Coa-independent reductive aldolases for enzymatic-catalyzed reductive aldol reactions

Abstract

The present invention relates to CoA-independent reductive aldolases derived from ene reductases (ERs) for preparing α-branched β'-hydroxy carbonyl compounds through enzymatic-catalyzed reductive aldol reaction by reacting α,β-unsaturated carbonyl donors with carbonyl acceptors in the presence of a polypeptide capable of catalyzing reductive CoA-independent aldol reactions and a cofactor, wherein the polypeptide is a modified ene reductase (ER). The present invention further relates to a method for preparing α-branched β'-hydroxy carbonyl compounds through enzymatic-catalyzed reductive aldol reaction.

Description

[0001] CoA-independent reductive aldolases for enzymatic-catalyzed reductive aldol reactions

[0002] Specification

[0003] The present invention relates to CoA-independent reductive aldolases derived from ene reductases (ERs) for preparing a-branched P’-hydroxy carbonyl compounds through enzymatic-catalyzed reductive aldol reaction by reacting a,p-unsaturated carbonyl donors with carbonyl acceptors in the presence of a polypeptide capable of catalyzing CoA-independent reductive aldol reactions and a cofactor, wherein the polypeptide is a modified ene reductase (ER). The present invention further relates to a method for preparing a-branched P’-hydroxy carbonyl compounds through enzymatic-catalyzed reductive aldol reaction in the presence of polypeptide capable of catalyzing CoA-independent reductive aldol reactions, wherein the polypeptide is a modified ene reductase (ER).

[0004] Background of the invention

[0005] C-C bond forming reactions allow the synthesis of complex molecules from simpler precursors and are a cornerstone of chemistry and biology. Among the most important C-C forming reactions are aldol reactions through which two carbonyl compounds are converted into a p-hydroxy functionalized carbonyl product. Many biologically relevant compounds display this structural element in their backbone, which makes these molecules highly relevant targets for organic synthesis. Reductive aldol reactions enable the direct coupling of a,p-unsaturated carbonyl compounds with carbonyl electrophiles. Although introduced to organic synthesis more than 30 years ago, a biocatalytic equivalent for this reaction type is still lacking.

[0006] In nature, C-C bond formations are catalyzed by aldolases, which provide an interesting alternative to purely synthetic chemical routes, because these enzymes can operate under mild conditions at high turnover rates and with exquisite diastereo- and enantioselectivity. Over the last years, various naturally occurring aldolases have been successfully evolved for improved activity under different conditions and engineered for increased promiscuity past their natural substrates for diverse applications in biosynthesis and organo-catalysis.

[0007] More than 30 years ago, reductive aldol reactions were introduced to organic chemistry by Revis and Hilty. The reductive aldol reactions enable the direct, catalytic coupling of a,[3-unsaturated carbonyl compounds, such as enoates or acrylates with carbonyl electrophiles, which has greatly extended the scope of C-C bond forming reactions. However, several challenges remain for application of these reactions in synthetic chemistry. The majority of reductive aldol reactions involve hydrosilanes as reductants which are relatively expensive and lead to unwanted adducts that incorporate silyl ether moieties. Other approaches employ precious and scarce transition metals as catalysts, which allow diastereomer- and enantioselective C-C couplings, but require enantiomerically pure organo ligands that are difficult to synthesize. In principle, above challenges could be overcome by the availability of suitable biocatalysts that would enable stereoselective reductive aldol reactions under mild and sustainable reaction conditions. However, so far, no enzyme has been described that is able to catalyze a reductive aldol reaction.

[0008] Isoda et al., Chem. Pharm Bull. 2014, Vol. 62, No. 10, 956-961 disclose an asymmetric reductive aldol-type reaction of a,[3-unsaturated esters with carbonyl compounds using an Rh catalyst and Et2Zn. A chiral zinc complex is generated from the a,[3-unsaturated ester as the key intermediate from Et2Zn and Wilkinson’s catalyst with diisopropyl l-(+)-tartrate to give a variety of enantioenriched [3-hydroxy esters.

[0009] Database UniProt excerpt, K. Saito et al., NADPH dehydrogenase 1 , EC=1.6.99.1 discloses old yellow enzyme 1 OYE1 (NADPH dehydrogenase 1 , EC=1.6.99.1) from Saccharomyces pastorianus (Saccharomyces cerevisiae x Saccharomyces eubayanus), Database UniProt excerpt, M. Miranda et al., Enoate Reductase, ER, EC=1.3.1.31 discloses old yellow enzyme 1 KYE1 (Enoate Reductase, ER, EC=1.3.1.31 ) from Kluyveromyces lactis, Database UniProt excerpt, K. Miura et al., N-Ethylmaleimide reductase, NEM reductase, EC=1.3.1.- discloses N- Ethylmaleimide reductase, nemA, reductase, EC=1.3.1.- from Escherichia coli (strain K12), and Database UniProt excerpt, H. Takami et al., NADPH dehydrogenase, EC=1.6.99.1 discloses namA (NADPH dehydrogenase, EC=1.6.99.1 ) from Geobacillus kaustophilus (strain HTA426).

[0010] Thus, it is the objective of the present invention to provide modified polypeptides capable of catalyzing reductive aldol reactions and a method for preparing a-branched P’-hydroxy carbonyl compounds by reductive aldol reaction with the use of a biocatalyst that enables stereoselective reductive aldol reactions under mild and sustainable reaction conditions. Thus, it is the objective of the present invention to provide a method for preparing a-branched P’-hydroxy carbonyl compounds by enzymatic-catalyzed reductive aldol reaction.

[0011] The objective of the present invention is solved by the teaching of the independent claims. Further advantageous features, aspects and details of the invention are evident from the dependent claims, the description, the figures, and the examples of the present application.

[0012] Brief description of the invention

[0013] Surprisingly, it has been found that a modification in the amino acid sequence of ene reductases (ERs), in particular an amino acid substitution of the amino acid serving as the active site proton donor, provides polypeptides capable of catalyzing CoA-independent reductive aldol reactions. ERs catalyze the NADPH- dependent reduction of a,[3-unsaturated carbonyl compounds by resolving the enolate intermediate through protonation using a conserved tyrosine as proton donor (Figure 1). The amino acid substitution of this conserved tyrosine leads to suppression of proton donation and establishes a reductive aldol reaction in the scaffold of ERs. Thus, it has been found that polypeptides capable of catalyzing CoA-independent reductive aldol reactions, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence as set forth in SEQ ID NO: 1 , resolve the above objective.

[0014] Surprisingly, it has been found that that a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, comprising performing an enzymatic-catalyzed reductive aldol reaction with an a,[3-unsaturated carbonyl donor and a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde, by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase (ER) comprising the amino acid sequence as set forth in SEQ ID NO: 1 , resolves the above objective.

[0015] To access CoA-independent reductive aldol reactions, the inventors have screened different ERs and identified one homolog, which the inventors further engineered for a selected model reaction in a proof-of-principle. Overall, the present invention expands the repertoire of known enzymatic transformations by establishing reductive aldol couplings as a new-to-nature reaction with great potential for different applications in synthetic biology, biocatalysis and organic synthesis. The inventors of the present invention have realized a CoA-independent reductive aldol reaction in the scaffold of ene reductases (ERs). Mutation of the active site proton donor in the ER homolog NemA from Escherichia coli allowed to establish and optimize the reductive aldol reaction of the model substrate cyclohexenone with formaldehyde in the ER family as a proof-of-principle. Overall, the present invention sets the theoretical and experimental foundation for the development of reductive aldolases, a new-to-nature enzymatic activity of great potential for bio- and organo-catalysis

[0016] The present invention is therefore directed to method for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; c) performing an enzymatic-catalyzed reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1.

[0017] In preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0018] In preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.

[0019] In preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23. In preferred embodiments, the polypeptide further comprises an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0020] In preferred embodiments, the polypeptide further comprises an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 29.

[0021] In preferred embodiments, the cofactor is NADPH.

[0022] In preferred embodiments, the a,[3-unsaturated carbonyl donor has the general formula

[0023] (la) (lb) wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0024] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0025] In preferred embodiments, the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated enone or a,[3-unsaturated enal, preferably wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated cycloenone.

[0026] In preferred embodiments, the a,[3-unsaturated donor is an optionally substituted cyclohexenone.

[0027] In preferred embodiments, the carbonyl acceptor has the general formula (II): wherein R4represents -H, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9,

[0028] -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H11, -CH(CH3)-C3H7, -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5,

[0029] -CH2-C(CH3)3I-CH(C2H5)2, -C2H4-CH(CH3)2, -C6HI3,

[0030] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9I

[0031] -CH2-CH(CH3)-C3H7I-CH(CH3)-CH2-CH(CH3)2I-Ph, or -CH2-Ph.

[0032] In preferred embodiments, the carbonyl acceptor is selected from the group comprising or consisting of formaldehyde, acetaldehyde and propionaldehyde, more preferably wherein the carbonyl acceptor is formaldehyde.

[0033] In preferred embodiments, the enzymatic-catalyzed reductive aldol reaction is performed at a pH of 10 and / or wherein the concentration of the carbonyl acceptors is about 250 mM.

[0034] Description of the invention

[0035] The present invention relates to a method for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0036] According to the present invention, the polypeptide capable of catalyzing CoA- independent reductive aldol reactions is a modified ene reductase. Thus, a polypeptide capable of catalyzing CoA-independent reductive aldol reactions does not correspond to a wild type ene reductase. Moreover, according to the present invention the polypeptide capable of catalyzing CoA-independent reductive aldol reactions is a modified ene reductase modified by at least one amino acid substitution of a conserved tyrosine. Thus, according to the invention, the 14thamino acid of to the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. Therefore, the present invention relates to a method for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,p-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine.

[0037] Thus, the polypeptide capable of catalyzing CoA-independent reductive aldol reactions does not comprise the amino acid sequence as set forth in SEQ ID NO: 6, wherein the the 14thamino acid is tyrosine. More preferably, the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is phenylalanine.

[0038] The term "polypeptide capable of catalyzing reductive aldol reactions", as used herein, refers to an enzyme having the enzymatic activity in which a,p-unsaturated carbonyl donors are coupled with carbonyl acceptors, such as aldehydes, and a-branched P’-hydroxy carbonyl compounds are formed. The term "polypeptide capable of catalyzing CoA-independent reductive aldol reactions", as used herein, refers to an enzyme having the enzymatic activity in which a,p-unsaturated carbonyl donors are coupled with carbonyl acceptors, such as aldehydes, to provide a-branched P’-hydroxy carbonyl compounds, wherein the a,p-unsaturated carbonyl donors do not have to be provided in form of Coenzyme A-thiosters.

[0039] Herein, a “polypeptide capable of catalyzing reductive aldol reactions” is also referred to as “reductive aldolase”. The term "reductive aldolase activity", as used herein, refers to the enzymatic activity of catalyzing reductive aldol reactions of a,p-unsaturated carbonyl donors with carbonyl acceptors, such as aldehydes. Herein, the “polypeptide capable of catalyzing CoA-independent reductive aldol reactions” is also referred to as “CoA-independent reductive aldolase”. The newly identified group of enzymes called CoA-independent reductive aldolases are able to couple a,[3-unsaturated carbonyl donors with carbonyl acceptors to form a-branched P’-hydroxy carbonyl compounds, wherein the a,[3-unsaturated carbonyl donor do not have to be provided in form of a CoA-thioster. a,[3-Unsaturated thioesters are intermediates in several enzymatic processes. Two prominent examples are coumaroyl-coenzyme A and crotonyl-coenzyme A. Consequently, the term "CoA-independent reductive aldolase activity", as used herein, refers to the enzymatic activity of catalyzing CoA-independent reductive aldol reactions of a,p-unsaturated carbonyl donors with carbonyl acceptors, such as aldehydes. According to the method of the present invention, the "polypeptide capable of catalyzing CoA-independent reductive aldol reactions" or the “CoA-independent reductive aldolase” is a modified ene reductase (ER).

[0040] The term “ene reductase” (ER), as used herein, refers to an enzyme catalyzing the reduction of a,[3-unsaturated compounds. Ene reductases (ER) include several families of enzymes that naturally catalyze NAD(P)H-dependent biocatalytic alkene reduction. An ER enzyme family is the FMN-containing Old Yellow Enzyme (OYE) family of oxidoreductases (EC 1.6.99.1 ). These enzymes catalyze the reduction of a,[3-unsaturated compounds, with a high specificity for activating groups containing aldehydes, ketones or nitro groups. Other families of ERs include the oxygen-sensitive FAD and [4Fe-4S]-containing clostridial Enoate Reductases (EnoR; EC 1.3.1.31 ), the leukotriene B4 dehydrogenase subfamily of medium chain dehydrogenases / reductases (MDR; EC 1.3.1 ) and the salutaridine / menthone reductase-like subfamily of short chain dehydrogenases / reductases (SDR; EC 1.1.1.207-8).

[0041] The term “modified ene reductase”, as used herein, refers to a “polypeptide capable of catalyzing CoA-independent reductive aldol reactions”. The modified ene reductases according to the present invention are derived from wild type “ene reductases” by amino acid modification in the peptide sequence. Thus, modified ene reducatases according to the present invention are not naturally occurring wild type ene reductases. The amino acid sequence of ene reductases may be modified by deletion, insertation and / or amino acid substitution, preferably by amino acid substitution. "Percentage of sequence identity" and "percentage homology" are used interchangeably herein to refer to comparisons among polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i.e. gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical amino acid residue occurs in both 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 and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical amino acid residue occurs in both sequences or an amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences.

[0042] "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 polypeptide sequence. Since two polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polypeptides are typically performed by comparing sequences of the polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence.

[0043] "Substantial identity" refers to a polypeptide sequence that has at least 80 percent sequence identity, at least 85 percent identity and 89 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window. For polypeptides, the term "substantial identity" means that two polypeptide sequences, when optimally aligned, share at least 80 percent sequence identity, preferably at least 89 percent sequence identity, at least 95 percent sequence identity or more (e.g., 99 percent sequence identity). Preferably, residue positions which are not identical differ by conservative amino acid substitutions.

[0044] "Deletion" refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference enzyme while retaining enzymatic activity and / or retaining the improved properties of an engineered enoyl-CoA carboxylase / reductase or engineered reductive aldolase. Deletions can be directed to the internal portions and / or terminal portions of the polypeptide.

[0045] "Insertion" refers to modification to the polypeptide by addition of one or more amino acids from the reference polypeptide. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.

[0046] "Amino acid substitution" refers to modification to the polypeptide by substitution of one or more amino acids from the reference polypeptide with other amino acids. Amino acid substitution can comprise substitutions of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference polypeptide while retaining enzymatic activity.

[0047] "Isolated polypeptide" refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it, e.g., protein, lipids, and nucleic acids. 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). The polypeptide may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the polypeptide capable of catalyzing CoA-independent reductive aldol reactions can be an isolated polypeptide. "Improved enzyme property" refers to a polypeptide capable of catalyzing CoA- independent reductive aldol reactions that exhibits an improvement in any enzyme property as compared to a reference polypeptide. For the polypeptides capable of catalyzing reductive aldol reactions described herein, the comparison is generally made to another engineered or modified ene reductase. Enzyme properties for which improvement is desirable include, but are not limited to, enzymatic activity (which can be expressed in terms of percent conversion of the substrate), thermo stability, solvent stability, pH activity profile, cofactor requirements, refractoriness to inhibitors (e.g., substrate or product inhibition), stereospecificity, and stereoselectivity (including enantioselectivity).

[0048] “Increased enzymatic activity” refers to an improved property of the polypeptides capable of catalyzing reductive aldol reactions, which can be represented by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of imine reductase) as compared to the reference polypeptide. Any property relating to enzyme activity may be affected, including the classical enzyme properties of Km, Vmax or kcat, changes of which can lead to increased enzymatic activity. Improvements in enzyme activity can be from about 1.2 times the enzymatic activity of the corresponding wild-type enzyme, to as much as 2 times, 5 times, 10 times, 20 times, 25 times, 50 times or more enzymatic activity than another engineered or modified ene reductase. The activity can be measured by any one of standard assays, such as by monitoring changes in properties of substrates, cofactors, or products. In some embodiments, the amount of products generated can be measured by Liquid Chromatography-Mass Spectrometry (LC-MS). Comparisons of enzyme activities are made using a defined preparation of enzyme, a defined assay under a set condition, and one or more defined substrates. Generally, when lysates are compared, the numbers of cells and the amount of protein assayed are determined as well as use of identical expression systems and identical host cells to minimize variations in amount of enzyme produced by the host cells and present in the lysates.

[0049] "Suitable reaction conditions" refer to those conditions in the biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, cofactor loading, temperature, pH, buffers, co-solvents, etc.) under which a polypeptide capable of catalyzing reductive aldol reactions is capable of catalyzing the reductive aldol reaction of a,[3-unsaturated carbonyl donors with carbonyl acceptors, such as aldehydes, to a-branched P’-hydroxy carbonyl compounds. Exemplary "suitable reaction conditions" are provided in the present disclosure and are illustrated by the Examples.

[0050] "Cofactor regeneration system" or "cofactor recycling system" refers to a set of reactants that participate in a reaction that reduces the oxidized form of the cofactor (e.g., NADP+to NADPH). Cofactors oxidized by the modified ene reductase catalyzed reductive aldol reaction are regenerated in reduced form by the cofactor regeneration system. Cofactor regeneration systems comprise a stoichiometric reductant that is a source of reducing hydrogen equivalents and is capable of reducing the oxidized form of the cofactor. The cofactor regeneration system may further comprise a catalyst, for example an enzyme catalyst that catalyzes the reduction of the oxidized form of the cofactor by the reductant. Cofactor regeneration systems to regenerate NADH from NAD+or NADPH from NADP+, respectively, are known in the art and may be used in the methods described herein.

[0051] "Alkyl" refers to saturated hydrocarbon groups of from 1 to 18 carbon atoms, either straight chained or branched, more preferably from 1 to 8 carbon atoms, and most preferably 1 to 6 carbon atoms. An alkyl with a specified number of carbon atoms is denoted as Ci-Cs alkyl and refers to a linear Ci-Cs alkyl of -CH3, -C2H5, -C3H7, — C4H9, -C5H11, -CeHi3, — C7Hi5, -CSHI7, -CH2-Ph,

[0052] -CH2-CH2-Ph or a branched Ci-Cs alkyl or preferably branched C3-C8 alkyl of -CH(CH3)2, -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3I-CH(CH3)-C3H7, -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5, -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5,

[0053] -CH(CH3)-C4H9, -CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2, -CH(CH3)-CH(CH3)-C2H5, -CH2-CH(CH3)-CH(CH3)2, -CH2-C(CH3)2-C2H5, -C(CH3)2-C3H7, -C(CH3)2-CH(CH3)2, -C2H4-C(CH3)3, -CH(CH3)-C(CH3)3I-C4H8-CH(CH3)2, -C3H6-CH(CH3)-C2H5, -C3H6-CH(CH3)-C2H5, -C2H4-CH(CH3)-C3H7, -CH2-CH(CH3)-C4H9, -CH(CH3)-C5HH, -CH(C2H5)-C4H9, -C2H4-CH(CH3)-C3H7, -CH2-CH(C2H5)-C3H7, -CH2-CH(CH3)-C4H9, -CH2-CH(CH3)-CH2-CH(CH3)2, -CH(C2H5)-CH2-CH(CH3)2, -CH(CH3)-C2H4-CH(CH3)2, -CH(CH3)-CH2-CH(CH3)-C2H5, -CH2-CH(CH3)-CH(CH3)-C2H5, -CH(CH3)-CH2-CH(CH3)-C2H5, -CH(CH3)-CH(C2H5)-C2H5, -CH(C2H5)-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)-C3H7, -C2H4-CH(CH3)-CH(CH3)2I-CH2-CH(C2H5)-CH(CH3)2,

[0054] -CH2-CH(CH3)-CH2-CH(CH3)2, -CH2-CH(CH3)-CH(CH3)-C2H5,

[0055] -C2H4-C(CH3)2-C2H5, -CH2-C(CH3)(C2H5)2, -CH2-C(CH3)2-C3H7I

[0056] -CH2-C(CH3)2-C3H7I-C(CH3)(C2H5)-C3H7, -C(CH3)2-C4H9I

[0057] -CH2-C(CH3)2-CH(CH3)2I-C(CH3)(C2H5)-CH(CH3)2, -C(CH3)2-CH2-

[0058] CH(CH3)2I-C(CH3)2-C(CH3)3, -C(CH3)2-CH(CH3)-C2H5, -C3H6-C(CH3)3,

[0059] C2H4-C(CH3)2-C2H5, -CH2-CH(CH3)-C(CH3)3, -CH(C2H5)-C(CH3)3, CH(CH3)-CH2-C(CH3)3, -CH(CH3)-C(CH3)2-C2H5, — C5H10— CH(CH3)2, C4H8-C(CH3)3, -C4H8-CH(CH3)-C2H5, -C4H8-CH(CH3)-C2H5, ■C3H6-C(CH3)2-C2H5, -C3H6-CH(C2H5)-C2H5, -C3H6-CH(CH3)-C3H7, C2H4-C(CH3)2-C3H7, -C2H4-CH(C2H5)-C3H7, -C2H4-CH(CH3)-C4H9ICH2-C(CH3)2-C4H9I-CH2-CH(C2H5)-C4H9, -CH2-CH(CH3)-C5HH , C(CH3)2-C5HH , -CH(CH3)-C6HI3, -CH(C3H7)-C4H9I■CH(C2H5)-C5HII , -CH2-C(CH3)(C2H5)-C3H7, C2H4-CH(CH3)-CH2-CH(CH3)2, -CH2-C(CH3)2-CH2-CH(CH3)2, CH2-CH(C2H5)-CH2-CH(CH3)2I-CH2-CH(CH3)-C2H4-CH(CH3)2, CH2-CH(CH3)-CH2-C(CH3)3I-CH2-CH(CH3)-CH2-CH(CH3)-C2H5, C(CH3)(C2H5)-CH2-CH(CH3)2I-CH(C3H7)-CH2-CH(CH3)2, ■CH(C2H5)-C2H4-CH(CH3)2, -CH(C2H5)-CH2-C(CH3)3, ■CH(C2H5)-CH2-CH(CH3)-C2H5, -CH2-CH(CH3)-C2H4-CH(CH3)2, C(CH3)2-C2H4-CH(CH3)2, -CH(C2H5)-C2H4-CH(CH3)2, -CH(CH3)-C3H6-CH(CH3)2, -CH(CH3)-C2H4-C(CH3)3, -CH(CH3)-C2H4-CH(CH3)-C2H5, -CH2-CH(CH3)-CH2-CH(CH3)-C2H5:-C(CH3)2-CH2-CH(CH3)-C2H5, -CH(CH3)-C2H4-CH(CH3)-C2H5:-CH(CH3)-CH2-C(CH3)2-C2H5, -CH(CH3)-CH2-CH(CH3)-C3H7:C2H4-CH(CH3)-CH(CH3)-C2H5, -CH2-C(CH3)2-CH(CH3)-C2H5:CH2-CH(C2H5)-CH(CH3)-C2H5, -CH2-CH(CH3)-CH2-CH(CH3)-C2H5:CH2-CH(CH3)-C(CH3)2-C2H5, -CH2-CH(CH3)-CH(C2H5)2:

[0060] -C3H6-CH(CH3)-CH(CH3)2, -C2H4-C(CH3)2-CH(CH3)2,

[0061] -C2H4-CH(C2H5)-CH(CH3)2, -C2H4-CH(CH3)-C(CH3)3,

[0062] -C2H4-CH(CH3)-CH(CH3)-C2H5, — C3He- C(CH3)2— C2H5

[0063] -C2H4— C(CH3)2— C3H7, — CH2— C(CH3)(C2H5)2, -C2H4-C(C2HS)3

[0064] -C2H4— C(CH3)2— C3H7, — CH2— C(CH3)2— C4H9, -C(C2HS)2-C3H7

[0065] -C(CH3)(C3H7)— C3H7, — C(CH3)(C2HS)- C4H9, -C(CH3)(-C2H5)-C4H9

[0066] -C(CH3)2-C5HH , -C2H4-C(CH3)2-CH(CH3)2, -CH2-C(CH3)2-C(CH3)3

[0067] -C(C2H5)2-CH(CH3)2, -C(CH3)(C3H7)-CH(CH3)2, -C(CH3)(C2H5)-C(CH3)3

[0068] -CH2-C(CH3)2-CH2-CH(CH3)2I-C(CH3)2-C2H4-CH(CH3)2

[0069] -C(CH3)2-CH2-C(CH3)3, -CH2-C(CH3)2-C(CH3)3, -C4H8-C(CH3)3, -C3H6-C(CH3)2-C2H5, -C2H4-C(CH3)2-C3H7, -C2H4-CH(CH3)-C(CH3)3,

[0070] -CH2-C(CH3)2-C(CH3)3.

[0071] "Alkylene" refers to a straight or branched chain divalent hydrocarbon radical having from 1 to 18 carbon atoms, more preferably from 1 to 8 carbon atoms, and most preferably 1 to 6 carbon atoms.

[0072] "Alkenyl" refers to groups of from 2 to 12 carbon atoms, preferably 2 to 8 carbon atoms (C2-Cs alkenyl), either straight or branched containing at least one double bond but optionally containing more than one double bond. As used herein, the term “linear or branched C2-C8alkenyl” refers to -CH=CH2, -CH2-CH=CH2,

[0073] -C(CH3)=CH2, -CH=CH-CH3, -C2H4-CH=CH2, -CH=CH-C2H5,

[0074] -CH2-C(CH3)=CH2I-CH(CH3)-CH=CH, -CH=C(CH3)2I-C(CH3)=CH-CH3I

[0075] -CH=CH-CH=CH2, -C3H6-CH=CH2, -C2H4-CH=CH-CH3,

[0076] -CH2-CH=CH-C2H5, -CH=CH-C3H7, -CH2-CH=CH-CH=CH2,

[0077] -CH=CH-CH=CH-CH3, -CH=CH-CH2-CH=CH2, -C(CH3)=CH-CH=CH2I

[0078] -CH=C(CH3)-CH=CH2I-CH=CH-C(CH3)=CH2I-C2H4-C(CH3)=CH2,

[0079] -CH2-CH(CH3)-CH=CH2I-CH(CH3)-CH2-CH=CH2I-CH2-CH=C(CH3)2I

[0080] -CH2-C(CH3)=CH-CH3I-CH(CH3)-CH=CH-CH3I-CH=CH-CH(CH3)2I

[0081] -CH=C(CH3)-C2H5, -C(CH3)=CH-C2H5, -C(CH3)=C(CH3)2I

[0082] -C(CH3)2-CH=CH2I-CH(CH3)-C(CH3)=CH2I-C(CH3)=CH-CH=CH2I

[0083] -CH=C(CH3)-CH=CH2I-CH=CH-C(CH3)=CH2I-C4H8-CH=CH2,

[0084] -C3H6-CH=CH-CH3, -C2H4-CH=CH-C2H5, -CH2-CH=CH-C3H7,

[0085] -CH=CH-C4H9, -C3H6-C(CH3)=CH2, -C2H4-CH(CH3)-CH=CH2I

[0086] -CH2-CH(CH3)-CH2-CH=CH2I-CH2-CH=CH-CH3I-CH(CH3)-C2H4-CH=CH2, -C2H4-CH=C(CH3)2, -C2H4-C(CH3)=CH-CH3, -CH2-CH(CH3)-CH=CH-CH3I-CH(CH3)-CH2-CH=CH-CH3I-C(C4H9)=CH2, -CH2-CH=CH-CH(CH3)2I

[0087] -CH2-CH=C(CH3)-C2H5, -CH2-C(CH3)=CH-C2H5, -CH(CH3)-CH=CH-C2H5,

[0088] -CH=CH-CH2-CH(CH3)2I-CH=CH-CH(CH3)-C2H5, -CH=C(CH3)-C3H7,

[0089] -C(CH3)=CH-C3H7, -CH2-CH(CH3)-C(CH3)=CH2I-CH(CH3)-CH2-

[0090] C(CH3)=CH2I-CH(CH3)-CH(CH3)-CH=CH2I-CH2-C(CH3)2-CH=CH2I

[0091] -C(CH3)2-CH2-CH=CH2I-CH2-C(CH3)=C(CH3)2I-CH(CH3)-CH=C(CH3)2I

[0092] -C(CH3)2-CH=CH-CH3I-CH(CH3)-C(CH3)=CH-CH3I-CH=C(CH3)-CH(CH3)2I

[0093] -C(CH3)=CH-CH(CH3)2I-C(CH3)=C(CH3)-C2H5, -CH=CH-C(CH3)3I

[0094] -C(CH3)2-C(CH3)=CH2I-CH(C2H5)-C(CH3)=CH2, -C(CH3)(C2H5)-CH=CH2,

[0095] -CH(CH3)-C(C2H5)=CH2, -CH2-C(C3H7)=CH2, -CH2-C(C2H5)=CH-CH3,

[0096] -CH(C2H5)-CH=CH-CH3, -C(C3H7)=CH-CH3, -C(C2H5)=CH-C2H5,

[0097] -C(C2H5)=C(CH3)2, -C[C(CH3)3]=CH2I-C[CH(CH3)(C2H5)]=CH2,

[0098] -C[CH2-CH(CH3)2]=CH2I-C2H4-CH=CH-CH=CH2, -CH2-CH=CH-CH2-CH=CH2, -CH=CH-C2H4-CH=CH2,

[0099] -CH2-CH=CH-CH=CH-CH3, -CH=CH-CH2-CH=CH-CH3,

[0100] -CH=CH-CH=CH-C2H5, -CH2-CH=CH-C(CH3)=CH2I

[0101] -CH2-CH=C(CH3)-CH=CH2I-CH2-C(CH3)=CH-CH=CH2I

[0102] -CH(CH3)-CH=CH-CH=CH2I-CH=CH-CH2-C(CH3)=CH2I

[0103] -CH=CH-CH(CH3)-CH=CH2I-CH=C(CH3)-CH2-CH=CH2I

[0104] -C(CH3)=CH-CH2-CH=CH2I-CH=CH-CH=C(CH3)2I

[0105] -CH=CH-C(CH3)=CH-CH3I-CH=C(CH3)-CH=CH-CH3I

[0106] -C(CH3)=CH-CH=CH-CH3I-CH=C(CH3)-C(CH3)=CH2I

[0107] -C(CH3)=CH-C(CH3)=CH2I-C(CH3)=C(CH3)-CH=CH2I

[0108] -CH=CH-CH=CH-CH=CH2, -C5HIO-CH=CH2, -C4H8-CH=CH-CH3,

[0109] -C3H6-CH=CH-C2H5, -C2H4-CH=CH-C3H7, -CH2-CH=CH-C4H9,

[0110] -C4H8-C(CH3)=CH2, -C3H6-CH(CH3)-CH=CH2I-C2H4-CH(CH3)-CH2-CH=CH2I

[0111] -CH2-CH(CH3)-C2H4-CH=CH2I-C3H6-CH=C(CH3)2I-C3H6-C(CH3)=CH-CH3,

[0112] -C2H4-CH(CH3)-CH=CH-CH3I-CH2-CH(CH3)-CH2-CH=CH-CH3I

[0113] -C2H4-CH=CH-CH(CH3)2I-C2H4-CH=C(CH3)-C2H5,

[0114] -C2H4-C(CH3)=CH-C2H5, -CH2-CH(CH3)-CH=CH-C2H5,

[0115] -CH2-CH=CH-CH2-CH(CH3)2I-CH2-CH=CH-CH(CH3)-C2H5,

[0116] -CH2-CH=C(CH3)-C3H7I-CH2-C(CH3)=CH-C3H7I

[0117] -C2H4-CH(CH3)-C(CH3)=CH2I-CH2-CH(CH3)-CH2-C(CH3)=CH2I

[0118] -CH2-CH(CH3)-CH(CH3)-CH=CH2I-C2H4-C(CH3)2-CH=CH2I

[0119] -CH2-C(CH3)2-CH2-CH=CH2I-C2H4-C(CH3)=C(CH3)2I

[0120] -CH2-CH(CH3)-CH=C(CH3)2I-CH2-C(CH3)2-CH=CH-CH3I

[0121] -CH2-CH(CH3)-C(CH3)=CH-CH3I-CH2-CH=C(CH3)-CH(CH3)2I

[0122] -CH2-C(CH3)=CH-CH(CH3)2I-CH2-C(CH3)=C(CH3)-C2H5,

[0123] -CH2-CH=CH-C(CH3)3I-CH2-C(CH3)2-C(CH3)=CH2I

[0124] -CH2-CH(C2H5)-C(CH3)=CH2, -CH2-C(CH3)(C2H5)-CH=CH2,

[0125] -CH2-CH(CH3)-C(C2H5)=CH2, -C2H4-C(C3H7)=CH2I-C2H4-C(C2H5)=CH-CH3,

[0126] -CH2-CH(C2H5)-CH=CH-CH3, -CH2-C(C4H9)=CH2, -CH2-C(C3H7)=CH-CH3I

[0127] -CH2-C(C2H5)=CH-C2H5, -CH2-C(C2H5)=C(CH3)2, -CH2-C[C(CH3)3]=CH2I

[0128] -CH2-C[CH(CH3)(C2H5)]=CH2, -CH2-C[CH2-CH(CH3)2]=CH2I

[0129] -C3H6-CH=CH-CH=CH2, -C2H4-CH=CH-CH2-CH=CH2,

[0130] -CH2-CH=CH-C2H4-CH=CH2, -C2H4-CH=CH-CH=CH-CH3,

[0131] -CH2-CH=CH-CH2-CH=CH-CH3, -CH2-CH=CH-CH=CH-C2H5,

[0132] -C2H4-CH=CH-C(CH3)=CH2I-C2H4-CH=C(CH3)-CH=CH2I

[0133] -C2H4-C(CH3)=CH-CH=CH2I-CH2-CH(CH3)-CH=CH-CH=CH2I

[0134] -CH2-CH=CH-CH2-C(CH3)=CH2I-CH2-CH=CH-CH(CH3)-CH=CH2I

[0135] -CH2-CH=C(CH3)-CH2-CH=CH2I-CH2-C(CH3)=CH-CH2-CH=CH2I -CH2-CH=CH-CH=C(CH3)2I-CH2-CH=CH-C(CH3)=CH-CH3,

[0136] -CH2-CH=C(CH3)-CH=CH-CH3, -CH2-C(CH3)=CH-CH=CH-CH3,

[0137] -CH2-CH=C(CH3)-C(CH3)=CH2, -CH2-C(CH3)=CH-C(CH3)=CH2,

[0138] -CH2-C(CH3)=C(CH3)-CH=CH2, -CH2-CH=CH-CH=CH-CH=CH2,

[0139] -C6HI2-CH=CH2, -C5HIO-CH=CH-CH3, -C4H8-CH=CH-C2H5,

[0140] -C3H6-CH=CH-C3H7, -C2H4-CH=CH-C4H9, -C5HIO-C(CH3)=CH2,

[0141] C4H8-CH(CH3)-CH=CH2, -C3H6-CH(CH3)-CH2-CH=CH2

[0142] C2H4-CH(CH3)-C2H4-CH=CH2I-C4H8-CH=C(CH3)2

[0143] C4H8-C(CH3)=CH-CH3, -C3H6-CH(CH3)-CH=CH-CH3

[0144] C2H4-CH(CH3)-CH2-CH=CH-CH3, -C3H6-CH=CH-CH(CH3)2

[0145] C3H6-CH=C(CH3)-C2H5, -C3H6-C(CH3)=CH-C2H5

[0146] C2H4-CH(CH3)-CH=CH-C2H5, -C2H4-CH=CH-CH2-CH(CH3)2

[0147] C2H4-CH=CH-CH(CH3)-C2H5, -C2H4-CH=C(CH3)-C3H7

[0148] C2H4-C(CH3)=CH-C3H7, -C3H6-CH(CH3)-C(CH3)=CH2

[0149] C2H4-CH(CH3)-CH2-C(CH3)=CH2, -C2H4-CH(CH3)-CH(CH3)-CH=CH2

[0150] C3H6-C(CH3)2-CH=CH2, -C2H4-C(CH3)2-CH2-CH=CH2

[0151] C3H6-C(CH3)=C(CH3)2, -C2H4-CH(CH3)-CH=C(CH3)2

[0152] C2H4-C(CH3)2-CH=CH-CH3, -C2H4-CH(CH3)-C(CH3)=CH-CH3

[0153] C2H4-CH=C(CH3)-CH(CH3)2, -C2H4-C(CH3)=CH-CH(CH3)2

[0154] C2H4-C(CH3)=C(CH3)-C2H5, -C2H4-CH=CH-C(CH3)3

[0155] C2H4-C(CH3)2-C(CH3)=CH2, -C2H4-CH(C2H5)-C(CH3)=CH2

[0156] C2H4-C(CH3)(C2H5)-CH=CH2, -C2H4-CH(CH3)-C(C2H5)=CH2

[0157] C3H6-C(C3H7)=CH2, -C3H6-C(C2H5)=CH-CH3

[0158] C2H4-CH(C2H5)-CH=CH-CH3, -C2H4-C(C4H9)=CH2

[0159] C2H4-C(C3H7)=CH-CH3, -C2H4-C(C2H5)=CH-C2H5

[0160] C2H4-C(C2H5)=C(CH3)2, -C2H4-C[C(CH3)3]=CH2

[0161] C2H4-C[CH(CH3)(C2H5)]=CH2, -C2H4-C[CH2-CH(CH3)2]=CH2

[0162] C4H8-CH=CH-CH=CH2, -C3H6-CH=CH-CH2-CH=CH2

[0163] C2H4-CH=CH-C2H4-CH=CH2, -C3H6-CH=CH-CH=CH-CH3 C2H4-CH=CH-CH2-CH=CH-CH3, -C2H4-CH=CH-CH=CH-C2H5C3H6-CH=CH-C(CH3)=CH2, -C3H6-CH=C(CH3)-H=CH2C3H6-C(CH3)=CH-CH=CH2, -C2H4-CH(CH3)-CH=CH-CH=CH2

[0164] C2H4-CH=CH-CH2-C(CH3)=CH2I-C2H4-CH=CH-CH(CH3)-CH=CH2

[0165] C2H4-CH=C(CH3)-CH2-CH=CH2I-C2H4-C(CH3)=CH-CH2-CH=CH2

[0166] C2H4-CH=CH-CH=C(CH3)2I-C2H4-CH=CH-C(CH3)=CH-CH3

[0167] C2H4-CH=C(CH3)-CH=CH-CH3, -C2H4-C(CH3)=CH-CH=CH-CH3

[0168] C2H4-CH=C(CH3)-C(CH3)=CH2, -C2H4-C(CH3)=CH-C(CH3)=CH2 -C2H4-C(CH3)=C(CH3)-CH=CH2, -CH=CH-Ph and

[0169] -C2H4-CH=CH-CH=CH-CH=CH2.

[0170] "Alkenylene" refers to a straight or branched chain divalent hydrocarbon radical having 2 to 12 carbon atoms and one or more carbon-carbon double bonds, more preferably from 2 to 8 carbon atoms, and most preferably 2 to 6 carbon atoms.

[0171] "Alkynyl" refers to groups of from 2 to 12 carbon atoms, preferably from 2 to 8 carbon atoms, either straight or branched containing at least one triple bond but optionally containing more than one triple bond, and additionally optionally containing one or more double bonded moieties. As used herein, the term “linear or branched C2-Cs alkynyl” refers to -C=CH, -C=C-CH3, -CH2-C=CH, -C2H4-CECH, -CH2-CEC-CH3, -C=C-C2H5, -C2H4-CEC-CH3, -CH2-CEC-C2H5, -C=C-C3H7, -CH(CH3)-CECH, -CH2-CH(CH3)-CECH, -CH(CH3)-CH2-CECH, -CH(CH3)-CEC-CH3, -C4H8-CECH,

[0172] -C3H6-CEC-CH3, -C2H4-CEC-C2H5, -CH2-CEC-C3H7, -C=C-C4H9,

[0173] -C2H4-CH(CH3)-CECH, -CH2-CH(CH3)-CH2-CECH, -CH(CH3)-C2H4-CECH,

[0174] -CH2-CH(CH3)-CEC-CH3, -CH(CH3)-CH2-CEC-CH3, -CH(CH3)-CEC-C2H5,

[0175] -CH2-CEC-CH(CH3)2, -CEC-CH(CH3)-C2H5, -CEC-CH2-CH(CH3)2,

[0176] -CEC-C(CH3)3, -C3H6-CECH, -CH(C2H5)-CEC-CH3, -C(CH3)2-CEC-CH3,

[0177] -CH(C2H5)-CH2-CECH, -CH2-CH(C2H5)-CECH, -C(CH3)2-CH2-CECH,

[0178] -CH2-C(CH3)2-CECH, -CH(CH3)-CH(CH3)-CECH, -CH(C3H7)-CECH,

[0179] -C(CH3)(C2H5)-CECH, -C=C-C=CH, -CH2-C=C-C=CH, -C=C-C=C-CH3,

[0180] -CH(CECH)2, -C2H4-C=C-C=CH, -CH2-C=C-CH2-C=CH,

[0181] -C=C-C2H4-C=CH, -CH2-C=C-C=C-CH3, -C=C-CH2-C=C-CH3,

[0182] -C=C-C=C-C2H5, -C=C-CH(CH3)-C=CH, -CH(CH3)-C=C-C=CH,

[0183] -CH(CECH)-CH2-CECH, -C(CECH)2-CH3, -CH2-CH(CECH)2,

[0184] -CH(C=CH)-C=C-CH3, -C5HIQ-C=CH, -C4H8-CEC-CH3, -C3H6-CEC-C2H5, -C2H4-CEC-C3H7, -CH2-CEC-C4H9, -C3H6-CH(CH3)-CECH,

[0185] -C2H4-CH(CH3)-CH2-CECH, -CH2-CH(CH3)-C2H4-CECH, -C2H4-CH(CH3)- C=C-CH3, -CH2-CH(CH3)-CH2-CEC-CH3, -CH2-CH(CH3)-CEC-C2H5, -C2H4-CEC-CH(CH3)2, -CH2-CEC-CH(CH3)-C2H5, -CH2-CEC-CH2-CH(CH3)2, -CH2-CEC-C(CH3)3, -CH2-CH(C2H5)-CEC-CH3, -CH2-C(CH3)2-CEC-CH3, -CH2-CH(C2H5)-CH2-CECH, -C2H4-CH(C2H5)-CECH,

[0186] -CH2-C(CH3)2-CH2-CECH, -C2H4-C(CH3)2-CECH,

[0187] -CH2-CH(CH3)-CH(CH3)-CECH, -CH2-CH(C3H7)-CECH,

[0188] -CH2-C(CH3)(C2H5)-CECH, -C3H6-C=C-C=CH, -C2H4-C=C-CH2-C=CH,

[0189] -CH2-C=C-C2H4-C=CH, -C2H4-C=C-C=C-CH3, -CH2-CEC-CH2-CEC-CH3, -CH2-C=C-C=C-C2H5, -CH2-CEC-CH(CH3)-CECH,

[0190] -CH2-CH(CH3)-CEC-CECH, -CH2-CH(CECH)-CH2-CECH,

[0191] -CH2-C(CECH)2-CH3, -C2H4-CH(CECH)2, -CH2-CH(CECH)-CEC-CH3, -C6HI2-C=CH, -C5HIQ-C=C-CH3, -C4H8-CEC-C2H5, -C3H6-CEC-C3H7,

[0192] -C2H4-CEC-C4H9, -C4H8-CH(CH3)-CECH, -C3H6-CH(CH3)-CH2-CECH, -C2H4-CH(CH3)-C2H4-CECH, -C3H6-CH(CH3)-CEC-CH3,

[0193] -C2H4-CH(CH3)-CH2-CEC-CH3, -C2H4-CH(CH3)-CEC-C2H5,

[0194] -C3H6-CEC-CH(CH3)2, -C2H4-CEC-CH(CH3)-C2H5, -C2H4-CEC-CH2-

[0195] CH(CH3)2I-C2H4-CEC-C(CH3)3, -C2H4-CH(C2H5)-CEC-CH3, -C2H4-

[0196] C(CH3)2-CEC-CH3, -C2H4-CH(C2H5)-CH2-CECH, -C3H6-CH(C2H5)-CECH, -C2H4-C(CH3)2-CH2-CECH, -C3H6-C(CH3)2-CECH, -C2H4-CH(CH3)-

[0197] CH(CH3)-CECH, -C2H4-CH(C3H7)-CECH, -C2H4-C(CH3)(C2H5)-CECH, -C4H8-C=C-C=CH, -C3H6-C=C-CH2-C=CH, -C2H4-C=C-C2H4-C=CH,

[0198] -C3H6-C=C-C=C-CH3, -C2H4-CEC-CH2-CEC-CH3, -C2H4-C=C-C=C-C2H5, -C2H4-CEC-CH(CH3)-CECH, -C=C-Ph, -C2H4-CH(CH3)-CEC-CECH, -C2H4-CH(CECH)-CH2-CECH, -C2H4-C(CECH)2-CH3, -C3H6-CH(CECH)2, and -C2H4-CH(CECH)-CEC-CH3.

[0199] "Cycloalkyl" refers to cyclic alkyl groups of from 3 to 12 carbon atoms, preferably from 3 to 8 carbon atoms, having a single cyclic ring or multiple condensed rings which can be optionally substituted with from 1 to 3 alkyl groups. Exemplary cycloalkyl groups include, but are not limited to, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, 1 -methylcyclopropyl, 2-methyl- cyclopentyl, 2-methylcyclooctyl, and the like, or multiple ring structures, including bridged ring systems, such as adamantyl. As used herein, “C3-C8cycloalkyl” refers to cyclo-C3Hs, cyclo-C4H7, cyclo-CsHg, cyclo-CeHn, cyclo-C7Hi3, and cyclo-CsHis.

[0200] "Cycloalkylalkyl" refers to an alkyl substituted with a cycloalkyl, i.e., cycloalkyl- alkyl- groups, preferably having from 1 to 6 carbon atoms in the alkyl moiety and from 3 to 12 carbon atoms in the cycloalkyl moiety. Such cycloalkylalkyl groups are exemplified by cyclopropylmethyl, cyclohexylethyl and the like.

[0201] "Aryl" refers to an unsaturated aromatic carbocyclic group of from 6 to 12 carbon atoms inclusively having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). Exemplary aryls include phenyl, pyridyl, naphthyl and the like. "Arylalkyl" refers to an alkyl substituted with an aryl, i.e., aryl-alkyl groups, preferably having from 1 to 6 carbon atoms in the alkyl moiety and from 6 to 12 carbon atoms inclusively in the aryl moiety. Such arylalkyl groups are exemplified by benzyl, phenethyl and the like. As used herein, the term “C6-C8arylalkyl” includes, but is not limited to -CH2-Ph and -C2H4Ph.

[0202] "Heteroalkyl, "heteroalkenyl," and “heteroalkynyl," refer to alkyl, alkenyl and alkynyl as defined herein in which one or more of the carbon atoms are each independently replaced with the same or different heteroatoms or heteroatom ic groups. Heteroatoms and / or heteroatom ic groups which can replace the carbon atoms include, but are not limited to, -O-, -S-, -S-O-, -NRa-, -PH-, -S(O)-, -S(O)2-,- S(O)NRa-, -S(O)2NRa-, and the like, including combinations thereof, where each Rais independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. "Heteroaryl" refers to an aromatic heterocyclic group of from 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl). As used herein, the term “C1-C10 heteroaryl” refers to aromatic residues with one or more heteroatoms such as 0, S, N. "Heteroarylalkyl" refers to an alkyl substituted with a heteroaryl, i.e., heteroaryl-alkyl-groups, preferably having from 1 to 6 carbon atoms in the alkyl moiety and from 5 to 12 ring atoms inclusively in the heteroaryl moiety. Such heteroarylalkyl groups are exemplified by pyridylmethyl and the like. "Heterocycloalkyl" refers to a saturated or unsaturated group having a single ring or multiple condensed rings, from 2 to 9 carbon ring atoms and from 1 to 4 hetero ring atoms inclusively selected from nitrogen, sulfur or oxygen within the ring. Such heterocyclic groups can have a single ring (e.g., piperidinyl or tetrahydrofuryl) or multiple condensed rings (e.g., indolinyl, dihydrobenzofuran or quinuclidinyl). Examples of heterocycles include, but are not limited to, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, pyrrolidine, indoline and the like.

[0203] "Oxy" refers to a divalent group -O-, which may have various substituents to form different oxy groups, including ethers and esters. "Alkoxy" or "alkyloxy" are used interchangeably herein to refer to the group -ORa, wherein Rais an alkyl group, including optionally substituted alkyl groups. "Aryloxy" as used herein refer to the group -ORawherein Rais an aryl group as defined above including optionally substituted aryl groups as also defined herein.

[0204] "Carboxy" refers to -COOH. "Carboxyalkyl" refers to an alkyl substituted with a carboxy group.

[0205] "Carbonyl" refers to a functional group with the formula C(O) or C=O, composed of a carbon atom double-bonded to an oxygen atom. A compound containing a carbonyl group is herein also referred to as a carbonyl compound. Substituted carbonyl refers to the group Ra-C(O)-Raor Ra-C=O-Rawhere each Rais independently selected from optionally substituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, carboxy, aryl, aryloxy, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Typical substituted carbonyl groups including acids, ketones, aldehydes, amides, esters, acyl halides, thioesters, and the like.

[0206] “a,0-unsaturated carbonyl compound” or “a,0-unsaturated carbonyl donor” refers to an organic compound with the general structure (O=CR)-Ca=Cp-R. Examples include enones and enals. In these compounds the carbonyl group is conjugated with an alkene. “a,0-unsaturated enal” refers to an organic compound containing both alkene and aldehyde functional groups, wherin the alkene is conjugated to the carbonyl group of the aldehyde. “a,0-unsaturated enone” refers to an organic compound containing both alkene and ketone functional groups, wherein the alkene is conjugated to the carbonyl group of the ketone.

[0207] "Amino" refers to the group -NH2. Substituted amino refers to the group -NHRa, NRaRa, and NRaRaRa, where each Rais independently selected from optionally substituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, carboxy, aryl, aryloxy, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Typical amino groups include, but are limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylysulfonylamino, furanyl-oxy- sulfamino, and the like. "Aminoalkyl" refers to an alkyl group in which one or more of the hydrogen atoms are replaced with an amino group, including a substituted amino group. "Aminocarbonyl" refers to a carbonyl group substituted with an amino group, including a substituted amino group, as defined herein, and includes amides. "Aminocarbonylalkyl" refers to an alkyl substituted with an aminocarbonyl group, as defined herein. "Halogen" or "halo" refers to fluoro, chloro, bromo and iodo. "Haloalkyl" refers to an alkyl group in which one or more of the hydrogen atoms are replaced with a halogen. Thus, the term "haloalkyl" is meant to include monohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls. As used herein, the expression "C1-C2 haloalkyl" includes 1 -fluoromethyl, difluoromethyl, trifluoromethyl, 1- fluoroethyl, 1 ,1 -difluoroethyl, 1 ,2-difluoroethyl, 1 ,1 ,1 trifluoroethyl, perfluoroethyl, etc.

[0208] "Hydroxy" refers to -OH. "Hydroxyalkyl" refers to an alkyl substituted with one or more hydroxy group.

[0209] "Thio" or "sulfanyl" refers to -SH. Substituted thio or sulfanyl refers to -S-Ra, where Rais an alkyl, aryl or other suitable substituent. "Alkylthio" refers to -SRa, where Rais an alkyl, which can be optionally substituted. Typical alkylthio group include, but are not limited to, methylthio, ethylthio, n-propylthio, and the like. "Alkylthioalkyl" refers to an alkyl substituted with an alkylthio group, -SRa, where Rais an alkyl, which can be optionally substituted. "Thiocarbonyl" refers to a carbonyl group substituted with a thio group, including a substituted thio group, as defined herein, and includes thioesters.

[0210] "Optionally substituted" as used herein with respect to the foregoing chemical groups means that positions of the chemical group occupied by hydrogen can be substituted with another atom, such as carbon, oxygen, nitrogen, or sulfur, or a chemical group, exemplified by, but not limited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy, alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido, cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocycle, (heterocycle)oxy, and (heterocyclyl)alkyl; where preferred heteroatoms are oxygen, nitrogen, and sulfur. However, it is clear to a skilled person that the term “can be substituted” refers to the replacement of a hydrogen atom by one of the above-mentioned chemical groups. One of ordinary skill in the art would understand that with respect to any chemical group described as optionally substituted, only sterically practical and / or synthetically feasible chemical groups are meant to be included.

[0211] The inventors have previously demonstrated that ECRs can be converted into reductive aldolases by exchanging the electrophile at the active site. However, in case of ECRs the carbonyl donor has to be provided in form of CoA-thioster. Thus, the inventors have focused on other enzymes, especially enzyme families that are CoA-independent. To that end, the inventors have identified ene reductases (ERs) as suitable enzymes for providing CoA independent reductive aldolases. ERs catalyze the NADPH-dependent reduction of a,|3 unsaturated carbonyls. In contrast to ECRs, ERs resolve the enolate intermediate not by reaction with an exogenous electrophile (CO2), but by protonation using a conserved tyrosine as proton donor. Thus, the inventors aimed at suppressing proton donation to establish a reductive aldol reaction in the scaffold of ERs.

[0212] The inventors have selected four phylogenetically diverse ERs for engineering efforts: Oye1 from Saccharomyces pastorianus (SEQ ID NO: 11 ), Kye1 from Kluyveromyces lactis (SEQ ID NO: 12) (72 % / 83 % identity / sim ilarity compared to Oye1 ), NemA from Escherichia coli (SEQ ID NO: 14) (34 % / 51 % identity / sim ilarity compared to Oye1), and NamA from Geobacillus kaustophilus (SEQ ID NO: 13) (22 % / 38 % identity / similarity compared to Oye1 ).

[0213] The inventors replaced the conserved tyrosine proton donor in all four ERs by phenylalanine and screened the resulting variants for the reaction of cyclohexenone with formaldehyde, which yields 2-(hydroxymethyl)cyclohexan-1 - one (2-HMC) as product. Notably, all mutants tested were able to produce detectable amounts of 2-HMC. However, NemA Tyr187Phe clearly outperformed all other enzymes, between 3- and 20-fold with respect to 2-HMC yield.

[0214] The amino acid sequence of SEQ ID NO: 1 reflects a conserved segment (SEQ ID NO: 6) in the amino acid sequence of the ene reductase enzymes identified by the inventors (SEQ ID Nos.: 7 - 10) as shown in Table 1 below, wherein the active site proton donor tyrosine has been substituted by phenylalanine (SEQ ID Nos.: 2 - 5) for suppressing proton donation to establish a reductive aldol reaction in the scaffold of ERs. The conserved sequence segment (SEQ ID Nos.: 6) has been identified by multiple sequence alignment of the amino acid sequences of the four ene reductases. The identified conserved site including the active site proton donor tyrosine has a length of 48 amino acids and has an amino acid sequence as set forth in SEQ ID NO: 6. The amino acids 100% conserved throughout all aligned sequences are listed in bold in Tables 1 and 2.

[0215] Table 1 Conserved amino acid sequence of modified ERs

[0216] Table 2 Conserved amino acid sequence of ERs

[0217] Thus, the present invention relates to a method for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0218] In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 96% sequence identity, more preferably at least 97% sequence identity, more preferably at least 98% sequence identity, more preferably at least 99% sequence identity, more preferably at least 99.5% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0219] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. In preferred embodiments, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is phenylalanine. Besides phenylalanine, other hydrophobic amino acids, such as tryptophane (W), valine (L), leucine (L), isoleucine (I) or methionine (M), may be used to replace the proton donating tyrosine at the active site. Thus, in preferred embodiments, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is selected from the group consisting of phenylalanine, tryptophane, valine, leucine, isoleucing, and methionine, preferably phenylalanine.

[0220] Thus, the polypeptide capable of catalyzing CoA-independent reductive aldol reactions is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the polypeptide includes amino acid sequences, wherein the conserved tyrosine at the 14thposition of the amino acid sequence as set forth in SEQ ID NO: 6 is substituted by other amino acids than phenylalanine, i.e. tryptophane (W), valine (V), leucine (L), isoleucine (I) or methionine (M) (Table 3).

[0221] Table 3 Conserved amino acid sequence of other modified ERs

[0222] In the following, the inventors further focused on NemA. Because NemA still produced cyclohexanone as major product, the inventors tested different pHs and formaldehyde concentrations to favor the reductive aldol reaction over ene reduction. By optimizing reaction conditions, the yield for 2-HMC successfully increased from initially 6 % (pH=8.0 and 50 mM formaldehyde) to 30 % (pH=10 and 250 mM formaldehyde). Notably, the fivefold increase in yield was accompanied by a nearly tenfold improved chemoselectivity of the enzyme (defined as specificity factor o = yieldaidoi / yieldreduction), which increased from o = 0.06 to o = 0.55. Having optimized the working conditions of the reaction, the inventors decided to resort to enzyme engineering to further improve chemoselectivity.

[0223] The inventors turned their attention to efforts to increase formaldehyde accommodation. In previous studies, targeting the conserved Trp103, had proven to be a successful engineering strategy to accommodate new substrates at the active site. The inventors therefore replaced Trp103 with smaller hydrophobic residues (alanine, valine or phenylalanine), or amino acids that could serve as hydrogen bond donors to better orient the formaldehyde electrophile (glutamine or asparagine). Although all variants showed decreased overall yields, several variants exhibited significantly higher specificities, among them also double variant Tyr187Phe Trp103Ala that reached a specificity of o = 2.2. MD simulations indicated that this variant behaved more rigid than the single mutant, lowering solvent accessible volume compared to NemA WT and NemA Tyr187Phe, which suppresses enolate protonation at the active site, but probably also reduces substrate accessibility, explaining the observed yield. Nevertheless, these results showed that the reductive aldol reaction can be improved through additional engineering efforts in the scaffold of ERs.

[0224] Table 4: Further conserved amino acid sequence of wild type and modified ERs

[0225] Thus, the wt ERs comprise an conserved amino acid sequence having least 95% sequence identity to the amino acid sequence AGXDXXEXHXAXGYLXXXFLXPXSNXRX1 DXYGGSXENRXRXXXEVXDA (SEQ ID NO: 6) consisting of 48 amino acids, wherein each X represents independently of each other exactly one amino acid. More preferably each X represents independently of each other exactly one proteinogenic amino acids and more preferably exactly one canonic amino acid.

[0226] Thus, the modified ERs comprise an conserved amino acid sequence having least 95% sequence identity to the amino acid sequence AGXDXXEXHXAXGFLXXXFLXPXSNXRX1 DXYGGSXENRXRXXXEVXDA (SEQ ID NO: 1 ) consists of 48 amino acids, wherein each X represents independently of each other exactly one amino acid. More preferably each X represents independently of each other exactly one proteinogenic amino acids and more preferably exactly one canonic amino acid. According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0227] Table 5: Conserved site of the protein sequence of ERs

[0228] Table 6: Conserved site of the protein sequence of ERs Given the similarity of the active structure, also other modified ene reductases from other bacteria may be used in the method of the present invention. Thus, further modified ene reductases, which may be used in the method of the present invention, may be derived from ene reductases in in many bacteria such as Escherichia, Synechocystis, Mycrocystis, Pseudomonas, Shewanella, Bacillus, Streptomyces, Deinococcus, Schizosaccharomyces, Thermotoga, Clostridium, Mycobacterium, Streptomyces, Sulfolobus, as well as their close relatives.

[0229] With other words, the present invention relates to a method for preparing an a- branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,p-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; c) performing an enzymatic-catalyzed CoA-independent reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a CoA independent reductive aldolase in the presence of a cofactor, wherein the CoA independent reductive aldolase is a modified ene reductase comprising an amino acid sequence segment having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0230] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0231] Formulated differently, the present invention relates to a method for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,p-unsaturated carbonyl donor, wherein the a,p-unsaturated carbonyl donor is an a,p-unsaturated enone or a,p-unsaturated enal; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; c) performing an enzymatic-catalyzed CoA-independent reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a CoA independent reductive aldolase in the presence of a cofactor, wherein the CoA independent reductive aldolase is a modified ene reductase comprising an amino acid sequence segment having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0232] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0233] Thus, the present invention preferably relates to a method for preparing an a- branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence AGXDXXEXHXAXG F LXXXF LXPXS NXRXDXYGGSXE N RXRXXXE VXDA (SEQ ID NO: 1 ), wherein each X represents independently of each other exactly one amino acid.

[0234] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 . The present invention relates to a method for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,p-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23.

[0235] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23.

[0236] The present invention relates to a method for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,p-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence AGXDXVEXHSAXGFLLXQFLXPXSNXRTDXYGGSXENRARXXFEVDA (SEQ ID NO: 23), wherein each X represents independently of each other exactly one amino acid. According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23.

[0237] The present invention relates to a method for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,p-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0238] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0239] The present invention relates to a method for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,p-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence AGFDVIEIHAAHGFLINEFLSPLSNRRQDEYGGSPENRYRFLGEVIDA (SEQ ID NO: 2), AGFDLVELHSAHGFLLHQFLSPSSNHRTDQYGGSVENRARLVLEWDA (SEQ ID NO: 3), AGADGVEIHSANGFLLNQFLDPHSNTRTDEYGGSIENRARFTLEVVDA (SEQ ID NO: 4), or

[0240] AGADGVEIHSANGFLLNQFLDPISNKRTDEYGGSIENRARFVLEVVDA (SEQ ID NO: 5).

[0241] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0242] The present invention relates to a method for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,p-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid as set forth in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0243] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0244] The present invention relates to a method for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,p-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence

[0245] AGFDLVELHSAHGFLLHQFLSPSSNHRTDQYGGSVENRARLVLEWDA (SEQ ID NO: 3), AGADGVEIHSANGFLLNQFLDPHSNTRTDEYGGSIENRARFTLEVVDA (SEQ ID NO: 4), or

[0246] AGADGVEIHSANGFLLNQFLDPISNKRTDEYGGSIENRARFVLEVVDA (SEQ ID NO: 5).

[0247] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0248] The present invention relates to a method for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,p-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0249] According to the present invention, 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is not tyrosine. Preferably, the 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is phenylalanine. According to the present invention, 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is not tyrosine. Preferably, the 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is phenylalanine. According to the present invention, 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is not tyrosine. Preferably, the 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is phenylalanine. According to the present invention, 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is not tyrosine. Preferably, the 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0250] The present invention relates to a method for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,p-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence MSFVKDFKPQALGDTNLFKPIKIGNNELLHRAVIPPLTRMRALHPGNIPNRDWAVEYYTQ RAQRPGTMIITEGAFISPQAGGYDNAPGVWSEEQMVEWTKIFNAIHEKKSFVWVQLWVL GWAAFPDNLARDGLRYDSASDNVFMDAEQEAKAKKANNPQHSLTKDEIKQYIKEYVQAA KNSIAAGADGVEIHSANGFLLNQFLDPHSNTRTDEYGGSIENRARFTLEWDALVEAIGHE KVGLRLSPYGVFNSMSGGAETGIVAQYAYVAGELEKRAKAGKRLAFVHLVEPRVTNPFL TEGEGEYEGGSNDFVYSIWKGPVIRAGNFALHPEVVREEVKDKRTLIGYGRFFISNPDLV DRLEKGLPLNKYDRDTFYQMSAHGYIDYPTYEEALKLGWDKK (SEQ ID NO: 15).

[0251] According to the present invention, 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is not tyrosine. Preferably, the 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15.

[0252] The present invention relates to a method for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence MSFMNFEPKPLADTDIFKPIKIGNTELKHRVVMPALTRMRALHPGNVPNPDWAVEYYRQ RSQYPGTMIITEGAFPSAQSGGYDNAPGVWSEEQLAQWRKIFKAIHDNKSFVWVQLWV LGRQAFADNLARDGLRYDSASDEVYMGEDEKERAIRSNNPQHGITKDEIKQYIRDYVDA AKKCIDAGADGVEIHSANGFLLNQFLDPISNKRTDEYGGSIENRARFVLEWDAWDAVG AERTSIRFSPYGVFGTMSGGSDPVLVAQFAYVLAELEKRAKAGKRLAYVDLVEPRVTSPF QPEFEGWYKGGTNEFVYSVWKGNVLRVGNYALDPDAAITDSKNPNTLIGYGRAFIANPD

[0253] LVERLEKGLPLNQYDRPSFYKMSAEGYIDYPTYEEAVAKGYKK (SEQ ID NO: 16).

[0254] According to the present invention, 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is not tyrosine. Preferably, the 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 16.

[0255] The present invention relates to a method for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence MSSEKLYSPLKVGAITAANRIFMAPLTRLRSIEPGDIPTPLMAEYYRQRASAGLIISEATQIS AQAKGYAGAPGIHSPEQIAAWKKITAGVHAENGHMAVQLWHTGRISHASLQPGGQAPV APSALSAGTRTSLRDENGQAIRVETSMPRALELEEIPGIVNDFRQAIANAREAGFDLVELH SAHGFLLHQFLSPSSNHRTDQYGGSVENRARLVLEVVDAGIEEWGADRIGIRVSPIGTFQ NTDNGPNEEADALYLIEQLGKRGIAYLHMSEPDWAGGEPYTDAFREKVRARFHGPIIGA GAYTVEKAETLIGKGLIDAVAFGRDWIANPDLVARLQRKAELNPQRAESFYGGGAEGYT DYPTL (SEQ ID NO: 17).

[0256] According to the present invention, 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is not tyrosine. Preferably, the 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 17. The present invention relates to a method for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence

[0257] MNTMLFSPYTIRGLTLKNRIVMSPMCMYSCDTKDGAVRTWHKIHYPARAVGQVG LIIVEATGVTPQGRISERDLGIWSDDHIAGLRELVGLVKEHGAAIGIQLAHAGRKSQ VPGEIIAPSAVPFDDSSPTPKEMTKADIEETVQAFQNGARRAKEAGFDVIEIHAAH GFLINEFLSPLSNRRQDEYGGSPENRYRFLGEVIDAVREVWDGPLFVRISASDYH PDGLTAKDYVPYAKRMKEQGVDLVDVSSGAIVPARMNVYPGYQVPFAELIRREA DIPTGAVGLITSGWQAEEILQNGRADLVFLGRELLRNPYWPYAAARELGAKISAP VQYERGWRF (SEQ ID NO: 18).

[0258] According to the present invention, 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is not tyrosine. Preferably, the 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 18.

[0259] In preferred embodiments, the a,[3-unsaturated carbonyl donor has the general formula wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0260] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0261] In some preferred embodiments, the a,[3-unsaturated carbonyl donor has the general formula (la) or (lb): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0262] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0263] In some preferred embodiments, the a,[3-unsaturated carbonyl donor has the general formula (la) or (lb): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0264] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0265] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the a,[3-unsaturated carbonyl donor has the general formula (la) or (lb):

[0266] (la) (lb) wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0267] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring

[0268] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0269] Thus, the present invention preferably relates to a method for preparing an a- branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,p-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the a,[3-unsaturated carbonyl donor has the general formula (la): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0270] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring

[0271] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0272] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the a,[3-unsaturated carbonyl donor has the general formula (lb): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0273] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring

[0274] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0275] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23. wherein the a,[3-unsaturated carbonyl donor has the general formula (la) or (lb):

[0276] (la) (lb) wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0277] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0278] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23.

[0279] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23. wherein the a,[3-unsaturated carbonyl donor has the general formula (la): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0280] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0281] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23.

[0282] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23. wherein the a,[3-unsaturated carbonyl donor has the general formula (lb): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0283] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0284] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23.

[0285] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. wherein the a,[3-unsaturated carbonyl donor has the general formula (la) or (lb): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0286] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0287] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. wherein the a,[3-unsaturated carbonyl donor has the general formula (la) or (lb): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0288] According to the present invention, 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is not tyrosine. Preferably, the 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is phenylalanine. According to the present invention, 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is not tyrosine. Preferably, the 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is phenylalanine. According to the present invention, 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is not tyrosine. Preferably, the 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is phenylalanine. According to the present invention, 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is not tyrosine. Preferably, the 167thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0289] In preferred embodiments, the a,[3-unsaturated carbonyl donor has the general formula (la): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0290] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0291] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the a,[3-unsaturated carbonyl donor has the general formula (la): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0292] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0293] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0294] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23, wherein the a,[3-unsaturated carbonyl donor has the general formula (la): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0295] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0296] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23.

[0297] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, wherein the a,[3-unsaturated carbonyl donor has the general formula (la): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0298] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0299] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0300] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, wherein the a,[3-unsaturated carbonyl donor has the general formula (la): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0301] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0302] According to the present invention, 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is not tyrosine. Preferably, the 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is phenylalanine. According to the present invention, 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is not tyrosine. Preferably, the 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is phenylalanine. According to the present invention, 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is not tyrosine. Preferably, the 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is phenylalanine. According to the present invention, 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is not tyrosine. Preferably, the 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0303] In preferred embodiments, the a,[3-unsaturated carbonyl donor has the general formula (lb): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0304] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0305] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the a,[3-unsaturated carbonyl donor has the general formula (lb): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0306] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring. According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0307] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23, wherein the a,[3-unsaturated carbonyl donor has the general formula (lb): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0308] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0309] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23.

[0310] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, wherein the a,[3-unsaturated carbonyl donor has the general formula (lb): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0311] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0312] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0313] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. wherein the a,[3-unsaturated carbonyl donor has the general formula (lb): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0314] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0315] According to the present invention, 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is not tyrosine. Preferably, the 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is phenylalanine. According to the present invention, 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is not tyrosine. Preferably, the 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is phenylalanine. According to the present invention, 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is not tyrosine. Preferably, the 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is phenylalanine. According to the present invention, 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is not tyrosine. Preferably, the 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0316] More preferably, the a,[3-unsaturated carbonyl donor has the general formula (la) or (lb): wherein R1and R2independently of each other represent -H, -NH2, cyclo-CsHs, cyclo-C4H7, cyclo-CsHg, cyclo-CeHn, cyclo-C7Hi3, cyclo- C8Hi5, -Ph, -CH2-Ph, -C2H4Ph, -CPh3, -CH3, -C2H5, -C3H7, -CH(CH3)2I-C4H9, -CH2-CH(CH3)2I-CH(CH3)-C2H5, -C(CH3)3I-C5H11, -CH(CH3)-C3H7, -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2I

[0317] -C(CH3)2-C2H5, -CH2-C(CH3)3I-CH(C2H5)2, -C2H4-CH(CH3)2, -C6HI3, -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9, -CH2-CH(CH3)- C3H7, -CH(CH3)-CH2-CH(CH3)2I-C7Hi5, -C8Hi7, -C(CH3)2-C3H7,

[0318] -CH(CH3)-CH(CH3)-C2H5,-CH2-CH(CH3)-CH(CH3)2, -CH2-C(CH3)2-C2H5, -CH(CH3)-C(CH3)3I-C(CH3)2-CH(CH3)2I-C2H4-C(CH3)3, -CH=CH2, -CH2-CH=CH2, -C(CH3)=CH2I-CH=CH-CH3, -C2H4-CH=CH2, -CH2-CH=CH-CH3, -CH=CH-C2H5, -CH=C(CH3)2I-CH2-C(CH3)=CH2I-CH(CH3)-CH=CH, -C(CH3)=CH-CH3I-CH=CH-CH=CH2, -C3H6-CH=CH2, -C2H4-CH=CH-CH3, -CH2-CH=CH-C2H5, -CH=CH-C3H7, -CH2-CH=CH-CH=CH2, -CH=CH-CH=CH-CH3, -C2H4-CH=CH-CH3, -CH2-CH=CH-C2H5, -CH2-CH=CH-CH=CH2, -CH=CH-CH=CH-CH3, -CH=CH-CH2-CH=CH2, -C(CH3)=CH-CH=CH2I-CH=C(CH3)-CH=CH2I-CH=CH-C(CH3)=CH2I-CH2-CH=C(CH3)2I-C(CH3)=C(CH3)2I-C2H4-CH=CH2, -CH=CH-C2H5, -CH=C(CH3)2I-CH2-CH=CH-CH3, -CH=CH-CH=CH2, C3H6-CH=CH2, -CH=CH-C3H7, -C4H8-CH=CH2, -CH=CH-C4H9, -C3H6-CH=CH-CH3, -CH2-CH=CH-C3H7, -C2H4-CH=CH-C2H5, -CH2-C(CH3)=C(CH3)2I -C2H4-CH=C(CH3)2, -CH2-CECH, -CECH, -CEC-CH3, -C2H4-CECH,

[0319] -CEC-C2H5, -CH2-CEC-CH3, -CEC-CH=CH2, -CH=CH-CECH,

[0320] -CEC-CECH, -C3H6-CECH, -CEC-C3H7, -C2H4-CEC-CH3,

[0321] -CH2-CEC-C2H5, -CH2-CEC-CH=CH2, -CH2-CH=CH-CECH,

[0322] -CH2-CEC-CECH, -CEC-CH=CH-CH3, -CH=CH-CEC-CH3,

[0323] -CEC-CEC-CH3, -CEC-CH2-CH=CH2, -CH=CH-CH2-CECH,

[0324] -CEC-CH2-CECH, -C(CH3)=CH-CH=CH2, -CH=C(CH3)-CH=CH2,

[0325] -CH=CH-C(CH3)=CH2, -C(CH3)=CH-CECH, -CH=C(CH3)-CECH,

[0326] -CEC-C(CH3)=CH2, -C4H8-CECH, -CEC-C4H9, -C3H6-CEC-CH3,

[0327] -CH2-CEC-C3H7, -C2H4Ph, -CH=CH-Ph, -CEC-Ph, -CH2NH2, -CH2OH,

[0328] -CH2SH, -CH2-CH2NH2, -CH2-CH2SH, -C6H4-OCH3, -C6H4-OH,

[0329] -CH2-CH2-OCH3, -CH2-CH2OH, -CH2-OCH3, -CH2-C6H4-OCH3, -CH2-C6H4-OH, -CH2R3, -CH2CH2R3, or -CH2CH2CH2R3; or wherein

[0330] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring, and

[0331] R3represents -NH2, -OH, -SH, -F, -Cl, -Br, -I, -CN, -N3, -OCN, -NCO, -SCN, or -NCS.

[0332] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the a,[3-unsaturated carbonyl donor has the general formula (la) or (lb): wherein R1and R2independently of each other represent -H, — NH2, cyclo-CsHs, cyclo-C4H7, cyclo-CsHg, cyclo-CeHn, cyclo-C7Hi3, cyclo-

[0333] C8Hi5, -Ph, -CH2-Ph, -C2H4Ph, -CPh3, -CH3, -C2H5,

[0334] -C3H7, -CH(CH3)2, -C4H9, -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3,

[0335] -C5H11, -CH(CH3)-C3H7, -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2,

[0336] -C(CH3)2-C2H5, -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3,

[0337] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9, -CH2-CH(CH3)-

[0338] C3H7, -CH(CH3)-CH2-CH(CH3)2, -C7H15, -C8Hi7, -C(CH3)2-C3H7,

[0339] -CH(CH3)-CH(CH3)-C2H5,-CH2-CH(CH3)-CH(CH3)2, -CH2-C(CH3)2-C2H5,

[0340] -CH(CH3)-C(CH3)3, -C(CH3)2-CH(CH3)2, -C2H4-C(CH3)3, -CH=CH2,

[0341] -CH2-CH=CH2, -C(CH3)=CH2I-CH=CH-CH3I-C2H4-CH=CH2,

[0342] -CH2-CH=CH-CH3, -CH=CH-C2H5, -CH=C(CH3)2, -CH2-C(CH3)=CH2I

[0343] -CH(CH3)-CH=CH, -C(CH3)=CH-CH3, -CH=CH-CH=CH2,

[0344] -C3H6-CH=CH2, -C2H4-CH=CH-CH3, -CH2-CH=CH-C2H5,

[0345] -CH=CH-C3H7, -CH2-CH=CH-CH=CH2, -CH=CH-CH=CH-CH3,

[0346] -C2H4-CH=CH-CH3, -CH2-CH=CH-C2H5, -CH2-CH=CH-CH=CH2,

[0347] -CH=CH-CH=CH-CH3, -CH=CH-CH2-CH=CH2, -C(CH3)=CH-CH=CH2I

[0348] -CH=C(CH3)-CH=CH2, -CH=CH-C(CH3)=CH2I-CH2-CH=C(CH3)2,

[0349] -C(CH3)=C(CH3)2, -C2H4-CH=CH2, -CH=CH-C2H5, -CH=C(CH3)2,

[0350] -CH2-CH=CH-CH3, -CH=CH-CH=CH2, C3H6-CH=CH2, -CH=CH-C3H7,

[0351] -C4H8-CH=CH2, -CH=CH-C4H9, -C3H6-CH=CH-CH3,

[0352] -CH2-CH=CH-C3H7, -C2H4-CH=CH-C2H5, -CH2-C(CH3)=C(CH3)2,

[0353] -C2H4-CH=C(CH3)2, -CH2-CECH, -CECH, -CEC-CH3, -C2H4-CECH,

[0354] -CEC-C2H5, -CH2-CEC-CH3, -CEC-CH=CH2, -CH=CH-CECH,

[0355] -CEC-CECH, -C3H6-CECH, -CEC-C3H7, -C2H4-CEC-CH3,

[0356] -CH2-CEC-C2H5, -CH2-CEC-CH=CH2, -CH2-CH=CH-CECH,

[0357] -CH2-CEC-CECH, -CEC-CH=CH-CH3, -CH=CH-CEC-CH3,

[0358] -CEC-CEC-CH3, -CEC-CH2-CH=CH2, -CH=CH-CH2-CECH,

[0359] -CEC-CH2-CECH, -C(CH3)=CH-CH=CH2I-CH=C(CH3)-CH=CH2I

[0360] -CH=CH-C(CH3)=CH2, -C(CH3)=CH-CECH, -CH=C(CH3)-CECH,

[0361] -CEC-C(CH3)=CH2, -C4H8-CECH, -CEC-C4H9, -C3H6-CEC-CH3,

[0362] -CH2-CEC-C3H7, -C2H4Ph, -CH=CH-Ph, -CEC-Ph, -CH2NH2, -CH2OH,

[0363] -CH2SH, -CH2-CH2NH2, -CH2-CH2SH, -C6H4-OCH3, -C6H4-OH,

[0364] -CH2-CH2-OCH3, -CH2-CH2OH, -CH2-OCH3I-CH2-C6H4-OCH3,

[0365] -CH2-C6H4-OH, -CH2R3, -CH2CH2R3, or -CH2CH2CH2R3; or wherein

[0366] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring, and R3represents -NH2, -OH, -SH, -F, -Cl, -Br, -I, -CN, -N3, -OCN, -NCO, -SCN, or -NCS.

[0367] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0368] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23, wherein the a,[3-unsaturated carbonyl donor has the general formula (la) or (lb): wherein R1and R2independently of each other represent

[0369] -H, — NH2, cyclo-CsHs, cyclo-C4H7, cyclo-CsHg, cyclo-CeHn, cyclo-C7Hi3, cyclo- CsHis, -Ph, -CH2-Ph, -C2H4Ph, -CPh3, -CH3, -C2H5,

[0370] -C3H7, -CH(CH3)2I-C4H9, -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3I-C5H11, -CH(CH3)-C3H7, -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2I

[0371] -C(CH3)2-C2H5, -CH2-C(CH3)3I-CH(C2H5)2, -C2H4-CH(CH3)2, -C6HI3,

[0372] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9, -CH2-CH(CH3)- C3H7, -CH(CH3)-CH2-CH(CH3)2I-C7Hi5, -C8Hi7, -C(CH3)2-C3H7,

[0373] -CH(CH3)-CH(CH3)-C2H5,-CH2-CH(CH3)-CH(CH3)2, -CH2-C(CH3)2-C2H5, -CH(CH3)-C(CH3)3, -C(CH3)2-CH(CH3)2, -C2H4-C(CH3)3, -CH=CH2I

[0374] -CH2-CH=CH2, -C(CH3)=CH2I-CH=CH-CH3I-C2H4-CH=CH2,

[0375] -CH2-CH=CH-CH3, -CH=CH-C2H5, -CH=C(CH3)2I-CH2-C(CH3)=CH2,

[0376] -CH(CH3)-CH=CH, -C(CH3)=CH-CH3I-CH=CH-CH=CH2,

[0377] -C3H6-CH=CH2, -C2H4-CH=CH-CH3, -CH2-CH=CH-C2H5,

[0378] -CH=CH-C3H7, -CH2-CH=CH-CH=CH2, -CH=CH-CH=CH-CH3,

[0379] -C2H4-CH=CH-CH3, -CH2-CH=CH-C2H5, -CH2-CH=CH-CH=CH2,

[0380] -CH=CH-CH=CH-CH3, -CH=CH-CH2-CH=CH2, -C(CH3)=CH-CH=CH2I

[0381] -CH=C(CH3)-CH=CH2I-CH=CH-C(CH3)=CH2I-CH2-CH=C(CH3)2I

[0382] -C(CH3)=C(CH3)2I-C2H4-CH=CH2, -CH=CH-C2H5, -CH=C(CH3)2I

[0383] -CH2-CH=CH-CH3, -CH=CH-CH=CH2, C3H6-CH=CH2, -CH=CH-C3H7,

[0384] -C4H8-CH=CH2, -CH=CH-C4H9, -C3H6-CH=CH-CH3,

[0385] -CH2-CH=CH-C3H7, -C2H4-CH=CH-C2H5, -CH2-C(CH3)=C(CH3)2I

[0386] -C2H4-CH=C(CH3)2I-CH2-CECH, -CECH, -CEC-CH3, -C2H4-CECH,

[0387] -CEC-C2H5, -CH2-CEC-CH3, -CEC-CH=CH2, -CH=CH-CECH,

[0388] -CEC-CECH, -C3H6-CECH, -CEC-C3H7, -C2H4-CEC-CH3,

[0389] -CH2-CEC-C2H5, -CH2-CEC-CH=CH2, -CH2-CH=CH-CECH,

[0390] -CH2-CEC-CECH, -CEC-CH=CH-CH3, -CH=CH-CEC-CH3,

[0391] -CEC-CEC-CH3, -CEC-CH2-CH=CH2, -CH=CH-CH2-CECH,

[0392] -CEC-CH2-CECH, -C(CH3)=CH-CH=CH2I-CH=C(CH3)-CH=CH2I

[0393] -CH=CH-C(CH3)=CH2I-C(CH3)=CH-CECH, -CH=C(CH3)-CECH,

[0394] -CEC-C(CH3)=CH2, -C4H8-CECH, -CEC-C4H9, -C3H6-CEC-CH3,

[0395] -CH2-CEC-C3H7, -C2H4Ph, -CH=CH-Ph, -C=C-Ph, -CH2NH2, -CH2OH,

[0396] -CH2SH, -CH2-CH2NH2I-CH2-CH2SH, -C6H4-OCH3, -C6H4-OH,

[0397] -CH2-CH2-OCH3I-CH2-CH2OH, -CH2-OCH3I-CH2-C6H4-OCH3,

[0398] -CH2-C6H4-OH, -CH2R3, -CH2CH2R3, or -CH2CH2CH2R3; or wherein

[0399] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring, and

[0400] R3represents -NH2, -OH, -SH, -F, -Cl, — Br, -CN, -N3, -OCN, -NCO, -SCN, or -NCS.

[0401] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23.

[0402] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. wherein the a,[3-unsaturated carbonyl donor has the general formula (la) or (lb): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0403] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

[0404] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0405] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, wherein the a,[3-unsaturated carbonyl donor has the general formula (la) or (lb): wherein R1and R2independently of each other represent

[0406] -H, — NH2, cyclo-C3H5, cyclo-C4H7, cyclo-CsHg, cyclo-CeHn, cyclo-C7Hi3, cyclo¬

[0407] C8Hi5, -Ph, -CH2-Ph, -C2H4Ph, -CPh3, -CH3, -C2H5,

[0408] -C3H7, -CH(CH3)2I-C4H9, -CH2-CH(CH3)2I-CH(CH3)-C2H5, -C(CH3)3I

[0409] -C5H11, -CH(CH3)-C3H7, -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2I

[0410] -C(CH3)2-C2H5, -CH2-C(CH3)3I-CH(C2H5)2, -C2H4-CH(CH3)2, -C6HI3,

[0411] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9, -CH2-CH(CH3)-

[0412] C3H7, -CH(CH3)-CH2-CH(CH3)2, -C7Hi5, -C8HI7, -C(CH3)2-C3H7,

[0413] -CH(CH3)-CH(CH3)-C2H5,-CH2-CH(CH3)-CH(CH3)2, -CH2-C(CH3)2-C2H5,

[0414] -CH(CH3)-C(CH3)3I-C(CH3)2-CH(CH3)2, -C2H4-C(CH3)3, -CH=CH2,

[0415] -CH2-CH=CH2, -C(CH3)=CH2I-CH=CH-CH3, -C2H4-CH=CH2,

[0416] -CH2-CH=CH-CH3, -CH=CH-C2H5, -CH=C(CH3)2I-CH2-C(CH3)=CH2I

[0417] -CH(CH3)-CH=CH, -C(CH3)=CH-CH3I-CH=CH-CH=CH2,

[0418] -C3H6-CH=CH2, -C2H4-CH=CH-CH3, -CH2-CH=CH-C2H5,

[0419] -CH=CH-C3H7, -CH2-CH=CH-CH=CH2, -CH=CH-CH=CH-CH3,

[0420] -C2H4-CH=CH-CH3, -CH2-CH=CH-C2H5, -CH2-CH=CH-CH=CH2, -CH=CH-CH=CH-CH3, -CH=CH-CH2-CH=CH2I-C(CH3)=CH-CH=CH2I

[0421] -CH=C(CH3)-CH=CH2, -CH=CH-C(CH3)=CH2, -CH2-CH=C(CH3)2I

[0422] -C(CH3)=C(CH3)2, -C2H4-CH=CH2I-CH=CH-C2H5, -CH=C(CH3)2I

[0423] -CH2-CH=CH-CH3, -CH=CH-CH=CH2, C3H6-CH=CH2, -CH=CH-C3H7,

[0424] -C4H8-CH=CH2, -CH=CH-C4H9, -C3H6-CH=CH-CH3,

[0425] -CH2-CH=CH-C3H7, -C2H4-CH=CH-C2H5, -CH2-C(CH3)=C(CH3)2I

[0426] -C2H4-CH=C(CH3)2, -CH2-CECH, -CECH, -CEC-CH3, -C2H4-CECH,

[0427] CEC-C2H5, -CH2-CEC-CH3, -CEC-CH=CH2, -CH=CH-CECH

[0428] CEC-CECH, -C3H6-CECH, -CEC-C3H7, -C2H4-CEC-CH3

[0429] -CH2-CEC-C2H5, -CH2-CEC-CH=CH2, -CH2-CH=CH-CECH,

[0430] -CH2-CEC-CECH, -CEC-CH=CH-CH3, -CH=CH-CEC-CH3,

[0431] -CEC-CEC-CH3, -CEC-CH2-CH=CH2, -CH=CH-CH2-CECH,

[0432] -CEC-CH2-CECH, -C(CH3)=CH-CH=CH2I-CH=C(CH3)-CH=CH2I

[0433] -CH=CH-C(CH3)=CH2I-C(CH3)=CH-CECH, -CH=C(CH3)-CECH,

[0434] -CEC-C(CH3)=CH2, -C4H8-CECH, -CEC-C4H9, -C3H6-CEC-CH3,

[0435] -CH2-CEC-C3H7, -C2H4Ph, -CH=CH-Ph, -CEC-Ph, -CH2NH2, -CH2OH,

[0436] -CH2SH, -CH2-CH2NH2I-CH2-CH2SH, -C6H4-OCH3, -C6H4-OH,

[0437] -CH2-CH2-OCH3I-CH2-CH2OH, -CH2-OCH3, -CH2-C6H4-OCH3, -CH2-C6H4-OH, -CH2R3, -CH2CH2R3, or -CH2CH2CH2R3; or wherein

[0438] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring, and

[0439] R3represents -NH2, -OH, -SH, -F, -Cl, -Br, -I, -CN, -N3, -OCN, -NCO, -SCN, or -NCS.

[0440] According to the present invention, 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is not tyrosine. Preferably, the 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is phenylalanine. According to the present invention, 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is not tyrosine. Preferably, the 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is phenylalanine. According to the present invention, 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is not tyrosine. Preferably, the 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is phenylalanine. According to the present invention, 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is not tyrosine. Preferably, the 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0441] More preferably, R1and R2independently of each other represent

[0442] -H, -NH2, -Ph, -CH2-Ph, -C2H4Ph, -CH3, -C2H5,

[0443] -C3H7, -CH(CH3)2, -C4H9, -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3,

[0444] -C5H11, -CH(CH3)-C3H7, -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2,

[0445] -C(CH3)2-C2H5, -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6HI3,

[0446] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9, -CH2-CH(CH3)- C3H7, -CH(CH3)-CH2-CH(CH3)2, -C(CH3)2-C3H7, -CH(CH3)-CH(CH3)-C2H5, -CH2-CH(CH3)-CH(CH3)2, -CH2-C(CH3)2-C2H5, -CH(CH3)-C(CH3)3,

[0447] -C(CH3)2-CH(CH3)2, -C2H4-C(CH3)3, -CH=CH2I-CH2-CH=CH2I-C(CH3)=CH2, -CH=CH-CH3, -C2H4-CH=CH2, -CH2-CH=CH-CH3, -CH=CH-C2H5, -CH=C(CH3)2, -CH2-C(CH3)=CH2, -CH(CH3)-CH=CH, -C(CH3)=CH-CH3,

[0448] -CH=CH-CH=CH2, -C3H6-CH=CH2, -C2H4-CH=CH-CH3, -CH2-CH=CH- C2H5, -CH=CH-C3H7, -CH2-CH=CH-CH=CH2, -CH=CH-CH=CH-CH3, -C2H4-CH=CH-CH3, -CH2-CH=CH-C2H5, -CH2-CH=C(CH3)2, -C(CH3)=C(CH3)2, -C2H4-CH=CH2, -CH=CH-C2H5, -CH=C(CH3)2, -CH2- CH=CH-CH3, -C3H6-CH=CH2, -CH=CH-C3H7, -C4H8-CH=CH2, -CH=CH- C4H9, -C3H6-CH=CH-CH3, -CH2-CH=CH-C3H7, -C2H4-CH=CH-C2H5, -CH2-C(CH3)=C(CH3)2, -C2H4-CH=C(CH3)2, -CH2-CECH, -CECH, -CEC-CH3, -C2H4-CECH, -CEC-C2H5, -CH2-CEC-CH3, -C3H6-CECH, -CEC-C3H7, -C2H4-CEC-CH3, -CH2-CEC-C2H5, -C4H8-CECH, -CEC-C4H9, -C3H6- CEC-CH3, -CH2-CEC-C3H7, -CH=CH-Ph, -CEC-Ph, -C6H4-OCH3, -CH2-CH2-OCH3, -CH2-OCH3I-CH2-C6H4-OCH3, -CH2R3, -CH2CH2R3, or -CH2CH2CH2R3; and or wherein

[0449] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring, and

[0450] R3represents -NH2, -OH, -SH, -F, -Cl, -Br, -I, -CN, -N3, -OCN, -NCO, -SCN, or -NCS.

[0451] Still more preferably, R1and R2independently of each other represent

[0452] -H, -NH2, -Ph, -CH2-Ph, -C2H4Ph, -CH3,

[0453] -C2H5, -C3H7, -CH(CH3)2I-C4H9, -CH2-CH(CH3)2I-CH(CH3)-C2H5,

[0454] -C(CH3)3I-CH=CH2I-CH2-CH=CH2I-C(CH3)=CH2I-CH=CH-CH3, -C2H4-CH=CH2, -CH2-CH=CH-CH3I-CH=CH-C2H5, -CH=C(CH3)2I-CH2-C(CH3)=CH2I-CH(CH3)-CH=CH, -C(CH3)=CH-CH3I

[0455] -CH=CH-CH=CH2I-CH2-CECH, -CECH, -CEC-CH3, -C2H4-CECH, -CEC-C2H5, -CH2-CEC-CH3, -C3H6-CECH, -CEC-C3H7, -C2H4-CEC-CH3, -CH2-CEC-C2H5, -C4H8-CECH, -CEC-C4H9, -C3H6-CEC-CH3, -CH2-CEC-C3H7, -CH=CH-Ph, -CEC-Ph, -CH2-CH2-OCH3, -CH2-OCH3, -CH2R3, -CH2CH2R3, or -CH2CH2CH2R3; and or wherein

[0456] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring, and

[0457] R3represents -NH2, -OH, -SH, -F, -Cl, -Br, -I, -CN, -N3, -OCN, -NCO, -SCN, or -NCS.

[0458] Even more preferably, R1and R2independently of each other represent -H, -NH2, -Ph, -CH2-Ph, -C2H4Ph, -CH3,

[0459] -C2H5, -C3H7, -CH(CH3)2I-CH=CH2I-CH2-CH=CH2I-C(CH3)=CH2I

[0460] -CH=CH-CH3I-CH2-CECH, -CECH, -CEC-CH3, -C2H4-CECH, -CEC-C2H5, -CH2-CEC-CH3, -C3H6-CECH, -CEC-C3H7, -C2H4-CEC-CH3, -CH2-CEC-C2H5, -C4H8-CECH, -CEC-C4H9, -C3H6-CEC-CH3, -CH2-CEC-C3H7, -CH=CH-Ph, -CEC-Ph, -CH2-CH2-OCH3, -CH2-OCH3, -CH2R3, -CH2CH2R3, or -CH2CH2CH2R3; and or wherein

[0461] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring, and

[0462] R3represents -NH2, -OH, -SH, -F, -Cl, -Br, -I, -CN, -N3, -OCN, -NCO, -SCN, or -NCS.

[0463] Still more preferably, R1and R2independently of each other represent

[0464] -H, -NH2, -Ph, -CH2-Ph, -C2H4Ph, -CH3,

[0465] -C2H5, -C3H7, -CH=CH2I-CH2-CH=CH2I-CH=CH-CH3I-CH2-CECH, -CECH, -CEC-CH3, -CH=CH-Ph, -CEC-Ph, -CH2R3, -CH2CH2R3, or -CH2CH2CH2R3; and or wherein

[0466] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring, and

[0467] R3represents -F, -Cl, -Br, -I. In preferred embodiments, the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated enone or a,[3-unsaturated enal, preferably wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated cycloenone. In more preferred embodiments, the a,[3-unsaturated carbonyl donor is an optionally substituted cyclohexenone.

[0468] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated enone or a,[3-unsaturated enal, preferably wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated cycloenone, more wherein wherein the a,[3-unsaturated carbonyl donor is an optionally substituted cyclohexenone.

[0469] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0470] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23, wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated enone or a,[3-unsaturated enal, preferably wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated cycloenone, more wherein wherein the a,[3-unsaturated carbonyl donor is an optionally substituted cyclohexenone.

[0471] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23.

[0472] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated enone or a,[3-unsaturated enal, preferably wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated cycloenone, more wherein wherein the a,[3-unsaturated carbonyl donor is an optionally substituted cyclohexenone. According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0473] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated enone or a,[3-unsaturated enal, preferably wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated cycloenone, more wherein wherein the a,[3-unsaturated carbonyl donor is an optionally substituted cyclohexenone.

[0474] According to the present invention, 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is not tyrosine. Preferably, the 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is phenylalanine. According to the present invention, 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is not tyrosine. Preferably, the 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is phenylalanine. According to the present invention, 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is not tyrosine. Preferably, the 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is phenylalanine. According to the present invention, 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is not tyrosine. Preferably, the 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0475] In further preferred embodiments, the carbonyl acceptor has the general formula (II): wherein R4represents -H, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9,

[0476] -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H11, -CH(CH3)-C3H7,

[0477] -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5,

[0478] -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3,

[0479] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9,

[0480] -CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2, -Ph, or -CH2-Ph.

[0481] Preferably, R4represents -H, -CH3, -C2H5, -CsH?, -CH(CH3)2, -C4H9, -CH2-CH(CH3)2, -CH(CH3)-C2H5. More preferably, R3represents -H, -CH3, -C2H5, - CsH?, -C4H9. More preferably, R3represents -H, -CH3, -C2H5, - CsH?.

[0482] More preferably, R4represents -H, -CH3, -C2H5. Thus, it is preferred that the carbonyl acceptor is selected from the group comprising or consisting of formaldehyde, acetaldehyde and propionaldehyde.

[0483] Most preferably, R4represents -H or -CH3. Thus, most preferred is that the carbonyl acceptor is formaldehyde or acetaldehyde.

[0484] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the carbonyl acceptor has the general formula (II): wherein R4represents -H, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9, -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H11, -CH(CH3)-C3H7, -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5,

[0485] -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3,

[0486] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9,

[0487] -CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2, -Ph, or -CH2-Ph.

[0488] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0489] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23. wherein the carbonyl acceptor has the general formula (II): wherein R4represents -H, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9,

[0490] -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H11, -CH(CH3)-C3H7,

[0491] -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5,

[0492] -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3,

[0493] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9,

[0494] -CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2, -Ph, or -CH2-Ph.

[0495] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23.

[0496] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. wherein the carbonyl acceptor has the general formula (II): wherein R4represents -H, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9,

[0497] -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H11, -CH(CH3)-C3H7,

[0498] -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5,

[0499] -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3,

[0500] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9,

[0501] -CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2, -Ph, or -CH2-Ph.

[0502] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0503] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, wherein the carbonyl acceptor has the general formula (II): wherein R4represents -H, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9,

[0504] -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H11, -CH(CH3)-C3H7,

[0505] -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5,

[0506] -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3,

[0507] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9,

[0508] -CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2, -Ph, or -CH2-Ph.

[0509] According to the present invention, 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is not tyrosine. Preferably, the 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is phenylalanine. According to the present invention, 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is not tyrosine. Preferably, the 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is phenylalanine. According to the present invention, 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is not tyrosine. Preferably, the 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is phenylalanine. According to the present invention, 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is not tyrosine. Preferably, the 168thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0510] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the a,[3-unsaturated carbonyl donor has the general formula (la) or (lb):

[0511] (la) (lb) wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring, and wherein the carbonyl acceptor has the general formula (II): wherein R4represents -H, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9,

[0512] -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H11, -CH(CH3)-C3H7,

[0513] -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5,

[0514] -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3,

[0515] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9,

[0516] -CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2, -Ph, or -CH2-Ph.

[0517] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 . Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23. wherein the a,[3-unsaturated carbonyl donor has the general formula (la) or (lb):

[0518] (la) (lb) wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0519] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring, and wherein the carbonyl acceptor has the general formula (II): wherein R4represents -H, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9,

[0520] -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H11, -CH(CH3)-C3H7,

[0521] -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5,

[0522] -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3,

[0523] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9,

[0524] -CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2, -Ph, or -CH2-Ph. According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23.

[0525] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, wherein the a,[3-unsaturated carbonyl donor has the general formula (la) or (lb):

[0526] (la) (lb) wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0527] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring, and wherein the carbonyl acceptor has the general formula (II): (II) wherein R4represents -H, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9, -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H11, -CH(CH3)-C3H7, -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5,

[0528] -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3,

[0529] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9,

[0530] -CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2, -Ph, or -CH2-Ph.

[0531] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0532] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. wherein the a,[3-unsaturated carbonyl donor has the general formula (la) or (lb): wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or wherein

[0533] R1and R2are linked to form an optionally substituted 4-membered to 10- membered ring, and wherein the carbonyl acceptor has the general formula (II):

[0534] According to the present invention, 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is not tyrosine. Preferably, the 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is phenylalanine. According to the present invention, 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is not tyrosine. Preferably, the 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is phenylalanine. According to the present invention, 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is not tyrosine. Preferably, the 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is phenylalanine. According to the present invention, 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is not tyrosine. Preferably, the 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated enone or a,[3-unsaturated enal, preferably wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated cycloenone, more wherein wherein the a,[3-unsaturated carbonyl donor is an optionally substituted cyclohexenone, and wherein the carbonyl acceptor has the general formula (II): wherein R4represents -H, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9,

[0535] -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H11, -CH(CH3)-C3H7,

[0536] -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5,

[0537] -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3,

[0538] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9,

[0539] -CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2, -Ph, or -CH2-Ph.

[0540] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 . Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23. wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated enone or a,[3-unsaturated enal, preferably wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated cycloenone, more wherein wherein the a,[3-unsaturated carbonyl donor is an optionally substituted cyclohexenone, and wherein the carbonyl acceptor has the general formula (II): wherein R4represents -H, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9,

[0541] -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H11, -CH(CH3)-C3H7,

[0542] -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5,

[0543] -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3,

[0544] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9,

[0545] -CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2, -Ph, or -CH2-Ph.

[0546] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23. Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated enone or a,[3-unsaturated enal, preferably wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated cycloenone, more wherein wherein the a,[3-unsaturated carbonyl donor is an optionally substituted cyclohexenone, and wherein the carbonyl acceptor has the general formula (II): wherein R4represents -H, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9,

[0547] -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H11, -CH(CH3)-C3H7,

[0548] -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5,

[0549] -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3,

[0550] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9,

[0551] -CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2, -Ph, or -CH2-Ph.

[0552] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0553] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated enone or a,[3-unsaturated enal, preferably wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated cycloenone, more wherein wherein the a,[3-unsaturated carbonyl donor is an optionally substituted cyclohexenone, and wherein the carbonyl acceptor has the general formula (II): wherein R4represents -H, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9,

[0554] -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H11, -CH(CH3)-C3H7,

[0555] -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5,

[0556] -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3,

[0557] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9,

[0558] -CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2, -Ph, or -CH2-Ph.

[0559] According to the present invention, 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is not tyrosine. Preferably, the 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is phenylalanine. According to the present invention, 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is not tyrosine. Preferably, the 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is phenylalanine. According to the present invention, 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is not tyrosine. Preferably, the 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is phenylalanine. According to the present invention, 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is not tyrosine. Preferably, the 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0560] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the enzymatic-catalyzed reductive aldol reaction is performed at a pH of 10 and / or wherein the concentration of the carbonyl acceptor is about 250 mM.

[0561] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 . Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23. wherein the enzymatic-catalyzed reductive aldol reaction is performed at a pH of 10 and / or wherein the concentration of the carbonyl acceptor is about 250 mM.

[0562] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23.

[0563] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, wherein the enzymatic-catalyzed reductive aldol reaction is performed at a pH of 10 and / or wherein the concentration of the carbonyl acceptor is about 250 mM.

[0564] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0565] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. wherein the enzymatic-catalyzed reductive aldol reaction is performed at a pH of 10 and / or wherein the concentration of the carbonyl acceptor is about 250 mM.

[0566] According to the present invention, 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is not tyrosine. Preferably, the 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is phenylalanine. According to the present invention, 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is not tyrosine. Preferably, the 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is phenylalanine. According to the present invention, 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is not tyrosine. Preferably, the 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is phenylalanine. According to the present invention, 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is not tyrosine. Preferably, the 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0567] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated enone or a,[3-unsaturated enal, preferably wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated cycloenone, more wherein wherein the a,[3-unsaturated carbonyl donor is an optionally substituted cyclohexenone, and wherein the carbonyl acceptor has the general formula (II): wherein R4represents -H, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9,

[0568] -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H11, -CH(CH3)-C3H7,

[0569] -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5,

[0570] -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3,

[0571] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9,

[0572] -CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2, -Ph, or -CH2-Ph, and wherein the enzymatic-catalyzed reductive aldol reaction is performed at a pH of 10 and / or wherein the concentration of the carbonyl acceptor is about 250 mM.

[0573] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0574] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23. wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated enone or a,[3-unsaturated enal, preferably wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated cycloenone, more wherein wherein the a,[3-unsaturated carbonyl donor is an optionally substituted cyclohexenone, and wherein the carbonyl acceptor has the general formula (II): wherein R4represents -H, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9, -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H11, -CH(CH3)-C3H7, -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5,

[0575] -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3, -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9,

[0576] -CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2, -Ph, or -CH2-Ph, and wherein the enzymatic-catalyzed reductive aldol reaction is performed at a pH of 10 and / or wherein the concentration of the carbonyl acceptor is about 250 mM.

[0577] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23.

[0578] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated enone or a,[3-unsaturated enal, preferably wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated cycloenone, more wherein wherein the a,[3-unsaturated carbonyl donor is an optionally substituted cyclohexenone, and wherein the carbonyl acceptor has the general formula (II): wherein R4represents -H, -CH3,

[0579] -CH2-CH(CH3)2, -CH(CH3)-C2H5, -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5,

[0580] -CH2-C(CH3)3I-CH(C2H5)2, -C2H4-CH(CH3)2, -C6HI3,

[0581] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9I

[0582] -CH2-CH(CH3)-C3H7I-CH(CH3)-CH2-CH(CH3)2I-Ph, or -CH2-Ph, and wherein the enzymatic-catalyzed reductive aldol reaction is performed at a pH of 10 and / or wherein the concentration of the carbonyl acceptor is about 250 mM.

[0583] According to the present invention, 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is not tyrosine. Preferably, the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0584] Thus, the present invention relates to a method for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the following steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; and c) performing an enzymatic-catalyzed reductive aldol reaction with the a,[3-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated enone or a,[3-unsaturated enal, preferably wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated cycloenone, more wherein wherein the a,[3-unsaturated carbonyl donor is an optionally substituted cyclohexenone, and wherein the carbonyl acceptor has the general formula (II): wherein R4represents -H, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9,

[0585] -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H11, -CH(CH3)-C3H7,

[0586] -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5,

[0587] -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3,

[0588] -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9,

[0589] -CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2, -Ph, or -CH2-Ph, and wherein the enzymatic-catalyzed reductive aldol reaction is performed at a pH of 10 and / or wherein the concentration of the carbonyl acceptor is about 250 mM.

[0590] According to the present invention, 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is not tyrosine. Preferably, the 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is phenylalanine. According to the present invention, 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is not tyrosine. Preferably, the 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is phenylalanine. According to the present invention, 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is not tyrosine. Preferably, the 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is phenylalanine. According to the present invention, 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is not tyrosine. Preferably, the 168thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is phenylalanine. In further preferred embodiments, the polypeptide is a modified ene reductase comprising an amino acid sequence having at least 98% sequence identity, more preferably at least 99% sequence identity, even more preferably 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0591] The present invention further relates to a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or one of the amino acid sequence as set forth in SEQ 87 - SEQ 121 .

[0592] The present invention further relates to a polypeptide comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or one of the amino acid sequence as set forth in SEQ 87 - SEQ 121 .

[0593] The present invention further relates to a polypeptide comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or one of the amino acid sequence as set forth in SEQ 87 - SEQ 121 .

[0594] The present invention further relates to a polypeptide comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or one of the amino acid sequence as set forth in SEQ 87 - SEQ 121 .

[0595] The present invention further relates to a polypeptide comprising an amino acid sequence having at least 99% or 99.5% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or one of the amino acid sequence as set forth in SEQ 87 - SEQ 121 .

[0596] The present invention further relates to a polypeptide comprising an amino acid sequence having 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or one of the amino acid sequence as set forth in SEQ 87 - SEQ 121 .

[0597] The present invention further relates to a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or one of the amino acid sequence as set forth in SEQ 87 - SEQ 121 for use in preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction.

[0598] The present invention further relates to a polypeptide comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or one of the amino acid sequence as set forth in SEQ 87 - SEQ 121 for use in preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction. The present invention further relates to a polypeptide comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or one of the amino acid sequence as set forth in SEQ 87 - SEQ 121 for use in preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction.

[0599] The present invention further relates to a polypeptide comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or one of the amino acid sequence as set forth in SEQ 87 - SEQ 121 for use in preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction.

[0600] The present invention further relates to a polypeptide comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or one of the amino acid sequence as set forth in SEQ 87 - SEQ 121 for use in preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction.

[0601] The present invention further relates to a polypeptide comprising an amino acid sequence having 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or one of the amino acid sequence as set forth in SEQ 87 - SEQ 121 for use in preparing an a- branched P’-hydroxy carbonyl compound by reductive aldol reaction.

[0602] The amino acid sequence as set forth in SEQ 87 - SEQ 121 are as follows:

[0603] Kits

[0604] The modified ene reductases or CoA-indpendent reductive aldolases used in the method according to the present invention for preparing a-branched P’-hydroxy carbonyl compound by reductive aldol reaction may be provided in the form of kits. The enzymes in the kits may be present individually or as a plurality of enzymes. The kits can further include reagents for carrying out the enzymatic reactions, substrates for assessing the activity of enzymes, as well as reagents for detecting the products. The kits can also include reagent dispensers and instructions for use of the kits.

[0605] The kits described herein can include arrays comprising a plurality of different polypeptides having the enzymatic activity of a polypeptide capable of catalyzing reductive aldol reactions at different addressable positions, wherein the different polypeptides are different variants of a reference sequence each having at least one different improved enzyme property.

[0606] In one embodiment the kit for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, comprises: a) a polypeptide capable of catalyzing CoA-independent reductive aldol reactions, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

[0607] In one embodiment the kit for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, comprises: a) a polypeptide capable of catalyzing CoA-independent reductive aldol reactions, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23.

[0608] In one embodiment the kit for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, comprises: a) a polypeptide capable of catalyzing CoA-independent reductive aldol reactions, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0609] In one embodiment the kit for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, comprises: a) a polypeptide capable of catalyzing CoA-independent reductive aldol reactions, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0610] In one embodiment the kit for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction further comprises the cofactor NADPH.

[0611] Thus, the present invention is also directed to a kit for preparing an a-branched P’- hydroxy carbonyl compound by reductive aldol reaction comprising: a) a polypeptide capable of catalyzing CoA-independent reductive aldol reactions, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 , b) NADPH.

[0612] In one embodiment the kit for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, comprises: a) a polypeptide capable of catalyzing CoA-independent reductive aldol reactions, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23, b) NADPH.

[0613] In one embodiment the kit for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, comprises: a) a polypeptide capable of catalyzing CoA-independent reductive aldol reactions, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, b) NADPH.

[0614] In one embodiment the kit for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, comprises: a) a polypeptide capable of catalyzing CoA-independent reductive aldol reactions, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, b) NADPH.

[0615] Thus, the present invention is also directed to a kit for preparing an a-branched |3’- hydroxy carbonyl compound by reductive aldol reaction comprising: a) a polypeptide capable of catalyzing CoA-independent reductive aldol reactions, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 , b) NADPH, c) a cofactor generation system for the conversion of NADP+to NADPH.

[0616] In one embodiment the kit for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, comprises: a) a polypeptide capable of catalyzing CoA-independent reductive aldol reactions, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23, b) NADPH, c) a cofactor generation system for the conversion of NADP+to NADPH.

[0617] In one embodiment the kit for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, comprises: a) a polypeptide capable of catalyzing CoA-independent reductive aldol reactions, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, b) NADPH, c) a cofactor generation system for the conversion of NADP+to NADPH.

[0618] In one embodiment the kit for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, comprises: a) a polypeptide capable of catalyzing CoA-independent reductive aldol reactions, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, b) NADPH, c) a cofactor generation system for the conversion of NADP+to NADPH.

[0619] Description of the Figures

[0620] Figure 1 shows comparison of chemical aldol and reductive aldol reactions as well as re-engineering of carboxylases and ene reductases to serve as reductive aldolases. Newly formed C-C bond is highlighted in green.

[0621] Figure 2 shows reductive aldol reaction catalyzed by ERs. (A) Reaction scheme of reductive aldol reaction as well as yields for the reductive aldol and reduction reaction are shown for different ERs. (B) Crystal structure of NemA Tyr187Phe (PDB: 8BPQ) with the inhibitor p-hydroxybenzaldehyde modeled into the active site by overlaying our crystal structure with the inhibitor containing structure from Saccharomyces pastorianus (PDB: 1 OYB). (C-E) Representative structures from MD simulations of (C) NemA wildtype (PDB: 8BPP) (D) NemA Tyr187Phe, and (E) NemA Tyr187Phe Trp103Ala. The FMN cofactor is shown in yellow, the reactive enolate and formaldehyde are shown in purple, the flexible [3-hairpin (Thr140- Thr146) is highlighted in blue, and solvent accessible volumes are depicted as colored surfaces.

[0622] Figure 3 shows pptimization of reaction conditions of the EcNemA catalyzed reductive aldol reaction.

[0623] Figure 4 shows reductive aldol and reduction product yields as well as selectivity of different variants of EcNemA.

[0624] Figure 5 shows conformational Analysis of NemA ene-reductase WT, Tyr187Phe, and Tyr187Phe Trp103Ala variants: (A) Normalized eigenvalues for the first 30 eigenvectors from the Principal Component Analysis. (B,C) Representation of the first and second principal components concentrating the main conformational changes on the [3-hairpin movements of residues Thr130 to Thr146. (D,E,F) Projection of all trajectories of the WT, Tyr187Phe, and the Tyr187Phe Trp103Ala simulations on the two principal components, respectively. Increased flexibility of a system corresponds to larger area covered by its configurations (blue points). The orange star corresponds to the representative structure used for the Accessible Surface Area analysis Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.

[0625] EXAMPLES

[0626] Materials and Methods

[0627] Chemicals

[0628] 2-cyclohexen-1 -one was purchased from Sigma Aldrich AG. NADPH Na4 (98%) was purchased from Carl Roth GmbH. 2-(hydroxymethyl)cyclohexan-1-one was purchased from Enamine. Solvents and salts were all analytical grade or better.

[0629] Cloning and mutagenesis

[0630] Ene reductase genes (for sequences see Table 7) were purchased from Twist Biosciences and cloned into a pET-16b vector C-terminal to a 10x His tag and a factor Xa cleavage site using Gibson assembly (Table 8). Variants of ene reductases were generated either by QuikChange® Site-Directed Mutagenesis Kit (Stratagene, La Jolla, USA) or by KLD ligation (NEB, M0554).

[0631] Table 7: Nucleotide sequences of Ene reductases used. Sequences are given in in the direction of 5’ to 3’ end.

[0632] Table 8: Nucleotide sequences of primers used for generation of ER mutants. Sequences are given in in the direction of 5’ to 3’ end. The method by which mutations were introduced is indicated. Converting CoA-independent ERs into reductive aldolases

[0633] Four phylogenetically diverse ERs have been selected for engineering efforts: 0ye1 from Saccharomyces pastorianus, Kye1 from Kluyveromyces lactis (72 % / 83 % identity / similarity compared to Oye1 ), NemA from Escherichia coli (34 % / 51 % identity / similarity), and NamA from Geobacillus kaustophilus (22 % / 38 % identity / similarity). As model substrate cyclohexenone was used, which was well accepted by all four enzymes (Table 9).

[0634] Table 9: Testing different substrates for their reaction with different ene reductases. Rates were estimated by monitoring the decrease in NADPH absorbance in a TECAN platereader at 50 nM enzyme, 10 mM substrate, and 500 pM NADPH, in a total assay volume of 200 pl. Assay buffer: 50 mM potassium phosphate, pH= 7.0.

[0635] The conserved tyrosine proton donor was replaced in all four ERs by phenylalanine and the resulting variants were screened for the reaction of cyclohexenone with formaldehyde, which yields 2-(hydroxymethyl)cyclohexan-1 - one (2-HMC) as product. Notably, all mutants tested were able to produce detectable amounts of 2-HMC. However, NemA Tyr187Phe clearly outperformed all other enzymes, between 3- and 20-fold with respect to 2-HMC yield (Figure 2A).

[0636] In the following, the focus was placed on NemA. Because NemA still produced cyclohexanone as major product, different pHs and formaldehyde concentrations were tested to favor the reductive aldol reaction over ene reduction (Figure 3). By optimizing reaction conditions, the yield for 2-HMC could be successfully increased from initially 6 % (pH=8.0 and 50 mM formaldehyde) to 30 % (pH=10 and 250 mM formaldehyde; Figure 2A, 3). Notably, the fivefold increase in yield was accompanied by a nearly tenfold improved chemoselectivity of the enzyme (defined as specificity factor o = yieldaidoi / yieldreduction), which increased from o = 0.06 to o = 0.55. Having optimized the working conditions of the reaction, the inventors decided to resort to enzyme engineering to further improve chemoselectivity.

[0637] Ene reductases were produced in, BL21 (DE3). Briefly, cells were transformed and grown over night at 37 °C in LB. Cultures were diluted 1 :100 into 0.5-1 L TB and incubated at 37 °C until optical density at 600 nm (OD600) between 0.6 and 1.3. Protein production was induced with 0.5 mM isopropyl (3-D-1- thiogalactopyranoside (IPTG), and incubation continued for 18-32 h at 25 °C. Cells were harvested by centrifugation and lysed by sonication (purifications for activity assays) or using a microfluidizer (for crystallography). To increase FMN loading, approximately 20 mg FMN was added into the lysate and incubated for 15-30 min on ice. NemA was purified from the clarified lysate by Ni-NTA affinity chromatography using wash buffer (25 mM K2HPO4, 25 mM KH2HPO4, 150 mM NaCI, 20 mM imidazole) and elution buffer (25 mM K2HPO4, 25 mM KH2HPO4, 150 mM NaCI, 250 mM imidazole) at pH 7.5. The eluate was concentrated and purged of salts and imidazole using Amicon Ultra-15 centrifugation units (Merck, Germany) with a molecular weight cutoff of 30 kDa by washing with phosphate- buffered saline (PBS). For crystallization, two additional purification steps were added: immediately after Ni-NTA purification, the eluate was loaded onto a HiPrep QHP 16 / 10 column (GE Healthcare, United States) equilibrated with a low-salt buffer (40 mM TRIS, pH 7.5). Affinity chromatography was conducted using a flow rate of 1 mL min-1with a gradient to 80% high-salt buffer (40 mM TRIS, 1.0 M NaCI, pH 7.5) in 60 min. Fractions were collected based on FMN absorbance at 340 nm and concentrated using Amicon Ultra-15 centrifugation units with a molecular weight cutoff of 30 kDa. The concentrated eluate was loaded onto a HiLoad 16 / 600 Superdex 200 pg column (GE Healthcare) equilibrated with 20 mM TRIS, 50 mM NaCI, pH 7.5. Fractions were again collected based on absorbance at 340 nm, pooled and concentrated to 30 mg / mL on 30 kDa Amicon Ultra-15 filters. The concentration of FMN-loaded protein was determined by measuring absorbance at 462 nm and using a molar extinction coefficient of 10600 M-1cm-1. Purified proteins were stored at -20°C.

[0638] Crystallization & Structure Determination

[0639] Crystal plates were set up using the sitting drop vapor diffusion method. After initial broad screening for crystallization conditions, we arrived at the following conditions for refinement plates: for NemA wildtype the reservoir solution contained 50-175 mM malic acid and 14-28% PEG 3350; and for NemA Tyr187Phe 0.2 M potassium bromide, 0.2 M potassium thiocyanate, 0.1 M Tris pH 7.8, 2-8 % w / v y-PGA (Na+ form, LM) and 1-5 % w / v PEG 3350. Protein concentrated to 30 mg / mL and well solution were mixed in equal volumes (0.2 pl of each). Crystals of NemA appeared after 3-7 days and were protected with 35% PEG4000 in crystallisation condition. Crystals of NemA Tyr187Phe appeared after 3-7 weeks and were soaked in; one part PEG200 and one part crystallization solution.

[0640] Data for NemA was collected at the beamline P13 (Deutsches Elektronen- Synchrotron, Hamburg, Germany), whereas data for NemA Tyr187Phe was collected at the beamline ID23-1 (European Synchrotron Radiation Facility, Grenoble, France). All images were processed using XDS. The dataset was scaled using the program suite ccp4. The Phenix software package was used to perform molecular replacement (PhaserMR) for phasing of the EcOYE dataset by using the structure of pentaerythritol tetranitrate reductase from Enterobacter cloacae (PDB 3P7Y_A) as search model. The refined structure of EcOYE was then used as search model for NemA Tyr187Phe. Initial models were built with Phenix.AutoBuild and refined with phenix. refine. Manual refinement and ligand modelling was done in COOT. Final B-factor refinement and water positioning was also performed via phenix. refine. Data collection and refinement statistics can be found in Table 10.

[0641] Table 10: Crystallographic data collection and refinement statistics.

[0642] Statistics for the highest resolution shell are shown in parentheses.

[0643] ER assays were run on a 200 pl scale in 50 mM potassium phosphate buffer (pH=6.0-8.0) or glycine-NaOH buffer (pH=9.0-10.0) at the following substrate concentrations: 1 mM cyclohexenone, 50-1000 mM formaldehyde and 4 mM NADPH. Reactions were shaken at 30°C and reached completion after 1 h.

[0644] Afterwards, reactions were quenched by addition of 20 pl formic acid and 380 pl of methanol. Then reactions were spun at 17,000 g for 15 min to remove enzyme from the solution. The supernatant was analyzed by LC-MS. Quantification of cyclohexanone and 2-HMC was performed using a HRES-LC- MS. The chromatographic separation was performed on a Thermo Scientific Vanquish HPLC System using a Kinetex Evo C18 column (150 x ,12mm, 100 A, 1.7 pm, Phenomenex) equipped with a 20 X 2.1 mm guard column of similar specificity at a constant eluent flow rate of 0.2 ml / min and a column temperature of 30 °C with eluent A being 0.1 % formic acid in water and eluent B being 0.1 % of formic acid in MeOH (Honeywell). The injection volume was 5 pl. The elution profile consisted of the following steps and linear gradients: 0 - 4 min constant at 0% B; 4 - 12 min from 0 to 90 % B; 12 - 15 min constant at 90 % B; 15 - 15.1 min from 90 to 0% B; 15.1 - 20 min constant at 0 % B. A Thermo Scientific ID-X Orbitrap mass spectrometer was used in positive mode with an electrospray ionization source and the following conditions: ESI spray voltage 3500 V, sheath gas at 35 arbitrary units, auxiliary gas at 7 arbitrary units, sweep gas at 0 arbitrary units, ion transfer tube temperature at 300°C. Detection was performed in full scan mode using the Orbitrap mass analyser at a mass resolution of 240 000 in the mass range 50 - 250 (m / z). Extracted ion chromatograms of the [M+H]+ forms were integrated using Tracefinder software (Thermo Scientific). Absolute concentrations were calculated based on an external calibration curve.

[0645] Computer simulations

[0646] Simulations for the NemA ene reductase and its mutants were based on the reported crystal structure modeling the cyclohexanolate and formaldehyde in the active site. Details about parameters, simulation setup, MD simulations and free energy calculations are provided in the Supporting Information.

[0647] Conformational dynamics of NemA

[0648] Simulation details:

[0649] Simulations were carried out in a dodecahedron box with 1 nm of distance between the protein and the side of the box. The system was solvated with water molecules described by the tip3p water model. To obtain a neutral box, Na and Cl ions were added reaching a total concentration of 154 mmol / L. The temperature of 298K and 1 bar pressure were maintained using the V-rescale thermostat and the Parrinello- Rahman barostat. The interaction of the protein were described by the CHARMM36m force field while Cgenff was used for the oxidized flavin mononucleotide (FMN), cyclohexanolate and formaldehyde. Long range electrostatic interactions were treated with particle mesh ewald and for Van-der- Waals interactions a cut-off of 1.2 nm was used. WT and Y187F system were simulated 10 times (10 replicas) for 500ns while the double mutant Y187F_W103A considered 3 replicas of 500ns. GROMACS2021 was used for all simulations. Mutations were created with the visualization programs ucsf chimera 1 .8 using the WT structure as template. To avoid diffusion of the enolate and formaldehyde out of the active site, three harmonic potentials with force constants of 1000 kJ / nm2were applied to the distance between the enolate and His182 and His185 and the formaldehyde with the enolate.

[0650] Accessible Surface Area Analysis:

[0651] The accessible surface area shown in Figure 3C-E of the manuscript was calculated with the Pymol package and the Pyvol plugin and the probe radius was set to a minimum of 1.4 A and a maximum of 3.4 A (similar to water and formaldehyde radius). The protein, the FMN coenzyme, and the substrate were considered in the calculation of the accessible surface area. The representative structure used in the analysis was obtained by cluster analysis of the backbone atoms RMSD with the gromos method using the concatenated trajectories of each system and a cut-off of 2 A (see Figure S18D-E orange star).

[0652] Principal Component Analysis:

[0653] To identify the largest conformational motions of the WT, Tyr187Phe, and the Tyr187Phe Trp103Ala variants, we performed principal component analysis on the combined trajectories of the backbone atom’s position of all three systems. Equilibration of the systems was monitored by the RMSD of the Caatom positions. Diagonalizing the covariance matrix, eigenvectors and eigenvalues were obtained from which the first two eigenvectors concentrate more than 25 % of the conformational motions (Figure 5A). Both eigenvectors describe [3-hairpin movements of residues Thr130 to Thr146 shown in blue in Figure 3C-E of the manuscript (Figure 5B-C).

[0654] Projection of the WT and Tyr187Phe trajectories on the first two principal components shows that the mutant explores a larger area evidencing increased flexibility (Figure 5D-E).

[0655] Compared to the Tyr187Phe variant (Figure 5E), the Tyr187Phe Trp103Ala double mutant presents a smaller explored area and is therefore more rigid (Figure 5F). Active site engineering of NemA to increase chemoselectivity

[0656] To guide further engineering efforts, we solved the crystals structures of NemA WT and variant Tyr187Phe at 3.1 A and 2.3 A resolution, respectively. For variant Tyr187Phe we obtained crystals only in the presence of crystallization agent Xo-4. Overall, the structures of WT and variant Tyr187Phe were essentially superimposable (rmsd 0.34) with only minor differences in the region between residues Asp275 and Gly278. However, MD simulations indicated a significantly increased flexibility of [3-hairpin Thr130 to Thr146 (Figure 2C-E, 5).

[0657] Inspection of the active site of variant Tyr187Phe, identified residue Tyr69 as potential alternative proton donor which could cause residual side product formation (Figure 2B). However, mutation of this residue to phenylalanine or leucine did not further improve the enzyme’s chemoselectivity (Figure S12). We thus turned our attention to efforts to increase formaldehyde accommodation. In previous studies, targeting the conserved Trp103 (Figure 2B), had proven to be a successful engineering strategy to accommodate new substrates at the active site. We therefore replaced Trp103 with smaller hydrophobic residues (alanine, valine or phenylalanine), or amino acids that could serve as hydrogen bond donors to better orient the formaldehyde electrophile (glutamine or asparagine). Although all variants showed decreased overall yields, several variants exhibited significantly higher specificities, among them also double variant Tyr187Phe Trp103Ala that reached a specificity of o = 2.2 (Figure 4). MD simulations indicated that this variant behaved more rigid than the single mutant, lowering solvent accessible volume compared to NemA WT and NemA Tyr187Phe (Figure 2C-E, 5E, F), which suppresses enolate protonation at the active site, but probably also reduces substrate accessibility, explaining the observed yield. Nevertheless, these results showed that the reductive aldol reaction can be in principle improved through additional engineering efforts in the scaffold of ERs.

Claims

Claims1. A method for preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the steps: a) providing an a,[3-unsaturated carbonyl donor; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; c) performing an enzymatic-catalyzed reductive aldol reaction with the a,p-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing CoA-independent reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 .

2. The method according to claim 1 , wherein the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 1 is phenylalanine.

3. The method according to claim 1 or 2, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

4. The method according to claim 3, wherein the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 is phenylalanine.

5. The method according to claim 1 - 4, wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.

6. The method according to claim 5, wherein the 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is phenylalanine, or wherein the 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is phenylalanine, or wherein the 187thamino acid of the amino acid sequence asset forth in SEQ ID NO: 17 is phenylalanine, or wherein the 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is phenylalanine.

7. The method according to claim 1 , wherein the polypeptide is a modified ene reductase comprising an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23.

8. The method according to claim 7, wherein the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 23 is phenylalanine.

9. The method according to claim 1 , wherein the polypeptide further comprises an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

10. The method according to claim 9, wherein the 14thamino acid of the amino acid sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 is phenylalanine.

11. The method according to claim 9 or 10, wherein the polypeptide further comprises an amino acid sequence having least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 29.

12. The method according to claim 1 , wherein the polypeptide comprises an amino acid sequence having at least 100% sequence identity to any one of the amino acid sequence as set forth in SEQ ID NO: 87 - 121.

13. The method according to any one of the claims 1 - 12, wherein the cofactor is NADPH.

14. The method according to any one of the claims 1 - 3, wherein the a,[3-unsaturated carbonyl donor has the general formula (la) or (lb):wherein R1and R2are independently of each other selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, amino, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; or whereinR1and R2are linked to form an optionally substituted 4-membered to 10- membered ring.

15. The method according to claim 14, whereinR1and R2independently of each represent -H, -NH2, cyclo-CsHs, cyclo-C4H?, cyclo-CsHg, cyclo-CeHn, cyclo-C7Hi3, cyclo-CsHis, -Ph, -CH2-Ph, -C2H4Ph, -CPh3, -CH3, -C2H5,-C3H7, -CH(CH3)2, -C4H9, -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H11, -CH(CH3)-C3H7, -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2,-C(CH3)2-C2H5, -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3, -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9, -CH2-CH(CH3)- C3H7, -CH(CH3)-CH2-CH(CH3)2, -C7H15, -C8Hi7, -C(CH3)2-C3H7,-CH(CH3)-CH(CH3)-C2H5, -CH2-CH(CH3)-CH(CH3)2, -CH2-C(CH3)2-C2H5, -CH(CH3)-C(CH3)3, -C(CH3)2-CH(CH3)2, -C2H4-C(CH3)3, -CH=CH2, -CH2-CH=CH2, -C(CH3)=CH2, -CH=CH-CH3, -C2H4-CH=CH2, -CH2-CH=CH-CH3, -CH=CH-C2H5, -CH=C(CH3)2, -CH2-C(CH3)=CH2, -CH(CH3)-CH=CH, -C(CH3)=CH-CH3, -CH=CH-CH=CH2, -C3H6-CH=CH2, -C2H4-CH=CH-CH3, -CH2-CH=CH-C2H5, -CH=CH-C3H7, -CH2-CH=CH-CH=CH2, -CH=CH-CH=CH-CH3, -C2H4-CH=CH-CH3, -CH2-CH=CH-C2H5, -CH2-CH=CH-CH=CH2,-CH=CH-CH=CH-CH3, -CH=CH-CH2-CH=CH2, -C(CH3)=CH-CH=CH2, -CH=C(CH3)-CH=CH2, -CH=CH-C(CH3)=CH2, -CH2-CH=C(CH3)2, -C(CH3)=C(CH3)2, -C2H4-CH=CH2, -CH=CH-C2H5, -CH=C(CH3)2, -CH2-CH=CH-CH3, -CH=CH-CH=CH2, -C3H6-CH=CH2, -CH=CH-C3H7, -C4H8-CH=CH2, -CH=CH-C4H9, -C3H6-CH=CH-CH3, -CH2-CH=CH-C3H7, -C2H4-CH=CH-C2H5, -CH2-C(CH3)=C(CH3)2, -C2H4-CH=C(CH3)2, -CH2-CECH, -CECH, -CEC-CH3, -C2H4-CECH, -CEC-C2H5, -CH2-CEC-CH3, -CEC-CH=CH2, -CH=CH-CECH, -CEC-CECH, -C3H6-CECH, -CEC-C3H7, -C2H4-CEC-CH3,-CH2-CEC-C2H5, -CH2-CEC-CH=CH2, -CH2-CH=CH-CECH, -CH2-CEC-CECH, -CEC-CH=CH-CH3, -CH=CH-CEC-CH3, -CEC-CEC-CH3, -CEC-CH2-CH=CH2, -CH=CH-CH2-CECH, -CEC-CH2-CECH, -C(CH3)=CH-CH=CH2I-CH=C(CH3)-CH=CH2,-CH=CH-C(CH3)=CH2, -C(CH3)=CH-CECH, -CH=C(CH3)-CECH, -CEC-C(CH3)=CH2, -C4H8-C=CH, -C=C-C4H9, -C3H6-CEC-CH3,-CH2-CEC-C3H7, -C2H4Ph, -CH=CH-Ph, -C^C-Ph, -CH2NH2, -CH2OH, -CH2SH, -CH2-CH2NH2, -CH2-CH2SH, -C6H4-OCH3, -C6H4-OH, -CH2-CH2-OCH3I-CH2-CH2OH, -CH2-OCH3I-CH2-C6H4-OCH3, -CH2-C6H4-OH, -CH2R3, -CH2CH2R3, or -CH2CH2CH2R3; or whereinR1and R2are linked to form an optionally substituted 4-membered to 10- membered ring; and whereinR3represents -NH2, -OH, -SH, -F, -Cl, -Br, -I, -CN, -N3, -OCN, -NCO, -SCN, or -NCS.

16. The method according to any one of the claims 1 - 15, wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated enone or a,[3-unsaturated enal, preferably wherein the a,[3-unsaturated carbonyl donor is an a,[3-unsaturated cycloenone.

17. The method according to any one of the Claims 1 - 16, wherein the a,[3-unsaturated donor is an optionally substituted cyclohexenone.

18. The method according to any one of the claims 1 - 17, wherein the carbonyl acceptor has the general formula (II):wherein R4represents -H, -CH3, -C2Hs, -C3H7, -CH(CH3)2, -C4H9, -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3I-C5H11, -CH(CH3)-C3H7, -CH2-CH(CH3)-C2H5, -CH(CH3)-CH(CH3)2I-C(CH3)2-C2H5, -CH2-C(CH3)3I-CH(C2H5)2, -C2H4-CH(CH3)2, -C6HI3,-C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9,-CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2I-Ph, or -CH2-Ph.

19. The method according to any one of the Claims 1 - 18, wherein the carbonyl acceptor is selected from the group comprising or consisting of formaldehyde, acetaldehyde and propionaldehyde, preferably wherein the carbonyl acceptor is formaldehyde.

20. The method according to any one of the Claims 1 - 13, wherein the enzymatic-catalyzed reductive aldol reaction is performed at a pH of 10 and / or wherein the concentration of the carbonyl acceptors is about 250 mM.

21. A polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or one of the amino acid sequence as set forth in SEQ 87 - SEQ 121 for use in preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction.

22. The polypeptide according to Claim 21 , wherein the 197thamino acid of the amino acid sequence as set forth in SEQ ID NO: 15 is phenylalanine, wherein the 196thamino acid of the amino acid sequence as set forth in SEQ ID NO: 16 is phenylalanine, wherein the 187thamino acid of the amino acid sequence as set forth in SEQ ID NO: 17 is phenylalanine, and wherein the 169thamino acid of the amino acid sequence as set forth in SEQ ID NO: 18 is phenylalanine.

23. The polypeptide according to Claim 21 , wherein the polypeptide comprises an amino acid sequence having at least 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or one of the amino acid sequence as set forth in SEQ 87 - SEQ 121 for use in preparing an a-branched P’-hydroxy carbonyl compound by reductive aldol reaction.

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