Systems and methods for the direct reduction of carboxylic acids to aldehydes without ATP or COA-based activation

Abstract

Provided is a cell free system, a whole-cell biomanufacturing system, or methods which comprise a polypeptide that encodes an aldehyde dehydrogenase that directly reduces carboxylic acid containing compounds to aldehyde containing compounds without ATP or CoA-based activation. Also provided are additional polypeptides that can convert aldehyde containing compounds into additional functional groups and which can recycle nicotinamide-based coenzymes.

Description

Atorney docket No. 00058-087W01SYSTEMS AND METHODS FOR THE DIRECT REDUCTION OF CARBOXYLIC ACIDS TO ALDEHYDES WITHOUT ATP OR COA-BASED ACTIVATIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U. S.C. § 119 from ProvisionalApplication Serial No. 63 / 689,609, filed August 30, 2024 the disclosure of which is incorporated herein by reference.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. DE-AR0001508 awarded by the U.S. Department of Energy . The Government has certain rights in the invention.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] Accompanying this filing is a Sequence Listing entitled, “00058-087W01.xml” created on August 29,2025 and having 238,062 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 is a cell free system, a whole-cell biomanufacturing system, or methods which comprise a polypeptide that encodes an aldehyde dehydrogenase that directly reduces carboxylic acid containing compounds to aldehyde containing compounds without ATP or CoA-based activation or CO2 loss. Also provided are additional polypeptides that can convert aldehyde containing compounds into additional functional groups and which can recycle nicotinamide-based coenzymes.BACKGROUND

[0005] In microorganism-based fermentation systems, carboxylic acids like aliphatic acids (e.g, acetic acid, butyric acid), hydroxy-acids (e.g , lactic acid), keto-acids (e.g, 2-keto- butyrate), and dicarboxylic acids (e.g., succinic acid, adipic acid) are produced as stable metabolic intermediates and waste products. Due to the thermodynamic inertness of these compounds, chemical upgrading of these molecules has proven difficult, and they are regularly discarded or sold as low-value commodity chemicals.Atorney docket No. 00058-087W01SUMMARY

[0006] In the studies presented herein, an aldehyde dehydrogenase was paired with an alcohol dehydrogenase to reduce carboxylic acids to aldehydes via aldehyde dehydrogenase and NADH, and aldehydes were reduced to alcohols via alcohol dehydrogenase and NADH. Oxidized NADH (NAD+) was regenerated to NADH via phosphite dehydrogenase from Pseudomonas stutzeri or glucose dehydrogenase from Bacillus subtilis. Additionally, as presented herein are exemplary methods where variations on this system are employed. In these variations, the second enzy me, the alcohol dehydrogenase listed above, is exchanged for a different enzy me to provide alternative modification to the aldehyde produced from carboxylic acid reduction. In other examples, the second enzyme is removed, and aldehydes produced from carboxylic acid reduction are unmodified or separated from the reaction system by gas stripping. One hundred seventy ALDH scaffolds were identified and characterized to exhibit the ability7to direct reduce the carboxylic acids to the respective aldehydes.

[0007] The disclosure further provides for both a cell-free system and a whole-cell biomanufacturing systems to facilitate the biotransformation of a substrate into a desired product, comprising a recombinantly engineered polypeptide of the disclosure having aldehyde dehydrogenase activity that can directly reduce carboxylic acids to aldehydes without ATP or CoA-based activation or CO2 loss.

[0008] In a particular embodiment, the disclosure provides for a cell-free system or whole-cell biomanufacturing system to facilitate the biotransformation of a carboxylic acid containing substrate into a desired product, comprising: a first polypeptide encoding an aldehyde dehydrogenase that directly reduces carboxylic acids to aldehydes without adenosine triphosphate (ATP) or coenzyme A (CoA)-based activation, wherein the aldehyde dehydrogenase can use reduced nicotinamide-adenine dinucleotide (NADH) as a cofactor; and a first hydride donor compound; optionally, a second polypeptide that encodes an enzyme capable of converting an aldehyde group into anon-aldehyde group; optionally, a second hydride donor compound and a third polypeptide that encodes an enzyme that reduces the first hydride donor compound using the second hydride donor compound. In another embodiment, the first polypeptide comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to a sequence presented in SEQ ID NO: 1 to 170. In yet another embodiment, the first polypeptideAtorney docket No. 00058-087W01 comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, is at least 95%, at least 97%, at least 98%, or at least 99% identical to a sequence presented in SEQ ID NO:1, SEQ ID NO:39, SEQ ID NO:2, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:99, SEQ ID NO:105, SEQ ID NO: 119, SEQ ID NO: 126, and SEQ ID NO:170. In a further embodiment, the first polypeptide is engineered to contain 1 to 10 amino acid substitutions in comparison to a sequence presented in SEQ ID NO: 1 to 170, wherein the amino acid substitution(s) improve the aldehyde dehydrogenase activity for directly reducing carboxylic acids to aldehydes. In yet a further embodiment, the first polypeptide has the sequence of SEQ ID NO:39 with at least the amino acid substitution of Y402F. In a particular embodiment, the first polypeptide has the sequence of SEQ ID NO:2 with at least an amino acid substitution selected from Q350N, Q350R, and Q350A. In another embodiment, the first polypeptide has the sequence of SEQ ID NO:31 with one or more amino acid substitutions selected from (a), (b), (c), (d), (e), and / or (f): (a) W185S, W185D, W185H, W185N, W185K, or W185F; (b) N186D; (c) E285D; (d) F288A; (e) Q366I, Q366A, Q366L, Q366M, or Q366N; and / or (!) F418A or F418H. In a further embodiment, the first polypeptide has the sequence of SEQ ID NO: 31 with at least an amino acid substitution selected from W 185N, W185K, and W185F. In another embodiment, the first polypeptide has the sequence of SEQ ID NO:1 with one or more amino acid substitutions selected from (a), (b) and / or (c): (a) W164F; (b) T302S; and / or (c) W173M, W173F, and W173Y. In another embodiment, the first polypeptide has the sequence of SEQ ID NO:99 with at least an ammo acid substitution selected from W173M, W173F, and W173Y. In yet another embodiment, the cell-free system or whole-cell biomanufacturing system comprises the second polypeptide that encodes an enzyme capable of converting an aldehyde group into anon-aldehyde group. In a certain embodiment, the second polypeptide encodes an enzyme capable of converting an aldehyde group into a non-aldehyde group, utilizes the first hydride donor compound as a cofactor. In a further embodiment, the second polypeptide encodes an alcohol dehydrogenase, or an acetolactate synthase. In another embodiment, the second polypeptide encodes an alcohol dehydrogenase that has a sequence that is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:172 or SEQ ID NO: 173. In yet another embodiment, the second polypeptide encodes an acetolactate synthase that has a sequence that is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 174. In a further embodiment, the cell-free system or whole-cell biomanufacturing system comprises the thirdAtorney docket No. 00058-087W01 polypeptide and the second hydride donor compound. In yet a further embodiment, the third polypeptide encodes a phosphite dehydrogenase that has a sequence that is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 171, and wherein the second hydride donor compound is phosphite. In a certain embodiment, the third polypeptide encodes a glucose dehydrogenase that has a sequence that is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 175, and wherein the second hydride donor compound is glucose. In another embodiment, the cell-free system or whole-cell biomanufacturing system further comprises 1,4-dithiothreitol. In yet another embodiment, the system is a cell-free system, and the cell-free system utilizes polypeptide(s) that have been isolated and purified. In a further embodiment, the cell-free system further comprises an organic solvent overlay that supports production of an aldehyde by the first polypeptide and / or a non-aldehyde product produced by the second polypeptide, and wherein the polypeptide(s) and hydride donor(s) are in an aqueous solution. In another embodiment, the organic overly comprises a solvent selected from anisole, hexane, isooctane, toluene, dodecane, oleyl alcohol, and ethoxybenzene. In yet another embodiment, the organic solvent overlay is used at a ratio of 1 : 10 to 10: 1 to the aqueous solution. In a further embodiment, the organic solvent overlay is used at a ratio of 1 : 1 to 10: 1 to the aqueous solution. In yet a further embodiment, the cell-free system or whole-cell biomanufacturing system further comprises an apparatus to isolate a desired product that is an aldehyde, wherein the apparatus comprises: a reactor that can be heated which comprises the product aldehyde, wherein the reactor is in fluid connection to one or more cooled condensers filled with a solvent, wherein in heating the reactor causes the product aldehyde to go in the gaseous phase; a gas supply that is in fluid connection with the reactor, that is configured to introduce positive gas flow7into the reactor, wherein the gas introduced into the reactor flows from the reactor to the one or more cooled condensers; and one of more cooled condensers filled with a solvent that are configured to cool the gaseous aldehyde into a liquid phase so that it is trapped by the solvent, wherein the aldehyde can then be isolated from the solvent. In a particular embodiment, the system is a whole-cell biomanufacturing system wherein vectors are used to express the polypeptide(s) in a microorganism. In another embodiment, the microorganism is selected from a bacterium or yeast. In yet another embodiment, the first hydride donor compound is selected from reduced nicotinamide-adenine dinucleotide (NADH), reduced nicotinamide-adenine dinucleotide phosphate (NADPH), carba-nicotinamide adenineAtorney docket No. 00058-087W01 dinucleotide (cNADH), reduced nicotinamide mononucleotide (NMNH), reduced 3- carbamoyl-l-(4-carboxy benzyl) pyridin-l-ium (BANAH), reduced 1 -benzylnicotinamide (BNAH), reduced 3-carbamoyl-l-(3-phenylpropyl)pyridin-l-ium (3PNAH), and reduced 3- carbamoyl-l-(4-methoxybenzyl)pyridin-l-ium (BNA OMeH). In another embodiment, the first hydride donor compound is cNADH or NMNH.

[0009] The disclosure also provides for methods or processes to facilitate the biotransformation of a substrate into a desired product, comprising a recombinantly engineered polypeptide of the disclosure having aldehyde dehydrogenase activity that can directly reduce carboxylic acids to aldehydes without ATP or CoA-based activation. In a particular embodiment, the disclosure also provides a method to convert a carboxylic acid containing substrate to an aldehyde containing product, the method comprising: contacting the carboxylic acid containing substrate with a first polypeptide that encodes an aldehyde dehydrogenase that directly reduces carboxylic acids to aldehydes without adenosine triphosphate (ATP) or coenzyme A (CoA)-based activation in the presence of a first hydride donor compound in an aqueous solution, optionally, providing a second hydride donor compound and a second polypeptide that encodes an enzyme that reduces the first hydride donor compound using the second hydride donor compound, wherein the aldehyde dehydrogenase can use reduced first hydride donor compound as a cofactor, and wherein the aldehyde dehydrogenase converts the carboxylic acid containing substrate to an aldehyde containing product. In another embodiment, the carboxylic acid containing substrate is selected from a linear aliphatic carboxylic acid, a branched aliphatic carboxylic acid, an aromatic carboxylic acid, a diacid, a hydroxyacid and formic acid. In yet another embodiment, the linear aliphatic carboxylic acid is selected from acetic acid, propanoic acid, butyric acid, hexanoic acid, and nonanoic acid. In a certain embodiment, the branched aliphatic carboxylic acid is selected from isobutyric acid, and isopentanoic acid. In another embodiment, the aromatic carboxylic acid is selected from benzoic acid, 2-furoic acid, cinnamic acid and vanillic acid. In yet another embodiment, the diacid is selected from succinic acid, and adipic acid. In a further embodiment, the hydroxyacid is selected from 4- hydroxybutyric acid, 3-hydroxybutyric acid, lactic acid, and 2-hydroxylpropanoic acid. In a certain embodiment, the first polypeptide comprises a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98%, or at least 99% identical to a sequence presented in SEQ ID NO: 1 to 170. In another embodiment, the first polypeptideAtorney docket No. 00058-087W01 comprises a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98%, or at least 99% identical to a sequence presented in SEQ ID NO: 1, SEQ ID NO:39, SEQ ID NO:2, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:99, SEQ ID NO:105, SEQ ID NO: 119, SEQ ID NO: 126, and SEQ ID NO: 170. In yet another embodiment, the first polypeptide is engineered to contain 1 to 10 amino acid substitutions in comparison to a sequence presented in SEQ ID NO: 1 to 170. wherein the amino acid substitution(s) improve the aldehyde dehydrogenase activity for directly reducing carboxylic acids to aldehydes. In yet a further embodiment, the first polypeptide has the sequence of SEQ ID NO:39 with at least the amino acid substitution of Y402F. In a particular embodiment, the first polypeptide has the sequence of SEQ ID NO:2 with at least an amino acid substitution selected from Q350N, Q350R, and Q350A. In another embodiment, the first polypeptide has the sequence of SEQ ID NO:31 with one or more amino acid substitutions selected from (a), (b), (c), (d), (e), and / or (f): (a) W185S, W185D, W185H, W185N, W185K, or W185F; (b) N186D; (c) E285D; (d) F288A; (e) Q366I, Q366A, Q366L, Q366M, or Q366N; and / or (f) F418A or F418H. In a further embodiment, the first polypeptide has the sequence of SEQ ID NO:31 with at least an amino acid substitution selected from W185N, W 185K, and W 185F. In another embodiment, the first polypeptide has the sequence of SEQ ID NO: 1 with one or more amino acid substitutions selected from (a), (b) and / or (c): (a) W164F; (b) T302S; and / or (c) W173M, W173F. and W173Y. In another embodiment, the first polypeptide has the sequence of SEQ ID NO: 99 with at least an amino acid substitution selected from W173M, W173F, and W173Y. In a certain embodiment, the method further comprises isolating the aldehydes during the reaction process. In another embodiment, the method further comprises overlaying an organic solvent on top of the aqueous solution to allow for the continuous extraction of the aldehydes. In yet another embodiment, the organic solvent is selected from anisole, hexane, isooctane, toluene, dodecane, oleyl alcohol, and ethoxybenzene. In a certain embodiment, the organic solvent is used at a ratio of 1 : 10 to 10: 1 to the aqueous solution. In another embodiment, the organic solvent is used at a ratio of 1: 1 to 10: 1 to the aqueous solution. In a further embodiment, the method further comprises isolating the aldehyde containing products by using an apparatus, the apparatus comprising: a reactor that can be heated which comprises the product aldehyde, wherein the reactor is in fluid connection to one or more cooled condensers filled with a solvent, wherein in heating the reactor causes the product aldehyde to go in the gaseous phase; a gas supply that is in fluid connection with theAttorncy docket No. 00058-087W01 reactor, that is configured to introduce positive gas flow into the reactor, wherein the gas introduced into the reactor flows from the reactor to the one or more cooled condensers; and one of more cooled condensers filled with a solvent that are configured to cool the gaseous aldehyde into a liquid phase so that it is trapped by the solvent, wherein the aldehyde can then be isolated from the solvent. In a particular embodiment, the method comprises the second hydride donor compound and the second polypeptide that encodes an enzyme that reduces the first hydride donor compound using the second hydride donor compound. In another embodiment, the second polypeptide encodes a phosphite dehydrogenase that has a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 171, and wherein the second hydride donor compound is phosphite. In yet another embodiment, the second polypeptide encodes a glucose dehydrogenase that has a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 175, and wherein the second hydride donor compound is glucose. In a further embodiment, the method further comprises converting the aldehyde containing product to non-aldehyde containing product, comprising: contacting the aldehyde containing product with a third polypeptide that that encodes an enzyme capable of converting an aldehyde group into a non-aldehyde group. In a certain embodiment, the third polypeptide that encodes an enzyme capable of converting an aldehyde group into a non-aldehyde group, utilizes the first hydride compound as a coenzyme or cofactor. In another embodiment, the third polypeptide encodes an alcohol dehydrogenase, or an acetolactate synthase. In yet another embodiment, the third polypeptide encodes an alcohol dehydrogenase that has a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 172 or SEQ ID NO: 173. In a further embodiment, the third polypeptide encodes an acetolactate synthase that has a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 174. In yet a further embodiment, the first hydride donor compound is selected from reduced nicotinamideadenine dinucleotide (NADH), reduced nicotinamide-adenine dinucleotide phosphate (NADPH), carba-nicotinamide adenine dinucleotide (cNADH), reduced nicotinamide mononucleotide (NMNH), reduced 3 -carbamoyl- l-(4-carboxy benzyl) pyridin-l-ium (BANAH), reduced 1-benzylni cotinamide (BNAH), reduced 3-carbamoyl-l-(3- phenylpropyl)pyridin-l-ium (3PNAH), and reduced 3-carbamoyl-l-(4-Attorney docket No. 00058-087W01 methoxybenzyl)pyridin-l-ium (BNA OMeH). In another embodiment, the first hydride donor compound is cNADH or NMNH.

[0010] The disclosure provides for a kit, system, composition, or method as substantially described in the specification and / or drawings.

[0011] 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 descnption and drawings, and from the claims.DESCRIPTION OF DRAWINGS

[0012] 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.

[0013] FIG. 1 provides a screening platform for carboxylic acid reduction system. Carboxylic acids are directly reduced to aldehyde by the candidate enzyme. An alcohol dehydrogenase reduces the aldehyde to an alcohol. Phosphite dehydrogenase regenerates NAD+ to NADH.

[0014] FIG. 2 presents a system for direct reduction of acid to aldehyde. Hexanoic acid is used as an example, but example can contain acetic acid, propanoic acid, butyric acid, hexanoic acid, isobutyric acid, isopentanoic acid, benzoic acid, 2-furoic acid, vanillic acid, 5- hydroxypentanoic acid, or formic acid. Aldehyde dehydrogenase performs the NADH- mediated reduction of carboxylic acid to aldehyde. Alcohol dehydrogenase performs the NADH-mediated reduction of aldehyde to alcohol.

[0015] FIG. 3 presents the use of an aldehyde dehydrogenase to produce hexanol from hexanoic acid. Aldehyde produced by the aldehyde dehydrogenase is reduced to an alcohol using an alcohol dehydrogenase. Aldehyde dehydrogenase sequences included in this figure: A4IT08 (SEQ ID NO:1), P51977 (SEQ ID NO:2), P54114 (SEQ ID NO:3), P46367 (SEQ ID NO:4), P40047 (SEQ ID NO:5), P54115 (SEQ ID N0:6), Q04458 (SEQ ID NO:7), A0A6P3Q7G7 (SEQ ID NO: 8), A0A5J5DF59 (SEQ ID NO: 9), A0A0D6KE96 (SEQ ID NO: 10), L8N0N6 (SEQ ID NO: 11), UPI000407F0B2 (SEQ ID NO: 12), A0A2Z4LU87 (SEQ ID NO: 13), A0A0S8BPB2 (SEQ ID NO: 14), A0A1F8MB45 (SEQ ID NO: 15), A0A346XWA7 (SEQ ID NO: 16), UPI000A40ADAD (SEQ ID NO: 17), A0A139NAP6 (SEQ ID NO: 18), UPI0001E31496 (SEQ ID NO: 19), A0A537X7T4 (SEQ ID NO:20), A0A2E8CRC4 (SEQ ID NO:21), A0A2V7BEQ0 (SEQ ID NO:22), A0A1T1H988 (SEQ IDAtorney docket No. 00058-087W01NO:23), A0A1F6LNY1 (SEQ ID NO:24), A0A7H4GQ81 (SEQ ID NO:25), R7Z1F3 (SEQ ID NO:26), P51647 (SEQ ID NO:27), P77674 (SEQ ID NO:28), P23883 (SEQ ID NO:29), UPI001 AE6B188 (SEQ ID NO:30), P05091 (SEQ ID NO:31), P25526 (SEQ ID NO:32), P25553 (SEQ ID NO:33), F5Z5S7 (SEQ ID NO:34), UPI00019FFBE0 (SEQ ID NO:35), Q9LRI6 (SEQ ID NO:36), C9DIJ2 (SEQ ID NO:37), Q65NX0 (SEQ ID NO:38), UPI00005BF137 (SEQ ID NO:39), A0A6H1TS81 (SEQ ID NO:40), A0A7L1RJ35 +A0A7L1RY59 partial concatenation (SEQ ID NO:41), A0A6N7YK66 (SEQ ID NO:42). A0A918MZJ5 (SEQ ID NO:43), A0A858QBL4 (SEQ ID NO:44), A0A1G9VJH9 (SEQ ID NO:45), A0A1B1YTA3 (SEQ ID NO:46), R4G109 (SEQ ID NO:47), UPI00234FF82E (SEQ ID NO:48), A0A2U3D9B3 (SEQ ID NO:49), A0A1W9L4H0 (SEQ ID NO:50), A0A3S0VUQ7 (SEQ ID NO:51), A0A177P8X5 (SEQ ID NO:52), A0A7Z0SSR0 (SEQ ID NO:53), A0A2X4UZ81 (SEQ ID NO:54), A0A849HPN9 (SEQ ID NO:55), A0A4R6PRX6 (SEQ ID NO:56), A0A251X6G7 (SEQ ID NO:57), A0A2E2EWH7 (SEQ ID NO:58), A0A3B0T5Y3 (SEQ ID NO:59), A0A3B1AP81 (SEQ ID NO:60), A0A443K8I0 (SEQ ID NO:61), A0A4P6V3E3 (SEQ ID NO:62), A0A7Y9PEX6 (SEQ ID NO:63), A0A0J0V889 (SEQ ID NO:65), A0A0K6GMR6 (SEQ ID NO:66), A0A142D425 (SEQ ID NO:67), UPI0007A942CE (SEQ ID NO:68), A0A178T558 (SEQ ID NO:69), A0A1I0TE05 (SEQ ID NO:70), UPI0009ADBF94 (SEQ ID NO:71), UPI00017E6B4A (SEQ ID NO:72), UPI000A26C654 (SEQ ID NO:73), A0A2M9T1Z9 (SEQ ID NO:74), A0A327YHK1 (SEQ ID NO:75), A0A4Q1RV81 (SEQ ID NO:76), A0A4R1QFD0 (SEQ ID NO:77), A0A6G9J022 (SEQ ID NO:78), UPI00050099C5 (SEQ ID NO:79), A0A7U3YC36 (SEQ ID NO:80), A0A7V9YZI5 (SEQ ID NO:81), A0A7W0BY09 (SEQ ID NO:82), A0A7W8JJ24 (SEQ ID NO:83), A0A7W9YP99 (SEQ ID NO:84). A0A7W9YPV4 (SEQ ID NO:85). A0A840DPH9 (SEQ ID NO: 86), UPI001363667A (SEQ ID NO: 87). C5D8G6 (SEQ ID NO:88), UPI0002BF8D7A (SEQ ID NO:89), M8CWA2 (SEQ ID NO:90), S5ZGT3 (SEQ ID NO:91), S7SR93 (SEQ ID NO:92), U2WRT1 (SEQ ID NO:93), UPI0004DFAE51 (SEQ ID NO:94), UPI001EEBB712 (SEQ ID NO:95), UPI0015843A2B (SEQ ID NO:96), UPI0014923DAD (SEQ ID NO:97). UPI00030EE752 (SEQ ID NO:98), UPI0005CD4570 (SEQ ID NO:99), UPI00228641DC (SEQ ID NO: 100), UPI00135864F6 (SEQ ID NO: 101), UPI00208DAAE2 (SEQ ID NO: 102), UPI001315BED2 (SEQ ID NO: 103), UPI000489A7CA (SEQ ID NO:104), UPI001FCBF132 (SEQ ID NO:105), UPI0001D589C3 (SEQ ID NO:106), M2XRT2 (SEQ ID NO:107), A0A3L7TS19 (SEQ ID NO:108),Attorney docket No. 00058-087W01A0A2A5G690 (SEQ ID NO:109), Q1JUP4 (SEQ ID NO:110), A0A1B1YTA3 (SEQ ID NO:46), C7A2A0 (SEQ ID N0: 112), A0A6J4GM31 (SEQ ID N0:113), Q58806 (SEQ ID NO: 114), Q4F895 (SEQ ID NO: 115), Q9VLC5 (SEQ ID NO: 1 16), A0QSN7 (SEQ ID NO: 117), 086447 (SEQ ID N0:118), Q84DC3 (SEQ ID N0:119), A0A077WVD9 (SEQ ID NO: 120), Q8KZT4 (SEQ ID NO: 121), Q56694 (SEQ ID NO: 122), WP_209124955.1 (SEQ ID NO: 123), A0A0S8B2A0 (SEQ ID NO: 149), J7MGD1 (SEQ ID NO: 158). S3NC75 (SEQ ID NO: 159). A0A4Q0HY84 (SEQ ID NO: 160). UPI001F4DFA4B (SEQ ID NO: 161). A0A7X1PME0 (SEQ ID NO: 162), UPI000C179C88 (SEQ ID NO:163), UPI000650692E (SEQ ID NO: 164), and G2IKV5 (SEQ ID NO: 170). ScADH (SEQ ID NO: 172) is the alcohol dehydrogenase in example.

[0016] FIG. 4 presents a single component dropout of carboxylic acid reduction system. Individual components were removed from the complete system, separately. ScADH is the alcohol dehydrogenase in example. Aldehyde dehydrogenase sequences included in this figure: P05091 (SEQ ID NO:31).

[0017] FIG. 5 demonstrates the use of an aldehyde dehydrogenase to produce ethanol from acetate. Aldehyde produced by the aldehyde dehydrogenase is reduced to an alcohol using an alcohol dehydrogenase. ScADH (SEQ ID NO: 172) is the alcohol dehydrogenase in example. Aldehyde dehydrogenase sequences included in this figure: A4IT08 (SEQ ID NO: 1), UPI001FCBF132 (SEQ ID NO: 105), and UPI0005CD4570 (SEQ ID NO:99).

[0018] FIG. 6 demonstrates the use of an aldehyde dehydrogenase to produce propanol from propionate. Aldehyde produced by the aldehyde dehydrogenase is reduced to an alcohol using an alcohol dehydrogenase. ScADH (SEQ ID NO: 172) is the alcohol dehydrogenase in example. Aldehyde dehydrogenase sequences included in this figure: A4IT08 (SEQ ID NO: 1). A0A023CJ10 (SEQ ID NO:64), A0A0J0V889 (SEQ ID NO:65), A0A0K6GMR6 (SEQ ID NO: 66), A0A142D425 (SEQ ID NO: 67), UPI0007A942CE (SEQ ID NO:68), A0A178T558 (SEQ ID NO:69), A0A1I0TE05 (SEQ ID NO:70), UPI0009ADBF94 (SEQ ID NO:71), UPI00017E6B4A (SEQ ID NO:72), UPI000A26C654 (SEQ ID NO:73), A0A2M9T1Z9 (SEQ ID NO:74). A0A327YHK1 (SEQ ID NO:75), A0A4Q1RV81 (SEQ ID NO:76), A0A4R1QFD0 (SEQ ID NO:77), A0A6G9J022 (SEQ ID NO:78), UPI00050099C5 (SEQ ID NO:79), A0A7U3YC36 (SEQ ID NO: 80), A0A7V9YZI5 (SEQ ID NO:81), A0A7W0BY09 (SEQ ID NO:82), A0A7W8JJ24 (SEQ ID NO:83), A0A7W9YP99 (SEQ ID NO: 84), A0A7W9YPV4 (SEQ ID NO: 85), A0A840DPH9 (SEQ IDAttorney docket No. 00058-087W01NO: 86), UPI001363667 A (SEQ ID NO:87). C5D8G6 (SEQ ID NO: 88), UPI0002BF8D7A (SEQ ID NO:89), M8CWA2 (SEQ ID NO:90), S5ZGT3 (SEQ ID NO:91), S7SR93 (SEQ ID NO:92), U2WRT1 (SEQ ID NO:93), UPI0004DFAE51 (SEQ ID NO:94), UPI001EEBB712 (SEQ ID NO:95), UPI0015843A2B (SEQ ID NO:96), UPI0014923DAD (SEQ ID NO:97), UPI00030EE752 (SEQ ID NO:98), UPI0005CD4570 (SEQ ID NO:99), UPI00228641DC (SEQ ID NO: 100). UPI00135864F6 (SEQ ID NO: 101). UPI00208DAAE2 (SEQ ID NO: 102). UPI001315BED2 (SEQ ID NO: 103). UP1000489A7CA (SEQ ID NO: 104), UPI001FCBF132 (SEQ ID NO:105), UPI0001D589C3 (SEQ ID NO:106), A0A1B1YTA3 (SEQ ID NO:46), A0A6J4GM31 (SEQ ID NO: 113), Q4F895 (SEQ ID NO: 115), Q9VLC5 (SEQ ID NO: 116), and Q8KZT4 (SEQ ID NO: 121).

[0019] FIG. 7 presents the use of an aldehyde dehydrogenase to produce butanol from butyrate. Aldehyde produced by the aldehyde dehydrogenase is reduced to an alcohol using an alcohol dehydrogenase. ScADH (SEQ ID NO: 172) is the alcohol dehydrogenase in example. Aldehyde dehydrogenase sequences included in this figure: A4IT08 (SEQ ID NO: 1), UPI001FCBF132 (SEQ ID NO: 105), and UPI0005CD4570 (SEQ ID NO:99).

[0020] FIG. 8 provides for the use of an aldehyde dehydrogenase to produce isobutanol from isobutyrate. Aldehyde produced by the aldehyde dehydrogenase is reduced to an alcohol using an alcohol dehydrogenase. TcADH (SEQ ID NO: 173) is the alcohol dehydrogenase in example. Aldehyde dehydrogenase sequences included in this figure: A41T08 (SEQ ID NO: 1). UPI001FCBF132 (SEQ ID NO: 105), and UP10005CD4570 (SEQ ID NO: 199).

[0021] FIG. 9 presents the use of an aldehyde dehydrogenase to produce benzy l alcohol from benzoic acid. Aldehyde produced by the aldehyde dehydrogenase is reduced to an alcohol using an alcohol dehydrogenase. TcADH (SEQ ID NO: 173) is the alcohol dehydrogenase in example. Aldehyde dehydrogenase sequences included in this figure: A4IT08 (SEQ ID NO: 1), UPI000A26C654 (SEQ ID NO:73), A0A7W0BY09 (SEQ ID NO:82), UPI0005CD4570 (SEQ ID NO:99), UPI001FCBF132 (SEQ ID NO:105), Q84DC3 (SEQ ID NO: 119). Q9HUR4 (SEQ ID NO: 124), P94358 (SEQ ID NO: 126), UPI001E1E4C76 (SEQ ID NO: 127), UPI001EIE9641 (SEQ ID NO: 128), A0A095VIR5 (SEQ ID NO: 129), A0A1L3SPG8 (SEQ ID NO:131), A0A1X7A4R9 (SEQ ID NO:132), A0A2A3MQQ0 (SEQ ID NO: 133), A0A2T7UV49 (SEQ ID NO: 134), A0A438AI09 (SEQ ID NO: 135), A0A916RQF6 (SEQ ID NO: 137), A0A947KSW4 (SEQ ID NO: 138),Atorney docket No. 00058-087W01A0A9X1IQQ9 (SEQ ID NO: 139), A0AA47IBM1 (SEQ ID NO: 140), A0AAE4JPI1 (SEQ ID NO: 141), UPI00141AD733 (SEQ ID NO: 142), UPI00160AC323 (SEQ ID NO: 143). UPI001C8C5927 (SEQ ID NO: 144), UPI002367CC2E (SEQ ID NO: 146), UPI002810EC0B (SEQ ID NO: 147), W3WRJ5 (SEQ ID NO: 148), A0A0S8B2A0 (SEQ ID NO: 149), UPI001CE2568F (SEQ ID NO: 150), UPI0027E04FF2 (SEQ ID NO: 151), UPI000BD54C6D (SEQ ID NO: 152). UPI003871E0C3 (SEQ ID NO: 153), A0A223E7X4 (SEQ ID NO: 154), UP100100965F3 (SEQ ID NO: 155). UPI0009BE91EE (SEQ ID NO:156), A3RJV6 (SEQ ID NO:157), J7MGD1 (SEQ ID NO:158), S3NC75 (SEQ ID NO: 159), A0A4Q0HY84 (SEQ ID NO: 160), UPI001F4DFA4B (SEQ ID NO: 161), A0A7X1PME0 (SEQ ID NO: 162), UPI000C179C88 (SEQ ID NO: 163), UPI000650692E (SEQ ID NO: 164), P54886 (SEQ ID NO: 165), Q1XGL7 (SEQ ID NO: 166), Q72IB9 (SEQ ID NO: 167). F5Z5S7 (SEQ ID NO:34), Q88QE9 (SEQ ID NO: 168), Q88CR0 (SEQ ID NO: 169), and G2IKV5 (SEQ ID NO: 170).

[0022] FIG. 10 provides for the use of an aldehyde dehydrogenase to produce 4- hydroxybenzyl alcohol from 4-hydroxybenzoic acid. Aldehyde produced by the aldehyde dehydrogenase is reduced to an alcohol using an alcohol dehydrogenase. TcADH (SEQ ID NO: 173) is the alcohol dehydrogenase in example. Aldehyde dehydrogenase sequences included in this figure: G2IKV5 (SEQ ID NO: 170), Q9HUR4 (SEQ ID NO: 124), Q8NMB0 (SEQ ID NO: 125), P94358 (SEQ ID NO: 126), A0A095VIR5 (SEQ ID NO: 129). A0A1L3SPG8 (SEQ ID NO: 131). A0A1X7A4R9 (SEQ ID NO: 132). A0A2A3MQQ0 (SEQ ID NO:133), A0A438AI09 (SEQ ID NO: 135), A0A916RQF6 (SEQ ID NO: 137), A0A947KSW4 (SEQ ID NO: 138), A0A9X1IQQ9 (SEQ ID NO: 139), AOAA47IBM1(SEQ ID NO:140), and UPI001C8C5927 (SEQ ID NO: 144).

[0023] FIG. 11 presents the use of an aldehyde dehydrogenase to produce vanillyl alcohol from vanillic acid. Aldehyde produced by the aldehyde dehydrogenase is reduced to an alcohol using an alcohol dehydrogenase. TcADH (SEQ ID NO: 173) is the alcohol dehydrogenase in example. Aldehyde dehydrogenase sequences included in this figure: G2IKV5 (SEQ ID NO: 170), P94358 (SEQ ID NO: 126), A0A095VIR5 (SEQ ID NO: 129), A0A0Q5IVT9 (SEQ ID NO: 130), A0A1X7A4R9 (SEQ ID NO: 132), A0A2A3MQQ0 (SEQ ID NO: 133), A0A7Z2V8C7 (SEQ ID NO: 136), A0A916RQF6 (SEQ ID NO: 137), A0A947KSW4 (SEQ ID NO: 138), A0A9X1IQQ9 (SEQ ID NO: 139), A0AA47IBM1 (SEQAtorney docket No. 00058-087W01ID NO: 140), A0AAE4JPI1 (SEQ ID NO: 141), UPI001C8C5927 (SEQ ID NO: 144), and UPI002367CC2E (SEQ ID NO: 146).

[0024] FIG. 12 provides the use of an aldehyde dehydrogenase to produce trans- cinnamyl alcohol from trans-cinnamic acid. Aldehyde produced by the aldehyde dehydrogenase is reduced to an alcohol using an alcohol dehydrogenase. TcADH (SEQ ID NO: 173) is the alcohol dehydrogenase in example. Aldehyde dehydrogenase sequences included in this figure: A41T08 (SEQ ID NO: 1), UPI000A26C654 (SEQ ID NO:73). A0A7W0BY09 (SEQ ID NO:82), UPI0005CD4570 (SEQ ID NO:99) and UPI001FCBF132 (SEQ ID NO: 105).

[0025] FIG. 13 presents the use of an aldehyde dehydrogenase to produce furfuryl alcohol from 2-furoic acid. Aldehyde produced by the aldehyde dehydrogenase is reduced to an alcohol using an alcohol dehydrogenase. TcADH (SEQ ID NO: 173) is the alcohol dehydrogenase in example. Aldehyde dehydrogenase sequences included in this figure: A4IT08 (SEQ ID NO: 1). UPI000A26C654 (SEQ ID NO: 73), A0A7W0BY09 (SEQ ID NO:82), UPI0005CD4570 (SEQ ID NO:99) and UPI001FCBF132 (SEQ ID NO: 105).

[0026] FIG. 14 presents the use of an aldehyde dehydrogenase to produce 1,5- pentanediol from 5-hydroxypentanoic acid. Aldehyde produced by the aldehyde dehydrogenase is reduced to an alcohol using an alcohol dehydrogenase. TcADH (SEQ ID NO: 173) is the alcohol dehydrogenase in example. Aldehyde dehydrogenase sequences included in this figure: A41T08 (SEQ ID NO: 1), UPI0005CD4570 (SEQ ID NO:99). and UPI001FCBF132 (SEQ ID NO:105).

[0027] FIG. 15 demonstrates the direct reduction of benzoic acid to benzaldehyde with subsequent enzy matic modification to condense benzaldehyde with pyruvate to form L- PAC.

[0028] FIG. 16 shows the production of L-phenylacetylcarbinol through the direct reduction of benzoic acid. An aldehyde dehydrogenase reduces benzoic acid to benzaldehyde. Benzaldehyde is subsequently modified by acetolactate synthase (SEQ ID NO: 174) to produce L-phenylacetylcarbinol. Aldehyde dehydrogenase sequences included in this figure: A4IT08 (SEQ ID NO: 1) and UPI0005CD4570 (SEQ ID NO: 99).

[0029] FIG. 17 provides a schematic depicting the aldehyde dehydrogenase-mediated reduction of hexanal without subsequent modification by a second enzyme. In this schematicAtorney docket No. 00058-087W01 the reduction of hexanoic acid to hexanal is depicted, however, any carboxylic acid can be applied.

[0030] FIG. 18 presents the direct reduction of hexanoic acid to hexanal mediated by the NADH-dependent aldehyde dehydrogenase. Aldehyde dehydrogenase sequence used in this figure: P51977 (SEQ ID NO: 2).

[0031] FIG. 19 presents the direct reduction of butyrate to butyraldehyde mediated by an aldehyde dehydrogenase. Aldehyde dehydrogenase sequences included in this figure: A4IT08 (SEQ ID NO:1), UPI001FCBF132 (SEQ ID NO: 105), and UPI0005CD4570 (SEQ ID NO:99).

[0032] FIG. 20 shows the direct reduction of isobutyrate to isobutyraldehyde mediated by an aldehyde dehydrogenase. Aldehyde dehydrogenase sequences included in this figure: A4IT08 (SEQ ID NO:1), UPI001FCBF132 (SEQ ID NO: 105), and UPI0005CD4570 (SEQ ID NO: 99).

[0033] FIG. 21 presents aldehyde dehydrogenase-mediated direct reduction of benzoic acid to benzaldehyde without subsequent modification by a second enzyme.

[0034] FIG. 22 presents an apparatus setup for gas stripping of volatile aldehydes from reaction vessel. Air is continuously flowed through the heated reaction vessel to strip volatile aldehydes produced from the direct reduction of carboxylic acids. Stripped aldehydes are subsequently trapped in cold vessels containing ethyl acetate. The cold ethyl acetate traps are maintained at 0 °C.

[0035] FIG. 23 demonstrates aldehyde dehydrogenase mediated direct reduction of benzoic acid to benzaldehyde without subsequent modification by a second enzy me. Benzaldehyde is removed from the reaction vessel by a continuous gas flow of air, and benzaldehyde stripped from the reaction by the air is sequestered in a subsequent ethyl acetate trap maintained at 0 °C. Aldehyde dehydrogenase sequences included in this figure: A4IT08 (SEQ ID NO: 1) and UPI0005CD4570 (SEQ ID NO: 99).

[0036] FIG. 24 presents a schematic of engineered aldehyde dehydrogenase mediated reduction of hexanoic acid to hexanal. A second enzyme, in this example and alcohol dehydrogenase, subsequently reduces hexanal to hexanol. In this example, hexanoic acid is shown as the carboxylic acid, however, any carboxylic acid can be used.

[0037] FIG. 25 presents an engineered aldehyde dehydrogenase-mediated direct reduction of hexanoic acid to hexanal with subsequent reduction of hexanal to hexanol by anAttorncy docket No. 00058-087W01 alcohol dehydrogenase. ScADH (SEQ ID NO: 172) is the alcohol dehydrogenase in example. Engineered aldehyde dehydrogenases in this figure are based on P51977 (SEQ ID NO:2).

[0038] FIG. 26 provides for the direct reduction of hexanoic acid to hexanal mediated by engineered aldehyde dehydrogenase P05091 (SEQ ID NO:31). Hexanal is subsequently reduced to hexanol by an alcohol dehydrogenase. ScADH (SEQ ID NO: 172) is the alcohol dehydrogenase in example. Engineered aldehyde dehydrogenases in this figure are based on P05091 (SEQ ID NO:31).

[0039] FIG. 27 presents the use of an engineered aldehyde dehydrogenase in the direct reduction of benzoic acid to benzaldehyde. Benzaldehyde is subsequently reduced to benzy l alcohol by an alcohol dehydrogenase. TcADH (SEQ ID NO: 173) is the alcohol dehydrogenase in example. Engineered aldehyde dehydrogenases in this figure are based on A4IT08 (SEQ ID NO: 1) and UPI0005CD4570 (SEQ ID NO: 99).

[0040] FIG. 28 presents the use of an engineered aldehyde dehydrogenase in the direct reduction of cinnamate to cinnamaldehyde. Cinnamaldehyde is subsequently reduced to trans-cinnamyl alcohol by an alcohol dehydrogenase. TcADH (SEQ ID NO: 173) is the alcohol dehydrogenase in example. Engineered aldehyde dehydrogenases in this figure are based on A4IT08 (SEQ ID NO: 1) and UPI0005CD4570 (SEQ ID NO:99).

[0041] FIG. 29 provides for the use of engineered aldehyde dehydrogenase variants built on A4IT08 for the reduction of the aliphatic carboxylic acids hexanoic acid and propionate. Aldehydes produced from the aldehyde dehydrogenase-mediated carboxylic acid reduction are subsequently7reduced to an alcohol using an alcohol dehydrogenase. ScADH (SEQ ID NO: 172) is the alcohol dehydrogenase in example. Engineered aldehyde dehydrogenases in this figure are based on A4IT08 (SEQ ID NO: 1).

[0042] FIG. 30 presents the use of engineered aldehyde dehydrogenase variants built on UPI00005BF137 (SEQ ID NO:39) for the reduction of the hexanoic acid. Aldehydes produced from the aldehyde dehydrogenase-mediated carboxylic acid reduction are subsequently reduced to an alcohol using an alcohol dehy drogenase. ScADH (SEQ ID NO: 172) is the alcohol dehydrogenase in example. Engineered aldehyde dehydrogenases in this figure are based on UPI00005BF137 (SEQ ID NO:39).

[0043] FIG. 31 presents the direct reduction of 4-hydroxybenzoic acid to 4- hydroxybenzaldehyde mediated by a mutant aldehyde dehydrogenase where the wild-ty pe aldehyde dehydrogenase is not able to reduce 4-hydroxybenzoic acid to 4-Atorney docket No. 00058-087W01 hydroxybenzaldehyde. 4-hydroxybenzaldehyde is subsequently reduced to 4- hydroxybenzylalcohol by an alcohol dehydrogenase. TcADH (SEQ ID NO: 173) is the alcohol dehydrogenase in example. Engineered aldehyde dehydrogenases in this figure are based on A4IT08 (SEQ ID NO:1).

[0044] FIG. 32 provides a schematic of an engineered aldehyde dehydrogenase- mediated Direct reduction of hexanoic acid to hexanal. Hexanoic acid reduction is shown as an example, and any carboxylic acid can be exchanged.

[0045] FIG. 33 presents the use of engineered aldehyde dehydrogenase P51977 to perform the direct reduction of hexanoic acid to hexanal without subsequent modification of hexanal by an additional enzyme. ScADH (SEQ ID NO: 172) is the alcohol dehydrogenase in example. Engineered aldehyde dehydrogenases in this figure are based on P51977 (SEQ ID NO:2).

[0046] FIG. 34 shows the use of a chemical reagent to support improved reduction of hexanoic acid to hexanal. 5 mM dithiothreitol was added to the reaction at t = 0. ScADH (SEQ ID NO: 172) is the alcohol dehydrogenase in example. The aldehyde dehydrogenase sequence used in this figure is A4IT08 (SEQ ID NO: 1).

[0047] FIG. 35 presents a schematic showing the use of an aldehyde dehydrogenase to produce aldehydes and alcohols from a carboxylic acid. Aldehydes produced from the direct reduction of a carboxylic acid to an aldehyde are extracted from the aqueous reaction or transformed by an alcohol dehydrogenase to an alcohol. Alcohol produced from this process in also extracted from the aqueous reaction.

[0048] FIG. 36 provides the use of an aldehyde dehydrogenase to produce benzy l alcohol from benzoic acid where an organic solvent is used to continuously extract the product formed from the aldehyde dehydrogenase-mediated reduction of a carboxylic acid. TcADH (SEQ ID NO: 173) is the alcohol dehydrogenase in example. The aldehyde dehydrogenase sequence used in this figure is P94358 (SEQ ID NO: 126).

[0049] FIG. 37 provides a schematic showing the use of an aldehyde dehydrogenase to produce aldehydes a carboxylic acid. Aldehydes produced from the direct reduction of a carboxylic acid to an aldehyde are extracted from the aqueous reaction.

[0050] FIG. 38 presents the use of an aldehyde dehydrogenase to produce benzaldehyde or benzyl alcohol from benzoic acid using an organic solvent overlay to continuously extract the reduced product. In some cases, aldehyde dehydrogenases produceAtorney docket No. 00058-087W01 benzyl alcohol without the inclusion of an alcohol dehydrogenase. In this example, the use of an organic overlay solvent enables aldehyde production. Aldehyde dehydrogenase sequences included in this figure: G2IKV5 (SEQ ID NO: 170), Q84DC3 (SEQ ID NO: 1 19), and P94358 (SEQ ID NO: 126).

[0051] FIG. 39 provides for the use of an aldehyde dehydrogenase to produce cinnamaldehyde from cinnamate using an organic solvent overlay to continuously extract the reduced product. In this example, the use of an organic solvent overlay enables aldehyde production. Aldehyde dehydrogenase sequences included in this figure: G2IKV5 (SEQ ID NO: 170), Q84DC3 (SEQ ID N0: 119), and P94358 (SEQ ID NO:126).

[0052] FIG. 40 presents the use of an aldehyde dehydrogenase to produce hexanal from hexanoic acid using an organic solvent overlay to continuously extract the reduced product. In this example, the organic solvent overlay improves aldehyde production. Aldehyde dehydrogenase sequence included in this figure: P51977 (SEQ ID NO:2).

[0053] FIG. 41 presents another use of an aldehyde dehydrogenase to produce hexanal from hexanoic acid using an organic solvent overlay to continuously extract the reduced product. In this example a 3: 1 organic solvent: aqueous ratio was used. In this example, the use of an organic solvent overlay improves aldehyde production. Aldehyde dehydrogenase sequences included in this figure: A4IT08 (SEQ ID NO: 1), UPI001FCBF132 (SEQ ID NO: 105), and UPI0005CD4570 (SEQ ID NO:99).

[0054] FIG. 42 provides for the use of an aldehyde dehydrogenase to produce propionaldehyde from propionate using an organic solvent overlay to continuously extract the reduced product. In this example, the use of an organic solvent overlay enables the production of an aldehyde, where the same reactions without an overlay were unable to produce an aldehyde. Aldehyde dehydrogenase sequences included in this figure: A4IT08 (SEQ ID NO: 1), UPI001FCBF132 (SEQ ID NO: 105), and UPI0005CD4570 (SEQ ID NO:99).

[0055] FIG. 43 presents the use of an aldehyde dehydrogenase to produce butyraldehyde from butyrate using an organic solvent overlay to continuously extract the reduced product. In this example, the use of an organic solvent overlay improves aldehyde production. Aldehyde dehydrogenase sequences included in this figure: A4IT08 (SEQ ID NO: 1), UPI001FCBF132 (SEQ ID NO: 105), and UPI0005CD4570 (SEQ ID NO:99).Atorney docket No. 00058-087W01

[0056] FIG. 44 provides the use of an aldehyde dehydrogenase to produce isobutyraldehyde from isobutyrate using an organic solvent overlay to continuously extract the reduced product. In this example the use of an organic solvent overlay improves production and improves the product specificity of the aldehyde dehydrogenase system. Aldehyde dehydrogenase sequences included in this figure: A4IT08 (SEQ ID NO: 1), UPI001FCBF132 (SEQ ID NO: 105), and UPI0005CD4570 (SEQ ID NO:99).

[0057] FIG. 45 shows a schematic depicting the use of an aldehyde dehydrogenase to produce an alcohol from a carboxylic acid where the redox cofactor is recycled using a chemical reagent. In this manifestation, dithionite is used as the chemical reagent to reduce oxidized redox cofactor to the reduced form. The recycled redox cofactor is subsequently used by the aldehyde dehydrogenase to perform the direct reduction of carboxylic acids to aldehydes. The aldehydes produced from the aldehyde dehydrogenase-mediated direct reduction of carboxylic acids is subsequently reduced by an alcohol dehydrogenase. In this example, the alcohol dehydrogenase also utilizes the reduced redox cofactor produced by the chemical reagent.

[0058] FIG. 46 presents the use of an aldehyde dehydrogenase to produce propanol from propionate where the redox cofactor is recycled using a chemical reagent. In this example the chemical reagent is dithionite. In this example, an alcohol dehydrogenase is added to reduce the aldehyde produced from the aldehyde dehydrogenase-mediated carboxylic acid reduction to produce and alcohol, and the alcohol dehydrogenase is using the chemical reagent recycled redox cofactor. ScADH (SEQ ID NO: 172) is the alcohol dehydrogenase in example. Aldehyde dehydrogenase sequences included in this figure: A4IT08 (SEQ ID NO: 1).

[0059] FIG. 47 provides a schematic of use of an aldehyde dehydrogenase to reduce a carboxylic acid to an aldehyde where the redox cofactor is recycled using a chemical reagent. In this manifestation, dithionite is used as the chemical reagent to reduce oxidized redox cofactor to the reduced form. The recycled redox cofactor is subsequently used by the aldehyde dehydrogenase to perform the direct reduction of carboxylic acids to aldehydes.

[0060] FIG. 48 presents the use of an aldehyde dehydrogenase to produce hexanal from hexanoic acid where the redox cofactor is recycled using a chemical reagent. In this example, no alcohol dehydrogenase was added in order to preserve aldehyde production. When dithionite is used as the cofactor recycling component, no enzymatic cofactorAtorney docket No. 00058-087W01 reduction is applied. When enzymatic cofactor reduction is applied, no dithionite is added. Aldehyde dehydrogenase sequences included in this figure: P51977 (SEQ ID NO:2).

[0061] FIG. 49 provides a schematic depicting the use of an aldehyde dehydrogenase to perform the direct reduction of a carboxylic acid to an aldehyde using a noncanonical cofactor. In this example the noncanonical redox cofactor is carba-nicotinamide adenine dinucleotide (cNADH).

[0062] FIG. 50 presents the use of an aldehyde dehydrogenase to produce hexanal from hexanoic acid where the redox cofactor is the noncanonical redox cofactor carba- nicotinamide adenine dinucleotide (cNADH). In this example, phosphite dehydrogenase is used to recycle the noncanonical redox cofactor. In this example, an organic solvent overlay of ethoxybenzene was added to continuously extract the aldehyde from the aqueous reaction mixture. Aldehyde dehydrogenase sequences included in this figure: A4IT08 (SEQ ID NO:1) and UPI001FCBF132 (SEQ ID NO:105)

[0063] FIG. 51 shows a schematic depicting the use of an aldehyde dehydrogenase to perform the direct reduction of a carboxylic acid, where the noncanonical redox cofactor nicotinamide mononucleotide (NMNH) is used by the aldehyde dehydrogenase is the redox cofactor used by the aldehyde dehydrogenase.

[0064] FIG. 52 presents the use of an aldehyde dehydrogenase to produce hexanal from hexanoic acid where the redox cofactor is the noncanonical redox cofactor nicotinamide mononucleotide. In this example, an organic solvent overlay of ethoxybenzene was added to continuously extract the aldehyde from the aqueous reaction mixture. In this example, an engineered glucose dehydrogenase is used to recycle the noncanonical redox cofactor. Aldehyde dehydrogenase sequences included in this figure: P15977 (SEQ ID NO:2).DETAILED DESCRIPTION

[0065] 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 dehydrogenase" includes a plurality of such dehydrogenases and reference to "the cofactor" includes reference to one or more cofactors and equivalents thereof known to those skilled in the art, and so forth.

[0066] 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 thisAtorney docket No. 00058-087W01 disclosure belongs. Although many methods and reagents are similar or equivalent to those described herein, the exemplary methods and materials are disclosed herein.

[0067] 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. The publications are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure. 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.

[0068] 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" and "polypeptide" are used interchangeably herein. "Amino acid" 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."

[0069] A particular amino acid sequence of a given protein (re., the polypeptide's "primary7structure," 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 activity7of the polypeptide or proteins encoded by that gene or polynucleotide sequence.

[0070] As used herein, "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 different amino acids that have similar biochemical properties (e.g., charge, hydrophobicity and size). One of skill will recognize that individual mutations, deletions or additions which alter, add or delete a single amino acid or aAtorney docket No. 00058-087W01 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 an engineered polypeptide disclosed herein. For example, the 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 an 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 engineered polypeptide. Moreover, there are many protein modeling programs available, including those specifically recited herein (e.g, Spartan and 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 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 an 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 (e.g. , SEQ ID NO: 33 or SEQ ID NO: 157). Similar reasoning applies for the structural stability' of an engineered polypeptide disclosed herein.

[0071] 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 conserv ative 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 Try ptophan (W).Atorney docket No. 00058-087W01

[0072] 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.

[0073] It is understood that the addition of sequences which do not alter the encoded activity7of 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, z.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.

[0074] 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" (z.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 poly peptides provided herein.

[0075] As used herein, "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. As referred to herein, "sequence similarity" means the extent to which nucleotide orAtorney docket No. 00058-087W01 protein sequences are related. The extent of similarity between two sequences can be based on percent sequence identity and / or conservation. "Sequence identity" herein means the extent to which two nucleotide or amino acid sequences are invariant. "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. 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.

[0076] 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 Gin for Vai, He, Leu or Met), aromatic amino acid for basic or acidic amino acids (e.g. . Phe, Tyr or Trp for Asp, Asn, Glu or Gin) 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 (1), 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), try ptophan (W), and histidine (H).

[0077] "Dehydrogenase." as used herein, refers to 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 an 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.Atorney docket No. 00058-087W01

[0078] A "protein" or "polypeptide", which terms are used interchangeably herein, refers to one or more chains of chemical building blocks called amino acids that are linked together by chemical bonds called peptide bonds. An "enzyme," as used herein, refers to any substance, preferably composed wholly or largely of a protein, that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reactions.

[0079] A protein has "homology" or is "homologous" to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein. Alternatively, a protein has homology to a second protein if the two proteins have "similar" amino acid sequences. (Thus, the term "homologous proteins" is defined to mean that the two proteins have similar amino acid sequences).

[0080] As used herein, two proteins (or a region of the proteins) are substantially homologous when the amino acid sequences have at least about 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g. , gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In one embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, typically at least 40%, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity " is equivalent to amino acid or nucleic acid "homology"). The percent identity between the tw o sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap. which need to be introduced for optimal alignment of the two sequences.

[0081] When "homologous" is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similarAttorney docket No. 00058-087W01 chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (see. e.g., Pearson et al., 1994, hereby incorporated herein by reference).

[0082] In some instances, “isozy mes” can be used that carry' out the same functional conversion / reaction, but which are so dissimilar in structure that they are ty pically determined to not be “homologous”.

[0083] A "native" or "wild-type" protein, enzyme, polynucleotide, gene, or cell, means a protein, enzyme, polynucleotide, gene, or cell that occurs in nature.

[0084] A "parent" protein, enzyme, polynucleotide, gene, or cell is any protein, enzyme, polynucleotide, gene, or cell, from which any other protein, enzy me, 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, respectively. A parent polynucleotide or gene encodes for a parent protein or enzyme. In a certain embodiment, a “parent” protein, enzy me, polynucleotide, gene, or cell, is a wild ty pe protein, enzy me, polynucleotide, gene, or cell.

[0085] A "parental microorganism" refers to a cell used to generate a recombinant microorganism. The term "parental microorganism" describes, in one embodiment, a cell that occurs in nature, i.e. , a "wild-type" cell that has not been genetically modified. The term "parental microorganism" further describes a cell that sen es as the “parent” for further engineering. In this latter embodiment, the cell may have been genetically engineered, but serves as a source for further genetic engineering. Culture conditions suitable for the growth and maintenance of a recombinant microorganism are known (see, e.g., "Culture of Animal Cells-A Manual of Basic Technique" by Freshney, Wiley-Liss, N.Y. (1994), Third Edition). The skilled artisan will recognize that such conditions can be modified to accommodate the requirements of each microorganism.

[0086] For example, a wild-type microorganism can be genetically modified to express or over express a polypeptide that encodes an aldehyde dehydrogenase that directly reduces carboxylic acid containing compounds to aldehyde containing compounds without ATP or CoA-based activation. This microorganism can act as a parental microorganism inAtorney docket No. 00058-087W01 the generation of a microorganism modified to express or over-express a second or third polypeptide, such as a polypeptide that encodes an enzyme capable of converting an aldehyde group into a non-aldehyde group, or a polypeptide that encodes an enz me that reduces nicotinamide-adenine dinucleotide (NAD+) to NADH via the hydride donor compound. As used herein, “express” or “over express” refers to the phenotypic expression of a desired gene product, such a polypeptide disclosed herein. In one embodiment, a naturally occurring gene in the organism can be engineered such that it is linked to a heterologous promoter or regulatory domain, wherein the regulatory domain causes expression of the gene, thereby modifying its normal expression relative to the wild-type organism. Alternatively, the organism can be engineered to remove or reduce a repressor function on the gene, thereby modifying its expression. In yet another embodiment, a cassette comprising the gene sequence operably linked to a desired expression control / regulatory element is engineered into the microorganism.

[0087] Accordingly, a parental microorganism functions as a reference cell for successive genetic modification events. Each modification event can be accomplished by introducing one or more nucleic acid molecules into the reference cell. The introduction facilitates the expression or over-expression of one or more target enzymes or the reduction or elimination of one or more target enzy mes. It is understood that the term "facilitates" encompasses the activation of endogenous polynucleotides encoding a target enzyme through genetic modification of e.g.. a promoter sequence in a parental microorganism. It is further understood that the term "facilitates" encompasses the introduction of exogenous polynucleotides encoding a target enz me into a parental microorganism.

[0088] Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of codons differing in their nucleotide sequences can be used to encode a given amino acid. The disclosure includes polynucleotides of any sequence that encode a polypeptide of the disclosure that comprises the same amino acid sequence of said polypeptide.

[0089] The disclosure provides for polynucleotides in the form of recombinant DNA expression vectors or plasmids, as described in more detail elsewhere herein, that encode one or more the polypeptides disclosed herein. Generally, such vectors can either replicate in the cytoplasm of the host microorganism or integrate into the chromosomal DNA of the host microorganism. In either case, the vector can be a stable vector (z.e., the vector remainsAtorney docket No. 00058-087W01 present over many cell divisions, even if only with selective pressure) or a transient vector (z.e., the vector is gradually lost by host microorganisms with increasing numbers of cell divisions). The disclosure provides DNA molecules in isolated (z.e., not pure, but existing in a preparation in an abundance and / or concentration not found in nature) and purified (z.e., substantially free of contaminating materials or substantially free of materials with which the corresponding DNA would be found in nature) form.

[0090] The disclosure provides a number of polypeptide sequences in SEQ ID NOs: l- 175, which can be used to design, synthesize and / or isolate polynucleotide sequences using the degeneracy of the genetic code or using publicly available databases to search for the coding sequences.

[0091] As will be understood by those of skill in the art, it can be advantageous to modify a coding sequence to enhance its expression in a particular host. The genetic code is redundant with 64 possible codons, but most organisms ty pically use a subset of these codons. The codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons. Codons can be substituted to reflect the preferred codon usage of the host, a process sometimes called "codon optimization" or "controlling for species codon bias."

[0092] Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (see also, Murray et al. (1989) Nucl. Acids Res. 17:477-508) can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S', cerevisiae and mammals are UAA and UGA. respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E. coli commonly use UAA as the stop codon (Dalphin et al. (1996) Nucl. Acids Res. 24: 216-218). Methodology7for optimizing a nucleotide sequence for expression in a plant is provided, for example, in U.S. Pat. No. 6,015,891, and the references cited therein.

[0093] As used herein "hydride donor compound" refers to a compound that can provide hydrogen atoms in a chemical reaction.

[0094] As used herein, “Isolated polypeptide” refers to a polypeptide which is separated from other contaminants that naturally accompany it, e.g., protein, lipids, andAtorney docket No. 00058-087W01 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). It is also understood that an isolated polynucleotide molecule encoding a polypeptide homologous to the enzymes described herein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence encoding the particular polypeptide, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into the polynucleotide by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. In contrast to those positions where it may be desirable to make a nonconservative amino acid substitution, in some positions it is preferable to make conservative amino acid substitutions.

[0095] As used herein, “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.

[0096] As used herein, “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 comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences and may further comprise a sequence that is divergent between the two sequences, sequenceAtorney docket No. 00058-087W01 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.

[0097] As used herein, "sequence identity" means that two polypeptide sequences are substantially identical (z.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 (z.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identify (or percentage of sequence similarity). Regarding polynucleotide sequences, the terms sequence identity and sequence similarity have comparable meaning as described for protein sequences, with the term "percentage of sequence identify " 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. In a particular embodiment, a polypeptide sequence will have about 80%, about 85% or more, about 90% or more, about 95% or more, and about 98% or more, sequence identity to another polypeptide sequence.

[0098] As applied to polypeptides, the term substantial identify 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 identify 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.Atorney docket No. 00058-087W01

[0099] 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 identity7or percent similarity7are optimized, BL50 Matrix 15: -5, k-tuple=2: joining penalty=40, optimization=28; gap penalty -12, gap length penalty=-2; and width=16.

[0100] 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 identity7or percent sequence similarity. It also plots a tree or dendrogram 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 a 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).

[0101] 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 aminoAtorney docket No. 00058-087W01 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).

[0102] Additional favorable polypeptides sequences for engineering can be identified by using sequence alignment. For example, sequences that have sequence alignment of at least 95% to a sequence of SEQ ID:1 to SEQ ID: 175 would provide for such sequences. The modified polypeptide may then be assayed for their efficiency in directly reducing carboxylic acids to aldehydes without ATP or CoA-based activation.

[0103] A "vector" generally refers to a polynucleotide that can be propagated and / or transferred between organisms, cells, or cellular components. Vectors include viruses, bacteriophage, pro-viruses, plasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes). BACs (bacterial artificial chromosomes), and PLACs (plant artificial chromosomes), and the like, that are "episomes," that is, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide- conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that are not episomal in nature, or it can be an organism which comprises one or more of the above polynucleotide constructs such as an agrobacterium or a bacterium.

[0104] The various components of an expression vector can vary widely, depending on the intended use of the vector and the host cell(s) in which the vector is intended to replicate or drive expression. Expression vector components suitable for the expression of genes and maintenance of vectors in E. coll, yeast, Streptomyces, and other commonly used cells are widely known and commercially available. For example, suitable promoters for inclusion in the expression vectors of the disclosure include those that function in eukaryotic or prokaryotic host microorganisms. Promoters can comprise regulatory sequences that allow for regulation of expression relative to the growth of the host microorganism or that cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus. For E. coli and certain other bacterial host cells, promoters derived from genes for biosynthetic enzymes, antibiotic-resistance conferring enzymes, and phage proteins can be used and include, for example, the galactose, lactose (lac), maltose, tryptophan (trp), betalactamase (bla), bacteriophage lambda PL, and T5 promoters. In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No. 4,551,433, which is incorporated herein byAtorney docket No. 00058-087W01 reference in its entirety), can also be used. For E. coll expression vectors, it is useful to include an E. coli origin of replication, such as from pUC, plP, pl, and pBR.

[0105] Thus, recombinant expression vectors contain at least one expression system, which, in turn, is composed of at least a portion of a gene coding sequences operably linked to a promoter and optionally termination sequences that operate to effect expression of the coding sequence in compatible host cells. The host cells are modified by transformation with the recombinant DNA expression vectors of the disclosure to contain the expression system sequences either as extrachromosomal elements or integrated into the chromosome.

[0106] In microorganism-based fermentation systems, carboxylic acids like aliphatic acids (e.g, acetic acid, butyric acid), hydroxy-acids (e.g , lactic acid), keto-acids (e.g, 2-keto- butyrate), and dicarboxylic acids (e.g.. succinic acid, adipic acid) are produced as stable metabolic intermediates and waste products. Due to the thermodynamic inertness of these compounds, chemical upgrading of these molecules has proven difficult, and they are regularly discarded or sold as low-value commodity chemicals. Nature has adopted pathway s to functionalize and upgrade these carbon-rich wastes to aldehydes. However, these pathways all use expensive sacrificial reagents like adenosine triphosphate (ATP), coenzyme-A (CoA), and ferredoxin. Alternatively, decarboxylation of keto-acids can produce aldehydes, but key carbon molecules are lost to carbon dioxide (CO2), leading to a critical loss in carbon yield.

[0107] This necessitates a means to upgrade carboxylic acids to aldehydes without the use of ATP, CoA, ferredoxin, or decarboxylation. Aldehyde dehydrogenases are enzymes which naturally catalyze the oxidation of aldehydes to their respective carboxylic acids using the nicotinamide adenine dinucleotide (NAD+) or nicotinamide adenine dinucleotide phosphate (NADP+) as the only co-substrate. However, since their initial discovery and characterization in the early 1900’s, they have been described and accepted by the scientific populous as irreversible enzymes which cannot perform the direct reduction of carboxylic acids to aldehydes using reduced NAD+(NADH) or reduced NADP+(NADPH). Provided herein are processes developed for the unnatural application of aldehyde dehydrogenases to perform the direct reduction of carboxylic acids to aldehydes, platforms for the usage of aldehyde dehydrogenases to produce chemical products, and the use of engineered aldehyde dehydrogenases which exhibit improved carboxylic acid reduction activity.

[0108] The major barrier to using ALDHs to directly reduce carboxylic acids to aldehydes is that this reaction is highly thermodynamically unfavorable, as such this reactionAtorney docket No. 00058-087W01 is not performed in the natural environment. For example, the change in Gibb’s free energy' of the reaction of the aliphatic carboxylic acid butyrate is ArG’ = +50 kJ / mol at pH 7.5 (butyrate + NADH => butanal + NAD++ H2O). Although previously thought to be impossible, ALDHs may be able to facilitate this reaction when subjected to unnatural reaction conditions.

[0109] It was postulated herein that direct reduction of carboxylic acids to aldehydes was possible but any observable activity by ALDHs would be very low. Therefore, an enzymatic screening and high value chemical production platform was developed (see FIG. 1). In this system, aldehyde dehydrogenase was paired with an alcohol dehydrogenase from Saccharomyces cerevisiae to reduce carboxylic acids to aldehydes via aldehyde dehydrogenase and NADH, and aldehydes were reduced to alcohols via alcohol dehydrogenase and NADH. Oxidized NADH (NAD+) was regenerated to NADH via phosphite dehydrogenase from Pseudomonas stutzeri (SEQ ID NO: 171) or glucose dehydrogenase from Bacillus subtilis (SEQ ID NO: 175). Disclosed herein are exemplary methods where variations on this system are employed. In these variations, the second enzyme, the alcohol dehydrogenase listed above, is exchanged for a different enzyme to provide alternative modification to the aldehyde produced from carboxylic acid reduction. In other examples, the second enzyme is removed, and aldehydes produced from carboxylic acid reduction are unmodified or separated from the reaction system by gas stripping. Using the ALDH screening platform, 170 ALDH scaffolds were identified and characterized to exhibit the ability to direct reduce the carboxylic acids to the respective aldehydes.

[0110] The disclosure provides compositions comprising a recombinantly engineered polypeptide disclosed herein that has aldehyde dehydrogenase activity, wherein the wild type or parent sequence has been changed to contain one or more amino acid substitutions as disclosed herein (e.g, see FIGs. 25-31) that increases the polypeptides activity for converting a carboxylic acid containing compound to an aldehyde containing compound.

[0111] Synthetic biochemistry, in which complex biochemical conversions are performed cell-free using a mixture of enzymes, affords potential advantages over traditional metabolic engineering including: a higher level of flexibility in pathway design; greater control over component optimization; more rapid design-build-test cycles; and freedom from cell toxicity of intermediates or products.Atorney docket No. 00058-087W01

[0112] The disclosure provides for a cell-free system or whole-cell biomanufacturing system to facilitate the biotransformation of a carboxylic acid containing substrate into a desired product, comprising: a first polypeptide encoding an aldehyde dehydrogenase that directly reduces carboxylic acids to aldehydes without adenosine triphosphate (ATP) or coenzyme A (CoA)-based activation, wherein the aldehyde dehydrogenase can use reduced nicotinamide-adenine dinucleotide (NADH) as a cofactor; and a first hydride donor compound; optionally, a second polypeptide that encodes an enzyme capable of converting an aldehyde group into a non-aldehyde group; optionally, a second hydride donor compound and a third polypeptide that encodes an enzyme that reduces the first hydride donor compound using the second hydride donor compound. In another embodiment, the first polypeptide comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to a sequence presented in SEQ ID NO: 1 to 170. In yet another embodiment, the first polypeptide comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, is at least 95%, at least 97%, at least 98%, or at least 99% identical to a sequence presented in SEQ ID NO: 1, SEQ ID NO:39, SEQ ID NO:2, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:99, SEQ ID NO:105, SEQ ID NO: 119, SEQ ID NO: 126, and SEQ ID NO:170. In a further embodiment, the first polypeptide is engineered to contain 1 to 10 amino acid substitutions in comparison to a sequence presented in SEQ ID NO: 1 to 170, wherein the amino acid substitution(s) improve the aldehyde dehydrogenase activity for directly reducing carboxylic acids to aldehydes. In yet a further embodiment, the first polypeptide has the sequence of SEQ ID NO:39 with at least the amino acid substitution of Y402F. In a particular embodiment, the first polypeptide has the sequence of SEQ ID NO:2 with at least an amino acid substitution selected from Q350N, Q350R, and Q350A. In another embodiment, the first polypeptide has the sequence of SEQ ID NO:31 with one or more amino acid substitutions selected from (a), (b), (c), (d), (e), and / or (f): (a) W185S, W185D, W185H, W185N, W185K, or W185F; (b) N186D; (c) E285D; (d) F288A; (e) Q366I, Q366A, Q366L, Q366M, or Q366N; and / or (!) F418A or F418H. In a further embodiment, the first polypeptide has the sequence of SEQ ID NO: 31 with at least an amino acid substitution selected from W 185N, W185K, and W185F. In another embodiment, the first polypeptide has the sequence of SEQ ID NO:1 with one or more amino acid substitutions selected from (a), (b) and / or (c): (a) W164F; (b) T302S; and / or (c) W173M, W173F, and W173Y. In another embodiment, theAtorney docket No. 00058-087W01 first polypeptide has the sequence of SEQ ID NO:99 with at least an amino acid substitution selected from W173M, W173F, and W173Y. In yet another embodiment the cell-free system or whole-cell biomanufacturing system comprises the second polypeptide that encodes an enzyme capable of converting an aldehyde group into anon-aldehyde group. In a certain embodiment, the second polypeptide encodes an enzyme capable of converting an aldehyde group into a non-aldehyde group, utilizes the first hydride donor compound as a cofactor. In a further embodiment, the second polypeptide encodes an alcohol dehydrogenase, or an acetolactate synthase. In another embodiment, the second polypeptide encodes an alcohol dehydrogenase that has a sequence that is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:172 or SEQ ID NO: 173. In yet another embodiment, the second polypeptide encodes an acetolactate synthase that has a sequence that is at least 95%. at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 174. In a further embodiment, the cell-free system or whole-cell biomanufacturing system comprises the third polypeptide and the second hydride donor compound. In yet a further embodiment, the third polypeptide encodes a phosphite dehydrogenase that has a sequence that is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 171, and wherein the second hydride donor compound is phosphite. In a certain embodiment, the third polypeptide encodes a glucose dehydrogenase that has a sequence that is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 175, and wherein the second hydride donor compound is glucose. In another embodiment, the cell-free system or whole-cell biomanufacturing system further comprises 1,4-dithiothreitol. In yet another embodiment, the system is a cell-free system, and the cell-free system utilizes polypeptide(s) that have been isolated and purified. In a further embodiment, the cell-free system further comprises an organic solvent overlay that supports production of an aldehyde by the first polypeptide and / or a non-aldehyde product produced by the second polypeptide, and wherein the polypeptide(s) and hydride donor(s) are in an aqueous solution. In another embodiment, the organic overly comprises a solvent selected from anisole, hexane, isooctane, toluene, dodecane, oleyl alcohol, and ethoxybenzene. In yet another embodiment, the organic solvent overlay is used at a ratio of 1 : 10 to 10: 1 to the aqueous solution. In a further embodiment, the organic solvent overlay is used at a ratio of 1 : 1 to 10: 1 to the aqueous solution. In yet a further embodiment, the cell-free system or whole-cell biomanufacturing system further comprises an apparatus to isolate a desired product that is an aldehyde, wherein the apparatusAtorney docket No. 00058-087W01 comprises: a reactor that can be heated which comprises the product aldehyde, wherein the reactor is in fluid connection to one or more cooled condensers filled with a solvent, wherein in heating the reactor causes the product aldehyde to go in the gaseous phase; a gas supply that is in fluid connection with the reactor, that is configured to introduce positive gas flow into the reactor, wherein the gas introduced into the reactor flows from the reactor to the one or more cooled condensers; and one of more cooled condensers filled with a solvent that are configured to cool the gaseous aldehyde into a liquid phase so that it is trapped by the solvent, wherein the aldehyde can then be isolated from the solvent. In a particular embodiment, the system is a whole-cell biomanufacturing system wherein vectors are used to express the polypeptide(s) in a microorganism. In another embodiment, the microorganism is selected from a bacterium or yeast. In yet another embodiment, the first hydride donor compound is selected from reduced nicotinamide-adenine dinucleotide (NADH), reduced nicotinamide-adenine dinucleotide phosphate (NADPH), carba-nicotinamide adenine dinucleotide (cNADH), reduced nicotinamide mononucleotide (NMNH), reduced 3- carbamoyl- 1 -(4-carboxy benzyl) pyridin-l-ium (BANAH), reduced I -benzylnicotinamide (BNAH), reduced 3-carbamoyl-l-(3-phenylpropyl)pyridin-l-ium (3PNAH), and reduced 3- carbamoyl-l-(4-methoxybenzyl)pyridin-l-ium (BNA OMeH). In another embodiment, the first hydride donor compound is cNADH or NMNH.

[0113] The disclosure also provides for methods or processes to facilitate the biotransformation of a substrate into a desired product, comprising a recombinantly engineered polypeptide of the disclosure having aldehyde dehydrogenase activity that can directly reduce carboxylic acids to aldehydes without ATP or CoA-based activation. In a particular embodiment, the disclosure also provides a method to convert a carboxylic acid containing substrate to an aldehyde containing product, the method comprising: contacting the carboxylic acid containing substrate with a first polypeptide that encodes an aldehyde dehydrogenase that directly reduces carboxylic acids to aldehydes without adenosine triphosphate (ATP) or coenzyme A (CoA)-based activation in the presence of a first hydride donor compound in an aqueous solution, optionally, providing a second hydride donor compound and a second polypeptide that encodes an enzyme that reduces the first hydride donor compound using the second hydride donor compound, wherein the aldehyde dehydrogenase can use reduced first hydride donor compound as a cofactor, and wherein the aldehyde dehydrogenase converts the carboxylic acid containing substrate to an aldehydeAtorney docket No. 00058-087W01 containing product. In another embodiment, the carboxylic acid containing substrate is selected from a linear aliphatic carboxylic acid, a branched aliphatic carboxylic acid, an aromatic carboxylic acid, a diacid, a hydroxyacid and formic acid. In yet another embodiment, the linear aliphatic carboxylic acid is selected from acetic acid, propanoic acid, buty ric acid, hexanoic acid, and nonanoic acid. In a certain embodiment, the branched aliphatic carboxylic acid is selected from isobutyric acid, and isopentanoic acid. In another embodiment, the aromatic carboxylic acid is selected from benzoic acid, 2-furoic acid, cinnamic acid and vanillic acid. In yet another embodiment, the diacid is selected from succinic acid, and adipic acid. In a further embodiment, the hydroxyacid is selected from 4- hydroxybutyric acid, 3-hydroxybutyric acid, lactic acid, and 2-hydroxylpropanoic acid. In a certain embodiment, the first polypeptide comprises a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98%, or at least 99% identical to a sequence presented in SEQ ID NO: 1 to 170. In another embodiment, the first polypeptide comprises a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98%, or at least 99% identical to a sequence presented in SEQ ID NO: 1, SEQ ID NO:39, SEQ ID NO:2, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:99, SEQ ID NO:105, SEQ ID NO: 119, SEQ ID NO:126, and SEQ ID NO: 170. In yet another embodiment, the first polypeptide is engineered to contain 1 to 10 amino acid substitutions in comparison to a sequence presented in SEQ ID NO: 1 to 170. wherein the amino acid substitution(s) improve the aldehyde dehydrogenase activity for directly reducing carboxylic acids to aldehydes. In yet a further embodiment, the first polypeptide has the sequence of SEQ ID NO:39 with at least the amino acid substitution of Y402F. In a particular embodiment, the first polypeptide has the sequence of SEQ ID NO:2 with at least an amino acid substitution selected from Q350N, Q350R, and Q350A. In another embodiment, the first polypeptide has the sequence of SEQ ID NO:31 with one or more amino acid substitutions selected from (a), (b), (c), (d), (e), and / or (f): (a) W185S, W185D, W185H, W185N, W185K, or W185F; (b) N186D; (c) E285D; (d) F288A; (e) Q366I, Q366A, Q366L, Q366M, or Q366N; and / or (f) F418A or F418H. In a further embodiment, the first polypeptide has the sequence of SEQ ID NO:31 with at least an amino acid substitution selected from W185N, W 185K, and W 185F. In another embodiment, the first polypeptide has the sequence of SEQ ID NO: 1 with one or more amino acid substitutions selected from (a), (b) and / or (c): (a) W164F; (b) T302S; and / or (c) W173M, W173F. and W173Y. In another embodiment, the first polypeptide has theAtorney docket No. 00058-087W01 sequence of SEQ ID NO: 99 with at least an amino acid substitution selected from W173M, W173F, and W173Y. In a certain embodiment, the method further comprises isolating the aldehydes during the reaction process. In another embodiment, the method further comprises overlaying an organic solvent on top of the aqueous solution to allow for the continuous extraction of the aldehydes. In yet another embodiment, the organic solvent is selected from anisole, hexane, isooctane, toluene, dodecane, oleyl alcohol, and ethoxybenzene. In a certain embodiment, the organic solvent is used at a ratio of 1 : 10 to 10: 1 to the aqueous solution. In another embodiment, the organic solvent is used at a ratio of 1: 1 to 10: 1 to the aqueous solution. In a further embodiment, the method further comprises isolating the aldehyde containing products by using an apparatus, the apparatus comprising: a reactor that can be heated which comprises the product aldehyde, wherein the reactor is in fluid connection to one or more cooled condensers filled with a solvent, wherein in heating the reactor causes the product aldehyde to go in the gaseous phase; a gas supply that is in fluid connection with the reactor, that is configured to introduce positive gas flow into the reactor, wherein the gas introduced into the reactor flows from the reactor to the one or more cooled condensers; and one of more cooled condensers filled with a solvent that are configured to cool the gaseous aldehyde into a liquid phase so that it is trapped by the solvent, wherein the aldehyde can then be isolated from the solvent. In a particular embodiment, the method comprises the second hydride donor compound and the second polypeptide that encodes an enzyme that reduces the first hydride donor compound using the second hydride donor compound. In another embodiment, the second polypeptide encodes a phosphite dehydrogenase that has a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 171, and wherein the second hydride donor compound is phosphite. In yet another embodiment, the second polypeptide encodes a glucose dehydrogenase that has a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:175, and wherein the second hydride donor compound is glucose. In a further embodiment, the method further comprises converting the aldehyde containing product to non-aldehyde containing product, comprising: contacting the aldehyde containing product with a third polypeptide that that encodes an enzyme capable of converting an aldehyde group into a non-aldehyde group. In a certain embodiment, the third polypeptide that encodes an enzy me capable of converting an aldehyde group into a non-aldehyde group, utilizes the first hydride compound as a coenzymeAtorney docket No. 00058-087W01 or cofactor. In another embodiment, the third polypeptide encodes an alcohol dehydrogenase, or an acetolactate synthase. In yet another embodiment, the third polypeptide encodes an alcohol dehydrogenase that has a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 172 or SEQ ID NO: 173. In a further embodiment, the third polypeptide encodes an acetolactate synthase that has a sequence that at least 75%. at least 80%. at least 85%. is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 174. In yet a further embodiment, the first hydride donor compound is selected from reduced nicotinamideadenine dinucleotide (NADH), reduced nicotinamide-adenine dinucleotide phosphate (NADPH), carba-nicotinamide adenine dinucleotide (cNADH), reduced nicotinamide mononucleotide (NMNH). reduced 3 -carbamoyl- l-(4-carboxy benzyl) pyridin-l-ium (BANAH), reduced 1 -benzylnicotinamide (BNAH), reduced 3-carbamoyl-l-(3- phenylpropyl)pyridin-l-ium (3PNAH), and reduced 3-carbamoyl-l-(4- methoxybenzyl)pyridin-l-ium (BNA OMeH). In another embodiment, the first hydride donor compound is cNADH or NMNH.

[0114] For use in the applications described herein, kits and articles of manufacture are also described herein. Such kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, synnges, and test tubes. The containers can be formed from a variety of materials such as glass or plastic. The kits can include the components for the extracting of products (e.g., aldehydes and alcohols) disclosed herein, including the organic solvents used as an overlay provided herein and / or one or more components of the reaction system presented in FIG. 22.

[0115] For example, the container(s) can comprise one or more polypeptides described herein, optionally in a composition or in combination with another agent as disclosed herein. The container(s) optionally have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits optionally comprise an identifying description or label or instructions relating to its use in the methods described herein.

[0116] A kit will typically comprise one or more additional containers, each with one or more of various materials (such as reagents, optionally in concentrated form, and / orAtorney docket No. 00058-087W01 devices) desirable from a commercial and user standpoint for use of polypeptides described herein, including the organic solvents used as an overlay as described herein. Non-limiting examples of such materials include, but are not limited to, buffers, diluents, filters, needles, syringes; carrier, package, container, vial and / or tube labels listing contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

[0117] A label can be on or associated with the container. A label can be on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself, a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g. , as a package insert. A label can be used to indicate that the contents are to be used for a specific therapeutic application. The label can also indicate directions for use of the contents, such as in the methods described herein. These other therapeutic agents may be used, for example, in the amounts indicated in the Physicians' Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art.

[0118] The disclosure further provides that the compositions, systems and methods described herein can be further defined by the following aspects (aspects 1 to 61):1. A cell-free system or whole-cell biomanufacturing system to facilitate the biotransformation of a carboxylic acid containing substrate into a desired product, comprising: a first polypeptide encoding an aldehyde dehydrogenase that directly reduces carboxylic acids to aldehydes without adenosine triphosphate (ATP) or coenzy me A (CoA)- based activation, wherein the aldehyde dehydrogenase can use reduced nicotinamide-adenine dinucleotide (NADH) as a cofactor; and a first hydride donor compound; optionally, a second polypeptide that encodes an enzyme capable of converting an aldehyde group into a non-aldehyde group; optionally, a second hydride donor compound and a third polypeptide that encodes an enzyme that reduces the first hydride donor compound using the second hydride donor compound.2. The cell-free system or whole-cell biomanufacturing system of aspect 1, wherein the first polypeptide comprises a sequence that is at least 75%, at least 80%, at leastAtorney docket No. 00058-087W0185%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to a sequence presented in SEQ ID NO: 1 to 170.3. The cell-free system or whole-cell biomanufacturing system of aspect 1 or aspect 2, wherein the first polypeptide comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, is at least 95%, at least 97%, at least 98%, or at least 99% identical to a sequence presented in SEQ ID NO: 1, SEQ ID NO:39. SEQ ID NO:2. SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:99, SEQ ID NO: 105, SEQ ID NO: 119. SEQ IDNO: 126, and SEQ ID NO: 170.4. The cell-free system or whole-cell biomanufacturing system of any one of aspects 1 to 3, wherein the first polypeptide is engineered to contain 1 to 10 amino acid substitutions in comparison to a sequence presented in SEQ ID NO: 1 to 170, wherein the amino acid substitution(s) improve the aldehyde dehydrogenase activity for directly reducing carboxylic acids to aldehydes.5. The cell-free system or whole-cell biomanufacturing system of any one of aspects 1 to 4, wherein the first polypeptide has the sequence of SEQ ID NO:39 with at least the amino acid substitution of Y402F.6. The cell-free system or whole-cell biomanufacturing system of any one of aspects 1 to 4, wherein the first polypeptide has the sequence of SEQ ID NO:2 with at least an amino acid substitution selected from Q350N, Q350R, and Q350A.7. The cell-free system or whole-cell biomanufacturing system of any one of aspects 1 to 4, wherein the first polypeptide has the sequence of SEQ ID NO:31 with one or more amino acid substitutions selected from (a), (b), (c), (d), (e), and / or (f):(a) W185S, W185D, W185H, W185N, W185K, or W185F;(b) N186D;(c) E285D;(d) F288A;(e) Q366I, Q366A, Q366L, Q366M. or Q366N; and / or(I) F418A or F418H.8. The cell-free system or whole-cell biomanufacturing system of aspect 7, wherein the first polypeptide has the sequence of SEQ ID NO:31 with at least an amino acid substitution selected from W185N, W185K, and W185F.9. The cell-free system or whole-cell biomanufacturing system of any one ofAtorney docket No. 00058-087W01 aspects 1 to 4, wherein the first polypeptide has the sequence of SEQ ID NO: 1 with one or more amino acid substitutions selected from (a), (b) and / or (c):(a) W164F;(b) T302S; and / or(c) W173M, W173F, and W173Y.10. The cell-free system or whole-cell biomanufacturing system of any one of aspects 1 to 4, wherein the first polypeptide has the sequence of SEQ ID NO:99 with at least an amino acid substitution selected from W173M, W173F, and W173Y.11. The cell-free system or whole-cell biomanufacturing system of any one of aspects 1 to 10, wherein the cell-free system or whole-cell biomanufacturing system comprises the second polypeptide that encodes an enzyme capable of converting an aldehyde group into a non-aldehyde group.12. The cell-free system or whole-cell biomanufacturing system of aspect 11, wherein the second polypeptide encodes an enzy me capable of converting an aldehyde group into a non-aldehyde group, utilizes the first hydride donor compound as a cofactor.13. The cell-free system or whole-cell biomanufacturing system of aspect 12, wherein the second polypeptide encodes an alcohol dehydrogenase, or an acetolactate synthase.14. The cell-free system or whole-cell biomanufacturing system of aspect 13, wherein the second polypeptide encodes an alcohol dehydrogenase that has a sequence that is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 172 or SEQ ID NO:173.15. The cell-free system or whole-cell biomanufacturing system of aspect 13, wherein the second polypeptide encodes an acetolactate synthase that has a sequence that is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 174.16. The cell-free system or whole-cell biomanufacturing system of of any one of aspects 1 to 15, wherein the cell-free system or whole-cell biomanufacturing system comprises the third polypeptide and the second hydride donor compound.17. The cell-free system or whole-cell biomanufacturing system of aspect 16, wherein the third polypeptide encodes a phosphite dehydrogenase that has a sequence that is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 171, and wherein the second hydride donor compound is phosphite.Atorney docket No. 00058-087W0118. The cell-free system or whole-cell biomanufacturing system of aspect 16, wherein the third polypeptide encodes a glucose dehydrogenase that has a sequence that is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 175, and wherein the second hydride donor compound is glucose.19. The cell-free system or whole-cell biomanufacturing system of of any one of aspects 1 to 18, wherein the cell-free system or whole-cell biomanufacturing system further comprises A-dithiothreitol.20. The cell-free system or whole-cell biomanufacturing system of any one of aspects 1 to 19, wherein the system is a cell-free system, and the cell-free system utilizes polypeptide(s) that have been isolated and purified.21. The cell-free system or whole-cell biomanufacturing system of any one of aspects 1 to 20, wherein the cell-free system further comprises an organic solvent overlay that supports production of an aldehyde by the first polypeptide and / or a non-aldehyde product produced by the second polypeptide, and wherein the polypeptide(s) and hydride donor(s) are in an aqueous solution.22. The cell-free system or whole-cell biomanufacturing system of aspect 21, wherein the organic overly comprises a solvent selected from anisole, hexane, isooctane, toluene, dodecane, oleyl alcohol, and ethoxybenzene.23. The cell-free system or whole-cell biomanufacturing system of aspect 21 or aspect 22, wherein the organic solvent overlay is used at a ratio of 1: 10 to 10: 1 to the aqueous solution.24. The cell-free system or whole-cell biomanufacturing system of any one of aspects 21 to 23, wherein the organic solvent overlay is used at a ratio of 1 : 1 to 10: 1 to the aqueous solution.25. The cell-free system or whole-cell biomanufacturing system of any one of aspects 1 to 25, wherein the cell-free system or whole-cell biomanufacturing system further comprises an apparatus to isolate a desired product that is an aldehyde, wherein the apparatus comprises: a reactor that can be heated which comprises the product aldehyde, wherein the reactor is in fluid connection to one or more cooled condensers filled with a solvent, wherein in heating the reactor causes the product aldehyde to go in the gaseous phase; a gas supply that is in fluid connection with the reactor, that is configured to introduceAtorney docket No. 00058-087W01 positive gas flow into the reactor, wherein the gas introduced into the reactor flows from the reactor to the one or more cooled condensers; and one of more cooled condensers filled with a solvent that are configured to cool the gaseous aldehyde into a liquid phase so that it is trapped by the solvent, wherein the aldehyde can then be isolated from the solvent.26. The cell-free system or whole-cell biomanufacturing system of any one of aspects 1 to 19, wherein the system is a whole-cell biomanufacturing system wherein vectors are used to express the polypeptide(s) in a microorganism.27. The cell-free system or whole-cell biomanufacturing system of aspect 26, wherein the microorganism is selected from a bacterium or yeast.28. The cell-free system or whole-cell biomanufacturing system of any one of aspects 1 to 27, wherein the first hydride donor compound is selected from reduced nicotinamide-adenine dinucleotide (NADH), reduced nicotinamide-adenine dinucleotide phosphate (NADPH), carba-nicotinamide adenine dinucleotide (cNADH), reduced nicotinamide mononucleotide (NMNH). reduced 3 -carbamoyl- 1 -(4-carboxybenzyl) pyridin- 1-ium (BANAH), reduced 1 -benzylnicotinamide (BNAH), reduced 3-carbamoyl-l-(3- phenylpropyl)pyridin-l-ium (3PNAH), and reduced 3-carbamoyl-l-(4- methoxybenzyl)py ridin- 1 -ium (BNA OMeH).29. The cell-free system or whole-cell biomanufacturing system of any one of aspects 1 to 28, wherein the first hydride donor compound is cNADH or NMNH.30. A method to convert a carboxylic acid containing substrate to an aldehyde containing product, the method comprising: contacting the carboxylic acid containing substrate with a first polypeptide that encodes an aldehyde dehydrogenase that directly reduces carboxylic acids to aldehydes without adenosine triphosphate (ATP) or coenzyme A (CoA)-based activation in the presence of a first hydride donor compound in an aqueous solution, optionally, providing a second hydride donor compound and a second polypeptide that encodes an enzyme that reduces the first hydride donor compound using the second hydride donor compound, wherein the aldehyde dehydrogenase can use reduced first hydride donor compound as a cofactor, and wherein the aldehyde dehydrogenase converts the carboxylic acid containing substrateAtorney docket No. 00058-087W01 to an aldehyde containing product.31. The method of aspect 30. wherein the carboxylic acid containing substrate is selected from a linear aliphatic carboxylic acid, a branched aliphatic carboxylic acid, an aromatic carboxylic acid, a diacid, a hydroxyacid and formic acid.32. The method of aspect 30, wherein the linear aliphatic carboxylic acid is selected from acetic acid, propanoic acid, butyric acid, hexanoic acid, and nonanoic acid.33. The method of aspect 30. wherein the branched aliphatic carboxylic acid is selected from isobutyric acid, and isopentanoic acid.34. The method of aspect 30, wherein the aromatic carboxylic acid is selected from benzoic acid, 2-furoic acid, cinnamic acid and vanillic acid.35. The method of aspect 30. wherein the diacid is selected from succinic acid, and adipic acid.36. The method of aspect 30, wherein the hydroxyacid is selected from 4- hydroxybutyric acid, 3-hydroxybutyric acid, lactic acid, and 2-hydroxylpropanoic acid.37. The method of any one of aspects 30 to 36, wherein the first polypeptide comprises a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98%, or at least 99% identical to a sequence presented in SEQ ID NO:1 to 170.38. The method of any one of aspects 30 to 37, wherein the first polypeptide comprises a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%. at least 98%. or at least 99% identical to a sequence presented in SEQ ID NO:1, SEQ ID NO:39, SEQ ID NO:2, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:99, SEQ ID NO:105, SEQ ID NO: 119, SEQ ID NO: 126, and SEQ ID NO: 170.39. The method of any one of claims of any one of aspects 30 to 38. wherein the first polypeptide is engineered to contain 1 to 10 amino acid substitutions in comparison to a sequence presented in SEQ ID NO: 1 to 170, wherein the amino acid substitution(s) improve the aldehyde dehydrogenase activity for directly reducing carboxylic acids to aldehydes.40. The method of any one of aspects 30 to 39, wherein the first polypeptide has the sequence of SEQ ID NO:39 with at least the amino acid substitution of Y402F.41. The method of any one of aspects 30 to 39, wherein the first polypeptide has the sequence of SEQ ID NO:2 with an amino acid substitution selected from Q350N, Q350R, and Q350A.42. The method of any one of aspects 30 to 39, wherein the first polypeptide hasAtorney docket No. 00058-087W01 the sequence of SEQ ID NO:31 but with one or more amino acid substitutions selected from(a), (b). (c), (d), (e), and / or (f):(a) W185S, W185D, W185H, W185N, W185K, or W185F;(b) N186D;(c) E285D;(d) F288A:(e) Q366I. Q366A, Q366L, Q366M. or Q366N; and / or(f) F418A or F418H.43. The method of aspect 42, wherein the first polypeptide has the sequence of SEQ ID NO:31 with an amino acid substitution selected from W185N, W185K, and W185F.44. The method of any one of aspects 30 to 39, wherein the first polypeptide has the sequence of SEQ ID NO:1 with one or more amino acid substitutions selected from (a),(b) and / or (c):(a) W164F;(b) T302S; and / or(c) W173M, W173F, W173Y.45. The method of any one of aspects 30 to 39, wherein the first polypeptide has the sequence of SEQ ID NO:99 with at least an amino acid substitution selected from W173M, W173F, and W173Y.46. The method of any one of aspects 30 to 45, wherein the method further comprises isolating the aldehydes during the reaction process.47. The method of any one of aspects 30 to 46, wherein the method further comprises overlaying an organic solvent on top of the aqueous solution to allow for the continuous extraction of the aldehydes.48. The method of aspect 47, wherein the organic solvent is selected from anisole, hexane, isooctane, toluene, dodecane, oleyl alcohol, and ethoxybenzene.49. The method of aspect 47 or aspect 48, wherein the organic solvent is used at a ratio of 1 : 10 to 10: 1 to the aqueous solution.50. The method of any one of aspects 47 to 49, wherein the organic solvent is used at a ratio of 1 : 1 to 10: 1 to the aqueous solution.51. The method of any one of aspects 30 to 50, wherein the method furtherAtorney docket No. 00058-087W01 comprises isolating the aldehyde containing products by using an apparatus, the apparatus comprising: a reactor that can be heated which comprises the product aldehyde, wherein the reactor is in fluid connection to one or more cooled condensers filled with a solvent, wherein in heating the reactor causes the product aldehyde to go in the gaseous phase; a gas supply that is in fluid connection with the reactor, that is configured to introduce positive gas flow into the reactor, wherein the gas introduced into the reactor flows from the reactor to the one or more cooled condensers; and one of more cooled condensers filled with a solvent that are configured to cool the gaseous aldehyde into a liquid phase so that it is trapped by the solvent, wherein the aldehyde can then be isolated from the solvent.52. The method of any one of aspects 30 to 51, wherein the method comprises the second hydride donor compound and the second polypeptide that encodes an enzyme that reduces the first hydride donor compound using the second hydride donor compound.53. The method of aspect 52. wherein the second polypeptide encodes a phosphite dehydrogenase that has a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 171, and wherein the second hydride donor compound is phosphite.54. The method of aspect 52 or aspect 53, wherein the second polypeptide encodes a glucose dehydrogenase that has a sequence that at least 75%. at least 80%. at least 85%, is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 175, and wherein the second hydride donor compound is glucose.55. The method of any one of aspects 30 to 55, wherein the method further comprises converting the aldehyde containing product to non-aldehyde containing product, comprising: contacting the aldehyde containing product with a third polypeptide that that encodes an enzy me capable of converting an aldehyde group into a non-aldehyde group.56. The method of aspect 55. wherein the third polypeptide that encodes an enzyme capable of converting an aldehyde group into a non-aldehyde group, utilizes the first hydride compound as a coenzyme or cofactor.57. The method of aspect 55 of aspect 56, wherein the third polypeptide encodes an alcohol dehydrogenase, or an acetolactate synthase.Atorney docket No. 00058-087W0158. The method of aspect 57, wherein the third polypeptide encodes an alcohol dehydrogenase that has a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 172 or SEQ ID NO: 173.59. The method of aspect 57, wherein the third polypeptide encodes an acetolactate synthase that has a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 174.60. The method of any one of aspects 30 to 59, wherein the first hydride donor compound is selected from reduced nicotinamide-adenine dinucleotide (NADH), reduced nicotinamide-adenine dinucleotide phosphate (NADPH), carba-nicotinamide adenine dinucleotide (cNADH), reduced nicotinamide mononucleotide (NMNH). reduced 3- carbamoyl-l-(4-carboxy benzyl) pyridin-l-ium (BANAH), reduced 1 -benzylnicotinamide (BNAH), reduced 3-carbamoyl-l-(3-phenylpropyl)pyridin-l-ium (3PNAH), and reduced 3- carbamoy 1- 1 -(4-methoxy benz l )pyri din- 1 -ium (BNA OMeH).61. The method of any one of aspects 30 to 60, wherein the first hydride donor compound is cNADH or NMNH.

[0119] The following examples are intended to illustrate but not limit the disclosure. While they are typical of those that might be used, other procedures know n to those skilled in the art may alternatively be used.EXAMPLES

[0120] The invention is illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.

[0121] Reactions for the direct reduction of the carboxylic acid hexanoic acid to hexanal followed by a subsequent reduction to the respective alcohol. Reactions performing the direct reduction of carboxylic acids to aldehydes followed by a subsequent reduction to the respective alcohol were performed in 100 mM sodium phosphate buffer (pH 7.0) containing 100 mM sodium phosphite, 3 mM NADH, 50 mM carboxylic acid, 0.75 g / L ALDH (unless otherwise noted). 0.75 g / L ScADH (purchased from Sigma- Aldrich), and 1 g / L TS-PTDH. Reactions were initiated by spiking ALDH into the remaining reaction components in a 2 mL glass vial sealed with a PTFE-lined cap. Reactions were incubated without shaking for 24 hours at 30 °C. To analyze the concentration of alcohol in the reactions, the samples were extracted by liquid-liquid extraction with ethyl acetate at aAtorney docket No. 00058-087W01 sample to ethyl acetate ratio of 1: 1. After extraction, the solution was centrifuged at 20,000 x g for 2 minutes. The ethyl acetate fraction was transferred to a gas chromatography vial for analysis. Due to low protein yield after purification, reactions containing A0A6P3Q7G7 (SEQ ID NO:8), Q9LRI6 (SEQ ID NO:36), R4G109 (SEQ ID NO:47), A0A1W9L4H0 (SEQ ID NO:50), A0A3S0VUQ7 (SEQ ID NO:51), or A0A4R6PRX6 (SEQ ID NO:56) contained 0.21 g / L, 0.28 g / L, 0.26 g / L 0.2 g / L. 0.26 g / L. or 0.21 g / L, respectively. Results of which are shown in FIG. 3, FIG. 4, FIG. 26, FIG. 29, and FIG. 30

[0122] Reactions for the direct reduction of acetic acid and butyric acid to aldehydes followed by a subsequent reduction to the respective alcohols. Acetic acid and butyric acid were directly reduced to their respective aldehydes and further reduced to their respective alcohols in reactions containing 100 mM sodium phosphate pH 7.0, 100 mM sodium phosphite, 3 mM NADH, 10 g / L ALDH, 5 g / L ScADH, and 1.5 g / L TS-PTDH. Sodium acetate or sodium butyrate were supplied to reactions at 750 mM. The reactions were performed a 2 mL glass vial and capped with a PTFE-lined cap. The reactions were incubated at 30 °C without shaking for 72 hours. Samples containing ethanol were extracted by mixing hexane:lg / mL sodium phosphate dibasic: sample at a 4: l: l ratio or by mixing ethoxybenzene: Ig / mL sodium phosphate dibasic: sample at a 2: 1 : 1 ratio. Samples containing butanol were extracted by vigorously shaking with ethyl acetate at a 4: 1 solvent to sample ratio. Samples were centrifuged at 20,000 x g for 2 minutes to separate the two fractions, and the organic layer was transferred to a GC vial for analysis. When detecting ethanol, low amounts of ethanol was found in control reactions without ALDH. Ultimately, this ethanol contamination was traced to the NADH purchased from Sigma Aldrich, which we theorized used ethanol in the final stages of the manufacturing process. Therefore, the amount of ethanol was subtracted from control reactions without ALDH (175 mg / L) from the amount of ethanol measured in samples containing ALDH to yield the final concentrations presented in FIG. 5. Results of which for butanol production are presented in FIG. 7.

[0123] Reactions for the direct reduction of propionic acid to propionaldehyde followed by a subsequent reduction to propanol. Reactions performing the direct reduction of propionate to propionaldehyde followed by a subsequent reduction to propanol were performed in 100 mM sodium phosphate buffer (pH 7.0) containing 100 mM sodium phosphite, 3 mM NADH, 0.75 g / L ScADH, and 1 g / L TS-PTDH. When 500 mM sodium propionate was supplied. 0.75 g / L ALDH was added. When 50 mM sodium propionate wasAtorney docket No. 00058-087W01 supplied, 0.3 g / L ALDH was added. Reactions were performed in a 2 mL glass vial sealed with a PTFE-lined cap. Reactions were incubated without shaking for 24 hours at 30 °C. To analyze the concentration of alcohol in the reactions, the samples were extracted by liquidliquid extraction with ethyl acetate at a sample to ethyl acetate ratio of 1: 1. After extraction, the solution was centrifuged at 20,000 x g for 2 minutes. The ethyl acetate fraction was transferred to a gas chromatography vial for analysis. The results of which are presented in FIG. 6 and FIG. 29.

[0124] Reactions for the direct reduction of isobutyric acid to isobutyraldehyde followed by a subsequent reduction to isobutanol. Reactions performing the direct reduction of isobutyric acid to isobutyraldehyde followed by a subsequent reduction to isobutanol were performed in 100 mM sodium phosphate pH 7.0. 100 mM sodium phosphite, 3 mM NADH, 10 g / L ALDH, 1 g / L TcADH, and 1.5 g / L TS-PTDH. Sodium isobutyrate was supplied to the reactions at 522 mM. The reactions were performed in a 2 mL glass vial capped with a PTFE-lined cap. The reactions were incubated at 30 °C without shaking for 96 hours. Samples were taken periodically to monitor alcohol production. Samples were extracted by vigorously shaking with ethyl acetate at a 4:1 solvent to sample ratio. Samples were centrifuged at 20,000 x g for 2 minutes to separate the two fractions, and the organic layer was transferred to a GC vial for analysis. Results of which are shown in FIG. 8.

[0125] Reactions for the direct reduction of aromatic substrates (benzoic acid, trans-cinnamic acid, 4-hydroxybenzoic acid, vanillic acid, and 2-furoic acid) to aldehydes followed by a subsequent reduction to the respective alcohols. Reactions with aromatic substrates contained 100 mM sodium phosphate pH 7, 100 mM sodium phosphite, 3 mM NADH, and 2 g / L TS-PTDH. ALDH was generally fed at 3 g / L unless otherwise noted due to low protein yield after purification. UPI001E1E9641 (SEQ ID NO: 128), A0A095VIR5 (SEQ ID NO: 129), and A0A0Q51VT9 (SEQ ID NO: 130) were supplied at 1.6 g / L. Q88CR0 was supplied at 0.3 g / L. When subsequent reduction of the respective aldehydes to alcohols was performed, TcADH was fed at 0.75 g / L. All reactions were performed at 37 °C in 2 mL screw-top GC vials with a reaction volume of 40 pL unless otherwise noted. To prepare reactions, a mastermix of all components except ALDH was prepared on ice. ALDH concentrations were normalized and added to a 96-well PCR plate. Mastermix was then spiked into the PCR plate containing ALDHs, mixed well, and transferred to a 2 mL glass PTFE-lined screw cap GC vial. Reactions were then incubated forAtorney docket No. 00058-087W0124 hours unless otherwise noted. Aromatic reduction reactions with engineered aldehyde dehydrogenases were sampled at 48 hours.

[0126] For aromatic acid reduction reactions using an engineered aldehyde dehydrogenase and where aldehyde was subsequently modified to an alcohol, 4- hydroxybenzoic acid (see FIG. 31) and trans-cinnamic acid (see FIG. 28) were supplied at a final concentration of 50 mM. Benzoic acid (see FIG. 27) was supplied at a final concentration of 100 mM. When non-engineered aldehyde dehydrogenases were used. 4- hydroxybenzoic acid (see FIG. 10) and vanillic acid (see FIG. 11) were fed at 25 mM, while benzoic acid (see FIG. 9) and 2-furoic acid (see FIG. 13) was fed at 50 mM.

[0127] To sample benzyl alcohol and trans-cinnamyl alcohol accumulation (see FIG. 9 and FIG. 12), reactions extracted by addition of n-hexane at a ratio of 2: 1 organic: aqueous. The reactions were then shaken well for 5 minutes to extract alcohol, centrifuged at 20,000 x g for 2 min to separate the organic layer, which was then removed for GC analysis. To sample 2-furfuryl alcohol production (see FIG. 13), the extraction procedure follows exactly as with benzyl alcohol, but reaction halt and extraction is performed with ethyl acetate at 1: 1 organic: aqueous. To sample 4-hydroxybenzyl alcohol (see FIG. 10) and vanillyl alcohol (see FIG. 11) production, reactions were halted via addition of a solution of 45% (v / v) methanol and 5% (v / v) formic acid at a ratio of 2: 1 termination solution: sample. Reactions were then mixed well, centrifuged at 20,000 x g for 20 minutes, and the supernatant was transferred to HPLC vial for analysis.

[0128] Aromatic compound (benzoic acid and trans-cinnamic acid) carboxylic acid reduction reactions using an organic solvent overlay. For aromatic compound (benzoic acid and trans-cinnamic acid) carboxylic acid reduction reactions using an organic solvent overlay, sodium benzoate (see FIG. 36 and FIG. 38) was fed at 75 mM and sodium trans-cinnamate (see FIG. 39) was fed at 35 mM. Aqueous phase of aldehyde accumulation reactions was prepared and initiated with the same protocol as alcohol production assay setup, with the omission of TcADH (see FIG. 38 and FIG. 39). When the aldehyde was subsequently reduced to an alcohol using an alcohol dehydrogenase (see FIG. 36). the aqueous phase of the reactions was also prepared and initiated with the same protocol as alcohol production assay without overlay. After transferring initiated aqueous reactions to GC vials, 3 times the aqueous reaction volume was added of organic solvent as overlay (anisole, phenetole, hexane, isooctane, toluene, or no overlay). Reactions were then capped andAtorney docket No. 00058-087W01 incubated for 24 hours at 37 °C before a fraction of the organic layer was removed for GC analysis.

[0129] Reactions producing 1,5-pentanediol from 5-hydroxypentanoic acid.Reactions producing 1,5-pentanediol from 5-hydroxypentanoic acid contained 100 mM sodium phosphate pH 7.0, 100 mM sodium phosphite, 3 mM NADH, 750 mM sodium 5- hydroxypentanoate, 10 g / L ALDH, and 1.5 g / L TS-PTDH. Reactions were performed in 2 mL glass vials at 30 °C. Intermittently, a portion of the reaction as removed and mixed with 250 mM H2SO4 at a 1 :3 sample:acid treatment ratio. After mixing the samples were centrifuged at 20,000 x g and transferred to HPLC vials for analysis (see FIG. 14).

[0130] Reactions producing L-phenylacetylcarbinol from benzoic acid. Reactions producing L-phenylacetylcarbinol contained 100 mM sodium phosphate pH 7.0, 100 mM sodium phosphite, 50 mM sodium pyruvate, 5 mM MgCH, 100 mM sodium benzoate, 3 mM NADH, 0.1 mM thiamine pyrophosphate, 4 g / L ALDH, 1 g / L TS-PTDH, and 1 g / L pyruvate decarboxylase. Reactions were prepared in a 96-well PCR plate and then transferred to a 2 mL glass vial. Reactions were incubated at 30 °C for 24 hours. Reactions were sampled by extraction by addition of ethyl acetate at a 1 : 1 ethyl acetate: sample ratio. Extracted samples were mixed well, followed by centrifugation at 20,000 x g for 2 minutes. The organic layer was transferred to a gas chromatography vial for analysis (see FIG. 16).

[0131] Reactions using gas-stripping to continuously extract benzaldehyde.Reactions using gas-stripping to continuously extract benzaldehyde from the reaction mixture contained 100 mM sodium phosphate pH 7.0, 100 mM sodium phosphite, 100 mM sodium benzoate, 3 mM NADH, 2 g / L ALDH, and 1 g / L TS-PTDH. Reactions were performed at a reaction volume of ImL in a 2 mL glass vial sealed with a PTFE-lined crimp cap. The reaction vessel was connected to a pressurized air source, flowing through the reaction vessel at a low flow rate. The reaction vessel was subsequently connected with needles pierced through each septa cap and tubing to three additional 2 mL GC vials containing 1 mL of ethyl acetate held at 0 °C. The final ethyl acetate trap vial was vented to atmosphere using a needle. Reactions were incubated at 37 °C and monitored for benzaldehyde concentration every 24 hours for three days. Ethyl acetate was transferred to a gas chromatography vial for analysis (see FIG. 23).

[0132] Reactions performing the direct reduction of hexanoic acid to hexanal with wild type and engineered P51977. Reactions performing the direct reduction ofAtorney docket No. 00058-087W01 hexanoic acid to hexanal with wild type and engineered P51977 were performed in 100 mM sodium phosphate buffer (pH 7.0) containing 100 mM sodium phosphite. 3 mM NADH, 500 mM sodium hexanoate, 0.75 g / L ALDH, and 1 g / L TS-PTDH. For alcohol production, reactions contained 0.75 g / L ScADH (see FIG. 25). For aldehyde production, ScADH was omitted (see FIG. 18 and FIG. 33). Reactions were initiated by spiking ALDH into the remaining reaction components in a 2 mL glass vial sealed with a PTFE-lined cap. Reactions were incubated without shaking for 24 hours at 30 °C. To analyze the concentration of hexanal in the reactions, the samples were extracted by liquid-liquid extraction with ethyl acetate at a sample to ethyl acetate ratio of 1 : 1. After extraction, the solution was centrifuged at 20.000 g for 2 minutes. The ethyl acetate fraction was transferred to a gas chromatography vial for analysis.

[0133] Reactions performing the direct reduction of isobutyric acid to isobutyraldehyde without subsequent modification. Reactions performing the direct reduction of isobutyric acid to isobutyraldehyde without subsequent modification were performed in 100 mM sodium phosphate pH 7.0, 100 mM sodium phosphite. 3 mM NADH, 10 g / L ALDH, and 1 g / L TS-PTDH. Sodium isobutyrate was supplied to the reactions at 522 mM. The reactions were performed in a 2 mL glass vial capped with a PTFE-lined cap. The reactions were incubated at 30 °C without shaking for 18 hours. Samples were extracted by vigorously shaking with ethoxybenzene at a 3: 1 solvent to sample ratio. Samples were centrifuged at 20,000 - g for 2 minutes to separate the two fractions, and the organic layer was transferred to a GC vial for analysis (see FIG. 20).

[0134] Reactions performing the direct reduction of butyric acid to butyraldehyde without subsequent modification. Reactions performing the direct reduction of butyric acid to butyraldehyde without subsequent modification were performed in 100 mM sodium phosphate pH 7.0, 100 mM sodium phosphite, 3 mM NADH, 10 g / L ALDH, and 1 g / L TS-PTDH. Sodium butyrate was supplied to the reactions at 750 mM. The reactions were performed in a 2 mL glass vial capped with a PTFE-lined cap. The reactions were incubated at 30 °C without shaking for 42 hours. Samples were extracted by vigorously- shaking with ethoxy benzene at a 3: 1 solvent to sample ratio. Samples were centrifuged at 20,000 x for 2 minutes to separate the two fractions, and the organic layer was transferred to a GC vial for analysis (see FIG. 19).Atorney docket No. 00058-087W01

[0135] Reactions performing the direct reduction of hexanoic acid to hexanal with subsequent reduction to hexanol where DTT was supplied to the reaction.Reactions performing the direct reduction of hexanoic acid to hexanal with subsequent reduction to hexanol where DTT was supplied to the reactions were performed in 100 mM sodium phosphate buffer (pH 7.0) containing 100 mM sodium phosphite, 3 mM NADH, 50 mM sodium hexanoate, 0.75 g / L ALDH, and 1 g / L TS-PTDH. In the condition where DTT was supplied, 5 mM DTT was added to the reactions. Reactions were initiated by spiking ALDH into the remaining reaction components in a 2 mL glass vial sealed with a PTFE-lined cap. Reactions were incubated without shaking for 24 hours at 30 °C. To analyze the concentration of hexanol in the reactions, the samples were extracted by liquid-liquid extraction with ethyl acetate at a sample to ethyl acetate ratio of 1: 1. After extraction, the solution was centrifuged at 20,000 * g for 2 minutes. The ethyl acetate fraction was transferred to a gas chromatography vial for analysis (see FIG. 34).

[0136] Reactions performing the direct reduction of hexanoic acid to hexanal without subsequent modification and addition of organic solvent overlay with P51977 (SEQ ID NO:2). Reactions performing the direct reduction of hexanoic acid to hexanal without subsequent modification and addition of organic solvent overlay with P51977 were performed in 100 mM sodium phosphate pH 7.0, 100 mM sodium phosphite, 500 mM sodium hexanoate, 3 mM NADH, 0.3 or 2.0 g / L P51977, and 1 g / L TS-PTDH. Reactions were performed at a 50 pL volume in a 2 mL glass vial sealed with a PTFE-lined cap. 50 pL of ethoxybenzene was added at t = 0. Reactions were incubated at 30 °C without shaking. A small portion of ethoxybenzene was removed at 16 and 24 hours for gas chromatography analysis (see FIG. 40).

[0137] Reactions performing the direct reduction of hexanoic acid to hexanal without subsequent modification and addition of organic solvent overlay with 4AIT08 (SEQ ID NO:1). Reactions performing the direct reduction of hexanoic acid to hexanal without subsequent modification and addition of organic solvent overlay with 4AIT08 (SEQ ID NO: 1), UPI0005CD4570 (SEQ ID NO:99), and UPI001FCBF132 (SEQ ID NO: 105) were performed in 100 mM sodium phosphate pH 7.0, 100 mM sodium phosphite, 50 mM sodium hexanoate, 3 mM NADH, 10 g / L ALDH, and 1 g / L TS-PTDH. Reactions were performed at a 50 pL volume in a 2 mL glass vial sealed with a PTFE-lined cap. 150 pL of ethoxybenzene was added at t = 0. Reactions were incubated at 30 °C without shaking for 42 hours. A smallAtorney docket No. 00058-087W01 portion of ethoxy benzene was transferred to a gas chromatography vial for analysis (see FIG. 41).

[0138] Reactions performing the direct reduction of propionic acid and butyric acid to their respective aldehyde without subsequent modification and addition of organic solvent overlay. Reactions performing the direct reduction of propionic acid and butyric acid to their respective aldehydes without subsequent modification and addition of organic solvent overlay were performed in 100 mM sodium phosphate pH 7.0. 100 mM sodium phosphite, 3 mM NADH, 10 g / L ALDH, and 1 g / L TS-PTDH. Sodium propionate or sodium butyrate were supplied to the reactions at 750 mM. Ethoxybenzene was added at the organic solvent overlay at a 3: 1 solvent: sample ratio at t = 0. The reactions were performed in a 2 mL glass vial capped with a PTFE-lined cap. The reactions were incubated at 30 °C without shaking for 42 hours. Ethoxybenzene was directly transferred to a GC vial for analysis (see FIG. 42 and FIG. 43).

[0139] Reactions performing the direct reduction of isobutyric acid to isobutyraldehyde without subsequent modification and addition of organic solvent overlay. Reactions performing the direct reduction of isobutyric acid to isobutyraldehyde without subsequent modification and addition of organic solvent overlay were performed in 100 mM sodium phosphate pH 7.0, 100 mM sodium phosphite, 3 mM NADH, 10 g / L ALDH, and 1.5 g / L TS-PTDH. Sodium isobutyrate was supplied to the reactions at 522 mM.Ethoxy benzene was added at the organic solvent overlay at a 3: 1 solventsample ratio at t = 0. The reactions were performed in a 2 mL glass vial capped with a PTFE-lined cap. The reactions were incubated at 30 °C without shaking for 38 hours. Ethoxybenzene was directly transferred to a GC vial for analysis (see FIG. 44).

[0140] Use of a chemical reagent to regenerate reduced cofactor for aldehyde dehydrogenase-mediated direct reduction of propionic acid to propanol. Reactions using dithionite in the reduction of propionic acid to propanol contained sodium carbonate pH 9.5 (100 mM), sodium propionate (500 mM), NAD+(3 mM), A4IT08 (1 g / L), ScADH (1 g / L), at 100 pL is degassed under argon. Under anaerobic conditions dithionite powder is added to a final concentration of 100 mM. The reactions were incubated at 30 °C for 24 hrs. After incubation, the samples were extracted in ethyl acetate for GC analysis (see FIG. 46).

[0141] Use of a chemical reagent to regenerate reduced cofactor for aldehyde dehydrogenase-mediated direct reduction of hexanoic acid to hexanal. Use of a chemicalAtorney docket No. 00058-087W01 reagent to regenerate reduced cofactor for aldehyde dehydrogenase-mediated direct reduction of hexanoate to hexanal. Reactions using dithionite in the reduction of hexanoic acid to hexanal contained Tris-Cl pH 8 (100 mM), sodium hexanoate (100 mM), NAD+(3 mM), P51977 (0.3 g / L). A 100 pL reaction is degassed with argon, and under anaerobic conditions di thionite powder is added to a final concentration of 100 mM. As a negative control, a reaction without dithionite powder addition was run in parallel. As a second control reaction, a reaction with enzymatic cofactor recycling was performed, comprised of Tris-Cl pH 8 (100 mM), hexanoate (100 mM), NAD+(3 mM), P51977 (Sheep, 0.3 g / L), dithionite (0 mM), TS- PTDH (1 g / L), phosphite (100 mM). Samples were extracted at a 1:1 ethoxy benzene (see FIG. 48).

[0142] Aldehyde dehydrogenase-mediated reduction of a carboxylic acid to an aldehyde using the noncanonical cofactor carba-nicotinamide adenine dinucleotide.

[0143] Reactions contain 100 mM sodium phosphate pH 7.0, lOOmM sodium phosphite, 3 mM cNAD+, 50 mM sodium hexanoate, 1.249 g / L A4IT08 (SEQ ID NO: 33) or 0.855 g / L UPI001FCBF132 (SEQ ID NO: 157), and 0.337 g / L of PTDH at a total reaction volume of 30 pL. An organic overlay of ethoxy benzene was added at a 3:1 volumetric ratio of overlay to reaction volume. Reactions were initiated in a PCR Plate by adding the corresponding ALDH to a master mix containing all other components. Reactions were mixed well and transferred to GC vials where 90 pL of ethoxy benzene was layered on top of each reaction. Reactions were sealed and allowed to incubate for 48h at 30°C before sampling. Each sample was run in triplicate, and a no cofactor control was used to account for background activity. Final production values are calculated by subtracting the no cofactor background activity. To sample, 20 pL of the ethoxy benzene overlay was removed from the top of the reaction and transferred to a fresh GC vial for analysis (see FIG. 50).

[0144] Aldehyde dehydrogenase-mediated reduction of a carboxylic acid to an aldehyde using the noncanonical cofactor nicotinamide mononucleotide. Reactions contained 100 mM sodium phosphate pH 7.0, 100 mM D-glucose, 15 mM NMN+, 100 mM sodium hexanoate, 1.36 g / L of P51977, and 0.085 g / L of engineered 1-glucose dehydrogenase (GDH Triple) at a total volume of 30 pL. An organic overlay of ethoxy benzene was added at a 3: 1 volumetric ratio of overlay to reaction volume. Reactions were initiated in a PCR Plate by adding aldehyde dehydrogenase to a master mix containing all other components. Reactions were mixed well and transferred to GC vials where 90 pL ofAtorney docket No. 00058-087W01 ethoxy benzene was layered on top of each reaction at t = 0. Reactions were sealed and allowed to incubate for 24h at 30 °C before sampling. Final production values are calculated by subtracting the no cofactor background activity. To sample, 30 pL of the ethoxy benzene overlay was removed from the top of the reaction and transferred to a fresh GC vial for analysis (see FIG. 52).

[0145] Molecular Cloning. ALDH genes were purchased from Integrated DNA Technologies as A-terminal poly hisiti dine tagged synthetic gene constructs with terminal homologous arms for DNA assembly. Gene constructs were codon optimized for expression in E. coli using Integrated DNA Technologies’ codon optimization tool. Synthetic gene constructs were assembled into the expression vector by Gibson assembly. Gibson assembled products were transformed into E. coll XLl-Blue (Agilent Technologies) and plated on 2* Yeast Extract Tryptone media (2xYT, 16 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl) containing 100 mg / L ampicillin and incubated at 37 °C overnight. Solidified media for cell plating contained 15 g / L agar. After incubation, a single cell colony was picked from the plate and inoculated into 4 mL 2x YT media containing 100 mg / L ampicillin in a cell culture tube. The cultures were shaken at 37 °C overnight. Plasmids were isolated from the cultures using the Qiagen QIAprep Spin Miniprep Kit. ALDH gene sequences were confirmed by Sanger sequencing. Alcohol dehydrogenase from Saccharomyces cerevisiae (ScADH) was purchased as a lyophilized powder from Sigma Aldrich.

[0146] Protein expression. Plasmids containing aldehyde dehydrogenase, phosphite dehydrogenase, TcADH, or acetolactate dehydrogenase were transformed into E. coli BL-21 (Invitrogen) and plated on 2x YT media containing 100 mg / L ampicillin and incubated at 30 °C overnight. A single cell colony was picked from the plate and inoculated into 4 mL 2x YT media containing 100 mg / L ampicillin in a cell culture tube and incubated overnight at 30 °C. 500 pL of the cell culture was inoculated into 100 mL of 2x YT media containing 200 mg / L ampicillin in a 250 mL baffled shake flask. The flasks were shaken at 37 °C until the ODeoo of the culture reached ~ 0.6. The flasks were then shaken at room temperature for 30 minutes to cool. After cooling, the protein expression was induced by spiking isopropyl P-D-l- thiogalatopyranoside (IPTG) to a final concentration of 0.5 mM. The flasks were then shaken at room temperature overnight. Cells were harvested by centrifugation at 4000 x g at 4 °C for 12 minutes.Attorney docket No. 00058-087W01

[0147] His-tagged proteins were purified from the cells using nickel-affinity resin (Hispur Ni-NTA Resin, ThermoFisher). Cell pellets were resuspended in ice-cold Binding Buffer containing 50 mM sodium phosphate buffer pH 7.7, 300 mM sodium chloride, 10 mM imidazole, and 0.03% Triton X-100. The resuspended cells were transferred to a pre-chilled 2 mL bead beating tube containing ~0.5 mL 0. 1 mm soda lime glass beads (BioSpec Products). The cells were disrupted by bead beading using a MP Biomedical FastPrep-24 by beating at 6 m / s for 35 seconds, followed by 2 minutes of cooling in an ice water bath. The lysis process was repeated five times for each sample. After lysis, the tubes were centrifuged at 20,000 x g at 4 °C for 20 minutes to clarify the lysates. The clarified lysates were transferred to clean, pre-chilled microcentrifuge tubes for protein immobilization. Ni-NTA resin was washed five times with Binding Buffer to remove any storage buffer. 150 pL of equivalent hydrated resin bed was added to each clarified lysate. The tubes were incubated at 4 °C for -1 hour using an end-over-end inverting rotator. The resin was separated from the lysate by centrifugation at 500 x g at 4 °C for 2 minutes. The resin was then transferred to a Zymo Research Zymo-Spin Pl column. The resins were washed twice with 300 pL of Wash Buffer containing 50 m sodium phosphate pH 7.7, 300 mM sodium chloride, 50 mM imidazole, and 0.03% Triton X- 100. The proteins were eluted with Elution Buffer containing 50 mM sodium phosphate buffer pH 7.7, 300 mM sodium chloride, and 250 mM imidazole. The purified proteins w ere mixed with a 50% glycerol solution to a final concentration of 20% glycerol for storage at -80 °C until use. Protein concentration was determined by Bradford Assay using a dilution series of bovine serum albumin as standards. Additionally, protein expression and purification can be scaled to larger volumes of culture (1.5L media in a 2L baffled flasks) and purified in the same manner. Engineered glucose dehydrogenase was expressed at 30 °C.

[0148] Gas Chromatography (GC) Method for Detection of Benzaldehyde when extracted into ethyl acetate. GC was operated in constant flow mode with a carrier gas flowrate of 2 mL / min. The inlet was maintained at 250 °C. The split ratio was 5: 1. The oven was held at 60 °C for 2 minutes, then ramped at 10 °C / min to 230 °C. and then held at 230 °C for 12 minutes. The flame-ionization detector was maintained at 260 °C with an air flowrate of 300 ml / min, H2 flowrate of 30 mL / min, and a makeup nitrogen gas flowrate of 25 mL / min. Helium was used as the carrier gas (see FIG. 23).

[0149] Gas Chromatography Method for Detection of Hexanal and HexanolWhen Extracted into Ethyl Acetate. GC was operated in constant flow mode with a carrierAtorney docket No. 00058-087W01 gas flowrate of 2 mL / min. 2 gi L of sample was injected into the instrument. The inlet was maintained at 250 °C. The split ratio was 5: 1. The oven was held at 100 °C for two minutes, then ramped at 20 °C / min to 230 °C, then held at 230 °C for 5 minutes. The flame-ionization detector was maintained at 300 °C with an air flowrate of 350 ml / min, H2 flowrate of 30 mL / min, and a makeup nitrogen gas flowrate of 25 mL / min. Helium was used as the carrier gas (see FIG. 3, FIG. 4. FIG.18. FIG. 25, FIG. 26, FIG. 29, FIG. 30, FIG. 33. and FIG. 34)

[0150] Gas Chromatography Method for Detection of Propanol, Butanol,Isobutanol, Propionaldehyde, Butyraldehyde, Isobutyraldehyde, and Hexanal (ethoxybenzene extracted samples only). GC was operated in constant flow mode with a earner gas flowrate of 2 mL / min. 1 pL of sample was injected into the instrument. The inlet was maintained at 250 °C. The split ratio was 10: 1. The oven was held at 65 °C for 4 minutes, then ramped at 100 °C / min to 230 °C, then held at 230 °C for 4.35 minutes. The flame-ionization detector was maintained at 300 °C with an air flowrate of 350 ml / min, H2 flowrate of 30 mL / min, and a makeup nitrogen gas flowrate of 25 mL / min. Helium was used as the earner gas (see FIG. 6, FIG. 7, FIG. 8, FIG. 19, FIG. 20, FIG. 29, FIG. 40, FIG. 41, FIG. 42, FIG. 43, FIG. 44, FIG. 46, FIG. 48, FIG. 50, and FIG. 52)

[0151] Gas Chromatography Method for Detection of L-phenylacetylcarbinol.GC was operated in constant flow mode with a carrier gas flowrate of 2 mL / min. 1 pL of sample was injected into the instrument. The inlet was maintained at 250 °C. The split ratio was 5: 1. The oven was held at 60 °C for 2 minutes, then ramped at 10 °C / min to 225 °C, then held at 225 °C for 12 minutes. The flame-ionization detector was maintained at 260 °C with an air flowrate of 300 mL / min, H2 flowrate of 30 mL / min, and a makeup nitrogen gas flowrate of 25 mL / min. Helium was used as the earner gas (see FIG. 16).

[0152] Gas Chromatography Methods for Detection of Ethanol. When ethoxybenzene was used as the extraction solvent for ethanol, the GC was operated in constant flow mode w ith a carrier gas flow rate of 2 mL / min. 1 pL of sample was injected into the instrument. The inlet was maintained at 250 °C. The split ratio was 10: 1. The oven was held at 40 °C for 5.5 minutes, then ramped at 70°C / min to 230 °C, then held at 230 °C for 3.3 minutes. The flame-ionization detector was maintained at 300 °C with an air flowrate of 350 ml / min, H2 flowrate of 30 mL / min, and a makeup nitrogen gas flowrate of 25 mL / min. Helium was used as the carrier gas. When hexane was used as the extraction solvent forAtorney docket No. 00058-087W01 ethanol, the GC was operated in constant flow mode with a carrier gas flow rate was 1 mL / min. 1 pL of sample was injected into the instrument. The inlet was maintained at 250 °C. The split ratio was 20: 1. The oven was held at 35 °C for 10 minutes, then ramped at 30°C / min to 230 °C, then held at 230 °C for 5 minutes. The flame-ionization detector was maintained at 300 °C with an air flowrate of 350 mL / min, H2 flowrate of 30 mL / min, and a makeup nitrogen gas flowrate of 20 mL / min. Helium was used as the carrier gas (see FIG. 5).

[0153] Gas Chromatography Method for Benzyl Alcohol, Benzaldehyde, T rans- Cinnamyl Alcohol, and Trans-Cinnamaldehyde. GC was operated in constant flow mode with a carrier gas flowrate of 2 mL / min. 1 pL of sample was injected into the instrument. The inlet was maintained at 250 °C. The split ratio was 5: 1. The oven was held at 120 °C for 2 minutes, then ramped at 10 °C / min to 230 °C. then held at 230 °C for 5 minutes. The flameionization detector was maintained at 300 °C with an air flowrate of 350 mL / min, H2 flowrate of 30 mL / min, and a makeup nitrogen gas flowrate of 25 mL / min. Helium was used as the carrier gas (see FIG. 9, FIG. 12, FIG. 27, FIG. 28, FIG. 36, FIG. 38, and FIG. 39)

[0154] Gas Chromatography Method for 2-Furfuryl Alcohol. GC was operated in constant flow mode with a carrier gas flowrate of 1.8 mL / min. 1 pL of sample was injected into the instrument. The inlet was maintained at 250 °C. The split ratio was 5: 1. The oven was held at 50 °C for 5 minutes, then ramped at 10 °C / min to 230 °C, then held at 230 °C for 12 minutes. The flame-ionization detector was maintained at 300 °C with an air flowrate of 350 ml / min, H2 flowrate of 30 mL / min, and a makeup nitrogen gas flowrate of 25 mL / min. Helium was used as the carrier gas (see FIG. 13).

[0155] HPLC Analysis of 4-Hydroxybenzyl Alcohol and Vanillyl Alcohol. HPLC analysis was carried out on an Agilent 1100 HPLC equipped with a diode array detector. For the detection of 4-hydroxybenzyl alcohol and vanillyl alcohol, a Phenomenex Luna Omega 5pm Polar C18 100A column (150 x 4.6 mm) was used for separation. Isocratic separation was performed using a mobile phase of 50:50 0.1% (v / v) formic acid:0.1% (v / v) formic acid in methanol at a flowrate of 1 mL / min. The column was maintained at 40 °C. The diode array detector was monitored at 240 nm. 5 pL of sample was injected (see FIG. 10, FIG. 11, and FIG. 31)

[0156] HPLC Analysis of 1,5- pentanediol. HPLC analysis was carried out on an Agilent 1100 HPLC equipped with a diode array detector. For the detection of 1,5- pentanediol, a Bio-Rad Fast Acid Analysis HPLC Column (100 x 7.8 mm) was used forAtorney docket No. 00058-087W01 separation. A mobile phase of 20 rnM sulfuric acid was flowed through the column at 1 mL / min. The column was maintained at 55 °C. The refractive index detector was maintained at 55 °C. 15 pL of sample was injected into the system (see FIG. 14).Example 1.

[0157] Use of aldehyde dehydrogenase to directly reduce a carboxylic acid to an aldehyde followed by a subsequent enzymatic modification by a second enzyme (see FIG. 2). In this example, the second enzyme is an alcohol dehydrogenase. In this example, the starting substrates include linear aliphatic acids (acetic acid, propanoic acid, butyric acid, hexanoic acid, nonanoic acid, etc.), branched aliphatic acids (isobuty ric acid, isopentanoic acid, etc.), aromatic carboxylic acids (such as benzoic acid. 2-furoic acid, vanillic acid), and formic acid. FIGs. 3-14 presents the results of the direct reduction of various substrates, without ATP or CoA-based activation, including, to hexanol (see FIGs. 3-4), to ethanol (see FIG. 5), to propanol (see FIG. 6), to butanol (see FIG. 7), to isobtutanol (see FIG. 8), to benzyl alcohol (see FIG. 9), to 4-hydroxybenzyl alcohol (see FIG. 10), to vanilyl alcohol (see FIG. 11), to cinnanyl alcohol (see FIG. 12), to furfuryl alcohol (see FIG. 13). and to 1,5-pentanediol (see FIG. 14)Example 2.

[0158] Use of aldehyde dehydrogenase to directly reduce a carboxylic acid to an aldehyde followed by a subsequent enzymatic modification by a second enzyme to condense the aldehyde with an additional molecule. The second enzyme can be any enzyme to further modify the aldehyde, but in this particular example, acetolactate synthase is used for demonstration purposes (see FIG. 15). For this example, benzoic acid is direct reduced to benzaldehyde by an aldehyde dehydrogenase, which is then modified by acetolactate synthase and pyruvate to form L-phenyl acetyl carbinol (L-PAC) (see FIG. 16).Example 3

[0159] Use of an aldehyde dehydrogenase to directly reduce a carboxylic acid to produce aldehydes without the use of additional enzymatic modification of the aldehyde (see FIG. 17). In this example, the starting substrates include linear aliphatic acids (acetic acid, propanoic acid, butyric acid, hexanoic acid, nonanoic acid, etc.), branched aliphatic acids (isobutyric acid, isopentanoic acid, etc.), aromatic carboxylic acids (such as benzoic acid, 2- furoic acid, vanillic acid), diacids (such as succinic acid, adipic acid, etc.), hydroxyacids (such as 4-hydroxylbutyric acid, 3-hydroxylbulyric acid, lactic acid, 2-hydroxylpropanoicAttorney docket No. 00058-087W01 acid, etc.) and formic acid. As shown in FIG. 18, hexanoic acid was directly reduced to hexanal by a NADH-dependent aldehyde dehydrogenase. FIG. 19 shows the direct reduction of butyric acid to butyraldehyde by an aldehyde dehydrogenase. FIG. 20 shows the direct reduction of isobutyric acid to isobutyraldehyde by an aldehyde dehydrogenase.Example 4

[0160] Use of an aldehyde dehydrogenase to directly reduce a carboxylic acid to produce aldehydes (see FIG. 21) with the use of in situ extraction method to improve production of aldehydes from the direct reduction of carboxylic acids (see FIG. 22). In this example, the starting substrates include linear aliphatic acids (acetic acid, propanoic acid, butyric acid, hexanoic acid, nonanoic acid, etc.), branched aliphatic acids (isobutyric acid, isopentanoic acid, etc.), aromatic carboxylic acids (such as benzoic acid, 2-furoic acid, vanillic acid), diacids (such as succinic acid, adipic acid, etc ), hydroxyacids (as 4- hydroxylbutyric acid, 3-hydroxylbutyric acid, lactic acid, 2-hydroxylpropanoic acid, etc.) and formic acid.

[0161] As shown in FIG. 22, gas is bubbled into the reactor to strip volatile aldehydes from the liquid phase into the gaseous air. Aldehydes in the gaseous phase are subsequently trapped using condensers filled with solvent. In this example the gas is air. In this example, the aldehyde dehydrogenase from Geobacillus thermodenitrificans A4IT08 (SEQ ID NO:33) reduces benzoic acid to benzaldehyde (see FIG. 23).Example 5

[0162] Use of an engineered aldehyde dehydrogenase to improve the rate of carboxylic acid reduction to aldehydes. In this example, the aldehyde dehydrogenase is additionally paired with a second enzyme, such as an alcohol dehydrogenase, to drive aldehyde production. In this example, enzymatically engineered variants of aldehyde dehydrogenase are paired with an alcohol dehydrogenase to first reduce carboxylic acid to aldehyde and subsequently reduce aldehyde to alcohol. In this example, the starting substrates include linear aliphatic acids (acetic acid, propanoic acid, butyric acid, hexanoic acid, nonanoic acid, etc.), branched aliphatic acids (isobutyric acid, isopentanoic acid, etc.), aromatic carboxylic acids (such as benzoic acid, 2-furoic acid, vanillic acid), diacids (such as succinic acid, adipic acid, etc.), hydroxyacids (such as 4-hydroxylbutyrate, 3- hydroxylbutyrate, lactate, 2-hydroxylpropanate, etc) and formic acid.Atorney docket No. 00058-087W01

[0163] For this example, the aldehyde dehydrogenase is additionally paired with a second enzyme, such as an alcohol dehydrogenase, to drive aldehyde production (see FIG. 24). As s ow n in FIGs. 25-26 and 30, enzymatically engineered variants of an aldehyde dehydrogenase are paired with an alcohol dehydrogenase to first reduce hexanoic acid to hexanal and subsequently reduce hexanal to hexanol. As shown in FIG. 27, enzymatically engineered variants of an aldehyde dehydrogenase are paired with an alcohol dehydrogenase to first reduce benzoic acid to benzaldehyde and subsequently reduce benzaldehyde to benzyl alcohol. As shown in FIG. 28, enzymatically engineered variants of an aldehyde dehydrogenase are paired with an alcohol dehydrogenase to first reduce trans-cinnamic acid to cinnamaldehyde and subsequently reduce cinnamaldehyde to trans-cinnamyl alcohol. As shown in FIG. 29, enzymatically engineered variants of an aldehyde dehydrogenase are paired with an alcohol dehydrogenase to first reduced aliphatic carboxylic acids, hexanoic acid and propanoic acid, to aldehydes, and subsequently reduce aldehydes to alcohols, hexanol and propanol.Example 6

[0164] Use of an engineered aldehyde dehydrogenase to enable the direct reduction of carboxylic acids to aldehydes when the wild-type enzyme does not exhibit the ability’ to reduce carboxylic acid to aldehyde. In this example, the starting substrates include linear aliphatic acids (acetic acid, propanoic acid, butyric acid, hexanoic acid, nonanoic acid, etc.), branched aliphatic acids (isobutyric acid, isopentanoic acid. etc.), aromatic carboxylic acids (such as benzoic acid, 2-furoic acid, vanillic acid), diacids (such as succinic acid, adipic acid, etc.), hydroxyacids (as 4-hydroxylbutyric acid, 3-hydroxylbutyric acid, lactic acid, 2- hydroxylpropanoic acid, etc.) and formic acid. An engineered version of an aldehyde dehydrogenase was used to directly reduce 4-hydroxybenzoic acid to 4- hydroxybenzaldehyde, where 4-hydroxybenzaldehyde is subsequently reduced to 4- hydroxybenzyl alcohol using an alcohol dehydrogenase (see FIG. 31).Example 7

[0165] Use of an engineered aldehyde dehydrogenase to improve the rate of carboxylic acid reduction activity of an aldehyde dehydrogenase without the pairing of an additional enzy me to further modify the aldehyde. In this example, the starting substrates include linear aliphatic acids (acetic acid, propanoic acid, butyric acid, hexanoic acid, nonanoic acid, etc.), branched aliphatic acids (isobutyric acid, isopentanoic acid, etc.),Atorney docket No. 00058-087W01 aromatic carboxylic acids (such as benzoic acid. 2-furoic acid, vanillic acid), diacids (such as succinic acid, adipic acid, etc.), hydroxyacids (as 4-hydroxylbutyric acid. 3-hydroxylbutyric acid, lactic acid, 2-hydroxylpropanoic acid, etc.) and formic acid. As shown in FIG. 32, engineered versions of an aldehyde dehydrogenase are applied to directly reduce hexanoic acid to hexanal. Engineered versions of an aldehy de dehydrogenase were also applied to directly reduce hexanoic acid to hexanal (see FIG. 33).Example 8

[0166] Use of an aldehyde dehydrogenase with a chemical reagent to improve activity of enzy matic reaction. In this example aldehyde dehydrogenase is additionally paired with the supplementation of a chemical reagent, in this example, dithiothreitol (DTT), to improve the rate of carboxylic acid reduction (see FIG. 34). In this example, the starting substrates include linear aliphatic acids (acetic acid, propanoic acid, butyric acid, hexanoic acid, nonanoic acid, etc.), branched aliphatic acids (isobuty ric acid, isopentanoic acid, etc.), aromatic carboxylic acids (such as benzoic acid. 2-furoic acid, vanillic acid), diacids (such as succinic acid, adipic acid, etc.), hydroxyacids (as 4-hydroxylbutyric acid. 3-hydroxylbutyric acid, lactic acid, 2-hydroxylpropanoic acid, etc.) and formic acid.Example 9

[0167] Use of an aldehyde dehydrogenase with an organic solvent overlay to continuously extract products derived from the direct reduction of a carboxylic acid mediated by an aldehyde dehydrogenase. In this example, aldehydes produced from the ALDH- mediated direct reduction of the carboxylic acid are subsequently reduced to an alcohol using an alcohol dehydrogenase. Aldehydes produced from the direct reduction of a carboxylic acid to an aldehyde are extracted from the aqueous reaction or transformed by an alcohol dehydrogenase to an alcohol. Alcohol produced from this process in also extracted from the aqueous reaction (see FIG. 35). As shown in FIG. 36, an aldehyde dehydrogenase is used to produce benzyl alcohol from benzoic acid where an organic solvent is used to continuously extract the product formed from the aldehyde dehydrogenase-mediated reduction of benzoic acid.Example 10

[0168] Use of an aldehyde dehydrogenase with an organic solvent overlay to continuously extract products derived from the direct reduction of a carboxylic acid mediated by an aldehyde dehydrogenase (see FIG. 37). In this example, alcohol dehydrogenase isAtorney docket No. 00058-087W01 omitted from the system to produce aldehydes. In some manifestations, the use of an organic solvent overlay supports improved aldehyde product specificity by reducing the amount of non-specific alcohol production in the system, relative to a system without an organic solvent overlay. In some manifestations, the use of an organic solvent overlay supports improved aldehyde production, relative to a system without an organic solvent overlay. In another manifestation, the use of an organic solvent overlay supports production of an aldehyde or alcohol when the system without an organic solvent overlay was unable to produce an aldehyde or an alcohol.

[0169] As shown in FIG. 38, use of an organic overlay promoted formation of benzaldehyde from benzoic acid, with anisole providing the best effect. As shown in FIG. 39, an overlay of anisole promoted the formation of cinnamaldehyde from trans-cinnamic acid. As shown in FIG. 40, an overlay of 1 :1 ethoxybenzene to sample, promoted the formation of hexanal from hexanoic acid. As shown in FIG. 41, an overlay of 3:1 organic solvent to sample, promoted the formation of hexanal from hexanoic acid. As shown in FIG. 42, an overlay of 3: 1 ethoxybenzene to sample, promoted the formation of propionaldehyde from propionic acid. As shown in FIG. 43, an overlay of 3: 1 ethoxybenzene to sample, promoted the formation of butanal from butanoic acid. As shown in FIG. 44, an overlay of 3: 1 ethoxybenzene to sample, promoted the formation of isobutanal from isobutyric acid, and an overlay of 5: 1 ethoxy benzene to sample, promoted even greater formation of isobutanal from isobutyric acid.Example 11

[0170] Use of an aldehyde dehydrogenase to produce a carboxylic acid to an alcohol where the redox cofactor is recycled using a chemical reagent (see FIG. 45). In this manifestation, dithionite is used as the chemical reagent to reduce oxidized redox cofactor to the reduced form. The recycled redox cofactor is subsequently used by the aldehyde dehydrogenase to perform the direct reduction of carboxylic acids to aldehydes. The aldehydes produced from the aldehyde dehydrogenase-mediated direct reduction of carboxylic acids is subsequently reduced by an alcohol dehydrogenase. In this example, the alcohol dehydrogenase also utilizes the reduced redox cofactor produced by the chemical reagent. As shown in FIG. 46, adding a redox cofactor, dithionite, greatly increased formation of propanol from the propanoic acid, by first reducing propanoic acid toAtorney docket No. 00058-087W01 propionaldehyde using an aldehyde dehydrogenase, and then reducing propionaldehyde to propanol using an alcohol dehydrogenase.Example 12

[0171] Use of an aldehyde dehydrogenase to reduce a carboxylic acid to an aldehyde where the redox cofactor is recycled using a chemical reagent (see FIG. 47). In this manifestation, dithionite is used as the chemical reagent to reduce oxidized redox cofactor to the reduced form. The recycled redox cofactor is subsequently used by the aldehyde dehydrogenase to perform the direct reduction of carboxylic acids to aldehydes. As shown in FIG. 48, adding a redox cofactor, dithionite, greatly increased formation of hexanal from the hexanoic acid using an aldehyde dehydrogenase.Example 13

[0172] Use of an aldehyde dehydrogenase to reduce a carboxylic acid to an aldehyde where the redox cofactor is not the natural redox cofactor NADH or NADPH, herein referred to as a noncanonical cofactor (see FIG. 49 and FIG. 51). In this example, a method of continuous product removal is applied to extract the aldehydes produced during the production process. In this example, an organic solvent overlay w as applied to continuously extract the aldehyde produced from the aldehyde dehydrogenase-mediated direct carboxylic acid reduction.

[0173] As shown in FIG. 50, adding a noncanonical cofactor, carba-nicotinamide adenine dinucleotide (cNADH), promoted formation of hexanal from the hexanoic acid using an aldehyde dehydrogenase. As shown in FIG. 52, adding a noncanonical cofactor, nicotinamide mononucleotide (NMNH), greatly promoted formation of hexanal from the hexanoic acid using an aldehyde dehydrogenase.

[0174] A number of embodiments have been described herein. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of this disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

Atorney docket No. 00058-087W01WHAT IS CLAIMED IS:

1. A cell-free system or whole-cell biomanufacturing system to facilitate the biotransformation of a carboxylic acid containing substrate into a desired product, comprising: a first polypeptide encoding an aldehyde dehydrogenase that directly reduces carboxylic acids to aldehydes without adenosine triphosphate (ATP) or coenzyme A (CoA)- based activation, wherein the aldehyde dehydrogenase can use reduced nicotinamide-adenine dinucleotide (NADH) as a cofactor; and a first hydride donor compound; optionally, a second polypeptide that encodes an enzyme capable of converting an aldehyde group into a non-aldehyde group; optionally, a second hydride donor compound and a third polypeptide that encodes an enzyme that reduces the first hydride donor compound using the second hydride donor compound.

2. The cell-free system or whole-cell biomanufacturing system of claim 1, wherein the first polypeptide comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to a sequence presented in SEQ ID NO:1 to 170.

3. The cell-free system or whole-cell biomanufacturing system of claim 2, wherein the first polypeptide comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%. is at least 95%. at least 97%. at least 98%. or at least 99% identical to a sequence presented in SEQ ID NO: 1, SEQ ID NO:39, SEQ ID NO:2, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:99, SEQ ID NO: 105, SEQ ID NO: 119, SEQ ID NO: 126, and SEQ ID NO: 170.

4. The cell-free system or whole-cell biomanufacturing system of claim 1. wherein the first polypeptide is engineered to contain 1 to 10 amino acid substitutions in comparison to a sequence presented in SEQ ID NO: 1 to 170, wherein the amino acid substitution(s) improve the aldehyde dehydrogenase activity for directly reducing carboxylic acids to aldehydes.Atorney docket No. 00058-087W015. The cell-free system or whole-cell biomanufacturing system of claim 4. wherein the first polypeptide has the sequence of SEQ ID NO:39 with at least the amino acid substitution ofY402F.

6. The cell-free sy stem or whole-cell biomanufacturing system of claim 4, wherein the first polypeptide has the sequence of SEQ ID NO:2 with at least an amino acid substitution selected from Q350N, Q350R. and Q350A.

7. The cell-free sy stem or whole-cell biomanufacturing system of claim 4. wherein the first polypeptide has the sequence of SEQ ID NO:31 with one or more amino acid substitutions selected from (a). (b), (c), (d), (e), and / or (f):(a) W185S, W185D, W185H. W185N, W185K, or W185F;(b) N186D;(c) E285D:(d) F288A;(e) Q366I, Q366A, Q366L, Q366M, or Q366N; and / or(f) F418A or F418H.

8. The cell-free system or whole-cell biomanufacturing system of claim 7, wherein the first polypeptide has the sequence of SEQ ID NO:31 with at least an amino acid substitution selected from W185N, W185K, and W185F.

9. The cell-free system or whole-cell biomanufacturing system of claim 4. wherein the first polypeptide has the sequence of SEQ ID NO:1 with one or more amino acid substitutions selected from (a), (b) and / or (c):(a) W164F;(b) T302S; and / or(c) W1731 L W173F, and W173Y.

10. The cell-free sy stem or w hole-cell biomanufacturing system of claim 4, wherein the first polypeptide has the sequence of SEQ ID NO:99 with at least an amino acid substitution selected from W173M, W173F, and W173Y.Atorney docket No. 00058-087W0111. The cell-free system or whole-cell biomanufacturing system of claim 1. wherein the cell-free system or whole-cell biomanufacturing system comprises the second polypeptide that encodes an enzyme capable of converting an aldehyde group into a non-aldehyde group.

12. The cell-free sy stem or whole-cell biomanufacturing system of claim 11, wherein the second polypeptide encodes an enzyme capable of converting an aldehyde group into a nonaldehyde group, utilizes the first hydride donor compound as a cofactor.

13. The cell-free system or whole-cell biomanufacturing system of claim 12, wherein the second polypeptide encodes an alcohol dehydrogenase, or an acetolactate synthase.

14. The cell-free system or whole-cell biomanufacturing system of claim 13, wherein the second polypeptide encodes an alcohol dehydrogenase that has a sequence that is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 172 or SEQ ID NO: 173.

15. The cell-free sy stem or whole-cell biomanufacturing system of claim 13, wherein the second polypeptide encodes an acetolactate synthase that has a sequence that is at least 95%. at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 174.

16. The cell-free system or whole-cell biomanufacturing system of claim 1, wherein the cell-free system or whole-cell biomanufacturing system comprises the third polypeptide and the second hydride donor compound.

17. The cell-free system or whole-cell biomanufacturing system of claim 16, wherein the third polypeptide encodes a phosphite dehydrogenase that has a sequence that is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 171, and wherein the second hydride donor compound is phosphite.

18. The cell-free sy stem or whole-cell biomanufacturing system of claim 16, wherein the third polypeptide encodes a glucose dehydrogenase that has a sequence that is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 175, and wherein the second hydride donor compound is glucose.Atorney docket No. 00058-087W0119. The cell-free system or whole-cell biomanufacturing system of claim 1. wherein the cell-free system or whole-cell biomanufacturing system further comprises 1 ,4-dithiothreitol.

20. The cell-free system or whole-cell biomanufacturing system of any one of claims 1 to 19, wherein the system is a cell-free system, and the cell-free system utilizes polypeptide(s) that have been isolated and purified.

21. The cell-free system or whole-cell biomanufacturing system of claim 20, wherein the cell-free system further comprises an organic solvent overlay that supports production of an aldehyde by the first polypeptide and / or a non-aldehyde product produced by the second polypeptide, and wherein the polypeptide(s) and hydride donor(s) are in an aqueous solution.

22. The cell-free sy stem or whole-cell biomanufacturing system of claim 21, wherein the organic overly comprises a solvent selected from anisole, phenetole, hexane, isooctane, toluene, dodecane, oleyl alcohol, and ethoxybenzene.

23. The cell-free sy stem or whole-cell biomanufacturing system of claim 21, wherein the organic solvent overlay is used at a ratio of 1 : 10 to 10: 1 to the aqueous solution.

24. The cell-free system or whole-cell biomanufacturing system of claim 21, wherein the organic solvent overlay is used at a ratio of 1 : 1 to 10: 1 to the aqueous solution.

25. The cell-free system or whole-cell biomanufacturing system of claim 1. wherein the cell-free system or whole-cell biomanufacturing system further comprises an apparatus to isolate a desired product that is an aldehyde, wherein the apparatus comprises: a reactor that can be heated which comprises the product aldehyde, wherein the reactor is in fluid connection to one or more cooled condensers filled with a solvent, wherein in heating the reactor causes the product aldehyde to go in the gaseous phase; a gas supply that is in fluid connection with the reactor, that is configured to introduce positive gas flow into the reactor, wherein the gas introduced into the reactor flows from the reactor to the one or more cooled condensers; andAtorney docket No. 00058-087W01 one of more cooled condensers filled with a solvent that are configured to cool the gaseous aldehyde into a liquid phase so that it is trapped by the solvent, wherein the aldehyde can then be isolated from the solvent.

26. The cell-free system or whole-cell biomanufacturing system of claim 1, wherein the system is a whole-cell biomanufacturing system wherein vectors are used to express the polypeptide(s) in a microorganism.

27. The cell-free system or whole-cell biomanufacturing system of claim 26, wherein the microorganism is selected from a bacterium or yeast.

28. The cell-free system or whole-cell biomanufacturing system of claim 1, wherein the first hydride donor compound is selected from reduced nicotinamide-adenine dinucleotide (NADH). reduced nicotinamide-adenine dinucleotide phosphate (NADPH), carba- ni cotinamide adenine dinucleotide (cNADH), reduced nicotinamide mononucleotide (NMNH), reduced 3-carbamoyl-l-(4-carboxybenzyl) pyridin-l-ium (BANAH), reduced 1- benzylnicotinamide (BNAH), reduced 3-carbamoyl-l-(3-phenylpropyl)pyridin-l-ium (3PNAH), and reduced 3-carbamoyl-l-(4-methoxybenzyl)pyridin-l-ium (BNA OMeH).

29. The cell-free system or whole-cell biomanufacturing system of claim 28, wherein the first hydride donor compound is cNADH or NMNH.

30. A method to convert a carboxylic acid containing substrate to an aldehy de containing product, the method comprising: contacting the carboxylic acid containing substrate with a first polypeptide that encodes an aldehyde dehydrogenase that directly reduces carboxylic acids to aldehydes without adenosine triphosphate (ATP) or coenzy me A (CoA)-based activation in the presence of a first hydride donor compound in an aqueous solution, optionally, providing a second hydride donor compound and a second polypeptide that encodes an enzyme that reduces the first hydride donor compound using the second hydride donor compound,Atorney docket No. 00058-087W01 wherein the aldehyde dehydrogenase can use reduced first hydride donor compound as a cofactor, and wherein the aldehyde dehydrogenase converts the carboxylic acid containing substrate to an aldehyde containing product.

31. The method of claim 30, wherein the carboxylic acid containing substrate is selected from a linear aliphatic carboxylic acid, a branched aliphatic carboxylic acid, an aromatic carboxylic acid, a diacid, a hydroxyacid and formic acid.

32. The method of claim 30, wherein the linear aliphatic carboxylic acid is selected from acetic acid, propanoic acid, butyric acid, hexanoic acid, and nonanoic acid.

33. The method of claim 30, wherein the branched aliphatic carboxylic acid is selected from isobutyric acid, and isopentanoic acid.

34. The method of claim 30, wherein the aromatic carboxylic acid is selected from benzoic acid, 2-furoic acid, cinnamic acid and vanillic acid.

35. The method of claim 30, wherein the diacid is selected from succinic acid, and adipic acid.

36. The method of claim 30, wherein the hydroxyacid is selected from 4-hydroxybutync acid, 3 -hydroxy butyric acid, lactic acid, and 2-hydroxylpropanoic acid.

37. The method of any one of claims 30 to 36, wherein the first polypeptide comprises a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98%, or at least 99% identical to a sequence presented in SEQ ID NO: 1 to 170.

38. The method of claim 37, wherein the first polypeptide comprises a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98%, or at least 99% identical to a sequence presented in SEQ ID NO: 1, SEQ ID NO:39, SEQ ID NO:2, SEQAttorney docket No. 00058-087W01ID N0:31, SEQ ID NO:34, SEQ ID NO:99, SEQ ID NO: 105, SEQ ID NO: 119, SEQ ID NO: 126, and SEQ ID NO: 170.

39. The method of any one of claims 30 to 36, wherein the first polypeptide is engineered to contain 1 to 10 amino acid substitutions in comparison to a sequence presented in SEQ ID NO: 1 to 170. wherein the amino acid substitution(s) improve the aldehyde dehydrogenase activity for directly reducing carboxylic acids to aldehydes.

40. The method of claim 39, wherein the first polypeptide has the sequence of SEQ ID NO:39 with at least the amino acid substitution of Y402F.

41. The method of claim 39, wherein the first polypeptide has the sequence of SEQ ID NO:2 with an amino acid substitution selected from Q350N, Q350R, and Q350A.

42. The method of claim 39, wherein the first polypeptide has the sequence of SEQ ID NO:31 but with one or more amino acid substitutions selected from (a), (b), (c), (d), (e), and / or (f):(a) W185S, W185D, W185H, W185N, W185K, or W185F;(b) N186D;(c) E285D;(d) F288A;(e) Q366I, Q366A, Q366L, Q366M, or Q366N; and / or(I) F418A or F418H.

43. The method of claim 42, wherein the first polypeptide has the sequence of SEQ ID NO:31 with an amino acid substitution selected from W185N, W185K, and W185F.

44. The method of claim 39, wherein the first polypeptide has the sequence of SEQ ID NO: 1 with one or more amino acid substitutions selected from (a), (b) and / or (c):(a) W164F;(b) T302S; and / or(c) W173M. W173F, W173Y.Atorney docket No. 00058-087W0145. The method of claim 39, wherein the first polypeptide has the sequence of SEQ ID NO:99 with at least an amino acid substitution selected from W173M, W173F. and W173Y.

46. The method of claim 30, wherein the method further comprises isolating the aldehydes during the reaction process.

47. The method of claim 46, wherein the method further comprises overlaying an organic solvent on top of the aqueous solution to allow for the continuous extraction of the aldehydes.

48. The method of claim 47, wherein the organic solvent is selected from anisole, phenetole, hexane, isooctane, toluene, dodecane, oleyl alcohol, and ethoxybenzene.

49. The method of claim 46, wherein the organic solvent is used at a ratio of 1 : 10 to 10: 1 to the aqueous solution.

50. The method of claim 46, wherein the organic solvent is used at a ratio of 1 : 1 to 10: 1 to the aqueous solution.

51. The method of claim 46, wherein the method further comprises isolating the aldehyde containing products by using an apparatus, the apparatus comprising: a reactor that can be heated which comprises the product aldehyde, wherein the reactor is in fluid connection to one or more cooled condensers filled with a solvent, wherein in heating the reactor causes the product aldehyde to go in the gaseous phase; a gas supply that is in fluid connection with the reactor, that is configured to introduce positive gas flow into the reactor, wherein the gas introduced into the reactor flows from the reactor to the one or more cooled condensers; and one of more cooled condensers filled with a solvent that are configured to cool the gaseous aldehyde into a liquid phase so that it is trapped by the solvent, wherein the aldehyde can then be isolated from the solvent.Atorney docket No. 00058-087W0152. The method of claim 30, wherein the method comprises the second hydride donor compound and the second polypeptide that encodes an enzyme that reduces the first hydride donor compound using the second hydride donor compound.

53. The method of claim 52, wherein the second polypeptide encodes a phosphite dehydrogenase that has a sequence that at least 75%, at least 80%, at least 85%, is at least 95%. at least 97%. at least 98% or at least 99% identical to SEQ ID NO:

171. and wherein the second hydride donor compound is phosphite.

54. The method of claim 52, wherein the second polypeptide encodes a glucose dehydrogenase that has a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 175, and wherein the second hydride donor compound is glucose.

55. The method of claim 30, wherein the method further comprises converting the aldehyde containing product to non-aldehyde containing product, comprising: contacting the aldehyde containing product with a third polypeptide that that encodes an enzy me capable of converting an aldehyde group into a non-aldehyde group.

56. The method of claim 55. wherein the third polypeptide that encodes an enzyme capable of converting an aldehyde group into a non-aldehyde group, utilizes the first hydride compound as a coenzy me or cofactor.

57. The method of claim 55. wherein the third polypeptide encodes an alcohol dehydrogenase, or an acetolactate synthase.

58. The method of claim 57, wherein the third polypeptide encodes an alcohol dehydrogenase that has a sequence that at least 75%, at least 80%, at least 85%, is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 172 or SEQ ID NO: 173.Attorney docket No. 00058-087W0159. The method of claim 57, wherein the third polypeptide encodes an acetolactate synthase that has a sequence that at least 75%. at least 80%. at least 85%. is at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 174.

60. The method of claim 30, wherein the first hy dride donor compound is selected from reduced nicotinamide-adenine dinucleotide (NADH), reduced nicotinamide-adenine dinucleotide phosphate (NADPH). carba-mcotinamide adenine dinucleotide (cNADH), reduced nicotinamide mononucleotide (NMNH), reduced 3-carbamoyl-l-(4-carboxybenzyl) pyridin-l-ium (BANAH), reduced 1 -benzylnicotinamide (BNAH), reduced 3-carbamoyl-l- (3-phenylpropyl)pyridin-l-ium (3PNAH), and reduced 3 -carbamoyl- 1 -(4- methoxybenzyl)py ridin- 1 -ium (BN A OMeH).

61. The method of claim 60, wherein the first hydride donor compound is cNADH or NMNH.

Images