Engineered acyltransferases with altered substrate specificity

Abstract

The present invention relates to the field of fatty acid synthesis and polyketone compound synthesis as well as protein engineering. In particular, it relates to providing engineered acyltransferases with altered substrate specificity, as well as means and methods of using the acyltransferases of the invention for polyketide synthesis.

Description

Field of the Invention

[0001] The present invention relates to the fields of fatty acid synthesis, polyketide synthesis, and protein engineering. Specifically, it relates to providing engineered acyltransferases with altered substrate specificities, and means and methods for using the acyltransferases of the present invention for polyketide synthesis. Background of the Invention

[0002] Polyketides represent a class of versatile and important biomolecules. In particular, polyketide antibiotics, immunosuppressants, antiparasitic agents, and antifungal agents, cholesterol-lowering agents, and anti-tumor agents represent important classes of biomolecules with outstanding therapeutic significance.

[0003] Despite having biomolecular targets, most polyketides are not currently used as therapeutic agents, and thus only a very small fraction of their vast potential is utilized for this particular class of substances. To make polyketides suitable for use in humans, they need to be tailored for specific functions. However, due to their large carbon skeletons with numerous functional groups and stereochemical features, organic synthesis is very challenging and economically unfeasible for this particular class of substances. Therefore, there is a great need in the pharmaceutical and biotechnology industries to provide systems that allow the synthesis of new and especially custom-made polyketides with tailored functions within a reasonable time.

[0004] Although the polyketide synthase (PKS) system has great potential for synthesizing numerous different bioactive compounds for drug design and other applications, PKS and fatty acid synthase (FAS) enzymes are restricted to substrates that bind to coenzyme A (CoA). CoA is composed of cysteamine (β-mercaptoethylamine), β-alanine, pantoic acid, an organic phosphoric anhydride, and 3'-phosphoadenosine. The sulfhydryl group of cysteamine can form a "high-energy" thioester bond with a carboxylic acid, thereby forming an "activated" acyl-CoA molecule.

[0005] However, CoA is an abundant molecule in typical production strains. Thus, during metabolic engineering, having enzymes with reduced or even eliminated specificity for CoA can be beneficial for the in vivo production of polyketides to shift product yields towards desired keto compounds or target polyketides using specially designed enzymes as engineered molecular machines. Additionally, using artificial pathways (relying on acyl substrates that bind to different coenzyme moieties) for the biosynthesis of target compounds avoids interference with natural metabolism, and new functions no longer require the identification and establishment of suitable natural metabolic pathways to produce the substrates.

[0006] To our knowledge, there are no known acyltransferases that cannot bind CoA or that can bind other coenzyme moieties. As in the present invention, re-engineering coenzyme binding properties is a highly challenging task and requires in-depth analysis, understanding, and integration of the structural and functional properties associated with the enzyme, as well as sequence-based information, in the context of substrate binding. In particular, because the nucleotide binding pocket forms only during or after substrate binding and follows the "induced fit" concept. Thus, the binding pocket is only visible in the crystal structure of the acyltransferase complexed with its respective CoA ester substrate. This includes the identification of charged residues involved in the electrostatic binding of the phosphate group.

[0007] Crystal structures of some FAS enzymes and PKS systems have been solved (Smith and Tsai 2007, Rittner et al. 2020). However, (to our knowledge) it has never been anticipated that the CoA binding pocket of an acyltransferase could be artificially redesigned such that it ideally does not use acyl-CoA substrates and accepts subunits that bind to different coenzyme moieties (e.g., synthetic coenzyme moieties).

[0008] Accordingly, an object of the present invention is to provide re-engineered acyltransferases, particularly those with multi-specificity for acyl moieties, for use in helping to produce polyketides and polyketide precursors by biotechnological means, in order to expand the currently available production methods. A particular object is to expand and re-define the substrate recognition, binding, and catalytic spectrum of acyltransferases to provide new acyltransferase enzymes (termed Selectases) that are capable of accepting a wide range of substrates and, ideally, excluding CoA simultaneously, to allow the synthesis of targeted polyketides (precursors), preferably the synthesis of keto compounds or polyketide building blocks at positions within the polyketide molecule where the synthetic moiety can be inserted or added during in vivo synthesis when produced by the host cell of interest, or during in vitro synthesis when produced in an artificial enzyme system, or by semi-synthetic methods after the production of the (poly)ketide scaffold. Definitions

[0009] As used herein, "acyltransferase" refers to an acyltransferase within and / or derived from a fatty acid synthase (FAS) or polyketide synthase (PKS), including malonyl-acetyl-transferases (MAT), wherein the acyltransferase of the present invention (also referred to herein as "selected enzyme") is a reprogrammed acyltransferase derived from FAS. The acyltransferase can selectively bind different coenzyme-bound acyl molecules and trans-esterify them onto an acyl carrier protein (ACP), for example as part of fatty acid or polyketide synthesis. While native acyltransferases (including FAS malonyl-acetyl-transferase (MAT)) are specific for coenzyme A (CoA)-bound acyl molecules, the acyltransferases of the present invention are artificially reprogrammed acyltransferases that no longer accept acyl-CoA substrates and instead bind and trans-esterify acyls bound to other coenzyme moieties (including synthetic coenzyme moieties). The acyltransferases of the present invention can be expressed as part of FAS or a sub-part thereof, as a separate molecule, and / or as part of a fusion protein.

[0010] An acyltransferase "derived from" a fatty acid synthase refers to an acyltransferase for which the corresponding wild-type MAT is part of the fatty acid synthase in its native state, wherein the acyltransferase of the present invention can be present as part of the fatty acid synthase or can be an isolated acyltransferase according to the present disclosure. For example, for the reprogrammed acyltransferase according to SEQ ID NO:57, the corresponding wild-type MAT sequence is SEQ ID NO:29, which is part of the fatty acid synthase according to SEQ ID NO:1; thus, the reprogrammed acyltransferase according to SEQ ID NO:57 is derived from the fatty acid sequence according to SEQ ID NO:1. A variety of wild-type MATs and the fatty acid synthases of which they are part in their native state are disclosed as SEQ ID NO:29 to 56 and SEQ ID NO:1 to 28, respectively. A person skilled in the art can readily determine, for example by sequence alignment, the acyltransferase within a given FAS sequence. The acyltransferase can also be any fragment, portion, and / or (sub)domain of the sequences according to SEQID NO:29 to 56 or the corresponding sequences of different FASs, as long as it has acyltransferase activity.

[0011] As used herein, "isolated acyltransferase" refers to an acyltransferase that does not exist as part of such a continuous polypeptide: the continuous polypeptide comprises the complete fatty acid synthase from which it is derived, optionally comprising no more than the acyltransferase and an optional linker domain (LD) or a portion thereof, and / or the ketoacyl synthase (KS) of the fatty acid synthase from which it is derived. The isolated acyltransferase can exist as a discrete enzyme, i.e., a polypeptide consisting of the acyltransferase. Preferably, the polypeptide comprising the isolated acyltransferase further comprises: at least one protein, domain or portion thereof that enhances stability, such as an LD or a portion thereof, where the LD can be derived from the same FAS, or can be derived from at least one different FAS and / or polyketide synthase (PSK) in the form of a fusion protein, or different proteins, domains or portions thereof, such as an MBP tag or a different tag. In certain embodiments, the isolated acyltransferase exists as part of such a polypeptide: the polypeptide comprises the acyltransferase, KS and an LD that links the acyltransferase to the KS, where the KS and LD can be derived from the same FAS, or can be derived from at least one different FAS and / or PKS in the form of a fusion protein. Generally, the isolated acyltransferase can exist as part of a fusion protein that further comprises at least one polypeptide that is not derived from the same FAS or not derived from any FAS, such as one or more different enzyme moieties and / or domains (including PKS functional domains), one or more tags, one or more binding domains, a synthetic zipper, etc.

[0012] As used herein, "amino acid replacement" refers to the amino acid present at a given position in the primary amino acid sequence of a polypeptide being different from the amino acid present at the same position in the primary amino acid sequence of a reference polypeptide (e.g., a wild-type polypeptide). Typically, an amino acid replacement is achieved by mutating the corresponding codon of the nucleic acid sequence encoding the polypeptide to a codon encoding a different amino acid at that position. Means and methods for replacing an amino acid at a desired position (e.g., but not limited to PCR-based cloning methods as disclosed herein) are well recognized in the art and are available to those skilled in the art.

[0013] As used herein, "fatty acid synthase" or "FAS" refers to type I fatty acid synthase of animals (including humans), which is a multifunctional protein (also known as a multi-enzyme) capable of catalyzing all steps of fatty acid synthesis. In nature, wild-type fatty acid synthase catalyzes multiple rounds of Claisen condensation reaction, in which acetyl-CoA and malonyl-CoA recognized and transferred by acyltransferase are used as substrates to synthesize fatty acids, and the acyltransferase is malonyl-acetyl-transferase (MAT). The large multifunctional protein contains the following functional domains: acyltransferase that transfers acyl substrates to acyl carrier protein (ACP), ketoacyl synthase (KS) that catalyzes the Claisen condensation reaction, ketoacyl reductase (KR), hydroxyacyldehydratase (DH), and enoyl reductase (ER) that produce a fully saturated acyl skeleton, and thioesterase (TE) that releases the synthesized fatty acids (Smith and Tsai 2007).

[0014] As used herein, "fusion protein" refers to a polypeptide produced by: linking at least two different polypeptides to form a continuous artificial polypeptide, and the at least two different polypeptides can each be a complete protein existing in nature, a part thereof, or a fully synthetic polypeptide. Generally, fusion proteins are produced by fusing the nucleic acid sequences of the polypeptides to be linked. The means and methods for cloning and expressing fusion proteins are well recognized in the art and are available to those skilled in the art. The fusion proteins of the present disclosure can, for example, contain one or more tags, such as affinity tags (including at least one His tag, strep tag, SNAP tag, HA tag, etc., or a combination thereof, such as an N-terminal His tag and a C-terminal positioned strep tag, or vice versa), epitope tags, fluorescent tags, solubilizing tags, synthetic zippers, etc. Cleavable tags that can be removed after production can also be used. Generally, the tags will be located at the N or C terminus of the polypeptide so as not to interfere with the function of the folded protein, but in some cases, the tags can also be located within the polypeptide sequence (e.g., forming discrete structural epitopes that do not interfere with the folding and function of the polypeptide into which the tag is inserted). In addition, one or more linker sequences can be used, including FAS and / or PKS LD, and / or other linkers (including synthetic linkers) or spacers or flexible linkers to connect different entities of the fusion molecule to each other so that the different entities can properly perform their functions (e.g., "GA" linkers or spacers of various lengths for constructing α-helical structures, one or more binding or docking domains, one or more dimerization domains, such as leucine zippers can be used).

[0015] Whenever the present disclosure refers to a percent identity of a nucleic acid or amino acid sequence, the identity is determined by comparing the sequence of interest (reference sequence (e.g., SEQ ID NO disclosed herein)) with another sequence (query sequence) over the entire length of the reference sequence. The identity is obtained by using the EMBOSS Water pairwise sequence alignment (nucleotide) program or the EMBOSS Water pairwise sequence alignment (protein) program for amino acid sequences (www.ebi.ac.uk / Tools / psa / emboss_water / ). Those tools for local sequence alignment provided by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI) use a modified Smith-Waterman algorithm (see www.ebi.ac.uk / Tools / psa / and Smith, T.F. & Waterman, M.S. “Identification of common molecular subsequences” Journal of Molecular Biology, 1981 147(1):195-197). When performing the alignment, the default parameters defined by the EMBL-EBI are used. Those parameters are (i) for amino acid sequences: matrix = BLOSUM62, gap open penalty = 10, and gap extension penalty = 0.5, or (ii) for nucleic acid sequences: matrix = DNAfull, gap open penalty = 10, and gap extension penalty = 0.5.

[0016] As used herein, “linker domain” or “LD” refers to both the linker sequences upstream and downstream of the acyltransferase within FAS or PKS, which together connect the acyltransferase to the KS, and these two subparts are also referred to as the KS-AT (or KS-MAT) linker and the post-AT linker. The linker domains disclosed herein may comprise or consist of two subparts from the same FAS or PKS, or the linker domain may be a hybrid, comprising or consisting of the KS-(M)AT linker and the post-AT linker, each derived from a different FAS or PKS.

[0017] The "negatively charged" amino acids used herein refer to aspartic acid, glutamic acid, histidine, or unnatural amino acids or amino acid analogs that carry a negative net charge at least under certain pH conditions. The terms "aspartate" and "glutamate" are used interchangeably herein with the terms "aspartic acid" or "glutamic acid", respectively, regardless of the protonation state.

[0018] Notably, histidine can be negatively or positively charged, depending on the pH. Additionally, at a pH of about 8.0, a neutral charge is also possible. Thus, depending on the pH and taking into account the pI, histidine can be considered a negatively charged residue, but it can also vary.

[0019] The "reference position" used herein refers to one of the conserved positions (i), (ii), (iii), (iv), (v), (vi), (vii), (viii), or (ix) defined in Table 1. For the acyltransferase derived from murine FAS according to, for example, SEQ ID NO:1, in the case where the numbering is based on the sequence of the acyltransferase domain itself (SEQ ID NO:29), the reference positions (i) to (ix) correspond to positions D160, T161, F184, R286, K186, K285, R300, A282, and G137, and in the case where the numbering is based on the sequence of the full murine FAS (SEQ ID NO:1), they correspond to positions D647, T648, F671, R773, K673, K772, R787, A769, and G624. For the acyltransferase derived from chicken FAS according to SEQ ID NO:14, as another example, in the case where the numbering is based on the sequence of the acyltransferase domain itself (SEQ ID NO:42), the reference positions (i) to (viii) correspond to positions D160, T161, F184, R286, K186, R285, K300, A282, and G137, and in the case where the numbering is based on the sequence of the full chicken FAS (SEQ ID NO:14), they correspond to positions D646, T647, F670, R772, K672, R771, K786, A768, and G623. When comparing different sequences of acyltransferases according to the present disclosure (e.g., SEQ ID NO:29 to 56), the position numbers corresponding to the same reference position are often the same (when the numbering is based on the acyltransferase domain itself), but they can also be different.

[0020] The reference positions disclosed herein are not limited to the sequences of SEQ ID NOs: 1 to 28 or SEQ ID NOs: 29 to 56. For example, the sequence alignments shown in Table 1 are well recognized in the art, and for any given FAS sequence or the acyltransferase sequence therein, a person skilled in the art can easily determine the positions of the reference positions (i), (ii), (iii), (iv), (v), (vi), (vii), (viii), and / or (ix) in the sequence, and further determine whether the residue at the reference position (i) is a threonine, serine, or asparagine residue, whether the residue at the reference position (ii) is a phenylalanine residue, whether the residue at the reference position (iii) is an arginine residue, whether the residue at the reference position (iv) is a lysine or arginine residue, whether the residue at the reference position (v) is a lysine or arginine residue, whether the residue at the reference position (vi) is a lysine or arginine residue, whether the residue at the reference position (vii) is an alanine residue, whether the residue at the reference position (viii) is a glycine residue, and / or whether the residue at the reference position (ix) is an aspartic acid or glutamic acid residue.

[0021] Whenever the present disclosure mentions "similar" residues in the context of SEQ ID NO: 29, it refers to the residues present at the positions corresponding to the same reference position in different sequences. For example, T161 of SEQ ID NO: 14 is a threonine residue similar to T161 of SEQ ID NO: 29, and R285 of SEQ ID NO 14 is an arginine residue similar to K285 of SEQ ID NO: 29. Although the numbering based on SEQ ID NO: 14 is the same as that based on SEQ ID NO: 29, the similar residues may not necessarily have the same number as the corresponding residues in SEQ ID NO: 29, depending on the individual sequence.

[0022] The terms "polypeptide" and "protein" are used interchangeably herein. Naturally, protein function (including enzyme activity) depends on other factors such as temperature, pH, salt, etc., and may require other proteins and / or cofactors, as known to a person skilled in the art. The polypeptides disclosed herein may start from an additional methionine residue produced by translation of the start codon, or alternatively start from a different additional residue, such as valine or leucine produced by translation of an alternative start codon. Also, the polypeptides disclosed herein that start with a methionine residue may exist without the methionine, for example, if used for N-terminal fusion with a tag or other polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1(Fig.1) shows a cartoon of the mammalian FAS homodimer structure with one of the highlighted malonyl - acetyl - transferases (MAT), and a corresponding cartoon showing the position of the CoA - binding channel within the MAT in cartoon form. The numbering of the active - site residues (S94, H196, and N251) is based on SEQ ID NO:29.

[0024] Figure 2 (Fig.2) shows the residues of murine MAT (SEQ ID NO:29) identified in structural and sequence - based analyses for targeted reprogramming of coenzyme A binding. The structural schematics of eight different identified residues are shown in black, with the positively charged residues (K186, K285, and R300) marked with a black “+”. The structural schematic of CoA is shown in gray, with the negatively charged phosphate group marked with a gray “ - ”. The π - stacking between CoA - adenine and residues F184 and R286 is denoted as “π”. The H - bond between residue T161 and CoA - adenine is represented by a dashed line and “H”.

[0025] Figure 3 (Fig.3) shows an exemplary scheme for reprogramming an acyl - transferase by engineering malonyl - acetyl - transferase (MAT) to have altered substrate specificity. In the upper left is a schematic of the KS - LD - MAT polypeptide capable of binding CoA. In the upper right is a schematic of the engineered KS - LD - acyl - transferase according to the present invention. In the lower right is an example of using the reprogrammed acyl - transferase of the present invention.

[0026] Figure 4 (Fig.4) shows the turnover rates of fifty exemplary mutants on a logarithmic scale. At fixed substrate and acceptor concentrations, the specific transfer from Mal - CoA to N - (2 - mercaptoethyl) - hexanamide was analyzed. The Y - axis shows the turnover in seconds -1 on a logarithmic scale. Mutant A38 was measured in two concentration groups, and mutant A48 did not give a detectable signal under the conditions used. The error bars shown reflect the standard deviation from triplicates.

[0027] Figure 5 (Fig.5) shows the turnover rate of malonyl moiety transfer from a non - CoA substrate. At fixed substrate and acceptor concentrations, the specific transfer from the non - CoA substrate 3 - oxo - 3 - [[2 - [(1 - oxohexyl)amino]ethyl]thio] - propanoic acid to N - (2 - hydroxyethyl) - hexanamide was analyzed. The Y - axis shows the enzyme - specific turnover in seconds -1 on a logarithmic scale. The measurements were performed in duplicates.

[0028] Figure 6 (Fig.6) shows the exemplary enzyme kinetics of the transfer of the malonyl moiety from Mal-CoA ( Figure 6A ) and 3-oxo-3-[[2-[(1-oxohexyl)amino]ethyl]thio]-propionic acid ( Figure 6B ) to ACP by mutant A35. The y-axis shows the turnover in seconds -1 and the x-axis shows the substrate concentration in μM. Apparent kinetic parameters at a fixed acceptor concentration of 85 μM ACP were determined by fitting the data points with the Michaelis-Menten function. The error bars shown reflect the standard deviation from triplicates. SUMMARY OF THE INVENTION

[0029] In a first aspect, there is provided an acyltransferase having an altered substrate specificity, wherein the acyltransferase comprises one or more amino acid replacements at the following reference positions: (i) T161 according to SEQ ID NO:29, or a similar threonine, serine or asparagine residue at said reference position as defined in Table 1, wherein the amino acid is replaced with alanine, glycine, valine, leucine, isoleucine, tyrosine, phenylalanine, tryptophan, aspartic acid or glutamic acid; and / or (ii) F184 according to SEQ ID NO:29, or a similar phenylalanine residue at said reference position as defined in Table 1, wherein the amino acid is replaced with alanine, glycine, valine, leucine, isoleucine, methionine, aspartic acid or glutamic acid; and / or (iii) R286 according to SEQ ID NO:29, or a similar arginine residue at said reference position as defined in Table 1, wherein the amino acid is replaced with alanine, glycine, valine, leucine, isoleucine, methionine, aspartic acid or glutamic acid; and / or (iv) K186 according to SEQ ID NO:29, or a similar lysine or arginine residue at said reference position as defined in Table 1, wherein the amino acid is replaced with a negatively charged amino acid or replaced with glycine, alanine, valine, leucine, isoleucine or methionine; and / or (v) K285 according to SEQ ID NO:29, or a similar lysine or arginine residue at said reference position as defined in Table 1, wherein the amino acid is replaced with a negatively charged amino acid; and / or (vi) R300 according to SEQ ID NO:29, or a similar lysine or arginine residue at said reference position as defined in Table 1, wherein the amino acid is replaced with a negatively charged amino acid; and / or (vii) A282 according to SEQ ID NO:29, or a similar alanine residue at said reference position as defined in Table 1, wherein the amino acid is replaced with valine, leucine, isoleucine, phenylalanine or tyrosine; and / or (viii) G137 according to SEQ ID NO:29, or a similar glycine residue at said reference position as defined in Table 1, wherein the amino acid is replaced with valine, leucine, isoleucine, phenylalanine or tyrosine.

[0030] In some embodiments of the first aspect, the amino acid substitution at reference position (i) is a substitution to leucine, alanine, tryptophan or valine, preferably a substitution to leucine or alanine, more preferably a substitution to leucine; and / or wherein the amino acid substitution at reference position (ii) is a substitution to leucine, alanine, glycine, valine or glutamic acid, preferably a substitution to leucine, alanine or glutamic acid, more preferably a substitution to leucine or alanine; and / or wherein the amino acid substitution at reference position (iii) is a substitution to leucine, alanine, glycine or valine, preferably a substitution to leucine or alanine; and / or wherein the amino acid substitution at reference position (iv) is a substitution to a negatively charged amino acid, or a substitution to alanine or valine, preferably a substitution to glutamic acid or aspartic acid; and / or wherein the amino acid substitution at reference position (v) is a substitution to glutamic acid or aspartic acid; and / or wherein the amino acid substitution at reference position (vi) is a substitution to glutamic acid or aspartic acid; and / or wherein the amino acid substitution at reference position (vii) is a substitution to leucine, isoleucine, phenylalanine or tyrosine; and / or wherein the amino acid substitution at reference position (viii) is a substitution to leucine, isoleucine, phenylalanine or tyrosine, preferably a substitution to leucine.

[0031] In some embodiments of the first aspect, the amino acid substitution at reference position (i) is a substitution to leucine, isoleucine, tyrosine, phenylalanine or glycine; and / or wherein the amino acid substitution at reference position (ii) is a substitution to leucine, alanine, isoleucine or methionine; and / or wherein the amino acid substitution at reference position (iii) is a substitution to leucine, isoleucine or methionine; and / or wherein the amino acid substitution at reference position (iv) is a substitution to alanine, glycine, valine, leucine, isoleucine or methionine; and / or wherein the amino acid substitution at reference position (vii) is a substitution to valine; and / or wherein the amino acid substitution at reference position (viii) is a substitution to valine.

[0032] In some preferred embodiments of the first aspect, the acyltransferase is an isolated acyltransferase derived from fatty acid synthase.

[0033] In some embodiments of the first aspect, the acyltransferase comprises amino acid replacements as defined above at reference positions (iv) and (i), or (iv) and (ii), or (iv) and (iii), or (iv) and (v), or (iv) and (vi), or (iv) and (vii), or (iv) and (viii), or at two or more of the reference positions among (i), (ii), (iii), (v), (vi), (vii), and / or (viii) and (iv), optionally wherein the amino acid replacement at reference position (iv) is an amino acid replacement to glutamic acid or aspartic acid.

[0034] In some embodiments of the first aspect, the acyltransferase comprises amino acid replacements as defined above at reference positions (i) and (ii), or (i) and (iii), or (i) and (iv), or (i) and (v), or (i) and (vi), or (i) and (vii), or (i) and (viii), or at two or more of the reference positions among (ii), (iii), (iv), (v), (vi), (vii), and / or (viii) and (i), optionally wherein the amino acid replacement at reference position (i) is an amino acid replacement to leucine, alanine, tryptophan, or valine, preferably to leucine or alanine, more preferably to leucine.

[0035] In some embodiments of the first aspect, the acyltransferase comprises the amino acid replacement as defined above at reference position (i), optionally wherein the amino acid replacement at reference position (i) is an amino acid replacement to leucine, alanine, tryptophan, or valine, preferably to leucine or alanine, more preferably to leucine.

[0036] In some embodiments, the acyltransferase further comprises the amino acid replacements as defined above at reference positions (ii) and (iii), optionally wherein the amino acid replacement at reference position (ii) is an amino acid replacement to leucine, alanine, glycine, valine, or glutamic acid, preferably to leucine, alanine, or glutamic acid, more preferably to leucine or alanine; optionally wherein the amino acid replacement at reference position (iii) is an amino acid replacement to leucine, alanine, glycine, or valine, preferably to leucine or alanine.

[0037] In some embodiments, the acyltransferase further comprises the amino acid replacement as defined above at reference (iv), optionally wherein the amino acid replacement at reference position (iv) is an amino acid replacement to glutamic acid or aspartic acid.

[0038] In some embodiments, the acyltransferase further comprises an amino acid substitution as defined above at reference (vi), optionally wherein the amino acid substitution at reference position (vi) is an amino acid substitution to glutamic acid or aspartic acid.

[0039] In some embodiments of the first aspect, the acyltransferase comprises an amino acid substitution to glutamic acid at reference positions (iv) and (vi), or an amino acid substitution to aspartic acid at reference positions (iv) and (vi), or an amino acid substitution to glutamic acid at reference position (iv) and an amino acid substitution to aspartic acid at reference position (vi), or an amino acid substitution to aspartic acid at reference position (iv) and an amino acid substitution to glutamic acid at reference position (vi).

[0040] In some embodiments of the first aspect, the acyltransferase comprises an amino acid substitution to glutamic acid or aspartic acid at reference position (iii), and optionally further comprises an amino acid substitution at the following reference positions: (ix) D160 according to SEQ ID NO:29, or a similar aspartic acid or glutamic acid residue at said reference position as defined in Table 1, wherein the amino acid is substituted to arginine, histidine, lysine, glutamine or asparagine. In some embodiments, the amino acid substitution at reference position (ix) is a substitution to arginine. In other embodiments, the amino acid substitution at reference position (ix) is a substitution to histidine, lysine, glutamine or asparagine.

[0041] In some embodiments of the first aspect, the acyltransferase is derived from a fatty acid synthase of an organism selected from: Vertebrata, including Aves and Mammalia, including Artiodactyla, Perissodactyla, Carnivora, Primates and Rodentia, preferably wherein the acyltransferase is derived from a fatty acid synthase comprising or consisting of: a wild-type amino acid sequence selected from any one of SEQ ID NOs: 1 to 28, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity thereto.

[0042] In some embodiments of the first aspect, the acyltransferase comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 57 to 82 or 111 to 133, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity thereto.

[0043] In a second aspect, there is provided a fusion protein comprising at least one acyltransferase as described in any embodiment of the first aspect, preferably wherein the fusion protein further comprises at least one linker domain and optionally at least one ketoacyl synthase domain.

[0044] In a third aspect, there is provided a nucleic acid encoding the acyltransferase of the first aspect or the fusion protein of the third aspect.

[0045] In a fourth aspect, there is provided an expression construct or vector comprising at least one nucleic acid of the third aspect.

[0046] In a fifth aspect, there is provided a cell comprising at least one acyltransferase of the first aspect; and / or at least one fusion protein of the second aspect; and / or at least one nucleic acid of the third aspect; and / or at least one expression construct or vector of the fourth aspect.

[0047] In a sixth aspect, there is provided a kit comprising at least one acyltransferase of the first aspect; and / or at least one fusion protein of the second aspect; and / or at least one nucleic acid of the third aspect; and / or at least one expression construct or vector of the fourth aspect; and / or at least one cell of the fifth aspect; optionally further comprising at least one container; and / or a set of reagents, including buffers, medium and / or substrates; and / or transfection means. The kit generally contains all the reagents, buffers, cofactors, etc. necessary to ensure the function of at least one acyltransferase in the assay of interest.

[0048] In a seventh aspect, there is provided the following uses for polyketide synthesis, fatty acid synthesis and / or non-ribosomal peptide synthesis: at least one acyltransferase as defined in the first aspect; and / or at least one fusion protein as defined in the second aspect; and / or at least one nucleic acid as defined in the third aspect; and / or at least one expression construct or vector as defined in the fourth aspect; and / or at least one cell as defined in the fifth aspect; and / or at least one kit as defined in the sixth aspect, optionally wherein it is a use for reacting at least two different substrates. Detailed Description

[0049] The present invention will now be described in detail based on a detailed description including non-limiting examples, the accompanying drawings, and the sequences provided in the sequence listing.

[0050] In a first aspect, there is provided an acyltransferase having altered substrate specificity, wherein the acyltransferase comprises one or more amino acid replacements at the following reference positions: (i) T161 according to SEQ ID NO:29, or a similar threonine, serine, or asparagine residue at said reference position as defined in Table 1, wherein the amino acid is replaced with alanine, glycine, valine, leucine, isoleucine, tyrosine, phenylalanine, tryptophan, aspartic acid, or glutamic acid; and / or (ii) F184 according to SEQ ID NO:29, or a similar phenylalanine residue at said reference position as defined in Table 1, wherein the amino acid is replaced with alanine, glycine, valine, leucine, isoleucine, methionine, aspartic acid, or glutamic acid; and / or (iii) R286 according to SEQ ID NO:29, or a similar arginine residue at said reference position as defined in Table 1, wherein the amino acid is replaced with alanine, glycine, valine, leucine, isoleucine, methionine, aspartic acid, or glutamic acid; and / or (iv) K186 according to SEQ ID NO:29, or a similar lysine or arginine residue at said reference position as defined in Table 1, wherein the amino acid is replaced with a negatively charged amino acid or replaced with glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, cysteine, serine, threonine, histidine, or proline; and / or (v) K285 according to SEQ ID NO:29, or a similar lysine or arginine residue at said reference position as defined in Table 1, wherein the amino acid is replaced with a negatively charged amino acid; and / or (vi) R300 according to SEQ ID NO:29, or a similar lysine or arginine residue at said reference position as defined in Table 1, wherein the amino acid is replaced with a negatively charged amino acid; and / or (vii) A282 according to SEQ ID NO:29, or a similar alanine residue at said reference position as defined in Table 1, wherein the amino acid is replaced with valine, leucine, isoleucine, phenylalanine, or tyrosine; and / or (viii) G137 according to SEQ ID NO:29, or a similar glycine residue at said reference position as defined in Table 1, wherein the amino acid is replaced with valine, leucine, isoleucine, phenylalanine, or tyrosine.

[0051] Wild-type acyltransferases from fatty acid synthase (i.e., not containing one or more of the amino acid replacements of the present invention) can transfer different acyl groups from acyl-CoA (CoA) substrates to acyl carrier proteins. Although it is multi-specific for a variety of different acyl groups, it is specific for CoA-bound substrates. The two different types of specificity (CoA specificity and acyl multi-specificity) are conferred by different residues of the enzyme. The residues at the reference positions are crucial for binding to the CoA moiety of the substrate, and the amino acid replacements of the present invention can be used to alter these binding properties and engineer acyltransferases that can bind to substrates covalently bound to moieties other than CoA (including synthetic coenzyme moieties). The acyltransferases with altered substrate specificity according to the present invention are acyltransferases with a reduced ability to transfer CoA-bound substrates, preferably substantially no ability to transfer CoA-bound substrates, while acyl multi-specificity can remain unchanged.

[0052] The residues at reference positions (i), (ii), and (iii) are key residues of the corresponding coenzyme binding pocket (see Figure 2 ), and interact with the nucleobase moiety of the CoA moiety through hydrogen bonds (reference position (i)) or π-stacking (reference positions (ii) and (iii)). Replacement of one or more amino acids at reference positions (i), (ii), and / or (iii) with alanine, glycine, and / or valine can be used to prevent H-bonds and / or π-stacking. Replacement of the amino acid at reference position (i) with leucine, isoleucine, tyrosine, phenylalanine, tryptophan, aspartic acid, or glutamic acid can be used for spatial manipulation and reprogramming of the binding to the coenzyme moiety, where replacement with tyrosine, phenylalanine, or tryptophan can also be used to stabilize the protein through π-stacking. Replacement of the amino acid at reference positions (ii) and / or (iii) with leucine, isoleucine, methionine, aspartic acid, or glutamic acid can be used for spatial manipulation of the binding to the coenzyme moiety.

[0053] The lysine or arginine residues at reference positions (iv), (v), and (vi) interact electrostatically with the negatively charged phosphate groups of CoA. Replacement of one or more residues at reference positions (iv), (v), and / or (vi) with one or more negatively charged residues can be used to engineer acyltransferases that in turn bind to one or more positively charged chemical groups of coenzyme moieties other than CoA (including synthetic coenzyme moieties).

[0054] Replacement of the amino acid at reference position (iv) with an amino acid residue that is not positively charged or uncharged at neutral pH and is optionally non-polar can be used to artificially eliminate the electrostatic interaction at this reference position. Such amino acid residues include, for example, alanine, glycine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, cysteine, serine, threonine, histidine, proline, especially alanine, glycine, valine, leucine, isoleucine or methionine. The experimental results show that the amino acid replacement at reference position (iv) is highly additive with the amino acid replacements at reference positions (i), (ii), (iii) and (vi), and indicate that for reference position (iv), the most important factor is to eliminate the naturally occurring positive charge at this position, and the exact nature of the amino acid replacement or the introduction of a negative charge at this position has a lower correlation compared to the elimination of the positive charge.

[0055] The alanine residue at reference position (vii) and the glycine residue at reference position (viii) form structural parts of the coenzyme binding pocket. Replacement of the residue at one and / or both of these positions can be used to manipulate the structural shape of the coenzyme binding pocket by modifying the spatial arrangement.

[0056] In the wild-type protein, the aspartic acid or glutamic acid residue at reference position (ix) forms an H-bond with the arginine residue at reference position (iii). In some embodiments where the arginine at reference position (iii) containing a hydrogen donor residue is replaced with an aspartic acid or glutamic acid containing a hydrogen acceptor residue, it is also beneficial to replace the aspartic acid or glutamic acid at reference position (ix) with an amino acid containing a hydrogen donor residue, preferably replaced with arginine, histidine, lysine, glutamine or asparagine. This allows the mutant residues (iii) and (ix) to form an H-bond with the reversed hydrogen donor / acceptor role and can be used to stabilize the engineered acyltransferase.

[0057] The preferred positions for engineering the selected enzyme of the present invention are reference positions (i), (ii), (iii), (iv), (v) and / or (vi) alone or in combination, even more preferably any combination of reference positions (ii) and (iii) alone or preferably in combination, or any combination of reference positions (i) and (iv) alone or in combination, preferably in combination with additional engineering at at least one other position.

[0058] In some embodiments of the first aspect, the amino acid substitution at reference position (i) is a substitution to leucine, alanine, tryptophan or valine, preferably a substitution to leucine or alanine, more preferably a substitution to leucine; and / or wherein the amino acid substitution at reference position (ii) is a substitution to leucine, alanine, glycine, valine or glutamic acid, preferably a substitution to leucine, alanine or glutamic acid, more preferably a substitution to leucine or alanine; and / or wherein the amino acid substitution at reference position (iii) is a substitution to leucine, alanine, glycine or valine, preferably a substitution to leucine or alanine; and / or wherein the amino acid substitution at reference position (iv) is a substitution to a negatively charged amino acid or a substitution to alanine or valine, preferably a substitution to glutamic acid or aspartic acid; and / or wherein the amino acid substitution at reference position (v) is a substitution to glutamic acid or aspartic acid; and / or wherein the amino acid substitution at reference position (vi) is a substitution to glutamic acid or aspartic acid; and / or wherein the amino acid substitution at reference position (vii) is a substitution to leucine, isoleucine, phenylalanine or tyrosine; and / or wherein the amino acid substitution at reference position (viii) is a substitution to leucine, isoleucine, phenylalanine or tyrosine, preferably a substitution to leucine.

[0059] In some embodiments of the first aspect, the amino acid substitution at reference position (i) is a substitution to leucine, isoleucine, tyrosine, phenylalanine or glycine; and / or wherein the amino acid substitution at reference position (ii) is a substitution to leucine, alanine, isoleucine or methionine; and / or wherein the amino acid substitution at reference position (iii) is a substitution to leucine, isoleucine or methionine; and / or wherein the amino acid substitution at reference position (iv) is a substitution to alanine, glycine, valine, leucine, isoleucine or methionine; and / or wherein the amino acid substitution at reference position (vii) is a substitution to valine; and / or wherein the amino acid substitution at reference position (viii) is a substitution to valine.

[0060] In some preferred embodiments of the first aspect, the acyltransferase is an isolated acyltransferase derived from fatty acid synthase.

[0061] In one embodiment, the isolated acyltransferase is a polypeptide or a part of a polypeptide that comprises or consists of the acyltransferase and does not comprise other parts and / or domains of the FAS from which it is derived, including LD. The isolated acyltransferase can be part of a fusion protein according to the second aspect.

[0062] In another embodiment, the isolated acyltransferase is part of a polypeptide that comprises an acyltransferase and LD or a portion thereof, or consists of an acyltransferase and LD or a portion thereof, and does not include other parts and / or domains of the FAS from which it is derived. The isolated acyltransferase can be part of a fusion protein according to the second aspect.

[0063] In another embodiment, the isolated acyltransferase is part of a polypeptide that comprises a portion of FAS or consists of a portion of FAS, the portion of FAS spanning a ketoacyl synthase (KS), an acyltransferase, and the LD linking the KS and the acyltransferase, and does not include other parts and / or domains of the FAS from which it is derived. Such a KS-LD-acyltransferase can be part of a fusion protein according to the second aspect.

[0064] In some embodiments of the first aspect, the acyltransferase comprises reference positions (iv) and (i), or (iv) and (ii), or (iv) and (iii), or (iv) and (v), or (iv) and (vi), or (iv) and (vii), or (iv) and (viii), or two or more of the amino acid replacements as defined above at reference positions among (iv) and (i), (ii), (iii), (v), (vi), (vii), and / or (viii), optionally wherein the amino acid replacement at reference position (iv) is an amino acid replacement to glutamate or aspartate.

[0065] In some embodiments of the first aspect, the acyltransferase comprises reference positions (i) and (ii), or (i) and (iii), or (i) and (iv), or (i) and (v), or (i) and (vi), or (i) and (vii), or (i) and (viii), or two or more of the amino acid replacements as defined above at reference positions among (i) and (ii), (iii), (iv), (v), (vi), (vii), and / or (viii), optionally wherein the amino acid replacement at reference position (i) is an amino acid replacement to leucine, alanine, tryptophan, or valine, preferably to leucine or alanine, more preferably to leucine.

[0066] In some preferred embodiments, the acyltransferase comprises the amino acid replacements as defined above at reference positions (i), (ii), (iii), (iv), and optionally also at reference position (vi).

[0067] In some embodiments of the first aspect, the acyltransferase comprises the amino acid replacement as defined above at the reference position (i), optionally wherein the amino acid replacement at the reference position (i) is the replacement of the amino acid with leucine, alanine, tryptophan or valine, preferably with leucine or alanine, more preferably with leucine.

[0068] In some embodiments, the acyltransferase further comprises the amino acid replacement as defined above at the reference positions (ii) and (iii), optionally wherein the amino acid replacement at the reference position (ii) is the replacement of the amino acid with leucine, alanine, glycine, valine or glutamic acid, preferably with leucine, alanine or glutamic acid, more preferably with leucine or alanine, optionally wherein the amino acid replacement at the reference position (iii) is the replacement of the amino acid with leucine, alanine, glycine or valine, preferably with leucine or alanine.

[0069] In some embodiments, the acyltransferase further comprises the amino acid replacement as defined above at the reference (iv), optionally wherein the amino acid replacement at the reference position (iv) is the replacement of the amino acid with glutamic acid or aspartic acid.

[0070] In some embodiments, the acyltransferase further comprises the amino acid replacement as defined above at the reference (vi), optionally wherein the amino acid replacement at the reference position (vi) is the replacement of the amino acid with glutamic acid or aspartic acid.

[0071] In some embodiments of the first aspect, the acyltransferase comprises the replacement of the amino acids at the reference positions (iv) and (vi) with glutamic acid, or the replacement of the amino acids at the reference positions (iv) and (vi) with aspartic acid, or the replacement of the amino acid at the reference position (iv) with glutamic acid and the replacement of the amino acid at the reference position (vi) with aspartic acid, or the replacement of the amino acid at the reference position (iv) with aspartic acid and the replacement of the amino acid at the reference position (vi) with glutamic acid.

[0072] In one embodiment of the first aspect, the acyltransferase comprises the replacement of the amino acid at the reference position (i) with aspartic acid and the replacement of the amino acid at the reference position (ii) with glutamic acid.

[0073] In one embodiment of the first aspect, the acyltransferase comprises the replacement of the amino acids at least at the reference positions (i), (ii) and (iii) with leucine.

[0074] In one embodiment of the first aspect, the acyltransferase comprises the replacement of the amino acids at the reference positions (i), (ii) and (iii) with alanine, and the replacement of the amino acid at the reference position (iv) with aspartic acid or glutamic acid.

[0075] In one embodiment of the first aspect, the acyltransferase comprises amino acids at reference positions (i), (ii), and (iii) replaced with alanine; and the amino acid at reference position (iv) replaced with aspartic acid and the amino acid at reference position (vi) replaced with aspartic acid, or the amino acid at reference position (iv) replaced with aspartic acid and the amino acid at reference position (vi) replaced with glutamic acid, or the amino acid at reference position (iv) replaced with glutamic acid and the amino acid at reference position (vi) replaced with aspartic acid, or the amino acid at reference position (iv) replaced with glutamic acid and the amino acid at reference position (vi) replaced with glutamic acid.

[0076] In one embodiment of the first aspect, the acyltransferase comprises amino acids at reference positions (i), (ii), and (iii) replaced with leucine; and the amino acid at reference position (iv) replaced with glutamic acid or aspartic acid.

[0077] In one embodiment of the first aspect, the acyltransferase comprises amino acids at reference positions (i), (ii), and (iii) replaced with leucine; and the amino acid at reference position (iv) replaced with aspartic acid and the amino acid at reference position (vi) replaced with aspartic acid, or the amino acid at reference position (iv) replaced with aspartic acid and the amino acid at reference position (vi) replaced with glutamic acid, or the amino acid at reference position (iv) replaced with glutamic acid and the amino acid at reference position (vi) replaced with aspartic acid, or the amino acid at reference position (iv) replaced with glutamic acid and the amino acid at reference position (vi) replaced with glutamic acid.

[0078] In one embodiment of the first aspect, the acyltransferase comprises the amino acid at reference position (i) replaced with leucine and the amino acids at reference positions (ii) and (iii) replaced with alanine; and the amino acid at reference position (iv) replaced with glutamic acid or aspartic acid.

[0079] In one embodiment of the first aspect, the acyltransferase comprises an amino acid substitution to leucine at the reference position (i), amino acid substitutions to alanine at the reference positions (ii) and (iii); and an amino acid substitution to aspartic acid at the reference position (iv) and an amino acid substitution to aspartic acid at the reference position (vi), or an amino acid substitution to aspartic acid at the reference position (iv) and an amino acid substitution to glutamic acid at the reference position (vi), or an amino acid substitution to glutamic acid at the reference position (iv) and an amino acid substitution to aspartic acid at the reference position (vi), or an amino acid substitution to glutamic acid at the reference position (iv) and an amino acid substitution to glutamic acid at the reference position (vi).

[0080] In one embodiment of the first aspect, the acyltransferase comprises an amino acid substitution to leucine at the reference position (i), an amino acid substitution to glutamic acid at the reference position (ii), and an amino acid substitution to alanine at the reference position (iii); and an amino acid substitution to glutamic acid or aspartic acid at the reference position (iv).

[0081] In one embodiment of the first aspect, the acyltransferase comprises an amino acid substitution to leucine at the reference position (i), an amino acid substitution to glutamic acid at the reference position (ii), and an amino acid substitution to alanine at the reference position (iii); and an amino acid substitution to aspartic acid at the reference position (iv) and an amino acid substitution to aspartic acid at the reference position (vi), or an amino acid substitution to aspartic acid at the reference position (iv) and an amino acid substitution to glutamic acid at the reference position (vi), or an amino acid substitution to glutamic acid at the reference position (iv) and an amino acid substitution to aspartic acid at the reference position (vi), or an amino acid substitution to glutamic acid at the reference position (iv) and an amino acid substitution to glutamic acid at the reference position (vi).

[0082] In one embodiment of the first aspect, the acyltransferase comprises an amino acid substitution to alanine at the reference position (i), an amino acid substitution to glutamic acid at the reference position (ii), and an amino acid substitution to alanine at the reference position (iii); and an amino acid substitution to glutamic acid or aspartic acid at the reference position (iv).

[0083] In one embodiment of the first aspect, the acyltransferase comprises an amino acid at the reference position (i) replaced with alanine, an amino acid at the reference position (ii) replaced with glutamic acid, and an amino acid at the reference position (iii) replaced with alanine; and an amino acid at the reference position (iv) replaced with aspartic acid and an amino acid at the reference position (vi) replaced with aspartic acid, or an amino acid at the reference position (iv) replaced with aspartic acid and an amino acid at the reference position (vi) replaced with glutamic acid, or an amino acid at the reference position (iv) replaced with glutamic acid and an amino acid at the reference position (vi) replaced with aspartic acid, or an amino acid at the reference position (iv) replaced with glutamic acid and an amino acid at the reference position (vi) replaced with glutamic acid.

[0084] In one embodiment of the first aspect, the acyltransferase comprises an amino acid at the reference position (i) replaced with aspartic acid, an amino acid at the reference position (ii) replaced with glutamic acid, and an amino acid at the reference position (iii) replaced with alanine; and an amino acid at the reference position (iv) replaced with glutamic acid or aspartic acid.

[0085] In one embodiment of the first aspect, the acyltransferase comprises an amino acid at the reference position (i) replaced with aspartic acid, an amino acid at the reference position (ii) replaced with glutamic acid, and an amino acid at the reference position (iii) replaced with alanine; and an amino acid at the reference position (iv) replaced with aspartic acid and an amino acid at the reference position (vi) replaced with aspartic acid, or an amino acid at the reference position (iv) replaced with aspartic acid and an amino acid at the reference position (vi) replaced with glutamic acid, or an amino acid at the reference position (iv) replaced with glutamic acid and an amino acid at the reference position (vi) replaced with aspartic acid, or an amino acid at the reference position (iv) replaced with glutamic acid and an amino acid at the reference position (vi) replaced with glutamic acid.

[0086] In one embodiment of the first aspect, the acyltransferase comprises an amino acid at the reference position (i) replaced with aspartic acid, an amino acid at the reference position (ii) replaced with alanine, and an amino acid at the reference position (iii) replaced with alanine; and an amino acid at the reference position (iv) replaced with glutamic acid or aspartic acid.

[0087] In one embodiment of the first aspect, the acyltransferase comprises an amino acid substitution to aspartic acid at the reference position (i), an amino acid substitution to alanine at the reference position (ii), and an amino acid substitution to alanine at the reference position (iii); and an amino acid substitution to aspartic acid at the reference position (iv) and an amino acid substitution to aspartic acid at the reference position (vi), or an amino acid substitution to aspartic acid at the reference position (iv) and an amino acid substitution to glutamic acid at the reference position (vi), or an amino acid substitution to glutamic acid at the reference position (iv) and an amino acid substitution to aspartic acid at the reference position (vi), or an amino acid substitution to glutamic acid at the reference position (iv) and an amino acid substitution to glutamic acid at the reference position (vi).

[0088] In one embodiment of the first aspect, the acyltransferase comprises an amino acid substitution to alanine at the reference position (i), an amino acid substitution to glutamic acid at the reference position (ii), and an amino acid substitution to aspartic acid at the reference position (iii); and an amino acid substitution to glutamic acid or aspartic acid at the reference position (iv), optionally further comprising an amino acid substitution to arginine at the reference position (ix).

[0089] In one embodiment of the first aspect, the acyltransferase comprises an amino acid substitution to alanine at the reference position (i), an amino acid substitution to glutamic acid at the reference position (ii), and an amino acid substitution to aspartic acid at the reference position (iii); and an amino acid substitution to aspartic acid at the reference position (iv) and an amino acid substitution to aspartic acid at the reference position (vi), or an amino acid substitution to aspartic acid at the reference position (iv) and an amino acid substitution to glutamic acid at the reference position (vi), or an amino acid substitution to glutamic acid at the reference position (iv) and an amino acid substitution to aspartic acid at the reference position (vi), or an amino acid substitution to glutamic acid at the reference position (iv) and an amino acid substitution to glutamic acid at the reference position (vi), optionally further comprising an amino acid substitution to arginine at the reference position (ix).

[0090] In some embodiments of the first aspect, the acyltransferase comprises an amino acid substitution at the reference position (iii) with glutamate or aspartate, and optionally also comprises amino acid substitutions at the following reference positions: (ix) D160 according to SEQ ID NO:29, or a similar aspartate or glutamate residue at said reference position as defined in Table 1, wherein the amino acid is substituted with an amino acid comprising a hydrogen donor residue, preferably arginine, histidine, lysine, glutamine or asparagine. In some embodiments, the amino acid substitution at the reference position (ix) is a substitution with arginine. In other embodiments, the amino acid substitution at the reference position (ix) is a substitution with histidine, lysine, glutamine or asparagine.

[0091] In some embodiments of the first aspect, the acyltransferase is derived from a fatty acid synthase of an organism selected from the group consisting of the phylum Chordata, including the classes Aves and Mammalia, including Artiodactyla, Perissodactyla, Carnivora, Primates and Rodentia, preferably wherein the acyltransferase is derived from a fatty acid synthase comprising or consisting of: a wild-type amino acid sequence selected from any one of SEQ ID NOs: 1 to 28, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity thereto.

[0092] In some embodiments of the first aspect, the acyltransferase comprises or consists of: an amino acid sequence selected from any one of SEQ ID NOs: 57 to 82 or 111 to 133, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity thereto.

[0093] In a second aspect, there is provided a fusion protein comprising at least one acyltransferase of any embodiment of the first aspect, preferably wherein the fusion protein further comprises at least one linker domain and optionally at least one ketoacyl synthase domain.

[0094] In some embodiments, the fusion protein may comprise a tag for purification, such as but not limited to at least one His tag, step tag, FLAG tag, myc tag, MBP tag, GST tag or a combination thereof.

[0095] The acyltransferase of the present invention is preferably linked to other functional PKS or FAS moieties and / or domains, in particular the ketosynthase domain, via FAS and / or PKS LD. Of course, the acyltransferase can also be linked to other functional moieties and / or domains via conventional polypeptide linkers known in the art (such as glycine linkers, GS linkers, PT linkers or other common linkers).

[0096] In some embodiments, the fusion protein can be a polyketide synthase (PKS) or a portion thereof, wherein at least one acyltransferase has been replaced by the acyltransferase of the present invention, and the PKS portion of the fusion protein may further comprise engineered modifications.

[0097] Type I PKS is a large multifunctional protein with a modular enzyme structure, where one module contains a set of functional enzyme domains involved in incorporating α-carboxy acyl extender units into the growing polyketide chain. A Type I PKS module contains at least three different functional domains: ketosynthase (KS), which is responsible for catalyzing the Claisen condensation between the extender unit and the growing polyketide chain; acyltransferase, which selectively binds the extender unit and transesterifies the extender to an acyl carrier protein (ACP); and ACP, which serves as the acceptor for the extender unit and the growing polyketide chain. Type I PKS can also contain additional domains, such as ketoreductase (KR) that reduces the β-keto to a hydroxyl group, dehydratase (DH) that removes the hydroxyl group and generates a double bond between the α and β carbons, enoyl reductase (ER) that reduces the double bond generated by the DH domain, and thioesterase (TE) that terminates the synthesis process. Type I PKS can be an iterative PKS that uses the same functional domains to catalyze multiple extension cycles, or it can be an assembly-line PKS that contains different modules, with each module catalyzing one extension step (Hertweck 2009, Cogan et al. 2021).

[0098] The Type II PKS system consists of discrete proteins, each individually containing the enzyme activities for polyketide synthesis.

[0099] Typically, the starter unit or extender unit is a CoA-bound acyl group, such as acetyl-CoA, malonyl-CoA, methylmalonyl-CoA or propionyl-CoA, which are selectively recognized by the acyltransferase and transferred to the ACP. By replacing the acyltransferase with the engineered acyltransferase of the present invention, the PKS system can be reprogrammed to incorporate extender units bound to other coenzyme moieties, including synthetic coenzyme moieties. The acyltransferase of the PKS can be replaced by the acyltransferase of the present invention using conventional cloning strategies available to those skilled in the art.

[0100] In a modular, assembly-line type PKS system, the acyltransferase of one or two or more or all of the modules can be replaced by the reprogrammed acyltransferase of the present invention, optionally comprising an adjacent LD or a part thereof, and / or KS, to produce a reprogrammed PKS system capable of using extender units conjugated with coenzyme moieties other than CoA.

[0101] The acyltransferase or fusion protein according to multiple embodiments of the first or second aspect can be purified using the protein purification methods disclosed herein or any other conventional protein purification methods.

[0102] In a third aspect, there is provided a nucleic acid encoding the acyltransferase of the first aspect or the fusion protein of the second aspect.

[0103] The nucleic acid can be double-stranded or single-stranded DNA or RNA, including mRNA. Nucleic acids that are not part of an expression construct or vector can contain other sequences and / or modifications for increased stability.

[0104] In a fourth aspect, there is provided an expression construct or vector comprising at least one nucleic acid of the third aspect.

[0105] The nucleic acid of the third aspect and the expression construct or vector of the fourth aspect can further comprise sequences for expression in a desired host cell, such as a transcription terminator and / or a promoter (including an inducible promoter) operably linked to the nucleic acid encoding the acyltransferase or fusion protein, selectable markers, sequences for replication in a desired host cell, and / or other regulatory sequences, including sequences that regulate transcription, RNA processing and / or stability and / or translation. The nucleic acid or the expression construct or vector can contain a cDNA sequence (without introns) encoding the acyltransferase and / or fusion protein of the present invention, for example for expression in prokaryotic cells. For a variety of cell types, recombinant protein expression and suitable vectors and expression systems, promoters and other regulatory sequences have been well established. Those skilled in the art are familiar with the details and requirements of recombinant protein expression in different cell types and can readily determine the appropriate components for selection for expression in a desired host cell. The vector backbone of the expression construct or vector can be, for example, the pET22b, pET300, pET301, pET28b or pGEX-6P-1 vector system, but is not limited thereto.

[0106] In certain embodiments of the third or fourth aspect, the nucleic acid or the expression construct or vector is codon-optimized, wherein the codon optimization can be adjusted according to the desired host cell. Codon optimization for different cell types is well recognized in the art and tools for codon optimization are readily available to those skilled in the art.

[0107] The nucleic acid of the third aspect and / or the expression construct or vector of the fourth aspect may be suitable for stable replication and maintenance in a desired host cell. The nucleic acid and / or the expression construct or vector may also be suitable for integration into the host genome.

[0108] In a fifth aspect, there is provided a cell comprising at least one acyltransferase of the first aspect; and / or at least one fusion protein of the second aspect; and / or at least one nucleic acid of the third aspect; and / or at least one expression construct or vector of the fourth aspect.

[0109] In one embodiment, at least one nucleic acid encoding at least one acyltransferase of the first aspect and / or at least one fusion protein of the second aspect may be stably integrated into the genome of the host cell (including chromosomal / nuclear genome and / or plastid genome), for example, by any conventional genome editing strategy and / or any conventional vector suitable for genome integration. In certain embodiments, at least one nucleic acid of the present invention may be integrated into the genome by replacing the endogenous nucleic acid encoding the acyltransferase.

[0110] In another embodiment, the endogenous gene of the host cell encoding the acyltransferase is mutated to encode the acyltransferase of the present invention, wherein the mutation may be carried out by any mutation strategy, preferably the SDN-1 (site-directed nuclease 1) mutation strategy.

[0111] In another embodiment, at least one nucleic acid of the third aspect and / or at least one expression construct or vector of the fourth aspect are present extrachromosomally in the host cell.

[0112] The acyltransferase of the present invention can be expressed in any cell expression system. Established systems include prokaryotic cells, including Escherichia coli strains, such as but not limited to a variety of well-established BL21 or BL21(DE3) expression strains and their derivatives, including for example Rosetta, BAP1 or NiCo21(DE3) strains, other Escherichia coli strains, such as Escherichia coli K12 strain JM83, or other prokaryotic cells such as Bacillus subtilis expression systems; eukaryotic expression systems, such as including Saccharomyces cerevisiae cells, Komagataella phaffii (formerly known as Pichia pastoris) cells, Aspergillus niger cells; or immortalized cell lines, such as insect cell expression systems (such as Sf-9 or Sf-21 cells); or mammalian cell lines, such as Chinese hamster ovary (CHO) cells, HEK 293 cells, etc.

[0113] In one embodiment, at least one endogenous acyltransferase of the cell is knocked out, knocked down or otherwise inactivated and supplemented with at least one acyltransferase and / or fusion protein of the present invention; and / or at least one nucleic acid encoding the same, expression construct and / or vector.

[0114] In some preferred embodiments, the cell contains a PKS system, wherein at least one acyltransferase has been replaced by the acyltransferase of the present invention. In certain embodiments, the cell contains a modular type I PKS system, wherein the acyltransferase of one or two or more or all PKS modules is replaced by the reprogrammed acyltransferase of the present invention, optionally containing an adjacent LD or a part thereof and / or KS of the present invention.

[0115] In a sixth aspect, there is provided a kit, which comprises at least one acyltransferase of the first aspect; and / or at least one fusion protein of the second aspect; and / or at least one nucleic acid of the third aspect; and / or at least one expression construct or vector of the fourth aspect; and / or at least one cell of the fifth aspect; optionally further comprising at least one container; and / or a set of reagents, including buffers, media and / or substrates; and / or transfection means. The kit generally contains all the reagents, buffers, cofactors, etc. required to ensure the function of at least one acyltransferase in the assay of interest.

[0116] In one embodiment, the kit of the sixth aspect is a kit for in vivo synthesis in at least one cell according to the fifth aspect.

[0117] In another embodiment, the kit of the sixth aspect comprises at least one purified or partially purified acyltransferase of the first aspect and / or at least one purified or partially purified fusion protein of the second aspect, at least one buffer, at least one reaction substrate comprising a coenzyme moiety other than CoA, and optionally other suitable reaction components.

[0118] In another embodiment, the kit of the sixth aspect comprises a lysate of at least one cell according to the fifth aspect, at least one reaction substrate comprising a coenzyme moiety other than CoA, at least one buffer, and optionally other suitable reaction components.

[0119] In another embodiment, the kit of the sixth aspect comprises at least one in vitro transcription / translation system, at least one reaction substrate comprising a coenzyme moiety other than CoA, and optionally at least one buffer and / or other suitable reaction components, wherein the in vitro transcription / translation system expresses at least one nucleic acid of the third aspect and / or at least one expression construct or vector of the fourth aspect.

[0120] In the seventh aspect, there is provided the following uses for polyketide synthesis, fatty acid synthesis, and / or nonribosomal peptide synthesis: at least one acyltransferase as defined in the first aspect; and / or at least one fusion protein as defined in the second aspect; and / or at least one nucleic acid as defined in the third aspect; and / or at least one expression construct or vector as defined in the fourth aspect; and / or at least one cell as defined in the fifth aspect; and / or at least one kit as defined in the sixth aspect, optionally wherein it is a use for reacting at least two different substrates.

[0121] Since the selected enzyme according to the first aspect comprises specifically reprogrammed coenzyme binding properties without the need to alter the amino acid residues involved in acyl moiety binding, the acyltransferases of the present invention can retain the native multi-specificity with respect to acyl moieties. Retaining acyl multi-specificity can be very important, for example, for using one acyltransferase for different acyl moieties as substrates, especially when using at least two different substrates for polyketide synthesis, fatty acid synthesis, and / or nonribosomal peptide synthesis.

[0122] As used herein, "polyketide synthesis" includes the synthesis of diketides and oligoketides.

[0123] In certain embodiments of the seventh or ninth aspect, the polyketide synthesis, fatty acid synthesis, and / or nonribosomal peptide synthesis can be the synthesis of a mixture of polyketides, and / or fatty acids and / or proteins with metabolites of other biosynthetic pathways.

[0124] In one embodiment, the synthesis of the seventh aspect also uses in vivo synthesis in at least one cell according to the fifth aspect: at least one suitable culture medium and at least one reaction substrate comprising a coenzyme moiety other than CoA, so that at least one cell represents a modified production strain.

[0125] In another embodiment, the synthesis of the seventh aspect uses the following in vitro synthesis: at least one purified or partially purified acyltransferase of the first aspect and / or at least one purified or partially purified fusion protein of the second aspect, at least one buffer, at least one reaction substrate comprising a coenzyme moiety other than CoA, and optionally some other suitable reaction components.

[0126] In another embodiment, the synthesis of the seventh aspect uses the following in vitro synthesis: a lysate of at least one cell according to the fifth aspect, at least one reaction substrate comprising a coenzyme moiety other than CoA, at least one buffer, and optionally some other suitable reaction components.

[0127] In another embodiment, the synthesis of the seventh aspect uses the following in vitro synthesis: at least one in vitro transcription / translation system, at least one reaction substrate comprising a coenzyme moiety other than CoA, and optionally at least one buffer and / or some other suitable reaction components, wherein the in vitro transcription / translation system expresses at least one nucleic acid of the third aspect and / or at least one expression construct or vector of the fourth aspect.

[0128] In the eighth aspect, a method is provided, which includes: (a) providing (i) 3-oxo-3-[[2-[(1-oxohexyl)amino]ethyl]thio]-propionic acid; and (iia) at least one acyltransferase as defined in the first aspect; and / or at least one fusion protein as defined in the second aspect; and / or at least one nucleic acid as defined in the third aspect; and / or at least one expression construct or vector as defined in the fourth aspect; and at least one acyl carrier protein (ACP), or a nucleic acid encoding the same; or (iib) at least one cell as defined in the fifth aspect, which further comprises at least one ACP; and (b) allowing at least one acyltransferase to transfer at least one malonyl moiety from 3-oxo-3-[[2-[(1-oxohexyl)amino]ethyl]thio]-propionic acid to at least one ACP; (c) optionally obtaining at least one acyl-ACP.

[0129] In the ninth aspect, the use of 3-oxo-3-[[2-[(1-oxohexyl)amino]ethyl]thio]-propionic acid in polyketide synthesis, fatty acid synthesis, and / or non-ribosomal peptide synthesis is provided.

[0130] In one embodiment, the synthesis of the ninth aspect further uses in vivo synthesis in at least one cell according to the fifth aspect: at least one suitable culture medium and at least one reaction substrate containing 3-oxo-3-[[2-[(1-oxohexyl)amino]ethyl]thio]-propionic acid, so that at least one cell represents a modified production strain.

[0131] In another embodiment, the synthesis of the ninth aspect further uses the following in vitro synthesis: at least one purified or partially purified acyltransferase of the first aspect and / or at least one purified or partially purified fusion protein of the second aspect, at least one buffer, and optionally some other suitable reaction components.

[0132] In another embodiment, the synthesis of the ninth aspect further uses the following in vitro synthesis: at least one lysate of a cell according to the fifth aspect, at least one buffer, and optionally some other suitable reaction components.

[0133] In another embodiment, the synthesis of the ninth aspect further uses the following in vitro synthesis: at least one in vitro transcription / translation system and optionally at least one buffer and / or some other suitable reaction components, the in vitro transcription / translation system expressing at least one nucleic acid of the third aspect and / or at least one expression construct or vector of the fourth aspect. Examples Example 1: Computer-Aided Design for Reprogramming Targeted Acyltransferase

[0134] Due to the modular nature of the PKS system and its inherent variability in acyl backbone reduction, the PKS system is an attractive enzyme platform for the synthesis of a large number of different available biomolecules, especially for drug design. A key aspect limiting the general and effective biotechnological use of PKS is the nature of the naturally occurring acyltransferases, which have relatively high specificity when it comes to the elongation subunit (Smith and Tsai 2007). The MAT domain of metazoan FAS enzymes has been shown to allow for the efficient transfer of different acyl substrates (Rittner et al. 2018).

[0135] However, while FAS MAT can thus provide a certain versatility in terms of the acyl groups of the substrates, both FAS MAT and PKS AT suffer from a crucial drawback: they are completely dependent on CoA-bound substrates. The dependence on acyl-CoA substrates severely limits the possibility of generating artificial biomolecules by in vivo synthesis using engineered PKS systems. CoA-bound molecules, especially acetyl-CoA and malonyl-CoA, are highly abundant and inevitable in any in vivo environment and thus interfere with any possible biosynthesis based on CoA-bound substrates. Although the MAT crystal structure has been solved (Rittner et al. 2020), it has not been anticipated that the key activating component of CoA can be replaced, let alone how the acyltransferase can be artificially reprogrammed to not use acyl-CoA substrates but specifically accept subunits bound to different coenzyme moieties (such as synthetic coenzyme moieties).

[0136] Based on the crystal structure and the position of the CoA-binding pocket therein (see Figure 1 ), it was speculated that target residues highly attractive for reprogramming native acyltransferases could be identified. However, mutagenesis experiments showed that amino acid replacements at certain residues did not allow any functional protein expression. Therefore, an iterative testing strategy combined with in silico screening and protein engineering techniques was applied to define acyltransferase mutants that could be manipulated in such a way as to effectively block CoA binding and at the same time allow binding of different coenzyme moieties while still allowing proper overall folding of the protein. In addition, to provide a high degree of versatility in the selection including the acyltransferase protein itself, in addition to structural analysis, FAS amino acid sequences from multiple genera were aligned. The alignment of 28 different exemplary sequences is shown in Table 1. After extensive analysis, eight different target residues were identified among those sequences showing a certain degree of identity (see Figure 2 ). For this purpose, multiple hotspots of mutagenic activity were defined and tested as further detailed below. Table 1 Example 2: Cloning and expression of engineered acyltransferases

[0137] The basic plasmid encoding the engineered murine acyltransferase was prepared from a synthetic DNA fragment containing the KS-LD-MAT fragment (including the Strep-tag and His-tag) and cloned into the pET22b vector using the restriction enzymes NdeI and XhoI or using the in-Fusion Snap Assembly Master Mix (Takara). Mutations were introduced at eight different positions corresponding to the reference positions according to the present invention by PCR using primers containing nucleotide variations. A plasmid encoding ACP was prepared similarly. Another ribosome binding site (RBS) and another gene encoding 4'-phosphopantetheinyl transferase Sfp were integrated into the same plasmid. The synthetic gene of ACP was ordered according to its native DNA sequence and codon-optimized for Escherichia coli.

[0138] The PCR mixture was treated with Dpn1 and then DNA was purified using electrophoresis. The DNA was stained and illuminated with Sybr Green DNA dye (Carl Roth). The appropriate fragment was separated with a scalpel and the DNA was purified using the GeneJet Gel Extraction Kit (ThermoFisher). The fragment was transferred to Stellar TM Competent Cells (Takara) chemically competent cells for ligation and amplification of the targeted plasmid. The plasmid was isolated from the cells using a standard commercial plasmid purification kit.

[0139] Transform the plasmid encoding MAT or the engineered acyltransferase into BL21(DE3) chemically competent cells (Agilent). Transfer the transformed cells directly to a pre-culture of 5 mL LB medium containing ampicillin as a selection marker and incubate overnight at 37 °C in an incubation shaker. Use the pre-culture to inoculate 100 to 250 mL of the main culture in 2×YT or TB medium in a 500 mL Erlenmeyer flask. Incubate the culture at 37 °C for 2 h and then induce the expression system by adding 250 μM IPTG and incubate for an additional 16 h at 20 °C. Lyse the cells enzymatically or by centrifugation in lysis buffer, resuspend in lysis buffer containing DNaseI, and lyse by sonication (Hielscher UP200t, 43% amplitude, 5 min, on / off time 20 s). After centrifugation, purify the soluble fraction by Ni-NTA (ThermoFisher) and Strep affinity chromatography (strep-tactin sepharose, IBALifesciences GmbH) according to the standard protocol. Purify ACP by size exclusion chromatography (ROTIDex-25, carl-Roth) instead of Strep affinity chromatography as a second chromatographic purification. Freeze the purified protein in aliquots in liquid nitrogen and store at -25 °C or -80 °C. Determine the protein concentration by the absorbance of the protein at 280 nm. Record the absorbance on a NanoDrop One C (ThermoFisher) and convert it to concentration using the extinction coefficient of the corresponding protein, which is calculated from its primary sequence without the N-terminal methionine in Benchling. Example 3: Kinetic analysis of the engineered acyltransferase using MMal-CoA

[0140] An enzyme-coupled assay was performed to analyze the transfer of the acyl moiety from CoA to ACP. Four solutions were prepared as 4× concentrated stocks in assay buffer. Solution 1 contained acyltransferase at a concentration of 0.01 μM to 10 μM, depending on the transfer kinetics for a good signal-to-noise ratio. Solution 2 contained the enzyme-coupled system, comprising 8 mM α-ketoglutarate, 1.6 mM NAD+, 1.6 mM TPP, and 60 mU / 100 μL αKGDH. Solution 3 contained methylmalonyl (MMal)-CoA at a concentration of 0.1 μM to 1,000 μM, and solution 4 contained ACP from murine FAS as a 240 μM standalone protein. 5 μL of solutions 1 to 3 were pipetted into a 384-well low-volume HiBase microplate (Greiner Bio-one) and manually mixed. The reaction was initiated by adding solution 4 with the dispenser system of a microplate reader (ClarioStar, BMG labtech). The reaction progress was recorded using the following settings: wavelength: 348 - 20 nm; emission: 476 - 20 nm; gain: 1500; focal height: 11.9 mm; flash: 17 times; orbital averaging: off.

[0141] The kinetic data of the exemplary mutants are shown in Table 2. Table 2 The listed SEQ ID NOs refer to the corresponding acyltransferase sequences. The mutations are indicated with reference to the wild-type MAT sequence SEQ ID NO:29. *Comparative data from Rittner et al. 2018 Example 4: Analysis of Engineered Acyltransferases with Mal-CoA

[0142] On the basis of previous studies, other engineered mutants were expressed, purified and analyzed. Fifty different exemplary mutants were screened for the transfer of the malonyl moiety from CoA to N-(2-mercaptoethyl)-hexanamide. Four solutions were prepared as 4× concentrated stocks in assay buffer. Solution 1 contained acyltransferase at a concentration of 0.01 μM to 10 μM, which was divided into four groups according to their respective turnover rates to achieve a good signal-to-noise ratio. Solution 2 contained an enzyme-coupled system, including 8 mM α-ketoglutarate, 1.6 mM NAD+, 1.6 mM TPP and 60 mU / 100 μL αKGDH. Solution 3 contained a fixed concentration of 200 μM malonyl (Mal)-CoA, and solution 4 contained 200 μM of the acceptor N-(2-mercaptoethyl)-hexanamide. 5 μL of solutions 1 to 3 were pipetted into a 384-well small-volume HiBase microplate (Greiner Bio-one) and mixed manually. The reaction was initiated by adding 5 μL of solution 4 with an automated multi-channel pipette (ClipTip). The reaction progress was recorded using the following settings: wavelength: 348 - 20 nm; emission: 476 - 20 nm; gain: 1700; focus height: 12.1 mm; flashes: 10 times; track average: off.

[0143] The specific turnover rates of all tested engineered mutants are shown in Figure 4 a logarithmic scale. The unit RFU per second was converted to turnover per second using the known enzyme concentration and the calibration curve with NADH. Table 3 Example 5: Verification of the Activity of Re-engineered Mutants

[0144] To verify the activity of the heavily engineered mutants with substrate specificity altered from the natural CoA substrate to a non-CoA substrate, an alternative method for quantifying the specific turnover rate of the non-CoA substrate was established. Such non-CoA substrates cannot be detected by enzyme-coupled assays. We used a thiol-sensitive fluorophore (ThioGlo4; Yi et al. 2009) to quantify the free thiol groups released from the substrate 3-oxo-3-[[2-[(1-oxohexyl)amino]ethyl]thio]-propionic acid after the malonyl moiety was specifically transferred by the enzyme to a non-thiol acceptor. Briefly, three solutions were prepared at twice the concentration or as a 4× concentrated stock solution in the assay buffer. Solution 1 contained acyltransferase at a concentration of 50 to 500 nM, depending on its purification yield, and was diluted twofold in the assay. Solution 2 contained a fixed concentration of 200 μM of the substrate 3-oxo-3-[[2-[(1-oxohexyl)amino]ethyl]thio]-propionic acid, and solution 3 contained 20 mM of the acceptor N-(2-hydroxyethyl)-hexanamide. 10 μL of solution 1 and 5 μL of solution 2 were pipetted into a 384-well small-volume HiBase microplate (Greiner Bio-one). The reaction was initiated by adding 5 μL of solution 4 with a multi-channel pipette (ClipTip) and incubated for 10 minutes at room temperature. The reaction was terminated by adding 2.5 μL of 3 M HCl, neutralized by adding 2.5 μL of 4 M NaOH, and 10 μL of the reaction mixture was transferred to a new well. The free thiol was detected by adding 10 μL of 120 μM ThioGlo in a buffer of 250 mM potassium phosphate, 1 mM EDTA, and 10% glycerol, mixing it, and performing fluorescence detection on a microplate reader (ClarioStar, BMG labtech) using the following settings: excitation: 400 - 20 nm; emission: 465 - 20 nm; gain: 700; focus height: 12.5 mm; flashes: 20 times; orbital average: off. Wells without any enzyme but with the substrate and acceptor at the mentioned concentrations were used as background and reflected non-enzyme-specific transacylation.

[0145] Results of the transfer of the malonyl moiety from the non-CoA substrate are shown in Figure 5 the Example 6: In-depth kinetic analysis

[0146] In-depth kinetic analysis results of a representative mutant (A35) are shown herein, which allows comparison of the enzyme kinetic parameters for the transfer of the malonyl moiety from Mal-CoA or 3-oxo-3-[[2-[(1-oxohexyl)amino]ethyl]thio]-propionic acid to ACP. Enzyme kinetics were determined using the established enzyme-coupled assay for Mal-CoA kinetics and a variant of the new thiol quantification assay using ThioGlo.

[0147] The enzyme-coupled assay was performed with a 4× concentrated stock solution as described in Example 3. The enzyme concentration of A35 in Solution 1 was 4.2 μM, the substrate concentration of Mal-CoA in Solution 3 was 8 to 400 μM, and the acceptor concentration of ACP in Solution 4 was 340 μM.

[0148] A variant of the thiol quantification assay in Example 5 was established to enable the separation of the released thiol group from 3-oxo-3-[[2-[(1-oxohexyl)amino]ethyl]thio]-propionic acid after acyl transfer from the thiol group of acceptor ACP. The resulting N-(2-mercaptoethyl)-hexanamide was extracted from the reaction wells by chloroform extraction. Thus, the assay was performed as described in Example 5, but after terminating the reaction with 3 μL of HCl, each well was extracted with 2×100 μL of chloroform. First, 50 μL of the lower phase was transferred to a new well, and then 100 μL of the lower phase was transferred to the same well. A correction factor of 1.33 was applied. The chloroform was removed by speedvac, and N-(2-mercaptoethyl)-hexanamide was dissolved in 50 μL of assay buffer. 10 μL of the reaction mixture was mixed with 10 μL of 120 μM ThioGlo in assay buffer, and fluorescence was detected on a microplate reader (ClarioStar, BMG labtech) using the following settings: excitation: 400 - 20 nm; emission: 465 - 20 nm; gain: 700; focus height: 12.5 mm; flashes: 20; orbital average: off.

[0149] The concentrations of the three solutions were prepared at twice the concentration (Solution 2) or 4× concentrated stock solution (Solutions 1 and 3) in assay buffer. Solution 1 contained acyltransferase at a concentration of 170 nM, Solution 2 contained the substrate 3-oxo-3-[[2-[(1-oxohexyl)amino]ethyl]thio]-propionic acid at a concentration of 20 to 1000 nM, and Solution 3 contained acceptor ACP at a concentration of 340 μM. Additionally, TCEP was added to the reaction mixture at a final concentration of 1 mM.

[0150] Exemplary kinetic analysis results of mutant A35 are shown in FIG. 6 and Table 4. Table 4 – Kinetic analysis results for Mal-CoA (A) and 3-oxo-3-[[2-[(1-oxohexyl)amino]ethyl]thio]-propionic acid (B) corresponding to FIG. 6. substrate acceptor <![CDATA[K m [μM]]]> <![CDATA[k cat [seconds -1＊ > <![CDATA[k cat / K m [seconds -1 M -1 > factor A ACP 25 0.01 4.07E+02 1 B ACP 30 5.75 1.90E+05 467

[0151] A detailed kinetic analysis comparing the use of non-CoA substrates with CoA substrates has been provided, and further in-depth studies of various engineered mutants (including competitive assays using both CoA and non-CoA substrates) have been conducted and some are ongoing. References Hertweck C. The biosynthetic logic of polyketide diversity. Angew Chem Int Ed Engl. 2009;48(26):4688 - 716. doi:10.1002 / anie.200806121. Rittner A, Paithankar KS, Huu KV, Grininger M. Characterization of the Polyspecific Transferase of Murine Type I Fatty Acid Synthase (FAS) and Implications for Polyketide Synthase (PKS) Engineering. ACS Chem Biol. 2018 Mar 16;13(3):723 - 732. doi:10.1021 / acschembio.7b00718. Rittner A, Paithankar KS, Himmler A, Grininger M. Type I fatty acid synthase trapped in the octanoyl - bound state. Protein Sci. 2020 Feb;29(2):589 - 605. doi:10.1002 / pro.3797. Smith S, Tsai SC. The type I fatty acid and polyketide synthases: a tale of two megasynthases. Nat Prod Rep. 2007 Oct;24(5):1041 - 72. doi:10.1039 / b603600g. Yi L, Li H, Sun L, Liu L, Zhang C, Xi Z. A Highly Sensitive Fluorescence Probe for Fast Thiol - Quantification Assay of Glutathione Reductase. Angew.Chem. 2009;121(22):4094 - 4097. https: / / doi.org / 10.1002 / ange.200805693.

Claims

1. An acyltransferase having an altered substrate specificity, wherein the acyltransferase comprises one or more amino acid replacements at the following reference positions: (i) T161 according to SEQ ID NO:29, or a similar threonine, serine or asparagine residue at the reference position as defined in Table 1, wherein the amino acid is replaced with alanine, glycine, valine, leucine, isoleucine, tyrosine, phenylalanine, tryptophan, aspartic acid or glutamic acid; and / or (ii) F184 according to SEQ ID NO:29, or a similar phenylalanine residue at the reference position as defined in Table 1, wherein the amino acid is replaced with alanine, glycine, valine, leucine, isoleucine, methionine, aspartic acid or glutamic acid; and / or (iii) R286 according to SEQ ID NO:29, or a similar arginine residue at the reference position as defined in Table 1, wherein the amino acid is replaced with alanine, glycine, valine, leucine, isoleucine, methionine, aspartic acid or glutamic acid; and / or (iv) K186 according to SEQ ID NO:29, or a similar lysine or arginine residue at the reference position as defined in Table 1, wherein the amino acid is replaced with a negatively charged amino acid or replaced with glycine, alanine, valine, leucine, isoleucine or methionine; and / or (v) according to K285 of SEQ ID NO:29, or a similar lysine or arginine residue at the said reference position as defined in Table 1, wherein the amino acid is replaced with a negatively charged amino acid; and / or (vi) R300 according to SEQ ID NO:29, or a similar lysine or arginine residue at the reference position as defined in Table 1, wherein the amino acid is replaced with a negatively charged amino acid; and / or (vii) A282 according to SEQ ID NO:29, or a similar alanine residue at the reference position as defined in Table 1, wherein the amino acid is replaced with valine, leucine, isoleucine, phenylalanine or tyrosine; and / or (viii) G137 according to SEQ ID NO:29, or a similar glycine residue at the reference position as defined in Table 1, wherein the amino acid is replaced with valine, leucine, isoleucine, phenylalanine or tyrosine.

2. The acyltransferase according to claim 1, wherein the amino acid replacement at reference position (i) is replaced with leucine, alanine, tryptophan or valine, preferably replaced with leucine or alanine, more preferably replaced with leucine; and / or wherein the amino acid replacement at reference position (ii) is replaced with leucine, alanine, glycine, valine or glutamic acid, preferably replaced with leucine, alanine or glutamic acid, more preferably replaced with leucine or alanine; and / or wherein the amino acid replacement at reference position (iii) is replaced with leucine, alanine, glycine or valine, preferably replaced with leucine or alanine; and / or wherein The amino acid substitution at reference position (iv) is a substitution with a negatively charged amino acid, or a substitution with alanine or valine, preferably a substitution with glutamic acid or aspartic acid; and / or wherein The amino acid substitution at reference position (v) is a substitution with glutamic acid or aspartic acid; and / or wherein The amino acid substitution at reference position (vi) is a substitution with glutamic acid or aspartic acid; and / or wherein The amino acid substitution at reference position (vii) is a substitution with leucine, isoleucine, phenylalanine or tyrosine; and / or wherein The amino acid substitution at reference position (viii) is a substitution with leucine, isoleucine, phenylalanine or tyrosine, preferably a substitution with leucine.

3. The acyltransferase according to claim 1, wherein The amino acid substitution at reference position (i) is a substitution with leucine, isoleucine, tyrosine, phenylalanine or glycine; and / or wherein The amino acid substitution at reference position (ii) is a substitution with leucine, alanine, isoleucine or methionine; and / or wherein The amino acid substitution at reference position (iii) is a substitution with leucine, alanine, isoleucine or methionine; and / or wherein The amino acid substitution at reference position (iv) is a substitution with alanine, glycine, valine, leucine, isoleucine or methionine; and / or wherein The amino acid substitution at reference position (vii) is a substitution with valine; and / or wherein The amino acid substitution at reference position (viii) is a substitution with valine.

4. The acyltransferase according to any one of the preceding claims, wherein the acyltransferase is an isolated acyltransferase derived from fatty acid synthase.

5. The acyltransferase according to claim 1, wherein the acyltransferase comprises amino acid substitutions as defined in any one of claims 1 to 3 at two or more of the reference positions (iv) and (i), or (iv) and (ii), or (iv) and (iii), or (iv) and (v), or (iv) and (vi), or (iv) and (vii), or (iv) and (viii), or at two or more of the reference positions (i), (ii), (iii), (v), (vi), (vii) and / or (viii) in combination with (iv), optionally wherein the amino acid substitution at reference position (iv) is a substitution with glutamic acid or aspartic acid.

6. The acyltransferase according to claim 1, wherein the acyltransferase comprises amino acid substitutions as defined in any one of claims 1 to 3 at two or more of the reference positions (i) and (ii), or (i) and (iii), or (i) and (iv), or (i) and (v), or (i) and (vi), or (i) and (vii), or (i) and (viii), or at two or more of the reference positions (ii), (iii), (iv), (v), (vi), (vii) and / or (viii) in combination with (i), optionally wherein the amino acid substitution at reference position (i) is as defined in claim 2.

7. The acyltransferase according to any one of the preceding claims, wherein the acyltransferase comprises the amino acid replacement as defined in claim 1 at the reference position (i), optionally wherein the amino acid replacement at the reference position (i) is as defined in claim 2.

8. The acyltransferase according to claim 7, wherein the acyltransferase further comprises the amino acid replacement as defined in claim 1 at the reference positions (ii) and (iii): optionally wherein the amino acid replacement at the reference position (ii) is as defined in claim 2; optionally wherein the amino acid replacement at the reference position (iii) is as defined in claim 2.

9. The acyltransferase according to claim 7 or 8, wherein the acyltransferase further comprises the amino acid replacement as defined in claim 1 at the reference position (iv), optionally wherein the amino acid replacement at the reference position (iv) is as defined in claim 2.

10. The acyltransferase according to any one of claims 7 to 9, wherein the acyltransferase further comprises the amino acid replacement as defined in claim 1 at the reference position (vi), optionally wherein the amino acid replacement at the reference position (vi) is as defined in claim 2.

11. The acyltransferase according to any one of the preceding claims, wherein the acyltransferase comprises the amino acid replacement at the reference positions (iv) and (vi) being glutamate, or the amino acid replacement at the reference positions (iv) and (vi) being aspartate, or the amino acid replacement at the reference position (iv) being glutamate and the amino acid replacement at the reference position (vi) being aspartate, or the amino acid replacement at the reference position (iv) being aspartate and the amino acid replacement at the reference position (vi) being glutamate.

12. The acyltransferase according to any one of the preceding claims, wherein the acyltransferase comprises the amino acid replacement at the reference position (iii) being glutamate or aspartate, and optionally further comprises the amino acid replacement at the following reference positions: (ix) D160 according to SEQ ID NO:29, or a similar aspartate or glutamate residue at the reference position as defined in Table 1, wherein the amino acid is replaced with arginine, histidine, lysine, glutamine or asparagine.

13. The acyltransferase according to any one of the preceding claims, wherein the acyltransferase is derived from a fatty acid synthase of an organism selected from the group consisting of Vertebrata, including Aves and Mammalia, including Artiodactyla, Perissodactyla, Carnivora, Primates, and Rodentia, optionally wherein the acyltransferase is derived from a fatty acid synthase comprising or consisting of a wild-type amino acid sequence selected from any one of SEQ ID NOs: 1 to 28, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity thereto.

14. The acyltransferase according to any one of the preceding claims, wherein the acyltransferase comprises or consists of the following: The amino acid sequence of any one of SEQ ID NOs: 57 to 82 or 111 to 133, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity thereto.

15. A fusion protein comprising at least one acyltransferase according to any one of the preceding claims, preferably wherein the fusion protein further comprises at least one linker domain and optionally at least one ketoacyl synthase domain.

16. A nucleic acid encoding the acyltransferase according to any one of claims 1 to 9 or the fusion protein according to claim 15.

17. An expression construct or vector comprising at least one nucleic acid according to claim 16.

18. A cell comprising at least one acyltransferase according to any one of claims 1 to 14; and / or at least one fusion protein according to claim 15; and / or at least one nucleic acid according to claim 16; and / or at least one expression construct or vector according to claim 17.

19. A kit comprising at least one acyltransferase according to any one of claims 1 to 14; and / or at least one fusion protein according to claim 15; and / or at least one nucleic acid according to claim 16; and / or at least one expression construct or vector according to claim 17; and / or at least one cell according to claim 18; optionally further comprising at least one container; and / or a set of reagents, including buffers, media and / or substrates; and / or transfection means.

20. The following uses for polyketide synthesis, fatty acid synthesis, and / or non-ribosomal peptide synthesis: at least one acyltransferase as defined in any one of claims 1 to 14; and / or at least one fusion protein as defined in claim 15; and / or at least one nucleic acid as defined in claim 16; and / or at least one expression construct or vector as defined in claim 17; and / or at least one cell as defined in claim 18; and / or at least one kit as defined in claim 19, optionally wherein it is a use for reacting at least two different substrates.

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