Methods to shift redox equilibrium using an orthogonal redox cofactor, and applications thereof

Abstract

Provided herein is use of a design principle for recombinantly engineering polypeptides having redox enzyme activity that have improved catalytic efficiency for a noncanonical cofactor while having reduced catalytic efficiency for a natural cofactor. The disclosure further provides utilizing the foregoing design principle for the construction of orthogonal metabolic systems that are not tied to natural metabolism by an organism. Further provided herein is the production of an orthogonal metabolic system that utilized NMN(H) as a driving force for the in vivo conversion of (m)-Bdo to (SS)-BDo or (RR)-Bdo with high purity.

Description

Attorney Docket No.00058-086WO1 METHODS TO SHIFT REDOX EQUILIBRIUM USING AN ORTHOGONAL REDOX COFACTOR, AND APPLICATIONS THEREOF CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 651,888, filed May 24, 2024, the disclosure of which is incorporated herein by reference in its entirety. STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with Government support under Grant No.1847705, awarded by the National Science Foundation, Grant No. DE-AR0001508 awarded by the U.S. Department of Energy, and Grant No. DP2 GM137427 awarded by the National Institutes of Health. The Government has certain rights in the invention. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] Accompanying this filing is a Sequence Listing entitled, “00058-086WO1.xml” created on May 23, 2025 and having 33,527 bytes of data, machine formatted on IBM-PC, MS-Windows operating system. The sequence listing is hereby incorporated by reference in its entirety for all purposes. TECHNICAL FIELD

[0004] Provided herein is use of a design principle for recombinantly engineering polypeptides having redox enzyme activity that have improved catalytic efficiency for a noncanonical cofactor while having reduced catalytic efficiency for a natural cofactor. The disclosure further provides utilizing the foregoing design principle for the construction of orthogonal metabolic systems that are not tied to natural metabolism by an organism. Further shown herein is the production of an orthogonal metabolic system that utilized NMN(H) as a driving force for the in vivo conversion of (m)-BDO to (SS)-BDO or (RR)-BDO with high purity. BACKGROUND

[0005] Biomanufacturing of renewable chemicals by engineered microbes or synthetic biochemistry has the potential to supplant many petroleum-derived chemical industries. However, many engineered biosynthetic pathways fail to proceed past laboratory scale, because of insufficient titer, productivity, or yield. One prevalent reason is because the chassis organisms that provide high energy cofactors also contain numerous competingAttorney Docket No.00058-086WO1 pathways, which drain these critical resources. Partitioning and balancing redox cofactors is a prevalent need in metabolic engineering. SUMMARY

[0006] Nature's two cofactors, nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), are held at different reduction potentials to drive catabolism and anabolism in opposite directions. In biomanufacturing, there is a need to flexibly control redox reaction direction that is decoupled from catabolism and anabolism. Nicotinamide mononucleotide (NMN+) has been developed as a noncanonical cofactor that is orthogonal to NAD(P)+. Presented herein is the development of Nox Ortho, an NMNH-specific oxidase, that was designed to modulate the NMNH:NMN+ratio together with an NMN+-specific glucose dehydrogenase (GDH Ortho). More importantly, the design principle discovered from engineering and modeling Nox Ortho was translatable onto six unrelated enzymes to create NMN(H)-orthogonal biocatalysts that exhibited a consistent ~103– 106-fold cofactor specificity switch from NAD(P)+to NMN+. These enzymes were assembled to produce stereo-pure 2,3-butanediol in a cell-free system and in Escherichia coli, that was enabled by NMN(H)'s distinct redox ratio being firmly set by its designated driving forces, which was decoupled from both NAD(H) and NADP(H).

[0007] In a particular embodiment, the disclosure provides a recombinantly engineered polypeptide having redox enzyme activity that has improved catalytic efficiency for a noncanonical cofactor while having reduced catalytic efficiency for a natural cofactor, the recombinantly engineered polypeptide comprising: a modified or mutated natural cofactor binding pocket that comprises: (1) amino acid substitutions or mutations that form new hydrogen bond(s) that inhibit or prevent entry by the natural cofactor into the binding pocket; and (2) amino acid substitutions or mutations that promote the binding of the noncanonical cofactor by introducing new polar contact(s) that interact specifically with polar group(s) or moieties of the noncanonical cofactor. In another embodiment, the modified or mutated natural cofactor binding pocket of the recombinantly engineered polypeptide comprises 3 to 10 amino acid mutations or substitutions in comparison to the sequence of a wild-type or parent polypeptide. In yet another embodiment, at least one of the substitutions or mutations introduces a new polar contact that interacts specifically with a polar group of the noncanonical cofactor, and wherein at least two of the substitutions or mutations form a new hydrogen bond that inhibits or prevents entry by the natural cofactor into the binding pocket.Attorney Docket No.00058-086WO1 In a further embodiment, the recombinantly engineered polypeptide encodes a Rossmann- fold containing protein, and the new polar contact that interacts specifically with a polar group of the noncanonical cofactor is found in Rossmann helix α1 or α2, and wherein the new hydrogen bond that inhibits or prevents entry by the natural cofactor into the binding pocket closes the space between Rossmann strands β2 and β3. In yet a further embodiment, the modified or mutated natural cofactor binding pocket does not comprise mutations or substitutions that restricts the size of the natural cofactor binding pocket by forming hydrophobic packing interactions which inhibit or prevent entry by the natural cofactor into the binding pocket. In a certain embodiment, the recombinantly engineered polypeptide has at least 15-fold increase in catalytic efficiency for the noncanonical cofactor in comparison to a wild-type or parent polypeptide. In another embodiment, the recombinantly engineered polypeptide has at least a 50-fold decrease in catalytic efficiency for the natural cofactor in comparison to a wild-type or parent polypeptide. In yet another embodiment, the noncanonical cofactor is selected from the group consisting of nicotinamide mononucleotide (NMNH), 1-phenyl-1,4,-dihydronicotinamide , 1-benzyl-1,4-dihydronicotinamide, 1-(4- hydroxyphenyl)1,4-dihydronicotinamide, 1-methyl-1,4-dihydronicotinamide, nicotinamide flucytosine dinucleotide, nicotinamide mononucleoside, 1‐butyl‐1,4,5,6‐tetrahydropyridine‐3‐ carboxamide, 1‐(1‐benzyl‐1,4,5,6‐tetrahydropyridin‐3‐yl) ethenone, 1-benzyl-1,4- dihydropyridine-3-carboxylic acid, and 1‐benzyl‐1,4,5,6‐tetrahydropyridine‐3‐carbonitrile. In a further embodiment, the noncanonical cofactor is NMNH. In yet a further embodiment, the natural cofactor is selected from NAD(H), FAD(H), and NADP(H). In a particular embodiment, the recombinantly engineered polypeptide has a redox enzyme activity selected from an oxidase, a dehydrogenase, and an oxidoreductase. In a further embodiment, the recombinantly engineered polypeptide comprises a sequence that is at least 95% identical to a sequence from an oxidase, a dehydrogenase, or an oxidoreductase. In yet a further embodiment, the recombinantly engineered polypeptide comprises a sequence that is at least 98% identical to a sequence from an oxidase, a dehydrogenase, or an oxidoreductase. In a certain embodiment, the oxidase is selected from glucose oxidase, NADPH oxidase, amine oxidase, and NADH oxidase. In a further embodiment, the oxidase is an NADH oxidase selected from Anaerocolumna aminovalerica, Bacillus subtilis, Enterococcus faecalis, Lacticaseibacillus rhamnosus, Lactiplantibacillus pentosus, Lactococcus cremoris, Lactococcus lactis, Levilactobacillus brevis, Methanobrevibacter smithii, StreptococcusAttorney Docket No.00058-086WO1 agalactiae, Streptococcus mutans, and Streptococcus pyogenes. In yet a further embodiment, the oxidase is an NADH oxidase having an amino acid sequence that is at least 98% identical to SEQ ID NO:6 or SEQ ID NO:7. In another embodiment, the recombinantly engineered polypeptide comprises a sequence that is at least 98% identical to SEQ ID NO:6 but comprises at least the substitutions of I159T, D178N, A179F, and I243E. In yet another embodiment, the dehydrogenase or the oxidoreductase is selected from phosphite dehydrogenase, meso-(2R,3S)-butanediol dehydrogenase ((m)-Bdh), (2R,3R)-butanediol dehydrogenase ((R)-Bdh), (2S,3S)-butanediol dehydrogenase ((S)-Bdh), alcohol dehydrogenase, acetoin reductase, diacetyl reductase, alcohol dehydrogenase, glutathione reductase, homoserine dehydrogenase, glucose dehydrogenase, glycerol dehydrogenase, propanediol-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, acetaldehyde dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, pyruvate dehydrogenase, oxoglutarate dehydrogenase, and formate dehydrogenase. In yet another embodiment, the dehydrogenase or the oxidoreductase is from the enzyme class of (m)-Bdh, (R)-Bdh, or (S)- Bdh having a sequence that is at least 95% identical to the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21. In a further embodiment, the recombinantly engineered polypeptide comprises a sequence that is at least 98% identical to the sequence of SEQ ID NO:1 but comprises at least the amino acid substitutions of L39Q, A92K, and M194T. In yet a further embodiment, the recombinantly engineered polypeptide comprises a sequence that is at least 98% identical to the sequence of SEQ ID NO:2 but comprises at least the amino acid substitutions of M189T, Y34Q, and A87K. In another embodiment, the recombinantly engineered polypeptide comprises a sequence selected from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26.

[0008] In a particular embodiment, the disclosure also provides an expression vector comprising a recombinantly engineered polypeptide of the disclosure. In a further embodiment, the expression vector is a plasmid, a viral vector, a cosmid, or an artificial chromosome. In yet a further embodiment, the expression vector is a plasmid that comprises elements for expressing the recombinantly engineered polypeptide in a bacterium or a yeast.

[0009] In a certain embodiment, the disclosure further provides a microorganism thatAttorney Docket No.00058-086WO1 comprises a recombinantly engineered polypeptide of the disclosure. In a further embodiment, the microorganism is a bacterium, fungus, or a yeast. In yet another embodiment, the microorganism is a strain of E. coli that is unable to catabolize glucose and has decreased ability to degrade noncanonical cofactors.

[0010] In a particular embodiment, the disclosure further provides a microorganism that comprises an expression vector comprising a recombinantly engineered polypeptide of the disclosure. In a further embodiment, the microorganism is a bacterium, fungus, or a yeast. In yet another embodiment, the microorganism is a strain of E. coli that is unable to catabolize glucose and has decreased ability to degrade noncanonical cofactors.

[0011] In a certain embodiment, the disclosure also provides a method of stereo- upgrading (2S,3R)-meso-butanediol ((m)-BDO) to (2S,3S)-butanediol ((SS)-BDO) or (2R,3R)-butanediol ((RR)-BDO) in a cell free system, comprising: providing the following purified polypeptides in a buffer system comprising a natural cofactor and a noncanonical cofactor: a polypeptide encoding a Bdh, Dar, or BudC enzyme that utilizes NAD(H) or NADP(H) as a natural cofactor, a polypeptide encoding an orthogonal (S)-Bdh comprising the sequence of SEQ ID NO:25 or a polypeptide encoding an orthogonal (m)-Bdh comprising the sequence of SEQ ID NO:26, a polypeptide encoding an Nox oxidase comprising the sequence of SEQ ID NO:8 or SEQ ID NO:24, and a polypeptide encoding a Gdh dehydrogenase comprising the sequence of SEQ ID NO:5 or SEQ ID NO:22; and introducing (m)-BDO into the buffer system that is then converted into (SS)-BDO or (RR)-BDO by the activities of the purified polypeptides. In another embodiment, the polypeptide encoding an orthogonal (S)-Bdh has the sequence of SEQ ID NO:25, and wherein the polypeptide encoding an orthogonal (m)-Bdh has the sequence of SEQ ID NO:26. In yet another embodiment, the polypeptide encoding a Nox oxidase has the sequence of SEQ ID NO:24. In a further embodiment, the polypeptide encoding a Gdh dehydrogenase has the sequence of SEQ ID NO:22. In yet a further embodiment, the natural cofactor is NADP(H) or NAD(H), and the noncanonical cofactor is NMN(H). In a particular embodiment, the method stereo- upgrades (m)-BDO to (SS)-BDO in a cell-free system and the method comprises: providing the following purified polypeptides in a buffer system comprising NAD(H) and NMN(H): a polypeptide encoding a Bdh, Dar, or BudC enzyme that utilizes NAD(H) as a natural cofactor, a polypeptide encoding an orthogonal (S)-Bdh that comprises the sequence of SEQ ID NO:25, a polypeptide encoding a Nox oxidase that comprises the sequence of SEQ IDAttorney Docket No.00058-086WO1 NO:24, a polypeptide encoding a Gdh dehydrogenase that comprises the sequence of SEQ ID NO:22; and introducing (m)-BDO into the buffer system that is then converted into (SS)- BDO by the activities of the purified polypeptides.

[0012] In a certain embodiment, the disclosure also provides a method to produce (2S,3S)-butanediol (SS)-BDO)) in vivo, comprising: co-expressing the following polypeptides in a recombinant microorganism: a recombinantly engineered polypeptide encoding a (R)- Bdh enzyme that utilizes NAD(H) as a natural cofactor, a polypeptide encoding an orthogonal (S)-Bdh hat comprises the sequence of SEQ ID NO:25, a polypeptide encoding a Nox oxidase that comprises the sequence of SEQ ID NO:7, a polypeptide encoding a Gdh dehydrogenase that comprises the sequence of SEQ ID NO:22, a polypeptide encoding a glucose transport facilitator that has a sequence of SEQ ID NO:23; and producing (SS)-BDO by incubating the recombinant microorganism with media containing glucose, (2R,3S)-meso- butanediol, and NMN(H). In a further embodiment, the recombinant microorganism is a strain of E. coli that is unable to catabolize glucose and has decreased ability to degrade noncanonical cofactors.

[0013] In a particular embodiment, the disclosure further provides a method to produce (2S,3S)-butanediol ((SS)-BDO) in vivo, comprising: co-expressing the following polypeptides in a recombinant microorganism: a recombinantly engineered polypeptide encoding a (R)-Bdh enzyme that utilizes NADP(H) as a natural cofactor, a polypeptide encoding an orthogonal (S)-Bdh hat comprises the sequence of SEQ ID NO:25, a polypeptide encoding a Nox oxidase that comprises the sequence of SEQ ID NO:8, a polypeptide encoding a Gdh dehydrogenase that comprises the sequence of SEQ ID NO:22, a polypeptide encoding a glucose transport facilitator that has a sequence of SEQ ID NO:23; and producing (SS)-BDO by incubating the recombinant microorganism with media containing glucose, meso-(2R,3S)-butanediol, and NMN(H). In another embodiment, the recombinant microorganism is a strain of E. coli that is unable to catabolize glucose and has decreased ability to degrade noncanonical cofactors.

[0014] In a certain embodiment, the disclosure provides a composition, method, or system as substantially disclosed in the specification and figures.

[0015] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.Attorney Docket No.00058-086WO1 BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the disclosure and, together with the detailed description, serve to explain the principles and implementations of the invention.

[0017] FIG.1A-I provides an overview of the four proposed BDO stereo-upgrading systems for testing the crosstalk of two orthogonal cofactors. (A, C) Stereo-upgrading systems of (m)-BDO to (SS)-BDO where NAD(H) (A) or NADP(H) (C) drives the oxidation step and NMN(H) drives the reduction step. (B, D) Stereo-upgrading systems of (m)-BDO to (RR)-BDO where NMN(H) drives the oxidation step and NAD(H) (B) or NADP(H) (D) drives the reduction step. (E) Illustration showing that, when crosstalk between the oxidation and reduction steps occurs, reactions are reversible and incomplete. The four systems above cannot be achieved with a single cofactor or multiple cross talking cofactors. Gray and light gray signify oxidation and reduction, respectively. The two-color cofactor pool boxes represent crosstalk and fully reversible reaction steps. (F) The typical stereospecificity and chiral substrate preference of Bdhs from different enzyme classes used. (G) Single-cofactor reaction of the Bdhs with no cofactor-recycling reactions driving each step. (H) Single- cofactor reaction with L. brevis Nox WT recycling NADH to NAD+to drive the oxidation step. (I) Single-cofactor reaction with B. subtilis GDH WT recycling NAD+to NADH to drive the reduction step. The reaction does not proceed past the first oxidation step. The substrate for all experiments is 5 g / L m-BDO and conversion is measured after 48 h at 30 °C. Bars represent the mean of three independent replicates with error bars of one s.d. White diamonds indicate the values of individual replicates.

[0018] FIG.2A-D demonstrates directed evolution of Ll Nox to exclude NADH. (A) Schematic of the high-throughput growth-based selection platform workflow for NMNH- using enzymes. The designed site-saturated mutagenesis library was introduced to the engineered E. coli strain MX502, where growth depends upon Nox NMNH oxidase activity through the ED pathway. Fast-growing variants were characterized by a specific activity assay. PP, pentose phosphate. (B) The apparent catalytic efficiencies of Ll Nox WT (gray) and Nox Ortho (light gray) toward NADH, NADPH and NMNH. The assay was performed at 37 °C in 50 mM Tris-Cl, pH 7.0 with varying concentrations of reduced cofactors. (C) Model of Nox Ortho binding pose with NMNH and FAD revealed novel hydrogen-bond formation. (D) Ll Nox WT (gray) and Nox Ortho (light gray) with NADH bound revealed aAttorney Docket No.00058-086WO1 conformational change in NADH-binding pose. Substitutions on Nox Ortho exclude NADH from its native binding mode to become exposed to solvent, consistent with the decreased catalytic efficiency for NADH observed. Data are presented as the mean of three independent replicates (n = 3) with error bars of one s.d. White diamonds represent individual replicates. Two-tailed t-tests of unpaired samples were conducted assuming unequal variances (**P < 0.01): Ll Nox WT versus Ll Nox Ortho (NADH, P = 0.00073; NMNH, P = 0.0017).

[0019] FIG.3A-B Specific activity of Ll Nox I159T and Ll Nox selection variants towards NMNH. (A) Comparison of Ll Nox WT and Ll Nox I159T. (B) Specific activity toward NMNH for candidate Ll Nox variants from selection. LL-9 (Ortho), renamed Nox Ortho, showed ~30-fold increase in NMNH activity compared to WT. One unit of enzymatic activity was defined as the quantity of enzyme required to consume 1 μmol of reduced cofactor (NMNH) per minute. Data are presented as mean of two independent replicates with individual replicates depicted as diamonds.

[0020] FIG.4A-C Fitting curves for the determination of apparent Michaelis-Menten kinetic parameters. Apparent Michaelis-Menten curve with hyperbolic or linear fit of (A). Kp (m)-Bdh WT and Ortho, (B). Ser (S)-Bdh WT and Ortho, (C). Ll Nox WT and Ortho with NAD(P)H and NMNH. The initial rate data of the amount of supplied cofactor consumed were fitted to a linear form of Michaelis-Menten equation where the enzyme could not be saturated with the cofactor. For Ser (S)-Bdh WT with NAD+, values are average of four independent replicates (n=4). For all other samples, values are average of three independent replicates (n=3). Error bars represent one standard deviation.

[0021] FIG.5 presents a model of Ll Nox WT with NMNH. Model of Ll Nox WT generated in Rosetta with equivalent constraints.

[0022] FIG.6A-C provides an estimation of the structural stability of Ll Nox WT and Nox Ortho taking docking structures as input. Snapshots extracted from 100 ns molecular dynamics simulations of (A) Ll Nox WT, and (B) Nox Ortho. (C) Evolution of the number of hydrogen bonds formed between the phosphate tail of NMNH and protein as a function of time. For clarity, water and ions are omitted and only polar hydrogen are shown. Hydrogen bonds are represented in black dashed line.

[0023] FIG.7A-E presents the engineering Bdhs to use NMN(H). (A) Predicted interactions of Kp (m)-Bdh Ortho with NMN+. (B) Apparent catalytic efficiencies of Kp (m)- Bdh WT (gray) and Kp (m)-Bdh Ortho (green) with each cofactor. The substrate was 50 mMAttorney Docket No.00058-086WO1 (m)-BDO with varying cofactor concentration. (C) Specific activities of six Bdh homologs based on analogous substitution transfer from Kp (m)-Bdh Ortho (Kp (m)-Bdh sites). Summary of measured chiral substrate and product preference in (R / S)-Ac feeding experiments (see FIG.9). NT, not tested. The substrate was 10 mM (R / S)-Ac with 0.2 mM reduced cofactor. One unit of enzymatic activity was defined as the quantity of enzyme required to consume 1 μmol of reduced cofactor (NADH, NADPH or NMNH) per minute. (D) Predicted interactions of Ser (S)-Bdh Ortho with NMN+. (E) Apparent catalytic efficiencies of Ser (S)-Bdh WT (gray) and Ser (S)-Bdh Ortho (purple) with each cofactor. The substrate for Ser (S)-Bdh assays was 50 mM (SS)-BDO with varying cofactor concentration. Bars are the average of three independent replicates (n = 3) with error bars of one s.d. for all samples except for Ser (S)-Bdh WT with NAD+(n = 4). Individual replicates are shown as white diamonds. All activity assays of Bdhs were conducted at 30 °C in 50 mM Tris-Cl at pH 8.0. Two-tailed t-tests of unpaired samples were conducted assuming unequal variances (*P < 0.05): WT versus Ortho catalytic efficiency (Kp (m)-Bdh: NAD+, P = 0.014; NADP+, P = 0.0014; NMN+, P = 0.0037; Ser (S)-Bdh: NAD+, P = 0.0012; NADP+, P = 0.0033; NMN+, P = 2.0 × 10−7); NMNH versus NADH specific activity (P = 0.0012, 0.00049, 0.00010, 0.035, 0.015 and 0.00079); NMNH-specific versus NADPH-specific activity (P = 0.00096, 0.00013, 4.4 × 10−6, 0.037, 0.014 and 0.0041).

[0024] FIG.8 Kp (m)-Bdh WT and Kp (m)-Bdh Ortho predicted interactions with NAD+. Compared to Kp (m)-Bdh WT, Kp (m)-Bdh Ortho adopts a nonnatural, nonproductive NAD+conformer. Despite the polar interaction introduced by M189T, the mutations of A87K and Y34Q resulted in a malicious effect of NAD+binding by forming a novel hydrogen bond interaction that excludes the adenine group of NAD+while removing the favorable hydrophobic interaction between the cofactor and side chains of A87 and Y34.

[0025] FIG.9A-C presents the purified protein cycling reactions of NMNH-active (S)-Bdh homologs. (A) Design of the cycling reactions. Racemic acetoin (1:1 mixture of enantiomers) is converted into 2,3-butanediol stereoisomers to test the stereospecificity of the engineered Bdh homologs. Reactions contain 1 M NaCl, 100 mM potassium phosphate pH 7.5, 200 mM D-glucose, 2 mM NMN+, 5 g / L racemic acetoin, Gdh Ortho, and one of the Bdh homolog variants with the corresponding mutations. (B) Concentration of each acetoin stereoisomer consumed after 4 h of cycling reaction. Since only one acetoin isomer decreased in each reaction, consumption is calculated as difference in each acetoin isomer’sAttorney Docket No.00058-086WO1 concentration where error bars represent standard deviation of the difference calculated by propagation of error. Based on observed acetoin isomer consumption, the substrate specificity was summarized for each mutant in the table. (C) Concentration of each butanediol stereoisomer formed in the same reaction. Only the mutant of Ser Bdh forms detectable (SS)- BDO. Both Kp (m)-Bdh Ortho and Ser (S)-Bdh Ortho are stereospecific for (S)-installation because the stereoisomer installed for all enzymes tested is an (S)-chiral center. Values are the average of three independent replicates (n=3) with error bars representing one standard deviation. Individual replicates are depicted as diamonds.

[0026] FIG.10A-L demonstrates orthogonal redox driving forces enable BDO stereo-upgrading in four cell-free systems. (A-C) (m)-BDO to (SS)-BDO system that pairs an NAD(H)-driven oxidation step and NMN(H)-driven reduction step. (D-F) (m)-BDO to (RR)- BDO system that pairs an NMN(H)-driven oxidation step and NAD(H)-driven reduction step. (G-I) (m)-BDO to (SS)-BDO system that pairs an NADP(H)-driven oxidation step and NMN(H)-driven reduction step. (J-L) (m)-BDO to (RR)-BDO system that pairs an NMN(H)- driven oxidation step and NADP(H)-driven reduction step. (A, D, G, J) Reaction pathway maps. (B, E, H, K) Concentration of BDO and Ac isomers. Samples were taken for gas chromatography (GC) at 24 h, 72 h, 72 h and 72 h, respectively. Bars represent the mean concentration of three or four independent replicates with error bars of one s.d. Individual replicates are shown as white diamonds. (C,F, I, L) Concentration ratio of each redox cofactor’s cognate reduced and oxidized species on a log10 scale. Samples were taken for redox ratio measurement at 48 h, 72 h, 48 h and 24 h, respectively. Samples for GC and redox ratio analysis were taken from the same reactions. Bars represent the ratio of concentration means for three or four independent replicates as depicted with error bars calculated by the propagation of error from the s.d. of oxidized and total cofactor concentration measurements. White diamonds represent the values of individually calculated replicates. Ortho represents the cofactor engineered variants. Gluc, gluconic acid. Two-tailed t-tests for paired samples of logarithm-transformed redox ratios were conducted assuming unequal variances (**P < 0.01): NMN(H) versus NAD(H) ratio to produce (SS)-BDO (P = 0.00027) and (RR)-BDO (P = 0.00024); NMN(H) versus NADP(H) ratio to produce (SS)-BDO (P = 0.000023) and (RR)- BDO (P = 0.000055).

[0027] FIG.11 provides Ser (S)-Bdh WT and Ser (S)-Bdh Ortho predicted interactions with NAD+. Similar to the models of Kp (m)-Bdh WT and Kp (m)-Bdh OrthoAttorney Docket No.00058-086WO1 docked with NAD+in FIG.8, Ser (S)-Bdh Ortho is predicted to adopt an unnatural conformer that is not favored by Ser (S)-Bdh WT, leading to the significantly lower catalytic efficiency with NAD+.

[0028] FIG.12A-G presents bioprospecting an NADP+-active (R)-Bdh for (m)-BDO oxidation. (A-C) BLAST hits of Bs (R)-Bdh homologs in the NCBI nonredundant protein database. Amino acid frequency distributions at (A) E200, (B) L201, or (C) R205 (Bs (R)- Bdh numbering) are shown. Frequency is normalized to 2000 sequences. (D) Specific activity of Bs (R)-Bdh homologs with NAD+and NADP+. Values are the average of two independent replicates for NAD+and three independent replicates for NADP+with error bars representing one standard deviation. “n.d.” denotes that no data was collected for that protein with NAD+. One unit of enzymatic activity was defined as the quantity of enzyme required to produce 1 μmol of reduced cofactor (NADH or NADPH) per minute. (E) Setup of the oxidation cycling assay to determine (R)-Bdh homolog activity and stereospecificity. (R)-Bdh activity generates (S)-Ac, (S)-Bdh activity generates (R)-Ac based on the chiral center destroyed. Four Bs Bdh homologs were tested for their stereospecificity in the removal of (R)-chiral centers against Bs (R)-Bdh control reaction. (F) (m)-BDO consumption relative to a no cofactor, no enzyme control reaction. (G) Acetoin isomer concentrations. All four bioprospected (R)-Bdhs tested are stereospecific for the (R)-chiral center. (F, G) Values are the average of three independent replicates except with Cbu Bdh which was performed in duplicate (n=2). Error bars represent one standard deviation.

[0029] FIG.13A-G presents the resting cell stereo-upgrading of (m)-BDO to (SS)- BDO in E. coli. (A) Gene deletions and reaction pathways in the whole-cell chassis, strain MX102 R0. (B, D-E) (m)-BDO to (SS)-BDO system using NAD(H)-driven oxidation step paired with an NMN(H)-driven reduction step in vivo. (B) Reaction pathway details of the NAD(H)-driven oxidation step. (D) Concentration of BDO and Ac stereoisomers when 0 mM or 10 mM NMN+was supplemented to the medium, sampled after 24 h at 30 °C. (D) Product purity of (SS)-BDO in different NMN+supplementation conditions. (C, F-G) (m)-BDO to (SS)-BDO system using NADP(H)-driven oxidation step paired with an NMN(H)-driven reduction step in vivo. (C) Reaction pathway details of the NADP(H)-driven oxidation step. (F) Concentration of BDO and Ac stereoisomers when 0 mM or 10 mM NMN+was supplemented to the medium, sampled after 152 h at 18 °C. (G) Product purity of (SS)-BDO in different NMN+supplementation treatments. Bars represent an average of three biologicalAttorney Docket No.00058-086WO1 replicates with error bars of one s.d. White diamonds represent the values of individual replicates. Two-tailed t-tests of paired samples were conducted assuming unequal variances (P < 0.05): effect of 0 mM versus 10 mM NMN+ for (SS)-BDO and (S)-Ac percentage purity: (D, E) P = 0.0015, 0.00047 and 0.0014; (F, G) P = 0.0078, 0.016 and 0.0082. Product purity was calculated as the percentage of (SS)-BDO in the total amount of products formed ((R)-Ac, (S)-Ac, (RR)-BDO, (SS)-BDO and (m)-BDO).6-P-Gluc, 6-phosphogluconate; Glucose-6P, glucose-6-phosphate; NaMN, nicotinic acid mononucleotide.

[0030] FIG.14A-C provides for detection of BDO stereo-upgrading products in E. coli whole cell using NAD(H) as the oxidant by GC. (A) Gas chromatograms of a representative biological replicate for the 10 mM NMN+(green trace) supplementation condition and 0 mM NMN+condition (red trace) relative to a mixed standard (blue trace) of (R)-Ac, (S)-Ac, (SS)-BDO, (RR)-BDO, and (m)-BDO. Traces plot signal (pA) by flame ionization detector (vertical axis) vs time (min). (B-C) Comparison of three biological replicates in the 10 mM NMN+and 0 mM NMN+supplementation conditions, respectively. Traces plot signal (pA) by flame ionization detector (vertical axis) vs time (min).

[0031] FIG.15A-C provides for detection of BDO stereo-upgrading products in E. coli whole cell using NADP(H) as the oxidant by GC. (A) Gas chromatograms of a representative biological replicate for the 10 mM NMN+(pink trace) supplementation condition and 0 mM NMN+condition (blue trace) relative to a low and high concentration mixed standard (red and green traces) of (R)-Ac, (S)-Ac, (SS)-BDO, (RR)-BDO, and (m)- BDO. Retention profiles of BDO isomers can front at high concentration. Peak assignments could be reliably assigned within the concentration ranges of samples injected. Traces plot signal (pA) by flame ionization detector (vertical axis) vs time (min). (B-C) Comparison of three biological replicates in the 10 mM NMN+and 0 mM NMN+supplementation conditions, respectively. Traces plot signal (pA) by flame ionization detector (vertical axis) vs time (min).

[0032] FIG.16A-F presents visualizing emerging design principles for noncanonical redox cofactor-specific enzyme design. (A-B) Alignment of previously experimentally resolved structures of Nox (A) and Bdh (B) that are similar to Ll Nox and Kp Dar, the enzymes engineered in this study (PDB 5VN0 and 1GEG, respectively). Green spheres represent the analogous sites substituted to block dinucleotide cofactors in Nox Ortho and Bdh Ortho. Red and blue spheres mark the site of substitution proposed to form an NMN(H)Attorney Docket No.00058-086WO1 polar interaction. The rotated view of Bdh displays all three sites of mutagenesis. (C) The same view of Bdh from (B) superimposed onto 617 members of its CATH superfamily. Dark regions result from multiple overlapping structures and are interpreted as a metric for structurally conserved backbone positions. (D) Magnified image of the substitutions present in Bdh Ortho. Helix α2 is highlighted as a promising design region and is roughly coaxial to Rossmann helix α1. (E) The original view of Nox is aligned to the CATH superposition model with the Nox backbone trace highlighted in red. Substitution I159T on Rossmann helix α1 is marked with a red sphere. The α1 axis is highlighted with a yellow line. (F) A minimal Rossmann motif of Bdh (PDB 1GEG) is annotated with coaxial helix axes and arrows as a reference for previously described sites of design.

[0033] FIG.17 presents specific activity of Nox Ortho for all three cofactors at application conditions. Nox Ortho is significantly more active for NMNH than NAD(P)H in the condition used for the in vitro RR-BDO production cascade. Assay at 37 °C and 50 mM Tris-Cl at pH 7.0 with listed concentration of reduced cofactor are provided. Bars represent the mean of three independent replicates with error bars of one standard deviation for NADH and NADPH condition. Bars represent the mean of two independent replicates for NMNH. Individual replicates are indicated by white diamonds. One unit of enzymatic activity was defined as the quantity of enzyme required to consume 1 μmol of reduced cofactor (NADH, NADPH or NMNH) per minute.

[0034] FIG.18A-B demonstrates sequence diversity amongst design templates and NAD(P)-binding Rossmann-like domain superfamily. (A) Paired distance matrix of the sequence percent identity based on the wildtype sequences of the (S)-installing Bdh variants and Ll Nox. (B) Distribution of pairwise sequence percent identity within the NAD(P)- binding Rossmann-like domain superfamily (CATH Superfamily 3.40.50.720) in the CATH database. DETAILED DESCRIPTION

[0035] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polynucleotide” includes a plurality of such polynucleotides and reference to “the oxidoreductase” includes reference to one or more oxidoreductases, and so forth.Attorney Docket No.00058-086WO1

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.

[0037] Also, the use of “or” means “and / or” unless stated otherwise. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting.

[0038] It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”

[0039] All publications mentioned herein are incorporated by reference in full for the purpose of describing and disclosing methodologies that might be used in connection with the description herein. Moreover, with respect to any term that is presented in one or more publications that is similar to, or identical with, a term that has been expressly defined in this disclosure, the definition of the term as expressly provided in this disclosure will control in all respects.

[0040] As used herein, a “natural cofactor” refers to a non-protein chemical compound or metallic ion that is normally required for an enzyme's activity as a catalyst. Natural cofactors can be either loosely or tightly bound to the enzyme and can directly participate in the reaction. For example, NAD+or NADP+are natural cofactors for glucose dehydrogenase that are required for the enzyme’s activity. In direct contrast, NMN+is not a natural cofactor for glucose dehydrogenase, and therefore is not required for the enzyme’s in vivo activity.

[0041] As used herein, an “noncanonical cofactor” refers to a chemical compound or metallic ion that is not normally required or associated with a particular enzyme's activity as a catalyst, but by the result of mutations or other changes, the enzyme’s activity towards the noncanonical cofactor can be greatly enhanced. For example, NMN+is not a natural cofactor for glucose dehydrogenase, but by introducing mutations into the polypeptide sequence for glucose dehydrogenase, the glucose dehydrogenase’s activity towards NMN+can be greatly enhanced.Attorney Docket No.00058-086WO1

[0042] As used herein, a "mutation" means any process or mechanism resulting in a mutant protein, enzyme, polynucleotide, gene, or cell. This includes any mutation in which a protein, enzyme, polynucleotide, or gene sequence is altered, and any detectable change in a cell arising from such a mutation. Typically, a mutation occurs in a polynucleotide or gene sequence, by point mutations, deletions, or insertions of single or multiple nucleotide residues. A mutation includes polynucleotide alterations arising within a protein-encoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences. A mutation in a gene can be "silent", i.e., not reflected in an amino acid alteration upon expression, leading to a "sequence- conservative" variant of the gene. This generally arises when one amino acid corresponds to more than one codon.

[0043] Non-limiting examples of a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenlyated (e.g., farnesylated, geranylgeranylated) amino acid, an acetylated amino acid, an acylated amino acid, a pegylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like. References adequate to guide one of skill in the modification of amino acids are replete throughout the literature. Example protocols are found in Walker (1998) Protein Protocols on CD-ROM (Humana Press, Towata, N.J.).

[0044] Recombinant methods for producing and isolating modified polypeptides of the disclosure are described herein. In addition to recombinant production, the polypeptides may be produced by direct peptide synthesis using solid-phase techniques (e.g., Stewart et al. (1969) Solid-Phase Peptide Synthesis (WH Freeman Co, San Francisco); and Merrifield (1963) J. Am. Chem. Soc.85: 2149-2154). Peptide synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.) in accordance with the instructions provided by the manufacturer.

[0045] A "reductase" refers to an enzyme that catalyzes the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor. A “oxidoreductase” refers to an enzyme allows an oxidation to occur as well as a reduction to occur. Examples of oxidoreductases and reductases include those enzymes that act on the CH-OH group of donors with NAD+or NADP+as an acceptor, including alcohol dehydrogenase (NAD) (EC 1.1.1.1), alcohol dehydrogenase (NADP) (ECAttorney Docket No.00058-086WO1 1.1.1.2), homoserine dehydrogenase (EC 1.1.1.3), aminopropanol oxidoreductase (EC 1.1.1.4), diacetyl reductase (EC 1.1.1.5), glycerol dehydrogenase (EC 1.1.1.6), propanediol- phosphate dehydrogenase (EC 1.1.1.7), glycerol-3-phosphate dehydrogenase (NAD+) (EC 1.1.1.8), D-xylulose reductase (EC 1.1.1.9), L-xylulose reductase (EC 1.1.1.10), lactate dehydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37), isocitrate dehydrogenase (EC 1.1.1.42), and HMG-CoA reductase EC (1.1.1.88); enzymes that act on the CH-OH group of donors with oxygen as an acceptor, including glucose oxidase (EC 1.1.3.4), L- gulonolactone oxidase (EC 1.1.3.8), thiamine oxidase (EC 1.1.3.23), xanthine oxidase (EC 1.1.3.32); enzymes that act on the aldehyde or oxo group of donors with NAD+or NADP+as an acceptor, including acetaldehyde dehydrogenase EC (1.2.1.10), glyceraldehyde 3- phosphate dehydrogenase (EC 1.2.1.12), pyruvate dehydrogenase (EC 1.2.1.51), oxoglutarate dehydrogenase (EC 1.2.4.2); enzymes that act on the CH-CH group of donors with NAD+or NADP+as an acceptor, including biliverdin reductase (EC 1.3.1.24); enzymes that act on CH- CH group of donors with oxygen as an acceptor, including protoporphyrinogen oxidase (EC 1.3.3.4); enzymes that act on the CH-NH2 group of donors, including monoamine oxidase (EC 1.4.3.4); enzymes that act on the CH-NH group of donors with NAD+or NADP+as an acceptor, including dihydrofolate reductase (EC 1.5.1.3), and methylenetetrahydrofolate reductase (EC 1.5.1.20); enzymes that act on the CH-NH group of donors with oxygen as an acceptor, sarcosine oxidase (EC 1.5.3.1), and dihydrobenzophenanthridine oxidase (EC 1.5.3.12); enzymes that act on other nitrogenous compounds as donors, including urate oxidase (EC 1.7.3.3), nitrite reductase (EC 1.7.99.3), and nitrate reductase (EC 1.7.99.4); enzymes that act on the sulfur group of donors, including glutathione reductase (EC 1.8.1.7), thioredoxin reductase (EC 1.8.1.9), and sulfite oxidase (EC 1.8.3.1); enzymes that act on the heme group of donors, including cytochrome c oxidase (EC 1.9.3.1); enzymes that act on diphenols and related substances as donors, including coenzyme Q - cytochrome c reductase (EC 1.10.2.2), catechol oxidase (EC 1.10.3.1), and laccase (EC 1.10.3.2); enzymes that act on peroxide as acceptor, including Cytochrome c peroxidase (EC 1.11.1.5), catalase (EC 1.11.1.6), myeloperoxidase (EC 1.11.1.7), thyroid peroxidase (EC 1.11.1.8), and glutathione peroxidase (EC 1.11.1.9); enzymes that act on single donors with incorporation of molecular oxygen, 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27), renilla-luciferin 2- monooxygenase (EC 1.13.12.5), cypridina-luciferin 2-monooxygenase (EC 1.13.12.6), Firefly luciferase (EC 1.13.12.7), watasenia-luciferin 2-monooxygenase (EC 1.13.12.8), andAttorney Docket No.00058-086WO1 oplophorus-luciferin 2-monooxygenase EC (1.13.12.13); enzymes that act on paired donors with incorporation of molecular oxygen, including aromatase (EC 1.14.14.1), CYP2D6 (EC 1.14.14.1), CYP2E1 (EC 1.14.14.1), CYP3A4 (EC 1.14.14.1), Cytochrome P450 oxidase, nitric oxide synthase (EC 1.14.13.39), phenylalanine hydroxylase (EC 1.14.16.1), and tyrosinase (EC 1.14.18.1); and other oxidoreductases, including superoxide dismutase (EC 1.15.1.1), nitrogenase (EC 1.18.6.1), and deiodinase (EC 1.97.1.10). The above listing, provides for the classification of the foregoing enzymes by in the International Union of Biochemistry and Molecular Biology's Enzyme Commission [EC] numbering system. In a particular embodiment, the disclosure provides for a recombinantly engineered polypeptide based upon an oxidoreductase disclosed above, that has been engineered to contain amino acid mutations so as to enable the efficient recycling of a noncanonical cofactor. In a further embodiment, the oxidoreductase is an oxidase. Examples of oxidases include, but are not limited to, glucose oxidase, NADPH oxidase, amine oxidase, and NADH oxidase.

[0046] “Dehydrogenase” means an enzyme belonging to the group of oxidoreductases that oxidizes a substrate by reducing an electron acceptor, usually NAD+ / NADP+or a flavin coenzyme such as FAD or FMN. They also catalyze the reverse reaction, for instance alcohol dehydrogenase not only oxidizes ethanol to acetaldehyde in animals but also produces ethanol from acetaldehyde in yeast. In another embodiment, the disclosure provides for a recombinantly engineered polypeptide based upon a dehydrogenase, that has been engineered to contain amino acid mutations so as to enable the efficient recycling of a noncanonical cofactor. In a particular embodiment, the dehydrogenase is a (S)-specific butanediol dehydrogenase (Bdh).

[0047] A "protein" or "polypeptide", which terms are used interchangeably herein, comprises one or more chains of chemical building blocks called amino acids that are linked together by chemical bonds called peptide bonds. An "enzyme" means any substance, preferably composed wholly or largely of protein, that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reactions. A "native" or "wild-type" protein, enzyme, polynucleotide, gene, or cell, means a protein, enzyme, polynucleotide, gene, or cell that occurs in nature.

[0048] An "amino acid sequence" is a polymer of amino acids (a protein, polypeptide, etc.) or a character string representing an amino acid polymer, depending on context. The terms "protein," "polypeptide," and "peptide" are used interchangeably herein. "Amino acid"Attorney Docket No.00058-086WO1 is a molecule having the structure wherein a central carbon atom is linked to a hydrogen atom, a carboxylic acid group (the carbon atom of which is referred to herein as a "carboxyl carbon atom"), an amino group (the nitrogen atom of which is referred to herein as an "amino nitrogen atom"), and a side chain group, R. When incorporated into a peptide, polypeptide, or protein, an amino acid loses one or more atoms of its amino acid carboxylic groups in the dehydration reaction that links one amino acid to another. As a result, when incorporated into a protein, an amino acid is referred to as an "amino acid residue."

[0049] A particular amino acid sequence of a given protein (i.e., the polypeptide's "primary structure," when written from the amino-terminus to carboxy-terminus) is determined by the nucleotide sequence of the coding portion of a mRNA, which is in turn specified by genetic information, typically genomic DNA (including organelle DNA, e.g., mitochondrial or chloroplast DNA). Thus, determining the sequence of a gene assists in predicting the primary sequence of a corresponding polypeptide and more particular the role or activity of the polypeptide or proteins encoded by that gene or polynucleotide sequence.

[0050] "Conservative amino acid substitution" or, simply, "conservative substitution" of a particular sequence refers to the replacement of one amino acid, or series of amino acids, with essentially identical amino acid sequences. One of skill will recognize that individual mutations, deletions or additions which alter, add or delete a single amino acid or a percentage of amino acids in an encoded sequence result in "conservative variations" where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid. For purposes of this disclosure a “conservative amino acid substitution” does significantly affect the catalytic activity towards a noncanonical cofactor and / or structural stability of a recombinantly engineered polypeptide disclosed herein. For example, the recombinantly engineered polypeptide of the disclosure may comprise conservative amino acid mutations in regions of the sequence that do not impact the binding site for the noncanonical cofactor, e.g., conservative amino acid changes on the surface of the protein. Further, the sequence of a recombinantly engineered polypeptide disclosed herein can be aligned with polypeptide sequence(s) from enzymes that have similar structures and / or catalytic activity in order to identify amino acids that likely do not affect the catalytic activity and / or structural stability of the recombinantly engineered polypeptide. Moreover, there are many protein modeling programs available, including those specifically recited herein (e.g., Spartan andAttorney Docket No.00058-086WO1 RosettaDesign), which can identify conservative amino acid mutations with a high degree of probability / certainty that would not significantly affect the catalytic activity and / or structural stability of an recombinantly engineered polypeptide disclosed herein (e.g., see Ng et al., Predicting Deleterious Amino Acid Changes Genome Res 11:863-874 (2001)). As such, it is expected that one of skill in the art could reasonably predict that the sequence for a recombinantly engineered polypeptide disclosed herein can comprise a percentage of conservative amino acid mutations, as is described more fully below, and still have similar or the same catalytic activity for the noncanonical cofactor as a polypeptide sequence specifically recited herein. Similar reasoning applies for the structural stability of a recombinantly engineered polypeptide disclosed herein.

[0051] Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, one conservative substitution group includes Alanine (A), Serine (S), and Threonine (T). Another conservative substitution group includes Aspartic acid (D) and Glutamic acid (E). Another conservative substitution group includes Asparagine (N) and Glutamine (Q). Yet another conservative substitution group includes Arginine (R) and Lysine (K). Another conservative substitution group includes Isoleucine, (I) Leucine (L), Methionine (M), and Valine (V). Another conservative substitution group includes Phenylalanine (F), Tyrosine (Y), and Tryptophan (W).

[0052] Thus, "conservative amino acid mutations" of a polypeptide sequence disclosed herein include mutations of a percentage, typically less than 5%, 6%, 7%, 8%, 9%, or 10%, of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative substitution group. Accordingly, a conservatively substituted variation of a polypeptide of the disclosure can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or any range that includes or is in between mutations with a conservatively substituted variation of the same conservative substitution group.

[0053] It is understood that the addition of sequences which do not alter the encoded activity of a nucleic acid molecule, such as the addition of a non-functional or non-coding sequence, is a conservative variation of the basic nucleic acid. The "activity" of an enzyme is a measure of its ability to catalyze a reaction, i.e., to "function", and may be expressed as the rate at which the product of the reaction is produced. For example, enzyme activity can be represented as the amount of product produced per unit of time or per unit of enzyme (e.g., catalytic efficiency), or in terms of affinity or dissociation constants.Attorney Docket No.00058-086WO1

[0054] One of skill in the art will appreciate that many conservative variations of the nucleic acid constructs which are disclosed yield a functionally identical construct. For example, as discussed above, owing to the degeneracy of the genetic code, "silent mutations" (i.e., mutations in a nucleic acid sequence which do not result in an alteration in an encoded polypeptide) are an implied feature of every nucleic acid sequence which encodes an amino acid. Similarly, "conservative amino acid mutations," in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed construct. Such conservative variations of each disclosed sequence are a feature of the polypeptides provided herein.

[0055] "Conservative variants" are proteins or enzymes in which a given amino acid residue has been changed without altering overall conformation and function of the protein or enzyme, including, but not limited to, replacement of an amino acid with one having similar properties, including polar or non-polar character, size, shape and charge. Amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity (or identity) between any two proteins of similar function may vary and can be, for example, at least 30%, at least 50%, at least 70%, at least 80%, or at least 90%, as determined according to an alignment scheme.

[0056] As referred to herein, "sequence similarity" means the extent to which nucleotide or protein sequences are related. The extent of similarity between two sequences can be based on percent sequence identity and / or conservation.

[0057] "Sequence alignment" means the process of lining up two or more sequences to achieve maximal levels of identity (and, in the case of amino acid sequences, conservation) for the purpose of assessing the degree of similarity. In a particular embodiment, Numerous methods for aligning sequences and assessing similarity / identity are known in the art such as, for example, the Cluster Method, wherein similarity is based on the MEGALIGN algorithm, as well as BLASTN, BLASTP, and FASTA (Lipman and Pearson, 1985; Pearson and Lipman, 1988). When using all of these programs, the preferred settings are those that results in the highest sequence similarity.

[0058] Non-conservative modifications of a particular polypeptide are those which substitute any amino acid not characterized as a conservative substitution. For example, any substitution which crosses the bounds of the six groups set forth above. These include mutations of basic or acidic amino acids for neutral amino acids, (e.g., Asp, Glu, Asn, or GlnAttorney Docket No.00058-086WO1 for Val, Ile, Leu or Met), aromatic amino acid for basic or acidic amino acids (e.g., Phe, Tyr or Trp for Asp, Asn, Glu or Gln) or any other substitution not replacing an amino acid with a like amino acid. Basic side chains include lysine (K), arginine (R), histidine (H); acidic side chains include aspartic acid (D), glutamic acid (E); uncharged polar side chains include glycine (G), asparagine(N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine (C); nonpolar side chains include alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W); beta-branched side chains include threonine (T), valine (V), isoleucine (I); aromatic side chains include tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H).

[0059] A "parent" protein, enzyme, polynucleotide, gene, or cell, is any protein, enzyme, polynucleotide, gene, or cell, from which any other protein, enzyme, polynucleotide, gene, or cell, is derived or made, using any methods, tools or techniques, and whether or not the parent is itself native or mutant. A parent polynucleotide or gene encodes for a parent protein or enzyme. In a certain embodiment, a “parent” protein, enzyme, polynucleotide, gene, or cell, is a wild type protein, enzyme, polynucleotide, gene, or cell.

[0060] “Isolated polypeptide” refers to a polypeptide which is separated from other contaminants that naturally accompany it, e.g., protein, lipids, and polynucleotides. The term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis).

[0061] “Substantially pure polypeptide” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure polypeptide composition will comprise about 60 % or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species.Attorney Docket No.00058-086WO1

[0062] “Reference sequence" refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence can be at least 20 nucleotide or amino acid residues in length, at least 25 nucleotide or residues in length, at least 50 nucleotides or residues in length, or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity.

[0063] "Sequence identity" means that two amino acid sequences are substantially identical (i.e., on an amino acid-by-amino acid basis) over a window of comparison. The term "sequence similarity" refers to similar amino acids that share the same biophysical characteristics. The term "percentage of sequence identity" or "percentage of sequence similarity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical residues (or similar residues) occur in both polypeptide sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity (or percentage of sequence similarity). For example, a polypeptide or polynucleotide sequence may have certain percentage sequence identity to another sequence, typically a wild type or reference sequence. The sequence polypeptides disclosed herein may be expressly defined herein by use of SEQ ID Nos, in the attached sequence listing which is incorporated by this application in-full. Specific examples of percentage of sequence similarity between two sequences being compared herein includes, but is not limited to, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, alternatively, the percentage of sequence similarity between two sequences being compared herein can be “at least” a percentage selected from any of the foregoing percentages up to an including 100% sequence similarity. With regard to polynucleotideAttorney Docket No.00058-086WO1 sequences, the terms sequence identity and sequence similarity have comparable meaning as described for protein sequences, with the term "percentage of sequence identity" indicating that two polynucleotide sequences are identical (on a nucleotide-by-nucleotide basis) over a window of comparison. As such, a percentage of polynucleotide sequence identity (or percentage of polynucleotide sequence similarity, e.g., for silent mutations or other mutations, based upon the analysis algorithm) also can be calculated. Maximum correspondence can be determined by using one of the sequence algorithms described herein (or other algorithms available to those of ordinary skill in the art) or by visual inspection.

[0064] As applied to polypeptides, the term substantial identity or substantial similarity means that two peptide sequences, when optimally aligned, such as by the programs BLAST, GAP or BESTFIT using default gap weights or by visual inspection, share sequence identity or sequence similarity. Similarly, as applied in the context of two nucleic acids, the term substantial identity or substantial similarity means that the two nucleic acid sequences, when optimally aligned, such as by the programs BLAST, GAP or BESTFIT using default gap weights (described elsewhere herein) or by visual inspection, share sequence identity or sequence similarity.

[0065] One example of an algorithm that is suitable for determining percent sequence identity or sequence similarity is the FASTA algorithm, which is described in Pearson, W. R. & Lipman, D. J., (1988) Proc. Natl. Acad. Sci. USA 85:2444. See also, W. R. Pearson, (1996) Methods Enzymology 266:227-258. Preferred parameters used in a FASTA alignment of DNA sequences to calculate percent identity or percent similarity are optimized, BL50 Matrix 15: -5, k-tuple=2; joining penalty=40, optimization=28; gap penalty -12, gap length penalty=-2; and width=16.

[0066] Another example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity or percent sequence similarity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, (1987) J. Mol. Evol.35:351-360. The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153, 1989. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing aAttorney Docket No.00058-086WO1 cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity (or percent sequence similarity) relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., (1984) Nuc. Acids Res.12:387-395).

[0067] Another example of an algorithm that is suitable for multiple DNA and amino acid sequence alignments is the CLUSTALW program (Thompson, J. D. et al., (1994) Nuc. Acids Res.22:4673-4680). CLUSTALW performs multiple pairwise comparisons between groups of sequences and assembles them into a multiple alignment based on sequence identity. Gap open and Gap extension penalties were 10 and 0.05 respectively. For amino acid alignments, the BLOSUM algorithm can be used as a protein weight matrix (Henikoff and Henikoff, (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919).

[0068] Biomanufacturing of renewable chemicals by engineered microbes or synthetic biochemistry has the potential to supplant many petroleum-derived chemical industries. However, many engineered biosynthetic pathways fail to proceed past laboratory scale because of insufficient titer, productivity or yield. One prevalent obstacle to these goals is redox imbalance. The chassis organisms that provide high-energy cofactors also contain numerous competing pathways, which drain these critical resources. However, most of these competing pathways must remain intact because life-essential biomass precursors are produced. The exogeneous addition of high-energy cofactors can potentially mitigate the issue; however, the cost and stability of natural cofactors pose concerns for large-scale applications. Thus, redox-dependent industrial bioprocesses must achieve a delicate balance among the cell’s redox homeostasis, a source of life-essential metabolites and the redox balance of a complete biotransformation.

[0069] Nature uses two different redox cofactors, nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), to provide separateAttorney Docket No.00058-086WO1 driving forces ensuring that catabolism and anabolism, respectively, proceed in opposite directions by maintaining a low reduced NAD+(NADH):NAD+ratio and a high reduced NADP+(NADPH):NADP+ratio. This principle has been recapitulated in cell-free systems.

[0070] Therefore, in whole-cell biomanufacturing, the dependence on NAD(H) and NADP(H) permanently ties the reaction direction of a desired redox reaction to either catabolism or anabolism. This does not allow flexible control of reaction equilibrium. In cell- free biomanufacturing, NADP(H) is avoided because of its formidable cost and low stability. This leaves NAD(H) as the sole electron carrier, which alone cannot support thermodynamically uphill oxidation and reduction reactions simultaneously without forming a futile cycle.

[0071] These limitations associated with relying on natural cofactors have motivated the development of noncanonical cofactors. As shown herein, the noncanonical redox cofactor, nicotinamide mononucleotide (NMN+), largely operates in an orthogonal fashion to NAD(P)+to precisely channel reducing power in Escherichia coli whole cells and crude lysates. NMN+is also a lower-cost alternative to NAD(P)+and can be leveraged to sustain industrially relevant total turnover numbers in cell-free reactions.

[0072] Nicotinamide mononucleotide (NMN+), a truncated version of the native nicotinamide cofactors, was found to be an efficient noncanonical redox cofactor. Compared with other simpler NAD+mimetics, NMN+can be produced renewably using low-cost feedstocks through biosynthetic pathways. More importantly, its polar structural features (particularly the phosphate) offer unique advantages for enzyme design.

[0073] Disclosed herein is a reduced NMN+(NMNH)-specific oxidase (Nox Ortho) derived from Lactobacillus lactis water-forming NADH oxidase (Ll Nox) through a high- throughput, growth-based selection. Together with the NMN+-specific glucose dehydrogenase (GDH Ortho), this disclosure provides for precise modulation of the NMNH:NMN+ratio. It was established herein that the NMN(H) cofactor pool can be held at a distinct redox ratio on demand (ranging from 70 to 0.07 for NMNH:NMN+), which is decoupled from both NAD(H) and NADP(H) redox ratios in vitro and in vivo. By designing (S)-specific butanediol (BDO) dehydrogenases (Bdhs) to specifically use NMN(H), this orthogonal driving force can be tapped into to produce chiral-pure 2,3-BDO with a high degree of completion, without interference by the NAD(P) / H reduction potentials in vitro and in vivo.Attorney Docket No.00058-086WO1

[0074] Additionally, the studies presented herein facilitate a path to readily broaden the applications of this unwavering redox driving force. The enzyme design principle discovered from directed evolution of Nox Ortho, namely, to restrict the cofactor-binding pocket with hydrogen bonding, was consistently translated onto six Bdh enzymes to create NMN(H)-orthogonal catalysts. On different enzyme scaffolds, this strategy consistently results in a 1.0 × 103–3.0 × 106-fold switch of cofactor specificity from NAD(H) or NADP(H) to NMN(H) relative to wild type (WT). Given the conserved positions of these amino acid substitutions, the disclosure provides a platform technology that can be used to produce additional redox enzymes that catalyze key steps in biomanufacturing where precise equilibrium control is needed. Accordingly, the enzyme design principles and strategies disclosed herein from the directed evolution of Nox Ortho can be applied to other enzymes.

[0075] The studies presented herein uncovered an enzyme design principle (polar contacts that bridge across the cofactor-binding pocket) as a reliable strategy to engineer NMN(H)-orthogonal biocatalysts. Parallel implementation of this design principle across diverse enzymes enables the construction of orthogonal metabolic systems. One of the most important design principles in cellular metabolism is presented herein, the chemical compartmentalization by specific coenzymes and quantified actual reduction potentials by the redox ratios of cofactors in thetwo-dimensional redox systems. The methodology presented herein on a synthetic redox metabolism is true to the seminal description of orthogonal metabolic organization present in nature.

[0076] The ability to manipulate the thermodynamic equilibrium of select metabolic processes leveraging redox ratio with low interference from natural dehydrogenases is an especially promising capability of NMN(H) as a noncanonical redox cofactor. Further refinements of this strategy would broadly enable flexible biomanufacturing goals envisioned by metabolic engineers. The ability to engineer enzymes for NMN(H) presently yields notably less catalytically efficient enzymes than their natural counterparts with the canonical cofactors. Yet, with these enzymes, it was shown herein that they these circuits can be integrated into whole cells with minimal effort to balance redox resource allocation. Recent efforts in protein engineering enabled by directed evolution, machine learning and high- throughput screening have greatly accelerated the rate of enzyme design and accordingly, it is envisaged that iteration upon these techniques will soon close this gap. As orthogonal redox enzymes improve, so do their derivative selection platforms and screening technologies.Attorney Docket No.00058-086WO1

[0077] It was shown herein that the reduction potential of NMNH:NMN+is disconnected to that of NADH:NAD+and NADPH:NADP+, in both cell-free preparations and E. coli whole cells. Partitioning and balancing redox cofactors are a prevalent need in metabolic engineering. Unlike NAD(P)+, noncanonical cofactors are not tied to natural metabolism. Given that their recycling enzymes, such as GDH Ortho and Nox Ortho, can be expressed in the desired cellular compartment, with tunable level and at a controllable timing, noncanonical redox systems can support precise spatiotemporal control. Therefore, they may be deployed to solve the prevalent problem of balancing redox in metabolic engineering with a much wider range of freedom. This disclosure presents the full infrastructure of using NMN+as an orthogonal redox cofactor with its designated electron source and electron sink. BDO stereo-upgrading was demonstrated herein by using NMN(H) to direct redox driving force. The ability to control the reduction potential in individual metabolic processes, in separate cellular or organ compartments, at the correct time and without interfering with global redox homeostasis, provides an important technology leap over existing processes.

[0078] The disclosure further provides a cell-free system or whole-cell biomanufacturing systems to facilitate the biotransformation of a substrate into a desired product, comprising a recombinantly engineered polypeptide of the disclosure that has improved catalytic efficiency towards a noncanonical cofactor, and one or more polypeptides or proteins that encode enzymes that can use the same noncanonical cofactor in a biotransformation reaction(s). For example, if NMN+(NHNH) is used as a noncanonical cofactor, then the cell-free system of the disclosure comprises a recombinantly engineered polypeptide disclosed herein that has improved catalytic efficiency towards NMN+. It should be understood that the recombinantly engineered polypeptides of the disclosure are not just limited to NMN+and in-fact can be engineered to have greater catalytic efficiencies for additional noncanonical cofactors, such as 1-phenyl-1,4,-dihydronicotinamide (PNA+), 1- benzyl-1,4-dihydronicotinamide (BNA+), 1-(4-hydroxyphenyl)1,4-dihydronicotinamide (HPNA+), 1-methyl-1,4-dihydronicotinamide (MNA+), nicotinamide flucytosine dinucleotide (NFCD+), nicotinamide mononucleoside (NR+), 1‐butyl‐1,4,5,6‐tetrahydropyridine‐3‐ carboxamide, 1‐(1‐benzyl‐1,4,5,6‐tetrahydropyridin‐3‐yl) ethenone, 1-benzyl-1,4- dihydropyridine-3-carboxylic acid, and 1‐benzyl‐1,4,5,6‐tetrahydropyridine‐3‐carbonitrile.

[0079] The disclosure further provides a whole cell biomanufacturing system to facilitate the biotransformation of a substrate into a desired product, comprising recombinantAttorney Docket No.00058-086WO1 microorganism (e.g., bacteria and yeast) that have been modified to express an recombinantly engineered polypeptide of the disclosure, i.e., a polypeptide that has improved catalytic efficiency towards an noncanonical cofactor and optionally express one or more polypeptides or proteins that encode enzymes that can use the same noncanonical cofactor in a biotransformation reaction(s). The recombinant microorganisms described herein, may further comprise one or more introduced mutations to affect the microorganisms’ metabolic or enzymatic pathway(s), including, but not limiting to, introducing mutation(s) that disrupts one or more metabolic or enzymatic pathways of the microorganism, introducing one or more polypeptides that results in overexpression of one or more metabolic or enzymatic pathways of the microorganism, introducing one or more mutations that results in shunting metabolites from one metabolic or enzymatic pathway to another in the microorganism, introducing feedback mechanisms to either repress or activate enzymatic or metabolic pathways in the microorganism, or any combination of the foregoing. In the Examples presented herein, it was shown a biomanufacturing system comprising a strain of E. coli that is unable to catabolize glucose and has decreased ability to degrade noncanonical cofactors was able to produce stereo-pure Bdo when co-expressing one or more recombinant polypeptides of the disclosure (e.g., Ser (S)-Bdh Ortho) with one or more polypeptides described in Table 3 or Table 4, or having a sequence that is similar to a sequence presented in SEQ ID NOs:1-28.

[0080] The disclosure further provides that the compositions and methods described herein can be further defined by the following aspects (aspects 1 to 30): 1. A recombinantly engineered polypeptide having redox enzyme activity that has improved catalytic efficiency for a noncanonical cofactor while having reduced catalytic efficiency for a natural cofactor, the recombinantly engineered polypeptide comprising: a modified or mutated natural cofactor binding pocket that comprises: (1) amino acid substitutions or mutations that form new hydrogen bond(s) that inhibit or prevent entry by the natural cofactor into the binding pocket; and (2) amino acid substitutions or mutations that promote the binding of the noncanonical cofactor by introducing new polar contact(s) that interact specifically with polar group(s) or moieties of the noncanonical cofactor. 2. The recombinantly engineered polypeptide of aspect 1, wherein the modified or mutated natural cofactor binding pocket of the recombinantly engineered polypeptide comprises 3, 4, 5, 6, 7, 8, 9, or 10, or a range thereof, of amino acid mutations or substitutionsAttorney Docket No.00058-086WO1 in comparison to the sequence of a wild-type or parent polypeptide, particularly 3 to 6 amino acid mutations or substitutions in comparison to the sequence of a wild-type or parent polypeptide 3. The recombinantly engineered polypeptide of aspect 2, wherein at least one of the substitutions or mutations introduces a new polar contact that interacts specifically with a polar group of the noncanonical cofactor, and wherein at least two of the substitutions or mutations form a new hydrogen bond that inhibits or prevents entry by the natural cofactor into the binding pocket. 4. The recombinantly engineered polypeptide of aspect 3, wherein the recombinantly engineered polypeptide encodes a Rossmann-fold containing protein, and the new polar contact that interacts specifically with a polar group of the noncanonical cofactor is found in Rossmann helix α1 or α2, and wherein the new hydrogen bond that inhibits or prevents entry by the natural cofactor into the binding pocket closes the space between Rossmann strands β2 and β3. 5. The recombinantly engineered polypeptide of any one of aspects 1 to 4, wherein the modified or mutated natural cofactor binding pocket does not comprise mutations or substitutions that restricts the size of the natural cofactor binding pocket by forming hydrophobic packing interactions which inhibit or prevent entry by the natural cofactor into the binding pocket. 6. The recombinantly engineered polypeptide of any one of aspects 1 to 5, wherein the recombinantly engineered polypeptide has at least 10-fold at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45- fold, or at least 50-fold, increase in catalytic efficiency for the noncanonical cofactor in comparison to a wild-type or parent polypeptide, particularly, wherein the recombinantly engineered polypeptide has at least 15-fold increase in catalytic efficiency for the noncanonical cofactor in comparison to a wild-type or parent polypeptide. 7. The recombinantly engineered polypeptide of any one of aspects 1 to 6, wherein the recombinantly engineered polypeptide has at least 50-fold, at least 100-fold, at least 1000-fold, or at least 10000-fold, decrease in catalytic efficiency for the natural cofactor in comparison to a wild-type or parent polypeptide, wherein the recombinantly engineered polypeptide has at least 50-fold decrease in catalytic efficiency for the natural cofactor in comparison to a wild-type or parent polypeptide.Attorney Docket No.00058-086WO1 8. The recombinantly engineered polypeptide of any one of aspects 1 to 7, wherein the noncanonical cofactor is selected from the group consisting of nicotinamide mononucleotide (NMNH), 1-phenyl-1,4,-dihydronicotinamide , 1-benzyl-1,4- dihydronicotinamide, 1-(4-hydroxyphenyl)1,4-dihydronicotinamide, 1-methyl-1,4- dihydronicotinamide, nicotinamide flucytosine dinucleotide, nicotinamide mononucleoside, 1‐butyl‐1,4,5,6‐tetrahydropyridine‐3‐carboxamide, 1‐(1‐benzyl‐1,4,5,6‐tetrahydropyridin‐3‐ yl) ethenone, 1-benzyl-1,4-dihydropyridine-3-carboxylic acid, and 1‐benzyl‐1,4,5,6‐ tetrahydropyridine‐3‐carbonitrile. 9. The recombinantly engineered polypeptide of any one of aspects 1 to 8, wherein the noncanonical cofactor is NMNH. 10. The recombinantly engineered polypeptide of any one of aspects 1 to 9, wherein the natural cofactor is selected from NAD(H), FAD(H), and NADP(H), particularly wherein the natural cofactor is NAD(H). 11. The recombinantly engineered polypeptide of any one of aspects 1 to 10, wherein the recombinantly engineered polypeptide has a redox enzyme activity selected from oxidase, dehydrogenase, and oxidoreductase. 12. The recombinantly engineered polypeptide of any one of aspects 1 to 11, wherein the recombinantly engineered polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to a sequence from an oxidase, a dehydrogenase, or an oxidoreductase, particularly wherein the recombinantly engineered polypeptide comprises a sequence that is at least 80% identical to a sequence from an oxidase, a dehydrogenase, or an oxidoreductase. 13. The recombinantly engineered polypeptide of any one of aspects 1 to 12, wherein the recombinantly engineered polypeptide comprises a sequence that is at least 97%, at least 98%, at least 99% identical to a sequence from an oxidase, a dehydrogenase, or an oxidoreductase, particularly wherein the recombinantly engineered polypeptide comprises a sequence that is at least 98% identical to a sequence from an oxidase, a dehydrogenase, or an oxidoreductase. 14. The recombinantly engineered polypeptide of aspect 12, wherein the oxidase is selected from glucose oxidase, NADPH oxidase, amine oxidase, and NADH oxidase. 15. The recombinantly engineered polypeptide of aspect 14, wherein the oxidase is a NADH oxidase selected from Anaerocolumna aminovalerica, Bacillus subtilis,Attorney Docket No.00058-086WO1 Enterococcus faecalis, Lacticaseibacillus rhamnosus, Lactiplantibacillus pentosus, Lactococcus cremoris, Lactococcus lactis, Levilactobacillus brevis, Methanobrevibacter smithii, Streptococcus agalactiae, Streptococcus mutans, and Streptococcus pyogenes. 16. The recombinantly engineered polypeptide of aspect 12, wherein the oxidase is an NADH oxidase having an amino acid sequence that is at least 95% identical to SEQ ID NO:6 or SEQ ID NO:7. 17. The recombinantly engineered polypeptide of any one of aspects 1 to 16, wherein the recombinantly engineered polypeptide comprises a sequence that is at least 98% identical to SEQ ID NO:6 but comprises at least the substitutions of I159T, D178N, A179F, and I243E. 18. The recombinantly engineered polypeptide of aspect 12, wherein the dehydrogenase or the oxidoreductase is selected from phosphite dehydrogenase, meso- (2R,3S)-butanediol dehydrogenase ((m)-Bdh), (2R,3R)-butanediol dehydrogenase ((R)-Bdh), (2S,3S)-butanediol dehydrogenase ((S)-Bdh), alcohol dehydrogenase, acetoin reductase, diacetyl reductase, alcohol dehydrogenase, glutathione reductase, homoserine dehydrogenase, glucose dehydrogenase, glycerol dehydrogenase, propanediol-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, acetaldehyde dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, pyruvate dehydrogenase, oxoglutarate dehydrogenase, and phosphite and formate dehydrogenase. 19. The recombinantly engineered polypeptide of aspect 18, wherein the dehydrogenase or the oxidoreductase is from the enzyme class of (m)-Bdh, (R)-Bdh, or (S)- Bdh having a sequence that is at least 95% identical to the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21. 20. The recombinantly engineered polypeptide of aspect 19, wherein the recombinantly engineered polypeptide comprises a sequence that is at least 98% identical to the sequence of SEQ ID NO:1 but comprises at least the amino acid substitutions of L39Q, A92K, and M194T. 21. The recombinantly engineered polypeptide of aspect 19, wherein the recombinantly engineered polypeptide comprises a sequence that is at least 98% identical toAttorney Docket No.00058-086WO1 the sequence of SEQ ID NO:2 but comprises at least the amino acid substitutions of M189T, Y34Q, and A87K. 22. The recombinantly engineered polypeptide of any one of aspects 1 to 21, wherein the recombinantly engineered polypeptide consists of, consists essentially of, or comprises a sequence selected from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26. 23. An expression vector comprising the recombinantly engineered polypeptide of any one of aspects 1 to 22. 24. The expression vector of aspect 23, wherein the expression vector is a plasmid, a viral vector, a cosmid, or an artificial chromosome. 25. The expression vector of aspect 23 or aspect 24, wherein the expression vector is a plasmid that comprises elements for expressing the recombinantly engineered polypeptide in a bacterium or a yeast. 26. A microorganism that comprises a recombinantly engineered polypeptide of any one of aspects 1 to 22. 27. The microorganism of aspect 26, wherein the microorganism is a bacterium, fungus, or a yeast. 28. The microorganism of aspect 26 or aspect 27, wherein the microorganism is a strain of E. coli that is unable to catabolize glucose and has decreased ability to degrade noncanonical cofactors. 29. A microorganism that comprises the expression vector of any one of aspects 23 to 25. 30. The microorganism of aspect 29, wherein the microorganism is a bacterium, fungus, or a yeast. 31. The microorganism of aspect 29 or aspect 30, wherein the microorganism is a strain of E. coli that is unable to catabolize glucose and has decreased ability to degrade noncanonical cofactors. 32. A method of stereo-upgrading (2S,3R)-meso-butanediol ((m)-BDO) to (2S,3S)-butanediol ((SS)-BDO) or (2R,3R)-butanediol ((RR)-BDO) in a cell free system, comprising: providing the following purified polypeptides in a buffer system comprising a natural cofactor and a noncanonical cofactor:Attorney Docket No.00058-086WO1 a polypeptide encoding a Bdh, Dar, or BudC enzyme that utilizes NAD(H) or NADP(H) as a natural cofactor, a polypeptide encoding an orthogonal (S)-Bdh comprising the sequence of SEQ ID NO:25 or a polypeptide encoding an orthogonal (m)-Bdh comprising the sequence of SEQ ID NO:26, a polypeptide encoding a Nox oxidase comprising the sequence of SEQ ID NO:8 or SEQ ID NO:24, and a polypeptide encoding a Gdh dehydrogenase comprising the sequence of SEQ ID NO:5 or SEQ ID NO:22; and introducing (m)-BDO into the buffer system that is then converted into (SS)-BDO or (RR)-BDO by the activities of the purified polypeptides. 33. The method of aspect 32, wherein the polypeptide encoding an orthogonal (S)- Bdh has the sequence of SEQ ID NO:25, and wherein the polypeptide encoding an orthogonal (m)-Bdh has the sequence of SEQ ID NO:26. 34. The method of aspect 32 or aspect 33, wherein the polypeptide encoding a Nox oxidase has the sequence of SEQ ID NO:24. 35. The method of any one of aspects 32 to 34, wherein the polypeptide encoding a Gdh dehydrogenase has the sequence of SEQ ID NO:22. 36. The method of any one of aspects 32 to 35, wherein the natural cofactor is NADP(H) or NAD(H), and the noncanonical cofactor is NMN(H). 37. The method of any one of aspect 32, wherein the method stereo-upgrades (m)-BDO to (SS)-BDO in a cell free system and the method comprises: providing the following purified polypeptides in a buffer system comprising NAD(H) and NMN(H): a polypeptide encoding a Bdh, Dar, or BudC enzyme that utilizes NAD(H) as a natural cofactor, a polypeptide encoding an orthogonal (S)-Bdh that comprises the sequence of SEQ ID NO:25, a polypeptide encoding a Nox oxidase that comprises the sequence of SEQ ID NO:24, a polypeptide encoding a Gdh dehydrogenase that comprises the sequence of SEQ ID NO:22; andAttorney Docket No.00058-086WO1 introducing m-BDO into the buffer system that is then converted into (SS)-BDO by the activities of the purified polypeptides. 38. A method to produce (2S,3S)-2,3-butanediol (SS)-BDO)) in vivo, comprising: co-expressing the following polypeptides in a recombinant microorganism: a recombinantly engineered polypeptide encoding a (R)-Bdh enzyme that utilizes NAD(H) as a natural cofactor, a polypeptide encoding an orthogonal (S)-Bdh hat comprises the sequence of SEQ ID NO:25, a polypeptide encoding a Nox oxidase that comprises the sequence of SEQ ID NO:7, a polypeptide encoding a Gdh dehydrogenase that comprises the sequence of SEQ ID NO:22, a polypeptide encoding a glucose transport facilitator that has a sequence of SEQ ID NO:23; and producing (SS)-BDO by incubating the recombinant microorganism with media containing glucose, meso-(2R,3S)-butanediol, and NMN(H). 39. The method of aspect 38, wherein the recombinant microorganism is a strain of E. coli that is unable to catabolize glucose and has decreased ability to degrade noncanonical cofactors. 40. A method to produce (2S,3S)-butanediol ((SS)-BDO) in vivo, comprising: co-expressing the following polypeptides in a recombinant microorganism: a recombinantly engineered polypeptide encoding a (R)-Bdh enzyme that utilizes NADP(H) as a natural cofactor, a polypeptide encoding an orthogonal (S)-Bdh hat comprises the sequence of SEQ ID NO:25, a polypeptide encoding a Nox oxidase that comprises the sequence of SEQ ID NO:8, a polypeptide encoding a Gdh dehydrogenase that comprises the sequence of SEQ ID NO:22, a polypeptide encoding a glucose transport facilitator that has a sequence of SEQ ID NO:23; andAttorney Docket No.00058-086WO1 producing (SS)-BDO by incubating the recombinant microorganism with media containing glucose, meso-(2R,3S)-butanediol, and NMN(H). 41. The method of aspect 40, wherein the recombinant microorganism is a strain of E. coli that is unable to catabolize glucose and has decreased ability to degrade noncanonical cofactors. EXAMPLES

[0081] Media and Growth Conditions. Culture for cloning was routinely performed with the E. coli XL-1 Blue strain and protein expression was generally carried out in the E. coli BL21 (DE3) strain (see Table 1). Unless otherwise noted all E. coli cultures were grown in 2x YT medium prepared from ready-to-use media granules at 31 g / L (Fisher Bioreagents 2x YT Broth granulated media: 16 g / L casein peptone, 10 g / L yeast extract, 5 g / L NaCl). When appropriate, selective media was prepared at working concentrations of ampicillin (100 mg / L), spectinomycin (50 mg / L), kanamycin (50 mg / L), or chloramphenicol (20 mg / L from an ethanol-solvated stock). Generally, strains were cultured at 37 °C with 250 rpm agitation on a 1-inch diameter orbit. Induction was initiated with final concentrations of 0.1% L- arabinose for strains containing the PBAD promoter and 0.5 mM isopropyl ß-D-1- thiogalactopyranoside (IPTG) for strains containing the PLlacO1 promoter, unless otherwise noted. Table 1. Strains and Plasmids. Strains Description XL-1 blue E. coli Cloning strain BL21 E. coli Protein expression strain (DE3) BW25113 E. coli F-, lacIqrrnBT14ΔlacZWJ1hsdR514 ΔaraBADAH33ΔrhaBADLD78MX102 MX102 R0MX502 BW25113 ΔpncC Δpgi Δzwf ΔnadR Δgnd + pLM106 + pLS502 Plasmids Description pCP20 Temperature-inducible yeast Flp recombinase gene controlled by λcIts857 in a temperature-sensitive replicon pDA063 PLlacO1:: Bs bdhA (Bs (R)-Bdh), ColE1 ori, AmpR, N-terminal 6x His tagAttorney Docket No.00058-086WO1 pDA091 PLlacO1:: Ecl dar Y34Q-A87K-M189T, ColE1 ori, AmpR, N-terminal 6x His tag pDA092 PLlacO1:: Kp dar (Kp (m)-Bdh WT), ColE1 ori, AmpR, N-terminal 6x His tag 6x 6x N-Attorney Docket No.00058-086WO1 pYZ35 PLlacO1:: Ll nox I159T-D178N-A179F-I243E (Nox Ortho), ColE1 ori, AmpR, N-terminal 6x His tagPb, Paraclostridium bifermentans; Cbo, Clostridium botulinum; Km, Khelaifiella massiliensis; Cs, Clostridium saccharoperbutylacetonicum; Cbu, Clostridium butyricum; Zp, Zymobacter palmae; Lb, Lactobacillus brevis strain ATCC 367; Tp, triphosphopyridine (engineered from Lb); Zm, Zymomonas mobilis; Ll, Lactobacillus lactis; Ft, Francisella tularensis; Ser, Serratia sp. AS13; Ka, Klebsiella aerogenes KCTC 2190; Ko, Klebsiella oxytoca KCTC 1686.

[0082] Buffers and Selection Media Recipes. His-binding buffer was prepared following the recipes in the His-Spin Protein Miniprep kit (Zymo research) at pH 7.7: 300 mM NaCl, 50 mM sodium phosphate pH 7.7, 10 mM imidazole, and 0.03% Triton X-100. His-Wash Buffer was prepared at pH 7.7: 300 mM NaCl, 50 mM sodium phosphate, 50 mM imidazole, and 0.03% Triton X-100. His-Elution Buffer was prepared at pH 7.7: 300 mM NaCl, 50 mM sodium phosphate, and 250 mM imidazole.1× phosphate buffered saline (PBS) was prepared as 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 1.8 mM potassium phosphate monobasic, and titrated to pH 7.4. Titration of all buffers was performed with concentrated HCl or 10 M NaOH as appropriate.

[0083] M9 Wash Buffer contained 1 mM MgSO4, 0.1 mM CaCl2, BD Difco M9 salts (Na2HPO46.78 g / L, KH2PO43g / L, NaCl 0.5 g / L, NH4Cl 1 g / L), and Sigma Aldrich trace metal mix A5 with Co (H3BO32860 µg / L, MnCl2 ^ 4H2O 1810 µg / L, ZnSO47H2O 222 µg / L, Na2MoO4, 2H2O 390 µg / L, CuSO4, 5H2O 79 µg / L, Co(NO3)2^ 6H2O 49 µg / L). M9 Selection Media and Expression Media were prepared as described in King et al. (Nat. Commun.13: 7282 (2022)). M9 Selection Media shared the same composition of M9 Wash Buffer with the inclusion of 20 g / L D-glucose, 1 mM nicotinamide, 0.1 mM IPTG, and 0.05% (w / v) L- arabinose, 200 mg / L ampicillin, 50 mg / L kanamycin, and 50 mg / L spectinomycin. For solid media M9 Selection Plates, 15 g / L agar was added in addition to the M9 Selection Media composition. Expression media consisted of 2xYT media supplemented with 20 g / L D- glucose, 1 mM nicotinamide, and antibiotics as above.Attorney Docket No.00058-086WO1

[0084] Plasmid Construction. Standard PCR reactions were performed using the PrimeSTAR Max DNA Polymerase (TaKaRa) or KOD One PCR Master Mix -Blue- (Toyobo). Splicing by overlap extension PCR was performed with the KOD Xtreme Hot Start DNA Polymerase (Novagen). Generally, plasmids were constructed by PCR amplification of the target gene, with appropriate ~20-30 bp overlapping regions for downstream Gibson assembly included in the primers. Constructs were circularized with vector backbone by Gibson assembly and introduced to XL-1 Blue Mix and Go chemical competent cells (Zymo Research), miniprepped (Qiagen), and sequenced (Laragen) as previously described. pDA063 was constructed by amplification of the bdhA gene from a Bacillus subtilis ATCC 6051 genome template. Plasmids containing previously reported genes were subcloned from plasmids described in Table 1.

[0085] Reported plasmid constructs from synthetic genes such as L. lactis nox were ordered from Integrated DNA Technologies (IDTDNA) as gBlocks gene fragments with codon optimization performed by the IDTDNA codon optimization webtool for E. coli K12. All bio prospected genes were prepared as synthetic DNA. DNA fragments were designed with the appropriate ~30 bp Gibson overlapping sequences on each end for direct circularization into vector backbones by Gibson assembly. Plasmids encoding mutants were built through site-directed mutagenesis as described in Zhang et al. (Serratia sp. T241. Sci. Rep.6:1–12 (2016)) or encoded in the synthetic DNA design.

[0086] Splicing by Overlap Extension PCR. A standard splicing-by-overlap extension PCR was performed with a KOD Xtreme Hot Start DNA Polymerase PCR reaction mixture (Novagen) at 25 µL total volume and 0.5 µL of each linear DNA fragment with the primers excluded. After a 7-cycle touchdown PCR followed by 5 cycles fixed at the desired annealing temperature, the reactions were removed from the thermocycler. Primers were added to appropriate concentration and a PCR reaction fixed at the annealing temperature was cycled 25-30 times. Once isolated, the vector and insert were circularized by Gibson isothermal assembly.

[0087] Strain Construction. Strain MX102 R0was constructed by removal of the kanamycin resistance marker using the pCP20 plasmid (see Table 1) from the previously reported strain.

[0088] Protein Expression and Purification. Expression of recombinant protein was performed by introduction of the target plasmid into BL21 (DE3) Mix and Go chemicalAttorney Docket No.00058-086WO1 competent cells (Zymo Research, Table 1). A single colony was inoculated into 4 mL 2×YT- 200 mg / L ampicillin and grown for ~14-16 h at 30 °C. Then, 2×YT with 200 mg / L ampicillin media was inoculated to 0.07 OD600 and incubated while shaking for approximately 1 h and 45 min at 37 ºC. The cultures were removed from the shaker and placed at room temperature for 30 min without shaking, induced with 0.5 mM IPTG, and then incubated while shaking at 30 ºC, 24 h. Cells were harvested by centrifugation at 2,500 × g, 4 ºC, 30 min. Supernatant was decanted, and cell pellets stored at -80 ºC until purification. Cell lysis was performed mechanically, and purification was performed following a modified version of the Zymo Research His-Protein Miniprep detailed in the Supporting Information. Protein quantification was achieved by Bradford assay relative to a standard curve of bovine serum albumin. Purified protein was stored with 20% glycerol at -80 ºC.

[0089] For large scale protein expression cultures, Pyrex #4985 non-baffled shake flasks with phenolic screw-tops were used in place of conical tubes. The flasks were filled to 40% capacity with 2×YT-200 mg / L ampicillin media and inoculated to 0.07 OD600. After induction at 0.5 mM IPTG, the flasks were incubated in a room temperature shaker at ~25 °C at 177 rpm on a 2-inch orbit for ~24 hours. Screw-top flasks were incubated with their caps loose. At harvest, centrifuge bottles were used to pellet the cells. The cell pellets were stored at -80 °C after removal of supernatant.

[0090] Small Scale Lysis by Glass Bead Homogenization. Each pellet generated from either 20 mL culture grown in the conical tubes or by 40 mL culture distributed from shake flasks was resuspended in His-Binding Buffer containing 1 mM AEBSF protease inhibitor as appropriate. For purification of Ll Nox, His-Binding Buffer for resuspension was supplemented with 1 mg / L DNAse and 0.15 mM flavin adenine dinucleotide (FAD). Cell pellets from 20 mL culture were resuspended in 600 µL His-Binding Buffer by vortexing and cell pellets from 40 mL culture were resuspended in 800 µL. Each resuspended cell pellet was transferred to a 2 mL bead beating tube containing 0.5 mL, 0.1 mm diameter glass beads (BioSpec). The bead beating tubes were homogenized in 4 cycles for 35 seconds each at 6.0 m / s in a FastPrep-24 Classic (MP Biomedicals) with 5 min rest on ice between cycles. Once lysed, the samples were clarified of debris and glass beads by centrifugation at 4 °C, 21,000 × g for 15 min. The soluble fractions of each were transferred to 100 µL of HisPur Ni-NTA resin slurry (Thermo Scientific) equilibrated in His-Binding Buffer.

[0091] Large Scale Lysis by French Pressure Cell Disruption. The wet cell weightAttorney Docket No.00058-086WO1 (WCW) of the cell pellet was measured and 1-2 mL of His-binding buffer containing 1 mg / L DNAse and optionally 1 mM AEBSF protease inhibitor was added to the pellets per gram WCW. The cells were fully resuspended on ice. Once fully resuspended the cell suspension was lysed by 3-4 passes through a Thermo Electron French Press with the standard 40K cell at 4 °C. Pressure was maintained at 1,000 psig with medium ratio selected and the sample was allowed to exit the outlet at ~15 drops / min. Once cell disruption was complete the lysate was clarified by centrifugation at 4 °C, 21,000 × g for 15 min at 4 °C. Once pooled, the soluble fraction was ready for purification by immobilized metal affinity chromatography. Generally, a ratio of 100 µL HisPur Ni-NTA resin slurry (Thermo Scientific) equilibrated in His-Binding Buffer was used per mL of clarified lysate.

[0092] Purification of Poly-Histidine Tagged Protein. All steps performed at 4 °C or on ice. Clarified lysate and Ni-NTA resin were combined. The mixture was allowed to incubate end-over-end mixing (15 rpm) at 4 °C for 15 min to 1 hour. After incubation, the resin and lysate mixture were centrifuged at 4 °C, 700 × g for 1 min and the supernatant discarded. Six resin bed-volumes (volume of solid resin, BV) of His-Binding Buffer were added to the resin and mixed gently to fully resuspend the resin bed. The entire slurry was transferred to a Thermo Scientific Pierce centrifuge column. The binding buffer was removed by centrifugation at 700 × g for the Pierce columns. The resin was washed twice with 6 BV His-Wash Buffer. After the resin had been washed twice to remove nonspecific proteins, 1.5- 2.5 BVs of His-Elution Buffer was added directly to the resin. After 10 min of gently mixing the final elution was collected in a clean 1.5 mL or 15 mL tubes by centrifugation at 700 × g for 2 min. The eluant was quantified by Bradford assay relative to a standard curve of bovine serum albumin. Glycerol was added to a final concentration of 20% and stored in single-use aliquots at -80 °C.

[0093] Salting-Out-Extraction of Butanediol. An aqueous phase was prepared by mixture of 100 µL of sample with 100 µL 1,000 g / L potassium phosphate dibasic solution. 200 µL extraction solution (80%: 20% ethyl acetate: ethanol with 200 mg / L 4-oxoisophorone as an internal standard) was added to the aqueous phase and the extractions were mixed vigorously at maximum speed on an analog vortexer. The phases were separated by centrifugation at 21,000 × g, 2 min.100 µL of the organic phase was stored in GC vials with low volume inserts at 4 °C until injection.

[0094] Determination of Retention Times. For BDO stereoisomers, an analyticalAttorney Docket No.00058-086WO1 standard of each prepared in ethyl acetate was injected and compared to reaction chromatograms. For Ac, both literature retention order on an equivalent column chemistry and a reaction mixture of an enzyme with published (S)-chiral center installing stereospecificity was used to generate pure (S)-acetoin from diacetyl and record a characteristic retention time. Integrated peak area for each analyte and internal standard was recorded for each sample. A calibration curve was generated for the response ratio (area analyte / area of internal standard) of standards containing known mixtures of all BDO and Ac stereoisomers with fixed internal standard concentration. Concentrations of each were interpolated from standard curves, calculated by linear fit. Percent conversion is calculated as the concentration of desired butanediol isomer divided by the sum of all analytes (Ac and BDO isomers).

[0095] Preparation and Purification of NMNH. NMNH was prepared enzymatically and purified as described in King et al.

[0096] Specific Activity Assay. Specific activity assays performed to characterize Kp (m)-Bdh homologs were done in the following conditions: 50 mM Tris-Cl at pH 8.0, R / S- Ac at 10 mM, and 0.2 mM reduced cofactor, 30 °C. Then, 100 µL reactions were prepared by addition of master mix to 10 µL of each enzyme. Enzyme stored in His-Elution Buffer with 20 % glycerol (from a 50 % glycerol stock). Enzyme dilutions were carried out with ice cold His-Elution Buffer. Reaction rates were recorded as the difference between substrate and the no-substrate control. The production or consumption of reduced cofactor was detected with a SpectraMax M3 spectrophotometer at 340 nm, where the extinction coefficient used for calculation of NAD(P)H reaction rate was 6.22 mM-1cm-1or 4.89 mM-1cm-1for NMNH. One unit of enzymatic activity was defined as the quantity of enzyme required to produce or consume 1 μmol of reduced cofactor (NADH, NADPH or NMNH) per minute under the enzyme assay conditions described.

[0097] Specific activity assays performed to characterize the Bs (R)-Bdh homologs were carried out in the following conditions: 50 mM glycine-NaOH pH 10.0, 10 mM (m)- Bdo, 4 mM oxidized cofactor. Specific activity assays of Nox variants were carried out in the following conditions: 50 mM Tris-Cl at pH 7.0 with 0.3 mM reduced cofactor at 37 °C. The reaction rate was calculated as the difference between a no cofactor control and with cofactor reaction to account for potential non-specific absorbance decrease at 340 nm in the absence of the addition of reduced cofactor. The dilution buffer for Nox was 50 mM Tris-Cl at pHAttorney Docket No.00058-086WO1 7.0. Unless otherwise noted, handling of enzymes, initiation of reaction, and calculation of reaction velocity was performed as described above.

[0098] Apparent Kinetic Parameter Determination. Apparent Michaelis-Menten kinetic parameters for cofactors were determined by nonlinear fit of recorded initial reaction rate data to the Michaelis-Menten equation below – where v0 is initial rate, ET is the total enzyme concentration, and C is the varying cofactor concentration. For Bdhs the kinetic parameters, kcat and Km, describe the apparent turnover number and apparent Michaelis constant at 50 mM substrate, respectively. ^^ ^^^^ ^^ ^ୡୟ^^ൌ ^^୫ ^ ^^For Ser (S)-Bdh the assay condition Tris-Cl pH 8.0, 50 mM (SS)-Bdo, andcofactor concentration varied. For Kp - same assay condition was performed but with 50 mM (m)-Bdo. For Ll Nox the assay condition was 37 °C, 50 mM Tris-Cl pH 7.0 with variable reduced cofactor concentration.

[0099] Under conditions where the enzyme could not be saturated with cofactor (Km≫ C) the initial rate data were instead recorded and fit to a linear form of the Michaelis- Menten equation to solve for catalytic efficiency, kcat / Km. ^^^^^ୡ^^ ^^ୟ^^ൌ

[0100] Gas(GC) was performed with an Agilent 6850 (Agilent Technologies) coupled to a flame-ionization detector. All gases were purchased from Airgas. The Agilent CP-ChiraSilDex CB (25 m × 0.25 mm ID × 0.25 µm film thickness × 5-inch cage) was used for all separations. A method was developed with the following parameters: The inlet was heated to 250 °C with a pressure of 25.0 psi. The injection volume was 1 µL. The inlet was set to split at a ratio of 20:1 with helium used as the carrier gas. The GC was operated in constant pressure mode at 25.0 psi. The oven program started at 80 °C that was held for 10 min, and then a 10 °C / min ramp to 110 °C, followed by a 20 °C / min ramp to 200 °C with a 2 min hold. The flame ionization detector was set to 275 °C, 40 mL / min H2, 350 mL / min air, with a makeup helium flow of 45 ml / min. The elution order observed is (R)-Ac, (S)-Ac, (SS)-Bdo, (RR)-Bdo, (m)-Bdo, and then internal standard, 4-oxoisophorone. Samples for GC analysis were processed by a salting-out-extraction with ethyl acetate as described above.Attorney Docket No.00058-086WO1

[0101] Purified Protein Cycling Reactions. Cycling reactions consisting of purified proteins were prepared similarly to previous research. Reactions were initiated by addition of a mixture of each reaction’s enzymes to a master mix containing all other components. For determination of enzyme stereospecificity, reaction mixtures contained 100 mM potassium phosphate (KPi) pH 7.5, 1 M NaCl, 200 mM D-glucose, 2 mM oxidized cofactor, and 5 g / L racemic acetoin. The final concentration of enzyme in the reaction was 11.7 µM Gdh Ortho and 28.1 µM of Bdh. All molar protein concentrations are reported based on the molecular weight of the monomers. When an enzyme was omitted from a reaction, an equivalent volume of His-Elution Buffer was added in its place. The 350 µL reactions were incubated in 15 mL conical tubes at a 45° angle, 30 °C, 250 rpm for 4 h prior to GC analysis as described above. Analogous reactions for (R)-Bdh bioprospecting are described further herein.

[0102] For the single cofactor model systems, 5 g / L m-BDO, 2 mM NAD+, 100 mM potassium phosphate, 200 mM 3-morpholinopropane-1-sulfonic acid (MOPS) pH 8.0, 10 µM Ser (S)-Bdh WT, 5 µM Bs (R)-Bdh were provided. Cofactor recycling enzymes consisted of 10 µM Lb Nox or 10 µM Gdh WT as appropriate.450 µL reactions were incubated while shaking for 48 h at 30 °C prior to sample analysis by GC.

[0103] For the (m)-BDO to (SS)-BDO reaction cascade using NAD+and NMN+(see FIG.1A and FIG.9A), the 900 µL reaction contained 300 mM Tris-Cl pH 8.0, 1 M NaCl, 200 mM D-glucose, 2 mM oxidized cofactor, and 5 g / L (m)-BDO. The protein ratio used was 10 µM Lb Nox, 5 µM Bs (R)-Bdh, 20 µM Gdh Ortho, and 60 µM Ser (S)-Bdh Ortho. For the NADP+and NMN+system (see FIG.1C and FIG.9G), the protein ratio used was 20 µM Tp Nox, 10 µM Cs (R)-Bdh, 20 µM Gdh Ortho, and 60 µM Ser (S)-Bdh Ortho and reaction volume was 700 µL. Reactions to produce (SS)-BDO were sampled after 24 h or 72 h at 30 °C, for GC analysis.

[0104] For the (m)-BDO to (RR)-BDO reaction cascade using NAD+and NMN+the 700 µL reactions contained 300 mM Tris-Cl pH 8.0, 1 M NaCl, 200 mM D-glucose, 2 mM NMN+, 0.2 mM NAD+and 2 g / L (m)-BDO. Because the determination of Kmfor Nox Ortho with NAD(P)H was technically limited, a specific activity assay of Nox Ortho with each cofactor was prototyped to guide system design (see FIG.12). The concentration of NAD(P)+in these systems was 0.2 mM. The protein ratio was 10 µM Nox Ortho, 60 µM Kp (S)-Bdh Ortho, 5 µM Bs Gdh, and 5 µM Bs (R)-Bdh. For the NADP+and NMN+system, NADP+was substituted for NAD+and 5 µM Cs (R)-Bdh was substituted for Bs (R)-Bdh. Reactions toAttorney Docket No.00058-086WO1 produce (RR)-BDO were sampled after 72 h at 30 °C, for GC analysis.

[0105] Standard Selection Protocol of Ll NOX Libraries and Control Plasmids into MX502. The strain construction and standard selection were performed as described in King et al. Plasmid pLS501 (XenA D116E) and pYZ10 (Ll Nox WT) served as positive and negative controls, respectively. Plasmid pYZ20 (Ll Nox I159T) was also transformed as library template control as a benchmark.

[0106] Selection of Ll Nox Library pYZ100. The library culture was obtained by following the standard selection protocol described above and was washed three times in M9 Wash Buffer and then diluted to a final concentration of ~105cells / mL in M9 Wash Buffer. This 40 µL of suspension was plated on each of seven M9 Selection Plates with 20 g / L D- glucose and 2 mM NMN+. The plates were incubated at 30 °C and monitored periodically for 10 days. After 10 days, six colonies from each experimental plate and colonies from the control plates were streaked onto fresh M9 Selection Plates to isolate variants and validate growth. After 7 days, plates were evaluated for growth and 18 colonies with diverse growth phenotypes were selected for sequencing and NMNH activity characterization (see Table 2). Colonies were cultured in 2×YT liquid media overnight and plasmids were extracted using the QIAprep Spin Miniprep kit (Qiagen). Table 2. Mutations Observed in Ll Nox Variants Obtained from Library Selection Asp 178 Ala 179 Ile 243 V i t i i inoAttorney Docket No.00058-086WO1 LL-10 CCT P TGT C TTG L LL-11 CCG P CTG L CAT H [00quantification. The Amplite Colorimetric NADH Assay Kit (AAT Bioquest) with some components from the redox ratio kits were reformulated to prepare an NMNH / NMN+kit. The NAD+:NADH ratio and NADP+:NADPH ratio quantification was performed according to the kit’s instructions with additional pre-assay sample processing steps to remove certain enzymes detailed herein. The NMNH:NMN+reformulated kit method is detailed further herein. Briefly, the difference between the NADH assay kit and the redox ratio kits is the addition of a selective reduced cofactor degradation step and cofactor specific enzymatic recycling. Therefore, the NMNH:NMN+ratio quantification kit was prepared by using the selective cofactor degradation solutions from the redox ratio kits with addition of a strictly NMN+-specific reducing enzyme, Gdh Ortho, to the final colorimetric assay master mix solution. Data for FIG.9C, F, I, and L which correspond to systems in FIG.1A-D were sampled at 48 h, 72 h, 48 h, and 24 h respectively.

[0108] Resting Cell Biotransformation. All necessary plasmids were inserted into strain MX102 R0by electroporation and plated on 2×YT with 200 mg / L ampicillin, 50 mg / L spectinomycin, and 50 mg / L kanamycin (ASK). Triplicate seed cultures of 4 mL 2×YT-ASK with 5 g / L D-mannitol were prepared from single colonies and grown at 37 °C for 16 h. Cell cultivation cultures were normalized to an OD600 of 0.04 in 50 mL 2×YT-ASK with 10 g / L D-mannitol and 0.5 mM 5-aminolevunic acid. The cells were incubated shaking for 4 h at 30 °C and then induced with 0.5 mM IPTG and 0.1% (w / v) L-arabinose. After induction, cells were incubated for 12 h while shaking at 30 °C before harvest. At harvest the OD600 of each culture was determined and the volume of cells necessary to prepare a final suspension of 100 OD600 in 3 mL final volume was transferred to 50 mL conical tubes and pelleted at 2,500 × gAttorney Docket No.00058-086WO1 for 10 min at room temperature. The pelleted cells were washed by gentle resuspension and centrifugation twice in 40 mL 100 mM potassium phosphate pH 7.5 at room temperature, then washed one more time in 10 mL of the same and transferred to 15-mL conical tubes. The conical tubes were pelleted a final time and resuspended to a total volume of 3 mL.

[0109] The resting cell reactions were initiated by the addition of 600 µL washed cell suspension to 2.4 mL concentrated reaction master mix in 15 mL conical tubes. The reaction mixture was composed of 200 mM D-glucose, 0 or 10 mM NMN+, 5 g / L (m)-BDO, 0.5 mM IPTG, 100 mM dibasic potassium phosphate, 200 mM MOPS, ASK, and 0.1% (w / v) L- arabinose. Resting cell reactions were then incubated horizontally at 250 rpm for 48 h at 30 °C. Resting cell reactions with expressed Tp Nox were instead incubated at 18 °C for 152 h.

[0110] Rosetta Molecular Simulations. The Ll Nox, Kp (m)-Bdh, and Ser (S)-Bdh structures were generated by Alphafold and subsequently relaxed using a backbone constrained FastRelax procedure. The NMN+, NAD+, and NADP+conformer library used was previously established. Initial coordinates for NMN+were obtained by aligning the Ll Nox structure to the homologous structure PDB: 2BC0. Initial coordinates for NMN+in Kp (m)-Bdh and Ser (S)-Bdh were obtained by alignment to the homologous structures PDB 1GEG and PDB 3A28, respectively.

[0111] The detailed method for Ll Nox is presented below. Bdhs were simulated in the same manner with the exclusion of flavin adenine dinucleotide (FAD+). For each simulation, one of the cofactors and FAD+were placed into the active site, and the enzyme- ligand complex was optimized. The EnzRepackMinimize protocol was applied to predict the WT structure bound with NMN+and amino acid substitutions that improve binding affinity for NMN+. This process involved Monte Carlo evaluation, sampling alternative rotamers, side chain substitutions, and backbone minimization to examine different binding pocket geometries. Restraints were imposed to maintain the cofactors in a catalytically competent geometry for hydride transfer and interactions with FAD+. After 2000 simulations for each batch, the top 20 outputs based on constraint score, protein-ligand interface energy score, and total system energy score were visually examined. For ligand docking simulation with mutations, the corresponding mutations were generated using the MutateResidue mover in RosettaScripts. Example run files, constraints, options, RosettaScripts XML, and ligand params files are available on Zenodo (doi.org / 10.5281 / zenodo.11478967).

[0112] MD simulations. The docking structures were picked as initial structures forAttorney Docket No.00058-086WO1 subsequent MD simulations. The CHARMM36m force field with cross term map correction and CHARMM general force field were selected for parameterizing proteins and cofactors, such as FAD and NMNH, respectively. For both WT and mutants (LLNox_LL9), structures were all centered in a periodic cubic box of 72 × 82 × 90 A and solvated using the TIP3P water model. Sodium and chloride ions were added for neutralization and the ionic concentration was maintained at 150 mM. Both systems were equilibrated using the NVT ensemble over 200 ps with Cα atoms of protein and heteroatoms of FAD and NMNH fixed. A 10-ns NPT simulation was performed with the same atomic constraints while decreasing from 100 to 0 kcal per mol per Å2gradually at the interval of 2 ns. Lastly, a 100-ns NPT simulation was collected without constraints. Throughout, temperature and pressure were held at 310 K and 1 atm using Langevin dynamics and Nose–Hoover–Langevin piston (excluding hydrogen atoms), respectively. Particle mesh Ewald summation85 with a 12-Å cutoff was used for electrostatic calculations. The integration time step was set to 2 fs using the SHAKE algorithm. All MD simulations were conducted using package suite NAMD 2.14. For hydrogen-bond analysis, the threshold distance between donor and acceptor and the donor–hydrogen–acceptor angle were 3 Å and 20°, respectively. Analysis was conducted over the phosphate group of NMNH and protein with VMD88. Only the last 60 ns of the trajectory was used. The input, topology and parameter files are available on Zenodo (doi.org / 10.5281 / zenodo.11478967).

[0113] Structural alignment of the Rossmann family. The structural alignment within the CATH superfamily 3.40.50.720 was performed with the >35 % sequence identity domain cluster representatives as calculated in the CATH database. Crystal structures and their specified chains were aggregated into a single PyMOL session. Structures with an NAD ligand were aligned with the PyMOL alignment tool according to their atomic coordinates. The pairwise root-mean-square deviation (r.m.s.d.) for all pairs was calculated using TM- align. On the basis of the pairwise r.m.s.d., the crystal structures were clustered using the SciPy python library and organized into eight groups with a cutoff distance of 33 on the resultant dendrogram. These eight groups were aligned using the PyMOL alignment tool to a structure with an NAD ligand from each group as the reference structure for that cluster. For visualization of the structural conservation, the backbones of the structures in the alignment are shown as transparent cartoon loops.

[0114] Code availability. The input files and source code for the Rosetta molecularAttorney Docket No.00058-086WO1 simulation and MD simulations are publicly accessible on Zenodo (doi.org / 10.5281 / zenodo.11478967).

[0115] Bioprospecting an (R)-Bdh with NADP+-Activity. A cofactor containing crystal structure of Bs (R)-Bdh was approximated by alignment of PDB: 6IE0 (Bs (R)-Bdh) with the cofactor bound crystal structure of greatest pairwise sequence identity in the protein databank (PDB: 1PL6). The atomic coordinates of the NAD+ligand was transferred to the Bs R-Bdh structure, and the 2’-hydroxyl replaced with phosphate in Pymol (Schrodinger). For bioprospecting cofactor specificity reversal homologs, it was hypothesized that homologs with mismatched residues at key positions on the loop that would contact the 2’-phosphate of NADP+would be the best targets. Of the top 2,000 protein-protein BLAST hits of Bs (R)-Bdh against the NCBI nonredundant protein database, 891 sequences were found to have at least one mismatch at positions E200, L201, and R205 of Bs (R)-Bdh (see FIG.10A-C). The sequences were further filtered to only include substitution of E200 to glutamine due to its potential to create favorable electrostatic interactions with the 2’-phosphate of NADP+. An E200Q hits table of 163 sequences was recorded and sorted by descending pairwise sequence identity. Interestingly, all hits for E200Q also had the substitution L201R. A representative sample of nine sequences were selected throughout the list (see Table 1, Table 3, and Table 4) for codon optimization, DNA synthesis, and plasmid cloning as further described herein. Table 3. Cofactor Preference and Database Identifiers for Primary Enzymes in Stereo- Upgrading Systems. Enzyme UniProt Accession Cofactor Preference Ser Bdh2 A0A7U3Z3T2 NAD(H) Kp Dar D7RP28 NAD(H) Bs BdhA O34788 NAD(H) NAD(H) and Cs Bdh M1MWX5 NADP(H) Bs Gdh P12310 NAD+and NADP+Ll Nox A2RIB7 NADH Lb Nox Q03Q85 NADH Tp Nox Mutated variant of Lb NoxaNADPH a Tp Nox (Lb Nox G159A-D177A-A178R-M179S-P184R)Attorney Docket No.00058-086WO1 Table 4. Database Identifiers for Wild Type Sequences of Other Enzymes Used in Bioprospecting Experiments. Enzyme Accession Enzyme Class Ecl Dar GenBank| JN035909 (m)-Bdh Kp BudC PDB| 1GEG (m)-Bdh Ka BudC NCBI Ref| WP_015366942 (m)-Bdh Ka Dar GenBank| VEC79507 (m)-Bdh Ko BudC GenBank| AEX06195 (m)-Bdh As Bdh GenBank| NLH91458 (R)-Bdh Cr Bdh GenBank| AEI90716 (R)-Bdh Ca Bdh NCBI Ref| WP_073006451 (R)-Bdh Pb Bdh NCBI Ref| WP_148550966 (R)-Bdh Cbo Bdh NCBI Ref| WP_075141790 (R)-Bdh Km Bdh NCBI Ref| WP_102401827 (R)-Bdh Cbu Bdh NCBI Ref| WP_104675707 (R)-Bdh Zp Bdh NCBI Ref| WP_027704711 (R)-Bdh PDB, Protein Data Bank; NCBI Ref, NCBI Reference Sequence Database; (m)-Bdh, Enzyme Class 1.1.1.B20 (meso-2,3-butanediol dehydrogenase); (S)-Bdh, Enzyme Class 1.1.1.76 ((2S,3S)-butanediol dehydrogenase); (R)-Bdh, Enzyme Class 1.1.1.4 ((2R,3R)-butanediol dehydrogenase).

[0116] Cycling Reaction for R-Bdh Homologs Stereospecificity. Reactions contained final concentrations of 100 mM potassium phosphate pH 7.5, 1 M NaCl, 200 mM D-glucose, 2mM cofactor, and 5 g / L m-BDO in a final volume of 450 µL. Each Nox was provided at 11.7 µM and Bdh homologs at 28.1 µM. Protein dilutions were carried out using His-Elution Buffer. Reactions were initiated by addition of 213 µL protein mixture to 237 µL concentrated reaction master mix. Reactions in 15 mL conical tube were incubated, shaking (250 rpm, 45° angle) at 30 °C for 10 hours then sampled as described in the Salting-Out- Extraction of Butanediol section above.

[0117] Reformulation of Commercial Redox Ratio Kits for NMNH / NMN+quantification. The Amplite Colorimetric NAD+ / NADH Ratio Assay Kit (AAT Bioquest) was used to measure NAD(H) and NAD+, the Amplite Colorimetric NADP+ / NADPH Ratio Assay Kit was used to measure NADP(H), and the Amplite Colorimetric NADH Assay Kit with some components from the other kits were reformulated to prepare an NMNH / NMN+kit. Briefly, a colorimetric method for quantification of NMN+and total NMN(H) wasAttorney Docket No.00058-086WO1 designed to function in the same way as the commercial kits. In principle, the kits are coupled cycling reactions between an enzyme with strict cofactor specificity and a chromogenic sensor with a maximum absorbance at 460 nm upon reduction by any reduced nicotinamide cofactor. Since only the cofactor recognized by the kit’s enzyme is recycled, only one cofactor is turned over repeatedly. Reduced species of the other cofactors, once oxidized by the probe, are not recycled so their contribution to the final absorbance at 460 nm becomes negligible when many turnovers of the target cofactor occur.

[0118] To measure only the reduced or oxidized species in a sample, one set of each sample is treated with a vendor supplied (AAT Bioquest) acid extraction solution and heat. In this condition the reduced cofactor is destroyed, the sample is neutralized, and then oxidized cofactor measured. The total pool of a given cofactor is measured by treatment with a vendor supplied control solution (AAT Bioquest) that does not degrade either form. The reduced cofactor is quantified as the difference between the total pool of a given nucleotide and oxidized form of that cofactor. Based on this principle, the Amplite Colorimetric NADH Assay Kit which uses the same probe to directly measure NADH without a recycling enzyme could be reformulated to measure the turnover of NMN(H) if a strictly NMN+-dependent enzyme and substrate were provided. Therefore, the previously engineered Gdh Ortho was chosen as the recycling enzyme with the NADH probe, assay buffer, and D-glucose. To measure multiple cofactors simultaneously, the samples were treated in parallel following the protocol for each kit. The protocol below is general to all kits as the kits only differ by the assay working solution formulation, appropriate dilution fold to stay within the linear dynamic range, and ratio of sample to master mix volume.

[0119] Samples of 75 µL reaction mixture from the (SS)-Bdo or (RR)-Bdo stereo- upgrading reactions were aliquoted into microcentrifuge tubes and immediately stored at -80 °C. When all samples were collected, the samples were removed from the freezer to thaw. Immediately once thawed, 150 µL 1x PBS was added to each sample. The samples were mixed by vortexing and centrifuged. To ensure that no enzymes in the reaction mixture can interfere with the assay readout, all protein greater than 10 kDa was removed from each sample by filtration of 200 µL sample through PALL Acroprep Advance 96 well 10K MWCO Omega short tip filtration plate (8034) by centrifugation at 1,500 × g for 10 min, 4 °C. A 96-well plate was placed underneath to collect the protein-free filtrate. Protein-free samples were processed in parallel to the standard curves.Attorney Docket No.00058-086WO1

[0120] A twelve-point standard curve for the range of 0-2 mM was generated for NAD+, NADP+, and NMN+in the same buffer composition as the samples. The standard curve was diluted three-fold in 1x PBS. Once it was determined that the NAD+or NADP+was entirely oxidized in all NMN(H) containing samples, two separate NMN+standard curves were prepared containing a constant concentration of either 2 mM NAD+or 2 mM NADP+. This was to ensure that any change in color at 460 nm attributable to the other cofactors was accounted for because while exceedingly small, some NAD(P)+activity with Gdh Ortho is possible.

[0121] Oxidized cofactor sample treatment: 15 µL of protein-free sample was combined with 15 µL of Component D: Extraction Solution using a multichannel pipette and mixed gently by pipetting. Extraction solution will degrade the reduced nucleotides. The samples were then incubated in a thermocycler for 15 min at 37 °C then cooled to 4 °C. Immediately after cooling the samples were removed from the thermocycler and 15 µL of Component E: Neutralization Solution was added and mixed by gentle pipetting with a multichannel.10 µL aliquots of the processed samples were made for each cofactor determination method (NAD+, NADP+, NMN+). The NAD+aliquots were diluted 10-fold in 1x PBS, NADP+33-fold in 1x PBS, and NMN+aliquots were diluted 8 / 3-fold in 1x PBS.50 µL of diluted NAD(P)+sample was transferred to fresh PCR tubes that would be used for initiation of the colorimetric cycling reaction. For NMN+, 20 µL of diluted sample was transferred to fresh PCR tubes for initiation of the assay reaction.

[0122] Total nucleotide pool (oxidized + reduced single cofactor) and standards sample treatment: 15 µL of protein-free sample was combined with 15 µL of Component F: Control Solution using a multichannel pipette and mixed gently by pipetting. Control solution does not degrade the reduced or oxidized nucleotides. The samples were then incubated in a thermocycler for 15 min at 37 °C then cooled to 4 °C. Immediately after cooling the samples were removed from the thermocycler and 15 µL of Component F: Control Solution was added and mixed by gentle pipetting with a multichannel.10 µL aliquots of the processed samples were made for each cofactor determination method (NAD(H), NADP(H), NMN(H)). The NAD(H) aliquots were diluted 10-fold in 1x PBS, NADP(H) 33-fold in 1x PBS, and NMN(H) aliquots were diluted 8 / 3-fold in 1x PBS.50 µL of diluted NAD(P)(H) sample was transferred to fresh PCR tubes that would be used for initiation of the colorimetric cycling reaction. For NMN(H), 20 µL of diluted sample was transferred to fresh PCR tubes forAttorney Docket No.00058-086WO1 initiation of the assay reaction.

[0123] Assay working solution preparation: Assay buffers were prepared with the following recipes with NADH or NADPH components from the corresponding commercial kit (AAT Bioquest). The same working solution was used for samples from either sample treatment protocol. Commercial recycling enzymes were resuspended in 500 µL 1x PBS from lyophilized powder. NAD(H) assay buffer was prepared as a bulk working solution and contained 40 µL Component B-II: NADH Probe Buffer per sample, 10 µL Component B-I: NADH Probe per sample, and 1.5 µL Component A: NAD / NADH Recycling Enzyme Mix per sample. NADP(H) assay buffer was prepared as a bulk working solution and contained 40 µL Component B-II: NADPH Probe Buffer per sample, 10 µL Component B-I: NADPH Probe per sample, and 1.5 µL Component A: NADP / NADPH Recycling Enzyme Mix per sample. NMN(H) assay buffer was prepared as a bulk working solution using components from the Amplite Colorimetric NADH Assay Kit and contained the following: 40 µL Component B: Assay Buffer per sample, 10 µL Component A: NADH Probe per sample, 10 µL of 200 g / L D-glucose per sample, 5 µL 1x PBS per sample, and 15 µL 67 µM Gdh Ortho per sample. Gdh Ortho was stored at -80 °C in high concentration as single use aliquots prior to addition to the working solution, dilution to 67 µM from stock was done with his-elution buffer as the diluent.

[0124] Reaction initiation: For NAD(P)(H) determination, 50 µL of NAD(P)(H) assay working solution was distributed to clean PCR tubes. For NMN(H) assay working solution, 80 µL was distributed to clean PCR tubes. To initiate the colorimetric recycling reaction assay, working solution was transferred to 20 µL treated samples by multichannel pipette, mixed gently by pipetting, and transferred to a 96 well visible plate at room temperature. The plate was incubated at room temperature without agitation or exposure to light for 0.5-3 hours then the endpoint absorbance at 460 nm was recorded.

[0125] Data Analysis: The 0 mM nucleotide standard for each nucleotide was used as the blank for all samples of that assay type. The equation of the best fit line to the corresponding standard curve was used to quantify the concentration of the target nucleotide in the samples. Reduced cofactor was calculated as [total pool of a single nucleotide] - [Oxidized]. Three replicate samples were processed per condition, per cofactor, and per treatment type (oxidized or total pool). Error of the total pool and oxidized speciesAttorney Docket No.00058-086WO1 concentrations is calculated as the standard deviation of three independent replicates. Error in the concentration of reduced species and redox ratio was calculated by propagation of error.

[0126] Stereo-upgrading as a test bed for orthogonal driving forces. BDO is an important biobased chiral chemical with broad industrial applications such as synthetic rubbers, fuels, and pharmaceuticals. BDO exists as three stereoisomers (see FIG.1): meso- (2S,3R)-butanediol ((m)-BDO), (2S,3S)-butanediol ((SS)-BDO), and (2R,3R)-butanediol ((RR)-BDO). The goal of the system disclosed herein is to convert (m)-BDO to either (SS)- BDO or (RR)-BDO with high purity, which is a value-added process.

[0127] The challenge in this process is that it requires two contradicting steps to both go to completion. First, an oxidation destroys a specific chiral center to yield acetoin (Ac). Second, a reduction installs a new chiral center (the four proposed designs are shown in FIG. 1A-D and discussed below). If these two reaction steps use the same cofactor, for example, NAD(H), then the NADH:NAD+ratio would need to be kept low for step one and high for step two. This is not feasible if both steps occur simultaneously in the same space (see FIG. 1E). When NAD(H) is incubated with 5 g / L (m)BDO and multiple Bdhs with different stereospecificities (the different enzyme classes are depicted in FIG.1F) in the absence of cofactor-recycling reactions to drive either step to completion, the result is a nearly equal distribution of (m)-BDO, (SS)-BDO and (RR)-BDO (see FIG.1G). This is because both oxidation and reduction steps suffer from the low driving force that stems from a suboptimal NADH:NAD+ratio and these enzymes operate in a fully reversible fashion. When an oxidation driving force is introduced by coupling to a water-forming NADH oxidase (Lactobacillus brevis Nox WT), the NADH:NAD+ratio is held low and the reaction only completes the first oxidation step on m-BDO, yielding a mixture of (S)-Ac and (R)-Ac (see FIG.1H). On the other hand, when a reduction driving force is introduced by NAD+- reducing GDH (Bacillus subtilis WT), the NADH:NAD+ratio is held high, and the reaction does not proceed past step one; (m)-BDO remains untransformed (see FIG.1I).

[0128] Broadly, current solutions to this problem are twofold. First, the two steps can be compartmentalized into separate cells with inverse NADH:NAD+ratios. This approach requires the intermediate to build up to a substantial level and readily diffuse across the cell membrane, which limits the scope of conversion. For example, complex and low- concentration intermediates in natural product biosynthetic pathways may not meet these requirements. In vitro, compartmentalization is also challenging to achieve. Second,Attorney Docket No.00058-086WO1 NADP(H) can be assigned as the reducing cofactor and NAD(H) can be assigned as the oxidizing cofactor. However, although many natural or engineered enzymes can have a preference between NAD(H) and NADP(H), most are promiscuous to both cofactors to a substantial degree because of the intrinsic high similarity between the two cofactors. Therefore, obtaining a strongly NAD(H)-specific enzyme for every oxidizing step and an NADP(H)-specific one for every reducing step is not always feasible, especially as the number of pathway steps increases. Furthermore, the reduction potential of NAD(H) and NADP(H) can become connected in vivo depending on the cell’s metabolic state and NADP(H) is too expensive to use at large scale in vitro.

[0129] The BDO-upgrading model system illustrates that, when limited to a single cofactor, metabolic pathways are unable to complete two contradicting steps no matter whether or how the driving force is applied. When two or more cofactors are used with enzymatic crosstalk among them (for example, NAD(H) and NADP(H)), the outcome resembles the single-cofactor system to some degree, dependent on the rate of crosstalk. This justifies a fundamental design principle in metabolism; orthogonality permits thermodynamically incompatible reactions to occur simultaneously.

[0130] In the design, NMN(H) flexibly was used as either the reducing or the oxidizing cofactor and demonstrate that it can be paired with either NAD(H) or NADP(H) as the opposing cofactor, without crosstalk in the same space. This results in a total of four combinations in cofactor use (see FIG.1A–D). Here, the enzymes were engineered to specifically destroy or install the (S)-chiral center to use NMN(H) (see FIG.1F). If NMN(H) is an orthogonal redox driving force that is insulated from both NAD(H) and NAD(P)H, two of the four cofactor combinations will yield pure (SS)-BDO (see FIG.1A, C) and the other two will yield pure (RR)-BDO (see FIG.1B, D). To demonstrate the versatility of the method compared to the two existing approaches mentioned above, tests were conducted both in vitro and in vivo in the same E. coli cells without compartmentalization.

[0131] Development of an orthogonal NMNH oxidase. For NMN(H) to drive either oxidation or reduction on demand, the design requires two orthogonal and complimentary NMN(H) cycling enzymes. GDH Ortho was used to maintain a high NMNH:NMN+ratio. To maintain a low NMNH:NMN+ratio, it was sought to engineer an orthogonal NMNH oxidase (Nox Ortho) based on the water-forming NADH oxidase. Water-forming oxidases (Nox) catalyze the reaction of reduced cofactor with oxygen and provide a byproduct-free and high-Attorney Docket No.00058-086WO1 driving-force reaction to rapidly recycle NAD(P)H to NAD(P)+in bioprocesses. Nox from L. pentosus (Lp Nox) has been engineered to accept NMNH and other noncanonical cofactors. However, Lp Nox still retains activity toward NADH and NADPH. As discussed above, cofactor promiscuity is undesirable because it breaks the insulation between separate driving forces.

[0132] A new Nox scaffold from L. lactis was utilized herein because of its high affinity for oxygen, broad operational pH and temperature ranges, and robust applications in vitro and in vivo. To enable NMNH activity, a similar rational design from Lp Nox was migrated to Ll Nox (namely, the I159T substitution), which resulted in a ~20-fold improved activity compared to WT with NMNH (see FIG.3A). This substitution is further discussed below.

[0133] With Ll Nox I159T as template, next was sought to further improve NMNH activity by using a high-throughput, growth-based selection platform (see FIG.2A). This is based on an engineered E. coli strain MX502 (BW25113 ΔpncC Δpgi Δzwf ΔnadR Δgnd + pLM106 + pLS502) (see Table 1) that metabolizes glucose exclusively by GDH Ortho, which relies on an NMNH-oxidizing enzyme for continuous function through the Entner– Doudoroff (ED) pathway instead of the conventional Embden–Meyerhof–Parnas (EMP) and the pentose phosphate pathways. Although this selection is not explicitly designed to yield orthogonal NMNH oxidases, it was postulated that orthogonal Nox variants would stand out in the evolution because they would not disturb the NAD(P)H pools essential for cell fitness and they can turn over NMNH more rapidly without NAD(P)H occupying their active sites as competitive substrates.

[0134] During growth selection, three positions (D178, A179, and I243) were subjected to site-saturated mutagenesis with degenerate codons (NNK). These conserved sites had a demonstrable effect on the cofactor preference of Lp Nox. The library yielded ~2 × 106independent transformants, sufficient to cover the theoretical library size, 203 = 8,000, by more than ten times. Selection was performed at 30 °C on agar plates of M9 minimal medium supplemented with 20 g l−1d-glucose and 2 mM NMN+. Plates were monitored for 10 days and 18 colonies were chosen for analysis on the basis of desirable growth phenotypes compared to controls (see Table 1). A specific activity assay was performed on sequenced variants (see Table 2 and FIG.3B). The best variant Ll Nox I159T-D178N-A179F; I243E (Nox Ortho; see Table 1) demonstrated a ~250-fold increase in the apparent catalyticAttorney Docket No.00058-086WO1 efficiency, kcat / Km, toward NMNH (see FIG.2B and FIG.4C). Nox Ortho was also found to have the most severely diminished NADH activity, a ~100-fold decrease in apparent catalytic efficiency compared to WT (see FIG.4B, see Table 5 and FIG.4C). Catalytic efficiency toward NADPH remained low for both WT and Ortho (see FIG.2B, see Table 5 and FIG. 4C). Table 5. Apparent Kinetic Parameters of Enzymes Engineered for NMN(H). Enzyme Variant Cofactor Km(mM) kcat(s–1) kcat / –Km(mM1s–1) NAD+0.20 ± 0.04 69 ± 1 355 ± 70 WT- NMN+6.7 ± 0.3 2.0 ± 0.3 0.30 ± 0.03 NAD+0.22 ± 0.03 20 ± 1 96 ± 20 WT NADP+1.5 ± 0.2 0.20 ± 0.01 0.13 ± 0.01 Ser (S)- NMN+5.6 ± 0.3 0.043 ± 0.004 0.0079 ± 0.001 Bdh NAD+1.6 ± 0.1 0.019 ± 0.001 0.012 ± 0.001 OrthoN+ 0.0020 ±0.0014 ± L39Q-A92K- M194TADP 1.4 ± 0.1 0.00010.0001 NMN+4.3 ± 0.4 0.38 ± 0.04 0.086 ± 0.002 NADH0.045 ±0.002 92 ± 2 2000 ± 90I159T-D178N-A179F- NADPH – – 20 ± 1 I243E NMNH – – 55 ± 4 “–“ denotes no data if the apparent Kmand kcatcould not be determined precisely. Apparent catalytic efficiency in these cases were instead calculated based on a linear fit to a modified Michaelis-Menten equation under the assumption Km≫ cofactor concentration. WT, wild type. See description for detailed derivation of equations used.

[0135] To understand Nox Ortho’s drastically switched cofactor preference, RosettaAttorney Docket No.00058-086WO1 docking was performed. The resultant models suggested that the I159T substitution stabilizes NMNH binding by introducing a novel polar interaction with the phosphate of NMNH, which was not seen when Ll Nox WT was modeled with NMNH (see FIG.2C and FIG.5). Taking these docking models as input, molecular dynamics (MD) simulations were conducted to further examine NMNH-binding stability in Ll Nox WT and Nox Ortho (see FIG.6). The evolution of hydrogen bonds formed between the phosphate of NMNH and each enzyme over time was evaluated (see FIG.6A-B) and it was observed that Nox Ortho established more hydrogen bonds with NMNH than Ll Nox WT during the last 60 ns of the MD simulation (see FIG.6C). While the number of hydrogen bonds formed by Ll Nox WT fluctuated between zero and one, Nox Ortho established two, three and occasionally four hydrogen bonds. This indicates an enhanced hydrogen-bond network at the Nox Ortho active site that supports NMNH stabilization.

[0136] D178N and I243E are predicted to form a new hydrogen bond (see FIG.2C- D). A179F potentially restricts the conformer choices of D178N, making the latter sample the hydrogen-bonding conformation more frequently (see FIG.2C-D). This hydrogen bond is predicted to dislodge NADH from the binding pocket into solvent (see FIG.2D). This prediction is supported by the high Km of Nox Ortho for NADH and NADPH, as these natural cofactors lose contact with the binding pocket (see Table 5 and FIG.4C; a high Km is inferred by nonsaturation kinetics at 2 mM reduced cofactor). Furthermore, at physiological concentrations of NADH and NADPH in E. coli (~0.1 – 1 mM), the high Km of Nox Ortho toward NADH and NADPH would imply that NAD(P)H occupies a small fraction of Nox Ortho active sites during the growth selection.

[0137] Reducing the size of the cofactor-binding pocket is an established approach to engineering enzymes to use smaller, noncanonical cofactors. Lp Nox and phosphite dehydrogenase (Ptdh) were engineered with bulky and hydrophobic residues at these conserved sites, which packed against one another to fill the cofactor-binding pocket and improve activity. Those packing interactions, in principle, should introduce the same steric hindrance as the hydrogen bond discovered here. Yet, those hydrophobic packing substitutions did not effectively block NAD(H) and NADP(H) binding like the hydrogen bond in Nox Ortho. One explanation may be that a hydrogen bond is stronger than a van der Waals contact, making the steric hindrance more durable to enzyme conformational shifts during catalysis. Alternatively, when hydrophobic substitutions fill the cofactor-bindingAttorney Docket No.00058-086WO1 pocket instead of polar contacts, the residual interaction between the hydrophobic surface of the flexible adenylyl moiety and the hydrophobic cleft can persist.

[0138] Development of NMN(H)-specific Bdhs. With GDH Ortho and Nox Ortho in hand, Bdhs were needed that can alter the (S)-chiral center using NMN(H) specifically (see FIG.1A–D). For a Bdh that uses NMN(H) as the oxidizing cofactor (see FIG.1B, D), the (m)-BDO dehydrogenase from Klebsiella pneumoniae, Kp (m)-Bdh, was chosen for its (S)- stereospecificity and high expression. Following the design principle derived from Nox Ortho engineering, an NMN(H)-specific variant Kp (m)-Bdh Ortho, a triple mutant (M189T-Y34Q- A87K) was generated. It was predicted that M189T would establish a novel polar contact with the phosphate of NMN+and that Y34Q and A87K would form a hydrogen bond to close off the binding pocket and prevent entry of NAD(P)+(see FIG.7A and FIG.8). This design was successful; the apparent catalytic efficiency of Kp (m)-Bdh Ortho for NMN+improved ~19-fold compared to WT (see FIG.7B, Table 5 and FIG.4A). Kp (m)-Bdh Ortho’s catalytic efficiency for NAD+and NADP+decreased 1.6 × 105-fold and 70-fold compared to WT, respectively. Together, Kp (m)Bdh Ortho features a 3.0 × 106-fold and 1.3 × 103-fold cofactor specificity switch compared to WT from NAD+or NADP+to NMN+, respectively, by catalytic efficiency (see Table 5 and FIG.4A). Bdh and Nox share little homology (~13% pairwise sequence identity) and are vastly different structurally. Success in engineering distinct enzyme families suggests that the design rules presented herein are unexpectedly universal.

[0139] Kp (m)-Bdh Ortho catalyzes the interconversion of (m)-BDO and (R)-Ac. For a Bdh that uses NMNH for reduction (see FIG.1A, C), the process requires that it accepts (S)-Ac as substrate to install an (S)-chiral center. To address this, six (S)-installing Bdhs from the (m)-Bdh and (S)-Bdh enzyme families were searched from the literature or by putative annotation: K. aerogenes KCTC 2190 BudC, K. aerogenes NCTC 8846 Dar, K. pneumoniae BudC56, K. oxytoca KCTC 1686 BudC, E. cloacae ssp. dissolvens SDM Dar57 and Serratia sp. AS13 (see Tables 1, 3, and 4). All of the enzymes were converted into NMN(H)- orthogonal enzymes en masse by mapping the same substitution pattern as applied to Kp (m)- Bdh Ortho onto them (six successful transfers; see FIG.7C). This streamlined process again underscores the translatability of the design principles disclose herein. All variants were tested by purified protein cycling reactions with NMN+(see FIG.9A). All of variants were found to install (S)-stereospecificity (see FIG.7C and FIG.9B-C), while onlyAttorney Docket No.00058-086WO1 Ser Bdh Ortho (L39Q; A92K; M194T) showed the desired substrate preference of converting (S)-Ac into (SS)-BDO. This variant was named as Ser (S)-Bdh Ortho.

[0140] Rosetta modeling of Ser (S)-Bdh Ortho supports the prediction that the hydrogen bond formed by A92K and L39Q, while not affecting NMN+binding (see FIG. 7D), pushes NAD+into a solvent-exposed, nonproductive binding pose (see FIG.11). Furthermore, M194T anchors NMN+by interacting with its phosphate (see FIG.7D). Corroborating these predictions, Ser (S)-Bdh Ortho (L39Q-A92K; M194T) features an 8.7 × 104-fold and 1.0 × 103-fold cofactor specificity switch compared to WT from NAD+and NADP+to NMN+, respectively, according to its apparent catalytic efficiency (see FIG.7E, Table 5 and Fig.4B).

[0141] Overall, all necessary parts of the four BDO stereo upgrading systems (see FIG.1A-D) were obtained: GDH Ortho to generate NMNH-reducing power, Nox Ortho to generate NMN+-oxidizing power and Kp (m)-Bdh Ortho and Ser (S)-Bdh Ortho to harness these orthogonal driving forces to manipulate (S)-chiral centers.

[0142] Orthogonal driving forces enable stereo-upgrading in vitro. Using the NMN(H)-specific enzymes, all four designs were systematically tested (see FIG.1A–D) using purified proteins in vitro (see FIG.10). GDH WT was used to reduce NAD(P)+and GDH Ortho was used to reduce NMN+. L. brevis Nox (Lb Nox), Tp Nox (an Lb Nox mutant; see Table 1) and Nox Ortho were used to oxidize NADH, NADPH, and NMNH, respectively. Bdhs of appropriate cofactor, substrate, and chiral specificity were chosen accordingly (see Tables 3 and 4). Natural cofactors, NAD(P)+, were supplemented at 2 mM or 0.2 mM and NMN+was supplemented at 2 mM. All systems behaved as intended and demonstrated NMN(H)’s distinct reduction potential compared to NAD(H) and NADP(H) in either oxidizing or reducing directions based on the redox ratio.

[0143] In the system that included Lb Nox, Bacillus subtilis (R)-Bdh (Bs (R)-Bdh), GDH Ortho, and Ser (S)-Bdh Ortho (see FIGs.10A and 1A), 5 g / L (m)-BDO was converted to ~3.8 g l−1 (SS)-BDO (77% conversion) (see FIG.10B). Here, NMN(H) provided the reducing power and a high NMNH:NMN+ratio of 3.7 ± 0.2 was observed (see FIG.10C), while NADH:NAD+was maintained at a low ratio of 0.10 ± 0.03. Alternatively, in the system with Nox Ortho, Kp (m)-Bdh Ortho, GDH WT and Bs (R)-Bdh (see FIGs.10D and 1B), 2 g / L (m)-BDO was converted to ~1.6 g / L (RR)-BDO (90% conversion) (see FIG.10E). Here, NMN(H) provided the oxidizing power with a low NMNH:NMN+ratio of 0.10 ± 0.03 versusAttorney Docket No.00058-086WO1 NADH:NAD+at 100 ± 30 (see FIG.10F). Both systems achieved high purity in producing the desired BDO isomers. The stark contrast between NMN(H) and NAD(H) redox ratios supports that these two cofactors are orthogonal to each other.

[0144] The further the distance between the two cofactors’ redox ratios, the more strongly they can deliver two opposing, insulated driving forces; this insulation is ensured by all enzymes’ strict cofactor specificities. Indeed, when comparing the (SS)-BDO-producing system (see FIGs.10A-C and 1A) to the (RR)-BDO-producing system (see FIGs.10D-F and 1B), it was observed that the closer NMN(H) versus NAD(H) redox ratios in the (SS)-BDO- producing system resulted in Bs (R)-Bdh and Ser (S)-Bdh Ortho retaining small residual ability to catalyze the reverse, unintended reactions, as evident by the slight buildup of side products (RR)-BDO, (R)-Ac and (S)-Ac. This could be because of the small side activity of Ser (S)-Bdh Ortho toward NAD+(see FIG.7E and Table 5), which penetrates the insulation and permits some slow leakage by crosstalk between reactions. This was not observed in the system based on Kp (m)-Bdh Ortho paired with NAD(H) (see FIGs.10D-F and 1B), which had superior specificity for NMN+(see FIG.7B and Table 5). This leakage phenomenon because of crosstalk between reactions by shared cofactor usage exists and is shown to be prevalent and profound between the natural cofactors, NAD(H) and NADP(H), in nature because of the presence of numerous promiscuous enzymes and the dynamic use of direct and indirect modes of cofactor exchange. The high structural deviancy of noncanonical cofactors promises to mitigate this.

[0145] After showcasing the BDO stereo-upgrading system’s capacity to orchestrate redox driving forces in complex mixtures using NAD(H) and NMN(H), next was sought to demonstrate that these principles are translatable to systems based on the other natural redox driving force, NADP(H), and thereby compatible with implementation in vivo. A system using NADP(H) paired with NMN(H) was also prepared. Previous studies described NADPH-dependent (R)-Bdhs but it was found their activity in m-BDO oxidation to be limited. Therefore, an NADP+-active (R)-Bdh was bioprospected from Clostridium saccharoperbutylacetonicum (Cs (R)-Bdh) on the basis of sequence homology to the readily reversible Bs (R)-Bdh (see FIG.12A-C). Subsequently, the specific activity with NADP+and stereospecific oxidation to destroy the (R)-chiral center of (m)-BDO was confirmed (see FIG. 12D-G).

[0146] The (SS)-BDO-producing system contained Tp Nox, Cs (R)-Bdh, GDH OrthoAttorney Docket No.00058-086WO1 and Ser (S)-Bdh Ortho (see FIG.10G) and produced ~3.9 g / L (SS)-BDO from 5 g / L (m)- BDO (86% conversion) (see FIG.10H). The redox ratios suggest that NMN(H) can be held far from equilibrium with NADP(H), with NMNH serving as a strong reducing power (NMNH:NMN+= 70 ± 20) (see FIG.10I), while the NADP(H) pool stays oxidized at 0.11± 0.03 (see FIG.10I). Unlike the (SS)-BDO-producing system based on NAD(H) (see FIG. 10A), the NAD(P) system (see FIG.10G) does not suffer byproduct formation, consistent with the Ser (S)-Bdh Ortho’s superior orthogonality toward NADP+(see FIG.7B, Table 5 and FIG.4B). The complementary stereo-upgrading system contained Nox Ortho, Kp (m)- Bdh Ortho, GDH WT and Cs (R)-Bdh (see FIG.10J) and produced ~1.9 g / L (RR)-BDO from 2 g / L (m)-BDO (89% conversion) (see FIG.10K). Here, with NMN(H) as the oxidizing power, the NMNH:NMN+ratio was expectedly low (0.07 ± 0.02), while the reducing power in this system, NADP(H), was high (NADPH:NADP+= 20 ± 0.6) (see FIG.10L). These results demonstrate ideal orthogonality between NADP(H) and NMN(H) for the system based on Kp (m)-Bdh Ortho.

[0147] Altogether, Kp (m)-Bdh Ortho, Ser (S)-Bdh Ortho and Nox Ortho, which share a unifying enzyme design principle for cofactor specificity, combine to form four different stereo-upgrading systems capable of remarkedly pure preparations of chiral BDO.

[0148] NMN(H) enables BDO stereo-upgrading in E. coli whole cells. To convert m-BDO to (SS)-BDO, a previously reported E. coli strain, MX102 R0was used (see Table 1). This organism cannot catabolize glucose, the sacrificial electron donor, and has decreased ability to degrade NMN+(see FIG.13A). NAD(H) was fist used as the oxidant and NMN(H) the reductant (see FIG.13B). Lb Nox, Bs (R)-Bdh, GDH Ortho, Ser (S)-Bdh Ortho and Zymomonas mobilis (Zm Glf) encoding a glucose transport facilitator were co-expressed from plasmids (see FIG.13B; pDA129, pDA131 and pSM10 in Table 1) in E. coli and prepared as resting cells. When cells were incubated with 200 mM glucose, 5 g / L (m)-BDO and 10 mM NMN+supplemented, 3.8 g l−1of (SS)-BDO was produced (see FIG. 13D and FIG.14). The product purity of cells supplemented with 10 mM NMN+also increased dramatically relative to cells without NMN+supplementation, with the former reaching 81% pure (SS)-BDO (see FIG.13E). When no NMN+was supplemented, a substantial amount of (S)-Ac was observed. This is indicative of undesirable reversible reactions caused by insufficient driving forces, as discussed above.

[0149] The system can also function with NADP(H) as the oxidant, when Lb Nox isAttorney Docket No.00058-086WO1 replaced with Tp Nox and Bs (R)-Bdh is replaced with Cs (R)-Bdh (see FIG.13C; pDA129, pDA162 and pSM10 in Table 1). The resting cells produced a final titer of 2.5 g / L (SS)-BDO when 10 mM NMN+was supplied (see FIG.13F and FIG.15). Without NMN+supplementation, very little (SS)-BDO was produced (0.40 g / L) even though a high concentration of (S)-Ac was formed (2.0 g / L), suggesting a lack of reducing power when NMN+was not supplied. Again, the product purity in the NMN+-supplied cells was greatly improved, from 7.7% pure with 0 mM NMN+to 50% pure (SS)-BDO with 10 mM NMN+(see FIG.13G). The primary impurity in this system was (m)-BDO, the starting material, which points to the first, oxidative step catalyzed by the NADP(H)-active Cs (R)-Bdh and driven by NADPH-specific Tp Nox as the major bottleneck. The equivalent cell-free system readily catalyzed the oxidation with NADP+(see FIG.10G-I). Yet, even in resting E. coli cells, the redox ratio of NADP(H) is challenging to invert toward efficient oxidation. The small total NADP(H) pool size could also impact the efficiency of this step; basal NADP(H) concentrations are on the order of 0.1 mM, whereas 2 mM NADP+was provided in the cell- free system (see FIG.10G-I).

[0150] Orthogonal metabolic systems can vastly simplify the optimization efforts required to achieve whole-cell biotransformation because the engineering efforts are focused on the heterologous pathway itself. However, the approach of orthogonal metabolic systems still relies upon what is presently specialized expertise in engineering enzymes for noncanonical cofactors; broad adoption of this versatile strategy to control metabolic flux hinges on the capability of a range of users to translate successful designs across the diverse biocatalytic repertoire that exists in nature.

[0151] Mapping design principles onto the Rossmann superfamily. In the design of (m)-Bdh Ortho and (S)-Bdh Ortho, it was observed that paired substitutions, predicted to form a polar contact that interrupts dinucleotide binding, translated amongst enzymes with low sequence homology (see FIG.18A). Yet, structural homology among these enzymes does occur at the Rossmann fold. It was sought to map the substitutions against Rossmann- fold-containing proteins that represent a diverse sequence and functional space.

[0152] First, the equivalent substitution sites of Nox Ortho and (m)-Bdh Ortho were located on their homologs, L. brevis Nox (Protein Data Bank (PDB) 5VN0)41 and Kp BudC (PDB 1GEG)56, respectively (see FIG.16A-B light gray, gray and dark gray spheres). The substitutions described as blocking dinucleotide cofactors are represented with light grayAttorney Docket No.00058-086WO1 spheres. The substitutions described as hydrogen-bonding partners to the terminal phosphate of NMN(H) are represented with dark gray or gray spheres.

[0153] Next, it was sought to project the substitution signatures of Nox Ortho and (m)-Bdh Ortho (see FIG.16A-B, respectively) onto the broad NAD(P)-binding Rossmann- like domain superfamily in the CATH (class, architecture, topology, homology) database (CATH superfamily 3.40.50.720) to understand site conservation. High conservation could indicate high generalizability of the design principles at these sites to build NMN+-orthogonal enzymes. In total, 617 experimentally determined protein structures—each a discrete structural neighborhood representative within the superfamily—were systematically superimposed (see FIG.16C), with most sharing <30% pairwise sequence similarity (see FIG.18B). Here, darkness of the backbone superposition was interpreted as a structural conservation metric, where darker regions are more conserved. Note that Nox Ortho’s or (m)- Bdh Ortho’s dinucleotide cofactor-blocking sites are in highly conserved areas (see FIG. 16D-E), which correspond to loops adjacent to β2 and β3 strands of the Rossmann fold (see FIG.16F). This suggests high translatability of the NAD(P)+-excluding strategy reported here.

[0154] Additional strategies reported here were analyzed to enable NMN(H) activity, namely, novel polar interactions to NMN(H). It was observed that I159T in Nox Ortho is in the highly conserved helix α1 in the Rossman fold (see FIG.16E, dark gray sphere, with helical axis as a light gray line, and FIG.16F, dark gray arrow), consistent with prior experimental success in this site’s translatability.

[0155] Alternatively, targeting the site represented by M189T of (m)-BDO Ortho (see FIG.16D, gray sphere) has robustly yielded high-NMN+-activity mutants across different proteins. This site was translated among all six NMN(H)-active Bdh variants reported herein (M189T; M192T in Table 5 and FIGs.7C and 16D), which share 47.3–99.6% sequence similarity (see FIG.18A), and the previously engineered GDH Ortho12 (position I195R). This site is located on a second α-helix coaxial to Rossmann helix α1 (see FIG.16D, light gray line, and FIG.16F, α2). This area is less conserved because it is peripheral to the core Rossmann domain (see FIG.16C-D). For enzyme scaffolds with this additional helix, it was envisioned strong translatability of the design principle based on this site to build polar contacts to NMN+.

[0156] Altogether, the design principles disclosed herein, can be translated to a broadAttorney Docket No.00058-086WO1 range of enzymes, are twofold. First, NMN(H) recognition can be initiated using sites on Rossmann helix α1 or α2 (see FIG.16). Second, NAD(H) and NADP(H) can be excluded by closing the space between Rossmann strands β2 and β3 (see FIG.16).

[0157] Orthogonal metabolic systems can vastly simplify the optimization efforts required to achieve whole cell biotransformation, because the engineering efforts can be primarily focused on the heterologous pathway itself. Here, the orthogonal system achieves high product purity with minimal alterations to the host’s native metabolism. This work establishes that NMN(H) reduction potential can be firmly enforced in vivo without the interference from the NAD(H) and NADP(H) redox state, which will deliver persistent driving force in the ever-changing cellular environment.

[0158] Certain embodiments of the invention have been described. It will be understood that various modifications may be made without departing from the spirit and scope of the invention. Other embodiments are within the scope of the following claims.

Claims

Attorney Docket No.00058-086WO1 WHAT IS CLAIMED IS:

1. A recombinantly engineered polypeptide having redox enzyme activity that has improved catalytic efficiency for a noncanonical cofactor while having reduced catalytic efficiency for a natural cofactor, the recombinantly engineered polypeptide comprising: a modified or mutated natural cofactor binding pocket that comprises: (1) amino acid substitutions or mutations that form new hydrogen bond(s) that inhibit or prevent entry by the natural cofactor into the binding pocket; and (2) amino acid substitutions or mutations that promote the binding of the noncanonical cofactor by introducing new polar contact(s) that interact specifically with polar group(s) or moieties of the noncanonical cofactor.

2. The recombinantly engineered polypeptide of claim 1, wherein the modified or mutated natural cofactor binding pocket of the recombinantly engineered polypeptide comprises 3 to 10 amino acid mutations or substitutions in comparison to the sequence of a wild-type or parent polypeptide.

3. The recombinantly engineered polypeptide of claim 2, wherein at least one of the substitutions or mutations introduces a new polar contact that interacts specifically with a polar group of the noncanonical cofactor, and wherein at least two of the substitutions or mutations form a new hydrogen bond that inhibits or prevents entry by the natural cofactor into the binding pocket.

4. The recombinantly engineered polypeptide of claim 3, wherein the recombinantly engineered polypeptide encodes a Rossmann-fold containing protein, and the new polar contact that interacts specifically with a polar group of the noncanonical cofactor is found in Rossmann helix α1 or α2, and wherein the new hydrogen bond that inhibits or prevents entry by the natural cofactor into the binding pocket closes the space between Rossmann strands β2 and β3.

5. The recombinantly engineered polypeptide of claim 1, wherein the modified or mutated natural cofactor binding pocket does not comprise mutations or substitutions thatAttorney Docket No.00058-086WO1 restricts the size of the natural cofactor binding pocket by forming hydrophobic packing interactions that inhibit or prevent entry by the natural cofactor into the binding pocket.

6. The recombinantly engineered polypeptide of claim 1, wherein the recombinantly engineered polypeptide has at least 15-fold increase in catalytic efficiency for the noncanonical cofactor in comparison to a wild-type or parent polypeptide.

7. The recombinantly engineered polypeptide of claim 1, wherein the recombinantly engineered polypeptide has at least a 50-fold decrease in catalytic efficiency for the natural cofactor in comparison to a wild-type or parent polypeptide.

8. The recombinantly engineered polypeptide of claim 1, wherein the noncanonical cofactor is selected from the group consisting of nicotinamide mononucleotide (NMNH), 1- phenyl-1,4,-dihydronicotinamide , 1-benzyl-1,4-dihydronicotinamide, 1-(4- hydroxyphenyl)1,4-dihydronicotinamide, 1-methyl-1,4-dihydronicotinamide, nicotinamide flucytosine dinucleotide, nicotinamide mononucleoside, 1‐butyl‐1,4,5,6‐tetrahydropyridine‐3‐ carboxamide, 1‐(1‐benzyl‐1,4,5,6‐tetrahydropyridin‐3‐yl) ethenone, 1-benzyl-1,4- dihydropyridine-3-carboxylic acid, and 1‐benzyl‐1,4,5,6‐tetrahydropyridine‐3‐carbonitrile.

9. The recombinantly engineered polypeptide of claim 8, wherein the noncanonical cofactor is NMNH.

10. The recombinantly engineered polypeptide of claim 1, wherein the natural cofactor is selected from NAD(H), FAD(H), and NADP(H).

11. The recombinantly engineered polypeptide of claim 1, wherein the recombinantly engineered polypeptide has a redox enzyme activity selected from an oxidase, a dehydrogenase, and an oxidoreductase.

12. The recombinantly engineered polypeptide of claim 11, wherein the recombinantly engineered polypeptide comprises a sequence that is at least 95% identical to a sequence from an oxidase, a dehydrogenase, or an oxidoreductase.Attorney Docket No.00058-086WO1 13. The recombinantly engineered polypeptide of claim 12, wherein the recombinantly engineered polypeptide comprises a sequence that is at least 98% identical to a sequence from an oxidase, a dehydrogenase, or an oxidoreductase.

14. The recombinantly engineered polypeptide of claim 13, wherein the oxidase is selected from glucose oxidase, NADPH oxidase, amine oxidase, and NADH oxidase.

15. The recombinantly engineered polypeptide of claim 14, wherein the oxidase is an NADH oxidase selected from Anaerocolumna aminovalerica, Bacillus subtilis, Enterococcus faecalis, Lacticaseibacillus rhamnosus, Lactiplantibacillus pentosus, Lactococcus cremoris, Lactococcus lactis, Levilactobacillus brevis, Methanobrevibacter smithii, Streptococcus agalactiae, Streptococcus mutans, and Streptococcus pyogenes.

16. The recombinantly engineered polypeptide of claim 13, wherein the oxidase is an NADH oxidase having an amino acid sequence that is at least 98% identical to SEQ ID NO:6 or SEQ ID NO:

7.

17. The recombinantly engineered polypeptide of claim 1, wherein the recombinantly engineered polypeptide comprises a sequence that is at least 98% identical to SEQ ID NO:6 but comprises at least the substitutions of I159T, D178N, A179F, and I243E.

18. The recombinantly engineered polypeptide of claim 12, wherein the dehydrogenase or the oxidoreductase is selected from phosphite dehydrogenase, meso- (2R,3S)-butanediol dehydrogenase ((m)-Bdh), (2R,3R)-butanediol dehydrogenase ((R)-Bdh), (2S,3S)-butanediol dehydrogenase ((S)-Bdh), alcohol dehydrogenase, acetoin reductase, diacetyl reductase, alcohol dehydrogenase, glutathione reductase, homoserine dehydrogenase, glucose dehydrogenase, glycerol dehydrogenase, propanediol-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, acetaldehyde dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, pyruvate dehydrogenase, oxoglutarate dehydrogenase, and formate dehydrogenase.Attorney Docket No.00058-086WO1 19. The recombinantly engineered polypeptide of claim 18, wherein the dehydrogenase or the oxidoreductase is from the enzyme class of (m)-Bdh, (R)-Bdh, or (S)-Bdh having a sequence that is at least 95% identical to the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:

21.

20. The recombinantly engineered polypeptide of claim 19, wherein the recombinantly engineered polypeptide comprises a sequence that is at least 98% identical to the sequence of SEQ ID NO:1 but comprises at least the amino acid substitutions of L39Q, A92K, and M194T.

21. The recombinantly engineered polypeptide of claim 19, wherein the recombinantly engineered polypeptide comprises a sequence that is at least 98% identical to the sequence of SEQ ID NO:2 but comprises at least the amino acid substitutions of M189T, Y34Q, and A87K.

22. The recombinantly engineered polypeptide of claim 1, wherein the recombinantly engineered polypeptide comprises a sequence selected from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:

26.

23. An expression vector comprising the recombinantly engineered polypeptide of any one of claims 1 to 22.

24. The expression vector of claim 23, wherein the expression vector is a plasmid, a viral vector, a cosmid, or an artificial chromosome.

25. The expression vector of claim 24, wherein the expression vector is a plasmid that comprises elements for expressing the recombinantly engineered polypeptide in a bacterium or a yeast.Attorney Docket No.00058-086WO1 26. A microorganism that comprises a recombinantly engineered polypeptide of any one of claims 1 to 22.

27. The microorganism of claim 26, wherein the microorganism is a bacterium, fungus, or a yeast.

28. The microorganism of claim 26, wherein the microorganism is a strain of E. coli that is unable to catabolize glucose and has decreased ability to degrade noncanonical cofactors.

29. A microorganism that comprises the expression vector of claim 23.

30. The microorganism of claim 29, wherein the microorganism is a bacterium, fungus, or a yeast.

31. The microorganism of claim 30, wherein the microorganism is a strain of E. coli that is unable to catabolize glucose and has decreased ability to degrade noncanonical cofactors.

32. A method of stereo-upgrading meso-(2S,3R)-butanediol ((m)-BDO) to (2S,3S)- butanediol ((SS)-BDO) or (2R,3R)-butanediol ((RR)-BDO) in a cell free system, comprising: providing the following purified polypeptides in a buffer system comprising a natural cofactor and a noncanonical cofactor: a polypeptide encoding a Bdh, Dar, or BudC enzyme that utilizes NAD(H) or NADP(H) as a natural cofactor, a polypeptide encoding an orthogonal (S)-Bdh comprising the sequence of SEQ ID NO:25 or a polypeptide encoding an orthogonal (m)-Bdh comprising the sequence of SEQ ID NO:26, a polypeptide encoding an Nox oxidase comprising the sequence of SEQ ID NO:8 or SEQ ID NO:24, and a polypeptide encoding a Gdh dehydrogenase comprising the sequence of SEQ ID NO:5 or SEQ ID NO:22; and introducing (m)-BDO into the buffer system that is then converted into (SS)-BDO or (RR)-BDO by the activities of the purified polypeptides.Attorney Docket No.00058-086WO1 33. The method of claim 32, wherein the polypeptide encoding an orthogonal (S)-Bdh has the sequence of SEQ ID NO:25, and wherein the polypeptide encoding an orthogonal (m)- Bdh has the sequence of SEQ ID NO:

26.

34. The method of claim 32, wherein the polypeptide encoding a Nox oxidase has the sequence of SEQ ID NO:

24.

35. The method of claim 32, wherein the polypeptide encoding a Gdh dehydrogenase has the sequence of SEQ ID NO:

22.

36. The method of claim 32, wherein the natural cofactor is NADP(H) or NAD(H), and the noncanonical cofactor is NMN(H).

37. The method of claim 32, wherein the method stereo-upgrades (m)-BDO to (SS)-BDO in a cell free system and the method comprises: providing the following purified polypeptides in a buffer system comprising NAD(H) and NMN(H): a polypeptide encoding a Bdh, Dar, or BudC enzyme that utilizes NAD(H) as a natural cofactor, a polypeptide encoding an orthogonal (S)-Bdh that comprises the sequence of SEQ ID NO:25, a polypeptide encoding a Nox oxidase that comprises the sequence of SEQ ID NO:24, a polypeptide encoding a Gdh dehydrogenase that comprises the sequence of SEQ ID NO:22; and introducing (m)-BDO into the buffer system that is then converted into (SS)-BDO by the activities of the purified polypeptides.

38. A method to produce (2S,3S)-butanediol (SS)-BDO)) in vivo, comprising: co-expressing the following polypeptides in a recombinant microorganism:Attorney Docket No.00058-086WO1 a recombinantly engineered polypeptide encoding a (R)-Bdh enzyme that utilizes NAD(H) as a natural cofactor, a polypeptide encoding an orthogonal (S)-Bdh hat comprises the sequence of SEQ ID NO:25, a polypeptide encoding a Nox oxidase that comprises the sequence of SEQ ID NO:7, a polypeptide encoding a Gdh dehydrogenase that comprises the sequence of SEQ ID NO:22, a polypeptide encoding a glucose transport facilitator that has a sequence of SEQ ID NO:23; and producing (SS)-BDO by incubating the recombinant microorganism with media containing glucose, meso-(2R,3S)-butanediol, and NMN(H).

39. The method of claim 38, wherein the recombinant microorganism is a strain of E. coli that is unable to catabolize glucose and has decreased ability to degrade noncanonical cofactors.

40. A method to produce (2S,3S)-butanediol (SS)-BDO)) in vivo, comprising: co-expressing the following polypeptides in a recombinant microorganism: a recombinantly engineered polypeptide encoding a (R)-Bdh enzyme that utilizes NADP(H) as a natural cofactor, a polypeptide encoding an orthogonal (S)-Bdh hat comprises the sequence of SEQ ID NO:25, a polypeptide encoding a Nox oxidase that comprises the sequence of SEQ ID NO:8, a polypeptide encoding a Gdh dehydrogenase that comprises the sequence of SEQ ID NO:22, a polypeptide encoding a glucose transport facilitator that has a sequence of SEQ ID NO:23; and producing (SS)-BDO by incubating the recombinant microorganism with media containing glucose, meso-(2R,3S)-butanediol, and NMN(H).Attorney Docket No.00058-086WO1 41. The method of claim 40, wherein the recombinant microorganism is a strain of E. coli that is unable to catabolize glucose and has decreased ability to degrade noncanonical cofactors.

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