbial organism" or "microorganism" are intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical.

The term "isolated" when used in reference to a microbial organism is intended to mean an organism that is substantially free of at least one component as the referenced microbial organism is found in nature. The term includes a microbial organism that is removed from some or all components as it is found in its natural environment. The term also includes a microbial organism that is removed from some or all components as the microbial organism is found in non-naturally occurring environments.

In some aspects the ADH variant gene is introduced into a cell with a gene disruption. The term "gene disruption," or grammatical equivalents thereof, is intended to mean a genetic alteration that renders the encoded gene product inactive or attenuated. The genetic alteration can be, for example, deletion of the entire gene, deletion of a regulatory sequence required for transcription or translation, deletion of a portion of the gene which results in a truncated gene product, or by any of various mutation strategies that inactivate or attenuate the encoded gene product. One particularly useful method of gene disruption is complete gene deletion because it reduces or eliminates the occurrence of genetic reversions. The phenotypic effect of a gene disruption can be a null mutation, which can arise from many types of mutations including inactivating point mutations, entire gene deletions, and deletions of chromosomal segments or entire chromosomes. Specific antisense nucleic acid compounds and enzyme inhibitors, such as antibiotics, can also produce null mutant phenotype, therefore being equivalent to gene disruption.

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

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

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

A variety of microorganism may be suitable for the incorporating the variant ADH, optionally with one or more other transgenes Such organisms include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human. Exemplary species are reported in U.S. Application Serial Number 13/975,678 (filed August 26, 2013), which is incorporated herein by reference, and include, for example, Escherichia coli, Saccharomyces cerevisiae, Saccharomyces kluyveri, Candida boidinii, Clostridium kluyveri, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium difficile, Clostridium botulinum, Clostridium tyrobutyricum, Clostridium tetanomorphum, Clostridium tetani, Clostridium propionicum, Clostridium aminobutyricum, Clostridium subterminale, Clostridium sticklandii, Ralstonia eutropha, Mycobacterium bovis, Mycobacterium tuberculosis, Porphyromonas gingivalis, Arabidopsis thaliana, Thermus thermophilus, Pseudomonas species, including Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas fluorescens, Homo sapiens, Oryctolagus cuniculus, Rhodobacter spaeroides, Thermoanaerobacter brockii, Metallosphaera sedula, Leuconostoc mesenteroides, Chloroflexus aurantiacus, Roseiflexus castenholzii, Erythrobacter, Simmondsia chinensis, Acinetobacter species, including Acinetobacter calcoaceticus and Acinetobacter baylyi, Porphyromonas gingivalis, Sulfolobus tokodaii, Sulfolobus solfataricus, Sulfolobus acidocaldarius, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus brevis, Bacillus pumilus, Rattus norvegicus, Klebsiella pneumonia, Klebsiella oxytoca, Euglena gracilis, Treponema denticola, Moorella thermoacetica, Thermotoga maritima, Halobacterium salinarum, Geobacillus stearothermophilus, Aeropyrum pernix, Sus scrofa, Caenorhabditis elegans, Corynebacterium glutamicum, Acidaminococcus fermentans, Lactococcus lactis, Lactobacillus plantarum,

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

Streptomyces griseus, Schizosaccharomyces pombe, Geobacillus thermoglucosidasius, Salmonella typhimurium, Vibrio cholera, Heliobacter pylori, Nicotiana tabacum, Oryza sativa, Haloferax mediterranei, Agrobacterium tumefaciens, Achromobacter denitrificans, Fusobacterium nucleatum, Streptomyces clavuligenus, Acinetobacter baumanii, Mus musculus, Lachancea kluyveri, Trichomonas vaginalis, Trypanosoma brucei, Pseudomonas stutzeri, Bradyrhizobium japonicum, Mesorhizobium loti, Bos taunts, Nicotiana glutinosa, Vibrio vulnificus, Selenomonas ruminantium, Vibrio parahaemolyticus, Archaeoglobus fulgidus, Haloarcula marismortui, Pyrobaculum aerophilum, Mycobacterium smegmatis MC2 155, Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium marinum M, Tsukamurella paurometabola DSM 20162, Cyanobium PCC7001, Dictyostelium discoideum AX4, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes.

In certain embodiments, suitable organisms include Acinetobacter baumannii Naval-82, Acinetobacter sp. ADP1, Acinetobacter sp. strain M-l, Actinobacillus succinogenes 130Z, Allochromatium vinosum DSM 180, Amycolatopsis methanolica, Arabidopsis thaliana, Atopobium parvulum DSM 20469, Azotobacter vinelandii DJ, Bacillus alcalophilus ATCC 27647, Bacillus azotoformans LMG 9581, Bacillus coagulans 36D1, Bacillus megaterium, Bacillus methanolicus MGA3, Bacillus methanolicus PBl, Bacillus methanolicus PB-1, Bacillus selenitireducens MLS 10 , Bacillus smithii, Bacillus subtilis , Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia multivorans, Burkholderia pyrrocinia, Burkholderia stabilis, Burkholderia thailandensis E264, Burkholderiales bacterium Joshi_001, Butyrate-producing bacterium L2-50, Campylobacter jejuni, Candida albicans, Candida boidinii, Candida methylica, Carboxydothermus hydrogenoformans, Carboxydothermus hydrogenoformans Z-2901, Caulobacter sp. AP07, Chloroflexus aggregans DSM9485, Chloroflexus aurantiacus J-10-fl, Citrobacter freundii, Citrobacter koseri ATCC BAA-895, Citrobacter youngae , Clostridium, Clostridium

acetobutylicum, Clostridium acetobutylicum ATCC 824, Clostridium acidurici, Clostridium aminobutyricum, Clostridium asparagiforme DSM 15981, Clostridium beijerinckii , Clostridium beijerinckii NCIMB 8052, Clostridium bolteae ATCC BAA-613, Clostridium carboxidivorans P7, Clostridium cellulovorans 743B, Clostridium difficile, Clostridium hiranonis DSM 13275, Clostridium hylemonae DSM 15053, Clostridium kluyveri, Clostridium kluyveri DSM 555, Clostridium ljungdahli, Clostridium ljungdahlii DSM 13528, Clostridium methylpentosum DSM 5476 , Clostridium pasteurianum, Clostridium pasteurianum DSM 525, Clostridium perfringens, Clostridium perfringens ATCC 13124, Clostridium perfringens str. 13, Clostridium

phytofermentans ISDg, Clostridium saccharobutylicum, Clostridium saccharoperbutylacetonicum, Clostridium saccharoperbutylacetonicum Nl-4, Clostridium tetani, Corynebacterium glutamicum ATCC 14067, Corynebacterium glutamicum R, Corynebacterium sp. U-96, Corynebacterium variabile, Cupriavidus necator N-1, Cyanobium PCC7001, Desulfatibacillum alkenivorans AK-01, Desulfitobacterium hafniense, Desulfitobacterium metallireducens DSM 15288, Desulfotomaculum reducens MI-1, Desulfovibrio africanus str. Walvis Bay, Desulfovibrio fructosovorans JJ, Desulfovibrio vulgaris str. Hildenborough, Desulfovibrio vulgaris str. Miyazaki F Dictyostelium discoideum AX4, Escherichia coli, Escherichia coli K-12 , Escherichia coli K-12 MG1655, Eubacterium hallii DSM 3353 , Flavobacterium frigoris, Fusobacterium nucleatum subsp.

polymorphum ATCC 10953 , Geobacillus sp. Y4.1MC1, Geobacillus themodenitrificans NG80-2, Geobacter bemidjiensis Bern, Geobacter sulfurreducens, Geobacter sulfurreducens PCA, Geobacillus stearothermophilus DSM 2334, Haemophilus influenzae, Helicobacter pylori, Homo sapiens, Hydrogenobacter thermophilus, Hydrogenobacter thermophilus TK-6, Hyphomicrobium denitrificans ATCC 51888, Hyphomicrobium zavarzinii, Klebsiella pneumoniae, Klebsiella pneumoniae subsp. pneumoniae MGH 78578, Lactobacillus brevis ATCC 367, Leuconostoc mesenteroides, Lysinibacillus fusiformis, Lysinibacillus sphaericus, Mesorhizobium loti

MAFF303099, Metallosphaera sedula, Methanosarcina acetivorans, Methanosarcina acetivorans C2A, Methanosarcina barkeri, Methanosarcina mazei TucOl, Methylobacter marinus,

Methylobacterium extorquens, Methylobacterium extorquens AMI, Methylococcus capsulatas, Methylomonas aminofaciens, Moorella thermoacetica, Mycobacter sp. strain JC1 DSM 3803, Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium bovis BCG, Mycobacterium gastri , Mycobacterium marinum M, Mycobacterium smegmatis, Mycobacterium smegmatis MC2 155, Mycobacterium tuberculosis, Nitrosopumilus salaria BD31, Nitrososphaera gargensis Ga9.2, Nocardia farcinica IFM 10152, Nocardia iowensis (sp. NRRL 5646), Nostoc sp. PCC 7120,

Ogataea angusta, Ogataea parapolymorpha DL-1 (Hansenula polymorpha DL-1), Paenibacillus peoriae KCTC 3763, Paracoccus denitrificans, Penicillium chrysogenum, Photobacterium profundum 3TCK, Phytofermentans ISDg, Pichia pastoris, Picrophilus torridus DSM9790, Porphyromonas gingivalis, Porphyromonas gingivalis W83, Pseudomonas aeruginosa PA01, Pseudomonas denitrificans, Pseudomonas knackmussii, Pseudomonas putida, Pseudomonas sp, Pseudomonas syringae pv. syringae B728a, Pyrobaculum islandicum DSM 4184, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii OT3, Ralstonia eutropha, Ralstonia eutropha HI 6, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodobacter sphaeroides ATCC 17025, Rhodopseudomonas palustris, Rhodopseudomonas palustris CGA009, Rhodopseudomonas palustris DX-1, Rhodospirillum rubrum, Rhodospirillum rubrum ATCC 11170, Ruminococcus obeum ATCC 29174, Saccharomyces cerevisiae, Saccharomyces cerevisiae S288c, Salmonella enterica, Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, Salmonella enterica typhimurium , Salmonella typhimurium, Schizosaccharomyces pombe, Sebaldella termitidis ATCC 33386 , Shewanella oneidensis MR-1, Sinorhizobium meliloti 1021, Streptomyces coelicolor, Streptomyces griseus subsp. griseus NBRC 13350, Sulfolobus acidocalarius, Sulfolobus solfataricus P-2, Synechocystis str. PCC 6803, Syntrophobacter fumaroxidans, Thauera aromatica, Thermoanaerobacter sp. X514, Thermococcus kodakaraensis, Thermococcus litoralis, Thermoplasma acidophilum, Thermoproteus neutrophilus, Thermotoga maritima, Thiocapsa roseopersicina, Tolumonas auensis DSM 9187, Trichomonas vaginalis G3, Trypanosoma brucei, Tsukamurella paurometabola DSM 20162, Vibrio cholera, Vibrio harveyi ATCC BAA-1116, Xanthobacter autotrophicus Py2, Yersinia intermedia, or Zea mays.

In some aspects the variant ADH gene is introduced into a cell engineered with increased of levels of 1 ,4-butanediol (BDO) or hydroxylbutyrate (4-HB) biosynthetic capability, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and/or nonorthologous gene displacements are present in the referenced microorganism that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes.

With the complete genome sequence available for now more than 550 species (with more than half of these available on public databases such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes, the identification of genes encoding the requisite BDO or 4-HB biosynthetic pathway as well as other known biosynthetic pathways for 1,3-butanediol (13BDO), butadiene, 6-amino caproic acid (6ACA),

hexamethyldiamine (HMD A), adipic acid or derivatives thereof, croytl alcohol, methyl vinyl carbinol, 3-buten-l-ol, succinic acid or derivatives thereof, n-propanol, isopropanol, propylene, methacrylic acid or derivatives thereof, methanol metabolic and/or formaldehyde assimilation activity for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations allowing biosynthesis of various target products including 1,3 -butanediol (13BDO), 1, 4-butanediol (BDO), 4-HB, butadiene, 6-amino caproic acid (6ACA),

hexamethyldiamine (HMD A), adipic acid or derivatives thereof, croytl alcohol, methyl vinyl carbinol, 3-buten-l-ol, succinic acid or derivatives thereof, n-propanol, isopropanol, propylene, methacrylic acid or derivatives thereof, metabolism of methanol and/or assimilation of formaldehyde described herein with reference to a particular organism such as E. coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.

Therefore, the engineered cell including the non-natural NAD ⁺ -dependent alcohol dehydrogenase, can include one or more genetic alterations, such as inserted transgenes, deletions, attenuation, mutations, etc., desired to increase levels of one or more intermediates or a product thereof, and include those genetic modifications as described in U.S. Application Serial Number 13/975,678 (filed August 26, 2013), which is incorporated herein by reference.

Exemplary alcohol metabolic pathway gene(s), such as described in U.S. Application Serial Number 13/975,678, encode a protein selected from the group consisting of a), a formate dehydrogenase (EM8), a formaldehyde activating enzyme (EM10), a formaldehyde dehydrogenase (EMU), a S-(hydroxymethyl)glutathione synthase (EM12), a glutathione-dependent formaldehyde dehydrogenase (EMI 3), a S-formylglutathione hydrolase (EM 14), a formate hydrogen lyase (EM15), and a hydrogenase (EM16), any or more can be coexpressed with the non-natural NAD ⁺ - dependent alcohol dehydrogenase in the engineered cell.

Other exemplary alcohol metabolic pathway gene(s), such as described in U.S. Application

Serial Number 13/975,678, encode an alcohol metabolic pathway gene(s) encoding a protein selected from the group consisting of a succinyl-CoA reductase (aldehyde forming) (EB3), a 4- hydroxybutyrate (4-HB) dehydrogenase (EB4), a 4-HB kinase (EB5), a phosphotrans-4- hydroxybutyrylase (EB6), a 4-hydroxybutyryl-CoA reductase (aldehyde forming) (EB7), a 1,4- butanediol dehydrogenase (EB8); a succinate reductase (EB9), a succinyl-CoA reductase (alcohol forming) (EB10), 4-hydroxybutyryl-CoA transferase (EB11), a 4-hydroxybutyryl-CoA synthetase (EB12), a 4-HB reductase (EB13), and a 4-hydroxybutyryl-CoA reductase (alcohol forming) (EB15), a succinyl-CoA transferase (EB1), and a succinyl-CoA synthetase (EB2A), any or more can be coexpressed with the non-natural NAD ⁺ -dependent alcohol dehydrogenase in the engineered cell. Target products obtained from, and product pathways suitable for producing in, host cells expressing the engineered NAD+-dependent methanol or ethanol dehydrogenases described herein include the following. Of particular interest are a target product obtained using pyruvate and acetyl-CoA as entry point or precursor to its product pathway(s), in part because the methanol metabolic pathway using the novel enzymes enables fixing the carbon of methanol into pathways to pyruvate and acetyl-CoA. Target products include (a) 1 ,4-butanediol and intermediates thereto, such as 4-hydroxybutanoic acid (4-hydroxybutanoate, 4-hydroxybutyrate, 4-HB), (b) butadiene and intermediates thereto, such as 1,4-butanediol, 1,3-butanediol, crotyl alcohol, 3-buten-2-ol (methyl vinyl carbinol) and 3-buten-l-ol, (c) 1,3-butanediol and intermediates thereto, such as 2,4- pentadienoate, crotyl alcohol or 3-buten-l-ol, (d) adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine and levulinic acid and their intermediates, e.g. 4-aminobutyryl-CoA, (e) methacrylic acid (2-methyl-2-propenoic acid) and its esters known collectively as methacrylates, such as methyl methacrylate, methyl methacrylate, 3-hydroxyisobutyrate and/or 2- hydroxyisobutyrate and their intermediates, (f) 1,2-propanediol (propylene glycol), n-propanol, 1,3-propanediol and glycerol, and their intermediates and (g) succinic acid and intermediates thereto.

1 ,4-butanediol and intermediates thereto, such as 4-hydroxybutanoic acid (4- hydroxybutanoate, 4-hydroxybutyrate, 4-HB), are target products that can be made by co- expressing the novel alcohol dehydrogenases described herein with a product pathway described herein as well as in the following documents. Suitable product pathways and enzymes, methods for screening and methods for isolating are found herein as well as in the following documents, incorporated herein by reference: WO2008115840A2 published 25 September 2008 entitled Compositions and Methods for the Biosynthesis of 1, 4-Butanediol and Its Precursors;

WO2010141780A1 published 9 December 2010 entitled Process of Separating Components of A Fermentation Broth ; WO2010141920A2 published 9 December 2010 entitled Microorganisms for the Production of 1, 4-Butanediol and Related Methods; WO2010030711A2 published 18 March 2010 entitled Microorganisms for the Production of 1, 4-Butanediol ; WO2010071697A1 published 24 June 2010 Microorganisms and Methods for Conversion of Syngas and Other Carbon Sources to Useful Products; WO2009094485A1 published 30 July 2009 Methods and Organisms for Utilizing Synthesis Gas or Other Gaseous Carbon Sources and Methanol; WO2009023493A1 published 19 February 2009 entitled Methods and Organisms for the Growth-Coupled Production of 1, 4-Butanediol; WO2008115840A2 published 25 September 2008 entitled Compositions and Methods for the Biosynthesis of 1, 4-Butanediol and Its Precursors; and International Application No. PCT/US 13/56725 filed 27 August 2013 entitled Microorganisms an Methods for Enhancing the Availability of Reducing Equivalents in the Presence of Methanol, and for Producing 1,4- Butanediol Related Thereto. Butadiene and intermediates thereto, such as 1 ,4-butanediol, 1,3-butanediol, crotyl alcohol, 3-buten-2-ol (methyl vinyl carbinol) and 3-buten-l-ol, are target products that can be made by co- expressing the novel alcohol dehydrogenases described herein with a product pathway described in the following documents. In addition to direct fermentation to produce butadiene, 1,3-butanediol, 1 ,4-butanediol, crotyl alcohol, 3-buten-2-ol (methyl vinyl carbinol) and 3-buten-l -ol can be separated, purified (for any use), and then dehydrated to butadiene in a second step involving metal-based catalysis. Suitable product pathways and enzymes, methods for screening and methods for isolating are found in the following documents, incorporated herein by reference: WO2011140171A2 published 10 November 201 lentitled Microorganisms and Methods for the Biosynthesis of Butadiene; WO2012018624A2 published 9 February 2012 entitled

Microorganisms and Methods for the Biosynthesis of Aromatics, 2,4-Pentadienoate and 1,3- Butadiene; WO2011140171A2 published 10 November 2011 entitled Microorganisms and Methods for the Biosynthesis of Butadiene; WO2013040383A1 published 21 March 2013 entitled Microorganisms and Methods for Producing Alkenes; WO2012177710A1 published 27 December 2012 entitled Microorganisms for Producing Butadiene and Methods Related thereto;

WO2012106516A1 published 9 August 2012 entitled Microorganisms and Methods for the Biosynthesis of Butadiene; WO2013028519A1 published 28 February 2013 entitled

Microorganisms and Methods for Producing 2,4-Pentadienoate, Butadiene, Propylene, 1,3- Butanediol and Related Alcohols; and United States Serial No. 61/799255 filed 15 March 2013.

1,3-butanediol and intermediates thereto, such as 2,4-pentadienoate, crotyl alcohol or 3- buten-l-ol, are target products that can be made by co-expressing the novel alcohol

dehydrogenases described herein with a product pathway described herein as well as in the following documents. Suitable product pathways and enzymes, methods for screening and methods for isolating are found herein as well as in the following documents, incorporated herein by reference: WO2011071682A1 published 16 June 2011 entitled Methods and Organisms for

Converting Synthesis Gas or Other Gaseous Carbon Sources and Methanol to 1, 3-Butanediol; WO2011031897A published 17 March 2011 entitled Microorganisms and Methods for the Co- Production of Isopropanol with Primary Alcohols, Diols and Acids; WO2010127319A2 published 4 November 2010 entitled Organisms for the Production of 1,3-Butanediol; WO2013071226A1 published 16 May 2013 entitled Eukaryotic Organisms and Methods for Increasing the Availability of Cytosolic Acetyl-CoA, and for Producing 1,3-Butanediol; WO2013028519A1 published 28 February 2013 entitled Microorganisms and Methods for Producing 2,4-Pentadienoate, Butadiene, Propylene, 1,3-Butanediol and Related Alcohols; WO2013036764A1 published 14 March 2013 entitled Eukaryotic Organisms and Methods for Producing 1,3-Butanediol; WO2013012975A1 published 24 January 2013 entitled Methods for Increasing Product Yields; WO2012177619A2 published 27 December 2012 entitled Microorganisms for Producing 1, 3-Butanediol and Methods Related Thereto; and United States Serial No. 61/799255 filed 15 March 2013..

Adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine and levulinic acid, and their intermediates, e.g. 4-aminobutyryl-CoA, are target products, useful for example for making nylon polymers, that can be made by co-expressing the novel alcohol dehydrogenases described herein with a product pathway described herein as well as in the following documents. Suitable product pathways and enzymes, methods for screening and methods for isolating are found herein as well as in the following documents, incorporated herein by reference: WO2010129936A1 published 11 November 2010 entitled Microorganisms and Methods for the Biosynthesis of Adipate, Hexamethylenediamine and 6-Aminocaproic Acid; WO2013012975A1 published 24 January 2013 entitled Methods for Increasing Product Yields; WO2012177721A1 published 27 December 2012 entitled Microorganisms for Producing 6-Aminocaproic Acid; WO2012099621A1 published 26 July 2012 entitled Methods for Increasing Product Yields; and application U.S. Serial No. 61/766,620 filed 19 February 2013 entitled Microorganisms an Methods for Enhancing the Availability of Reducing Equivalents in the Presence of Methanol, and for Producing Adipate, 6- Aminocaproate, Hexamethylenediamine or Caprolactam Related Thereto.

Methacrylic acid (2-methyl-2-propenoic acid; used in the preparation of its esters known collectively as methacrylates, such as methyl methacrylate, which is used most notably in the manufacture of polymers), methacrylate ester such as methyl methacrylate, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate and their intermediates are target products, useful for example for making polymers, that can be made by co-expressing the novel alcohol dehydrogenases described herein with a product pathway described herein as well as in the following documents. Suitable product pathways and enzymes, methods for screening and methods for isolating are found herein as well as in the following documents, incorporated herein by reference: WO2012135789A2 published 4 October 2012 entitled Microorganisms for Producing Methacrylic Acid and

Methacrylate Esters and Methods Related Thereto; WO2009135074A2 published 5 November 2009 entitled Microorganisms for the Production of Methacrylic Acid; and application U.S. Serial No. 61/766,660 filed 19 February 2013 entitled Microorganisms an Methods for Enhancing the Availability of Reducing Equivalents in the Presence of Methanol, and for Producing 3- Hydroxyisobutyate or Methacrylic Acid Related Thereto.

1 ,2-propanediol (propylene glycol), n-propanol, 1,3-propanediol and glycerol, and their intermediates are target products, useful for example for making polymers, that can be made by co- expressing the novel alcohol dehydrogenases described herein with a product pathway described herein as well as in the following documents. Suitable product pathways and enzymes, methods for screening and methods for isolating are found herein as well as in the following documents, incorporated herein by reference: WO2009111672A1 published 9 November 2009 entitled Primary Alcohol Producing Organisms; WO2011031897A1 17 March 2011 entitled

Microorganisms and Methods for the Co-Production of Isopropanol with Primary Alcohols, Diols and Acids; WO2012177599A2 published 27 December 2012 entitled Microorganisms for Producing N-Propanol 1, 3-Propanediol, 1, 2-Propanediol or Glycerol and Methods Related Thereto; and application U.S. Serial No. 61/766,635 filed 19 February 2013

entitled Microorganisms an Methods for Enhancing the Availability of Reducing Equivalents in the Presence of Methanol, and for Producing 1 ,2-Propanediol, n-Propanol, 1,3 -Propanediol, or Glycerol Related Thereto.

Succinic acid and intermediates thereto (useful to produce products including polymers, e.g. PBS, 1,4-butanediol, tetrahydrofuran, pyrrolidone, solvents, paints, deicers, plastics, fuel additives, fabrics, carpets, pigments, and detergents) are target products that can be made by co- expressing the novel alcohol dehydrogenases described herein with a product pathway described herein as well as in the following documents. Suitable product pathways and enzymes, methods for screening and methods for isolating are found herein as well as in the following documents, incorporated herein by reference: EP1937821A2 published 2 July 2008 entitled Methods and Organisms for the Growth-Coupled Production of Succinate; and application U.S. Serial No. 61/766,635 filed 19 February 2013 entitled Microorganisms and Methods for Enhancing the Availability of Reducing Equivalents in the Presence of Methanol, and for Producing Succinate Related Thereto.

Target products obtained from, and product pathways suitable for producing in, host cells co-expressing the engineered NAD+-dependent methanol or ethanol dehydrogenases described herein include the following.

Butadiene and intermediates thereto, such as 1,4-butanediol, 1,3-butanediol, crotyl alcohol, 3-buten-2-ol (methyl vinyl carbinol) and 3-buten-l-ol, are target products that can be made by co- expressing the novel alcohol dehydrogenases described herein with a product pathway described in the following documents. In addition to direct fermentation to produce butadiene, 1,3-butanediol, 1,4-butanediol, crotyl alcohol, 3-buten-2-ol (methyl vinyl carbinol) and 3-buten-l -ol can be separated, purified (for any use), and then dehydrated to butadiene in a second step involving metal-based catalysis. Suitable product pathways and enzymes, methods for screening and methods for isolating are found in: WO2011140171A2 published 10 November 201 lentitled

Microorganisms and Methods for the Biosynthesis of Butadiene; WO2012018624 A2 published 9 February 2012 entitled Microorganisms and Methods for the Biosynthesis of Aromatics, 2, 4- Pentadienoate and 1, 3 -Butadiene; O2011140171 A2 published 10 November 2011 entitled Microorganisms and Methods for the Biosynthesis of Butadiene; WO2013040383A1 published 21 March 2013 entitled Microorganisms and Methods for Producing Alkenes; WO2012177710A1 published 27 December 2012 entitled Microorganisms for Producing Butadiene and Methods Related thereto; WO2012106516A1 published 9 August 2012 entitled Microorganisms and Methods for the Biosynthesis of Butadiene; WO2013028519A1 published 28 February 2013 entitled Microorganisms and Methods for Producing 2,4-Pentadienoate, Butadiene, Propylene, 1,3- Butanediol and Related Alcohols; and United States Serial No. 61/799255 filed 15 March 2013.

In some embodiments, the disclosure provides organisms comprising a MDH variant and that are engineered to improve the availability of reducing equivalents or utilizing formaldehyde resulting from methanol via a formaldehyde assimilation pathway (FAB), which can be used for the production of target product molecules. It will be recognized by one skilled in the art that any product molecule that utilizes reducing equivalents in its production can exhibit enhanced production through other biosynthetic pathways.

BDO is a valuable chemical for the production of high performance polymers, solvents, and fine chemicals. It is the basis for producing other high value chemicals such as tetrahydrofuran (THF) and gamma-butyrolactone (GBL). The value chain is comprised of three main segments including: (1) polymers, (2) THF derivatives, and (3) GBL derivatives. In the case of polymers, BDO is a comonomer for polybutylene terephthalate (PBT) production. PBT is a medium performance engineering thermoplastic used in automotive, electrical, water systems, and small appliance applications. Conversion to THF, and subsequently to polytetramethylene ether glycol (PTMEG), provides an intermediate used to manufacture spandex products such as LYCRA ^® fibers. PTMEG is also combined with BDO in the production of specialty polyester ethers (COPE). COPEs are high modulus elastomers with excellent mechanical properties and oil/environmental resistance, allowing them to operate at high and low temperature extremes. PTMEG and BDO also make thermoplastic polyurethanes processed on standard thermoplastic extrusion, calendaring, and molding equipment, and are characterized by their outstanding toughness and abrasion resistance. The GBL produced from BDO provides the feedstock for making pyrrolidones, as well as serving the agrochemical market. The pyrrolidones are used as high performance solvents for extraction processes of increasing use, including for example, in the electronics industry and in pharmaceutical production. Accordingly, provided herein is bioderived BDO produced according to the methods described herein and biobased products comprising or obtained using the bioderived BDO.

In numerous engineered pathways, realization of maximum product yields based on carbohydrate feedstock is hampered by insufficient reducing equivalents or by loss of reducing equivalents to byproducts. Methanol is a relatively inexpensive organic feedstock that can be used to generate reducing equivalents by employing one or more methanol metabolic enzymes as shown in FIG. 3a. The reducing equivalents produced by the metabolism of methanol can then be used to power the glucose to BDO production pathways, for example, as shown in FIG. 2. IN FIG. 2, the organism comprises at least one exogenous nucleic acid encoding a BDOPE expressed in a sufficient amount to produce BDO. In certain embodiments, the BDOPE is selected from the group consisting of a succinyl-CoA transferase (EBl) or a succinyl-CoA synthetase (EB2A) (or succinyl-CoA ligase); a succinyl-CoA reductase (aldehyde forming) (EB3); a 4-hydroxybutyrate (4-HB) dehydrogenase (EB4); a 4-HB kinase (EB5); a

phosphotrans-4-hydroxybutyrylase (EB6); a 4-hydroxybutyryl-CoA reductase (aldehyde forming) (EB7); a 1,4-butanediol dehydrogenase (EB8); a succinate reductase (EB9); a succinyl-CoA reductase (alcohol forming) (EB10); a 4-hydroxybutyryl-CoA transferase (EBl 1) or a 4- hydroxybutyryl-CoA synthetase (EB12); a 4-HB reductase (EB13); a 4-hydroxybutyryl-phosphate reductase (EB14); and a 4-hydroxybutyryl-CoA reductase (alcohol forming) (EB15).

Enzymes, genes and methods for engineering pathways from succinate and succinyl-CoA to various products, such as BDO, into a microorganism, are now known in the art (see, e.g., U.S. Publ. No. 2011/0201089). A set of BDOPEs represents a group of enzymes that can convert succinate to BDO as shown in FIG. 2. The additional reducing equivalents obtained from the MDH pathway, as disclosed herein, improve the yields of all these products when utilizing carbohydrate-based feedstock. For example, BDO can be produced from succinyl-CoA via previously disclosed pathways (see for example, Burk et al., WO 2008/115840). Exemplary enzymes for the conversion succinyl-CoA to BDO include EB3 (FIG. 2, Step B), EB4 (FIG. 2, Step C), EB5 (FIG. 2, Step D), EB6 (FIG. 2, Step E), EB7 (FIG. 2, Step F), EB8 (FIG. 2, Step G), EB10 (FIG. 1, Step I), EBl 1 (FIG. 2, Step J), EB12 (FIG. 2, Step J), EB14 (FIG. 2, Step L), EB13 (FIG. 2, Step K), and EBl 5 (FIG. 2, Step M). EB9 (FIG. 2, Step H) can be additionally useful in converting succinate directly to the BDOP intermediate, succinate semialdehyde.

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

1 C ₆ H ₁₂ 0 ₆ → 1.09 C ₄ H ₁₀ O ₂ + 1.64 C0 ₂ + 0.55 H ₂ 0

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

10 CH ₃ OH + 3 C ₆ H ₁₂ 0 ₆ = 6 C ₄ H ₁₀ O ₂ + 8 H ₂ 0 + 4 C0 ₂

In a similar manner, the maximum theoretical yields of succinate and 4-HB can reach 2 mol/mol glucose using the reactions shown in FIGS. 1 and 2.

C ₆ H ₁₂ 0 ₆ + 0.667 CH ₃ OH + 1.333 C0 ₂ → 2 C ₄ H ₆ 0 ₄ + 1.333 H ₂ 0 C ₆ H ₁₂ 0 ₆ + 2 CH ₃ OH→ 2 C ₄ H ₈ 0 ₃ + 2 H ₂ 0

In other embodiments, the organism having a MDH protein, either alone or in combination with a BDOP, as provided herein, may further comprises a formaldehyde assimilation pathway (FAP) that utilizes formaldehyde, e.g., obtained from the oxidation of methanol, in the formation of intermediates of certain central metabolic pathways that can be used, for example, in the formation of biomass. In certain embodiments, the organism further comprises a FAP, wherein said organism comprises at least one exogenous nucleic acid encoding a formaldehyde assimilation pathway enzyme (FAPE) expressed in a sufficient amount to produce an intermediate of glycolysis and/or a metabolic pathway that can be used in the formation of biomass. In one embodiment, the FAPE is expressed in a sufficient amount to produce an intermediate of glycolysis. In another embodiment, the FAPE is expressed in a sufficient amount to produce an intermediate of a metabolic pathway that can be used in the formation of biomass. In some of the embodiments, the FAP comprises a hexulose-6-phosphate (H6P) synthase (EF1), a 6-phospho-3-hexuloisomerase (EF2), a

dihydroxyacetone (DHA) synthase (EF3) or a DHA kinase (EF4). In one embodiment, the FAP comprises an EF1 and an EF2. In one embodiment, the intermediate is a H6P, a fructoses- phosphate (F6P), or a combination thereof. In other embodiments, the FAP comprises an EF3 or an EF4. In one embodiment, the intermediate is a DHA, a DHA phosphate, or a combination thereof. In certain embodiments, the organism comprises two exogenous nucleic acids, each encoding a FAPE.

Also provided herein are exemplary pathways, which utilize formaldehyde produced from the oxidation of methanol (e.g., as provided in FIG. 3A, step J) in the formation of intermediates of certain central metabolic pathways that can be used for the formation of biomass. One exemplary FAP that can utilize formaldehyde produced from the oxidation of methanol (e.g., as provided in FIG. 3 A) is shown in FIG. 3b, which involves condensation of formaldehyde and D-ribulose-5- phosphate to form H6P by EF1 (FIG. 3b, step A). The enzyme can use Mg ²⁺ or Mn ²⁺ for maximal activity, although other metal ions are useful, and even non-metal-ion-dependent mechanisms are contemplated. H6P is converted into F6P by EF2 (FIG. 3b, step B). Another exemplary pathway that involves the detoxification and assimilation of formaldehyde produced from the oxidation of methanol (e.g., as provided in FIG. 3a) is shown in FIG. 3c and proceeds through DHA. EF3 is a special transketolase that first transfers a glycoaldehyde group from xylulose-5-phosphate to formaldehyde, resulting in the formation of DHA and G3P, which is an intermediate in glycolysis (FIG. 3c, step A). The DHA obtained from DHA synthase is then further phosphorylated to form DHA phosphate by a DHA kinase (FIG. 3c, step B). DHAP can be assimilated into glycolysis and several other pathways. Rather than converting formaldehyde to formate and on to CO ₂ off-gassed, the pathways provided in FIGS 3b and 3c show that carbon is assimilated, going into the final product. Thus, in one embodiment, an organism having a MDH protein, either alone or in combination with a BDOP, as provided herein, further comprises a FAP that utilizes formaldehyde, e.g., obtained from the oxidation of methanol, in the formation of intermediates of certain central metabolic pathways that can be used, for example, in the formation of biomass. In some embodiments, the FAP comprises 3 A or 3B, wherein 3 A is an EF1 and 3B is an EF2 In other embodiments, the FAP comprises 4A or 4B, wherein 4A is an EF3 and 4B is an EF4.In certain embodiments, provided herein is a organism having a MDH protein, wherein said organism comprises at least one exogenous nucleic acid encoding an EM9 expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol and/or expressed in a sufficient amount to convert methanol to formaldehyde. In some embodiments, the organism comprises at least one exogenous nucleic acid encoding an EM9 expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol. In other embodiments, the organism comprises at least one exogenous nucleic acid encoding an EM9 expressed in a sufficient amount to convert methanol to formaldehyde. In some embodiments, the microbial organism further comprises a FAP. In certain embodiments, the organism further comprises at least one exogenous nucleic acid encoding a FAPE expressed in a sufficient amount to produce an intermediate of glycolysis. In certain embodiments, the FAPE is selected from the group consisting of an EF1, an EF2, an EF3 and an EF4.

Exemplary enzymes suitable for the reactions described herein to metabolize methanol for either or both reducing equivalents or carbon include the following, with respect to Figure 3A, particualry as regards to Steps J, L, I, G, H, M, N and O.

FIG. 3, Step G - Formate Hydrogen Lyase (EM15)

An EM 15 enzyme can be employed to convert formate to carbon dioxide and hydrogen. An exemplary EM15 enzyme can be found in Escherichia coli. The E. coli EM15 consists of hydrogenase 3 and formate dehydrogenase-H (Maeda et al., Appl Microbiol Biotechnol 77:879-890

(2007) ). It is activated by the gene product oifhlA. (Maeda et al., Appl Microbiol Biotechnol 77:879-890 (2007)). The addition of the trace elements, selenium, nickel and molybdenum, to a fermentation broth has been shown to enhance EM15 activity (Soini et al., Microb.Cell Fact. 7:26

(2008) ). Various hydrogenase 3, EM8 and transcriptional activator genes are shown below.

Protein GenBank ID GI number Organism

hycA NP 417205 16130632 Escherichia coli K-12 MG1655 hycB NP 417204 16130631 Escherichia coli K-12 MG1655 hycC NP 417203 16130630 Escherichia coli K-12 MG1655 hycD NP 417202 16130629 Escherichia coli K-12 MG1655 hycE NP 417201 16130628 Escherichia coli K-12 MG1655 hycF NP 417200 16130627 Escherichia coli K-12 MG1655 hycG NP 417199 16130626 Escherichia coli K-12 MG1655 hycH NP 417198 16130625 Escherichia coli K-12 MG1655 hycl NP 417197 16130624 Escherichia coli K-12 MG1655 fdhF NP 418503 16131905 Escherichia coli K-12 MG1655

fhlA NP 417211 16130638 Escherichia coli K-12 MG1655

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

Additional EM 15 systems have been found in Salmonella typhimurium, Klebsiella pneumoniae, Rhodospirillum rubrum, Methanobacterium formicicum (Vardar-Schara et al., Microbial Biotechnology 1 : 107-125 (2008)).

FIG. 3, Step H - Hydrogenase (EM16)

Hydrogenase enzymes can convert hydrogen gas to protons and transfer electrons to acceptors such as ferredoxins, NAD+, or NADP+. Ralstonia eutropha HI 6 uses hydrogen as an energy source with oxygen as a terminal electron acceptor. Its membrane-bound uptake [NiFe]- hydrogenase is an "02-tolerant" EM16 (Cracknell, et al. Proc Nat Acad Sci, 106(49) 20681-20686 (2009)) that is periplasmically-oriented and connected to the respiratory chain via a b-type cytochrome (Schink and Schlegel, Biochim. Biophys. Acta, 567, 315-324 (1979); Bernhard et al., Eur. J. Biochem. 248, 179-186 (1997)). R. eutropha also contains an 0 ₂ -tolerant soluble EM16 encoded by the Hox operon which is cytoplasmic and directly reduces NAD+ at the expense of hydrogen (Schneider and Schlegel, Biochim. Biophys. Acta 452, 66-80 (1976); Burgdorf, J. Bact. 187(9) 3122-3132(2005)). Soluble EM16 enzymes are additionally present in several other organisms including Geobacter sulfurreducens (Coppi, Microbiology 151, 1239-1254 (2005)), Synechocystis str. PCC 6803 (Germer, J. Biol. Chem., 284(52), 36462-36472 (2009)), and Thiocapsa roseopersicina (Rakhely, Appl. Environ. Microbiol. 70(2) 722-728 (2004)). The Synechocystis enzyme is capable of generating NADPH from hydrogen. Overexpression of both the Hox operon from Synechocystis str. PCC 6803 and the accessory genes encoded by the Hyp operon from Nostoc sp. PCC 7120 led to increased EM 16 activity compared to expression of the Hox genes alone (Germer, J. Biol. Chem. 284(52), 36462-36472 (2009)). Protein GenBank ID GI Number Organism

HoxF NP 942727.1 38637753 Ralstonia eutropha HI 6

HoxU NP 942728.1 38637754 Ralstonia eutropha HI 6

HoxY NP 942729.1 38637755 Ralstonia eutropha HI 6

HoxH NP 942730.1 38637756 Ralstonia eutropha HI 6

HoxW NP 942731.1 38637757 Ralstonia eutropha HI 6

Hoxl NP 942732.1 38637758 Ralstonia eutropha HI 6

HoxE NP 953767.1 39997816 Geobacter sulfurreducens

HoxF NP 953766.1 39997815 Geobacter sulfurreducens

HoxU NP 953765.1 39997814 Geobacter sulfurreducens

HoxY NP 953764.1 39997813 Geobacter sulfurreducens

HoxH NP 953763.1 39997812 Geobacter sulfurreducens

GSU2717 NP 953762.1 39997811 Geobacter sulfurreducens

HoxE NP 441418.1 16330690 Synechocystis str. PCC 6803

HoxF NP_441417.1 16330689 Synechocystis str. PCC 6803

Unknown function NP 441416.1 16330688 Synechocystis str. PCC 6803

HoxU NP 441415.1 16330687 Synechocystis str. PCC 6803

HoxY NP 441414.1 16330686 Synechocystis str. PCC 6803

Unknown function NP 441413.1 16330685 Synechocystis str. PCC 6803

Unknown function NP 441412.1 16330684 Synechocystis str. PCC 6803

HoxH NP 441411.1 16330683 Synechocystis str. PCC 6803

HypF NP 484737.1 17228189 Nostoc sp. PCC 7120

HypC NP 484738.1 17228190 Nostoc sp. PCC 7120

HypD NP 484739.1 17228191 Nostoc sp. PCC 7120

Unknown function NP 484740.1 17228192 Nostoc sp. PCC 7120

HypE NP 484741.1 17228193 Nostoc sp. PCC 7120

HypA NP 484742.1 17228194 Nostoc sp. PCC 7120

HypB NP 484743.1 17228195 Nostoc sp. PCC 7120

HoxlE AAP50519.1 37787351 Thiocapsa roseopersicina

HoxlF AAP50520.1 37787352 Thiocapsa roseopersicina

HoxlU AAP50521.1 37787353 Thiocapsa roseopersicina

HoxlY AAP50522.1 37787354 Thiocapsa roseopersicina

HoxlH AAP50523.1 37787355 Thiocapsa roseopersicina

The genomes of E. coli and other enteric bacteria encode up to four EM16 enzymes (Sawers, G., Antonie Van Leeuwenhoek 66:57-88 (1994); Sawers et al., J Bacteriol. 164: 1324-1331 (1985); Sawers and Boxer, Eur.J Biochem. 156:265-275 (1986); Sawers et al., J Bacteriol.

168:398-404 (1986)). Given the multiplicity of enzyme activities E. coli or another host organism can provide sufficient EM 16 activity to split incoming molecular hydrogen and reduce the corresponding acceptor. Endogenous hydrogen-lyase enzymes of E. coli include hydrogenase 3, a membrane-bound enzyme complex using ferredoxin as an acceptor, and hydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3 and 4 are encoded by the hyc and hyf gene clusters, respectively. EM16 activity in E. coli is also dependent upon the expression of the hyp genes whose corresponding proteins are involved in the assembly of the EMI 6 complexes (Jacobi et al., Arch.Microbiol 158:444-451 (1992); Rangarajan et al., J Bacteriol. 190:1447-1458 (2008)). The M. thermoacetica and Clostridium ljungdahli EMI 6s are suitable for a host that lacks sufficient endogenous EM 16 activity. M. thermoacetica and C. ljungdahli can grow with CO ₂ as the exclusive carbon source indicating that reducing equivalents are extracted from H ₂ to enable acetyl-CoA synthesis via the Wood-Ljungdahl pathway (Drake, H. L., J Bacteriol. 150:702-709 (1982); Drake and Daniel, Res Microbiol 155:869-883 (2004); Kellum and Drake, J Bacteriol. 160:466-469 (1984)). M. thermoacetica has homologs to several hyp, hyc, and hyf genes from E. coli. These protein sequences encoded for by these genes are identified by the following GenBank accession numbers. In addition, several gene clusters encoding EM 16 functionality are present in M. thermoacetica and C. ljungdahli (see for example US 2012/0003652).

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

Moth 2183 YP 431015 83591006 Moorella thermoacetica

Moth 2184 YP 431016 83591007 Moorella thermoacetica

Moth 2185 YP 431017 83591008 Moorella thermoacetica

Moth 2186 YP 431018 83591009 Moorella thermoacetica

Moth 2187 YP 431019 83591010 Moorella thermoacetica

Moth 2188 YP 431020 83591011 Moorella thermoacetica

Moth 2189 YP 431021 83591012 Moorella thermoacetica

Moth 2190 YP 431022 83591013 Moorella thermoacetica

Moth 2191 YP 431023 83591014 Moorella thermoacetica

Moth 2192 YP 431024 83591015 Moorella thermoacetica

Moth 0439 YP 429313 83589304 Moorella thermoacetica

Moth 0440 YP 429314 83589305 Moorella thermoacetica

Moth 0441 YP 429315 83589306 Moorella thermoacetica

Moth 0442 YP 429316 83589307 Moorella thermoacetica

Moth 0809 YP 429670 83589661 Moorella thermoacetica

Moth 0810 YP 429671 83589662 Moorella thermoacetica

Moth 0811 YP 429672 83589663 Moorella thermoacetica

Moth 0812 YP 429673 83589664 Moorella thermoacetica

Moth 0814 YP 429674 83589665 Moorella thermoacetica

Moth 0815 YP 429675 83589666 Moorella thermoacetica

Moth 0816 YP 429676 83589667 Moorella thermoacetica

Moth 1193 YP 430050 83590041 Moorella thermoacetica

Moth 1194 YP 430051 83590042 Moorella thermoacetica

Moth 1195 YP 430052 83590043 Moorella thermoacetica

Moth 1196 YP 430053 83590044 Moorella thermoacetica

Moth 1717 YP 430562 83590553 Moorella thermoacetica

Moth 1718 YP 430563 83590554 Moorella thermoacetica

Moth 1719 YP 430564 83590555 Moorella thermoacetica

Moth 1883 YP 430726 83590717 Moorella thermoacetica

Moth 1884 YP 430727 83590718 Moorella thermoacetica

Moth 1885 YP 430728 83590719 Moorella thermoacetica

Moth 1886 YP 430729 83590720 Moorella thermoacetica

Moth 1887 YP 430730 83590721 Moorella thermoacetica

Moth 1888 YP 430731 83590722 Moorella thermoacetica

Moth 1452 YP 430305 83590296 Moorella thermoacetica

Moth 1453 YP 430306 83590297 Moorella thermoacetica

Moth 1454 YP 430307 83590298 Moorella thermoacetica

Genes encoding EMI 6 enzymes from C. ljungdahli are shown below.

CLJU c28670 ADK15915.1 300436148 Clostridium ljungdahli

CLJU c28660 ADK15914.1 300436147 Clostridium ljungdahli

CLJU c28650 ADK15913.1 300436146 Clostridium ljungdahli

CLJU c28640 ADK15912.1 300436145 Clostridium ljungdahli

In some cases, EMI 6 encoding genes are located adjacent to a CODH. In Rhodospirillum rubrum, the encoded CODH/hydrogenase proteins form a membrane-bound enzyme complex that has been indicated to be a site where energy, in the form of a proton gradient, is generated from the conversion of CO and H ₂ 0 to C0 ₂ and H ₂ (Fox et al., JBacteriol. 178:6200-6208 (1996)). The CODH-I of C. hydrogenoformans and its adjacent genes have been proposed to catalyze a similar functional role based on their similarity to the R. rubrum CODH/hydrogenase gene cluster (Wu et al., PLoS Genet. I :e65 (2005)). The C. hydrogenoformans CODH-I was also shown to exhibit intense CO oxidation and C0 ₂ reduction activities when linked to an electrode (Parkin et al., J Am.Chem.Soc. 129: 10328-10329 (2007)).

Some EMI 6 and CODH enzymes transfer electrons to ferredoxins. Ferredoxins are small acidic proteins containing one or more iron-sulfur clusters that function as intracellular electron carriers with a low reduction potential. Reduced ferredoxins donate electrons to Fe-dependent enzymes such as ferredoxin-NADP ⁺ oxidoreductase, pyruvate :ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (OFOR). The H. thermophilus gene fdxl encodes a [4Fe-4S]-type ferredoxin that is required for the reversible carboxylation of 2-oxoglutarate and pyruvate by OFOR and PFOR, respectively (Yamamoto et al., Extremophiles 14:79-85 (2010)). The ferredoxin associated with the Sulfolobus solfataricus 2-oxoacid: ferredoxin reductase is a monomeric dicluster [3Fe-4S][4Fe-4S] type ferredoxin (Park et al. 2006). While the gene associated with this protein has not been fully sequenced, the N-terminal domain shares 93% homology with the zfx ferredoxin from S. acidocaldarius. The E. coli genome encodes a soluble ferredoxin of unknown physiological function,/^. Some evidence indicates that this protein can function in iron-sulfur cluster assembly (Takahashi and Nakamura, 1999). Additional ferredoxin proteins have been characterized in Helicobacter pylori (Mukhopadhyay et al. 2003) and

Campylobacter jejuni (van Vliet et al. 2001). A 2Fe-2S ferredoxin from Clostridium

pasteurianum has been cloned and expressed in ?. coli (Fujinaga and Meyer, Biochemical and Biophysical Research Communications, 192(3): (1993)). Acetogenic bacteria such as Moorella thermoacetica, Clostridium carboxidivorans P7, Clostridium ljungdahli and Rhodospirillum rubrum are predicted to encode several ferredoxins, listed below.

Protein GenBank ID GI Number Organism

fdxl BAE02673.1 68163284 Hydrogenobacter thermophilus

M11214.1 AAA83524.1 144806 Clostridium pasteurianum

Zfx AAY79867.1 68566938 Sulfolobus acidocalarius

Fdx AAC75578.1 1788874 Escherichia coli

hp_0277 AAD07340.1 2313367 Helicobacter pylori

fdxA CAL34484.1 112359698 Campylobacter jejuni

Moth 0061 ABC 18400.1 83571848 Moorella thermoacetica

Moth 1200 ABC19514.1 83572962 Moorella thermoacetica

Moth 1888 ABC20188.1 83573636 Moorella thermoacetica

Moth 2112 ABC20404.1 83573852 Moorella thermoacetica

Moth 1037 ABC19351.1 83572799 Moorella thermoacetica

CcarbDRAFT 4383 ZP 05394383.1 255527515 Clostridium carboxidivorans P7

CcarbDRAFT 2958 ZP 05392958.1 255526034 Clostridium carboxidivorans P7

CcarbDRAFT 2281 ZP 05392281.1 255525342 Clostridium carboxidivorans P7

CcarbDRAFT 5296 ZP 05395295.1 255528511 Clostridium carboxidivorans P7

CcarbDRAFT 1615 ZP 05391615.1 255524662 Clostridium carboxidivorans P7

CcarbDRAFT 1304 ZP 05391304.1 255524347 Clostridium carboxidivorans P7 cooF AAG29808.1 11095245 Carboxydothermus hydrogenoformans fdxN CAA35699.1 46143 Rhodobacter capsulatus

Rru A2264 ABC23064.1 83576513 Rhodospirillum rubrum

Rru A1916 ABC22716.1 83576165 Rhodospirillum rubrum

Rru A2026 ABC22826.1 83576275 Rhodospirillum rubrum

cooF AAC45122.1 1498747 Rhodospirillum rubrum

fdxN AAA26460.1 152605 Rhodospirillum rubrum

Alvin 2884 ADC63789.1 288897953 Allochromatium vinosum DSM 180

Fdx YP 002801146.1 226946073 Azotobacter vinelandii DJ

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

Fdx CAA12251.1 3724172 Thauera aromatica

CHY 2405 YP 361202.1 78044690 Carboxydothermus hydrogenoformans

Fer YP 359966.1 78045103 Carboxydothermus hydrogenoformans

Fer AAC83945.1 1146198 Bacillus subtilis

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

CLJU c00930 ADK13195.1 300433428 Clostridium ljungdahli

CLJU cOOOlO ADK13115.1 300433348 Clostridium ljungdahli

CLJU c01820 ADK13272.1 300433505 Clostridium ljungdahli CLJU c 17980 ADK14861.1 300435094 Clostridium ljungdahli

CLJU c 17970 ADK14860.1 300435093 Clostridium ljungdahli

CLJU c22510 ADK15311.1 300435544 Clostridium ljungdahli

CLJU c26680 ADK15726.1 300435959 Clostridium ljungdahli

CLJU c29400 ADK15988.1 300436221 Clostridium ljungdahli

Ferredoxin oxidoreductase enzymes transfer electrons from ferredoxins or flavodoxins to NAD(P)H. Two enzymes catalyzing the reversible transfer of electrons from reduced ferredoxins to NAD(P)+ are ferredoxin:NAD+ oxidoreductase (EC 1.18.1.3) and ferredoxin :NADP+ oxidoreductase (FNR, EC 1.18.1.2). Ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) has a noncovalently bound FAD cofactor that facilitates the reversible transfer of electrons from

NADPH to low-potential acceptors such as ferredoxins or flavodoxins (Blaschkowski et al., Eur. J. Biochem. 123:563-569 (1982); Fujii et al., 1977). The Helicobacter pylori FNR, encoded by HP1164 (fqrB), is coupled to the activity of pyruvate: ferredoxin oxidoreductase (PFOR) resulting in the pyruvate-dependent production of NADPH (St et al. 2007). An analogous enzyme is found in Campylobacter jejuni (St Maurice et al., J. Bacteriol. 189:4764-4773 (2007)). A

ferredoxin:NADP+ oxidoreductase enzyme is encoded in the E. coli genome by fpr (Bianchi et al. 1993). Ferredoxin:NAD+ oxidoreductase utilizes reduced ferredoxin to generate NADH from NAD+. In several organisms, including E. coli, this enzyme is a component of multifunctional dioxygenase enzyme complexes. The ferredoxin :NAD+ oxidoreductase of E. coli, encoded by hcaD, is a component of the 3-phenylproppionate dioxygenase system involved in involved in aromatic acid utilization (Diaz et al. 1998). NADH:ferredoxin reductase activity was detected in cell extracts of Hydrogenobacter thermophilus, although a gene with this activity has not yet been indicated (Yoon et al. 2006). Additional ferredoxin:NAD(P)+ oxidoreductases have been annotated in Clostridium carboxydivorans P7. The NADH-dependent reduced ferredoxin: NADP oxidoreductase of C. kluyveri, encoded by nfnAB, catalyzes the concomitant reduction of ferredoxin and NAD+ with two equivalents of NADPH (Wang et al, J Bacteriol 192: 5115-5123 (2010)). Finally, the energy-conserving membrane-associated Rnf-type proteins (Seedorf et al, PNAS 105:2128-2133 (2008); and Herrmann, J. Bacteriol 190:784-791 (2008)) provide a means to generate NADH or NADPH from reduced ferredoxin.

CcarbDRAFT 2639 ZP 05392639.1 255525707 Clostridium carboxidivorans P7

CcarbDRAFT 2638 ZP 05392638.1 255525706 Clostridium carboxidivorans P7

CcarbDRAFT 2636 ZP 05392636.1 255525704 Clostridium carboxidivorans P7

CcarbDRAFT 5060 ZP 05395060.1 255528241 Clostridium carboxidivorans P7

CcarbDRAFT 2450 ZP 05392450.1 255525514 Clostridium carboxidivorans P7

CcarbDRAFT 1084 ZP 05391084.1 255524124 Clostridium carboxidivorans P7

RnfC EDK33306.1 146346770 Clostridium kluyveri

RnfD EDK33307.1 146346771 Clostridium kluyveri

RnfG EDK33308.1 146346772 Clostridium kluyveri

RnfE EDK33309.1 146346773 Clostridium kluyveri

RnfA EDK33310.1 146346774 Clostridium kluyveri

RnfB EDK3331 1.1 146346775 Clostridium kluyveri

CLJU_cl l410 (RnfB) ADK14209.1 300434442 Clostridium ljungdahlii

CLJU c 11400 (RnfA) ADK14208.1 300434441 Clostridium ljungdahlii

CLJU cl 1390 (RnfE) ADK14207.1 300434440 Clostridium ljungdahlii

CLJU cl 1380 (RnfG) ADK14206.1 300434439 Clostridium ljungdahlii

CLJU cl 1370 (RnfD) ADK14205.1 300434438 Clostridium ljungdahlii

CLJU_cl l 360 (RnfC) ADK14204.1 300434437 Clostridium ljungdahlii

MOTH 1518 (NfnA) YP 430370.1 83590361 Moorella thermoacetica

MOTH_1517(NfhB) YP 430369.1 83590360 Moorella thermoacetica

CHY 1992 (NfnA) YP 360811.1 78045020 Carboxydothermus

hydrogenoformans

CHY 1993 (NfnB) YP 360812.1 78044266 Carboxydothermus

hydrogenoformans

CLJU C37220 (NfnAB) YP 003781850.1 300856866 Clostridium ljungdahlii

FIG.3, Step I - Formate Dehydrogenase (EM8)

Formate dehydrogenase (FDH; EM8) catalyzes the reversible transfer of electrons from formate to an acceptor. Enzymes with FDH activity utilize various electron carriers such as, for example, NADH (EC 1.2.1.2), NADPH (EC 1.2.1.43), quinols (EC 1.1.5.6), cytochromes (EC 1.2.2.3) and EMI 6s (EC 1.1.99.33). FDH enzymes have been characterized from Moorella thermoacetica (Andreesen and Ljungdahl, J Bacteriol 1 16:867-873 (1973); Li et al., J Bacteriol 92:405-412 (1966); Yamamoto et al., J Biol Chem. 258: 1826-1832 (1983). The loci, Moth_2312 is responsible for encoding the alpha subunit of EM 8 while the beta subunit is encoded by Moth_2314 (Pierce et al., Environ Microbiol (2008)). Another set of genes encoding EM8 activity with a propensity for CO ₂ reduction is encoded by Sfum_2703 through Sfum_2706 in

Syntrophobacter fumaroxidans (de Bok et al., Eur J Biochem. 270:2476-2485 (2003)); Reda et al., PNAS 105 : 10654-10658 (2008)). A similar set of genes presumed to carry out the same function are encoded by CHY 0731 , CHY 0732, and CHY 0733 in C. hydrogenoformans (Wu et a\., PLoS Genet l :e65 (2005)). EM8s are also found many additional organisms including C.

carboxidivorans P7, Bacillus methanolicus, Burkholderia stabilis, Moorella thermoacetica ATCC 39073, Candida boidinii, Candida methylica, and Saccharomyces cerevisiae S288c. The soluble EM8 from Ralstonia eutropha reduces NAD ⁺ (JdsG, -B, -A, -C, -D) (Oh and Bowien, 1998), Several EM8 enzymes have been identified that have higher specificity for NADP as the cofactor as compared to NAD. This enzyme has been deemed as the NADP-dependent formate dehydrogenase and has been reported from 5 species of the Burkholderia cepacia complex. It was tested and verified in multiple strains of Burkholderia multivorans, Burkholderia stabilis, Burkholderia pyrrocinia, and Burkholderia cenocepacia (Hatrongjit et al., Enzyme and Microbial Tech., 46: 557-561 (2010)). The enzyme from Burkholderia stabilis has been characterized and the apparent K _m of the enzyme were reported to be 55.5 mM, 0.16 niM and 1.43 mM for formate, NADP, and NAD respectively. More gene candidates can be identified using sequence homology of proteins deposited in Public databases such as NCBI, JGI and the metagenomic databases.

FIG. 3, Step J - Methanol Dehydrogenase (EM9)

NAD+ dependent EM9 enzymes (EC 1.1.1.244) catalyze the conversion of methanol and NAD+ to formaldehyde and NADH. See the present invention as described herein.

FIG. 3, Step L - Formaldehyde Dehydrogenase (EMU) Oxidation of formaldehyde to formate is catalyzed by EMI 1. A NAD+ dependent EMI 1 enzyme is encoded by fdhA of Pseudomonas putida (Ito et al, J Bacteriol 176: 2483-2491 (1994)). Additional EMI 1 enzymes include the NAD+ and glutathione independent EMI 1 from

Hyphomicrobium zavarzinii (Jerome et al, Appl Microbiol Biotechnol 77:779-88 (2007)), the glutathione dependent EMI 1 oiPichia pastoris (Sunga et al, Gene 330:39-47 (2004)) and the NAD(P)+ dependent EMU of Methylobacter marinus (Speer et al, FEMS Microbiol Lett, 121(3):349-55 (1994)).

In addition to the EMI 1 enzymes listed above, alternate enzymes and pathways for converting formaldehyde to formate are known in the art. For example, many organisms employ glutathione-dependent formaldehyde oxidation pathways, in which formaldehyde is converted to formate in three steps via the intermediates S-hydroxymethylglutathione and S-formylglutathione (Vorholt et al, J Bacteriol 182:6645-50 (2000)). The enzymes of this pathway are EM12 (EC 4.4.1.22), EM13 (EC 1.1.1.284) and EM14 (EC 3.1.2.12).

FIG. 3, Step M - Spontaneous or S-(hydroxymethyl)glutathione Synthase (EM12)

While conversion of formaldehyde to S-hydroxymethylglutathione can occur

spontaneously in the presence of glutathione, it has been shown by Goenrich et al (Goenrich, et al., J Biol Chem 277(5);3069-72 (2002)) that an enzyme from Paracoccus denitrificans can accelerate this spontaneous condensation reaction. The enzyme catalyzing the conversion of formaldehyde and glutathione was purified and named glutathione-dependent formaldehyde-activating enzyme (Gfa). The gene encoding it, which was named gfa, is located directly upstream of the gene for EMI 3, which catalyzes the subsequent oxidation of S-hydroxymethylglutathione. Putative proteins with sequence identity to Gfa from P. denitrificans are present also in Rhodobacter sphaeroides, Sinorhizobium meliloti, and Mesorhizobium loti.

FIG. 3, Step N - Glutathione-Dependent Formaldehyde Dehydrogenase (EM13) EM 13 (GS-FDH) belongs to the family of class III alcohol dehydrogenases. Glutathione and formaldehyde combine non-enzymatically to form hydroxymethylglutathione, the true substrate of the GS-FDH catalyzed reaction. The product, S-formylglutathione, is further metabolized to formic acid.

FIG. 3, Step O - S-formylglutathione Hydrolase (EM14)

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

Exemplary enzymes for the methods of using formaldehyde produced from the oxidation of methanol in the formation of intermediates of central metabolic pathways for the formation of target product or biomass are further described, particularly with respect to Figures 3B and 3C.

Provided herein are exemplary pathways, which utilize formaldehyde produced from the oxidation of methanol (see, e.g., FIG. 3, step J) in the formation of intermediates of certain central metabolic pathways that can be used for the formation of biomass. Exemplary MMPs for enhancing the availability of reducing equivalents, as well as the producing formaldehyde from methanol (step J), are provided in FIG. 3.

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

Another exemplary pathway that involves the detoxification and assimilation of formaldehyde produced from the oxidation of methanol {e.g., as provided in FIG. 3) is shown in FIG. 3C and proceeds through DHA. EF3 is a special transketolase that first transfers a glycoaldehyde group from xylulose-5 -phosphate to formaldehyde, resulting in the formation of DHA and G3P, which is an intermediate in glycolysis (FIG. 3C, step A). The DHA obtained from DHA synthase is then further phosphorylated to form DHA phosphate by a DHA kinase (FIG. 3C, step B). DHAP can be assimilated into glycolysis and several other pathways.

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

Both of the EF1 and EF2 enzymes are found in several organisms, including methanotrops and methylotrophs where they have been purified (Kato et al., 2006, BioSci Biotechnol Biochem. 70(1):10-21. In addition, these enzymes have been reported in heterotrophs such as Bacillus subtilis also where they are reported to be involved in formaldehyde detoxification (Mitsui et al., 2003, AEM 69(10):6128-32, Yasueda et al. , 1999. J Bac 181(23):7154-60. Genes for these two enzymes from the methyl otrophic bacterium Mycobacierium gastri MB 19 have been fused and E. coli steins harboring the hps-phi construct showed more efficient utilization of formaldehyde (Orita et al., 2007, Appl Microbiol Biotechnol. 76:439-445). In some organisms, these two enzymes naturally exist as a fused version that is bifunctional.

[0100] Exemplary candidate genes for H6P synthase are:

[0101] Exemplary gene candidates for EF2 are:

[0102] Candidates for enzymes where both of these functions have been fused into a open reading frame include the following.

FIG. 3C, Step A - Dihydroxyacetone synthase (EF3)

Another exemplary pathway that involves the detoxification and assimilation of formaldehyde produced from the oxidation of methanol (e.g., as provided in FIG. 3) is shown in FIG. 3C and proceeds through DHA. EF3 is a special transketolase that first transfers a glycoaldehyde group from xylulose-5 -phosphate to formaldehyde, resulting in the formation of DHA and G3P, which is an intermediate in glycolysis (FIG. 3C, step A). The DHA obtained from DHA synthase is then further phosphorylated to form DHA phosphate by a DHA kinase (FIG. 3C, step B). DHAP can be assimilated into glycolysis and several other pathways.

The EF3 enzyme in Candida boidinii uses thiamine pyrophosphate and Mg ²⁺ as cofactors and is localized in the peroxisome. The enzyme from the methanol-growing carboxydobacterium, Mycobacter sp. strain JC1 DSM 3803, was also found to have DHA synthase and kinase activities (Ro et al., 1997, JBac 179(19):6041-7). DHA synthase from this organism also has similar cofactor requirements as the enzyme from C. boidinii. The K _m s for formaldehyde and xylulose 5 -phosphate were reported to be 1.86 mM and 33.3 microM, respectively. Several other mycobacteria, excluding only Mycobacterium tuberculosis, can use methanol as the sole source of carbon and energy and are reported to use EF3 (Part et al., 2003, JBac 185(1): 142-7.

FIG. 3C, Step B - Dihydroxyacetone (DHA) kinase

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

In Escherichia coli, DHA kinase is composed of the three subunits DhaK, DhaL, and DhaM and it functions similarly to a phosphotransferase system (PTS) in that it utilizes phosphoenolpyruvate as a phosphoryl donor (Gutknecht et al., 2001, EMBO J. 20(10):2480-6). It differs in not being involved in transport. The phosphorylation reaction requires the presence of the EI and HPr proteins of the PTS system. The DhaM subunit is phosphorylated at multiple sites. DhaK contains the substrate binding site (Garcia-Alles et al., 2004, 43(41): 13037-45; Siebold et al., 2003, PNAS. 100(14):8188-92). The K _M for DHA for the E. coli enzyme has been reported to be 6 μΜ. The K subunit is similar to the N-terminal half of ATP -dependent EF4 of Citrobacter freundii and eukaryotes.

Exemplary DHA kinase gene candidates for this step are:

Suitable purification and/or assays to test, e.g., for the production of BDO can be performed using well known methods. Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art.

The BDO or other target molecules may separated from other components in the culture using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, evaporation, filtration, membrane filtration (including reverse osmosis, nanofiltration, ultrafiltration, and microfiltration), membrane filtration with diafiltration, membrane separation, reverse osmosis, electrodialysis, distillation, extractive distillation, reactive distillation, azeotropic distillation, crystallization and recrystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, carbon adsorption, hydrogenation, and ultrafiltration. All of the above methods are well known in the art.

Examples of target molecule isolation processes include distillation for 13BDO, 14BDO, butadiene, methyl vinyl carbinol, 3-buten-l-ol, n-propanol, isopropanol, propylene, and crotyl alcohol; crystallization for 6ACA (alternatively it can be converted to caprolactam and then purified via distillation as a final step), HMD A, adipic acid or derivatives thereof, succinic acid or derivatives thereof, or any of crystallization, distillation, or extraction for methacrylic acid or derivatives thereof.

Target molecules such asl3BDO, 14BDO, butadiene, methyl vinyl carbinol n-propanol, isopropanol, propylene, crotyl alcohol; 3-buten-l-ol, 6ACA, HMDA, adipic acid or derviaties thereof, succinic acid or derivatives thereof, or methacrylic acid or derivatives thereof are chemicals used in commercial and industrial applications. In some embodiments, BDO and/or 4- HB are used in various commercial and industrial applications. Non- limiting examples of such applications include production of plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like. Moreover, BDO and/or 4-HB are also used as a raw material in the production of a wide range of products including plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like.

Accordingly, in some embodiments, provided are biobased plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like, comprising one or more bioderived BDO and/or 4-HB or bioderived BDO and/or 4-HB intermediate thereof produced by an organism provided herein or produced using a method disclosed herein.

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

In some embodiments, the disclosure provides plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like, comprising bioderived BDO and/or 4-HB or bioderived BDO and/or 4-HB intermediate thereof, wherein the bioderived BDO and/or 4-HB or bioderived BDO and/or 4-HB intermediate thereof includes all or part of the BDO and/or 4-HB or BDO and/or 4-HB intermediate thereof used in the production of plastics, elastic fibers, polyurethanes, polyesters, including

polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like,. Thus, in some aspects, the disclosure provides a biobased plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and

polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like, comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% bioderived BDO and/or 4-HB or bioderived BDO and/or 4-HB intermediate thereof as disclosed herein. Additionally, in some aspects, the disclosure provides a biobased plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like, wherein the BDO and/or 4-HB or BDO and/or 4-HB intermediate thereof used in its production is a combination of bioderived and petroleum derived BDO and/or 4-HB or BDO and/or 4-HB intermediate thereof. For example, a biobased plastics, elastic fibers, polyurethanes, polyesters, including

polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like, can be produced using 50% bioderived BDO and/or 4-HB and 50% petroleum derived BDO and/or 4-HB or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like, using the bioderived BDO and/or 4-HB or bioderived BDO and/or 4-HB intermediate thereof of the disclosure are well known in the art. In one embodiment, the product is a plastic. In one embodiment, the product is an elastic fiber. In one embodiment, the product is a polyurethane. In one embodiment, the product is a polyester. In one embodiment, the product is a polyhydroxyalkanoate. In one embodiment, the product is a poly-4-HB. In one embodiment, the product is a co-polymer of poly-4-HB. In one embodiment, the product is a poly(tetramethylene ether) glycol. In one embodiment, the product is a polyurethane-polyurea copolymer. In one embodiment, the product is a spandex. In one embodiment, the product is an elastane. In one embodiment, the product is a Lycra™. In one embodiment, the product is a nylon.

In some embodiments, provided herein is a culture medium comprising bioderived BDO. In some embodiments, the bioderived BDO is produced by culturing an organism having a MDH protein and BDOP, as provided herein. In certain embodiments, the bioderived BDO has a carbon- 12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source. In one embodiment, the culture medium is separated from a organism having a MDH protein and BDOP.

In other embodiments, provided herein is a bioderived BDO. In some embodiments, the bioderived BDO is produced by culturing an organism having a MDH protein and BDOP, as provided herein, n some embodiments, the bioderived BDO has an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%. In certain embodiments, the bioderived BDO is a component of culture medium.

In certain embodiments, provided herein is a composition comprising a bioderived BDO provided herein, for example, a bioderived BDO produced by culturing an orgaism having a MDH protein and BDOP, as provided herein. In some embodiments, the composition further comprises a compound other than said bioderived BDO. In certain embodiments, the compound other than said bioderived BDO is a trace amount of a cellular portion of an organism having a MDH protein and a BDOP, as provided herein.

In some embodiments, provided herein is a biobased product comprising a bioderived BDO provided herein. In certain embodiments, the biobased product is a plastic, elastic fiber, polyurethane, polyester, polyhydroxyalkanoate, poly-4-HB, co-polymer of poly-4-HB, poly(tetramethylene ether) glycol, polyurethane-polyurea copolymer, spandex, elastane, Lycra™, or nylon. In certain embodiments, the biobased product comprises at least 5% bioderived BDO. In certain embodiments, the biobased product is (i) a polymer, THF or a THF derivative, or GBL or a GBL derivative; (ii) a plastic, elastic fiber, polyurethane, polyester, polyhydroxyalkanoate, poly-4- HB, co-polymer of poly-4-HB, poly(tetramethylene ether) glycol, polyurethane-polyurea copolymer, spandex, elastane, Lycra™, or nylon; (iii) a polymer, a resin, a fiber, a bead, a granule, a pellet, a chip, a plastic, a polyester, a thermoplastic polyester, a molded article, an injection- molded article, an injection-molded part, an automotive part, an extrusion resin, an electrical part and a casing; and optionally where the biobased product is reinforced or filled and further where the biobased product is glass-reinforced or -filled or mineral-reinforced or -filled; (iv) a polymer, wherein the polymer comprises polybutylene terephthalate (PBT); (v) a polymer, wherein the polymer comprises PBT and the biobased product is a resin, a fiber, a bead, a granule, a pellet, a chip, a plastic, a polyester, a thermoplastic polyester, a molded article, an injection-molded article, an injection-molded part, an automotive part, an extrusion resin, an electrical part and a casing; and optionally where the biobased product is reinforced or filled and further where the biobased product is glass-reinforced or -filled or mineral-reinforced or -filled; (vi) a THF or a THF derivative, wherein the THF derivative is polytetramethylene ether glycol (PTMEG), a polyester ether (COPE) or a thermoplastic polyurethane; (viii) a THF derivative, wherein the THF derivative comprises a fiber; or (ix) a GBL or a GBL derivative, wherein the GBL derivative is a pyrrolidone. In certain embodiments, the biobased product comprises at least 10% bioderived BDO. In some embodiments, the biobased product comprises at least 20% bioderived BDO. In other

embodiments, the biobased product comprises at least 30% bioderived BDO. In some

embodiments, the biobased product comprises at least 40% bioderived BDO. In other

embodiments, the biobased product comprises at least 50% bioderived BDO. In one embodiment, the biobased product comprises a portion of said bioderived BDO as a repeating unit. In another embodiment, provided herein is a molded product obtained by molding the biobased product provided herein. In other embodiments, provided herein is a process for producing a biobased product provided herein, comprising chemically reacting said bioderived-BDO with itself or another compound in a reaction that produces said biobased product. In certain embodiments, provided herein is a polymer comprising or obtained by converting the bioderived BDO. In other embodiments, provided herein is a method for producing a polymer, comprising chemically or enzymatically converting the bioderived BDO to the polymer. In yet other embodiments, provided herein is a composition comprising the bioderived BDO, or a cell lysate or culture supernatant thereof.

In some embodiments, the carbon feedstock and other cellular uptake sources such as phosphate, ammonia, sulfate, chloride and other halogens can be chosen to alter the isotopic distribution of the atoms present in BDO and/or 4-HB or any BDO and/or 4-HB pathway intermediate. The various carbon feedstock and other uptake sources enumerated above will be referred to herein, collectively, as "uptake sources." Uptake sources can provide isotopic enrichment for any atom present in the product BDO and/or 4-HB or BDO and/or 4-HB pathway intermediate, or for side products generated in reactions diverging away from a BDO and/or 4-HB pathway. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens. The same holds true for the MMPs and FAPs, as well as intermediates thereof, provided herein. In some embodiments, the uptake sources can be selected to alter the carbon-12, carbon-13, and carbon- 14 ratios. In some embodiments, the uptake sources can be selected to alter the oxygen-16, oxygen-17, and oxygen-18 ratios. In some embodiments, the uptake sources can be selected to alter the hydrogen, deuterium, and tritium ratios. In some embodiments, the uptake sources can selected to alter the nitrogen- 14 and nitrogen- 15 ratios. In some embodiments, the uptake sources can be selected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35 ratios. In some embodiments, the uptake sources can be selected to alter the phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In some embodiments, the uptake sources can be selected to alter the chlorine-35, chlorine-36, and chlorine-37 ratios.

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

environmental or atmospheric carbon source, such as CO ₂ , which can possess a larger amount of carbon- 14 than its petroleum-derived counterpart.

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

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

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

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

The biobased content of a compound is estimated by the ratio of carbon- 14 ( ¹⁴ C) to carbon- 12 ( ¹² C). Specifically, the Fraction Modern (Fm) is computed from the expression: Fm = (S- B)/(M-B), where B, S and M represent the ¹⁴ C/ ¹² C ratios of the blank, the sample and the modern reference, respectively. Fraction Modern is a measurement of the deviation of the ¹⁴ C/ ¹² C ratio of a sample from "Modern." Modern is defined as 95% of the radiocarbon concentration (in AD 1950) of National Bureau of Standards (NBS) Oxalic Acid I (i.e., standard reference materials (SRM) 4990b) normalized to per mil (Olsson, The use of Oxalic acid as a Standard, in, Radiocarbon Variations and Absolute Chronology, Nobel Symposium, 12th Proc, John Wiley & Sons, New York (1970)). Mass spectrometry results, for example, measured by ASM, are calculated using the internationally agreed upon definition of 0.95 times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalized to per mil. This is equivalent to an absolute (AD 1950) ¹⁴ C/ ¹² C ratio of 1.176 ± 0.010 x 10 ^"12 (Karlen et al., Arkiv Geofysik, 4:465-471 (1968)). The standard calculations take into account the differential uptake of one isotope with respect to another, for example, the preferential uptake in biological systems of C ¹² over C ¹³ over C ¹⁴ , and these corrections are reflected as a Fm corrected for δ ¹³ .

An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of 1955 sugar beet. Although there were 1000 lbs made, this oxalic acid standard is no longer commercially available. The Oxalic Acid II standard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of 1977 French beet molasses. In the early 1980's, a group of 12 laboratories measured the ratios of the two standards. The ratio of the activity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). The isotopic ratio of HOx II is -17.8 per mille. ASTM D6866-11 suggests use of the available Oxalic Acid II standard SRM 4990 C (Hox2) for the modern standard (see discussion of original vs. currently available oxalic acid standards in Mann, Radiocarbon, 25(2) :519-527

(1983)). A Fm = 0% represents the entire lack of carbon-14 atoms in a material, thus indicating a fossil (for example, petroleum based) carbon source. A Fm = 100%, after correction for the post- 1950 injection of carbon-14 into the atmosphere from nuclear bomb testing, indicates an entirely modern carbon source. As described herein, such a "modern" source includes biobased sources.

As described in ASTM D6866, the percent modern carbon (pMC) can be greater than 100% because of the continuing but diminishing effects of the 1950s nuclear testing programs, which resulted in a considerable enrichment of carbon-14 in the atmosphere as described in ASTM D6866-11. Because all sample carbon-14 activities are referenced to a "pre-bomb" standard, and because nearly all new biobased products are produced in a post -bomb environment, all pMC values (after correction for isotopic fraction) must be multiplied by 0.95 (as of 2010) to better reflect the true biobased content of the sample. A biobased content that is greater than 103% suggests that either an analytical error has occurred, or that the source of biobased carbon is more than several years old.

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

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

Accordingly, in some embodiments, provided are BDO and/or 4-HB or a BDO and/or 4-

HB pathway intermediate thereof that has a carbon-12, carbon-13, and carbon-14 ratio that reflects an atmospheric carbon, also referred to as environmental carbon, uptake source. For example, in some aspects the BDO and/or 4-HB or a BDO and/or 4-HB intermediate thereof can have an Fm value of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or as much as 100%. In some such embodiments, the uptake source is CO ₂ . In some embodiments, provided is BDO and/or 4- HB or a BDO and/or 4-HB intermediate thereof that has a carbon-12, carbon-13, and carbon-14 ratio that reflects petroleum-based carbon uptake source. In this aspect, the BDO and/or 4-HB or a BDO and/or 4-HB intermediate thereof can have an Fm value of less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2% or less than 1%. In some embodiments, provided is BDO and/or 4-HB or a BDO and/ or 4-HB intermediate thereof that has a carbon-12, carbon-13, and carbon-14 ratio that is obtained by a combination of an atmospheric carbon uptake source with a petroleum-based uptake source. Using such a combination of uptake sources is one way by which the carbon-12, carbon-13, and carbon-14 ratio can be varied, and the respective ratios would reflect the proportions of the uptake sources.

Further, the disclosure relates, in part, to biologically produced BDO and/or 4-HB or BDO and/or 4-HB intermediate thereof as disclosed herein, and to the products derived therefrom, wherein the BDO and/or 4-HB or a BDO and/or 4-HB intermediate thereof has a carbon-12, carbon-13, and carbon-14 isotope ratio of about the same value as the CO ₂ that occurs in the environment. For example, in some aspects, provided are a bioderived BDO and/or 4-HB or a bioderived BDO and/or 4-HB intermediate thereof having a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the CO ₂ that occurs in the environment, or any of the other ratios disclosed herein. It is understood, as disclosed herein, that a product can have a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the CO ₂ that occurs in the environment, or any of the ratios disclosed herein, wherein the product is generated from bioderived BDO and/or 4-HB or a bioderived BDO and/or 4-HB intermediate thereof as disclosed herein, wherein the bioderived product is chemically modified to generate a final product. Methods of chemically modifying a bioderived product of BDO and/or 4-HB, or an intermediate thereof, to generate a desired product are well known to those skilled in the art, as described herein. Also provided are plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG)(also referred to as PTMO, polytetramethylene oxide) and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like, having a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the CO ₂ that occurs in the environment, wherein the plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like, are generated directly from or in combination with bioderived BDO and/or 4-HB or a bioderived BDO and/or 4-HB intermediate thereof as disclosed herein.

Those skilled in the art will understand that an organism can be engineered that secretes the biosynthesized compounds when grown on a carbon source such as a methanol alone or combined with other carbohydrates. Such compounds include, for example, BDO and any of the intermediate metabolites in the BDOP. All that is required is to engineer in one or more of the required enzyme or protein activities to achieve biosynthesis of the desired compound or intermediate including, for example, inclusion of some or all of the BDO biosynthetic pathways. Accordingly, provided herein is an organism that produces and/or secretes BDO when grown on a carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the BDOP when grown on a carbohydrate or other carbon source. The BDO producing microbial organisms provided herein can initiate synthesis from an intermediate. The same holds true for intermediates in the formaldehyde assimilation.

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

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

In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of from 200: 1 to 1 :200. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of from 100: 1 to 1 : 100. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of from 100: 1 to 5: 1. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of from 50: 1 to 5 : 1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 100: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 90: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 80: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 70: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 60: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 50: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 40: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 30: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 20: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 10: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 5 : 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 2: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 : 1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 : 100. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :90. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :80. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :70. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :60. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :50. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :40. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :30. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :20. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 : 10. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :5. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1 :2. In certain embodiments of the ratios provided above, the sugar is a sugar-containing biomass.

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

In certain embodiments, the carbon source comprises a mixture of methanol and formate, and a sugar (e.g., glucose). In certain embodiments, sugar is provided for sufficient strain growth. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of from 200: 1 to 1 :200. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of from 100:1 to 1 :100. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of from 100: 1 to 5: 1. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of from 50: 1 to 5 : 1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 100:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 90:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 80: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 70: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 60: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 50: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 40: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 30: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 20: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 10: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 5 : 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 2: 1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 : 1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 : 100. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :90. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :80. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :70. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :60. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :50. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :40. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :30. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :20. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 : 10. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :5. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1 :2. In certain embodiments of the ratios provided above, the sugar is a sugar-containing biomass.

1. An engineered cell either (a) expressing a non-natural NAD ⁺ -dependent alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase and capable of at least two fold greater conversion of methanol or ethanol to_formaldehyde or acetaldehyde, respectively, as compared to an engineered cell expressing the corresponding alcohol dehydrogenase without amino acid substitution or (b) expressing a first sequence that is a non-natural NAD ⁺ -dependent alcohol dehydrogenase comprising at least one amino acid substitution capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to an engineered cell expressing a second sequence that is a non-natural NAD ⁺ -dependent alcohol dehydrogenase, wherein the first and second sequences differ with regards to the at least one amino acid substitution.

2. The engineered cell of embodiment 1 further comprising one or more metabolic pathway transgene(s) encoding a protein of a metabolic pathway that promotes production of a target product or intermediate thereof.

3. The engineered cell of embodiments 1 or 2, wherein expression of the non-natural alcohol dehydrogenase provides an increased amount of reducing equivalents for an increase in a target product and/or for increased fixation of carbon from the formaldehyde into a target product.

4. The engineered cell of embodiment any of the previous embodiments further comprising a transgene encoding an enzyme to convert the formaldehyde to formate thereby generating reducing equivalents useful to product the target product and/or able to fix carbon of formate into the target product.

5. The engineered cell of any of the previous embodiments wherein the target product is selected from the group consisting of a diol, 1 ,4-butadiol, 1,3-butadiol, butadiene, succinate, adipate, HMD A, 6-aminocaproic acid (6ACA), or an intermediate compound thereof.

6. The engineered cell of any of the previous embodiments further comprising one or more alcohol metabolic pathway gene(s) encoding a protein selected from the group consisting of a), a formate dehydrogenase (EM8), a formaldehyde activating enzyme (EM10), a formaldehyde dehydrogenase (EMI 1), a S-(hydroxymethyl)glutathione synthase (EM12), a glutathione- dependent formaldehyde dehydrogenase (EM13), a S-formylglutathione hydrolase (EM14), a formate hydrogen lyase (EM15), and a hydrogenase (EM16).

7. The engineered cell of any of the previous embodiments further comprising one or more alcohol metabolic pathway gene(s) encoding a protein selected from the group consisting of a succinyl-CoA reductase (aldehyde forming) (EB3), a 4-hydroxybutyrate (4-HB) dehydrogenase

(EB4), a 4-HB kinase (EB5), a phosphotrans-4-hydroxybutyrylase (EB6), a 4-hydroxybutyryl-CoA reductase (aldehyde forming) (EB7), a 1 ,4-butanediol dehydrogenase (EB8); a succinate reductase (EB9), a succinyl-CoA reductase (alcohol forming) (EB10), 4-hydroxybutyryl-CoA transferase (EB11), a 4-hydroxybutyryl-CoA synthetase (EBl 2), a 4-HB reductase (EB13), and a 4- hydroxybutyryl-CoA reductase (alcohol forming) (EBl 5), a succinyl-CoA transferase (EBl), and a succinyl-CoA synthetase (EB2A).

8. A composition comprising the cell of any of the previous embodiments, or a cell extract thereof.

9. The composition of embodiment 8 wherein the composition is a cell culture composition, optionally comprising a target product or intermediate thereof.

10. A cell culture composition comprising a target product or intermediate thereof produced by the cell of any of the previous embodiments.

11. A composition comprising a target product or intermediate thereof produced by the cell of any of the previous embodiments, optionally comprising cell debris and/or residual culture medium.

12. The composition of embodiment 11 comprising target product or intermediate thereof. which is at least 50%, 60%, 70%, 80%, 90%, 95%, 96, 97, 98, 99 or 99.9% pure in the composition.

13. The composition of embodiment 11 or 12 comprising a detectable trace amount of a nucleic acid encoding the non-natural NAD ⁺ -dependent alcohol dehydrogenase, or a detectable trace amount of a metabolic pathway intermediate or product not produced in the corresponding original cell absent expression of the non-natural NAD+-dependent alcohol dehydrogenase.

14. The composition of embodiment 11 wherein the metabolic pathway intermediate or product is 4-hydroxybutyrate (4-HB) and 1,3 propanediol (1,3-PDO).

15. The composition of embodiment 14 comprising an amount of 1,4-butanediol or 1,3- butanediol in the range of 70-90 % (vol/vol) and an amount of water in the range of 10-30 %

(vol/vol).

16. A method for increasing the conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, comprising a step of (a) culturing an engineered cell expressing a NAD ⁺ -dependent non-natural alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase in a culture medium comprising methanol or ethanol, where in said culturing the cell provides at least two fold greater conversion of the methanol or ethanol to formaldehyde or acetaldehyde respectively, as compared to an engineered cell expressing the corresponding alcohol dehydrogenase without amino acid substitution.

17. A method for increasing the conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, comprising a step of (a) providing a reaction composition having a pH in the range of 6 - 8, the composition comprising a NAD -dependent non-natural alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase and methanol or ethanol, where in the composition said NAD ⁺ -dependent non-natural alcohol dehydrogenase provides at least two fold greater conversion of methanol or ethanol to a formaldehyde or acetaldehyde respectively, as compared to the corresponding alcohol dehydrogenase without amino acid substitution.

18. A nucleic acid encoding a NAD ⁺ -dependent non-natural alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase capable, when expressed in a cell, of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde respectively, as compared to the corresponding alcohol dehydrogenase without amino acid substitution.

19. An expression construct comprising the nucleic acid of embodiment 18.

20. A NAD ⁺ -dependent non-natural alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase capable, when expressed in a cell, of at least two fold greater conversion of a methanol or ethanol to formaldehyde or acetaldehyde respectively, as compared to the corresponding alcohol dehydrogenase without amino acid substitution.

21. The subject matter of any of the previous embodiments wherein the methanol is converted to formaldehyde.

22. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase is capable of at least three fold greater, of at least four fold, of at least five fold, of at least six fold, of at least seven fold, at least 8 fold, at least 9 fold, at least 10 fold, or at least 11 fold, conversion of methanol or ethanol to a formaldehyde or acetaldehyde, respectively, in vivo, as compared to the corresponding alcohol dehydrogenase without amino acid substitution.

23. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase is capable of at least three fold greater, of at least four fold, of at least five fold, of at least six fold, of at least seven fold, at least 8 fold, at least 9 fold, at least 10 fold, or at least 11 fold, conversion of methanol or ethanol to a formaldehyde or acetaldehyde, respectively, in vitro, as compared to the corresponding alcohol dehydrogenase without amino acid substitution.

24. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase is capable of an increase in conversion of methanol or ethanol to a formaldehyde or acetaldehyde respectively, as compared to the corresponding alcohol dehydrogenase without amino acid substitution, in the range of two fold to twelve fold greater, in the range of two fold to eleven fold greater, in the range of two fold to ten fold greater, in the range of two fold to nine fold greater, in the range of two fold to eight fold greater, in the range of two fold to seven fold greater, in the range of two fold to six fold greater, in the range of two fold to five fold greater, or in the range of two fold to four fold greater.

25. A NAD ⁺ -dependent_non-natural alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase having a catalytic efficiency (k _cat /K _m ) for the conversion of methanol to formaldehyde of 8.6 X 10 ^"4 or greater.

26. The subject matter of any of the previous embodiments comprising an activator protein that is an Act Nudix hydrolase.

27. A method of producing a target product or its intermediate comprising culturing the engineered cell of embodiment any of embodiments 1-7 in a culture medium comprising methanol or ethanol to produce the the target product (TP) or its intermediate (INT).

28. The method of embodiment 27 further comprising a step of isolating or purifying target product (TP) or its intermediate (INT).

29. The method of embodiment 28 wherein the step of isolating or purifying comprises one or more of continuous liquid-liquid extraction, pervaporation, evaporation, filtration, membrane filtration (including reverse osmosis, nanofiltration, ultrafiltration, and microfiltration), membrane filtration with diafiltration, membrane separation, reverse osmosis, electrodialysis, distillation, extractive distillation, reactive distillation, azeotropic distillation, crystallization and

recrystalhzation, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, carbon adsorption, hydrogenation, and ultrafiltration.

30. The method of embodiment 29 selcted from the group consisting of: (a) target product:

1 ,4-butanediol, purification: distillation; (b) target product: 1,3-butanediol, purification: distillation; (c) target produc Butadiene, purification: distillation; (d) target product: 6-AminoCaproic Acid, purification:crystallization, (a) target product: caprolactam, purification: distillation as a final step; (e) target product: hexamethylenediame (HMD A), purification:crystallization; (f) target product: Adipic acid, purification: crystallization (adipic acid crystals); (g) target product: Crotyl alcohol, purification:distillation (h) target product: methyl vinyl carbinol, purification: distillation; (i) target product succinic acid-crystallization (succinic acid crystals); (J) target product :n-propanol, purification:distillation; (k) target product: isopropanol, purification: distillation; (1) target product propylene, purification:distillation; (m) target product: methacrylic acid,

purification:crystallization, distillation, or extraction (n) target product: methylmethacrylate (MMA) or another ester, purification: distillation or crystallization.

31. The method of any of embodiments 31 wherein the step of isolating or purifying further comprises distillation.

32. The method of embodiments 28-31 wherein the target product is a diol.

33. The method of embodiments 28-32 wherein the target product is a diol is 1,4- butanediol or 1,3-butanediol. 34. The method of embodiments 28-32 comprising purifying the target product to at least 50%, 60%, 70%, 80%, 90%, 95%, 96, 97, 98, 99 or 99.9% purity in a composition.

35. A method of preparing a polymer comprising obtaining a target product produced by the engineered cell of any of embodiments 1-7 or method of any of the embodiments 27-34 and polymerizing the target product, optionally with one or more other monomeric compounds, to provide a polymeric product.

36. The method of embodiment 35 further comprising a step of isolating or purifying the polymeric product.

37. The method of embodiments 35 or 36 comprising purifying the polymer product to at least 50%, 60%, 70%, 80%, 90%, 95%, 96, 97, 98, 99 or 99.9% purity in a composition.

38. A polymer prepared according to the method of any of embodiments 35-37.

39. The polymer of embodiment 38 which is a homopolymer or copolymer.

40. The polymer of embodiment 39 that is selected from the group consisting of polybutylene terephthalate (PBT) and polybutylene succinate (PBS).

41. A composition comprising a polymer blend comprising the polymer of any ones of embodiments 38-40.

42. An article comprising the polymer or composition any one of embodiments 38-41.

43. The article of embodiment 42 which is a plastic article.

44. The article of embodiment 17d or 17e which is molded, extruded, or shaped from the polymer or composition any one of embodiments 41-43.

45. A biobased product comprising target product produced by the engineered cell of any of embodiments 1-7 or the polymer of any ones of embodiments 38-40 wherein said biobased product is

(i) a polymer, THF or a THF derivative, or GBL or a GBL derivative;

(ii) a plastic, elastic fiber, polyurethane, polyester, polyhydroxyalkanoate, poly-4-HB, co-polymer of poly-4-HB, poly(tetramethylene ether) glycol, polyurethane- polyurea copolymer, spandex, elastane, Lycra™, or nylon;

(iii) a polymer, a resin, a fiber, a bead, a granule, a pellet, a chip, a plastic, a polyester, a thermoplastic polyester, a molded article, an injection-molded article, an injection- molded part, an automotive part, an extrusion resin, an electrical part and a casing; and optionally where the biobased product is reinforced or filled and further where the biobased product is glass-reinforced or -filled or mineral-reinforced or -filled;

(iv) a polymer, wherein the polymer comprises polybutylene terephthalate (PBT);

(v) a polymer, wherein the polymer comprises PBT and the biobased product is a resin, a fiber, a bead, a granule, a pellet, a chip, a plastic, a polyester, a thermoplastic polyester, a molded article, an injection-molded article, an injection-molded part, an automotive part, an extrusion resin, an electrical part and a casing; and optionally where the biobased product is reinforced or filled and further where the biobased product is glass-reinforced or -filled or mineral-reinforced or -filled; a THF or a THF derivative, wherein the THF derivative is polytetramethylene glycol (PTMEG), a polyester ether (COPE) or a thermoplastic polyurethane;

(viii) a THF derivative, wherein the THF derivative comprises a fiber; or

(ix) a GBL or a GBL derivative, wherein the GBL derivative is a pyrrolidone;

wherein said biobased product optionally comprises at least 5%, at least 10%, at least 20%, at least 30%, at least 40% or at least 50% bioderived BDO; and/or wherein said biobased product optionally comprises a portion of said bioderived BDO as a repeating unit.

46. A molded product obtained by molding the biobased product of embodiment 10.

47. A process for producing the biobased product of embodiment 45, comprising chemically reacting said bioderived BDO with itself or another compound in a reaction that produces said biobased product.

48. A polymer comprising or obtained by converting the bioderived BDO of embodiment

45.

49. A method for producing a polymer, comprising chemically of enzymatically converting the bioderived BDO of embodiment 45 to the polymer.

50. A composition comprising the bioderived BDO of embodiment 45, or a cell lysate or culture supernatant thereof.

51. A method of producing an intermediate of glycolysis and/or an intermediate of a metabolic pathway that can be used in the formation of biomass, comprising culturing the engineered cell of any one of embodiments 1-7 under conditions and for a sufficient period of time to produce the intermediate, and optionally wherein the intermediate is consumed to provide a reducing equivalent or to incorporate into BDO or target product.

52. The method of embodiment 51, wherein the organism is cultured in a medium comprising biomass, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, starch, glycerol, methanol, carbon dioxide, formate, methane, or any combination thereof as a carbon source.

53. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase has sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to an NAD ⁺ -dependent_alcohol dehydrogenase template selected from the group consisting of SEQ ID NO: l (MDH MGA3 17392), EIJ77596.1, AAA22593.1, EIJ77618.1, EIJ78790.1, EIJ80770.1, EIJ78397.1, EIJ83020.1, EFI69743.1, YP_004860127.1, YP_001699778.1, ZP l 1313277.1, ZP_05587334.1, YP_004681552.1, AGF87161, YP 002138168.1, YP 359772.1, YP 001343716.1, ZP 16224338.1, AAC45651.1, YP_007491369.1, YP_002434746, ΥΡ_005052855, ΝΡ_561852.1, ΥΡ_001447544,

YP 001113612.1, YP 011618, ZP 01220157.1, YP 003990729.1, ZP 07335453.1, NP 717107, ΥΡ 003310546.1, ZP 10241531.1, YP 001337153.1, YP 026233.1, ΥΡ 694908, YP 725376.1, YP 001663549, EKC54576, YP 001126968.1 or a fragment of said template having said dehydrogenase activity with an amino-terminal deletion, carboxy-terminal deletion, or both, the fragment having a sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to the template.

54. The subject matter of any of the previous embodiments said template is selected from the group consisting of EIJ77596.1, EIJ78397.1, EFI69743.1, YP 001699778.1, YP 002138168.1, YP_359772.1, YP_005052855, NP_561852.1, YP_001447544, ZP 01220157.1, YP 003990729.1, ZP 10241531.1, and YP 026233.1.

55. The subject matter of any of the previous embodiments wherein the alcohol dehydrogenase is a methanol dehydrogenase.

56. The subject matter of embodiment 55 wherein the methanol dehydrogenase is from bacteria.

57. The subject matter of embodiment 56 wherein the methanol dehydrogenase is from Bacillus.

58. The subject matter of embodiment 57 wherein the methanol dehydrogenase is from

Bacillus methanolicus MGA3 or Bacillus methanolicus PB1.

59. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase has a sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to any one of the templates of embodiment 53, and wherein the dehydrogenase comprises one or more amino acid substitutions based on formula: R ^J XR ² , where R ¹ is an original amino acid at position X of the template, and R ² is the variant amino acid that replaces R ¹ at a position on the template corresponding to X, wherein XR ² is selected from the group consisting of (a) 11T, 38N, 42Q, 48D, 531, 56K, 60E, 61A, 63F, 65Q, 70N, 711, 71T, 71V, 74S, 81G, 84R, 86K, 87K, 94V, 99P, 99T, 103V, 106L, 107S, 108V, 108W, 109Y, 112K, 112R, 115H, 116F, 117D, 117Q, 117Y, 120H, 120R, 121A, 121D, 121E, 121L, 121M, 121R, 121S, 121T, 121V, 121W, 121Y, 122A, 122P, 123D, 1231, 123L, 123R, 123Y, 1241, 124L, 124R, 125C, 125G, 125W, 126G, 126V, 127C, 127R, 128A, 128R, 128S, 129A, 129M, 129P, 129S, 130F, 1301, 130Y, 134T, 143T, 145M, 146N, 147R, 148A, 148F, 148G, 1481, 148T, 148V, 148W, 149L, 149M, 149T, 149V, 150A, 1501, 152M, 155V, 157N, 158E, 158H, 158K, 158W, 161A, 161G, 161Q, 161S, 161V, 163F, 163N, 163Q, 163T, 164G, 164N, 165G, 181R, 184T, 186M, 190A, 190S, 199V, 217K, 226M, 256C, 267H, 269S, 270M, 270S, 270Y, 296S, 298H, 300T, 302V, 312V, 316V, 323M, 333L, 336L, 337C, 343D, 344A, 344G, 345E, 350K, 354M, 355D, 3551, 355K, 358G, 360A, 360G, 360K, 360R, 360S, 361N, 361R, 363K, and 379M or group consisting of (b) 38N, 60E, 711, 71V, 87K, 99T, 103V, 107S, 108V, 108W, 109Y, 115H, 116F, 117D, 117Q, 121D, 121E, 121L, 121M, 121R, 121 S, 121T, 121V, 121W, 121Y, 122P,

123D, 1231, 123L, 123R, 123Y, 1241, 124L, 125C, 125G, 125V, 125W, 126G, 127C, 127R, 128A, 128R, 128S, 129A, 129M, 129P, 129S, 129V, 130F, 1301, 130Y, 134T, 143T, 146N, 149L, 149M, 149T, 149V, 150A, 157N, 158E, 158H, 158K, 158W, 163Q, 164N, 267H, 270M, 270S, 270Y, 345E, 355D, 360G, 360K, 360R, 360S, and 361R.

60. The subject matter of embodiment 59 wherein XR ² is selected from the group consisting of 107S, 121D, 123D, 1231, 123L, 123R, 123Y, 129A, 129M, 129P, 129S, 129V, 130F, 1301, 130Y, 143T, 146N, 149L, 149M, 149T, 149V, 158E, 158H, 158K, 158W, 267H, 270M, 270S, 270Y, 355D, 360G, 360K, 360R, and 360S

61. The subject matter of embodiment 60 wherein R ^J XR ² is selected from the group consisting of (a) SI IT, D38N, H42Q, E48D, N53I, E56K, D60E, V61A, I63F, P65Q, D70N, P71I, P71T, P71V, T74S, D81G, K84R, E86K, N87K, I94V, S99P, S99T, A103V, I106L, G107S, L108V, L108W, V109Y, N112K, N112R, R115H, II 16F, N117D, Nl 17Q, Nl 17Y, Q120H, Q120R, G121A, G121D, G121E, G121L, G121M, G121R, G121 S, G121T, G121V, G121W, G121Y, V122A, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, S124R, V125C, V125G, V125W, E126G, E126V, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T145M, T146N, S147R, L148A, L148F, L148G, L148I, L148T, L148V, L148W, A149L, A149M, A149T, A149V, V150A, V150I, T152M, A155V, K157N, V158E, V158H, V158K, V158W, P161A, P161G, P161Q, P161 S, P161V, I163F, I163N, I163Q, I163T, D164G, D164N, E165G, K181R, A184T, L186M, T190A, T190S, I199V, Q217K, L226M, G256C, Q267H, G269S, G270M, G270S, G270Y, T296S, R298H, A300T, I302V,G312V, A316V, I323M, F333L, P336L, S337C, G343D, V344A, V344G, K345E, E350K, K354M, N355D, N355I, N355K, E358G, V360A, V360G, V360K, V360R, V360S, C361N, C361R, Q363K, and K379M or (b) D38N, D60E, P71I, P71V, N87K, S99T, A103V, G107S, L108V, L108W, V109Y, R115H, I116F, Nl 17D, Nl 17Q, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, V125C, V125G, V125W, E126G, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T146N, A149L, A149M, A149T, A149V, V150A, K157N, V158E, V158H, V158K, V158W, I163Q, D164N, Q267H, G270M, G270S, G270Y, K345E, N355D, V360G, V360K, V360R, V360S, and C361R.

62. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase has sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to any one of the templates of embodiment 53, and comprises a original amino acid at all positions that are not substituted at amino acid position numbers of group (a) 11, 38, 42, 48, 53, 56, 60, 61, 63, 65, 70, 71, 74, 81, 84, 86, 87, 94, 99, 103, 106, 107, 108, 109, 112, 115, 116, 117, 117, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 145, 146, 147, 148, 149, 150, 152, 155, 157, 158, 161, 163, 164, 165, 181, 184, 186, 190, 199, 217, 226, 256, 267, 269, 270, 296, 298, 300, 302, 312, 316, 323, 333, 336, 337, 343, 344, 345, 350, 354, 355, 358, 360, 361, 363 and 379; or of group (b) 38, 60, 71, 87, 99, 103, 107, 108, 109, 115, 116, 117, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 146, 149, 150, 157, 158, 163, 164, 267, 270, 345, 355, 360, and 361.

63. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase has sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to any one of the templates of embodiment 53, and comprises a original amino acid at all positions that are not amino acid position numbers 107, 121, 123, 129, 130, 143, 146, 149,158, 267, 270, 355, 360.

64. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises two, three, four, five, six, seven, eight, nine, ten, eleven or twelve, amino acid substitutions selected from the group consisting of: . (a) SI IT, D38N, H42Q, E48D, N53I, E56K, D60E, V61A, I63F, P65Q, D70N, P71I, P71T, P71V, T74S, D81G, K84R, E86K, N87K, I94V, S99P, S99T, A103V, I106L, G107S, L108V, L108W, V109Y, N112K, Nl 12R, Rl 15H, II 16F, Nl 17D, Nl 17Q, Nl 17Y, Q120H, Q120R, G121A, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122A, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, S124R, V125C, V125G, V125W, E126G, E126V, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T145M, T146N, S147R, L148A, L148F, L148G, L148I, L148T, L148V, L148W, A149L, A149M, A149T, A149V, V150A, V150I, T152M, A155V, K157N, V158E, V158H, V158K, V158W, P161A, P161G, P161Q, P161S, P161V, I163F, I163N, I163Q, I163T, D164G, D164N, E165G, K181R, A184T, L186M, T190A, T190S, I199V, Q217K, L226M, G256C, Q267H, G269S, G270M, G270S, G270Y, T296S, R298H, A300T, I302V,G312V, A316V, I323M, F333L, P336L, S337C, G343D, V344A, V344G, K345E, E350K, K354M, N355D, N355I, N355K, E358G, V360A, V360G, V360K, V360R, V360S, C361N, C361R, Q363K, and K379M or (b) D38N, D60E, P71I, P71V, N87K, S99T, A103V, G107S, L108V, L108W, V109Y, R115H, II 16F, N117D, N117Q, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, V125C, V125G, V125W, E126G, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T146N, A149L, A149M, A149T, A149V, V150A, K157N, V158E, V158H, V158K, V158W, I163Q, D164N, Q267H, G270M, G270S, G270Y, K345E, N355D, V360G, V360K, V360R, V360S and C361R.

65. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a set of amino acid substitutions selected from the group consisting of (a) D70N, L148G, P161G, V360A; (b) D70N, L148G, V360A, C361N; (c) D70N, L148V, V150I, P161A, V360G; (d) D70N, L148V, V360G; (e) D70N, P161A, V360A; (f) D70N, P161V, V360G, C361N; (g) D70N, V150L P161A, V360A; (h) D70N, V150L P161V, V360G, C361N; (i) E48D, L148V, P161A, V360A; (J) L148G, P161A, V360A, C361N; (k) L148G, P161A, V360G; (1) L148G, P161A, V360G, C361N; (m) L148G, P161G, V360A; (n) L148G, P161G, V360G, C361N; (o) L148G, V360A, C361N; (p) L148G, V360G, C361N; (q) L148L P161G, V360G; (r) L148I, P161V, V360G; (s) L148T, V150L V360A; (t) L148T, V360G; (u) L148V, P161A, V360A; (v) L148V, V150I, P161A, V360A; (w) L148V, V150L P161A, V360A, C361N; (x) L148V, V150L P161A, V360G; (y) L148V, V150L P161A, V360G, C361N; (z) L148V, V150L P161A, V360G, C361N; (aa) L148V, V150I, P161G, V360A; (ab) L148V, V150I, P161V, V360G, C361N; (ac) L148W, P161A, V360A, C361N; (ad) Nl 12K , S147R, P161A, V360A; (ae) P161A, Q217K, V360A, C361N; (af) P161A, V360A, C361N; (ag) P161A, V360G; (ah) P161V, E358G, V360G; (ai) P161V, V360A, C361N; (aj) L148W, P161A, V360A, C361N; (ak) N112K , S147R, P161A, V360A; (al) P161A, Q217K, V360A, C361N; (am) P161A, V360A, C361N; (an) P161A, V360G; (ao) P161V, E358G, V360G; (ap) P161V, V360A, C361N; (aq) P161V, V360G; (ar) P65Q, L148G, V150I, P161A, V360G, C361N; (as) S147R, L148A, V150L P161A, V360G; (at) S147R, L148F, V150I, P161G, V360G; (au) S147R, L148V, P161G, V360A; (av) P161V, V360G; (aw) P65Q, L148G, V150I, P161A, V360G, C361N; (ax) S147R, L148A, V150L P161A, V360G; (ay) S147R, L148F, V150L P161G, V360G; (az) S147R, L148V, P161G, V360A; (aaa) S147R, L148V, P161V, V360G; (aab) S147R, L148V, V150L P161A, C361N; (aac) S147R, L148V, V150I, P161G, V360G; (aad) S147R, P161A, V360A; (aae) S147R, P161A, V360A, C361N; (aaf) S147R, P161A, V360G; (aag) S147R, P161V, V360G; (aah) S147R, P161V, V360G, C361N; (aai) S147R, V150I, P161V, V360A; (aaj) S147R, V150L V360A, C361N; (aak) T145M, L148L V360G; (aal) V150L I302V, V360G, C361N; (aam) V150I, P161A, C361N; (aan) V150L P161G, V360A, C361N; (aao) V150L P161G, V360G; (aap) V150I, P161G, V360G, C361N; (aaq) V150I. P161V, C361N; (aar) V150I, P161V, K354R, V360A, C361N; (aas) V150L P161V, V360A, C361N; (aat) V150L P161V, V360G, C361N; (aau) V150L V360A, C361N; (aav) V150I, V360G; (aaw) SI IT, T74S, G269S, V344A; (aax) K84R, I163T; (aay) V122A, I163N; (aaz) G107S, F333L; (aaaa) V129M, T152M, G343D; (aaab) I63F, N355K; (aaac) G107S, F333L; (aaad) E86K, S99T, A149V; (aaae) N53I, V158E; (aaaf) N355I, K379M; (aaag) H42Q, G107S; (aaah) Q120H, I163N; (aaai) A149V, I323M; (aaaj) G107S, F333L; (aaak) D164G, K181R; (aaal) A155V, R298H, N355D; (aaam) N123D, E165G; (aaan) I163F, L186M; (aaao) G121A, T296S; (aaap) I94V, S99P, N123I; (aaaq) E126V, V129M, V344G; (aaar) Q120R, S143T; (aaas) G256C, A316V; (aaat) P161Q, G312V; (aaau) L226M, A300T, V360A; (aaav) S337C, E350K, N355D, Q363K; (aaaw) D81G, V158E; (aaax) I106L, Nl 17Y, E126V; (aaay) G107S, G121D; (aaaz) V61A, V158E; (aaaaa) N53I, V158E; (aaaab) Nl 17Y, T190S; (aaaac) S124R, I199V; (aaaad) K354M, C361R; (aaaae) A184T, C361R; (aaaag) E56K, Q267H; (aaaag) S124R, El 26G; (aaaah) T190A, N355K; (aaaai) P71T, F333L; (aaaaj) G107S, F333L; and (aaaak) N123I, P336L, (aaaal) D38D/A149V, (aaaam) D38N/V163V, (aaaan) D73D/L108V, (aaaao) G121R/P161 S, and (aaaap) N112R/P161S.

66. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of

TNA and VTNAF (SEQ ID NO: 79).

67. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of

VEV and GVEVA (SEQ ID NO: 80).

68. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of

DIA, PDIAD (SEQ ID NO: 81), DVA, and PDVAD (SEQ ID NO: 82).

69. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of

EKC and QEKCD (SEQ ID NO: 83).

70. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of

STH and GSTHD (SEQ ID NO: 84).

71. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of

TVK and DTVKA (SEQ ID NO: 85).

72. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of SLV,

GVV, GWV, GLY, ISLVA (SEQ ID NO: 86), IGVVA (SEQ ID NO: 87), IGWVA (SEQ ID NO: 88), and IGLYA (SEQ ID NO: 89)

73. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of ΗΓΝ, RFN, RID, RIQ, GHIND (SEQ ID NO: 90), GRFND (SEQ ID NO: 91), GRIDD (SEQ ID NO: 92), and GRIQD (SEQ ID NO: 93). 74. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of DVNSVEKPVV (SEQ ID NO: 94), EVNSVEKPVV (SEQ ID NO: 95), LVNSVEKPVV (SEQ ID NO: 96), MVNSVEKPVV (SEQ ID NO: 97), RVNSVEKPVV (SEQ ID NO: 98), SVNSVEKPW (SEQ ID NO: 99), TVNSVEKPW (SEQ ID NO: 100), WNSVEKPVV (SEQ ID NO: 101),

WVNSVEKPW (SEQ ID NO: 102), YVNSVEKPW (SEQ ID NO: 103), GPNSVEKPVV (SEQ ID NO: 104), GVDSVEKPW (SEQ ID NO: 105), GVISVEKPW (SEQ ID NO: 106),

GVLSVEKPW (SEQ ID NO: 107), GVRSVEKPVV (SEQ ID NO: 108), GVYSVEKPW (SEQ ID NO: 109). GVNIVEKPVV (SEQ ID NO: 110), GVNLVEKPW (SEQ ID NO: 111),

GVNSCEKPVV (SEQ ID NO: 112), GVNSGEKPVV (SEQ ID NO: 113), GVNSWEKPVV (SEQ ID NO: 114), GVNSVGKPW (SEQ ID NO: 115), GVNSVECPVV (SEQ ID NO: 116), GVNSVERPVV (SEQ ID NO: 117), GVNSVEKAW (SEQ ID NO: 118). GVNSVEKRVV (SEQ ID NO: 119). GVNSVEKSW (SEQ ID NO: 120). GVNSVEKPAV (SEQ ID NO: 121).

GVNSVEKPMV (SEQ ID NO: 122). GVNSVEKPPV (SEQ ID NO: 123). GVNSVEKPSV (SEQ ID NO: 124). GVNSVEKPVF (SEQ ID NO: 125). GVNSVEKPVI (SEQ ID NO: 126), and GVNSVEKPVY (SEQ ID NO: 127).

75. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of TETT (SEQ ID NO: 128), SETN (SEQ ID NO: 129), GTETTS (SEQ ID NO: 130), and GSETNS (SEQ ID NO: 131).

76. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of LLVI (SEQ ID NO: 132), LMVI (SEQ ID NO: 133), LTVI (SEQ ID NO: 134), LVVI (SEQ ID NO: 135), and LAAI (SEQ ID NO: 136).

77. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of NVKMPVID (SEQ ID NO: 137), KEKMPVID (SEQ ID NO: 138), KHKMPVID (SEQ ID NO: 139), KKKMPVID (SEQ ID NO: 140), KWKMPVID (SEQ ID NO: 141), KVKMPVQD (SEQ ID NO: 142), and KVKMPVIN (SEQ ID NO: 143).

78. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of HVGG (SEQ ID NO: 144), QVGM (SEQ ID NO: 145), QVGS (SEQ ID NO: 146), and QVGY (SEQ ID NO: 147).

79. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of VEE and GVEEE (SEQ ID NO: 148). 80. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of

DAYEDVC (SEQ ID NO: 149), NAYEDGC (SEQ ID NO: 150), NAYEDKC (SEQ ID NO: 151), and NAYEDRC (SEQ ID NO: 152), and NAYEDSC (SEQ ID NO: 153), and NAYEDVR (SEQ ID NO: 154).

81. A nucleic acid encoding the non-natural alcohol dehydrogenase of any of embodiments

53-80.

82. A non-natural alcohol dehydrogenase has sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to an NAD ⁺ -dependent alcohol dehydrogenase template selected from the group consisting of SEQ ID NO: l (MDH MGA3_17392), EIJ77596.1, AAA22593.1, EIJ77618.1, EIJ78790.1, EIJ80770.1, EIJ78397.1, EIJ83020.1, EFI69743.1, YP_004860127.1, YP_001699778.1, ZP_11313277.1, ZP_05587334.1, YP 004681552.1, AGF87161, YP 002138168.1, YP 359772.1, YP 001343716.1,

ZP_16224338.1, AAC45651.1, YP_007491369.1, YP_002434746, YP_005052855,

NP 561852.1, YP 001447544, YP 001113612.1, YP 011618, ZP 01220157.1, YP 003990729.1, ZP_07335453.1, NP_717107, YP 003310546.1, ZP l 0241531.1, YP_001337153.1,

YP 026233.1, YP 694908, YP 725376.1, YP 001663549, EKC54576, YP 001126968.1 or a fragment of said template having said dehydrogenase activity with an amino-terminal deletion, carboxy-terminal deletion, or both, the fragment having a sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to the template.

83. A non-natural alcohol dehydrogenase of embodiment 82 wherein said template is selected from the group consisting of EIJ77596.1, EIJ78397.1, EFI69743.1, YP 001699778.1, YP_002138168.1, YP_359772.1, YP_005052855, NP_561852.1, YP_001447544, ZP_01220157.1, YP 003990729.1, ZP 10241531.1, and YP 026233.1.

84. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase has a sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to any one of the templates of embodiment 53, and wherein the dehydrogenase comprises one or more amino acid substitutions based on formula: R ^J XR ² , where R ¹ is a original amino acid at position X of the template, and R ² is the variant amino acid that replaces R ¹ at a position on the template corresponding to X, wherein XR ² is selected from the group consisting of (a) 11T, 38N, 42Q, 48D, 531, 56K, 60E, 61A, 63F, 65Q, 70N, 711, 71T, 71V, 74S, 81G, 84R, 86K, 87K, 94V, 99P, 99T, 103V, 106L, 107S, 108V, 108W, 109Y, 112K, 112R, 115H, 116F, 117D, 117Q, 117Y, 120H, 120R, 121A, 121D, 121E, 121L, 121M, 121R, 121 S, 121T, 121V, 121W, 121Y, 122A, 122P, 123D, 1231, 123L, 123R, 123Y, 1241, 124L, 124R, 125C, 125G, 125W, 126G, 126V, 127C, 127R, 128A, 128R, 128S, 129A, 129M, 129P, 129S, 130F, 1301, 130Y, 134T, 143T, 145M, 146N, 147R, 148A, 148F, 148G, 1481, 148T, 148V, 148W, 149L, 149M, 149T, 149V, 150A, 1501, 152M, 155V, 157N, 158E, 158H, 158K, 158W, 161A, 161G, 161Q, 161 S, 161V, 163F, 163N, 163Q, 163T, 164G, 164N, 165G, 181R, 184T, 186M, 190A, 190S, 199V, 217K, 226M, 256C, 267H, 269S, 270M, 270S, 270Y, 296S, 298H, 300T, 302V, 312V, 316V, 323M, 333L, 336L, 337C, 343D, 344A, 344G, 345E, 350K, 354M, 355D, 3551, 355K, 358G, 360A, 360G, 360K, 360R, 360S, 361N, 361R, 363K, and 379M or the group consisting of (b) 38N, 60E, 711, 71V, 87K, 99T, 103V, 107S, 108V, 108W, 109Y, 115H, 116F, 117D, 117Q, 121D, 121E, 121L, 121M, 121R, 121 S, 121T, 121V, 121W, 121Y, 122P,

123D, 1231, 123L, 123R, 123Y, 1241, 124L, 125C, 125G, 125V, 125W, 126G, 127C, 127R, 128A, 128R, 128S, 129A, 129M, 129P, 129S, 129V, 130F, 1301, 130Y, 134T, 143T, 146N, 149L, 149M, 149T, 149V, 150A, 157N, 158E, 158H, 158K, 158W, 163Q, 164N, 267H, 270M, 270S, 270Y, 345E, 355D, 360G, 360K, 360R, 360S, and 361R.

85. A non-natural alcohol dehydrogenase of embodiment 82 wherein XR ² is selected from the group consisting of 107S, 121D, 123D, 1231, 123L, 123R, 123Y, 129 A, 129M, 129P, 129S, 129V, 130F, 1301, 130Y, 143T, 146N, 149L, 149M, 149T, 149V, 158E, 158H, 158K, 158W, 267H, 270M, 270S, 270Y, 355D, 360G, 360K, 360R, and 360S

86. A non-natural alcohol dehydrogenase of embodiment 82 wherein R ^J XR ² is selected from the group consisting of (a) SI IT, D38N, H42Q, E48D, N53I, E56K, D60E, V61A, I63F, P65Q, D70N, P71I, P71T, P71V, T74S, D81G, K84R, E86K, N87K, I94V, S99P, S99T, A103V, 1106L, Gl 07S, L 108 V, L 108 W, V 109Y, N 112K, N 112R, Rl 15H, 1116F, N 117D, N 117Q, N 117Y, Q120H, Q120R, G121A, G121D, G121E, G121L, G121M, G121R, G121 S, G121T, G121V, G121W, G121Y, V122A, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, S124R, V125C, V125G, V125W, E126G, E126V, K127C, K127R, P128A, P128R, P128S, V129A,

V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T145M, T146N, S147R, L148A, L148F, L148G, L148I, L148T, L148V, L148W, A149L, A149M, A149T, A149V, V150A, V150I, T152M, A155V, K157N, V158E, V158H, V158K, V158W, P161A, P161G, P161Q, P161S, P161V, I163F, I163N, I163Q, I163T, D164G, D164N, E165G, K181R, A184T, L186M, T190A, T190S, I199V, Q217K, L226M, G256C, Q267H, G269S, G270M, G270S, G270Y, T296S,

R298H, A300T, I302V,G312V, A316V, I323M, F333L, P336L, S337C, G343D, V344A, V344G, K345E, E350K, K354M, N355D, N355I, N355K, E358G, V360A, V360G, V360K, V360R, V360S, C361N, C361R, Q363K, and K379M; or the group consisting of (b) D38N, D60E, P71I, P71V, N87K, S99T, A103V, G107S, L108V, L108W, V109Y, Rl 15H, II 16F, Nl 17D, Nl 17Q, G121D, G121E, G121L, G121M, G121R, G121 S, G121T, G121V, G121W, G121Y, V122P,

N123D, N123I, N123L, N123R, N123Y, S124I, S124L, V125C, V125G, V125W, E126G, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T146N, A149L, A149M, A149T, A149V, V150A, K157N, V158E, V158H, V158K, V158W, I163Q, D164N, Q267H, G270M, G270S, G270Y, K345E, N355D, V360G, V360K, V360R, V360S, and C361R.

87. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase has sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to any one of the templates of embodiment 53, and comprises an original amino acid at all positions that are not substituted at amino acid position numbers of group (a) 11, 38, 42, 48, 53, 56, 60, 61, 63, 65, 70, 71, 74, 81, 84, 86, 87, 94, 99, 103, 106, 107, 108, 109, 112, 115, 116, 117, 117, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 145, 146, 147, 148, 149, 150, 152, 155, 157, 158, 161, 163, 164, 165, 181, 184, 186, 190, 199, 217, 226, 256, 267, 269, 270, 296, 298, 300, 302, 312, 316, 323, 333, 336, 337, 343, 344, 345, 350, 354, 355, 358, 360, 361, 363 and 379; or of group (b) 38, 60, 71, 87, 99, 103, 107, 108, 109, 115, 116, 117, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 146, 149, 150, 157, 158, 163, 164, 267, 270, 345, 355, 360, and 361.

88. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase has sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to any one of the templates of embodiment 53, and comprises an original amino acid at all positions that are not amino acid position numbers 107, 121, 123, 129, 130, 143, 146, 149,158, 267, 270, 355, 360.

89. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises two, three, four, five, six, seven, eight, nine, ten, eleven or twelve amino acid substitutions selected from the group consisting of: (a) SI IT, D38N, H42Q, E48D, N53I, E56K, D60E, V61A, I63F, P65Q, D70N, P71I, P71T, P71V, T74S, D81G, K84R, E86K, N87K, I94V, S99P, S99T, A103V, I106L, G107S, L108V, L108W, V109Y, N112K, Nl 12R, Rl 15H, II 16F, Nl 17D, Nl 17Q, Nl 17Y, Q120H, Q120R, G121A, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122A, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, S124R, V125C, V125G, V125W, E126G, E126V, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T145M, T146N, S147R, L148A, L148F, L148G, L148I, L148T, L148V, L148W, A149L, A149M, A149T, A149V, V150A, V150I, T152M, A155V, K157N, V158E, V158H, V158K, V158W, P161A, P161G, P161Q, P161 S, P161V, I163F, I163N, I163Q, I163T, D164G, D164N, E165G, K181R, A184T, L186M, T190A, T190S, I199V, Q217K, L226M, G256C,

Q267H, G269S, G270M, G270S, G270Y, T296S, R298H, A300T, I302V,G312V, A316V, I323M, F333L, P336L, S337C, G343D, V344A, V344G, K345E, E350K, K354M, N355D, N355I, N355K, E358G, V360A, V360G, V360K, V360R, V360S, C361N, C361R, Q363K, and K379M or the group consisting of (b) D38N, D60E, P71I, P71V, N87K, S99T, A 103V, G107S, LI 08V, L108W, V109Y, R115H, II 16F, Nl 17D, N117Q, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121 W, G121Y, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, V125C, V125G, V125W, E126G, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T146N, A149L, A149M, A149T, A149V, V150A, K157N, V158E, V158H, V158K, V158W, I163Q, D164N, Q267H, G270M, G270S, G270Y, K345E, N355D, V360G, V360K, V360R, V360S and C361R.

90. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a set of amino acid substitutions selected from the group consisting of (a) D70N, L148G, P161G, V360A; (b) D70N, L148G, V360A, C361N; (c) D70N, L148V, V150I, P161A, V360G; (d) D70N, L148V, V360G; (e) D70N, P161A, V360A; (f) D70N, P161V, V360G, C361N; (g) D70N, V150I, P161A, V360A; (h) D70N, V150I, P161V, V360G, C361N; (i) E48D, L148V, P161A, V360A; (J) L148G, P161A, V360A, C361N; (k) L148G, P161A, V360G; (1) L148G, P161A, V360G, C361N; (m) L148G, P161G, V360A; (n) L148G, P161G, V360G, C361N; (o) L148G, V360A, C361N; (p) L148G, V360G, C361N; (q) L148I, P161G, V360G; (r) L148I, P161V, V360G; (s) L148T, V150I, V360A; (t) L148T, V360G; (u) L148V, P161A, V360A; (v) L148V, V150I, P161A, V360A; (w) L148V, V150I, P161A, V360A, C361N; (x) L148V, V150I, P161A, V360G; (y) L148V, V150I, P161A, V360G, C361N; (z) L148V, V150I, P161A, V360G, C361N; (aa) L148V, V150I, P161G, V360A; (ab) L148V, V150I, P161V, V360G, C361N; (ac) L148W, P161A, V360A, C361N; (ad) Nl 12K , S147R, P161A, V360A; (ae) P161A, Q217K, V360A, C361N; (af) P161A, V360A, C361N; (ag) P161A, V360G; (ah) P161V, E358G, V360G; (ai) P161V, V360A, C361N; (aj) L148W, P161A, V360A, C361N; (ak) N112K , S147R, P161A, V360A; (al) P161A, Q217K, V360A, C361N; (am) P161A, V360A, C361N; (an) P161A, V360G; (ao) P161V, E358G, V360G; (ap) P161V, V360A, C361N; (aq) P161V, V360G; (ar) P65Q, L148G, V150I, P161A, V360G, C361N; (as) S147R, L148A, V150I, P161A, V360G; (at) S147R, L148F, V150I, P161G, V360G; (au) S147R, L148V, P161G, V360A; (av) P161V, V360G; (aw) P65Q, L148G, V150I, P161A, V360G, C361N; (ax) S147R, L148A, V150I, P161A, V360G; (ay) S147R, L148F, V150I, P161G, V360G; (az) S147R, L148V, P161G, V360A; (aaa) S147R, L148V, P161V, V360G; (aab) S147R, L148V, V150I, P161A, C361N; (aac) S147R, L148V, V150I, P161G, V360G; (aad) S147R, P161A, V360A; (aae) S147R, P161A, V360A, C361N; (aaf) S147R, P161A, V360G; (aag) S147R, P161V, V360G; (aah) S147R, P161V, V360G, C361N; (aai) S147R, V150I, P161V, V360A; (aaj) S147R, V150I, V360A, C361N; (aak) T145M, L148I, V360G; (aal) V150I, I302V, V360G, C361N; (aam) V150I, P161A, C361N; (aan) V150I, P161G, V360A, C361N; (aao) V150L P161G, V360G; (aap) V150I, P161G, V360G, C361N; (aaq) V150I, P161V, C361N; (aar) V150I, P161V, K354R, V360A, C361N; (aas) V150I, P161V, V360A, C361N; (aat) V150I, P161V, V360G, C361N; (aau) V150I, V360A, C361N; (aav) V150I, V360G; (aaw) SI IT, T74S, G269S, V344A; (aax) K84R, I163T; (aay) V122A, I163N; (aaz) G107S, F333L; (aaaa) V129M, T152M, G343D; (aaab) I63F, N355K; (aaac) G107S, F333L; (aaad) E86K, S99T, A149V; (aaae) N53I, V158E; (aaaf) N355I, K379M; (aaag) H42Q, G107S; (aaah) Q120H, I163N; (aaai) A149V, I323M; (aaaj) G107S, F333L; (aaak) D164G, K181R; (aaal) A155V, R298H, N355D; (aaam) N123D, E165G; (aaan) I163F, L186M; (aaao) G121A, T296S; (aaap) I94V, S99P, N123I; (aaaq) E126V, V129M, V344G; (aaar) Q120R, S143T; (aaas) G256C, A316V; (aaat) P161Q, G312V; (aaau) L226M, A300T, V360A; (aaav) S337C, E350K, N355D, Q363K; (aaaw) D81G, V158E; (aaax) I106L, N117Y, E126V; (aaay) G107S, G121D; (aaaz) V61A, V158E; (aaaaa) N53I, V158E; (aaaab) Nl 17Y, T190S; (aaaac) S124R, I199V; (aaaad) K354M, C361R; (aaaae) A184T, C361R; (aaaag) E56K, Q267H; (aaaag) S124R, El 26G; (aaaah) T190A, N355K; (aaaai) P71T, F333L; (aaaaj) G107S, F333L; and (aaaak) N123I, P336L, (aaaal) D38D/A149V, (aaaam) D38N/V163V, (aaaan) D73D/L108V, (aaaao) G121R/P161 S, and (aaaap) N112R/P161S.

91. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of

TNA and VTNAF (SEQ ID NO: 79).

92. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of

VEV and GVEVA (SEQ ID NO: 80).

93. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of

DIA, PDIAD (SEQ ID NO: 81), DVA, and PDVAD (SEQ ID NO: 82).

94. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of

EKC and QEKCD (SEQ ID NO: 83).

95. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of

STH and GSTHD (SEQ ID NO: 84).

96. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of

TVK and DTVKA (SEQ ID NO: 85).

97. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of SLV, GVV, GWV, GLY, ISLVA (SEQ ID NO: 86), IGVVA (SEQ ID NO: 87), IGWVA (SEQ ID NO: 88), and IGLYA (SEQ ID NO: 89).

98. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of ΗΓΝ, RFN, RID, RIQ, GHIND (SEQ ID NO: 90), GRFND (SEQ ID NO: 91), GRIDD (SEQ ID NO: 92), and GRIQD (SEQ ID NO: 93).

99. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of DVNSVEKPVV (SEQ ID NO: 94), EVNSVEKPVV (SEQ ID NO: 95), LVNSVEKPVV (SEQ ID NO: 96), MVNSVEKPVV (SEQ ID NO: 97), RVNSVEKPVV (SEQ ID NO: 98), SVNSVEKPW (SEQ ID NO: 99), TVNSVEKPW (SEQ ID NO: 100), WNSVEKPVV (SEQ ID NO: 101), WVNSVEKPW (SEQ ID NO: 102), YVNSVEKPW (SEQ ID NO: 103), GPNSVEKPVV (SEQ ID NO: 104), GVDSVEKPW (SEQ ID NO: 105), GVISVEKPW (SEQ ID NO: 106),

GVLSVEKPW (SEQ ID NO: 107), GVRSVEKPVV (SEQ ID NO: 108), GVYSVEKPW (SEQ ID NO: 109). GVNIVEKPVV (SEQ ID NO: 110), GVNLVEKPW (SEQ ID NO: 111),

GVNSCEKPVV (SEQ ID NO: 112), GVNSGEKPVV (SEQ ID NO: 113), GVNSWEKPVV (SEQ ID NO: 114), GVNSVGKPW (SEQ ID NO: 115), GVNSVECPVV (SEQ ID NO: 116), GVNSVERPVV (SEQ ID NO: 117), GVNSVEKAW (SEQ ID NO: 118). GVNSVEKRVV (SEQ ID NO: 119). GVNSVEKSW (SEQ ID NO: 120). GVNSVEKPAV (SEQ ID NO: 121).

GVNSVEKPMV (SEQ ID NO: 122). GVNSVEKPPV (SEQ ID NO: 123). GVNSVEKPSV (SEQ ID NO: 124). GVNSVEKPVF (SEQ ID NO: 125). GVNSVEKPVI (SEQ ID NO: 126), and GVNSVEKPVY (SEQ ID NO: 127).

100. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of TETT (SEQ ID NO: 128), SETN (SEQ ID NO: 129), GTETTS (SEQ ID NO: 130), and GSETNS (SEQ ID NO: 131).

101. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of LLVI (SEQ ID NO: 132), LMVI (SEQ ID NO: 133), LTVI (SEQ ID NO: 134), LVVI (SEQ ID NO: 135), and LAAI (SEQ ID NO: 136).

102. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of NVKMPVID (SEQ ID NO: 137), KEKMPVID (SEQ ID NO: 138), KHKMPVID (SEQ ID NO: 139), KKKMPVID (SEQ ID NO: 140), KWKMPVID (SEQ ID NO: 141), KVKMPVQD (SEQ ID NO: 142), and KVKMPVIN (SEQ ID NO: 143). 103. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of HVGG (SEQ ID NO: 144), QVGM (SEQ ID NO: 145), QVGS (SEQ ID NO: 146), and QVGY (SEQ ID NO: 147).

104. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of VEE and GVEEE (SEQ ID NO: 148).

105. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of

DAYEDVC (SEQ ID NO: 149), NAYEDGC (SEQ ID NO: 150), NAYEDKC (SEQ ID NO: 151), and NAYEDRC (SEQ ID NO: 152), and NAYEDSC (SEQ ID NO: 153), and NAYEDVR (SEQ ID NO: 154).

106. A nucleic acid encoding the non-natural alcohol dehydrogenase of any of embodiments 82-105.

107. An expression construct comprising the nucleic acid of 106.

108. An engineered cell comprising the nucleic acid or expression construct of embodiments 106 or 107.

109. The engineered cell of embodiment 108 further comprising one or more alcohol metabolic pathway gene(s) encoding a protein selected from the group consisting of a), a formate dehydrogenase (EM8), a formaldehyde activating enzyme (EM 10), a formaldehyde dehydrogenase (EMU), a S-(hydroxymethyl)glutathione synthase (EM12), a glutathione-dependent formaldehyde dehydrogenase (EMI 3), a S-formylglutathione hydrolase (EM 14), a formate hydrogen lyase (EM15), a hydrogenase (EM16).

110. The engineered cell of embodiment 108 further comprising one or more alcohol metabolic pathway gene(s) encoding a protein selected from the group consisting of a succinyl-

CoA reductase (aldehyde forming) (EB3), a 4-hydroxybutyrate (4-HB) dehydrogenase (EB4), a 4- HB kinase (EB5), a phosphotrans-4-hydroxybutyrylase (EB6), a 4-hydroxybutyryl-CoA reductase (aldehyde forming) (EB7), a 1 ,4-butanediol dehydrogenase (EB8); a succinate reductase (EB9), a succinyl-CoA reductase (alcohol forming) (EB10), 4-hydroxybutyryl-CoA transferase (EB11), a 4- hydroxybutyryl-CoA synthetase (EB12), a 4-HB reductase (EB13), and a 4-hydroxybutyryl-CoA reductase (alcohol forming) (EB15), a succinyl-CoA transferase (EB1), and a succinyl-CoA synthetase (EB2A).

111. The engineered cell of embodiment 108-110 which is bacteria.

112. The transgenic bacteria of embodiment 111 which is Bacillus.

113. A method for increasing the conversion of a methanol or ethanol to a dehydrogenated product of the alcohol comprising a step of (a) culturing the engineered cell of any of embodiments 108-112 in a culture medium comprising a methanol or ethanol, where in said culturing the cell provides at least two fold greater conversion of the methanol or ethanol to a dehydrogenated product of the alcohol, as compared to an engineered cell expressing a corresponding alcohol dehydrogenase without amino acid substitution.

114. A method for increasing the conversion of a methanol or ethanol to a dehydrogenated product of the alcohol comprising a step of (a) providing a reaction composition having a pH in the range of 6 - 8, the composition comprising a non-natural alcohol dehydrogenase of any of embodiments 82-105, where in the composition said culturing the cell provides at least two fold greater conversion of the methanol or ethanol to a dehydrogenated product of the alcohol, as compared to an engineered cell expressing a corresponding alcohol dehydrogenase without amino acid substitution.

115. A method of providing a diol comprising culturing the engineered cell of any of embodiments 108-112 in a culture medium comprising a methanol or ethanol to provide the diol.

116. The method of embodiment 115 wherein the diol is 1,4 butanediol.

117. A method of preparing a polymer comprising obtaining a monomer product produced by the engineered cell or method of any of embodiments 108-116 and polymerizing the monomer to provide a polymeric product.

118. A polymer prepared according to the method of embodiment 117.

119. A method of screening for a non-natural alcohol dehydrogenase having increased activity, optionally at least 2 fold, optionally at least 4 fold or greater activity, compared to its unmodified counterpart, comprising (1) creating one or more non-natural alcohol dehydrogenases selected from SEQ ID NO: l and non-natural alcohol dehydrogenases having a sequence identity of 45% or greater to SEQ ID NO: 1 having a substitution at a position other than an amino acid position selected from group (a) 11, 38, 42, 48, 53, 56, 60, 61, 63, 65, 70, 71, 74, 81, 84, 86, 87, 94, 99, 103, 106, 107, 108, 109, 112, 115, 116, 117, 117, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 145, 146, 147, 148, 149, 150, 152, 155, 157, 158, 161, 163, 164, 165, 181, 184, 186, 190, 199, 217, 226, 256, 267, 269, 270, 296, 298, 300, 302, 312, 316, 323, 333, 336, 337, 343, 344, 345, 350, 354, 355, 358, 360, 361, 363 and 379; or of group (b) 38, 60, 71, 87, 99, 103, 107, 108, 109, 115, 116, 117, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 146, 149, 150, 157, 158, 163, 164, 267, 270, 345, 355, 360, and 361, or a position corresponding thereto, (2) assay the created enzyme for the activity and (3) selecting those having increased activity, optionally at least 2 fold, optionally at least 4 fold, or greater activity compared to the unmodified counterpart.

Examples

Assay for testing activity of methanol dehydrogenase in vitro A high-throughput screening assay was used to evaluate lysates for methanol

dehydrogenase (MeDH) oxidation activity of methanol and other alcohol substrates. Lysates were prepared by a commercial chemical reagent from Escherichia coli cells that contained a plasmid harboring a MeDH library variant and an integrated chromosomal copy of the activator protein. An aliquot of the lysate was applied to a 384-well assay plate. To initiate the alcohol oxidation reaction, a substrate-buffer mix (pH 7.6 or pH 8.5) containing 0.5 M methanol or other alcohol, 0.5 mM NAD, 5 mM MgCl ₂ , 10 μΜ l-methoxy-5-methylphenazinium methylsulfate (1-methoxy PMS), & 1 mM 3-(4, 5-dimethylthiazol-2-yl)-2, 5 -diphenyltetrazolium bromide (MTT) was added. Initial rates were monitored via absorbance at 560 nm. MeDH variants that showed higher activity than the wild-type control were evaluated for further characterization.

Formaldehyde assay

A strain lacking frmA, frmB, frmR (the genes responsible for formaldehyde utilization in E. coli) was created using Lamba Red recombinase technology. Plasmids expressing methanol dehydrogenases were transformed into the strain, then grown to saturation in LB medium

+antibiotic at 37°C with shaking. Cultures were adjusted by OD and then diluted 1 : 10 into M9 medium + 0.5% glucose + antibiotic and cultured at 37°C with shaking for 6-8h until late log phase. Methanol was added to 2% v/v and the cultures were further incubated for 30 min with shaking at 37°C. Cultures were spun down and the supernatant was assayed for formaldehyde produced using DetectX Formaldehyde Detection kit from Arbor Assays, MI according to manufacturer's instructions.

Formate assay

The assay was developed to evaluate in vivo activity of methanol dehydrogenases by measuring the formate production in a host strain containing the first two steps of the MeOH pathway but lacking formate dehydrogenases (hycE, fdnGHI, fdoGHI, fdhF) that convert formate to CO ₂ . Plasmids expressing methanol dehdyrogenases were transformed into this strain. Strains were inoculated from colonies or glycerol stocks in LB+antibiotics in 96-deep well plates. The plates were sealed with breathable culture films and shaken at 37°C at 800 rpm. Overnight cultures were centrifuged at 5250 rpm for 10 minutes to pellet the cells. Cells were resuspended in 1 ml M9 medium in plates that were sealed with breathable culture films and shaken at 37 degree at 800 rpm. Samples were taken for a time course study and formate concentrations were measured using the formate kit based on instructions provided by the manufacturer.

Assay for purification of methanol dehydrogenases and for characterizing their activity

Cells expressing methanol dehydrogenase are cultured at 37°C in LB containing 2 mM MgS0 ₄ . Once harvested, cells are lysed in BugBuster Protein Extraction Reagent (Novagen) supplemented with 15 kU/mL lysozyme (Novagen), 25 U/mL bezonase (Novagen), IX Pierce

Protease Inhibitors (Thermo Scientific), 0.5 mM tris(2-carboxyethyl)phosphine hydrochloride, and 2 mM MgS04. Lysates are clarified via centrifugation and purified on a 5 mL StrepTrap HP column (GE Healthcare Life Sciences). The column is prepared in and washed with 100 mM MOPS pH 7.5, 0.2 M NaC12, 2 mM MgS0 ₄ , 0.5 mM TCEP (buffer A). The purified proteins are eluted with buffer A containing 0.3 mg/mL desthiobiotin.

DNA 2.0 gene synthesis

Methanol dehydrogenase gene candidates were synthesized after optimizing for codon usage by DNA 2.0 (Welch et al., PloS One 2009, 4(9):e7002, Design parameters to control synthetic gene expression in Escherichia coli).

In vivo labeled assay for conversion of methanol to CO ₂

Strains with functional reductive TCA branch and pyruvate formate lyase deletion were grown aerobically in LB medium overnight, followed by inoculation of M9 high-seed media containing IPTG and aerobic growth for 4hrs. These strains had methanol dehydrogenase/ ACT pairs in the presence and absence of formaldehyde dehydrogenase or formate dehydrogenase. At this time, strains were pelleted, resuspended in fresh M9 medium high-seed media containing 2% ¹³ CH ₃ OH, and sealed in anaerobic vials. Head space was replaced with nitrogen and strains grown for 40 hours at 37°C. Following growth headspace was analyzed for 13-C0 ₂ . Media was examined for residual methanol as well as BDO and byproducts.

All constructs expressing MeDH mutants and MeDH/ACT pairs grew to slightly lower ODs than strains containing empty vector controls. This is likely due to the high expression of these constructs.

Description of the NAD-dependent methanol dehydrogenase/ activator protein, its expression and use

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

The enzyme has been characterized as a multisubunit complex built from 43 kDa subunits containing one Zn and 1 -2 Mg atoms per subunit. Electron microscopy and sedimentation studies determined it to be a decamer, in which two rings with five-fold symmetry are stacked on top of each other (Vonck et al., J. Biol. Chem. 266, p. 3949-3954, 1991). It is described to contain a tightly but not covalently bound cofactor and requires exogenous NAD ⁺ as e ^" -acceptor to measure activity in vitro. A strong increase (10-40-fold) of in vitro activity was observed in the presence of an activator protein (Act), which is a homodimer (21 kDa subunits) and contains one Zn and one Mg atom per subunit.

The mechanism of the activation was investigated by Kloosterman et al. (J. Biol. Chem.

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

The stoichiometric ratio between Act and MeDH is not well defined in the literature.

Kloosterman et al. (J. Biol. Chem. 277, p. 34785-34792, 2002) determine the ratio of dimeric Act to decameric MeDH for full in vitro activation to be 10:1. In contrast, Arfman et al. (J. Biol. Chem. 266, 3955-3960, 1991) determined a ratio of 3: 1 in vitro for maximum and a 1 :6 ratio for significant activation, but observe a high sensitivity to dilution. Based on expression of both proteins in Bacillus, the authors estimate the ratio in vivo to be around 1 : 17.5. In vitro experiments with purified activator protein (2317A) and methanol dehydrogenase (2315 A) have showed the ratio of "act" to methanol dehydrogenase to be 10:1. This in vitro test was done with 5 M methanol, 2 niM NAD and 10 uM methanol dehydrogenase 2315 A at pH 7.4.

The sequence of the activator protein from Bacillus methanolicus MGA3 (locus tag:

MGA3 09170, GI number: 387591061, Accession number: EIJ83380) used in the assays is shown below: MGKLFEEKTIKTEQIFSGRVVKLQVDDVELPNGQTSKREIVRHPGAVAVIAITNENKIVM V EQYRKPLEKSrVEIPAGKLEKGEDPRITALRELEEETGYECEQMEWLISFATSPGFADEI IHIY VAKGLSKKENAAGLDEDEFVDLIELTLDEALQYIKEQRIYDSKTVIAVQYLQLQEALKNK .

2315 Stability assay and data

The thermostability of methanol dehydrogenase 2315A and the corresponding activator protein 2317A were assessed and melting temperatures were found to be 62 and 75°C, respectively. The melting temperatures were measured using a Protein thermal shift assay from

Applied biosystems. The assay provides relative thermal stabilities (melting temperatures) of purified proteins. It relies on a proprietary fluorescent dye that binds to hydrophobic regions of denatured proteins upon heating in the RT-PCR machine. The relative melting temperature is calculated from the slope of the fluorescence signal peak.

Current promoter and plasmid for overexpression

Methanol dehydrogenase 2315 was expressed with several constitutive and inducible promoters of varying strengths. The figure below shows the expression levels of two MeDH variants when expressed under three promoters: pi 19, pl04 and pl07. The two variants that were expressed were 2315L and 2315B. 2315B was a mutant constituted based on a mutation S97G identified from Hektor et al (ibid).

MDH protein concentrations Methanol dehydrogenase is a very soluble protein. SDS-PAGE analysis of soluble proteins from lysates of E. coli strains expressing different variants of the WT 2315A are shown. Specifically, the left panel shows the gel run on the lysates and the right panel shows the gel run on supernatant for the WT enzyme 2315A, compared with the variants 2315L and R, a variant from Hektor et al. called 2315B, and an empty vector.

The cells were lysed using Bugbuster as described previously. The amount of protein was quantified using the Image Lab 3.0 software from BioRad. The WT protein was estimated to be ~ 27% of the total protein.

Background on plasmids and promoters

Vector backbones were obtained from Dr. Rolf Lutz of Expressys

(http://www.expressys.de/). The vectors and strains are based on the pZ Expression System developed by Dr. Rolf Lutz and Prof. Hermann Bujard (Lutz,R. & Bujard,H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/Il-I2 regulatory elements. Nucleic Acids Res 25, 1203-1210 (1997).). Art available promoters PI 19, pi 04, pi 07, pi 19 provided varying levls of enzyme expression as desired. Vectors obtained were pZE131uc, pZA331uc, pZS* 131uc and pZE221uc and contained the luciferase gene as a stuffer fragment. To replace the luciferase stuffer fragment with a lacZ-alpha fragment flanked by appropriate restriction enzyme sites, the luciferase stuffer fragment was first removed from each vector by digestion with EcoRI and Xbal. The lacZ-alpha fragment was PCR amplified from pUC 19 with the following primers :

lacZalpha-RI

5 ' GACGAATTCGCTAGCAAGAGGAGAAGTCGACATGTCCAATTCACTGGCCGTCGTTTTAC3 '

(SEQ ID NO: 155) lacZalpha 3 ' BB

5' -GACCCTAGGAAGCTTTCTAGAGTCGACCTATGCGGCATCAGAGCAGA-3'

(SEQ ID NO: 156)

This generated a fragment with a 5' end of EcoRI site, Nhel site, a Ribosomal Binding Site, a Sail site and the start codon. The 3 ' end of the fragment contained the stop codon, Xbal, Hindlll, and Avrll sites. The PCR product was digested with EcoRI and Avrll and ligated into the base vectors digested with EcoRI and Xbal (Xbal and Avrll have compatible ends and generate a non- site). Because Nhel and Xbal restriction enzyme sites generate compatible ends that can be ligated together (but generate a site after ligation that is not digested by either enzyme), the genes cloned into the vectors could be "Biobricked" together

(http://openwetware.Org/wiki/Synthetic_Biology:BioBricks). Briefly, this method enables joining an unlimited number of genes into the vector using the same 2 restriction sites (as long as the sites do not appear internal to the genes), because the sites between the genes are destroyed after each addition. Initially, expression was low from these vectors, and they were subsequently modified using the Phusion® Site-Directed Mutagenesis Kit (NEB, Ipswich, MA, USA) to insert the spacer sequence AATTAA between the EcoRI and Nhel sites. This eliminated a putative stem loop structure in the RNA that bound the RBS and start codon.

All vectors have the pZ designation followed by letters and numbers indicating the origin of replication, antibiotic resistance marker and promoter/regulatory unit. The origin of replication is the second letter and is denoted by E for ColEl, A for pl5A and S for pSClOl (as well as a lower copy number version of pSClOl designated S*) - based origins. The first number represents the antibiotic resistance marker (1 for Ampicillin, 2 for Kanamycin, 3 for Chloramphenicol). The final number defines the promoter that regulated the gene of interest (1 for PLtetO-1, 2 for PLlacO- 1 and 3 for PAllacO-1) and each of these promoters became activated by its corresponding inducer molecule (pLtetO can be induced by tetracycline; pLlacO-1 and pAllacO-1 can be induced by IPTG). Three base vectors, pZS*13S, pZA33S and pZE13S, were then designed and constructed to serve as "inducible" plasmid vectors.

In addition to the "inducible" promoters mentioned above, a set of "constitutive" promoters were sampled from the Registry (http://partsregistry.org/). Each of these "constitutive" promoters was then introduced into the pZS*13S vector backbone to replace the pAllacO-1 inducible promoter via Sequence and Ligation Independent Cloning (SLIC) method described by Li & Eledge (Nature Methods 2007, 4:251-256). Of these sampled "constitutive" promoters (plOO, pl04, pl05, pl07, pl08, pi l l, pi 15 & pi 19), experiments were carried out to establish an order of promoter strength that was verified by protein expression levels. For the work discussed here, we employed both "inducible" and "constitutive" plasmid vectors, modified for the biobricks and SLIC insertions as discussed above. To further fine-tune protein expression levels of some overly expressed proteins, ribosomal binding site (RBS) in between promoter and gene coding sequence was modified accordingly using the RBS calculator (https://salis.psu.edu/software/).

Mutagenesis techniques - Error prone-PCR

EpPCR (Pritchard et al., J Theor.Biol. 234:497-509 (2005)) introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions by the addition of Mn ²⁺ ions, by biasing dNTP concentrations, or by other conditional variations. The five step cloning process to confine the mutagenesis to the target gene of interest involves: 1) error-prone PCR amplification of the gene of interest; 2) restriction enzyme digestion; 3) gel purification of the desired DNA fragment; 4) ligation into a vector; 5) transformation of the gene variants into a suitable host and screening of the library for improved performance. This method can generate multiple mutations in a single gene simultaneously, which can be useful to screen a larger number of potential variants having a desired activity. A high number of mutants can be generated by EpPCR, so a high-throughput screening assay or a selection method, for example, using robotics, is useful to identify those with desirable characteristics. Mutagenesis techniques - Site saturation mutagenesis

In Site Saturation Mutagenesis, the starting materials are a supercoiled dsDNA plasmid containing an insert and two primers which are degenerate at the desired site of mutations (Kretz et al., Methods Enzymol. 388:3-11 (2004)). Primers carrying the mutation of interest, anneal to the same sequence on opposite strands of DNA. The mutation is typically in the middle of the primer and flanked on each side by approximately 20 nucleotides of correct sequence. The sequence in the primer is N N or NNK (coding) and MNN (noncoding) (N = all 4, K = G, T, M = A, C). After extension, Dpnl is used to digest dam-methylated DNA to eliminate the wild-type template. This technique explores all possible amino acid substitutions at a given locus (that is, one codon). The technique facilitates the generation of all possible replacements at a single-site with no nonsense codons and results in equal to near-equal representation of most possible alleles. This technique does not require prior knowledge of the structure, mechanism, or domains of the target enzyme. If followed by shuffling or Gene Reassembly, this technology creates a diverse library of recombinants containing all possible combinations of single-site up-mutations. The usefulness of this technology combination has been demonstrated for the successful evolution of over 50 different enzymes, and also for more than one property in a given enzyme.

Combinatorial mutants

MeDH structure model and structures for comparison

To design a library of mutations for improving the catalytic rates of 2315, genes of several MeDHs as well as various Fe-dependent ADH genes were aligned. The described

structure/function relationships for the Fe-dependent ADHs (40-47% sequence identity to MeDHs) were used to identify regions of functional importance within the MeDH sequence. An alignment of the identified regions is presented in Figure 4.

Blast search using the MeDH sequence against the PDB structure database found several structures of Fe-dependent alcohol dehydrogenases with sequence identities between 40 and 47%.

Similarities are spread out over the whole length of the protein. Given this similarity to known structures, the MeDH sequence was used to generate a 3 -dimensional model using the web- based iTasser structure prediction tool (Roy et al, Nature Protocols, 5: 725 -738 (2010).). The following three structures were specifically used for alignment and comparison with the MeDH from Bacillus:

30X4: ADH-2 from Zymomonas mobilis (sequence identity 47%)

1RRM: Lactaldehyde reductase from Escherichia coli (FucO, sequence identity 43%) 3BFJ: l,3-PDO oxidoreductase from Klebsiella pneumoniae (DhaT, sequence identity 47%) The Zymomonas enzyme was crystallized with bound cofactor and the structure was well analyzed including the annotation of certain amino acid residues for metal, cofactor and proposed substrate binding (EtOH modeled into structure, Moon et al., J. Mol. Biol. 407, p 413-424, 2011). Like the Lactaldehyde reductase (Montella et al., J. Bact. 187, p. 4957-4966, 2005) from £. coli, the Zymomonas ADH is a homodimer. In contrast, the Klebsiella enzyme (Marcal et al., J. Bact. 191, p. 1143-1151, 2009) was found to be a decamer with a structure that resembles the MeDH appearance in electron microscopy studies.

Sequence comparison shows that all four coordination residues of the Fe-dependent ADHs are conserved in the MeDH structure. Two of these four residues are in a histidine-rich sequence (residues 258-290) suggested by Hektor et al. (J. Biol. Chem. 277, p. 46966-46973, 2002) as a putative metal binding site. As Fe and Zn share very similar binding characteristics, the same amino acids responsible for the Fe-binding in the ADHs may coordinate the Zn-atom of MeDH. From the alignment, the following four amino acids are likely to constitute the metal binding site in MeDH (numbering transferred to Genomatica gene ID 2315): D193, H197, H262, H276

Amino acids considered important for cofactor binding in the Zymomonas ADH are mostly conserved in the MeDH sequence. The respective residues are listed below for Genomatica gene ID 2315. If the respective amino acid differs, the Zymomonas ADH residue is noted in parentheses: D38, D70(N), G97, S98, T137, T138, T146, L148(F), L178

To cast a wide net, residues in a distance of 8 A or less from the CI atom of propanediol were identified and are listed together with the annotated Zymomonas residues below. Residue numbers were transferred to Genomatica gene ID 2315 and the respective amino acids in the original protein are given in parentheses.

From Zymomonas ADH-2: L148(F149), V150(I151), P161(A162), F253(F254),

L258(L259), H266(H267), D359(D360), V360(A361), C361(C362)

From £. coli FucO (<8 A distance): T141(T144), G142(A145), S143(A146), L148(N151), A149(Y152), H266(H267)

Mapping of the suggested mutagenesis sites onto the structure model of MeDH shows that the residues selected as target sites line the entrance to the active site of the monomer. Positions 253, 258, 266, and 359 were found to be strictly conserved, suggesting that they are more likely to be essential for function and were therefore eliminated from the list of residues identified for mutations.

For the remaining five residues, amino acids for substitution were selected based on their occurrence in related sequences. Only for position 148 which was annotated with having a role in limiting the substrate size as well as positioning the nicotinamide ring in the active site, a full panel of amino acids (NNK) is proposed. Narrowing down the pool of substitutions in the other four positions made it possible to include additional target sites while maintaining a reasonable library size. The following three sites were added based on their proposed function and location in Fe- dependent ADHs: D70, T145, S147. When comparing the variation for the respective positions in a sequence alignment it was noted that one of the residues is homolog to residue 160 in an in house tested alcohol dehydrogenase. As a P160G mutation increased the activity of this alcohol dehydrogenase, a glycine was added to the list of substitutions in the respective MeDH position. The table below summarizes the final list of targeted residues and substitutions (positions based on gene 2315A):

Table 1

Amino acids mutations with respect to SEQ ID NO: 1, providing greater than two fold activity when present as single mutations:

G121Y 3.1 1.5 G121 121Y 121

V122P 2.5 2.3 V122 122P 122

N123L 2.7 1.8 N123 123L 123

N123R 2.7 1.6 N123 123R 123

N123Y 3.0 1.8 N123 123Y 123

SI 241 3.4 0.9 S124 1241 124

S124L 2.4 1.2 S124 124L 124

V125C 2.6 3.2 V125 125C 125

V125G 2.6 3.3 V125 125G 125

V125W 2.7 3.9 V125 125W 125

E126G 4.0 0.6 E126 126G 126

K127C 2.6 3.9 K127 127C 127

K127R 2.5 3.3 K127 127R 127

P128A 2.3 3.0 P128 128A 128

P128R 2.4 3.3 P128 128R 128

V129A 3.7 1.9 V129 129A 129

V129M 4.7 1.4 V129 129M 129 X

V129P 2.8 1.3 V129 129P 129

V129S 3.0 1.5 V129 129S 129

V130F 2.1 1.4 V130 130F 130

V130Y 2.0 2.0 V130 130Y 130

A134T 4.4 0.0 A134 134T 134

A149T 5.9 0.8 A149 149T 149

V150A 3.0 1.2 V150 150A 150

K157N 3.6 0.8 K157 157N 157

V158E 2.6 4.3 V158 158E 158 X

V158H 2.2 2.8 V158 158H 158

V158K 2.0 2.6 V158 158K 158

V158W 2.5 4.0 V158 158W 158

D164N 3.7 0.7 D164 164N 164

G270S 2.8 2.9 G270 270S 270 X

K345E 4.1 0.6 K345 345E 345

N355D 3.3 1.7 N355 355D 355 X

C361R 3.0 0.8 C361 361R 361

D38N 5.4 7.3 D38 38N 38 +

P71I 7.6 2.5 P71 711 71 +

P71V 6.8 3.5 P71 71V 71 +

G107S 5.1 2.5 G107 107S 107 + X

LI 08V 6.4 3.9 L108 108V 108 +

L108W 7.4 4.8 L108 108W 108 +

N117Q 3.3 4.3 N117 117Q 117 +

N123D 3.0 2.0 N123 123D 123 + X

N123I 3.1 2.5 N123 1231 123 + X

P128S 3.1 3.7 P128 128S 128 +

VI 301 3.4 2.3 V130 1301 130 + X

S143T 3.8 2.4 S143 143T 143 + X

T146N 3.4 2.0 T146 146N 146 + X A149L 4.8 3.0 A149 149L 149 +

A149M 4.6 2.7 A149 149M 149 +

A149V 4.9 2.9 A149 149V 149 + X

I163Q 4.4 2.1 1163 163Q 163 +

Q267H 6.3 4.3 Q267 267H 267 + X

G270M 4.3 4.2 G270 270M 270 +

G270Y 4.2 4.2 G270 270Y 270 +

V360G 4.9 2.1 V360 360G 360 + X

V360K 4.6 2.6 V360 360K 360 +

V360R 4.6 3.4 V360 360R 360 + X

V360S 4.5 3.5 V360 360S 360 +

Table 2

Additional combination mutations with respect to SEQ ID NO: 1

(generated from rationale design)

L148W, P161A, V360A, C361N 5.7 1.06

N112K , S147R, P161A, V360A 9.7 5.40

P161A, Q217K, V360A, C361N 6.7 4.15

P161A, V360A, C361N 8.0 3.58

P161A, V360G 7.4 4.8

P161V, E358G, V360G 6.0 4.96

P161V, V360A, C361N 9.7 5.2

P161V, V360G 6.4 4.0

P65Q, L148G, VI 501, PI 61 A, V360G, 5.5 2.32 C361N

S147R, L148A, V150I, P161A, V360G 5.6 4.07

S147R, L148F, V150I, P161G, V360G 6.7 6.0

S147R, L148V, P161G, V360A 6.3 4.4

S147R, L148V, P161V, V360G 8.9 6.15

S147R, L148V, V150I, P161A, C361N 5.8 3.10

S147R, L148V, V150I, P161G, V360G 6.7 4.17

S147R, P161A, V360A 6.3 5.53

S147R, P161A, V360A, C361N 6.6 3.6

S147R, P161A, V360G 5.7 5.22

S147R, P161V, V360G 10.0 5.84

S147R, P161V, V360G, C361N 5.7 2.11

S147R, V150I, P161V, V360A 5.7 5.13

S147R, V150I, V360A, C361N 5.6 4.66

T145M, L148I, V360G 8.0 3.48

V150I, I302V, V360G, C361N 7.8 4.01

V150I, P161A, C361N 5.8 3.42

V150I, P161G, V360A, C361N 5.6 2.88

V150I, P161G, V360G 5.6 4.28

V150I, P161G, V360G, C361N 7.1 3.26

V150I, P161V, C361N 5.7 3.9

V150I, P161V, K354R, V360A, C361N 8.8 4.24

V150I, P161V, V360A, C361N 6.0 5.04

V150I, P161V, V360G, C361N 5.6 4.9

V150I, V360A, C361N 6.4 4.43

V150I, V360G 6.8 6.79

Table 3

Additional combination mutations with respect to SEQ ID NO: 1

(generated from epPCR)

Mutations In Vitro Assay— Average of Triplicates

SI IT, T74S, G269S, V344A 5.75

K84R, I163T 5.38

V122A, I163N 5.01

G107S, F333L 4.55

V129M, T152M, G343D 4.45

I63F, N355K 4.44

G107S, F333L 4.42 E86K, S99T, A149V 4.41

N53I, V158E 4.38

N355I, K379M 4.30

H42Q, G107S 4.08

Q120H, I163N 4.06

A149V, I323M 4.04

G107S, F333L 3.69

D164G, K181R 3.68

A155V, R298H, N355D 3.66

N123D, E165G 3.65

I163F, L186M 3.65

G121A, T296S 3.63

194 V, S99P, N123I 3.62

E126V, V129M, V344G 3.60

Q120R, S143T 3.58

G256C, A316V 3.56

P161Q, G312V 3.52

L226M, A300T, V360A 3.49

S337C, E350K, N355D, Q363K 3.43

D81G, V158E 3.42

I106L, N117Y, E126V 3.40

G107S, G121D 3.36

V61A, V158E 3.31

N53I, V158E 3.28

N117Y, T190S 3.16

S124R, I199V 3.13

K354M, C361R 2.97

A184T, C361R 2.86

E56K, Q267H 2.85

S124R, E126G 2.79

T190A, N355K 2.77

P71T, F333L 2.75

G107S, F333L 2.74

N123I, P336L 2.71

Table 4

Additional combination mutations with respect to SEQ ID NO: 1:

N112R/P161 S 7 3.2

Table 5

Putative motifs and roles of the amino acid positions

with respect to SEQ ID NO:l.

Table 6

In vivo assays showing formaldehyde (HCHO) production by various NNOMO comprising a plasmid expressing a methanol dehydrogenase

ZP 10819291. YP 00686325 YP 0013371

1 <1 8 >1 53 >20

Empty vector 2.33 NP_394301.1 >1 YP 694908 >20

ZP 10750164.

1 >1 NP 717107 >20

YP 023929.1 >1 AAC45651 >10

ZP 08977641. ZP 1131327

1 <1 7.1 >10

ZP 10117398. ZP 1622433

1 <1 8.1 >10

YP 00410804 YP 0011136

5.1 <1 12 >10

ZP 09753449. YP 0048601

1 <1 27 >10

YP 0033105

Empty vector 0.17 46 >10

YP 0013437

16 >10

NP 717107 >10

YP 0024347

46 >10

Empty vector 0.11

TABLE 7

Wild-type enzymology

TABLE 8

Wild type and variant enzymology

Activity using methanol was determined.

"Assays were performed at pH 7.6, 37 °C in the presence of 2 mM NAD.

TABLE 9

Wild type and variant enzymology

1,4-Butanediol-dependent steady-state kinetic parameters for wild-type and variant methanol dehydrogenase."

"Assays were performed at pH 7.6, 37 °C in the presence of 2 mM NAD.

TABLE 10

Substitution templates

PB1

+ 9/10 EIJ80770.1 387588449 Bacillus 385 62 79 1 methanolicus

MGA3

++ 11/12 EIJ78397.1 387586073 Bacillus 385 61 78 1 methanolicus

PB1

+ 13/14 EIJ83020.1 387590701 Bacillus 385 61 79 1 methanolicus

MGA3

++ 15/16 EFI69743.1 298729190 Lysinibacillus 401 56 74 5 fusiformis

+ 17/18 YP 0048601 347752562 Bacillus 386 56 76 1

27.1 coagulans

36D1

++ 19/20 YP 0016997 169829620 Lysinibacillus 402 54 73 5

78.1 sphaericus

+ 21/22 ZP 1131327 410459529 Bacillus 386 54 73 1

7.1 azotoformans

LMG 9581

n.d 23/24 ZP 0558733 257139072 Burkholderia 390 54 70 2

4.1 thailandensis

E264

+ 25/26 YP 0046815 339322658 Cupriavidus 390 53 70 2

52.1 necator N-l

n.d 27/28 AGF87161 451936849 uncultured 393 53 71 3 organism

++ 29/30 YP 0021381 197117741 Geobacter 387 52 71 1

68.1 bemidjiensis

Bern

++ 31/32 YP 359772. 78043360 Carboxydother 383 52 72 0

1 mus

hydrogenoform

ans Z-2901

+ 33/34 YP 0013437 152978087 Actinobacillus 385 51 71 1

16.1 succinogenes

130Z

+ 35/36 ZP 1622433 421788018 Acinetobacter 390 51 70 2

8.1 baumannii

Naval-82

+ 37/38 AAC45651.1 2393887 Clostridium 385 51 69 1 pasteurianum

DSM 525

n.d 39/40 YP 0074913 452211255 Methanosarcin 386 51 71 1

69.1 a mazei TucOl

n.d 41/42 YP 0024347 218885425 Desulfovibrio 393 50 70 3

46 vulgaris str.

'Miyazaki F'

++ 43/44 YP 0050528 374301216 Desulfovibrio 393 49 70 3

55 africanus str.

Walvis Bay

++ 45/46 NP 561852. 18309918 Clostridium 385 49 68 1

1 perfringens str.

13

++ 47/48 YP 0014475 156976638 Vibrio 382 49 69 0

44 campbellii ATCC BAA- 1116

+ 49/50 YP 0011136 134300116 Desulfotomacu 388 49 70 2

12.1 lum reducens

MI-1

n.d 51/52 YP_011618 46580810 Desulfovibrio 393 49 70 3 vulgaris str.

Hildenborough

++ 53/54 ZP 0122015 90412151 Photobacterium 382 48 69 0

7.1 profundum

3TCK

++ 55/56 YP 0039907 312112413 Geobacillus sp. 384 48 67 1

29.1 Y4.1MC1

+ 57/58 ZP 0733545 303249216 Desulfovibrio 393 48 69 3

3.1 fructosovorans

JJ

+ 59/60 NP_717107 24373064 Shewanella 382 48 66 0 oneidensis MR- 1

+ 61/62 YP 0033105 269122369 Sebaldella 384 48 68 1

46.1 termitidis

ATCC 33386

++ 63/64 ZP 1024153 390456003 Paenibacillus 384 47 67 1

1.1 peoriae KCTC

3763

+ 65/66 YP 0013371 152972007 Klebsiella 387 47 67 1

53.1 pneumoniae

subsp.

pneumoniae

MGH 78578

++ 67/68 YP 026233. 49176377 Escherichia 383 46 64 0

1 coli

+ 69/70 YP_694908 110799824 Clostridium 382 46 69 0 perfringens

ATCC 13124

n.d 71/72 YP 725376. 113866887 Ralstonia 366 46 60 15

1 eutropha HI 6

n.d 73/74 YP 0016635 167040564 Thermoanaerob 389 45 68 2

49 acter sp. X514

n.d 75/76 EKC54576 406526935 human gut 384 37 55 3 metagenome

n.d 77/78 YP 0011269 138896515 Geobacillus 387 27 44 7

68.1 themodenitrific

ans NG80-2

Percent identity is given based on global alignment to SEQ ID NO: 1.

TABLE 11

Template Polypeptides

Sequences of Template Polypeptides indicating exemplary substitutions, their positions and their corresponding positions in other template sequences: NO:

1 387585261 MTTNFFIPPASVIGRGAVKEVGTRLKQIGAKKALIVTDAFLHSTGLSEEVAKNIREAGVD

VAIFPKAQPDPADTQVHEGVDVFKQENCDSLVSIGGGSSHDTAKAIGLVAANGGRIND

YQGVNSVEKPVVPVVAITTTAGTGSETTSLAVITDSARKVKM PVIDEKITPTVAIVDPEL

M VKKPAGLTI ATGM DALSH Al EAYVAKGATPVTDAFAIQAM KLI N EYLPKAVAN GEDI E

AREKMAYAQYMAGVAFNNGGLGLVHSISHQVGGVYKLQHGICNSVNMPHVCAFNLI

AKTERFAHIAELLGENVAGLSTAAAAERAIVALERINKSFGIPSGYAEMGVKEEDIE LLAK

N AYE DVCTQS N P RVPTVQDI AQI 1 K N AM

3 143175 MTNFFIPPASVIGRGAVKEVGTRLKQIGAKKALIVTDAFLHSTGLSEEVAKNIREAGLDV

AIFPKAQPDPADTQVHEGVDVFKQENCDALVSIGGGSSHDTAKAIGLVAANGGRINDY

QGVNSVEKPVVPVVAITTTAGTGSETTSLAVITDSARKVKMPVIDEKITPTVAIVDP ELM

VKKPAGLTIATGMDALSHAI EAYVAKGATPVTDAFAIQAM KLIN EYLPKAVANGEDIEA

REAMAYAQYMAGVAFNNGGLGLVHSISHQVGGVYKLQHGICNSVN MPHVCAFNLIA

KTERFAHIAELLGENVSGLSTAAAAERAIVALERYNKNFGIPSGYAEMGVKEEDIEL LAKN

AFEDVCTQSN PRVATVQDIAQIIKNAL

5 387585284 MTQRNFFIPPASVIGRGAVKEVGTRLKQIGATKALIVTDAFLHGTGLSEEVAKNIREAGL

DAVIFPKAQPDPADTQVHEGVDIFKQEKCDALVSIGGGSSHDTAKAIGLVAANGGRIN DYQGVNSVEKPVVPVVAITTTAGTGSETTSLAVITDSARKVKMPVIDEKITPTVAIVDPE LM VKKPAGLTI ATGM DALSHAIEAYVAKRATPVTDAFAI QAM KLI NEYLPRAVAN GEDI EAREAMAYAQYMAGVAFNNGGLGLVHSISHQVGGVYKLQHGICNSVNMPHVCQFN LIARTERFAHIAELLGENVSGLSTASAAERAIVALQRYNKNFGIPSGYAEMGVKEEDIEL L A N N AY QD VCTLD N PRVPTVQDIAQIIKNAL

7 387586466 MTKTKFFIPSSTVFGRGAVKEVGARLKAIGATKALIVTDAFLHSTGLSEEVAKN IREAGLD

VVIFPKAQPDPADTQVHEGVEVFKQEKCDALVSIGGGSSHDTAKGIGLVAANGGRIND

YQGVNSVEKQVVPQIAITTTAGTGSETTSLAVITDSARKVKMPVIDEKITPTVAIVD PEL

MVKKPAGLTIATGM DALSHAIEAYVAKRATPVTDAFAIQAM KLIN EYLPKAVANGEDIE

AREAMAYAQYMAGVAFNNGGLGLVHSISHQVGGVYKLQHGICNSVVMPHVCQFNLI

ARTERFAHIAELLGENVSGLSTASAAERTIAALERYNRNFGIPSGYKAMGVKEEDIE LLAN

NAMQDVCTLDNPRVPTVQDIQQIIKNAL

9 387588449 MKNTQSAFYMPSVNLFGAGSVNEVGTRLAGLGVKKALLVTDAGLHSLGLSEKIAGIIRE

AGVEVAIFPKAEPNPTDKNVAEGLEAYNAENCDSIVTLGGGSSHDAGKAIALVAANGG

TIHDYEGVDVSKKPMVPLIAINTTAGTGSELTKFTIITDTERKVKMAIVDKHVTPTL SIND

PELMVGMPPSLTAATGLDALTHAIEAYVSTGATPITDALAIQAIKIISKYLPRAVAN GKDI

EAREQMAFAQSLAGMAFNNAGLGYVHAIAHQLGGFYNFPHGVCNAILLPHVCRFNLI

SKVERYAEIAAFLGENVDGLSTYEAAEKAIKAIERMARDLNIPKGFKELGAKEEDIE TLAK

NAMN DACALTN PRKPKLEEVIQIIKNAM

11 387586073 MTNTQSIFYIPSVNLFGPGSVNEVGTRLAGLGVKKALLVTDAGLHGLGLSEKIASIIREA G

VEVLIFPKAEPN PTDKNVAEGLEVYNAENCDSIVTLGGGSSHDAGKGIALVAANGGTIY

DYEGVDKSKKPMVPLIAINTTAGTGSELTRFTIITDTERKVKMAIVDKHVTPTLSIN DPEL

MVGMPPSLTAATGLDALTHAIEAYVSTAATPITDALAIQAIKIISKYLPRAFANGKD MEA

REQMAFAQSLAGMAFNNASLGYVHAIAHQFGGFYNFPHGVCNAILLPHVCRFNLISK V

ERFAEIAALLGENVAGLSTREAAEKGIKAIERMAKDLNIPRGFKELGAKEEDIVTLA ENA

MKDATALTN PRKPKLEEVIQIIKNAM

13 387590701 MTNTQSAFFMPSVNLFGAGSVNEVGTRLADLGVKKALLVTDAGLHGLGLSEKISSIIRA

AGVEVSIFPKAEPN PTDKNVAEGLEAYNAENCDSIVTLGGGSSHDAGKAIALVAANGG

KIHDYEGVDVSKEPMVPLIAINTTAGTGSELTKFTIITDTERKVKMAIVDKHVTPTL SIND

PELMVGMPPSLTAATGLDALTHAIEAYVSTGATPITDALAIQAIKIISKYLPRAVAN GKDI

EAREQMAFAQSLAGMAFNNAGLGYVHAIAHQLGGFYNFPHGVCNAVLLPYVCRFNLI

SKVERYAEIAAFLGENVDGLSTYDAAEKAIKAIERMAKDLN IPKGFKELGAKEEDIETLAK

NAMKDACALTNPRKPKLEEVIQIIKNAM

15 298729190 MSDVLKQFVM PKTN LFGPGAIQEVGKRLN DLEVKKTLI VTDEGLH KLG LSEQI AN 1 ITAA

GIDVAIFPKAEPNPTDQN IEDGISVYHAENCDSIVSLGGGSAHDAAKGIGLIASNGGRIH DYEGVDKSQNPLVPLIAINTTAGTASEMTRFTIITDTARKVKMAIVDKHVTPLLSINDPE LMIGLPPALTAATGVDALTHAIESFVSTNATPITDACAEKVLQLIPEYLPRAYANGADIE A REQMVYAQFLAGMAFNNASLGYVHAIAHQLGGFYN LPHGVCNAILLPHVCRFNVTAR TERFARIAELLGENVEGLSKRDAAEKAITAIEKLSQDLN IPSGFRELGAKDEDIEILAKNALL DVCAETNPRKATLEDIKQIITNAMGPIVKKEESLEAVALS

347752562 MLTGLRTDFQMPSVNLFGQGTAEEIGNRLKNLGCRRPLIVTDEGLHQLGYSEKIAAYIKE

AGLEVAIYPKAEPNPTDKNVEDGLKTYHEENCDSIVSLGGGSAHDCAKGIGLVAANGG

KIHDYEGLDRSEKPMVPLVAINTTAGTASEMTKFTIITDTSRKVKMAIVDKHVTPVL SIN

DPLLMVGMPPSLTAATGLDALTHAVEAYVSTAATPVTDACAIKAIQIIPQYLPKAVA NG

NDMEAREQMVYAQYLAGMAFNNASLGYVHAIAHQFGGFYNLPHGVCNAILLPHVCR

FNLIARKERFAEIAVALGEKTDSLSVDEAAEKAITAIERLAAQLNIPKGFKELGAKE EDIEIL

AQHAMQDACAATN PRKPTQKEVEAI 1 KAAM

169829620 MSDVLKQFVM PKKN LFGPGAIQEVGKH LN DLEVKKTLI VTDEG LH KLGLSEQI AN 1 ITAA

GIDVAIFPKAEPNPTDQN IEDGIADYHAESCDSIVSLGGGSAHDAAKGIGLIASNGGRIQ

DYEGVDKSQNPLVPLIAINTTAGTASEMTRFTIITDTARKVKMAIVDKHVTPLLSIN DSE

LMIGLPPALTAATGVDALTHAIESFVSTNATPITDACAEKVLQLVPEFLPRAYANGA DLE

AREQMVYAQFLAGMAFNNASLGYVHAIAHQLGGYYNLPHGVCNAILLPHVCRFNVTA

RTERFARIAELLGENVTGLSKRDAAEKAISAIEKLSKDLNIPSGFRELGAKDEDIEI LAKNA

MLDVCAETNPRKATLDDIKQIITNAMGPIVKKEESLEAVAAL5

410459529 MANQKVYGFFMPTVN LMGVGAVNEAGPRIKALGCNKSLLVTDKGL5KMGVAEEIAN I

IGQAGVEVSIFDGAEPN PTDLNVEAGLKQYRELGCDSIISLGGGSSHDCAKGIGLVASNG GTIHDYEGVDMSKEPMIPLVAINTTAGTASEMTRFCIITDTSRKIKMAIVDKHTTPLISI N DPILTVKM PAG LTAATGM DALTHAI EAYVSTDATPITDACALQTI RLVSQN LRAA VANG EDI DARN NMCYAQFLGGMAFNNASLGYVHAIAHQLGGFYNLPHGVCNAVLLPHVERF N LI AKPERFVDIAIALGENVSGLPTRAAAEI ALTAI ETLAKDVGIPGSLTELGVKEEDIPLLA EN AM RDACSFTN PRKATLDDVQGM 1 RAAL

257139072 MSYLNIAQRTDSFFIPCVTLIGPGCARETGVRAKSLGAKKALIVTDAGLHKMGLSEIVAG

HIRDAGLQAVIFAGAEPNPTDVNVHDGVERFQREGCDFIVSLGGGSSHDCAKGIGLVT

AGGGHIRDYEGIDKSTVPMTPLISINTTAGTAAEMTRFCIITNSSN HVKMAIVDWRCTP

LIAIDDPCLMVAMPPALTAATGMDALTHAVEAYVSTAATPITDACAEKAIALIGEWL PK

AVANGESM EARAAMCYAQYLAGMAFNNASLGYVHAMAHQLGGFYNLPHGVCNAIL

LPHVCEFN LIAAPERFATIASLLGVNTAGSSTVDAARAGHAAIPRLSASIGIPAGLAALGV

RVEDHEVMASNAQKDACMLTNPRKATLAQVIAIFAAAM

339322658 MTHLNIANRVDSFFIPCVTLFGPGCARETGARARSLGARKALIVTDAGLHKMGLSEVVA

GHIREAGLQAVIFPGAEPNPTDVNVHDGVKLFEREECDFIVSLGGGSSHDCAKGIGLVT AGGGHIRDYEGIDKSTVPMTPLISINTTAGTAAEMTRFCIITNSSN HVKMAIVDWRCTP LIAIDDPSLMVAMPPALTAATGMDALTHAIEAYVSTAATPITDACAEKAIVLIAEWLPKA VANGDSMEARAAMCYAQYLAGMAFNNASLGYVHAMAHQLGGFYNLPHGVCNAILL PHVSEFNLIAAPERYARIAELLGENIGGLSAHDAAKAAVSAIRTLSTSIGIPAGLAGLGV KA DDH EVM ASN AQKD ACM LTN P RKATLAQVM Al FAAAM

451936849 MSLVNYLQLADRTDGFFIPSVTLVGPGCVKEVGPRAKMLGAKRALIVTDAGLHKMGLS

QEIADLLRSEGI DSVIFAGAEPN PTDINVHDGVKVYQKEKCDFIVSLGGGSSHDCAKGIG LVTAGGGHIRDYEGVDKSKVPMTPLIAINTTAGTASEMTRFCIITNTDTHVKMAIVDW RCTPLVAIDDPRLMVKMPPALTAATGMDALTHAVEAYVSTAATPITDTCAEKAIELIGQ WLPKAVANGDWM EARAAMCYAQYLAGMAFNNASLGYVHAMAHCjLGGFYNLPHG VCNAILLPHVCQFNLIAATERYARIAALLGVDTSGMETREAALAAIAAIKELSSSIGIPR GL SELG VKAADH KVM AEN AQKDACM LTN P RKATLEQVI GIFEAAM

197117741 MALGEQTYGFYIPTVSLMGIGSAKETGGQIKALGASKALIVTDKGLSAMGVADKIKSQV

EEAGVSAVIFDGAEPNPTDINVHDGVKVYQDNGCDAIISLGGGSSHDCAKGIGMVIGN

GGHIRDLEGVNKTTKPMPAFVAINTTAGTASEMTRFCIITNTDTHVKMAIVDWRCTP

NVAINDPLLMVGKPAALTAATGMDALTHAVEAYVSTIATPITDACAIKAIELIAEFL SKAV

ANGEDLEARDKMAYAEYLAGMAFNNASLGYVHSMAHCjLGGFYNLPHGVCNAILLPA

VSQYNLIACPKRFADIAKALGENIDGLSVTEAGQKAIDRIRTLSASIGIPTGLKALN VKEAD

LTIMAENAKKDACQFTNPRKATLEQVVQIFKDAM 78043360 MKTYRFYMPPVSLMGIGCLKEAGEEIKKLGFKKALIVTDKVLVKIGLVNKLTEILDNEGI E

YVIFDETKPN PTVKNVEDGLKMLKEN NCDFLISFGGGSPHDCAKGIGLVATNGGSIKDY

EGVNKSAKPMLPLVAVNTTAGTASEMTRFSIITDEDRHVKMAIVDWHVTPIMAVN DP

ELMVEM PKALTAATGMDALTHAIEAYVSIDATPVTDAAALKAIELIFKYLKRAVENGKDI

EARDKMAYAEYLAGVAFNNAGLGYVHAMAHQLGGFYDLPHGVCNAVLLPHVQAYNL

QVVPERFIDIAKAMGINVENLTAKEAGEKVLEAIKNLSREIGIPSGLKELGVKEEDL KTLAE

NALKDACGFTNPKQASLDDIIRIFKEAM

152978087 MSTYYFLPTRNVFGENAVEEVGTLMKSLGGNN PLIVTDAFLAKNGMADQLAAVLSNA

GLKPVIFGGAEPNPTDKNVEEGIVFYNEHGCDSIISLGGGSSHDCAKGIGLIASNGGRIQ

DYEGVDRSHNAMVPLMAVNTTAGTASEITRFCIITDTARKVKMAIVDWRITPQIAVN D

PLLM KG M PPSLTAATGM DALTH Al EAYVSTAAN PLTDAAALM AITM IQQYLPKAVAN

GDYMKARDKMAYAQYLAGIAFNNASLGYVHAMAHQLGGFYNLPHGVCNAILLPYVEE

FNLIGN LN RFRDIAKAMGENIDGLCTDDAALKAIGAIRRLSKQVGIPANLQLLGVKPEDF

DVM AEN AM KDVCM LTN P RKATKQQVI El FQRAYDG D

421788018 MAFKNIADQTNGFYIPCVSLFGPGCAKEIGTKAQN LGAKKALIVTDEGLFKFGVADLIAS

YLTEAGVASHIFPGAEPN PTDINVHNGVNAYN ENGCDFIVSLGGGSSHDCAKGIGLVTA

GGGHIRDYEGIDKSKVPMTPLIAVNTTAGTASEMTRFCIITNTDTHVKMAIVDWRCT P

LIAIDDPKLMIAKPAGLTAATGMDALTHAVEAYVSTAANPITDACAEKAITMISQWL QP

AVANGENIEARDAMSYAQYLAGMAFNNASLGYVHAMAHQLGGFYNLPHGVCNAILL

PHVCEFNLIACPDRYAKIAELMGVNTHGLTVTEAAYAAIDAIRKLSSLIGIPSGLTE LGVKT

EDLAVM AE N AQKD ACM LTN P RKAN H AQVVEI F KAAL

2393887 MRMYDFLAPNVNFMGAGAIKLVGERCKILGGKKALIVTDKFLRNMEDGAVAQTVKYIK

EAGIDVAFYDDVEPNPKDTNVRDGLKVYRKENCDLIVTVGGGSSHDCGKGIGIAATHE

GDLYDYAGIETLTNPLPPIVAVNTTAGTGSEVTRHCVITNTKTKIKFVIVSWRNLPL VSIN

DPILMIKKPAGLTAATGM DALTH AIESYVSKDANPVTDALAIQAIKLI AN N LRQAVALGE

NLEAREN MAYASLLAGMAFNNANLGYVHAMAHQLGGLYDMAHGVANAMLLPHVE

RYN LISNPKKFADIAEFMGENIEGLSVMEAAEKAIDAMFRLSKDVGIPASLKEMGVNEG

DFEYMAKMALKDGNAFSN PRKGN EKDI VKIFREAF

452211255 MIEKMTYTYLNPKIALMGPGCVNGIGTHAKDLGGTKALIVSGKSRHGKELAADIRRILER

AGIEAAIFPGADPN PTDTSVMEGADIYRKENCNMIVAVGGGSPMDCAKAIGIVVYNG

GRINDYEGVGKVTRGIPPLITVNTTAGTASEMTSFTIITDTERHIKMAIVDPRITPD VAV

NDPELMVSMPPALTAATGM DALTH AVEAYVSTMATPTTDAAAIKAIELISKYLPEAVLH

GEDIRARDMMAHAEYLAGIAFNNASLGYVHSMAHCjLGGFYDLPHGVCNAILLPYVE M

YNKQVCPERFADIAKAMGEKVEGLSPEEAADKAI EAIKKLAAEIGIPSGLKELGAREEDLE

LLAENAMQDVCRLTNPRELSKEDIIEIYRKAL

218885425 MAVQEQVYGFFIPSVTLIGIGASKAIPEKIKALGGSKPLIVTDMGIVKAGILKQITDLLD AA

KMAYSVYDETIPN PTDDNVHKGVEVYKKN KCDSLITLGGGSSHDCGKGIGLVIANGGKI

HDFEGVDKSFKPMPPYVAVNTTAGTASEMTRFCIITDTSRKVKMAIVDWRVTPSIAL D

DPLLMMGM PPALTAATGM DALTH AVEAYVSTIATPMTDACAEQAITLIATFLRRAVA

NGRDIEARERMCFAQYLAGMAFNNASLGHVHAMAHCjLGGFYDLPHGECNAILLPHV

SQFN LIAKLDRFARIAELMGEN ISGLSVRDAAEKAICAIKRLSADVGI PAGLVALGKRYGK

DVKAKDIAIMTKNAQKDACGLTNPRCPTDADVAAIYEAAM

374301216 MAVREQVYGFFIPSVTLIGIGASKEIPN KIRDLGGKKPLIVTDQGIVKAGI LKMITDHMDK

AGMQYSVYDKTIPNPTDNNVAEGVEVYKKEGCDSLITLGGGSSHDCGKGVGLVVSNG

GKIHDYEGVDKSTKPLPPYVAVNTTAGTASEMTRFCIITDTSRKVKMAIVDWRVTPG IA

LDDPLLMVGM PPALTAATGMDALTHAVEAYVSTIATPMTDACAEKAISLIFTFLRRATA

NGQDIEAREGMCFAQYLAGMAFNNASLGHVHAMAHCiLGGFYDLPHGECNAILLPHV

EKYN LIAKVERFGKMAEIMGEN IQGMSPRAAAEKCLDAIRQLSQDVGIPSGLIELGKRY

GKNVKKEDIDTMTGNAQKDACGFTNPRCPSDKDVKAIYEAAL

18309918 MRMYDYLVPSVNFMGANSISVVGERCKILGGKKALIVTDKFLRGLKGGAVELTEKYLKE

AGIEVAYYDGVEPN PKDTNVKDGLKIFQDENCDMIVTVGGGSSHDCGKGIGIAATHEG DLYDYAGIETLTNPLPPIVAVNTTAGTASEVTRHCVITNTKTKVKFVIVSWRN LPLVSIN D PMLMVGKPAGLTAATGMDALTHAVEAYVSKDANPVTDAAAIQAIKLISSNLRQAVAL GENLVARENMAYGSLLAGMAFNNANLGYVHAMAHQLGGLYDM PHGVANAMLLPH VCKYN LISNPQKFADIAEFMGENIEGLSVMDAAQKAIDAMFRLSTDIGIPAKLRDMGVK EEDFGYMAEMALKDGNAFSNPRKGNERDIVEIFKAAF

156976638 MTSAFFIPTVNLMGAGCLKDATDSIQSQGFKKGLIVTDKILNQIGVVKQVQDLLAERDV

ETVVFDGTQPNPTISNVNDGLALLTDN ECDFVISLGGGSPHDCAKGIALVASNGGKIAD

YEGVDQSAKPMMPLIAINTTAGTASEMTRFCIITDEERHIKMAIVDKHTTPLISVND PEL

MLAKPASLTAATGMDALTHAIEAYVSIAATPITDAVAIKAIELIQAYLRTAVKNGED LEAR

EQMAYAQFMAGMAFNNASLGYVHAMAHQLGGFYDLPHGVCNAILLPHVQRYNAQ

VCPERLRDVAKAMGVNVEDMSAEAGAAAAIDAIVTLAKDVGIPAGIKELGAKLEDIP TL

ADNALKDACGFTN PKQATHEEISKIFEEAM

134300116 MTVGEQVFGYFIPTVNLMGVGAHKEIPDQVKVLGGSNVLIVTDAFLGRPGGMADDIK

GMLEAENIKVTIYAGAEPNPTDVNVHDGLKVYQECGADMI LSLGGGSSHDCAKGIGIV

ATNGGNIRDYEGINKSSKAMPPFIAVNTTAGTASEMTRFCIITNTSN HVKMAIVDWRC

TPNIAINDPLLMAGM PPALTAATGMDALTHAIEAYVSVAATPVTDSAALMAIKLISQYL

RAAVANGENMEARDKMAYAEFLGGMAFNNASLGYVHAMAHQLGGFYN LPHGVCN

AILLPHVEAFNLIACPERFVDIAVAMGENVEGLSVRDAADKALSAIRKLSADVGIPA GLTE

LGVKEEDLKTM AENAM KDACALTN PRKATLN DIVGIYKTAL

46580810 MAVQEQVYGFFIPRVTLIGIGASKAIPEKIKALGGSKPLIVTDMGIVKAGILKQITDLLD AA

KMAYSVYDETIPN PTDDNVHKGVDVYKKNKCDSLITLGGGSSHDCGKGIGLVVANGGK

IHDFEGVDKSTQRMPPYLAVNTTAGTASEMTRFCIITDTSRKVKMAIVDWRVTPN IAL

DDPLLMLGMPPALTAATGM DALTHAVEAYVSTIATPMTDACAEQAITLIATFLRRAVA

NGQDLEARERMCFAQYLAGMAFNNASLGHVHAMAHQLGGFYDLPHGECNAILLPHV

SKFNLIAKLDRYARIAQLMGENIAGLSTREAAERAISAIKCL5TDVGIPAGLVALGK RYGK

DVKAADIAIMTKNAQKDACGLTN PRCPTDADVAAIYEAAL

90412151 MSSAFFIPSVN LMGAGCLTEAADAVKAHGFKKALIVTDKVLNQIGVVKQVVDLLAERN

VEAVVFDGTQPN PTMGNVEAGLALLKANECDFVISLGGGSPHDCAKGIALVASNGGSI

SDYEGVDVSAKPQLPLVAINTTAGTASEMTRFCIITDEARHIKMAIVDKNTTPLMSV ND

PELMLAKPASLTAATGMDALTHAIEAYVSTAATPITDAVAIKAMELIQAHLRTAVN DGQ

NLEAREQMAYAQFMAGMAFNNASLGYVHAMAHQLGGFYDLPHGVCNAVLLPHVQ

RYNAKVCPERLRDVAKAMGVNVEAMTADQGADAALEAIQVLSKDVGIPAGLKDLGAK

NEDISILADNALKDACGFTNPKQATHEEISEIFAAAM

312112413 MSNAHVFYVPSTNLMGRGCLAKVGPFIKEFGFKKALVVTDKFLHKSGIAGKVLAVLDEI

GVNYVVYDDVKPNPTTKNVYAGADLFKKNECDFLVSVGGGSPQDTAKAIGLYVTNGG

DIRDYEGVNKTKNKSVPIVAVNTTAGTSSEFTINYVITDEERNVKMVMVDKNSLVTI SV

NDPELMVDKPAALTAATGMDALTHAIEAVVTPGSYTVTDATALAAIEIIFNYLPRAV KN

GHDIEAREQMAYAMFLVGIAFNNAGLGMVHAMAHQLGGMYDLPHGVCNAMLLPIV

ERENAKRDPRKFRAIAKAAGIDVTGKTDEQCAEEVIEAIKALSREIGIPSKL5ELGV DEVDL

EKLAN NALKDACAPGNPFQPTKEEVISMFKEIL

303249216 MAVREQVYGFFIPSVTLIGIGAAKQIPEKIKALGGTKPLIVTDKGVVKVGVCKMITDLLD A

AGMKYHIYDETIPNPTDENVHKGVEVYKKEGCDSLITLGGGSSHDCGKGIGLVISNGGKI

HDYEGVDKSSKPFMPYLAVNTTAGTASEMTRFCIITDLSRHVKMAIVDWRVTPHIAI D

DPVLMVGMPPALTASTGMDALTHAVEAFVSTIAN PMTDACAIEAIKLIFKYLRKAVAN

GQDMEAREGMCFAEYLAGMAFNNASLGHVHAMAHQLGGFYDLPHGECNAILLPHV

ESYNLIAKVEKFAEMAKIMGENIEGMAPRDAAELCLKAIRQLSVDVGIPAGLVELGK RY

G K D VK A A D 1 PT M TG N AQK D ACG LTN PRCPTDKDVAAI YTA A L

24373064 MAAKFFIPSVNVLGKGAVDDAIGDIKTLGFKRALIVTDKPLVNIGLVGEVAEKLGQNGIT

STVFDGVQPN PTVGNVEAGLALLKANQCDFVISLGGGSPHDCAKGIALVATNGGSIKD

YEGLDKSTKPQLPLVAINTTAGTASEMTRFCIITDEARHIKMAIVDKHTTPILSVND PEL

MLKKPASLTAATGMDALTHAVEAYVSIAAN PITDACAIKAIELIQGNLVNAVKQGQDIE

AREQMAYAQFLAGMAFN NASLGYVHAMAHQLGGFYDLPHGVCNALLLPHVQEYNA

KVVPHRLKDIAKAMGVDVAKMTDEQGAAAAITAIKTLSVAVNIPEN LTLLGVKAEDIPT

LADNALKDACGFTNPKQATHAEICQIFTNAL

269122369 MKVSRRIYWPAVTLIGPGCVKEIGGDIKDLGLKKALVVTDNVLVKIGVVKKVTDVLDESG INYVVVDDIQPN PTMKNIHDGLNTYKSENCDFVISIGGGSPQDAGKAIGLLATNGGEIK

DYEGINMSKHKSVPIIAINTTAGTASEVTINYVITN EDTHIKMVMVDKNCLASIAVSDPE

LMTGKPADLTAATGMDALTHAIEAYVSTGAYELTDVLALEAVKLIGESLEDAVKDGN NI

EARSKMAYASYIAGMSFNNAGLGYVHSMAHQLGGFYNLPHGVCNAILLPHVEKFNSA

NTGDKLRKVAEILGENVEGLSVEEANAKAIEAIMKLSERVGIPKGLKELGVKEEDFK VMA

ENALKDVCAGTNPREVTLEDTIALYKEAL

390456003 MTGTSKFMMPGMSLMGSGALADAGTEIGKLGYTNALIVTDKPLVDIGIVKKVTSVLESI

NVKSVVYSGTQPNPTVTNVNEGLELLSQSKCDFIISLGGGSPHDCAKGIALLASNGGQI

GDYEGVDKSTKPSFPLIAINTTAGTASEMTMFCIITDEERHIKMAIVDNHTTPLIAV NDP

DLMMAMPKSLTAATGM DALTHSIEAYVSTNATPITDACAIKAIELIRDN LARAVDDGN

DVEARSQMAYAEFLAGMAFNNAGLGFVHAMAHQLGGFYNLPHGVCNAILLPHVERY

NAKASAERLTDIARALGENTDGVTPEQGAN LALQAIEKLAKRVNIPSGLEELGVKREDFT

VLAAN ALKDACGVTN PVQPTQQEVI Al FEQAM

152972007 MSYRMFDYLVPNVNFFGPNAISVVGERCQLLGGKKALLVTDKGLRAIKDGAVDKTLHY

LREAGIEVAIFDGVEPN PKDTNVRDGLAVFRREQCDIIVTVGGGSPHDCGKGIGIAATH

EG DLYQYAGI ETLTN PLPP 1 VA VNTTAGTASEVTRH CVLTNTETKVKFVI SWRN LPSVS

INDPLLMIGKPAALTAATGMDALTHAVEAYISKDAN PVTDAAAMQAIRLIARNLRQAV

ALGSNLQAREN MAYASLLAGMAFNNAN LGYVHAMAHQLGGLYDMPHGVANAVLLP

HVARYNLIAN PEKFADIAELMGENITGLSTLDAAEKAIAAITRLSMDIGIPQH LRDLGVKE

ADFPYMAEMALKDGNAFSNPRKGN EQEIAAIFRQAF

49176377 MAASTFFIPSVNVIGADSLTDAMNMMADYGFTRTLIVTDNMLTKLGMAGDVQKALE

ERN IFSVIYDGTQPNPTTENVAAGLKLLKENNCDSVISLGGGSPHDCAKGIALVAANGG

DIRDYEGVDRSAKPQLPMIAINTTAGTASEMTRFCIITDEARHIKMAIVDKHVTPLL SVN

DSSLMIGMPKSLTAATGM DALTHAIEAYVSIAATPITDACALKAVTMIAEN LPLAVEDGS

NAKAREAMAYAQFLAGMAFNNASLGYVHAMAHQLGGFYNLPHGVCNAVLLPHVQV

FNSKVAAARLRDCAAAMGVNVTGKNDAEGAEACINAIRELAKKVDIPAGLRDLNVKE E

D F A V LAT N A LK D ACG FTN P 1 QATH EEIVAIYRAAM

110799824 MSYKFFMPAISLMGADCLKDAGDQVGELGFKKALIVTDKVLGQIGIVKKVTDVLDNKNI

EYAIYDETKPNPTVKNVN DGLALLKEKECDFVISLGGGSAHDCAKGIALLATNGGEIKDY

EGVDKSKKPQLPMVGINTTAGTGSEMTLFAIITDEERHIKMALVDKHLTPIIAVN DPI L

MLAMPKSLTAATGMDALTHAIEAYVSTAATPITDACAEKAIELISNYLVNAVENGQD VE

ARDMMAYAEYLAGMAFNNASLGYVHAMAHQLGGFYNLPHGVCNAILLPHVQEYNK

STSASRLAKIAKIMGGNIEGLTDEQGADLCIDMIKSLSQTIGIPEGLGVLGVKESDF ETLAT

NALN DACSLTN PRKGNLEEVIAIFKKAM

113866887 MRARPARAPKRKAQERPSSSRM PACTRWGYPKPSRGTSARQGFRPLIFPGAEPN PTD

VNVHDGVKLFEQEGCDFIVSLGGGSSHDCAKGIGLVTAGGGHIRDYEGIDKSTVPMTP

LISINTTAGTAAEMTRFCllTNSSN HVKMAi DWRCTPLIAIDDPRLMVAMPPALTAAT

GMDALTHAVEAYVSTAATPITDACAEKAIALIGEWLPKAVANGNSLEARAAMCYAQY L

AGMAFNNASLGYVHAMAHQLGGLYNLPHGVCNAILLPHVSEFNLIAAPERFAKIAEL L

GENVASLSTSDAAKAAISAIRALAASIGIPAGLASLGVKAEDHEVMAHNAQKDACML T

N P RRATTAQVI Al F AAAM

167040564 MKIFKFHMPPINLIGVGCLKDVGREIKKLGFKKGIIVTDKVLVRAGLVNNVISVLEEEGI EY

VVFDETKPNPTIKNVTNGLKLLIEN KCDFIISCGGGSAHDCAKGIGLIAKEKNFIDEVERL

DKVKCGGWNSALLLPLVAINTTAGTGSEVTKFAIITDEEKRIKMPIVDWRITPLIAV NDP

LLM IGMPKSLTAASGMDALTHAIEAYISIDANPFTDALALKAIEIIFNYLKRAVENGNDIE

AREKMAYAEFLAGIAFNNAGLGYVHAMAHQLGGFYDLPHGVCNAVLLPHVLEYNLEA

VQN KLIYIAKAMGIDVDKLTTKEIGGKIIESINQL5QEIGIPSRLKELGVKEEDIKELSQNAL

KDVCGFTNPKKATLEDIIN IFKSAM

406526935 MGNRIILN GTSYFGRGARENVITELRN RNFTKALVVTDKN LLDAHVTN LVTDVLDKNDF

SYQIYSDIKPN PTTLNVQEGVTFCRNSKADVIIAVGGGSAIDTAKAISIIMTN PEHFDVISL DGAVETKN AGMPI IALPTTAGTAAEVTI NYVITN PVGPKKM VCVDPH Dl PI VAI 1 DQDL MEKMPKSLAASTGM DALTHAMEGYTTKAAWLMTDMFHLNAMALIYKNLEKAVNLK DRDAIDNVGYGQYIAGMGFSNVGLGIVHSMAHSLGAFFDTPHGLANALLLPHVLKFN GKICPDLFRNMGRAMGLDMDN LTDDEAVDKVVDAVRSLAIKIGIPQTLKEIGIKKEDLP M LAHQAI DDVCTAGN PRN VTEQDI LALYQEAYE

138896515 MQNFTFRN PTKLIFGRGQIEQLKEEVPKYGKKVLLVYGGGSIKRNGLYDEVMSLLTDIGA

EVVELPGVEPN PRL5TVKKGVDICRREGIEFLLAVGGGSVIDCTKAIAAGAKFDGDPWE

FITKKATVTEALPFGTVLTLAATGSEMNAGSVITNWETKEKYGWGSPVTFPQFSILD PT

YTMTVPKDHTVYGIVDMMSHVFEQYFHHTPNTPLQDRMCEAVLKTVIEAAPKLVDDL

ENYELRETIMYSGTIALNGFLQMGVRGDWATHDIEHAVSAVYDIPHAGGLAILFPNW

MKHVLDENVSRFAQLAVRVFDVDPTGKTERDVALEGIERLRAFWSSLGAPSRLADYG I

GEEN LELMADKAMAFGEFGRFKTLNRDDVLAILRASL

consensus M(T _; K)(N )(T )( )(SJ _; R)(NAI, )F(F _; Y)(l,M)P(P _; S)(AN _; S)(N _; SJ)(V _; L)(FJ)G(R _;

A _; P)G(A _; S)V(K _; N)EVG(T _; A)RL(K _; A)(Q _; G _; D _; A)(I _; L)G(AN)(K _; T)KAL(IJ)VTDA(F,G)LH(

G^SXTJlGLSEf E^KXVJXA^SXK^S^GXNJllRfEA^AGfV^LKD _j EXV XAN^S^LllFPKAfQ

_; E)P( N)P(AJ)D(T _; K)( N)V(H _; A)EG(V _; L)(D _; E)(VAI)(F _; Y)( _; N)( A)E(N,K)CD(S _;

AXLTlVfSJXl.DGGGSSHDiT.AXG.AlKiA.OKG.AlLVAANGGiRJ.KlKN.H. YlDYiQ

.ElGVfN.DKSN.KKV.KE.KKK.EKP.QKMNlVPiLV.QXl.VlAKNjnTAGTGSE iT.L)

T(S,K,R)(F,L)(A,T)(V,I)ITD(S,T)(A,E)RKVKM(P,A)(V,I)(IN)D( E,K)(H,K)(I,V)TPT(V,L)

(A^SlUV^NlDPELMVfK^GXK^MlPfPA^IG.SlLTf lA^ATGfM^LlDAUSJlHAIEAYVfA^S)

(KJXG^RA^ATPfVJlTDAfF^LlAIQAfMJlKf LJlUN^SXE^KlYLPfR^KlAfV^FlANGfE^K)

DfLMlEAREf A^KlMAfY^FlAQfY^SXM^LlAGfMNlAFNNfGA^IG^lLGfY^LlVHfSA^

XSAiHQlV^UFlGGiFNlYfK^N XF^XP^QlHGflNlCNfSA^l JXN^LNXM^LlPlH^Yl C

(R AlFNLXA^SXK^RXTNlERfF^YlAfH^EllAfEA^l FlLGENVfSADlGLSTfA^Y^RXS,

A _; E _; D)AAE(R _; K)(AJ _; G)XKNA)A(LJ)(E _; Q)R(M _; YJ)(NA( _; R)( N _; S)(F _; L)(G _; N)IP(S

V.AXCJXT.AXLQXT.S.DlNPRfV.KXP.AXT.KXV.LXQ.EXD.EXl .VXA.I.QlQIIKNAi

M,L).