Methods for acyl group transfer and polyketide synthesis

Abstract

The present invention relates to the fields of fatty acid- and polyketide synthesis, and protein engineering. In particular, it relates to methods of using altered acyltransferases having an altered coenzyme specificity for acyl group transfer and polyketide synthesis.

Description

[0001]Munich, 2 July 2024 Our Ref.: KM 5619-03WO CMC / SEG Applicant: kez.biosolutions GmbH Serial Number: New Application kez.biosolutions GmbH Am Mühlenberg 11, 14476 Potsdam Novel methods for acyl group transfer and polyketide synthesis Technical field The present invention relates to the fields of fatty acid- and polyketide synthesis, and protein engineering. In particular, it relates to methods of using altered acyltransferases having an altered coenzyme specificity for acyl group transfer and polyketide synthesis. 5 Background Polyketides represent a versatile and important class of biomolecules. Particularly polyketide antibiotics, immunosuppressants, antiparasitics, as well as antifungal, cholesterol-lowering, and antitumoral agents represent important classes of biomolecules of outstanding therapeutic interest. 10 Despite having a biomolecular target, most polyketides are currently not used as therapeutics so that only a tiny fraction of their great potential is used for this particular substance class. In order to make them applicable for the human body, polyketides need to be tailored with certain functionalities. However, organic synthesis is very challenging and economically unfeasible for this particular substance class, due to their large carbon 15 scaffolds with many functional groups and stereochemical properties. There is thus a great need in the pharmaceutical and biotechnological industry to provide systems allowing the synthesis of new and particularly custom-made polyketides with tailored functionalities in a reasonable time. While polyketide synthase (PKS) systems bear a vast potential for synthetizing myriads of 20 different biologically active compounds for drug design and other application, PKS and fatty acid synthase (FAS) enzymes are limited to substrates bound to Coenzyme A (CoA). CoA is composed of cysteamine (β-mercaptoethylamine), β-alanine, pantoic acid, *20250219276*  - 2 - organophosphate anhydride and a 3′-phosphoadenosine. The thiol group of the cysteamine can form a “high energy” thioester bond with carboxylic acids thereby forming “activated” acyl-CoA molecules. CoA, however, is an abundant molecule in typical production strains so that naturally 5 present CoA-bound substrates can interfere with polyketide synthesis. Consequently, having enzymes available with a decreased or even abolished specificity for CoA could be advantageous for the in vivo production of polyketides during metabolic engineering to shift the product yield towards a desired ketide or polyketide compound of interest using specifically designed enzymes as engineered molecular machines. In addition, using an 10 artificial pathway (relying on acyl substrates bound to a different coenzyme moiety) for the biosynthesis of a target compound, avoids interference with the natural metabolism and a new functionality does no longer require identifying and establishing a suitable natural metabolic pathway to produce the substrate. It was thus an object of the present invention to provide methods of acyl group transfer as well as polyketide synthesis that overcome 15 these problems associated with CoA-bound substrates and to expand the currently available production methods. Crystal structures of some FAS enzymes and PKS systems have been solved (Smith and Tsai 2007, Rittner et al.2020). However, it has (to our knowledge) never been envisioned to use acyltransferases with an altered CoA binding pocket that, ideally, does not use acyl- 20 CoA substrates and accepts subunits bound to different (non-CoA) coenzyme moieties, such as synthetic coenzyme moieties. It was a particular object to expand and re-define the substrate recognition, binding and catalysis spectrum of acyltransferases to provide new methods allowing the use of a wide range of substrates, ideally while excluding CoA, to allow for targeted polyketide(- 25 precursor) synthesis, preferably the synthesis of ketides or polyketide building blocks comprising positions within the polyketide molecule, where synthetic moieties can be inserted or added already during synthesis in vivo when produced by a host cell of interest or in vitro when produced in an artificial enzymatic system, or by semi-synthetic processes after production of the (poly-)ketide scaffold. 30 Summary of the Invention In a first aspect, there is provided a method comprising: (a) providing (i) at least one non- CoA acyltransferase substrate; and (ii) at least one altered acyltransferase ; and (iii) at least one acyl carrier protein (ACP); (b) allowing the at least one acyltransferase to transfer the - 3 - acyl group of the at least one non-CoA acyltransferase substrate to the at least one ACP; (c) obtaining at least one acyl-ACP; wherein the at least one altered acyltransferase has a CoA-binding pocket that is altered to reduce CoA acyltransferase substrate transfer activity. In one embodiment of the first aspect, step (b) is performed in at least one cell comprising 5 the components (i), (ii), and (iii) of step (a). In one embodiment of the first aspect, step (b) is performed outside of a cell. In one embodiment of the first aspect, step (b) the at least one altered acyltransferase is provided in a cell-free system and / or as a purified or partially purified protein or as a lysate comprising the altered acyltransferase, optionally wherein the at least one altered 10 acyltransferase is provided as a fusion protein further comprising at least one linker domain and optionally at least one ketoacyl synthase domain. In a second aspect, there is provided a method for polyketide synthesis, the method comprising: (a) providing (i) at least one non-CoA acyltransferase substrate; and (ii) optionally at least one CoA acyltransferase substrate; and (ii) a polyketide synthase (PKS) 15 system, wherein the PKS system comprises at least one altered acyltransferase; (b) allowing polyketide synthesis, wherein the at least one non-CoA acyltransferase substrate, and optionally the at least one CoA acyltransferase substrate, is / are used as a substrate in the polyketide synthesis; (c) obtaining at least one polyketide, and optionally purifying said at least one polyketide; wherein the at least one altered acyltransferase has a CoA-binding 20 pocket that is altered to reduce CoA acyltransferase substrate transfer activity. In one embodiment of the second aspect, the PKS system is a PKS type I system, wherein one, two, three or more PKS modules comprise and / or interact with at least one altered acyltransferase. In one embodiment of the second aspect, the PKS system is a PKS type II system, wherein 25 the individual catalytic proteins of the PKS system interact with at least one altered acyltransferase. In one embodiment of the second aspect, the loading domain, the first, second, third, fourth, fifth, six, seventh, eighth, ninth, tenth, eleventh, thirteenth, fourteenths, fifteenth, and / or sixteenth PKS module and / or one or more subsequent PKS modules comprise(s) and / or 30 interact(s) with at least one altered acyltransferase. - 4 - In one embodiment of the second aspect, the at least one altered acyltransferase is part of at least one multifunctional PKS type I polypeptide, optionally through replacement of at least one of the PKS acyltransferases. In one embodiment of the second aspect, the at least one altered acyltransferase is 5 provided as a separate polypeptide, optionally as a fusion protein further comprising at least one linker domain and optionally at least one ketoacyl synthase domain. In one embodiment of the second aspect, the polyketide synthesis, or part of the polyketide synthesis, is performed in at least one cell comprising the PKS system or parts thereof. In one embodiment of the second aspect, the polyketide synthesis, or part of the polyketide 10 synthesis, is performed outside of a cell. In one embodiment of the second aspect, the at least one altered acyltransferase is provided in a cell-free system and / or as a purified or partially purified protein or as a lysate comprising the altered acyltransferase, optionally wherein the at least one altered acyltransferase is provided as a fusion protein further comprising at least one linker domain 15 and optionally at least one ketoacyl synthase domain. In one embodiment of the first or second aspect, wherein the at least one altered acyltransferase has a CoA-binding pocket that is altered to reduce CoA acyltransferase substrate transfer activity and optionally to increase transfer activity for the at least one non-CoA acyltransferase substrate. 20 In one embodiment of the first or second aspect, the at least one altered acyltransferase has a CoA-binding pocket that is altered to transfer the at least one non-CoA acyltransferase substrate at a higher rate than the equivalent CoA acyltransferase substrate with the same acyl group and / or, in case at least one CoA acyltransferase substrate is provided in step (a), the at least one CoA acyltransferase substrate provided 25 in step (a). In one embodiment of the first or second aspect, the at least one non-CoA substrate comprises or consists of (ii) a non-CoA coenzyme; and (i) an acyl group; optionally wherein part (i) and part (ii) are linked via a thioester. - 5 - In one embodiment of the first or second aspect, the non-CoA coenzyme has a neutral net charge or a positive net charge. In one embodiment of the first or second aspect, the positive net charge is a single, two- fold, three-fold or four-fold positive net charge. 5 In one embodiment of the first or second aspect, the non-CoA coenzyme does not comprise a negative charge. In one embodiment of the first or second aspect, the non-CoA coenzyme does not comprise a negative charge that is within 10 to 25 angstrom of the high energy bond, preferably the ester bond, more preferably the thioester bond, linking the non-CoA coenzyme to the acyl 10 group. In one embodiment of the first or second aspect, the acyl group comprises or consist of a malonyl group, an acetyl group, a methylmalonyl group, a propionyl group, or a derivative thereof. In a third aspect, there is provided a use of at least altered acyltransferase as defined in 15 the first or second aspect, wherein the at least one altered acyltransferase originates from a PKS; and / or at least one non-CoA acyltransferase substrate as defined in the second or fifths aspect; and / or at least one compound as defined in the sixth aspect and optionally at least one CoA acyltransferase substrate for polyketide synthesis, fatty acid synthesis, and / or nonribosomal peptide synthesis. 20 In a fourth aspect, there is provided a kit comprising at least one altered acyltransferase as defined in the first or second aspect; and / or at least one non-CoA acyltransferase substrate as defined in the second or fifth aspect; and / or at least one compound as defined in the sixth aspect, optionally further comprising at least one CoA acyltransferase substrate, and / or at least one container, and / or a set of reagents including buffer and / or medium, 25 and / or means of transfection. A kit will usually comprise all agents, buffers, co-factors etc. necessary to guarantee the functionality of the at least one acyltransferase in an assay of interest. In a fifth aspect there is provided a non-CoA polyketide substrate comprising or consisting of (i) a non-CoA coenzyme; and (ii) an acyl group; wherein part (i) and part (ii) are linked 30 via a thioester , optionally wherein a) the non-CoA coenzyme has a neutral net charge or - 6 - a positive net charge, further optionally , wherein the positive net charge is a single, two- fold, three-fold or four-fold positive net charge; and / or b) wherein the non-CoA coenzyme does not comprise a negative charge. In one embodiment of the fifth aspect, the non-CoA coenzyme comprises or consist of a 5 compound according to formula (1): wherein R1 represents the linked acyl group, wherein the acyl group is not a malonyl group. In a sixth aspect there is provided a compound according to formula (1): 10 wherein R1 is hydrogen. In one embodiment of the fifth or sixth aspect, the acyl group comprises or consist of a malonyl group, an acetyl group, a methylmalonyl group, a propionyl group, or a derivative 15 thereof. In another embodiment of the fifth or sixth aspect, the acyl group is an acyl group according to any one of formulas 4 to 19, wherein the asterisk denotes the non-CoA coenzyme: - 8 - 5 10 (19). Definitions An “acyltransferase” or “AT” as used herein refers to a fatty acid synthase (FAS) acyltransferase or polyketide synthase (PKS) acyltransferase having an Enzyme Commission number selected from EC 2.3.1.39 ([acyl-carrier-protein] S- 15 malonyltransferase) or EC 2.3.1.38 ([acyl-carrier-protein] S-acetyltransferase). An acyltransferase can selectively bind and trans-esterify different coenzyme-bound acyl molecules onto an acyl carrier protein (ACP), for instance as part of fatty acid or polyketide synthesis. While natural acyltransferases are specific for coenzyme A (CoA)-bound acyl molecules (“CoA acyltransferase substrates”), an acyltransferase of the present invention 20 is an artificially altered acyltransferase that no longer accepts acyl-CoA substrates and can instead bind and trans-esterify acyl groups bound to other coenzyme moieties, including synthetic coenzyme moieties (“non-CoA acyltransferase substrates”). An acyltransferase of the present invention may be expressed as part of a FAS and / or PKS, or a sub-part - 9 - thereof, as a separate molecule and / or as part of a fusion protein. An “altered acyltransferase” as used herein, refers to an acyltransferase having an altered CoA-binding pocket according to the present invention leading to a reduced CoA acyltransferase substrate transfer activity. 5 An acyltransferase “originating from a FAS” refers to an acyltransferase for which the corresponding, wild type acyltransferase (AT) is part of said fatty acid synthase in nature, wherein an acyltransferase of the present invention may be present as part of said fatty acid synthase or may be an isolated acyltransferase according to the present disclosure. For instance, for the altered acyltransferase according to SEQ ID NO: 57 the corresponding 10 wild type malonyl-acetyl-transferase (MAT) sequence is SEQ ID NO: 29, which is part of the fatty acid synthase according to SEQ ID NO: 1; hence the altered acyltransferase according to SEQ ID NO: 57 originates from the fatty acid sequence according to SEQ ID NO: 1. Various wild type ATs and the fatty acid synthases they are part of in nature, are disclosed as SEQ ID NOs: 29 to 56 and SEQ ID NOs: 1 to 28, respectively. An 15 acyltransferase “originating from a PKS” refers to an acyltransferase for which the corresponding, wild type acyltransferase is part of said PKS in nature, wherein an acyltransferase of the present invention may be present as part of said PKS or may be an isolated acyltransferase according to the present disclosure. For instance, for the altered acyltransferase according to SEQ ID NO: 154 the corresponding wild type AT sequence is 20 SEQ ID NO: 139, which is part of module 6 of the 6-deoxyerythronolide-B synthase EryA3, a PKS in Saccharopolyspora erythraea; hence the altered acyltransferase according to SEQ ID NO: 154 originates from the 6-deoxyerythronolide-B synthase EryA3. The skilled person can easily determine, for instance through sequence and / or structural alignments, the acyltransferase within a given FAS or PKS sequence. An acyltransferase may also be 25 any fragment, part and / or (sub)domain of a FAS or PKS acyltransferase, as long as it has acyltransferase activity. An “isolated acyltransferase” as used herein, refers to an altered acyltransferase that is not present as part of a continuous polypeptide comprising the complete FAS or PKS from which it originates, optionally not comprising more than the acyltransferase and optionally 30 the linker domain (LD), or a part thereof, and / or the ketoacyl synthase (KS) of the FAS or PKS from which it originates. An isolated acyltransferase may be present as a discrete enzyme, i.e. a polypeptide consisting of said acyltransferase. Preferably, a polypeptide comprising an isolated acyltransferase further comprises at least one protein, domain or part thereof to increase the stability, such as an LD, or a part thereof, wherein the LD may 35 originate from the same FAS or PKS or may originate, in form of a fusion protein, from at - 10 - least one different FAS and / or PKS, or a different protein, domain or part thereof, for example an MBP-tag or a different tag. In certain embodiments, an isolated acyltransferase is present as part of a polypeptide comprising the acyltransferase, a KS as well as a LD connecting the acyltransferase and the KS, wherein the KS, the LD may originate from the 5 same FAS or PKS or may originate, in form of a fusion protein, from at least one different FAS and / or PKS. Generally, an isolated acyltransferase may be present as part of a fusion protein further comprising at least one polypeptide that does not originate from the same FAS or PKS or does not originate from any FAS or PKS, such as one or more different enzyme parts and / or domains, including FAS and / or PKS functional domains, one or more 10 tags, one or more binding domains, synthetic zippers and / or the like. An “amino acid exchange” as used herein, refers to the presence of an amino acid at a given position of the primary amino acid sequence of a polypeptide that differs from the amino acid present at said position of the primary amino acid sequence of a reference polypeptide, such as a wild type polypeptide. Commonly, amino acid exchanges are 15 achieved by mutating the respective codon of a nucleic acid sequence encoding the polypeptide into a codon encoding a different amino acid at said position. Means and methods to exchange an amino acid at a desired position, for instance but not limited to PCR-based cloning methods as disclosed herein, are well established in the field and available to the skilled person. 20 The terms “CoA-binding pocket” and “CoA-binding region” are used interchangeably herein and refers to the region of the acyltransferase that binds (when not altered according to the present invention) to the CoA moiety for and / or during the acyl group transfer and that comprises one or more or all of the reference positions according to the present invention. An altered CoA-binding pocket according to the present invention is a CoA binding pocket 25 that has been altered to reduce the CoA acyltransferase substrate transfer activity and optionally to increase the substrate transfer activity for at least one desired non-CoA acyltransferase substrate. An altered CoA binding pocket may be achieved by one or more amino acid exchanges within the CoA-binding pocket and / or by one or more amino acid exchanges outside of the CoA-binding pocket altering the structure of the CoA-binding 30 pocket. The skilled person can use the natural CoA acyltransferase substrate(s) in methods known in the art and / or described herein measuring the acyl group transfer and / or the release of free CoA directly or indirectly in order to verify a reduced CoA acyltransferase substrate transfer activity. For polyspecific acyltransferases, the skilled person can use one of the natural CoA acyltransferase substrates as an exemplary CoA acyltransferase 35 substrate to determine whether the CoA acyltransferase substrate transfer activity is - 11 - reduced. In embodiments specifying a percentage of reduced CoA acyltransferase substrate transfer activity, it is sufficient for polyspecific acyltransferases that the transfer activity of one of the natural CoA acyltransferase substrate is reduced to the specified percentage range. A “natural CoA acyltransferase substrate” is the substrate or one of the 5 substrates of a given acyltransferase without the altered CoA-binding pocket according to the present invention. Altering of the CoA-binding pocket according to the present invention may be used in combination with one or more further mutations changing the acyl specificity of the acyltransferase (in addition to the altered coenzyme specificity according to the present invention). In this case, the natural CoA acyltransferase substrate(s) is / are the CoA 10 acyltransferase substrate(s) of that mutated acyltransferase with a changed acyl specificity but without the altered CoA-binding pocket. A reduced CoA acyltransferase substrate transfer activity according to the present invention describes a change in the coenzyme-binding properties due to the altered CoA- binding region, thereby leading to an altered coenzyme specificity. A reduced CoA 15 acyltransferase substrate transfer activity according to the present invention does not include a reduction of substrate transfer activity due to a change in acyl specificity or an overall inactivation of the AT, for example due to denaturing. However, in certain embodiments, the at least one altered acyltransferase may have an altered acyl specificity in addition to the reduced CoA acyltransferase substrate transfer activity. A non-CoA 20 acyltransferase substrate may be used to verify the non-CoA acyltransferase substrate activity (for instance S-Malonyl-N-hexanoylcysteamine as shown in example 4). In preferred embodiments, an acyltransferase having an altered CoA-binding pocket is an acyltransferase having an amino acid exchange to reduce the CoA acyltransferase substrate transfer activity at 1, 2, 3, 4, 5, 6, 7 or 8 reference position(s) according to table 25 1 to table 5. A “fatty acid synthase” or “FAS” as used herein, refers to a fatty acid synthase of type I, being a multi-functional protein, also called a multi-enzyme, capable of catalyzing all steps of fatty acid synthesis or a fatty acid synthase of type II comprising multiple monofunctional enzymes for fatty acid synthesis. In nature, wild type fatty acid synthases catalyze multiple 30 rounds of Claisen condensation reactions with acetyl-CoA and malonyl-CoA recognized and transferred by an acyltransferase being a malonyl-acetyl-transferase (MAT). The large multi-functional proteins comprise the functional domains of an acyltransferase transferring the acyl-substrates onto an acyl carrier protein (ACP), a ketoacyl synthase (KS) catalyzing the Claisen condensation reaction, a ketoacyl reductase (KR), a hydroxyacyl dehydratase - 12 - (DH) and an enoyl reductase (ER) creating a fully saturated acyl backbone and a thioesterase (TE) releasing the synthesized fatty acid. (Smith and Tsai 2007). A “fusion protein” as used herein refers to a polypeptide that is created by joining at least two different polypeptides, which may each be a complete protein existing in nature, a part 5 thereof, or a fully synthetic polypeptide, to form one continuous artificial polypeptide. Commonly, a fusion protein is produced by fusing the nucleic acid sequences of the polypeptides to be joined. Means and methods to clone and express fusion proteins are well-established in the field and available to the skilled person. Fusion proteins of the present disclosure may for instance comprise one or more tags, such as affinity tags, 10 including at least one His-tag, strep-tag, SNAP-tag, HA-tag and the like, or combinations thereof, e.g., an N-terminal His-tag and a C-terminally located strep-tag, or vice versa, epitope tags, fluorescence tags, solubilization tags, synthetic zippers or the like. A cleavable tag that can be removed after production may be used as well. Usually, tags will be located at the N- or C-terminus of a polypeptide not to disturb the function of the folded 15 protein, but in certain cases, a tag may also be located within a polypeptide sequence (e.g., forming a discrete structural epitope not disturbing the fold and function of the polypeptide the tag is inserted in). Further, one or more linker sequences may be used, including a FAS and / or PKS LD, and / or other linkers (including synthetic linkers) or spacers or flexible linkers to connect the different entities of a fusion molecule with each other so that the 20 different entities can exert there function appropriately (e.g., “GA” linkers or spacers of various length building alpha-helical structures may be used, one or more binding or docking domains, one or more dimerization domains, such as leucine zippers). Whenever the present disclosure relates to the percentage of the identity of nucleic acid or amino acid sequences, this identity is determined by a comparison of a sequence of 25 interest, the reference sequence (e.g., a SEQ ID NO as disclosed herein), to another sequence, the query sequence, over the entire length of the reference sequence. Identity is obtained by using the EMBOSS Water Pairwise Sequence Alignments (nucleotide) program or the EMBOSS Water Pairwise Sequence Alignments (protein) program (www.ebi.ac.uk / Tools / psa / emboss_water / ) for amino acid sequences. Those tools 30 provided by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI) for local sequence alignments 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, 1981147 (1):195-197). When conducting an alignment, the default parameters defined by the EMBL-EBI are used. 35 Those parameters are (i) for amino acid sequences: Matrix = BLOSUM62, gap open - 13 - penalty = 10 and gap extend penalty = 0.5 or (ii) for nucleic acid sequences: Matrix = DNAfull, gap open penalty = 10 and gap extend penalty = 0.5. A ”linker domain” or “LD” as used herein refers to both linker sequences upstream and downstream of an acyltransferase within a FAS or PKS that – together – link the 5 acyltransferase to the KS, both sub-parts also being referred to as KS-AT (or KS-MAT) Linker and post-AT Linker. A linker domain as disclosed herein may comprise or consist of both sub-parts from the same FAS or PKS or a linker domain may be hybrid comprising or consisting of a KS-(M)AT Linker and a post-AT Linker, each stemming from a different FAS or PKS. 10 A “negatively charged” amino acid as used herein, refers to aspartate, glutamate, histidine or a non-natural amino acid or amino acid analog carrying a negative net charge at least at certain pH conditions. The terms “aspartate” and “glutamate” are used interchangeably with the terms “aspartic acid” and “glutamic acid”, respectively, herein, irrespective of the protonation state. Notably, histidine can be negatively or positively charged depending on 15 the pH. Further, a neutral charge already at pH ~ 8.0 is possible. Therefore, depending on the pH and in view of the pI, histidine can be qualified as negatively charged residue, but also differently. A “reference position” as used herein, refers to one of the conserved positions (i), (ii), (iii), (iv), (v), (vi), (vii) or (viii) as defined in Tables 1 to Table 5. For an acyltransferase 20 originating, for example, from a murine FAS according to SEQ ID NO: 1, the reference positions (i) to (viii) correspond to the positions D160, T161, F184, R286, K186, K285, R300, A282, and G137 with the numbering based on the sequence of the acyltransferase domain itself (SEQ ID NO: 29), which corresponds to positions D647, T648, F671, R773, K673, K772, R787, A769, and G624 with the numbering based on the sequence of the 25 complete murine FAS (SEQ ID NO: 1). For an acyltransferase originating from a chicken FAS according to SEQ ID NO: 14, as a further example, the reference positions (i) to (viii) correspond to the positions D160, T161, F184, R286, K186, R285, K300, A282, and G137 with the numbering based on the sequence of the acyltransferase domain itself (SEQ ID NO: 42), which corresponds to positions D646, T647, F670, R772, K672, R771, K786, 30 A768 and G623 with the numbering based on the sequence of the complete chicken FAS (SEQ ID NO: 14). When comparing different sequences of acyltransferases according to the present disclosure (e.g. SEQ ID NOs: 29 to 56 or SEQ ID NOs: 134 to 153) numbers of positions corresponding to the same reference position may be identical (when the numbering is based on the acyltransferase itself) or may differ. - 14 - 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 or SEQ ID NOs: 134 to 153. Sequence alignments, such as shown in Table 1 to Table 5, are well-established in the field and the skilled person can easily determine for any given acyltransferase sequence the positions of reference 5 positions (i), (ii), (iii), (iv), (v), (vi), (vii) and / or (viii) within that sequence and further determine whether one or more of the reference positions are present in that sequence. As the structure of the protein fold is well conserved, the skilled person may additionally use structural alignments, for instance based on x-ray cristallography, (cryo-)EM and / or alphafold structures, in combination with sequence alignments in certain cases, such as for 10 sequences that are less well conserved, to determine the reference positions. Those may, for instance, be a threonine, serine, glutamine or asparagine residue at reference position (i), a phenylalanine, arginine, lysine, glutamate or histidine residue at reference position (ii), an arginine, glutamate, aspartate or histidine residue at reference position (iii), a lysine, glutamine or arginine residue at reference position (iv), a lysine or arginine residue at 15 reference position (v), a lysine or arginine residue at reference position (vi), an alanine, serine, valine or glycine residue at reference position (vii) and / or a glycine, alanine, proline or serine residue at reference position (viii). Whenever the present disclosure refers to an “analogous” residue in the context to SEQ ID NO: 29, it refers to the residue present in a different sequence at the position that 20 corresponds to the same reference position. For example, T161 of SEQ ID NO: 14 is an analogous threonine residue to T161 of SEQ ID NO: 29 and R285 of SEQ ID NO 14 is an analogous arginine residue to K285 of SEQ ID NO: 29. While the numbering based on SEQ ID NO: 14 is identical to the numbering based on SEQ ID NO: 29, an analogous residue may not necessarily have the identical number as the respective residue in SEQ ID NO: 29, 25 depending on the individual sequence. A “polyketide synthase” or “PKS” as used herein, refers to a type I PKS, being a large multifunctional protein that has a modular enzymatic structure, in which one module comprises a set of functional enzymatic domains involved in the incorporation of an α- carboxyacyl extender unit into a growing polyketide chain, or a type II PKS comprising 30 discrete monofunctional proteins for polyketide synthesis. A type I PKS module comprises at least three different functional domains: a ketoacyl synthase (KS), which is responsible for catalyzing the Claisen condensation between an extender unit and the growing polyketide chain; an acyltransferase, which selectively binds an extender unit and trans- esterifies the extender onto an acyl carrier protein (ACP); the ACP serves as the acceptor 35 for the extender unit and the growing polyketide chain. A type I PKS may further comprise - 15 - additional domains, such as a ketoacyl reductase (KR), which reduces the ß-ketone to a hydroxyl group, a dehydratase (DH) which removes the hydroxyl group and creates a double bond between the α and ß carbon, an enoyl reductase (ER) which reduces the double bond generated by the DH domain, and a thioesterase (TE) terminating the 5 synthesis process. A type I PKS may be an iterative PKS, catalyzing multiple elongation cycles repeatedly using the same functional domains, or it may be an assembly-line PKS comprising different modules, each of which catalyzes one elongation step (Hertweck 2009, Cogan et al.2021). “Polyketide synthesis” as used herein includes synthesis of diketides and oligoketides. PKS systems comprising at least one altered acyltransferase according 10 to the present invention may consist of a single continuous polypeptide chain or may comprise of two or more polypeptide chains comprising the various PKS domains. In some embodiments, PKS systems comprising an altered acyltransferase according to the present invention may comprise only PKS domains all originating from the same PKS, wherein the at least one altered acyltransferase according to the present invention may 15 also originate from the same PKS or may originate from a FAS or different PKS. In other embodiments, PKS systems comprising at least one altered acyltransferase according to the present invention may comprise PKS domains from different PKS. For example and without limitation, PKS modules from different PKS may be combined to form a synthetic PKS system. 20 The terms “polypeptide” and “protein” are used interchangeably herein. Naturally, protein functionality, including enzymatic activity, is dependent on further factors, such as temperature, pH, salts, and the like and may require further proteins and / or co-factors as it is known to the skilled person. Polypeptides disclosed herein may start with an additional methionine residue resulting from translation of the start codon, or alternatively with a 25 different additional residue, such as valine or leucine resulting from translation of an alternative start codon, likewise polypeptides disclosed herein as beginning with a methionine residue may also be present without said methionine e.g. if used in context of an N-terminal fusion to a tag or other polypeptide. Brief description of the Drawings 30 Figure 1 (Fig.1) shows a cartoon of the structure of a mammalian FAS homodimer with one of the malonyl-acetyl-transferases (MAT) highlighted as an exemplary AT, together with a corresponding cartoon showing the position of the CoA binding channel within a MAT as a cartoon. The numbering of the residues of the active site (S94, H196 and N251) is based on SEQ ID NO: 29. - 16 - Figure 2 (Fig.2) shows the residues of a murine MAT (SEQ ID NO: 29) as an exemplary acyltransferase. Structural sketches of the eight different identified residues are shown in black with the positively charged residues (K186, K285 and R300) being marked with a black “ ”. A structural sketch of the CoA is shown in gray, with the negatively charged 5 phosphate groups being marked with a gray “-“. Pi-stacking between the CoA-Adenine and the residues F184 and R286 are indicated as “π”. H-bonding between residue T161 and the CoA-Adenine is indicated with a dotted line and an “H”. Figure 3 (Fig.3) shows an exemplary scheme of engineering an acyltransferase into an altered acyltransferase having coenzyme specificity. In the top left is a sketch of a KS-LD- 10 AT polypeptide capable of binding CoA. In the top right is a sketch of an altered KS-LD-AT according to the present invention. On the bottom right are examples of using an altered acyltransferase of the present invention. Figure 4 (Fig.4) shows turnover rates for the transfer of malonyl moieties from a non-CoA substrate. Specific transfers from the non-CoA substrate S-Malonyl-N-hexanoylcysteamine 15 to N-(2-hydroxyethyl)-hexanamide were analyzed at fixed substrate and acceptor concentrations. The Y-axis shows enzyme-specific turnover in s-1. Measurements were performed in duplets. Figure 5 (Fig. 5) shows turnover rates of DEBSAT6 variants for the transfer of methylmalonyl moieties from methylmalonyl-CoA (dark gray) or KEZ (light gray) to ACP. 20 The Y-axis shows enzyme-specific turnover in s-1. Measurements were performed in at least triplets. Figure 6 (Fig.6) shows a structural alignment of DEBS AT6 in grey (PDB code: 8g4u) and mouse MAT in black (PDB code: 5my0). The alignment was performed with Pymol (and was specified to the range of the 8 reference positions). 25 Figure 7 (Fig.7) shows the turnover rates in s-1for exemplary non-CoA acyltransferase substrates. Numbers 4 to 12 denote acyl residues according to the respective formula as described herein. Figure 8A (Fig.8A) shows a scheme of PKS module and the corresponding PKS module after introduction of an altered acyltransferase of the present invention. “LD” denotes a 30 Linker Domain according to the present invention and “Sel” denotes a Selectase, i.e. an altered acyltransferase according to the present invention. - 17 - Figure 8B (Fig. 8B) shows the turnover rates in min-1of a CoA and a non-CoA acyltransferase substrate using the PKS module according to Fig.8A. Each left bar shows results of the CoA acyltransferase substrate and each right bar shows results of the non- CoA acyltransferase substrate. 5 Figure 9 (Fig. 9) shows turnover rates of AVESAT variants for the transfer of 2- Methylbutyryl-(“2-MeBu”)-acyl moieties using CoA (dark gray) or KEZ (light gray) coenzyme to ACP. The Y-axis shows enzyme-specific turnover in s-1. Measurements were performed in at least triplets. Figure 10 (Fig. 10) shows turnover rates of RAPSAT6 variants for the transfer of 10 Mehylmalonyl-(“MM”)-acyl moieties using CoA (dark gray) or KEZ (light gray) coenzyme to ACP. The Y-axis shows enzyme-specific turnover in s-1. Measurements were performed in at least triplets. Figure 11 (Fig.11) shows turnover rates of RAPSAT14 variants for the transfer of Malonyl- (“Mal”)-acyl moieties using CoA (dark gray) or KEZ (light gray) coenzyme to ACP. The Y- 15 axis shows enzyme-specific turnover in s-1. Measurements were performed in at least triplets. Figure 12 (Fig.12) shows turnover rates of Nid AT variants for the transfer of Ethylmalonyl -(“EtM”)-acyl moieties using CoA (dark gray) or KEZ (light gray) coenzyme to ACP. The Y- axis shows enzyme-specific turnover in s-1. Measurements were performed in at least 20 triplets. Figure 13 (Fig.13) shows turnover rates of PIKS complete module 6 with AT6 variants for the transfer of MM-acyl moieties using CoA (dark gray) or KEZ (light gray) coenzyme to ACP. The Y-axis shows enzyme-specific turnover in s-1. Measurements were performed in at least triplets. 25 Figure 14 (Fig.14) shows turnover rates of KirCII AT variants for the transfer of EtM-acyl moieties using CoA (dark gray) or KEZ (light gray) coenzyme to ACP. The Y-axis shows enzyme-specific turnover in s-1. Measurements were performed in at least triplets. Figure 15 (Fig.15) shows turnover rates of AVESAT variant B82 for the transfer of the natural CoA substrate (dark gray) and various non-CoA substrates (light gray) to ACP. The - 18 - Y-axis shows enzyme-specific turnover in s-1. Measurements were performed in at least triplets. Figure 16 (Fig.16) shows turnover rates of Nid AT variants for the transfer of the natural CoA substrate (dark gray) and KEZ with MM- (light grey) and EtM-(off-white) acyl moieties 5 to ACP. The Y-axis shows enzyme-specific turnover in s-1. Measurements were performed in at least triplets. Figure 17 (Fig.17) shows turnover rates of C. elegans AT variants for the transfer of Mal- acyl moieties using CoA (dark gray) or KEZ (light gray) coenzyme to ACP. The Y-axis shows enzyme-specific turnover in s-1. Measurements were performed in at least triplets. 10 Detailed description The present invention will now be described in detail based on the detailed description, including non-limiting Examples, the attached drawings and the sequences as provided with the sequence listing. In a first aspect, there is provided a method comprising: (a) providing (i) at least one non- 15 CoA acyltransferase substrate; and (ii) at least one altered acyltransferase ; and (iii) at least one acyl carrier protein (ACP); (b) allowing the at least one acyltransferase to transfer the acyl group of the at least one non-CoA acyltransferase substrate to the at least one ACP; (c) obtaining at least one acyl-ACP; wherein the at least one altered acyltransferase has a CoA-binding pocket that is altered to reduce CoA acyltransferase substrate transfer activity. 20 In one embodiment of the first aspect, step (b) is performed in at least one cell comprising the components (i), (ii), and (iii) of step (a). In one embodiment of the first aspect, step (b) is performed outside of a cell. In one embodiment of the first aspect, step (b) the at least one altered acyltransferase is provided as a purified or partially purified protein or as a lysate comprising the altered 25 acyltransferase, optionally wherein the at least one altered acyltransferase is provided as a fusion protein further comprising at least one linker domain and optionally at least one ketoacyl synthase domain. In a second aspect, there is provided a method for polyketide synthesis, the method comprising: (a) providing (i) at least one non-CoA acyltransferase substrate; and (ii) - 19 - optionally at least one CoA acyltransferase substrate; and (ii) a polyketide synthase (PKS) system, wherein the PKS system comprises at least one altered acyltransferase; (b) allowing polyketide synthesis, wherein the at least one non-CoA acyltransferase substrate, and optionally the at least one CoA acyltransferase substrate, is / are used as a substrate in 5 the polyketide synthesis; (c) obtaining at least one polyketide, and optionally purifying said at least one polyketide; wherein the at least one altered acyltransferase has a CoA-binding pocket that is altered to reduce CoA acyltransferase substrate transfer activity. In one embodiment of the first or second aspect, the at least one altered acyltransferase comprises one or more amino acid exchange(s) at reference position(s) (i) T161 according 10 to SEQ ID NO: 29, or an analogous amino acid, such as a threonine, serine, glutamine or asparagine residue, at said reference position as defined in Table 1, wherein the amino acid is exchanged to alanine, glycine, valine, leucine, isoleucine, tyrosine, phenylalanine, tryptophan, aspartate or glutamate; and / or (ii) F184 according to SEQ ID NO: 29, or an analogous amino acid, such as a phenylalanine, arginine or histidine residue, at said 15 reference position as defined in Table 1, wherein the amino acid is exchanged to alanine, glycine, valine, leucine, isoleucine, methionine, aspartate or glutamate; and / or (iii) R286 according to SEQ ID NO: 29, or an analogous amino acid, such as an arginine or histidine residue, at said reference position as defined in Table 1, wherein the amino acid is exchanged to alanine, glycine, valine, leucine, isoleucine, methionine, aspartate or 20 glutamate; and / or (iv) K186 according to SEQ ID NO: 29, or an analogous amino acid, such as a lysine or arginine residue, at said reference position as defined in Table 1, wherein the amino acid is exchanged to a negatively charged amino acid or to 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 an 25 analogous amino acid, such as a lysine or arginine residue, at said reference position as defined in Table 1, wherein the amino acid is exchanged to a negatively charged amino acid; and / or (vi) R300 according to SEQ ID NO: 29, or an analogous amino acid, such as a lysine or arginine residue, at said reference position as defined in Table 1, wherein the amino acid is exchanged to a negatively charged amino acid, and / or (vii) A282 according 30 to SEQ ID NO: 29, or an analogous amino acid, such as an alanine residue, at said reference position as defined in Table 1, wherein the amino acid is exchanged to valine, leucine, isoleucine, phenylalanine or tyrosine; and / or (viii) G137 according to SEQ ID NO: 29, or an analogous amino acid, such as a glycine residue, at said reference position as defined in Table 1, wherein the amino acid is exchanged to valine, leucine, isoleucine, 35 phenylalanine or tyrosine. - 20 - Wild type (i.e. without comprising the one or more amino acid exchanges of the present invention) FAS and PKS acyltransferases can be polyspecific for multiple different acyl groups or specific for a particular acyl group. However, all of them are specific for CoA- bound substrates. The two different types of specificity, the CoA specificity and the acyl 5 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 exchanges of the present invention can be used to alter these binding properties and alter acyltransferases so that they can bind to substrates covalently bound to moieties other than CoA, including synthetic coenzyme moieties. Acyltransferases having altered substrate 10 specificity according to the present invention are acyltransferases having a reduced ability, preferably essentially no ability, to transfer CoA-bound substrates, while the acyl specificity may be unaltered. In certain embodiments, the altered acyltransferase is an acyl specific altered acyltransferase. In preferred embodiment, the at least one altered acyltransferase is an acyl polyspecific acyltransferase. As the at least one acyltransferase comprises 15 specifically reprogrammed coenzyme-binding properties due to the altered CoA-binding region without the necessity for exchanging amino acid residues involved in the acyl moiety binding, the at least one altered acyltransferase of the present invention can retain the natural polyspecificity regarding the acyl moiety. Retaining the acyl polyspecificity can be of great importance for methods according to the first or second aspect, e.g. for using one 20 acyltransferase for different acyl-moieties as substrates, in particular when using at least two different substrates for polyketide synthesis, fatty acid synthesis and / or non-ribosomal peptide synthesis. The residues at reference positions (i), (ii) and (iii) are crucial residues for the binding pocket of the respective coenzyme (c.f. Fig.2 for MAT as exemplary AT) and interact with 25 the nucleobase portion of the CoA moiety through hydrogen bonding (reference position (i)) or pi-stacking (reference positions (ii) and (iii)). One or more amino acid exchange(s) at reference positions (i), (ii) and / or (iii) to alanine, glycine and / or valine may be used to prevent the H-bonding and / or Pi-stacking. An amino acid exchange at reference position (i) to leucine, isoleucine, tyrosine, phenylalanine, tryptophan, aspartate or glutamate may 30 be used to sterically manipulate and reprogram the binding to coenzyme moieties, wherein an exchange to tyrosine, phenylalanine or tryptophan further may be used to stabilize the protein through pi-stacking. The amino acid exchange(s) at reference position (ii) and / or (iii) to leucine, isoleucine, methionine, aspartate or glutamate may be used to sterically manipulate the binding to coenzyme moieties. - 21 - Lysine or arginine residues at reference positions (iv), (v) and (vi) interact electrostatically with the negatively charged phosphate groups of CoA. Exchanging one or more residues at reference positions (iv), (v) and / or (vi) to one or more negatively charged residue(s) may be used to alter acyltransferases so that they, instead, bind to one or more positively 5 charged chemical groups of coenzyme moieties other than CoA, including synthetic coenzyme moieties. An amino acid exchange at reference position (iv) to amino acid residues that are, at a neutral pH, not positively charged or uncharged, and optionally non-polar, such as alanine, glycine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, 10 cysteine, serine, threonine, histidine, proline, in particular alanine, glycine, valine, leucine, isoleucine or methionine may be used to artificially eliminate the electrostatic interaction at this reference position. Experimental results show that the effect of amino acid exchanges at reference position (iv) are highly additive with amino acid exchanges at reference position, (i), (ii), (iii) and (vi) and suggest that the most important factor in respect to 15 reference position (iv) is the elimination of the naturally occurring positive charge at this position, while the exact nature of the amino acid exchange or the introduction of a negative charge at this position are less relevant than the elimination of the positive charge. An alanine residue at reference position (vii) and a glycine residue at reference position (viii) form structural parts of the coenzyme binding pocket. Exchanging the residues at one 20 and / or both of these positions may be used to manipulate the structural shape of the coenzyme binding pocket through modification of the steric arrangement. Preferred positions for altering an AT of the present invention are reference positions (i), (ii), (iii), (iv), (v) and / or (vi), alone or in combination, even more preferred is a combination of reference positions (ii) and (iii), preferably in combination with at least one further amino 25 acid exchange at at least one other position, or a combination of reference positions (i) and (iv), preferably in combination with at least one further amino acid exchange at at least one other position, or a combination of reference positions (iv) and (vi), preferably in combination with at least one further amino acid exchange at least one other position. In some embodiments of the first or second aspect, an amino acid exchange at reference 30 position (i) is an exchange to leucine, alanine, tryptophan, or valine, preferably to leucine or alanine, more preferably to leucine; and / or wherein an amino acid exchange at reference position (ii) is an exchange to leucine, alanine, glycine, valine, or glutamate, preferably to leucine, alanine or glutamate, more preferably to leucine or alanine; and / or wherein an - 22 - amino acid exchange at reference position (iii) is an exchange to leucine, alanine, glycine, or valine, preferably to leucine or alanine; and / or wherein an amino acid exchange at reference position (iv) is an exchange to a negatively charged amino acid, or to alanine or valine, preferably to glutamate or aspartate; and / or wherein an amino acid exchange at 5 reference position (v) is an exchange to glutamate or aspartate; and / or wherein an amino acid exchange at reference position (vi) is an exchange to a negatively charged amino acid, or to alanine or valine, preferably to glutamate or aspartate; and / or wherein an amino acid exchange at reference position (vii) is an exchange to leucine, isoleucine, phenylalanine or tyrosine; and / or wherein an amino acid exchange at reference position (viii) is an exchange 10 to leucine, isoleucine, phenylalanine or tyrosine, preferably to leucine. In some embodiments of the first or second aspect, an amino acid exchange at reference position (i) is an exchange to leucine, isoleucine, tyrosine, phenylalanine or glycine; and / or wherein an amino acid exchange at reference position (ii) is an exchange to leucine, alanine, isoleucine or methionine; and / or wherein an amino acid exchange at reference 15 position (iii) is an exchange to leucine, isoleucine or methionine; and / or wherein an amino acid exchange at reference position (iv) is an exchange to alanine, glycine, valine, leucine, isoleucine or methionine; and / or wherein an amino acid exchange at reference position (vi) is an exchange to alanine, glycine, valine, leucine, isoleucine or methionine and / or wherein an amino acid exchange at reference position (vii) is an exchange to valine; and / or wherein 20 an amino acid exchange at reference position (viii) is an exchange to valine. In some embodiments of the first or second aspect, the at least one altered acyltransferase comprises amino acid exchanges 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 (iv) and two or more of reference positions of (i), (ii), (iii), (v), (vi), (vii) and / or (viii), 25 optionally wherein the amino acid exchange at reference position (iv) is an amino acid exchange to glutamate or aspartate. In some embodiments of the first or second aspect, the at least one altered acyltransferase comprises amino acid exchanges 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 (i) and 30 two or more of reference positions of (ii), (iii), (iv), (v), (vi), (vii) and / or (viii), optionally wherein the amino acid exchange at reference position (i) is an amino acid exchange to leucine, alanine, tryptophan, or valine, preferably to leucine or alanine, more preferably to leucine. - 23 - In preferred embodiments of the first or second aspect, the at least one altered acyltransferase comprises amino acid exchanges as defined above at reference positions (i), (ii), (iii), (iv), and optionally also at reference position (vi). In some embodiments of the first or second aspect, the at least one altered acyltransferase 5 comprises amino acid exchanges as defined above at reference positions: (i), optionally wherein the amino acid exchange at reference position (i) is an amino acid exchange to leucine, alanine, tryptophan, or valine, preferably to leucine or alanine, more preferably to leucine. In some embodiments of the first or second aspect, the at least one altered acyltransferase 10 further comprises amino acid exchanges as defined above at reference positions: (ii), optionally wherein the amino acid exchange at reference position (ii) is an amino acid exchange to leucine, alanine, glycine, valine, or glutamate, preferably to leucine, alanine or glutamate, more preferably to leucine or alanine; and (iii), optionally wherein the amino acid exchange at reference position (iii) is an amino acid exchange to leucine, alanine, 15 glycine, or valine, preferably to leucine or alanine. The experimental data shows that reference positions IV and Vi are of high relevance in particular for altered acyltransferases originating from a PKS. Therefore, it is preferred that the at least one altered acyltransferase comprises an amino acid exchange as defined herein at reference position (iv) and / or at reference position (vi), in particular for 20 embodiments of altered acyltransferase originating from a PKS.In some embodiments of the first or second aspect, the at least one altered acyltransferase further comprises an amino acid exchange as defined above at reference (iv), optionally wherein the amino acid exchange at reference position (iv) is an amino acid exchange to glutamate or aspartate. In some embodiments of the first or second aspect, the at least one altered acyltransferase 25 further comprises an amino acid exchange as defined above at reference (vi), optionally wherein the amino acid exchange at reference position (vi) is an amino acid exchange to glutamate or aspartate. In some embodiments of the first or second aspect, the at least one altered acyltransferase comprises amino acid exchanges to glutamate at reference positions (iv) and (vi), or amino 30 acid exchanges to aspartate at reference positions (iv) and (vi), or an amino acid exchange to glutamate at reference position (iv) and an amino acid exchange to aspartate at - 24 - reference position (vi), or an amino acid exchange to aspartate at reference position (iv) and an amino acid exchange to glutamate at reference position (vi). In one embodiment of the first or second aspect, the at least one altered acyltransferase comprises an amino acid exchange to aspartate at reference position (i) and an amino acid 5 exchange to glutamate at reference position (ii). In one embodiment of the first aspect, the at least one altered acyltransferase comprises an amino acid exchange to leucine at least at reference positions (i), (ii) and (iii). In one embodiment of the first or second aspect, the at least one altered acyltransferase comprises an amino acid exchange to alanine at reference positions (i), (ii) and (iii), and an 10 amino acid exchange to aspartate or glutamate at reference position (iv). In one embodiment of the first or second aspect, the at least one altered acyltransferase comprises an amino acid exchange to alanine at reference positions (i), (ii) and (iii); and an amino acid exchange to aspartate at reference position (iv) and an amino acid exchange to aspartate at reference position (vi) or an amino acid exchange to aspartate at reference 15 position (iv) and an amino acid exchange to glutamate at reference position (vi) or an amino acid exchange to glutamate at reference position (iv) and an amino acid exchange to aspartate at reference position (vi) or an amino acid exchange to glutamate at reference position (iv) and an amino acid exchange to glutamate at reference position (vi). In one embodiment of the first or second aspect, the at least one altered acyltransferase 20 comprises an amino acid exchange to leucine at reference positions (i), (ii) and (iii); and an amino acid exchange to glutamate or aspartate at reference position (iv). In one embodiment of the first or second aspect, the at least one altered acyltransferase comprises an amino acid exchange to leucine at reference positions (i), (ii) and (iii); and an amino acid exchange to aspartate at reference position (iv) and an amino acid exchange 25 to aspartate at reference position (vi) or an amino acid exchange to aspartate at reference position (iv) and an amino acid exchange to glutamate at reference position (vi) or an amino acid exchange to glutamate at reference position (iv) and an amino acid exchange to aspartate at reference position (vi) or an amino acid exchange to glutamate at reference position (iv) and an amino acid exchange to glutamate at reference position (vi). - 25 - In one embodiment of the first or second aspect, the at least one altered acyltransferase comprises an amino acid exchange to leucine at reference positions (i) and an amino acid exchange to alanine at reference positions (ii) and (iii); and an amino acid exchange to glutamate or aspartate at reference position (iv). 5 In one embodiment of the first or second aspect, the at least one altered acyltransferase comprises an amino acid exchange to leucine at reference positions (i) and an amino acid exchange to alanine at reference positions (ii) and (iii); and an amino acid exchange to aspartate at reference position (iv) and an amino acid exchange to aspartate at reference position (vi) or an amino acid exchange to aspartate at reference position (iv) and an amino 10 acid exchange to glutamate at reference position (vi) or an amino acid exchange to glutamate at reference position (iv) and an amino acid exchange to aspartate at reference position (vi) or an amino acid exchange to glutamate at reference position (iv) and an amino acid exchange to glutamate at reference position (vi). In one embodiment of the first or second aspect, the at least one altered acyltransferase 15 comprises an amino acid exchange to leucine at reference positions (i) and an amino acid exchange to glutamate at reference position (ii) and an amino acid exchange to alanine at reference position (iii); and an amino acid exchange to glutamate or aspartate at reference position (iv). In one embodiment of the first or second aspect, the at least one altered acyltransferase 20 comprises an amino acid exchange to leucine at reference positions (i) and an amino acid exchange to glutamate at reference position (ii) and an amino acid exchange to alanine at reference position (iii); and an amino acid exchange to aspartate at reference position (iv) and an amino acid exchange to aspartate at reference position (vi) or an amino acid exchange to aspartate at reference position (iv) and an amino acid exchange to glutamate 25 at reference position (vi) or an amino acid exchange to glutamate at reference position (iv) and an amino acid exchange to aspartate at reference position (vi) or an amino acid exchange to glutamate at reference position (iv) and an amino acid exchange to glutamate at reference position (vi). In one embodiment of the first or second aspect, the at least one altered acyltransferase 30 comprises an amino acid exchange to alanine at reference positions (i) and an amino acid exchange to glutamate at reference position (ii) and an amino acid exchange to alanine at reference position (iii); and an amino acid exchange to glutamate or aspartate at reference position (iv). - 26 - In one embodiment of the first or second aspect, the at least one altered acyltransferase comprises an amino acid exchange to alanine at reference positions (i) and an amino acid exchange to glutamate at reference position (ii) and an amino acid exchange to alanine at reference position (iii); and an amino acid exchange to aspartate at reference position (iv) 5 and an amino acid exchange to aspartate at reference position (vi) or an amino acid exchange to aspartate at reference position (iv) and an amino acid exchange to glutamate at reference position (vi) or an amino acid exchange to glutamate at reference position (iv) and an amino acid exchange to aspartate at reference position (vi) or an amino acid exchange to glutamate at reference position (iv) and an amino acid exchange to glutamate 10 at reference position (vi). In one embodiment of the first or second aspect, the at least one altered acyltransferase comprises an amino acid exchange to aspartate at reference positions (i) and an amino acid exchange to glutamate at reference position (ii) and an amino acid exchange to alanine at reference position (iii); and an amino acid exchange to glutamate or aspartate at 15 reference position (iv). In one embodiment of the first or second aspect, the at least one altered acyltransferase comprises an amino acid exchange to aspartate at reference positions (i) and an amino acid exchange to glutamate at reference position (ii) and an amino acid exchange to alanine at reference position (iii); and an amino acid exchange to aspartate at reference position 20 (iv) and an amino acid exchange to aspartate at reference position (vi) or an amino acid exchange to aspartate at reference position (iv) and an amino acid exchange to glutamate at reference position (vi) or an amino acid exchange to glutamate at reference position (iv) and an amino acid exchange to aspartate at reference position (vi) or an amino acid exchange to glutamate at reference position (iv) and an amino acid exchange to glutamate 25 at reference position (vi). In one embodiment of the first or second aspect, the at least one altered acyltransferase comprises an amino acid exchange to aspartate at reference positions (i) and an amino acid exchange to alanine at reference position (ii) and an amino acid exchange to alanine at reference position (iii); and an amino acid exchange to glutamate or aspartate at 30 reference position (iv). In one embodiment of the first or second aspect, the at least one altered acyltransferase comprises an amino acid exchange to aspartate at reference positions (i) and an amino acid exchange to alanine at reference position (ii) and an amino acid exchange to alanine - 27 - at reference position (iii); and an amino acid exchange to aspartate at reference position (iv) and an amino acid exchange to aspartate at reference position (vi) or an amino acid exchange to aspartate at reference position (iv) and an amino acid exchange to glutamate at reference position (vi) or an amino acid exchange to glutamate at reference position (iv) 5 and an amino acid exchange to aspartate at reference position (vi) or an amino acid exchange to glutamate at reference position (iv) and an amino acid exchange to glutamate at reference position (vi). In one embodiment of the first or second aspect, the at least one altered acyltransferase comprises an amino acid exchange to alanine at reference positions (i) and an amino acid 10 exchange to glutamate at reference position (ii) and an amino acid exchange to aspartate at reference position (iii); and an amino acid exchange to glutamate or aspartate at reference position (iv). In one embodiment of the first or second aspect, the at least one altered acyltransferase comprises an amino acid exchange to alanine at reference positions (i) and an amino acid 15 exchange to glutamate at reference position (ii) and an amino acid exchange to aspartate at reference position (iii); and an amino acid exchange to aspartate at reference position (iv) and an amino acid exchange to aspartate at reference position (vi) or an amino acid exchange to aspartate at reference position (iv) and an amino acid exchange to glutamate at reference position (vi) or an amino acid exchange to glutamate at reference position (iv) 20 and an amino acid exchange to aspartate at reference position (vi) or an amino acid exchange to glutamate at reference position (iv) and an amino acid exchange to glutamate at reference position (vi). In preferred embodiments of the first or second aspect, the at least one altered acyltransferase is at least one isolated acyltransferase originating from a FAS or PKS. 25 In one embodiment of the first or second aspect, the at least one isolated acyltransferase is a polypeptide or part of a polypeptide comprising or consisting of the acyltransferase, without comprising further parts and / or domains, including a LD, of the FAS or PKS from which it originates. The isolated acyltransferase may be part of a fusion protein. In another embodiment of the first or second aspect, the at least one isolated 30 acyltransferase is part of a polypeptide comprising or consisting of the acyltransferase and a LD, or a part thereof, without comprising further parts and / or domains of the FAS or PKS from which it originates. The isolated acyltransferase may be part of a fusion protein. - 28 - In another embodiment of the first or second aspect, the at least one isolated acyltransferase is part of a polypeptide comprising or consisting of the portion of a FAS or PKS spanning the ketoacyl synthase (KS), the acyltransferase and the LD connecting the KS and the acyltransferase, without comprising further parts and / or domains of the FAS or 5 PKS from which it originates. Such a KS-LD-acyltransferase may be part of a fusion protein. In another embodiment of the first or second aspect, the at least one isolated acyltransferase comprises at least the acyltransferase and the next subsequent ketoacyl synthase (KS) and Acyl carrier protein (ACP) originating from the same PKS or FAS, optionally further comprising the linker regions in between. 10 In another embodiment of the first or second aspect, the at least one isolated acyltransferase is part of a polypeptide comprising or consisting of the portion of a FAS or PKS comprising at least the acyltransferase and the next subsequent ketoacyl synthase (KS) and Acyl carrier protein (ACP), optionally including the linker regions in between, without comprising further parts and / or domains of the FAS or PKS from which it originates. 15 In another embodiment of the first or second aspect, the at least one isolated acyltransferase is part of a polypeptide comprising or consisting of the portion of a FAS or PKS spanning the acyltransferase until and including the next subsequent ketoacyl synthase (KS) and Acyl carrier protein (ACP) and regions in between, without comprising further parts and / or domains of the FAS or PKS from which it originates. 20 In certain embodiments of the first or second aspect, the at least one altered acyltransferase is provided as at least one fusion protein comprising at least one altered acyltransferase according to the present invention, preferably wherein the fusion protein further comprises at least one linker domain and optionally at least one ketoacyl synthase domain. 25 In some embodiments of the first or second aspect, the at least one fusion protein may comprise a tag for purification, for example but not limited to at least one His-tag, step-tag, FLAG-tag, myc-tag, MBP-tag, GST-tag, or a combination thereof. The acyltransferase of the present invention of preferably linked to other functional PKS or FAS parts and / or domains, in particular a ketoacyl synthase domain, via a FAS and / or PKS 30 LD. Of course, the acyltransferase may also be linked to other functional parts and / or - 29 - domains by conventional polypeptide linkers known in the art, such as glycine linkers, GS linkers, PT-linkers or other commonly used linkers. In some embodiments of the first or second aspect, the at least one fusion protein may be a polyketide synthase (PKS), or a part thereof, in which at least one acyltransferase has 5 been replaced by an acyltransferase of the present invention, wherein the PKS portion of the fusion protein may comprise further engineered modifications. In one embodiment of the first or second aspect, the acyltransferase originates from a FAS. In certain embodiments, the AT originates form a FAS of an organism selected from vertebrata, including aves and mammalia, including artiodactyla, perissodactyla, carnivora, 10 primates, rhabditida and rodentia, preferably wherein the acyltransferase originates from a fatty acid synthase comprising or consisting of a wild type amino acid sequence selected from any one of SEQ ID NO: 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. 15 In other embodiments, the acyltransferase originates from a PKS. In certain embodiments, the AT originates form a PKS of an organism selected from Streptomyces venezuelae, Saccharopolyspora erythraea, Streptomyces rapamycinicus, Treptomyces rapamycinicus, Treptomyces caelestis, Caenorhabditis elegans and Streptomyces avermitilis, preferably wherein the acyltransferase originates from a PKS comprising or consisting of a wild type 20 amino acid sequence selected from any one of SEQ ID NO: 225 to 232, 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. In some embodiments of the first or second aspect, the altered acyltransferase comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 57 to 82 or 111 to 133, 25 154 to 165, 171 to 180, 182 to 184, 186 to 188, 190 to 192, 194 to 196, 198 to 200, or 202 to 204, or 206 to 208, or 219 to 221 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. SEQ ID NOs: 57 to 82 and 111 to 154 are exemplary mutants of the mouse MAT. SEQ ID NOs: 154 to 165 are exemplary mutants of 30 Saccharopolyspora erythraea DEBSAT6. - 30 - In one embodiment of the second aspect, the PKS system is a PKS type I system, wherein one, two, three or more PKS modules comprise and / or interact with at least one altered acyltransferase. In one embodiment of the second aspect, the PKS system is a PKS type II system, wherein 5 one, two, three or more PKS modules interact with at least one altered acyltransferase. Usually, the starter unit or the extender unit is a CoA-bound acyl group, such as acetyl- CoA, malonyl-CoA, methylmalonyl-CoA or propionyl-CoA, which are selectively recognized and transferred to the ACP by the acyltransferase. By exchanging the acyltransferase to an altered acyltransferase of the present invention, a PKS system may be reprogrammed 10 to incorporate starter and / or extender units bound to other coenzyme moieties, including synthetic coenzyme moieties. The acyltransferase of a PKS may be replaced by an altered acyltransferase of the present invention through regular cloning strategies available to the skilled person. In modular, assembly-line PKS systems, the acyltransferase of one, or two, or more, or all 15 modules may be replaced by an altered acyltransferase of the present invention, optionally including the adjacent LD, or a part thereof, and / or KS, to produce a reprogrammed PKS system capable of using starter and / or extender units bound by coenzyme moieties other than CoA. In one embodiment of the first or second aspect, the at least one altered acyltransferase is 20 part of at least one multifunctional PKS type I polypeptide, optionally through replacement of at least one of the PKS acyltransferases. In one embodiment of the second aspect, the at least one altered acyltransferase is provided as at least one separate protein, optionally as a fusion protein as defined above, comprising the ketoacyl synthase domain of the FAS or PKS from which the altered 25 acyltransferase originates and / or the linker domain of the FAS or PKS from which the altered acyltransferase originates or a different linker as described herein or known in the art. In one embodiment of the second aspect, the loading domain, the first, second, third, fourth, fifth, six, seventh, eighth, ninth, tenth, eleventh, thirteenth, fourteenths, fifteenth, and / or 30 sixteenth PKS module and / or one or more subsequent PKS modules comprise(s) and / or interact(s) with at least one altered acyltransferase. In a PKS module comprising at least - 31 - one altered acyltransferase, the original acyltransferase of that PKS module may be replaced by an altered acyltransferase. In preferred embodiments, the cell comprises a PKS system in which at least one acyltransferase has been replaced with an altered acyltransferase of the present invention. 5 In certain embodiments, the cell comprises a modular type I PKS system in which the acyltransferase of one, or two, or more, or all PKS modules has been replaced by an altered acyltransferase optionally including the adjacent LD, or a part thereof, and / or KS, of the FAS or PKS from which the altered acyltransferase originates. In another embodiment, replacement of a PKS acyltransferase with an altered 10 acyltransferase may be the replacement of only the original acyltransferase with only the altered acyltransferase. In another embodiment, replacement of a PKS acyltransferase with an altered acyltransferase may be the replacement of the original acyltransferase and the adjacent linker domain of that PKS with the altered acyltransferase and adjacent linker domain of 15 the FAS or PKS from which the altered acyltransferase originates or a different linker as described herein or known in the art. In another embodiment, replacement of a PKS acyltransferase with an altered acyltransferase may be the replacement of the original acyltransferase, the adjacent linker domain and adjacent ketoacyl synthase domain of that PKS with the altered 20 acyltransferase, the ketoacyl synthase domain of the FAS or PKS from which the altered acyltransferase originates and the linker domain of the FAS or PKS from which the altered acyltransferase originates or a different linker as described herein or known in the art. In one embodiment, the first original acyltransferase, the adjacent linker domain and adjacent ketoacyl synthase domain of a triketide lactone synthase or of an engineered 25 triketide lactone synthase, for example, Pik 167 (see Zhang et al., 2023) is replaced with: (i) an altered acyltransferase and the adjacent post AT-Linker; or (ii) an altered acyltransferase the adjacent linker domain and adjacent ketoacyl synthase domain. In certain embodiments, the altered acyltransferase, post-AT linker or linker domain and / or the ketoacyl synthase domain originate from the mouse FAS. In embodiments, where no 30 KS domain is used, a linker domain may be used instead of a post AT linker. In one embodiment, the altered acyltransferase comprises an amino acid exchange to leucine at reference positions (i), (ii) and (iii) and an amino acid exchange to aspartate at reference - 32 - position (iv), optionally being an altered comprising or consisting of an amino acid sequence of any of SEQ ID NO: 122, 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. In one embodiment, the triketide lactone synthase or an 5 engineered triketide lactone synthase, for example, Pik 167, comprises or consists of an of an amino acid sequence of SEQ ID NO: 169, 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. In one embodiment of the second aspect, the at least one altered acyltransferase is 10 provided as a separate polypeptide, optionally as a fusion protein further comprising at least one linker domain and optionally at least one ketoacyl synthase domain. In one embodiment of the second aspect, the polyketide synthesis, or part of the polyketide synthesis, is performed in at least one cell comprising the PKS system or parts thereof. In embodiments of the first or second aspect, relating to performing the method or parts 15 thereof inside at least one cell, the at least one altered acyltransferase may be provided as at least one a nucleic acid encoding the at least one altered acyltransferase or a fusion protein comprising the same. The nucleic acid may be double and / or single stranded DNA and / or RNA, including mRNA. DNA and RNA, including naturally occurring nucleosides / nucleotides, modified and / or 20 non-natural nucleosides / nucleotides and natural and artificial (phosphothioate) linkages of nucleosides / nucleotides. A nucleic acid that is not part of an expression construct or vector may comprise other sequences and / or modification for improving the stability. In certain embodiments, the nucleic acid is comprised by an expression construct or vector comprising at least one nucleic acid of the third aspect. 25 The nucleic acid, expression construct or vector may further comprise sequences for expression in the desired host cell, such as a promoter, including inducible promoters, and / or a transcription terminator operably linked to the nucleic acid encoding the acyltransferase or the fusion protein, a selectable marker, a sequence for replication in a desired host cell, and / or further regulatory sequences, including sequences regulatory for 30 transcription, RNA processing and / or stability, and / or translation. The nucleic acid or expression construct or vector may comprise a cDNA sequence, devoid of introns, encoding the acyltransferase and / or fusion protein of the present invention, e.g. for - 33 - expression in prokaryotic cells. Recombinant protein expression, as well as suitable vectors and expression systems, promoters and further regulatory sequences are well-established for a multitude of cell types. The skilled person is well-aware of the particulars and requirements for recombinant protein expression in different cell types and can easily 5 determine suitable components to choose for expression in the desired host cell. Vector backbones for an expression construct or vector may for example be pET22b, pET300, pET301, pET28b, pCDF, pKC1139, pETDuet or pGEX-6P-1 vector systems, but are not limited thereto. The nucleic acid, expression construct or vector may be codon optimized, wherein the 10 codon optimization may be adapted according to the desired host cell. Codon optimizations for different cell types are well-established in the art and tools for codon optimization are readily available to the skilled person. The nucleic acid, expression construct or vector may be suitable for stable replication and maintenance in the desired host cell. The nucleic acid and / or expression construct or vector 15 may further be suitable for integration into the host genome. In one embodiment of the first or second aspect, at least one nucleic acid encoding the at least one altered acyltransferase or at least one fusion protein comprising the same may be stably integrated into the genome, including the chromosomal / nuclear genome and / or the plastid genome of the at least one cell, e.g. by any conventional genome editing strategy 20 and / or any conventional vector suitable for genome integration. In certain embodiments, the at least one nucleic acid may be integrated into the genome by replacing an endogenous nucleic acid encoding an acyltransferase. In another embodiment of the first or second aspect, at least one endogenous gene of the at least one cell encoding an acyltransferase is mutated to encode an altered 25 acyltransferase of the present invention, wherein the mutation may be performed by any mutation strategy, preferably a SDN-1 (site directed nuclease 1) mutation strategy. In another embodiment of the first or second aspect, at least one nucleic acid and / or at least one expression construct or vector encoding the at least one altered acyltransferase or at least one fusion protein encoding the same is present extrachromosomally in the at 30 least one cell. - 34 - In one embodiment, at least one endogenous acyltransferase of the at least one cell is knocked out, knocked down or otherwise inactivated and complemented with the at least one altered acyltransferase and / or fusion protein comprising the same. In one embodiment of the second aspect, the polyketide synthesis, or part of the polyketide 5 synthesis, is performed outside of a cell. An altered acyltransferase or fusion protein comprising the same of the various embodiments of the first aspect, may be purified using protein purification methods disclosed herein or any other conventional protein purification methods. The at least one altered acyltransferase of the present invention may be expressed in any 10 cellular expression system, established systems include prokaryotic cells, including Escherichia coli strains, such as but not restricted to various different well-established BL21 or BL21(DE3) expression strains as well as derivatives thereof, including for example Rosetta, BAP1 or NiCo21(DE3) strains, other E. coli strains, such as E. coli K12 strain JM83 or other prokaryotic cells such as Bacillus subtilis expression systems, eukaryotic 15 expression systems, for example including Saccharomyces cerevisiae cells, Komagataella phaffii (formerly called Pichia pastoris) cells, Aspergillus niger cells, or immortalized cell lines, such as insect cell expression systems, for example Sf-9 or Sf-21 cells, or mammalian cell lines, such Chinese hamster Ovary (CHO) cells, HEK 293 cells and the like. In one embodiment of the second aspect, the at least one altered acyltransferase is 20 provided as a purified or partially purified protein or as a lysate comprising the altered acyltransferase, optionally wherein the at least one altered acyltransferase is provided as a fusion protein further comprising at least one linker domain and optionally at least one ketoacyl synthase domain. In one embodiment of the second aspect, the method comprises the synthesis of at least 25 two different polyketides and the additional step: (d) forming at least one covalent bond between the at least two different polyketides to obtain at least one combined polyketide. The formation of the at least one covalent bond may, for instance, be done using click chemistry as known in the art. In one embodiment of the first or second aspect, the CoA acyltransferase substrate transfer 30 activity is reduced to ≤50%, ≤40%, or ≤30%, preferably to ≤20% or ≤10%, more preferably to ≤5%, ≤2%, most preferably ≤1%, In one embodiment, the substrate transfer activity is - 35 - measured and compared as Vmax (maximum velocity). In another embodiment, the substrate transfer activity is measured and compared as Kcat / KM (specificity constant). In one embodiment of the first or second aspect, the CoA acyltransferase substrate transfer activity is ≤50%, ≤40%, or ≤30%, preferably ≤20% or ≤10%, more preferably ≤5%, ≤2%, or 5 ≤1% of the transfer activity regarding the at least one non-CoA acyltransferase substrate provided in step (a). In one embodiment, the substrate transfer activity is measured and compared as Vmax (maximum velocity). In another embodiment, the substrate transfer activity is measured and compared as Kcat / KM(specificity constant). In one embodiment of the first or second aspect, wherein the at least one altered 10 acyltransferase has a CoA-binding pocket that is altered to reduce CoA acyltransferase substrate transfer activity and to increase transfer activity for the at least one non-CoA acyltransferase substrate. In one embodiment of the first or second aspect, the at least one altered acyltransferase has a CoA-binding pocket that is altered to transfer the at least one non-CoA 15 acyltransferase substrate at a higher rate than the equivalent CoA acyltransferase substrate with the same acyl group and / or, in case at least one CoA acyltransferase substrate is provided in step (a), the at least one CoA acyltransferase substrate provided in step (a). In one embodiment of the first or second aspect, the at least one non-CoA substrate 20 comprises or consists of (ii) a non-CoA coenzyme; and (i) an acyl group; optionally wherein part (i) and part (ii) are linked via an ester, preferably a thioester. It is important that the non-CoA coenzyme and the carboxy group of the acyl group form a high energy bond, the cleavage of which drives the transfer reaction of the acyl group to the ACP to form acyl - ACP. Preferably the high energy bond is a thioester. Thus, the non-CoA coenzyme 25 preferably has a thiol group to form a thioester with the acyl group. In one embodiment of the first or second aspect, the non-CoA coenzyme has a neutral net charge or a positive net charge. In one embodiment, the positive net charge is a single, two-fold, three-fold or four-fold positive net charge. In one embodiment of the first or second aspect, the non-CoA coenzyme does not comprise 30 a negative charge. - 36 - In one embodiment, the non-CoA coenzyme has a molar mass of ≤800 g / mol. In one embodiment, the non-CoA coenzyme is a peptide. In certain embodiment of the method or parts thereof being performed inside at least one cell, the peptide may be encoded by at least one nucleic acid molecule that is translated inside the at least one cell. 5 In certain embodiments, the peptide binds to the acyl group via an amino acid side chain, preferably via a thiol group of an amino acid side chain, for example the thiol group of cysteine. In one embodiment, the non-CoA coenzyme comprises or consist of a compound according to formula (1): 10 (1). wherein R1 is denotes the acyl group to which the non-CoA coenzyme is bound. The non- CoA coenzyme according to formula (1) is also referred to as “KEZ1” herein. In certain embodiments, the at least one acyltransferase originates from a FAS and the 15 non-CoA coenzyme comprises or consist of a compound according to formula (1) and / or the acyl group is an acyl group according to any one of formulas 4 to 19. In other embodiments, the at least one acyltransferase originates from a PKS and the non- CoA coenzyme comprises or consist of a compound according to formula (1) and / or the acyl group is an acyl group according to any one of formulas 4 to 19. 20 In one embodiment, the non-CoA acyltransferase substrate is provided as a complete preformed substrate. In another embodiment, the non-CoA acyltransferase substrate is provided as non-CoA coenzyme and carboxylic acid as separate molecules, which are reacted to form the at least one non-CoA acyltransferase substrate. In certain embodiment of the method or parts thereof being performed inside at least one cell, the non-CoA 25 coenzyme and carboxylic acid may be introduced as separate molecules into the at least one cell and reacted to form the at least one non-CoA acyltransferase substrate within said at least one cell. - 37 - In one embodiment of the first or second aspect, the acyl group comprises or consist of a malonyl group, an acetyl group, a methylmalonyl group, a propionyl group, or a derivative thereof. Although each acyltransferase has a natural specificity for a certain acyl moiety, it is well 5 known that some acyltransferases also accept other substrates that are, in nature, CoA- bound. Furthermore, the natural specificity of an acyltransferase is rather an apparent specificity, wherein the set of natural acyl moieties may be extended by using synthetic acyl moieties, provided as non-CoA substrates for the altered acyl transferases of the present invention. 10 The non-CoA substrates may be chosen for each acyltransferase and its mutants in the sense that the acyl moiety of the non-CoA acyl-thioester is structurally related to the respective natural acyl moiety of the natural CoA substrate. In general, a non-CoA extender substrate is only used for ATs in elongation modules and non-CoA starter substrates are only used for ATs in the loading modules. 15 As an example, it is well known that DEBS AT6, naturally transferring MM-CoA, cannot except Mal-CoA, but it is able to use EtM-CoA and larger substrates (Englund 2023). That implies that the non-CoA substrates used with the DEBS AT6 and its mutants must feature an alkyl chain, at least a methyl group. As another example, Raps AT14 naturally uses Mal- CoA as a substrate and discriminates against MM-CoA. In this case, non-CoA substrates, 20 which contain a methyl or longer alkyl group are not suitable for this transferase and its mutants. Instead, non-CoA substrates are chosen that contain, for example, halogen atoms in the ^-position instead of one or both hydrogen atoms. As another example, Ans AT8 transfers isobutylmalonyl-CoA. The same principles apply for this transferase and its mutants as for the DEBS AT6, but non-CoA extender substrates featuring longer alkyl25 moieties, like e.g. hexanyl or even branched alkyl chains, like e.g. 2-methylbutylmalonyl- CoA could be transferred more efficiently due to its natural acyl specificity. As a final example, the AVES AT0 (acyltransferase of the loading module) naturally transfers 2- methylbutyryl moieties, but is also capable to transfer other acyl moieties like isobutyryl, acetyl, 3-methylbutyryl, pentanoyl, butyryl and propionyl (Wang 2015). This shows that a 30 variety of non-CoA substrates can be used with this acyltransferase and its mutants and even longer acyl chains, branched acyl chains and functionalized acyl chains including halogenations. It would not make sense to use this transferase with non-CoA elongation substrates like malonyl or methylmalonyl moieties. - 38 - All acyl chains can also be functionalized on each position, which makes this approach so promising, as minimal invasive mutations in the acyltransferase can enable the regioselective chemobiosynthetic synthesis of a library of polyketide derivatives when non- CoA thioesters are employed. 5 In another embodiment of the first or second aspect, the acyl group is an acyl group according to any one of formulas 4 to 19, wherein the asterisk denotes the non-CoA coenzyme: 10 15 20 - 40 - Starter substrates may fulfil the following formula: R1: non-CoA coenzyme, preferably ≤800 g / mol; R2: C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, cycloalkyl, heterocycloalkyl, benzyl, benzyl, heterobenzyl. All R2 may be 5 functionalized at one or more positions with R3. R3: halogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, halogen, aryl, heteroaryl, benzyl, heterobenzyl, benzoyl, heterobenzoyl, -CN, -OCH3, -OR4, -C(=O)R4, -SR4, - S(=O)R4, -S(=O)2R4, -NO2, -NR4R5, -C(=O)OR4, -azide, -propargyl, -O(C=O)R4, - OC(=O)OR4, C(=O)NR4R5, -OC(=O)NR4R5. All R3 may be functionalized at one or more 10 positions with R4. R4: hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, benzyl, heterobenzyl, benzoyl, heterobenzoyl. All R4 may be functionalized at one or more positions with R5. R5: hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, benzyl, 15 heterobenzyl, benzoyl, heterobenzoyl. All R5 may be functionalized at one or more positions with R6. R6: hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, benzyl, heterobenzyl, benzoyl, heterobenzoyl, halogen, propargyl, azide, -CN, -NO2, -OH, -SH, -NH2, -COOH, - C(=O)H, -S(=O)H, -SO2H, -OCH3, SCH3. 20 Extender substrates may fulfil the following formula: - 41 - R1: non-CoA coenzyme, preferably ≤800 g / mol; R3, R4 = Cycloalkyl, heterocycloalkyl, All R3R4 may be functionalized at one or more positions with R4. R3: hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, benzyl, heterobenzyl, benzoyl, heterobenzoyl, halogen, -CN, -OCH3, -OR6, -C(=O)R6, -SR6, -S(=O)R6, - 5 S(=O)2R6, -NO2, -NR6R7, -C(=O)OR6, -azide, -propargyl, -O(C=O)R6, -OC(=O)OR7, C(=O)NR6R7, -OC(=O)NR6R7, All R3 may be functionalized at one or more positions with R4. R4 = as R3 R5: halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, benzyl, heterobenzyl, benzoyl,10 heterobenzoyl, -azide, halogen, -CN, -OCH3, -OR6, -C(=O)R6, -SR6, -S(=O)R6, - S(=O)2R6, -NO2, -NR6R7, -C(=O)OR6, -azid, -propargyl, -O(C=O)R6, -OC(=O)OR7, C(=O)NR6R7, -OC(=O)NR6R7, All R5 may be functionalized at one or more positions with R8. R6: hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, benzyl, 15 heterobenzyl, benzoyl, heterobenzoyl. All R3 may be functionalized at one or more positions with R8. R7: hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, benzyl, heterobenzyl, benzoyl, heterobenzoyl. All R7 may be functionalized at one or more positions with R8. 20 R8: hydrogen, halogen, alkyl, alkenyl, aryl, heteroaryl, benzyl, heterobenzyl, halogen, propargyl, -azid, -CN, -NO2, -OH, -SH, -NH2, -COOH, -C(=O)H, -S(=O)H, -SO2H, -OCH3, SCH3. In one embodiment, the non-CoA acyltransferase substrate is 3-Oxo-3-[[2-[(1- oxohexyl)amino]ethyl]thio]-propanoic acid (N-hexanoylcysteamine). 25 In one embodiment of the first or second aspect, the non-CoA acyltransferase substrate is as defined in the fifth or sixth aspect. In a third aspect, there is provided a use of at least altered acyltransferase as defined in the first or second aspect, wherein the at least one altered acyltransferase originates from - 42 - a PKS; and / or at least one non-CoA acyltransferase substrate as defined in the second aspect, and optionally at least one CoA acyltransferase substrate for polyketide synthesis, fatty acid synthesis, and / or nonribosomal peptide synthesis. In certain embodiments of the third aspect, the polyketide synthesis, fatty acid synthesis 5 and / or non-ribosomal peptide synthesis may be a synthesis of polyketide, and / or fatty acid and / or protein hybrids with metabolites of other biosynthetic synthesis pathways. In one embodiment, synthesis of the third aspect is in vivo synthesis in at least one cell comprising the at least one altered acyltransferase or fusion protein comprising the same as defined in the first or second aspect, further using at least one suitable medium and at 10 least one non-CoA acyltransferase substrate as defined in the first or second aspect, the at least one cell thus representing an engineered production strain. In another embodiment, synthesis of the third aspect is in vitro synthesis using at least one purified or partially purified altered acyltransferase or fusion protein comprising the same as defined in the first or second aspect, at least one non-CoA acyltransferase substrate as 15 defined in the first or second aspect, optionally at least one CoA acyltransferase substrate and further optionally at least one buffer and / or further suitable reaction components. In another embodiment, synthesis of the third aspect is in vitro synthesis using lysate of at least one cell comprising at least one altered acyltransferase or fusion protein comprising the same as defined in the first or second aspect, at least one non-CoA acyltransferase 20 substrate as defined in the first or second aspect, optionally at least one CoA acyltransferase substrate and further optionally at least one buffer and / or further suitable reaction components. In another embodiment, synthesis of the third aspect is in vitro synthesis using at least one in vitro transcription / translation system expressing at least one nucleic acid and / or at least 25 one expression construct or vector encoding the at least one altered acyltransferase or fusion protein comprising the same as defined in the first or second aspect, at least one non-CoA acyltransferase substrate as defined in the first or second aspect, optionally at least one CoA acyltransferase substrate and further optionally at least one buffer and / or further suitable reaction components. 30 In another embodiment, synthesis of the third aspect is in vitro synthesis further using at least one purified or partially purified altered acyltransferase or fusion protein comprising - 43 - the same as defined in the first or second aspect, at least one non-CoA acyltransferase substrate as defined in the first or second aspect, optionally at least one CoA acyltransferase substrate and further, optionally, at least one buffer and / or further suitable reaction components. 5 In another embodiment, synthesis of the third aspect is in vitro synthesis using lysate of at least one cell comprising the at least one altered acyltransferase or fusion protein comprising the same as defined in the first or second aspect, further using at least one non- CoA acyltransferase substrate as defined in the first or second aspect, optionally at least one CoA acyltransferase substrate, at least one buffer and optionally further suitable 10 reaction components. In another embodiment, synthesis of the third aspect is in vitro synthesis using at least one in vitro transcription / translation system expressing at least one nucleic acid and / or at least one expression construct or vector encoding the at least one altered acyltransferase or fusion protein comprising the same as defined in the first or second aspect, at least one 15 non-CoA acyltransferase substrate as defined in the first or second aspect, optionally at least one CoA acyltransferase substrate and further optionally at least one buffer and / or further suitable reaction components. In a fourth aspect, there is provided a kit comprising at least one altered acyltransferase as defined in the first or second aspect; and / or at least one non-CoA acyltransferase substrate 20 as defined in the second aspect; optionally further comprising at least one CoA acyltransferase substrate, and / or at least one container, and / or a set of reagents including buffer and / or medium, and / or means of transfection. A kit will usually comprise all agents, buffers, co-factors etc. necessary to guarantee the functionality of the at least one acyltransferase in an assay of interest. 25 In one embodiment, the kit of the fourth aspect is a kit for in vivo synthesis in at least one cell. In another embodiment, the kit of the fourth aspect comprises at least one purified or partially purified altered acyltransferase of the first or second aspect and / or at least one purified or partially purified fusion protein comprising the same, at least one non-CoA 30 acyltransferase substrate, optionally at least one CoA acyltransferase substrate and further optionally at least one buffer and / or further suitable reaction components. - 44 - In another embodiment, the kit of the fourth aspect comprises lysate of at least one cell, comprising the at least one altered acyltransferase or fusion protein comprising the same as defined in the first or second aspect, the kit further comprising at least one non-CoA acyltransferase substrate as defined in the first or second aspect, optionally at least one 5 CoA acyltransferase substrate, at least one buffer and optionally further suitable reaction components. In another embodiment, the kit of the fourth aspect comprises at least one in vitro transcription / translation system expressing at least one nucleic acid and / or at least one expression construct or vector encoding the at least one altered acyltransferase or fusion 10 protein comprising the same as defined in the first or second aspect, at least one non-CoA acyltransferase substrate as defined in the first or second aspect, optionally at least one CoA acyltransferase substrate and further optionally at least one buffer and / or further suitable reaction components. In a fifth aspect there is provided a non-CoA polyketide substrate comprising or consisting 15 of (i) a non-CoA coenzyme; and (ii) an acyl group; wherein part (i) and part (ii) are linked via a thioester, optionally wherein a) the non-CoA coenzyme has a neutral net charge or a positive net charge, further optionally, wherein the positive net charge is a single, two-fold, three-fold or four-fold positive net charge; and / or b) wherein the non-CoA coenzyme does not comprise a negative charge. 20 In one embodiment of the fifth aspect, the non-CoA coenzyme comprises or consist of a compound according to formula (1): (1), wherein R1 represents the linked acyl group, wherein the acyl group is not a malonyl group. 25 In a sixth aspect there is provided a compound according to formula (1): - 45 - (1), wherein R1 is hydrogen or an acyl group. In one embodiment of the fifth or sixth aspect, the acyl group comprises or consist of a 5 malonyl group, an acetyl group, a methylmalonyl group, a propionyl group, or a derivative thereof. In embodiments of the sixth aspect, wherein R1 is a hydrogen, the compound, being a non- CoA coenzyme without an acyl group, can be provided as part of or in preparation of the methods of the present invention together with at least one acyl group donor, typically a 10 carboxylic acid comprising the desired acyl group, to form at least one non-CoA substrate as described herein. For example and without limitation, the compound of the sixths aspect, wherein R1 is a hydrogen, and at least one acyl group donor may both be provided to at least one cell comprising a PKS system comprising at least one altered acyltransferase of the present invention, so that at least one non-CoA substrate is formed within that cell for 15 in vivo polyketide synthesis. In another embodiment of the fifth or sixth aspect, the acyl group is an acyl group according to formula (2): R2: C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, cycloalkyl, heterocycloalkyl, benzyl, benzyl, 20 heterobenzyl. All R2 may be functionalized at one or more positions with R3. R3: halogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, halogen, aryl, heteroaryl, benzyl, heterobenzyl, benzoyl, heterobenzoyl, -CN, -OCH3, -OR4, -C(=O)R4, -SR4, - - 46 - S(=O)R4, -S(=O)2R4, -NO2, -NR4R5, -C(=O)OR4, -azide, -propargyl, -O(C=O)R4, - OC(=O)OR4, C(=O)NR4R5, -OC(=O)NR4R5. All R3 may be functionalized at one or more positions with R4. R4: hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, benzyl, 5 heterobenzyl, benzoyl, heterobenzoyl. All R4 may be functionalized at one or more positions with R5. R5: hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, benzyl, heterobenzyl, benzoyl, heterobenzoyl. All R5 may be functionalized at one or more positions with R6. 10 R6: hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, benzyl, heterobenzyl, benzoyl, heterobenzoyl, halogen, propargyl, azide, -CN, -NO2, -OH, -SH, -NH2, -COOH, - C(=O)H, -S(=O)H, -SO2H, -OCH3, SCH3. In another embodiment of the fifth or sixth aspect, the acyl group is an acyl group according to formula (3): 15 R3, R4 = Cycloalkyl, heterocycloalkyl, All R3R4 may be functionalized at one or more positions with R4. R3: hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, benzyl, heterobenzyl, benzoyl, heterobenzoyl, halogen, -CN, -OCH3, -OR6, -C(=O)R6, -SR6, -S(=O)R6, - 20 S(=O)2R6, -NO2, -NR6R7, -C(=O)OR6, -azide, -propargyl, -O(C=O)R6, -OC(=O)OR7, C(=O)NR6R7, -OC(=O)NR6R7, All R3 may be functionalized at one or more positions with R4. - 47 - R4 = as R3 R5: halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, benzyl, heterobenzyl, benzoyl, heterobenzoyl, -azide, halogen, -CN, -OCH3, -OR6, -C(=O)R6, -SR6, -S(=O)R6, - S(=O)2R6, -NO2, -NR6R7, -C(=O)OR6, -azid, -propargyl, -O(C=O)R6, -OC(=O)OR7, 5 C(=O)NR6R7, -OC(=O)NR6R7, All R5 may be functionalized at one or more positions with R8. R6: hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, benzyl, heterobenzyl, benzoyl, heterobenzoyl. All R3 may be functionalized at one or more positions with R8. 10 R7: hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, benzyl, heterobenzyl, benzoyl, heterobenzoyl. All R7 may be functionalized at one or more positions with R8. R8: hydrogen, halogen, alkyl, alkenyl, aryl, heteroaryl, benzyl, heterobenzyl, halogen, propargyl, -azid, -CN, -NO2, -OH, -SH, -NH2, -COOH, -C(=O)H, -S(=O)H, -SO2H, -OCH3, 15 SCH3. In another embodiment of the fifth or sixth aspect, the acyl group is an acyl group according to any one of formulas 4 to 19, wherein the asterisk denotes the non-CoA coenzyme: 20 25 - 49 - (17) 5 (19). In a seventh aspect, there is provided a use of at least one compound as defined in the fifth or sixth aspect for polyketide synthesis, fatty acid synthesis, and / or non-ribosomal peptide synthesis. In certain embodiments of the seventh aspect, the polyketide synthesis, fatty acid synthesis 10 and / or non-ribosomal peptide synthesis may be a synthesis of polyketide, and / or fatty acid and / or protein hybrids with metabolites of other biosynthetic synthesis pathways. In one embodiment, synthesis of the seventh aspect is in vivo synthesis in at least one cell comprising and at least one compound as defined in the fifth or sixth aspect as non-CoA acyltransferase substrate(s), optionally at least one altered acyltransferase or fusion 15 protein comprising the same as defined in the first or second aspect, optionally at least one CoA acyltransferase substrate, further using at least one suitable medium, the at least one cell thus representing an engineered production strain. In another embodiment, synthesis of the seventh aspect is in vitro synthesis using at least one compound as defined in the fifth or sixth aspect as non-CoA acyltransferase 20 substrate(s), at least one purified or partially purified altered acyltransferase or fusion protein comprising the same, optionally as defined in the first or second aspect, optionally at least one CoA acyltransferase substrate and further optionally at least one buffer and / or further suitable reaction components. In another embodiment, synthesis of the seventh aspect is in vitro synthesis using at least 25 one compound as defined in the fifth or sixth aspect as non-CoA acyltransferase substrate(s), lysate of at least one cell comprising at least one altered acyltransferase or - 50 - fusion protein comprising the same, optionally as defined in the first or second aspect, optionally at least one CoA acyltransferase substrate and further optionally at least one buffer and / or further suitable reaction components. In another embodiment, synthesis of the seventh aspect is in vitro synthesis using at least 5 one compound as defined in the fifth or sixth aspect as non-CoA acyltransferase substrate(s), at least one in vitro transcription / translation system expressing at least one nucleic acid and / or at least one expression construct or vector encoding the at least one altered acyltransferase or fusion protein comprising the same, optionally as defined in the first or second aspect, optionally at least one CoA acyltransferase substrate and further 10 optionally at least one buffer and / or further suitable reaction components. In another aspect there is provided a triketide lactone synthase or an engineered triketide lactone synthase, for example, a Pik 167, wherein the first original acyltransferase, the adjacent linker domain and adjacent ketoacyl synthase domain is replaced with: (i) an altered acyltransferase and the adjacent post-AT Linker; or with (ii) an altered 15 acyltransferase and the adjacent linker domain and adjacent ketoacyl synthase domain. In certain embodiments, the altered acyltransferase, post-AT linker or linker domain and / or the ketoacyl synthase domain originate from the mouse FAS. In embodiments, where no KS domain is used, a linker domain may be used instead of a post AT linker. In one embodiment, the altered acyltransferase comprises an amino acid exchange to leucine at 20 reference positions (i), (ii) and (iii) and an amino acid exchange to aspartate at reference position (iv), optionally being an altered comprising or consisting of an amino acid sequence of SEQ ID NO: 122, 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. 25 In one embodiment, the triketide lactone synthase or an engineered triketide lactone synthase, for example, a Pik 167, comprises or consists of an of an amino acid sequence of SEQ ID NO: 169, 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. 30 - 51 - Examples Example 1: In silico design for targeted acyltransferase reprogramming Due to their modular nature and their innate variability in acyl backbone reduction, PKS systems are an enticing enzymatic platform to be exploited for synthesizing a plethora of 5 different useful biomolecules, in particular for drug design. A crucial aspect limiting a versatile and effective biotechnological use of PKS is the nature of naturally occurring acyltransferases, which have a relatively high specificity when it comes to the extender subunits (Smith and Tsai 2007). The MAT domains of metazoan FAS enzymes were shown to allow an efficient transfer of different acyl substrates (Rittner et al.2018). 10 However, while a FAS MAT might therefore offer a certain versatility regarding the acyl group of the substrate, both FAS MATs and PKS ATs suffer from a crucial shortcoming: they are fully dependent on CoA-bound substrates. The dependency on Acyl-CoA substrates strongly limited the possibility for artificial biomolecule production using an engineered PKS system for in vivo synthesis. CoA-bound molecules, in particular Acetyl- 15 CoA and Malonyl-CoA, are highly abundant and unavoidable in any in vivo setting, thereby interfering with any possible biosynthesis based on CoA-bound substrates. While the MAT crystal structure is solved (Rittner et al.2020), it has not been envisioned that the crucial activating component of CoA may be replaced, let alone how an acyltransferase may be artificially reprogrammed so that it does not use Acyl-CoA substrates and, instead, 20 specifically accepts subunits bound to different coenzyme moieties, such as synthetic coenzyme moieties. Based on the crystal structure and the position of the CoA binding pocket therein (see Fig.1), it was speculated that target residues that are highly attractive to reprogramming a natural acyltransferase could be identified. However, mutation experiments showed that 25 amino acid exchanges at certain residues do not allow any functional protein expression. Therefore, an iterative testing strategy and protein engineering techniques combined with in silico searches were applied to define acyltransferase mutants that can be manipulated in a way to efficiently block CoA binding and –at the same time – that allow binding of different coenzyme moieties, while still allowing correct overall folding of the protein. 30 Moreover, to offer a high versatility including the choice of acyltransferase protein itself, alignments of FAS amino acid sequences from various genera were performed in addition to the structural analysis. An alignment of 28 different example sequences is shown in Table 1. After extensive analysis, eight different target residues have been identified (see Fig.2) in those sequences showing a certain degree of identity. To this end, various hot - 52 - spots for mutational activities were defined that were tested in the following as further detailed below. To verify the identified hot sports in PKS ATs, four exemplary alignments were performed, grouped by the type of PKS ATs and using the FAS MAT as reference sequence. Correct alignment of the active site residues from the catalytic dyad (BB of a 5 third residue involved) were used to judge the results. Table 1 - FAS ATs (viii) (i) SEQ 1 Mus musculus 621 AAVGLSWEECKQRCPAGVVPACHNSEDTVT SEQ 2 Rattus norvegicus 621 AAVGLSWEECKQRCPPGVVPACHNSEDTVT 10 SEQ 3 Cricetulus griseus 621 AAVGLSWEECKQRCPSGVVPACHNSEDTVT SEQ 4 Homo sapiens 621 AAVGLSWEECKQRCPPGVVPACHNSKDTVT SEQ 5 Macaca mulatta 621 AAVGLSWEECKQRCPPGVVPACHNSKDTVT SEQ 6 Bos taurus 621 AAVGLTWEECKQRCPPGIVPACHNCIDTVT SEQ 7 Capra hircus 620 AAVGLTWEECKQRCPPGIVPACHNSIDTVT 15 SEQ 8 Equus caballus 621 AAVGLSWEECKQRCPPGVVPACHNSKDTVT SEQ 9 Felis catus 761 AAVGLSWEECKQRCPPGVVPACHNSEDTVT SEQ 10 Sus scrofa 621 AAVGLSWEECKQRCPPGIVPACHNSKDTVT SEQ 11 Cuculus canorus 615 AAVGLSWEECKQICPPNVVPACHNSEDTVT SEQ 12 Calypte anna 622 AAVGLTWEECKQQCPPNVVPACHNSEDTVT 20 SEQ 13 Anas platyrhynchos 620 AAVGLTWEECKQRCPPNVVPACHNSEDTVT SEQ 14 Gallus gallus 620 AAVGLTWEECKQRCPPNVVPACHNSEDTVT SEQ 15 Nestor notabilis 615 AAVGLTWEECKQRCPPNVVPACHNSEDTVT SEQ 16 Podiceps cristatus 622 AAVGLTWEECKQCCPPNVVPACHNSEDTVT SEQ 17 Balearica regulorum gibbericeps 624 AAVGLTWEECKQCCPPNVVPACHNSEDTVT 25 SEQ 18 Phaethon lepturus 621 AAVGLTWEECKQCCPPNVVPACHNSEDTVT SEQ 19 Phoenicopterus ruber 246 AAVGLTWEECKQCCPPNVVPACHNSEDTVT SEQ 20 Ooceraea biroi 650 AAVGLSWEDAQKMCPPNVSPACHNSMDSVT SEQ 21 Anopheles funestus 669 AAVGLSWEECKQKLPKDVIPACHNSADSVT SEQ 22 Anopheles darlingi 677 AAVGLSWEDCKQKLPKDVIPACHNSSDSVT 30 SEQ 23 Glossina austeni 780 AAVGLSWEEAHKRLPADCFPACHNSAENCT SEQ 24 Drosophila melanogaster 761 AAVGLSWEDAHSRVPSDCFPVCHNSEDNCT SEQ 25 Brugia malayi 633 AAVGLSWEEAGKRCPEGVIPACHNAADSVT SEQ 26 Caenorhabditis elegans 661 AAVGLTWEQVKEQAPPGVVAACHNGADSVT SEQ 27 Caenorhabditis briggsae 778 AAVGLTWEEVKAQAPPGVVAACHNGADSVT 35 SEQ 28 Caenorhabditis remanei 634 AAVGLTWEEVKAQAPKGVVAACHNGADSVT (ii) (iv) SEQ 1 ISGPQAAVNEFVEQLKQEGVFAKEVRTGGLAFHSYFMEGIAPTLLQALKKVIREPRPRSARWLSTS SEQ 2 ISGPQAAVNEFVEQLKQEGVFAKEVRTGGLAFHSYFMEGIAPTLLQALKKVIREPRPRSARWLSTS SEQ 3 ISGPQAAVSEFVEQLKQEGVFAKEVRTGGLAFHSYFMEGIAPMLLQALKKVIREPRPRSARWLSTS 40 SEQ 4 ISGPQAPVFEFVEQLRKEGVFAKEVRTGGMAFHSYFMEAIAPPLLQELKKVIREPKPRSARWLSTS SEQ 5 ISGPQASVFEFMEQLRKEGVFAKEVRTGGMAFHSYFMEAIAPPLLQALKKVIREPKPRSARWLSTS SEQ 6 ISGPQASMLEFVQQLKQEGVFAKEVRTGGMAFHSYFMDAIAPMLLQQLKKVIREPQPRSPRWLSTS SEQ 7 ISGPQASMLEFVKQLKQEGVFAKEVQTGGMAFHSYFMDAIAPTLLQQLKKVIREPQLRSPRWLSTS SEQ 8 ISGPQAAVSEFVEQLKQEGVFAKEVRTGGLAFHSYFMDSISHTLLQALKKVIREPRPRSVRWLSTS 45 SEQ 9 ISGPQAEVAAFVAELKREGVFAKEVRTGGMAFHSYFMDSIAPTLLQALKKVIREPRPRSARWLSTS SEQ 10 ISGPQAAMSEFLQQLKREDVFVKEVRTGGIAFHSYFMESIAPTLLRQLRKVILDPKPRSKRWLSTS SEQ 11 VSGPLGAVSEFVAKLKKDGVFAKEVRTSGVAFHSHYMASIAPVLLSALKKVIPHPKPRSPRWISTS SEQ 12 ISGPLATVSEFVAKLKKAGVFAKEVRSAGVAFHSHYMASIAPVLLSALKKVIPHPKPRSARWISTS SEQ 13 ISGPLDSVNEFVAKLKKDGVFAKEVRSAGVAFHSYYMASIAPALLSALRKVIPHPKPRSARWISTS 50 SEQ 14 VSGPLDSVSEFVTKLKKDGVFAKEVRRAGVAFHSYYMASIAPALLSALKKVIPHPKPRSARWISTS SEQ 15 VSGPLESVTEFVAKLKKDGVFAKEVRSAGVAYHSHYMASIAPVLLNALKKVIPHPKPRSARWISTS SEQ 16 ISGPLDSVHEFVAKLKKDGVFAKEVRSAGVAFHSYYMASIAPVLLSALKKVIPHPKPRSARWISTS SEQ 17 VSGPLDSVNEFVAKLKKDGVFAKEVRSAGVAFHSYYMASIAPVLLSALKKVIPHPKPRSARWISTS SEQ 18 VSGPLDSVNEFVAKLKKDGVFAKEVRSAGVAFHSYYMASIAPVLLSALKKVIPHPKPRSARWISTS 55 SEQ 19 VSGPLDSVREFVAKLKKDGVFAKEVRSAGVAFHSYYMASIAPVLLSALKKVIPHPKPRSARWISTS SEQ 20 ISGPTKDVLKFVEELKSKNIFAKLVRSSGIAFHSKYIASAGPKLRASLDKIIPNPKQRSAKWISSS - 53 - SEQ 21 ISGPVNSVGKVIADLNAQGIFAKGVKSSGIAFHSRYIADAAPKLRKSLDKIIPNPKNRTPRWISTS SEQ 22 ISGPVASVGKVIADLNAQGIFAKGVKSSGIAFHSRYIADAAPKLRKSLDKIIPNPKPRTQRWISTS SEQ 23 ISGPEESIDAVCQKLTAEGVFARAVKSSGYAFHSKYIADAGPKLRKSLEKVIPNAKNRSPRWISSS SEQ 24 ISGPEASIEALVAKLNAEGVFAKAVNSSGYAFHSKYIAEAGPKLRKSLEKIIPNAKNRTARWISTS 5 SEQ 25 ISGDAEKIKEFVEELKKEDIFGKLVDSSGIPFHSPAMLKVKDKMLKAMRTSVPNPKPRSSRWISTS SEQ 26 ISGDAEGVATFCAQLKEKDIFAKVVDTSGIPFHSPAMLAVQDEMIECMRTAVPEPKPRSSKWISTS SEQ 27 ISGDAEGVASFCAQLKEKEIFAKVVDTSGIPFHSPAMLAVKDEMIESMRTAVPEPKPRSSKWISTS SEQ 28 ISGDAEGVATFCAQLKEKDIFAKVVDTSGIPFHSPAMLAVKDEMIESMRTAVPEPKPRSSKWISTS 10 (vii) (v)(iii) SEQ 1 IPEAQWQSSLARTSSAEYNVNNLVSPVLFQEALWHIPEHAVVLEIAPHALLQAVLKRGVKSSCTII SEQ 2 IPEAQWQSSLARTSSAEYNVNNLVSPVLFQEALWHVPEHAVVLEIAPHALLQAVLKRGVKPSCTII SEQ 3 IPEAQWQSSLARTSSAEYNVNNLVSPVLFQEALWHVPEHAVLLEIAPHALLQAVLKRGVKSSCTII SEQ 4 IPEAQWHSSLARTSSAEYNVNNLVSPVLFQEALWHVPEHAVVLEIAPHALLQAVLKRGLKPSCTII 15 SEQ 5 IPEAQWHSSLARTSSAEYNVNNLVSPVLFQEALCHVPEHAVVLEVAPHALLQAVLKRGLKPGCTII SEQ 6 IPETQWQESLARTFSAEYNVNNLVSPVLFQEALWRVPEDAVVLEIAPHALLQAVLKRGLKSSCTII SEQ 7 IPESQWHESLARTFSAEYNVNNLVSPVLFQEALWHVPENAVVLEIAPHALLQAILKRGLQPSCTII SEQ 8 IPEAQWQSSLARTFSAEYNVNNLVSPVLFQEALCHVPEHAVVLEIAPHALLQAVLKRGLKPSCTIV SEQ 9 IPEAQWQGSLARTFSAEYNVNNLVSPVLFQEALWHVPGDAVVLEIAPHALLQAVLKRGLKSSCTIV 20 SEQ 10 IPEAQWQGSLARTFSAEYSVNNLVSPVLFQEALQHVPAHAVVVEIAPHALLQAVLKRSLESSCTII SEQ 11 IPESQWQSDLARYSSAEYHVNNLVNPVLFHEGLSHVPGNALVVEIAPHALLQAILKRSLKPTCIIL SEQ 12 IPESQWQSDLAKYSSAEYHVNNLVSPVLFHEGLKHIPENAVVVEIAPHALLQAILKRTLKPTCTIL SEQ 13 IPESQWQSDLARNSSAEYYVNNLVSPVLFHEGLKHIPENAVVVEIAPHALLQAILRRSLKPSCTIL SEQ 14 IPESQWQSDLARNSSAEYHVNNLVNPVLFHEGLKHIPENAVVVEIAPHALLQAILRRTLKPTCTIL 25 SEQ 15 IPESQWQSDLARSCSAEYLVNNLVNPVLFHDGLKHVPENAVVVEIAPHALLQAILRRTLKPTCTIL SEQ 16 IPESQWQSDLARNSSAEYHVNNLVNPVLFHEGLKHVPENAVVVEIAPHALLQAILRRTLKPTCTIL SEQ 17 IPESQWQSDLARNSSAEYHVNNLVNPVLFHEGLKHVPENAVVVEIAPHALLQAILRRTLKPTCTIL SEQ 18 IPESQWQSDLARNSSAEYHVNNLVNPVLFHEGLKHIPENAVVVEIAPHALLQAILRRTLKPTCTIL SEQ 19 IPESQWQSDLARNSSAEYHVNNLVNPVLFQEGLKHIPENAVVVEIAPHALLQAILRRALKPTCTIL 30 SEQ 20 IPEAAWGSPLAQVSSSAYHVNNLLSPVLFQEAIAHIPDNAITIEIAPHCLLQAILRRSLPSTVTNV SEQ 21 IPEESWPTPLAQQSSSAYHVNNLLSPVLFSEGLKHVPANAICIEIAPHGLLQAILKRALGKDATNL SEQ 22 IPEESWGTALAQQSSSAYHVNNLLSPVLFAEGLKHVPANAICIEIAPHGLLQAILKRALGKEATNL SEQ 23 IPEVAWNTAIAQQASAAYHVNNLLSPVLFHQALQHVPKNAICIEVAPTGLLQAILKRSLGNETTNL SEQ 24 IPESAWNTPVAKQSSAAYHVNNLLSPVLFHEALQHVPKNAISVEIAPHGLLQAILKRALGPDATNL 35 SEQ 25 IPESNWENELAQMCSADYHTNNAVSPVLFYEALQKIPANAVTIEIAPHCLMHSILRRSLQKTCTNV SEQ 26 IPEDDWESDLAATCSAEYHVHNACSPVLFYEAIQKIPANAVTIEMAPHSLMQAILRRSLQKTVTNV SEQ 27 IPEEDWESDLAATCSAEYHVHNACSPVLFYEALQKIPANAVTIEMAPHSLMQAILRRSLQKTVTNV SEQ 28 IPEEDWESDLAATCSAEYHVHNACSPVLFYEALQKIPANAVTIEMAPHSLMQAILRRSLMKTVTNV 40 (vi) SEQ 1 PLM-KR---DHKDNLEFFLTNLGKVHLTGINVNPNALFPPVEFP--APRGTPLISPHI 834 SEQ 2 PLM-KR---DHKDNLEFFLTNLGKVHLTGIDINPNALFPPVEFP--VPRGTPLISPHI 834 SEQ 3 PLM-KR---DHKDNLEFFLTNLGKVHLTGIDVNPNALFPPVDFP--APRGTPLISPHI 834 SEQ 4 PLM-KK---DHRDNLEFFLAGIGRLHLSGIDANPNALFPPVEFP--APRGTPLISPLI 834 45 SEQ 5 PLM-KK---DHRDNLEFFLTGIGRLHLSGIDANPNALFPPVEFP--APRGTPLISPLI 834 SEQ 6 PLM-KK---DHRDNLEFFLSNVGQLYLTGIDVNPNGLFPPVEFP--APRGTPLISPHI 834 SEQ 7 PLM-KK---DHRDNLEFFLSNVGQLYLTGIDVNPNGLFPPVEFP--APRGTPLISPHI 833 SEQ 8 PLM-KK---DHRDNLEFFLSNVGRLHLMGIDVNPNGLFPPVEFP--APRGTPLISPHI 834 SEQ 9 PLM-KK---DQRDNLEFFLSNVGKLHLLGFDVNPNGLLPPVEFP--VPRGTPLISPHI 974 50 SEQ 10 PLM-KK---DHRDNLEFFLSNVGRLHLAGVSVNPNGLFPPVEFP--APRGTPLISPHX 834 SEQ 11 PLM-KK---EHKNNLEFFLTQLGKFHMSGVNFNGNNLFPRVEYP--VPVETPLISPHI 828 SEQ 12 PLM-KK---DHKNNLEFFLTQAGKIHLTGINVLGNNLFPPVEYP--VPVGTPLISPYI 835 SEQ 13 PLM-KK---DQKNNLEFFLTQAGKIHLTGINVLGNNMFPSVEYP--VPVGTPLISPYI 833 SEQ 14 PLM-KK---DHKNNLEFFLTQTGKIHLTGINVLGNNLFPPVEYP--VPVGTPLISPYI 833 55 SEQ 15 PLM-KK---DQKNNLEFFLTQTGKIHLTGINVLGNNLFPLVEYP--VPVGTPLISPYI 828 SEQ 16 PLM-KK---DHKNNLEFFLTQTGKIHLTGINVLGNNLLPLVEYP--VPVGTPLISPYI 835 SEQ 17 PLM-KK---EHKNNLEFFLTQTGKIHLTGINVLGNNLFPLVEYP--VPVGTPLISPYI 837 SEQ 18 PLM-KK---EHKNNLEFFLTQTGKIHLTGINVLGNNLFPLVEYP--VPVGTPLISPYI 834 SEQ 19 PLM-KK---EHKNNLEFFLTQTGKIHLTGINVLGNNLFPLVEYP--VPVGTPLISPYI 459 60 SEQ 20 SLH-MR---DHTNNLAFLLSNIGKLYMAGAQPNISKLYPPVSFP--VGRGTPMIGPLV 863 SEQ 21 SLM-KR---DHANNMIFLLSNLGKLYAAGVQPQVQKLYRPITYP--VGRGTPMLNSLV 882 SEQ 22 SLM-KR---DHDNNLIFLLSNLGKLYAAGAQPQVQKLYPPITYP--VGRGTPMLNSLV 890 SEQ 23 SLI-KR---DYENNPEFFLASIGKLYAAGAQPQIMTLSKPISYP--VGRGTPMLGCKV 993 - 54 - SEQ 24 SLV-KR---GHENNVEFFLTNVGKLFAAGAQPQVLTLVRPISYP--VGRGTPMLNSKV 974 SEQ 25 GLINMK---EKDRELESFLQALGKIYQTGITIHIEALYPAIQYP--VPIGTPMISPMW 847 SEQ 26 GLM-NRPKSENDDELESFLGSLGKIYQAGVNIQITELYPGGQYKGVVPKGTPMIGPMW 879 SEQ 27 GLM-NKPKSENDNELEGFLGSLGKIYQAGVNIQISELYPGGQYKGVVPKGTPMIGPMW 996 5 SEQ 28 GLM-NKPKSENDDELESFLGSLGKIYQAGVNIQISELYPGGQYKGVVPKGTPMIGPMW 852 Catalytic positions are indicated as underlined, reference positions are indicated as bold with numbering of the reference positions shown on top. Table 2 - PKS specific cisATs 10 SEQ 29 NKRPLWFICSGMGTQWRGMGLSLMR-LDSFRESILRSDEAVKP-LGVKVSDLLLSTDE------- SEQ 134 ----AVFVFPGQGWQWAGMAVDLLDTSPVFAAALRECADALEPHLDFEVIPFLRAEAARREQDAA SEQ 135 ----SVFVFPGQGAQWEGMARELLP-VPVFAESIAECDAVLSEVAGFSVSEVLEPRPD------A SEQ 136 ----VVFLFPGQGSQWAGMGAELLSSSPVFAGKIRACDESMAPMQDWKVSDVLRQAPG------A SEQ 137 ----PVLVFPGQGAQWVGMARDLLESSEVFAESMSRCAEALSPHTDWKLLDVVRGDGG------P 15 SEQ 138 ----VAMVFPGQGAQWQGMARDLLRESQVFADSIRDCERALAPHVDWSLTDLLS---G------A SEQ 139 ----VVFVFPGQGAQWEGMARGLLS-VPVFAESIAECDAVLSEVAGFSASEVLEQRPD------A SEQ 140 ----VVFVFPGQGWQWLGMGSALRDSSIVFAERMAECAPALREFVDWDLFTVLDD---------P SEQ 141 ----AVFVFPGQGSQRAGMGEELAAAFPVFARIHQQVWDLLDV--------------------PD SEQ 142 ----VAFLFDGQGTQRLGMGKELYDSYPAFARAWDTVSAGFDKHLDHSLTDVCFGEGGST---TA 20 SEQ 143 ----AVLVFPGQGAQWAGMGVELAQASPVFAARLEECFGEIRRWVDWDPAQVLAD---------A SEQ 144 ----TAFLYSGQGAQHPGMGRQLYETFPTFATALNNTLDALDPHLDHPLRDIMWAPPHTP---QA SEQ 29 RTFDDIVHAFVSLTAIQIALIDLLTSVGLKPDGIIGHSLGEVACGYADGCLSQREAVLAAYWRGQ SEQ 134 LSTERVDVVQPVMFAVMVSLASMWRAHGVEPAAVIGHSQGEIAAACVAGALSLDDAARVVALRSR 25 SEQ 135 PSLERVDVVQPVLFAVMVSLARLWRACGAVPSAVIGHSQGEIAAAVVAGALSLEDGMRVVARRSR SEQ 136 PGLDRVDVVQPVLFAVMVSLAELWRSYGVEPAAVVGHSQGEIAAAHVAGALTLEDAAKLVVGRSR SEQ 137 DPHERVDVLQPVLFSIMVSLAELWRAHGVTPAAVVGHSQGEIAAAHVAGALSLEAAAKVVALRSQ SEQ 138 RPLDRVDVVQPALFAVMVSLAALWRSHGVEPAAVVGHSQGEIAAAHVAGALTLEDAAKLVAVRSR SEQ 139 PSLERVDVVQPVLFSVMVSLARLWGACGVSPSAVIGHSQGEIAAAVVAGVLSLEDGVRVVALRAK 30 SEQ 140 AVVDRVDVVQPASWRMMVSLAAVWQAAGVRPDAVIGHSQGEIAAACVAGAVSMRDAARIVTLRSE SEQ 141 LEVNETGYAQPALFAMQVALFGLLESWGVRPDAVIGHSVGELAAAYVSGVWSLEDACTLVSARAR SEQ 142 GLVDDTLYAQAGIFAMEAALFGLLEDWGVRPDFVAGHSIGEATAAYASGMLSLENVTTLIVARGR SEQ 143 DALESIERLQPVAFAVGVALAALWESVGVRPAAVVGHSQGEVAAACVAGVLSLADAVRVVVLRSR SEQ 144 HLLNQTTYTQPALFALQTALTHLLTSFGIHPHYLAGHSLGEITAAHTAGILTLTDAATLITQRAH 35 (viii) (i) SEQ 29 CIKDAHLPPGSMAAVGLSWEECKQR-----CPAGVVPACHNSEDTVTISGPQAAVNEFVEQLKQ SEQ 134 VIA-TMPGNKGMASIAAPAGEVRAR-----IGDRVEIAAVNGPRSVVVAGDSDELDRLVASCTT SEQ 135 AVR-AVAGRGSMLSVRGGRSDVEKLLADDSWTGRLEVAAVNGPDAVVVAGDAQAAREFLEYCEG 40 SEQ 136 LMR-SLSGEGGMAAVALGEAAVRER--LRPWQDRLSVAAVNGPRSVVVSGEPGALRAFSEDCAA SEQ 137 VLR-ELDDQGGMVSVGASRDELETV--LARWDGRVAVAAVNGPGTSVVAGPTAELDEFFAEAEA SEQ 138 VLR-RLGGQGGMASFGLGTEQAAER--IGRFAGALSIASVNGPRSVVVAGESGPLDELIAECEA SEQ 139 ALR-ALAGKGGMVSLAAPGERARAL--IAPWEDRISVAAVNSPSSVVVSGDPEALAELVARCED SEQ 140 AIARGLAGRGAMASVALPAQDVELV-------DGAWIAAHNGPASTVIAGTPEAVDHVLTAHEA 45 SEQ 141 LMQ-ALPAGGVMVAVPVSEDEARAV-----LGEGVEIAAVNGPSSVVLSGDEAAVLQAAEGL-- SEQ 142 ALR-TTPPGAM-VALRAGEEEVREF--LSRTGAALDLAAVNSPEAVVVSGEPEPVADFEAAWTA SEQ 143 LFAAELWGRGAIAAVGLPAEVVRER--IASLGGGLEVSADNGPASCAVAGPGQVLEEFVERLRG SEQ 144 LMQ-TLPETGAMTAINATPEEITPH-----LTPHTAIAAINSPTSTVISGNAQDIQHITHHFTT 50 (ii) (iv) SEQ 29 EGVFAKEVRTGGLAFHSYFMEGIAPTLLQALKKVIREPRPRSARWLSTSIPEAQWQSSLARTSSA SEQ 134 ECIRAKRLAV-DYASHSSHVETIRDALHAELGEDFH-PLPGFVPFFSTVTG--RWTQ-PDEL-DA SEQ 135 VGIRARAIPV-DYASHTAHVEPVRDELVQALA-GIT-PRRAEVPFFSTLTG--DFLD-GTEL-DA SEQ 136 EGIRVRDIDV-DYASHSPQIERVREELLETTG-DIA-PRPARVTFHSTVES--RSMD-GTEL-DA 55 SEQ 137 REMKPRRIAV-RYASHSPEVARIEDRLAAELG-TIT-AVRGSVPLHSTVTG--EVID-TSAM-DA SEQ 138 EGITARRIPV-DYASHSPQVESLREELLTELA-GIS-PVSADVALYSTTTG--QPID-TATM-DT SEQ 139 EGVRAKTLPV-DYASHSRHVEEIRETILADLD-GIS-ARRAAIPLYSTLHG--ERRD-GADM-GP SEQ 140 RGVRVRRITV-DYASHTPHVELIRDELLDITS-DSS-SQAPVVPWLSTVDG--SWVD--SPL-DV SEQ 141 --GKWTRLAT-SHAFHSARMEPMLEEFRAVAE-GLT-YRTPQVSMA----------V-GDQVTTA - 55 - SEQ 142 SGREARKLKV-RHAFHSRHVEAVLDEFRTALE-SLK-FRAPALPVVSTVTG--RLID-QDEMGTP SEQ 143 EGVRARVIAT-TVASHSQMVEPLREQLLEMLG-PIT-PEAGRVPVYSTVTG--GVLA-GSEL-GA SEQ 144 LGRKTRPLTV-SHAFHSPLLDPILTPLNTLAH-TLT-HHPAHTPLITNTT-----AT-PTHTLTP 5 (vii) (v)(iii) (vi) SEQ 29 EYNVNNLVSPVLFQEALWHIPEH--AVVLEIAPHALLQAVLKRGV------KSSCTIIPLMKRDH SEQ 134 GYWYRNLRRTVRFADAVRALAEQGYRTFLEVSAHPILTAAIEEIGD---GSGADLSAIHSLRRGD SEQ 135 GYWYRNLRHPVEFHSAVQALTDQGYATFIEVSPHPVLASSVQETLDDA---ESDAAVLGTLERDA SEQ 136 RYWYRNLRETVRFADAVTRLAESGYDAFIEVSPHPVVVQAVEEAVEEADG-AEDAVVVGSLHRDG 10 SEQ 137 SYWYRNLRRPVLFEQAVRGLVEQGFDTFVEVSPHPVLLMAVEETAEHA---GAEVTCVPTLRREQ SEQ 138 AYWYANLREQVRFQDATRQLAEAGFDAFVEVSPHPVLTVGIEATLDSALPADAGACVVGTLRRDR SEQ 139 RYWYDNLRSQVRFDEAVSAAVADGHATFVEMSPHPVLTAAVQEIAAD-------AVAIGSLHRDT SEQ 140 EYWYRNLREPVGFHPAVGQLQAEGDTVFVEVSASPVLLQAMDD----------DVVTVATLRRDD SEQ 141 EYWVRQVRDTVRFGEQVASYED---AVFVELGADRSLARLVDG----VAMLHGDHEIQAAIG--- 15 SEQ 142 EYWLRQVRRPVRFQDAVRELAEQGVGTFVEVGPSGALAS--AG----VECLGGDASFHAVLRPRS SEQ 143 EYWFANARRPVDFQGAVRALLADGRTAFIEVSPHPVLTMAVQDILDAT---GTSGVAVGSLRRGE SEQ 144 THWAHHARQPVQFANTITHLHHAGVTTYLEIGPDTTLTAIAPH----NLPPTTHTTFTPTLRKNT SEQ 29 KDNLEFFLTNLGKVHLTG- 20 SEQ 134 G-SLADFGEALSRAFAAGV SEQ 135 G-DADRFLTALADAHTRGV SEQ 136 G-DLSAFLRSMATAHVSGV SEQ 137 S-GPHEFLRNLLRAHVHGV SEQ 138 G-GLADFHTALGEAYAQGV 25 SEQ 139 A-E-EHLIAELARAHVHGV SEQ 140 G-DATRMLTALAQAYVHG- SEQ 141 ---------ALAHLYVNG- SEQ 142 P-EDVCLMTAIAELHAGG- SEQ 143 G-GPDRFMRSAGEAFTAG- 30 SEQ 144 P-EPDTLLTAIATTHTHN- Catalytic positions are indicated as underlined, reference positions are indicated as bold with numbering of the reference positions shown on top. SEQ ID NO: 29: mouse MAT, SEQ ID NO: 134: DEBS1AT1, SEQ ID NO: 135: DEBS1AT2, SEQ ID NO: 136: DEBS2AT3, 35 SEQ ID NO: 137: DEBS2AT4, SEQ ID NO: 138: DEBS3AT5, SEQ ID NO: 139: DEBS3AT6, SEQ ID NO: 140: RAPS3AT14, SEQ ID NO: 141: RAPS3AT12, SEQ ID NO: 142: RAPS2AT6, SEQ ID NO: 143: FluAAT1, SEQ ID NO: 144: FluAAT2. Table 3 - PKS cisATs with unusual specificities SEQ 29 NKRPLWFICSGMGTQWRGMGLSLMR-LDSFRESILRSDEAVKPL-GVKVSDLLLSTDER------ 40 SEQ 145 ----LAFLFTGQGAQTPGMGRGLCAAWPAFREAFDRCVALFDRELDRPLCEVMWAEPGSA--ESL SEQ 146 ----PVMVFPGQGGQWVGMGARLLDESPVFAARIAECEQALSAYVDWSLTDVLRGDG--S----- SEQ 147 ----VVFVFPGQGSQWAGMAEGLLERSGAFRSAADSCDAALRPYLGWSVLSVLRGEPDAP----- SEQ 149 ----AVFVFPGHGAQWPGMARRLFDDFPVFRESVLQCADAFAEFVDWSLLDVLRDEEGAP----- SEQ 150 ----VAFLFSGFGSESVGMGRELYETEPAFREAMDRCADLLAPHLPRRLTDVLYPARDAAGGAAA 45 SEQ 148 ----VAFVFPGQGSQWLGMGAELLASSSVFAAAMAECDAALGDYVGWSVIDVIRQDPAAP----- SEQ 29 TFDDIVHAFVSLTAIQIALIDLLTSVGLKPDGIIGHSLGEVACGYADGCLSQREAVLAAYWRGQC SEQ 145 LLDQTAFTQPALFTVEYALTALWRSWGVEPELVAGHSAGELVAACVAGVFSLEDGVRLVAARGRL SEQ 146 ELARIDVVQPVLWAVMVALAAVWADQGIEPAAVVGHSQGEIAAACVVGAISLDEAARIVAVRSVL 50 SEQ 147 SLDRVDVVQPVLFTMMVSLAAVWRALGVEPAAVVGHSQGEIAAAHVAGALSLDDSARIVALRSRA SEQ 149 PLHRVDVVQPALFTMMVSLAALWRSYGVEPSAVVGHSQGEIAAAYVAGALDLRDAARIVATRGKA SEQ 150 SLGDLSYAQPALFALEYCLAELWKSWGITPSAVVGHSLGECVAACVAGVFSLEDALTLVAARGRL SEQ 148 DPNLIEVVQPSLFAVHVSLAALWQHVGVRPAAVVGHSQGEIAAAVVSGALSLSDGARVIVARSAL 55 (viii) (i) (ii) SEQ 29 IKDAHLPPGSMAAVGLSWEECKQRCP---AGVVPACHNSEDTVTISGPQAAVNEFVEQLKQEGVF - 56 - SEQ 145 M-QGLSAGGAMVSLGAPEAEVAAAVAPHAAWVSIAAVNGPEQVVIAGVEQAVQAIAAGFAARGVR SEQ 146 L-RQLSGRGGMASLGMGQEQAADLID-GHPGVVVAAVNGPSSTVISGPPEGIAAVVADAQERGLR SEQ 147 W-LGLAGKGGMVAVPMPAEELRPRLVTWGDRLAVAAVNSPGSCAVAGDPEALAELVALLTGEGVH SEQ 149 W-LTLAGTGGMASVALPRAEAAERLRPFGHRLDIAAVNDPRSVTVAGDLDALEEFLTGLETEGVR 5 SEQ 150 M-ESLAGEGETFLVSADEATVRRVIAS--DPVSIGSINGPANIVISGAPAGVKSVVERLSQEGIE SEQ 148 LAEELLGKGAMAWIGTSADDVEDRLAQWADRLSVAGRNSPRAVTVVGETEALHELVAGCEADGIR (iv) SEQ 29 AKEVRTGGLAFHSYFMEGIAPTLLQALKKVIREPRPRSAR--WLSTSIPEAQWQSSLARTSSAEY 10 SEQ 145 TKRL-HVSHASHSPLMEPMLEEFGRVA----ASVTYRRPSVSLVSNLSGK----VVTDELSAPGY SEQ 146 ARAV-ASDVAGHGPQLDAILDQLTEGL----AGIRPAATDVAFYSTVTAGH---LTDTTELDTAY SEQ 147 ARPIPGVDTAGHSPQVDALRAHLLEVL----APVAPRPADIPFYSTVTGG----LLDGTELDATY SEQ 149 VRRVRQIVGAGHTAHVDALRDQLIETL----APTAPRSAPIAFCSTVTGG----LLDTAGLDHHY SEQ 150 VKKL-DVRRAAHSPLMDPMLEAFGKVA----RSIRYARPTIDLVANLTGE----VAGEEIATPEY 15 SEQ 148 TRIV-GSSVASHCAQIEPLRDRLLAMF----DEVTPRAARVPFYSSVTGT----VIDTTGMDAEY (vii) (v)(iii) (vi) SEQ 29 NVNNLVSPVLFQEALWHIPE---HAVVLEIAPHALLQAVLKRGVKS---SCTIIPLMKRDHKDNL SEQ 145 WVRHVREAVRFADGVKALHEA-GAGTFLEVGPKPTLLGLLPACLPEA--EPTLLASLRAGREEAA 20 SEQ 146 WVRNVRRTVRFADTIDALLAD-GYRLFIEVSPHPVLNLALEGLIERAAVPATVVPTLRRDHGDTT SEQ 147 WYRNMREPVEFERATRALIAD-GHDVFLETSPHPMLAVALEQTVTDAGTDAAVLGTLRRRHGGPR SEQ 149 WYRNARRTVLFEQAVRTLAEQ-GYGPFLEISAHPMFTVAVQETLEDAGVGAAVLATLRRDEGGPD SEQ 150 WCRQIRETVRMSACLRTLHDALGFEVFLELGPSPALVWNGMQCVPKR--SGAWIASLRPGRPDRA SEQ 148 WYRNAREPVDLEAAVRALLAD-GYAFFVELSAHPVLTVPVQETAEAVGADVAAVGSLRRDDGGPR 25 SEQ 29 EFFLTNLGKVHLTG SEQ 145 GV-LEALGRLWAAG SEQ 146 QL-ARAAAHAFAAG SEQ 147 AL-ALAVCRAFAHG 30 SEQ 149 RF-LRAAAEAHTAG SEQ 150 QI-LAALASLYANG SEQ 148 RF-LTSMAEGFVRG Catalytic positions are indicated as underlined, reference positions are indicated as bold 35 with numbering of the reference positions shown on top. SEQ ID NO: 29: mouse MAT, SEQ ID NO: 145: EpoDAT4, SEQ ID NO: 146: MonAIVAT5, SEQ ID NO: 147: NidA3AT5, SEQ ID NO: 148: DivDAT6, SEQ ID NO: 149: SfaIAT13 SEQ ID NO: 150: LeuAAT2. Table 4 - PKS loading cisATs SEQ 29 NKRPLWFICSGMGTQWRGMGLSLMRL-DSFRESILRSDEAVKPLGVKVSDLLLS-T-DERTFDDI 40 SEQ 151 ----VVFVFPGQGPQWPGMGRELLDASDVFRESVRACEAAFAPYVDWSVEQVLRDSPDAPGLDRV SEQ 152 ----VVFVFPGQGAQWAGMAGELLGESRVFAAAMDACARAFEPVTDWTLAQVLDSPE---QSRRV SEQ 29 VHAFVSLTAIQIALIDLLTSVGLKPDGIIGHSLGEVACGYADGCLSQREAV-LAAYWRGQCIKDA SEQ 151 DVVQPTLFAVMISLAALWRSQGVEPCAVLGHSLGEIAAAHVSGGLSLADAARVVTLWSQAQTT-- 45 SEQ 152 EVVQPALFAVQTSLAALWRSFGVTPDAVVGHSIGELAAAHVCGAAGAADAARAAALWSREMIP-- (viii) (i) (ii) SEQ 29 HLPPGSMAAVGLSWEECK-------QRCPAGVVPACHNSEDTVTISGPQAAVNEFVEQLKQEGVF SEQ 151 LAGTGALVSVAATPDELLPRIAPWTEDNPARLAVAAVNGPRSTVVSGAREAVADLVADLTAAQVR 50 SEQ 152 LVGNGDMAAVALSADEIEPRIARWDDD----VVLAGVNGPRSVLLTGSPEPVARRVQELSAEGVR (iv) SEQ 29 AKEVRTGGLAFHSYFMEGIAPTLLQALKKVIREPRPRSARWLSTSIPEAQWQSSL------ARTS SEQ 151 TRMIPV-DVPAHSPLMYAIEERVVSGLL------------PITPRPSRIPFHSSVTGGRLDTREL 55 SEQ 152 AQVINV-SMAAHSAQVDDIAEGMRSALA------------WFAPGGSEVPFYASLTGGAVDTREL - 57 - (vii) (v)(iii) SEQ 29 SAEYNVNNLVSPVLFQEALWHIPE--HAVVLEIAPHALLQAVLKRGVK--------SSCTIIPLM SEQ 151 DAAYWYRNMSSTVRFEPAARLLLQQGPKTFVEMSPHPVLTMGLQELAPDLGDTTGTADTVIMGTL SEQ 152 VADYWRRSFRLPVRFDEAIRSALEVGPGTFVEASPHPVLAAALQQTLDAEGS-----SAAVVPTL 5 (vi) SEQ 29 KRDHKDNLEFFLTNLGKVHLTG- SEQ 151 RRG-QGTLDHFLTSLAQLRGHG- SEQ 152 QRG-QGGMRRFLLAAAQAFTGGV 10 Catalytic positions are indicated as underlined, reference positions are indicated as bold with numbering of the reference positions shown on top. SEQ ID NO: 29: mouse MAT, SEQ ID NO: 151: AVES1AT0, SEQ ID NO: 152: DEBS1AT0. 15 Table 5 - PKS transAT SEQ 29 NKRPLWFICSGMGTQWRGMGLSLMRLDSFRES-ILRSDEAVKPLG-VKVSD-LLLSTDERTFDDI SEQ 153 ----LVFVFAGQGAQWDGMGLELLDTEPVFGAALRRCDERVRELAGFSVIQQLRAGPAMSRLGEI 20 SEQ 29 VHAFVSLTAIQIALIDLLTSVGLKPDGIIGHSLGEVACGYADGCLSQREAVLAAYWRGQCIKDAH SEQ 153 DVLQPTMVSLQIALVALWRSWRVEPDAVTGHSMGEISAGYAAGALTLDDALLIACRRSALLRRI- (viii) (i) (ii) (iv) SEQ 29 LPPGSMAAVGLSWEECKQRC---PAGVVPACHNSEDTVTISGPQAAVNEFVEQLKQEGVFAKEVR 25 SEQ 153 AGRGALATTELSPEAAHALAASSGGRICVAGENSPRSTVLAGDTATLTALVEDLDRRGVYCRMVR SEQ 29 TGGLAFHSYFMEGIAPTLLQALKKVIREPRPRSARWLSTSIPEAQWQSSLARTSSAEYNVNNLVS SEQ 153 G-TVASHSHYVDELRDDLAGALRPLSPV--PSRVPFYSTVTAAPV----PGTDLGPAYWMRNLRE 30 (vii) (v)(iii) (vi) SEQ 29 PVLFQEALWHI--PEHAVVLEIAPHALLQAVLKRGVKSSC---TIIPLMKRDHKDNLEFFLTNLG SEQ 153 PVRLAAATGRLAEDGHEIFVEVSTHPVLLSSLRQTLESAGRPGEVLPSGRRR--TERRAMLSSLG SEQ 29 KVHLTG 35 SEQ 153 TLFTYG Catalytic positions are indicated as underlined, reference positions are indicated as bold with numbering of the reference positions shown on top. SEQ ID NO: 29: mouse MAT, SEQ ID NO: 153: KirCII. 40 Example 2: Cloning and expression of altered acyltransferases The basic plasmid encoding altered murine acyltransferases was prepared from a synthetic DNA fragment, containing a KS-LD-AT fragment with an inactive KS domain including Strep- and His-tags and was cloned into a pET22b vector using the restriction enzymes NdeI and XhoI, or with the in-Fusion Snap Assembly Master Mix (Takara). Mutations in the 45 eight different positions corresponding to the reference positions according to the present invention were introduced by PCR using primers harboring the nucleotide variations. The plasmid encoding for the ACP was prepared similarly. An additional ribosome binding site (RBS) and another gene encoding for the 4’-phosphopantetheinyl transferase (PPT) Sfp - 58 - was integrated into the same plasmid. The synthetic gene for the ACP was ordered with its native DNA sequence and codon optimized for E. coli. The basic plasmid encoding altered DEBS module 6 acyltransferase (DEBS AT6) was prepared from genomic DNA of Saccharopolyspora erythraea (NRRL2338), containing a 5 KS-LD-AT fragment with an inactive KS domain including His-tags and was cloned into a pET22b vector using the restriction enzymes NdeI and NheI, or with the in-Fusion Snap Assembly Master Mix (Takara). Mutations in five different positions corresponding to the reference positions according to the present invention were introduced by PCR using primers harboring the nucleotide variations. The plasmid encoding for the ACP was 10 prepared similarly. An additional ribosome binding site (RBS) and another gene encoding for the 4’-phosphopantetheinyl transferase superfamily protein from Streptomyces platensis was integrated into the same plasmid. The synthetic gene for the ACP and the PPT was ordered with its native DNA sequence and codon optimized for E. coli. The basic plasmid encoding for Nid AT5 (txid36816 NRRL:2418) and Ans AT8 15 (Streptomyces sp. CNH-189) were ordered as synthetic DNA and the codons were optimized for E. coli using the algorithm from Thermofisher. The primary sequence was received from (Englund, E. et al.) and cloned into a pEZ22b vector, containing a KS-LD-AT fragment including a C-terminal His-Tag using the in-Fusion Snap Assembly Master Mix (Takara). The KS-LD fragment originates from DEBS M6 with an inactive KS domain. 20 Mutations in five different positions corresponding to the reference positions according to the present invention were introduced by PCR using primers harboring the nucleotide variations. The basic plasmid encoding for the Caenorhabditis elegans (C. elegans) FAS was ordered as synthetic DNA and the codons were optimized for E. coli using the algorithm from 25 Thermofisher. The synthetic DNA was cloned into a pEZ22b vector, containing a N-terminal twin-Strep Tag and a C-terminal His-Tag using the in-Fusion Snap Assembly Master Mix (Takara). Mutations in five different positions corresponding to the reference positions according to the present invention were introduced by PCR using primers harboring the nucleotide variations. 30 The basic plasmid encoding for the avermectin AT0 (AVES AT0, acyltransferase from the loading module) originating from Streptomyces avermitilis was ordered as synthetic DNA and the codons were optimized for E. coli using the algorithm from Thermofisher. The synthetic DNA was cloned into a pEZ22b vector, containing a N-terminal twin-Strep Tag - 59 - and a C-terminal His-Tag using the in-Fusion Snap Assembly Master Mix (Takara). Mutations in four different positions corresponding to the reference positions according to the present invention were introduced by PCR using primers harboring the nucleotide variations. 5 The basic plasmid encoding KirCII-AT (AM746336) was prepared from genomic DNA of Streptomyces collinus (DSM 40733; CP006259.1) into a pEZ22b vector including a C- terminal His-Tag using the in-Fusion Snap Assembly Master Mix (Takara). Mutations in five different positions corresponding to the reference positions according to the present invention were introduced by PCR using primers harboring the nucleotide variations. 10 The basic plasmids for RapsAT14 and RapsAT6 were prepared from genomic DNA of Streptomyces rapamycinicus DSM 41530 (QYCY01000001) into a pEZ22b vector, containing a KS-LD-AT fragment including a N-terminal twin-Strep Tag and a C-terminal His-Tag using the in-Fusion Snap Assembly Master Mix (Takara). Mutations in five different positions corresponding to the reference positions according to the present invention were 15 introduced by PCR using primers harboring the nucleotide variations The basic plasmid for piksAIV was prepared from genomic DNA of Streptomyces venezuelae (LN881739, DSM41110) into a pEZ22b vector, containing a C-terminal His- Tag using the in-Fusion Snap Assembly Master Mix (Takara). Mutations in three of the different positions corresponding to the reference positions according to the present 20 invention were introduced by PCR using primers harboring the nucleotide variations. The basic plasmid for DEBS3 was prepared from genomic DNA Saccharopolyspora erythraea (NRRL2338), into a pCDF vector, containing a C-terminal His-Tag using the in-Fusion Snap Assembly Master Mix (Takara). Mutations in four of the different positions corresponding to the reference positions according to the present invention were 25 introduced by PCR using primers harboring the nucleotide variations. The chimeric proteins consisting of parts of Piks LDD-M1 as well as Piks M5 were cloned from Streptomyces venezuelae (LN881739, DSM41110) into a pCDF vector, containing a N-terminal His-Tag using the in-Fusion Snap Assembly Master Mix (Takara). The PCR mixtures were treated with Dpn1 prior to DNA purification using electrophoresis. 30 DNA was stained and illuminated with Sybr Green DNA Dye (Carl Roth). Appropriate fragments were isolated with a scalpel and DNA was purified with the GeneJet Gel - 60 - Extraction kit (ThermoFisher). The fragments were transferred to Stellar™ Competent Cells (Takara) chemical competent cells for ligation and amplification of the targeted plasmids. The plasmids were isolated from cells using a standard commercial plasmid purification kit. The plasmids encoding the MAT, DEBS AT6 or altered acyltransferases were expressed 5 according to the literature (Rittner et al.2018; Chen et al.2006). Briefly, the plasmids were transformed into BL21(DE3) chemical competent cells (Pfeifer et al.2001). Transformed cells were directly transferred to a pre-culture of 5 mL LB-medium containing ampicillin as a selection marker, which was grown over night at 37 °C in an incubation shaker. The pre- culture was used to inoculate a 100-250 mL main-culture or 2 L culture (ACPs) in 2xYT or 10 TB medium in a 500 mL or 5 L Erlenmeyer flask. The cultures were grown for 2 hours at 37 °C and then the expression system was induced by adding 250 µM IPTG and were grown for another 16 hours at 20 °C (16 °C for AVES AT variants). The cells were lysed enzymatically in lysis buffer or centrifuged, re-suspended in lysis buffer containing DNaseI and lysed by sonification (Hielscher UP200t, 43% amplitude, 5 minutes, on / off time 15 20 seconds). After centrifugation, the soluble fraction was purified by Ni-NTA (ThermoFisher) and Strep affinity chromatography (strep-tactin sepharose, IBA Lifesciences GmbH), if a Strep-tag was present, following the standard protocol. The DEBS AT6 variants and ACPs were purified by size-exclusion chromatography (ROTI Dex-25, carl-Roth) instead of Strep affinity chromatography as a second chromatographic 20 purification. Purified proteins were frozen in aliquots in liquid nitrogen and stored at -80 °C. Protein concentrations were determined by their absorbance at 280 nm. The absorbance was recorded on a NanoDrop OneC(ThermoFisher) and converted to concentration using the extinction coefficient of the respective protein, calculated from their primary sequence without the N-terminal methionine in Benchling. 25 List of Sequences used herein if not indicated elsewhere: Complete PKS sequences PiksAIV SEQ ID NO: 211 DEBS3 SEQ ID NO: 212 RapB SEQ ID NO: 213 30 rapC SEQ ID NO: 214 NidA3 SEQ ID NO: 215 C. elegans FASn-1 SEQ ID NO: 26 AveA1 SEQ ID NO: 217 PKS ATs and their variants (interchangeably also referred to as mutants) 35 B11 to B19 in that order SEQ ID NOs: 154 to 162 - 61 - B37 to B39 in that order SEQ ID NOs: 163 to 165 B45 to B54 in that order SEQ ID NOs: 171 to 180 KirCII SEQ ID NO: 181 (in addition to SEQ ID NO 153) B75 to B77 in that order SEQ ID NOs: 182 to 184 5 RapsAT6 SEQ ID NO: 185 B66 to B68 in that order SEQ ID NOs: 186 to 188 RapsAT14 SEQ ID NO: 140 B69 to B71 in that order SEQ ID NOs: 190 to 192 Nid-AT5 SEQ ID NO: 193 10 B63 to B65 in that order SEQ ID NOs: 194 to 196 Ans-AT8 SEQ ID NO: 197 B60 to B62 in that order SEQ ID NOs: 198 to 200 C. elegans FASn-1 SEQ ID NO: 201 B72 to B72 in that order SEQ ID NOs: 202 to 204 15 pikAIV AT6 SEQ ID NO: 205 F95 to F97 in that order SEQ ID NOs: 206 to 208 F16 SEQ ID NO: 209 F84 SEQ ID NO: 210 AVES SEQ ID NO: 218 20 B80 to B82 in that order SEQ ID NOs: 219 to 221 Example 3: Kinetic analysis of altered acyltransferases using MM-CoA An enzyme-coupled assay was performed to analyze the transfer of acyl-moieties from CoA to the ACP. Four solutions were prepared as 4x concentrated stocks in the assay 25 buffer (50 mM sodium phosphate buffer, 10 % (v / v) glycerol, 1 mM EDTA, pH 7.6). Solution 1 contained the murine acyltransferase at the concentration range of 0.01 µM-10 µM and the acyltransferase DEBS AT6 at the concentration range of 0.8 µM-3.2 µM, depending on the transfer kinetics to achieve good signal to noise ratios. Solution 2 contained the enzyme-coupled system including 8 mM α-ketoglutaric acid, 1.6 mM NAD+, 1.6 mM TPP 30 and 20 mU / 100 µL αKGDH. Solution 3 contained the methylmalonyl (MM)-CoA at concentrations between 0.1 µM and 1,000 µM or at a fixed concentration of 200 µM for the DEBS AT6 and solution 4 contained the ACP from murine FAS as a standalone protein at 240 µM or the ACP6 from DEBS as a standalone protein at 264 µM.5 µL of Solutions 1-3 were pipetted into a 384-well Small Volume HiBase Microplates (Greiner Bio-one) and 35 mixed manually. The reactions were started by adding solution 4 with the dispenser system of the microplate reader (ClarioStar, BMG labtech) or an electronic multichannel pipette - 62 - (ThermoScientific, E1-ClipTip). The reaction progress was recorded using the following settings: 348-20 nm; emission: 476-20 nm; gain: 1500; focal height: 11.9 mm; flashes: 17; orbital averaging: off. Calibration was performed with reduced Nicotinamide adenine dinucleotide (NADH) under assay conditions. 5 Results of the methylmalonyl transfer of DEBS AT6 and variants thereof from MM-CoA (50 µM) to ACP6 (66 µM) are shown in Figure 5. Data show the success of the engineering approach as most of the mutants have reduced, drastically reduced or even abolished transfer for MM-CoA. Kinetic data for exemplary FAS AT mutants is shown in Table 6. 10 Table 6 (WT)29* none 0.62 4.000 6.5E+06(WT)29 none 0.53 1.297 2.5E+06A0157 T161A 1.47 0.051 3.5E+04A0258 T161Y 38.78 0.428 1.1E+04A0359 T161A F184A R286A 3.41 0.021 6.2E+03A04 60 T161A F184A K186A R286A 7.24 0.013 1.8E+03A0561 K186E 14.16 0.101 7.2E+03A06- A282W Not determinedA0762 K186D 18.63 0.093 5.0E+03A0863 K285D 0.11 0.133 1.2E+06A1065 R300D 5.06 0.582 1.2E+05A1166 R300E 3.33 0.834 2.5E+05A1267 K186D R300D 66.23 0.019 2.9E+02A1368 K186D R300E 685.98 0.190 2.8E+02A1469 K186E R300D 218.27 0.051 2.3E+02A1570 K186E R300E 58.39 0.030 5.2E+02Listed SEQ ID NOs refer to corresponding acyltransferase sequences. Mutations are indicated in reference to the wild type MAT sequence SEQ ID NO: 29. *Comparative data from Rittner et al.2018 Example 4: Validation of altered acyltransferase activity 15 To verify the re-engineered mutant activity, having a substrate specificity altered from natural CoA substrates to non-CoA substrates, two alternative methods to quantify specific turnover rates for non-CoA substrates were established. Such non-CoA substrates are not detectable by the enzyme-coupled assay. We used a thiol-sensitive fluorophore (ThioGlo4; - 63 - Yi et al.2009) to quantify the released free thiol groups from the substrate S-Malonyl-N- hexanoylcysteamine or S-Methylmalonyl-N-hexanoylcysteamine after the enzyme-specific transfer of the malonyl moiety to a non-thiol acceptor or the methylmalonyl moiety to DEBS ACP6. 5 Briefly, three solutions were prepared in either a twofold concentration or as 4x concentrated stocks in the assay buffer. Solution 1 contained the murine acyltransferase variants at concentrations between 50-500 nM depending on their purified yields and were diluted twofold in the assay. Solution 2 contained a fixed concentration of the substrate S- Malonyl-N-hexanoylcysteamine at 200 µM and solution 3 contained the acceptor N-(2- 10 hydroxyethyl)-hexanamide at 20 mM. 10 µL of Solutions 1 and 5 µL of Solution 2 were pipetted into a 384-well Small Volume HiBase Microplates (Greiner Bio-one). The reactions were started by adding 5 µL of solution 4 with a spichannel pipette (ClipTip) and incubated for 10 min at RT. The reactions were stopped by adding 2.5 µL 3 M HCl, neutralized by adding 2.5 µL 4 M NaOH and 10 µL of the reaction mixture was transferred to new wells. 15 Free thiols were detected by adding 10 µL of 120 µM ThioGlo in the buffer 250 mM potassium phosphate, 1 mM EDTA and 10 % glycerol, mixing and fluorescence detection at the microplate reader (ClarioStar, BMG labtech) using the following settings: 400-20 nm; emission: 465-20 nm; gain: 700; focal height: 12.5 mm; flashes: 20; orbital averaging: off. Wells not containing any enzyme, but the substrate and acceptor in the mentioned 20 concentrations served as the background and reflect non-enzyme specific transacylation. Results of the malonyl transfer from the non-CoA substrate are shown in Figure 4. Alternatively, three solutions were prepared in either a twofold concentration or as 4x concentrated stocks in the assay buffer (50 mM sodium phosphate buffer, 10 % (v / v) glycerol, 1 mM EDTA, pH 7.6). Only solution 3 contained additionally 0.1-1 mM 25 TCEP(Tris(2-carboxyethyl)phosphine hydrochloride). Solution 1 contained the DEBS AT6 acyltransferase variants at 2-3.6 µM and was diluted fourfold in the assay. Solution 2 contained the acceptor DEBS3 ACP6 at 120 µM and was diluted twofold in the assay. Solution 3 contained a fixed concentration of the substrate S-Methylmalonyl-N- hexanoylcysteamine at 200 µM and was diluted fourfold in the assay.12.5 µL of Solutions 30 1 and 25 µL of solution 3 were pipetted into a 96-well plate and then centrifuged for 1 min at 1000 rpm. The reactions were started by adding 12.5 µL of Solution 2 and mixing with the electronic multichannel pipette (ThermoScientific, E1-ClipTip) and incubated for 10 min at RT. The reactions were stopped by adding 50 µL acetonitrile (ACN). The reaction mixture was mixed and 10 µL of brine (saturated NaCl solution) was added. Then again 50 µL ACN - 64 - was added, and the mixture was mixed. After phase separation the lower phase was discarded by removing 110 µL with the electronic multichannel pipette. Then 10 µL of the remaining liquid was transferred to a 384-well Small Volume HiBase Microplates (Greiner Bio-one). The free thiols were detected by adding 10 µL freshly prepared 200 µM ThioGlo 5 in the assay buffer. The reaction mixture was mixed, and the fluorescence was detected at the microplate reader (ClarioStar, BMG labtech) directly and after 5 min of incubation. The following setting were used: 400-20 nm; emission: 465-20 nm; gain: 700; focal height: 12.5 mm; flashes: 20; orbital averaging: off. Wells not containing any enzyme, but the substrate and acceptor in the mentioned concentrations served as the background and were 10 subtracted from the measurements. Results of the methylmalonyl transfer from the non-CoA substrate (50 µM) to DEBS ACP6 (60 µM) are shown in Figure 5. Data show that the goal of the engineering approach was achieved for the DEBS AT6 domain. Some variants have excellent ratios between slow turnover rates for methylmalonyl moieties provided by CoA esters and fast turnover rates 15 for acyl moieties provided by synthetic non-CoA esters without affecting the protein fold. Example 5: Kinetic analysis of altered acyltransferases from various origins using 2-MeBu-CoA, Mal-CoA, MM-CoA and EtM-CoA The same assay as described in example 3 was used for a set of acyltransferases and their respective mutants. Changes in solution 1 solution 3 and solution 4 are stated in Table 7. 20 Like in example 3 solution 1 contained the acyltransferase and their respective mutants. Results for the transfer of acyl moieties from CoA substrates to ACP as stated in Table 7 are shown in Figures 9 to 14. Data show that the goal of the engineering approach was achieved for all ATs, by reducing transfer activity for the respective acyl-CoA Table 7 - 65 - Example 6: Validation of altered acyltransferases from various origins using non- CoA substrates with the 2-MeBu, Mal-, MM- and EtM-acyl moieties To demonstrate the universality of the engineering approach of the present invention, 5 different acyltransferases were chosen from various origins with differing specificities. To validate the shifted specificity of the altered acyltransferases towards non-CoA substrates, the assay described in example 4 (second part) was used to demonstrate the transfer of acyl moieties from non-CoA substrates to ACPs. Changes in solution 1 solution 2 and solution 3 are stated in the Table 8. Like in example 4, solution 1 contained the 10 acyltransferase and their respective mutants. Table 8 - 66 - Results for the transfer of acyl moieties from non-CoA substrates to ACP, as stated in Table 8, are shown in Figures 9-14. Data show that the goal of the engineering approach was achieved for all AT domains proving the general applicability of the herein presented 5 invention. Already with a minimal set of mutations, variants could be generated for all examples, which have the intended properties of accepting acyl moieties from synthetic non-CoA substrates, but almost not from CoA-esters. The engineering was very fast and predictable following our rules for mutating the reference positions and the skilled persons can therefore easily implement this strategy for their targets of interest. 10 Example 7: Exploiting natural acyl specificities of various acyltransferases to transfer synthetic non-CoA substrates Turnover rates for the transfer of various synthetic non-CoA substrates to ACP (DEBSACP6) were determined for variant B82 of the Aves-AT0 loading domain to compare with the remaining rate of this variant to transfer the 2-MeBu-moiety from CoA to ACP 15 (DEBSACP6). Assay conditions were as described in example 4 (second part) and example 6. The non-CoA substrates were S-2-Methylbutyryl-N-hexanoylcysteamine (2- MeBu-), S-2-Propionyl-N-hexanoylcysteamine (Pr-), S-2-Butyryl-N-hexanoylcysteamine (Bu-), S-2-Hexanoyl-N-hexanoylcysteamine (Hx-), S-2-Benzoyl-N-hexanoylcysteamine (Bz-) and S-2-Phenylacetyl-N-hexanoylcysteamine (PhAc-) as solution 3 containing 20 200 µM of the non-CoA substrate. The data are shown in Figure 15 and demonstrate that all acyl moieties from non-CoA substrates, except S-2-Phenylacetyl-N- hexanoylcysteamine, are transferred faster than the “natural” 2-MeBu-moiety from a CoA- ester, which can be exploited when a polyketide shall be derivatized at the position originating from the starter substrate. 25 To demonstrate that the DEBS AT6 domain can be engineered with the present invention to transfer synthetic fluorinated non-CoA substrates much faster than the natural substrate - 67 - MM-CoA, select variants from example 3 were investigated to transfer S- Fluoromethylmalonyl-N-hexanoylcysteamine to the ACP (DEBS ACP6). This approach is especially useful, when a regioselective insertion of a fluorine atom is intended into a polyketide scaffold to improve chemical and pharmaceutical properties. The assay was 5 performed as described in example 4 (second part) with the same conditions and S- Fluoromethylmalonyl-N-hexanoylcysteamine, as solution 3 with a concentration of 200 µM. Finally, to demonstrate the power of the herein described engineering approach, variants of Nid-AT5 were analyzed with the non-CoA substrate S-Methylmalonyl-N- hexanoylcysteamine and the turnover rate of the transfer of the acyl moiety was compared 10 to the turnover rate of the ethylmalonyl moiety from a CoA substrate. This is especially challenging as the Nid-AT5 domain has been evolved by nature to not accept methylmalonyl moieties, which are present in the cell where the niddamycin biosynthesis takes place. The assay was performed as described in example 4 (second part) with the same conditions and S-Methylmalonyl-N-hexanoylcysteamine, as solution 3 with a 15 concentration of 200 µM. The data are shown in Figure 16 and demonstrate that variants B63 and B64 indeed transfer methylmalonyl moieties faster from non-CoA substrates than ethylmalonyl moieties from CoA. Although, the measured transfer rate is relatively low, it confirms the feasibility of the approach. This paves the way to utilize these variants for the transfer of various other extender moieties from synthetic non-CoA substrates and exploit 20 the general polyspecificity of the domain, which has been described in literature by e.g. Englund et al., regardless of the presence of any CoA substrates in the production strain. (Englund 2023) Example 8: Chemobiosynthetic derivatization of triketide lactones in-vitro using AT mutants 25 It is well established that triketide lactones can be produced from two elongation reactions with lactonization in two modular systems (including a substrate loading mechanism), e.g. in an engineered assembly line from the pikromycin synthase (Miyazawa et al, 2021, Zhang et al 2023). By mutation of one AT domain, e.g. the module 6 AT, according to the present invention, it is possible to derivatize regioselectively the triketide lactone product when30 using non-CoA acyl-thioester in the presence of the natural extender CoA substrate MM- CoA. This is possible without further tedious mutagenesis approaches or domain swapping, which may destabilize the protein fold, by exploiting the ability of all ATs to transfer a certain spectrum of acyl-moieties, which e.g. can be observed in Englund et al.. (Englund 2023). The reaction can be tracked by measuring the consumption of Nicotinamide adenine - 68 - dinucleotide phosphate (NADPH) photometrically, as the first module contains a ketoreductase domain. Briefly, 1-6 µM of the enzyme containing the Loading didomain of pikromycin and the first extender module (KEZ0F16 or KEZ0F84) and 1-6 µM of the enzyme containing the Piks 5 module 6 with an AT6 variant (KEZ0F95 or KEZ0F96 or KEZ0F97) were combined with 60 µM NADPH, 100-300 µM MM-CoA and optionally 100-300 µM of a non-CoA acyl- thioester like e.g. S-Fluoromethylmalonyl-N-hexanoylcysteamine or S-Ethylmalonyl-N- hexanoylcysteamine. NADPH consumption was measured fluorometrically with the microplate reader (ClarioStar, BMG labtech) to track the turnover rate of the enzymes. The 10 reaction progress was recorded using the following settings: 348-20 nm; emission: 476-20 nm; gain: 1300; focal height: 12.1 mm; flashes: 50; orbital averaging: off. Calibration was performed with reduced Nicotinamide adenine dinucleotide phosphate (NADPH) under assay conditions. Example 9: Production of triketide lactone derivatives in-vitro using AT variants 15 Similar to example 8, triketide lactone formation can be analyzed by HPLC-MS analysis. In a competitive reaction set-up using MM-CoA and non-CoA acyl-thioesters, the introduction of either the methyl moiety from MM-CoA or an acyl moiety from the non-CoA substrate can be determined by the mass information. The reaction contains the same material as example 8, but the concentrations can be20 adjusted to the following concentrations to produce more triketide lactone (derivatives): 4- 15 µM of the enzyme containing the Loading didomain of pikromycin and the first extender module (KEZ0F16 or KEZ0F84), 4-15 µM of the enzyme containing the Piks module 6 with an AT6 variant (KEZ0F95 or KEZ0F96 or KEZ0F97), 500-4000 µM NADPH, 1500- 20000 µM MM-CoA and 1500-20000 µM of a non-CoA acyl-thioester like e.g. S- 25 Fluoromethylmalonyl-N-hexanoylcysteamine or S-Ethylmalonyl-N-hexanoylcysteamine. The reaction mixtures were acidified with 3 M HCl to pH 3 and extracted two to three times with the equal amount of ethyl acetate (EE). The organic solvent was evaporated under reduced pressure and the extract was analyzed by HPLC-MS for the masses of the triketide lactone corresponding to the introduction of methyl, fluoromethyl and / or ethyl moieties. The 30 product can optionally be purified using HPLC or other chromatography strategies like normal phase chromatography using silica gel. - 69 - Alternatively, a chemical substrate can be used to prime the Piks module 6. It is well established in literature that a NDK-SNAC or an active thioester of a pentaketide substrate can be used for polyketide synthesis (Rittner and Joppe 2022). The NDK-SNAC or the active thioester of pentaketide is used at 1-20 mM, replacing the first extender module. 5 Example 10: Production of a triketide lactones derivative library in-vitro using AT mutants The same concept as in examples 8 and 9 is used to produce different triketide lactones by using other non-CoA acyl-thioesters instead of S-Fluoromethylmalonyl-N- hexanoylcysteamine or S-Ethylmalonyl-N-hexanoylcysteamine. This allows to generate a 10 library of triketide lactones in-vitro with control of the regioselectivity. The biosynthesis can be followed by tracking the NADPH consumption similar to example 8 and by analyzing the reaction mixture by HPLC-MS similar to example 9. During the reaction the non-CoA substrate competes with the MM-CoA. The HPLC-MS reveals, if the methyl or the acyl moiety is converted from MM-CoA or the non-CoA acyl-thioester. 15 Example 11: Chemobiosynthetic derivatization of triketide lactones in-vitro using exchanges of AT mutants Similar to examples 8 and 9, triketide lactone derivatives can be produced, if the AT domain in the Piks module 6 is replaced by another acyltransferase variant, according to the present invention. For example, the piks-AT6 can be exchanged by mutants of the Nid- 20 AT5, Ans-AT8 or raps-AT14. Hereby, the acyltransferases can be swapped with or without the adjacent linker domain. This allows the production of different triketide lactone derivatives, which would not be possible with the specificity of the Piks AT6. The procedure is the same as in examples 8 and 9, but the described Piks-M6 AT swaps are used instead of the Piks-AT6 variants. The reaction can be supplemented with different non-CoA 25 substrates, as for example S-Ethylmalonyl-N-hexanoylcysteamine. Example 12: Chemobiosynthetic derivatization of triketide lactones in-vitro using exchanges of AT mutants combined with ACP and KS exchanges Similar to example 11, the acytransferases of the first elongation module (either the loading Didomain or the extender AT domain of the KEZ0F16 or KEZ0F84 constructs) can be 30 swapped simultaneously with the downstream ACP and KS domains to install domains having the matching specificity to be able to utilize the foreign acyl moiety for the - 70 - condensation reaction provided by the altered acyltransferase variant. The procedure is the same as in examples 8 and 9, but the described swaps are used instead of the first elongation module together with the WT of Piks module 6. The acyltransferases can be exchanged with or without the adjacent linker domain. The reaction is supplemented with 5 different non-CoA substrates, as for example S-Ethylmalonyl-N-hexanoylcysteamine, which allows the synthesis of different triketide lactone derivatives. Example 13: Chemobiosynthetic derivatization of triketide lactones in-vitro using DEBS AT mutants The same concept as in examples 8-11 is transferred to a different enzyme system. For 10 this example, the enzyme DEBS3 is used, which consists of two elongation modules, module 5 and module 6. In this example, the DEBS-AT6 is mutated, according to the present invention. Due to the presence of a ketoreductase in each module, a different triketide lactone scaffold can be produced. Mutations in the DEBS-AT6 domain with the addition of non-CoA aclyl-thioesters during catalysis allow the general regioselective 15 chemobiosynthetic derivatization of the triketide lactone in-vitro, by exploiting the ability of all ATs to transfer a certain spectrum of acyl-moieties, which e.g. can be observed in Englund et al.. (Englund 2023). It is well established in the literature that NDK-SNAC or an active thioester of a pentaketide can be used to prime DEBS3 (Rittner and Joppe 2022). A possible assay could look like 20 the following. Briefly, 1-6 µM of the DEBS3 mutants were combined with 4-20 mM of the priming substrate (NDK-SNAC or the active thioester of a pentaketide), 60 µM NADPH, 100-300 µM MM-CoA and optionally 100–300 µM of a non-CoA acyl-thioester like S- Fluoromethylmalonyl-N-hexanoylcysteamine or S-Ethylmalonyl-N-hexanoylcysteamine. NADPH consumption was measured fluorometrically with the microplate reader 25 (ClarioStar, BMG labtech) to track the turnover rate of the enzymes. The reaction progress was recorded using the following settings: 348-20 nm; emission: 476-20 nm; gain: 1300; focal height: 12.1 mm; flashes: 50; orbital averaging: off. Calibration was performed with reduced NADPH under assay conditions. Alternatively, DEBS3 can be primed with a didomain of DEBS ACP4 with an upstream 30 fused malonyl-acetyl transferase (MAT), which can originate for example from the mouse FAS. As the MAT is a promiscuous enzyme, it can load the ACP4 with various acyl moieties from CoA acyl-thioester or non-CoA acyl-thioester. In the assay, the priming NDK-SNAC or the active thioester of a pentaketide is replaced by 1-6 µM of the MAT-ACP4 construct. - 71 - Additionally, the thioesterase of DEBS3 can be replaced by other thioesterase domains, for example from the Piks with or without their upstream ACP, to match the required specificities and improve yields. Furthermore, similar to the concept of example 11, the DEBS AT6 can be exchanged by 5 variants of the Nid-AT5, Ans-AT8 or raps-AT14, according to the present invention. This allows the production of different triketide lactone derivatives (Englund 2023). The procedure is the same as in example 11, but the described DEBS-M6 swaps are used instead of the DEBS-AT6 variants. The acyltransferase can be exchanged with or without the adjacent linker domain. The reaction can be supplemented by different non-CoA acyl- 10 thioesters, like e.g. S-Ethylmalonyl-N-hexanoylcysteamine. Example 14: Production of triketide lactones in-vitro using DEBS AT mutants Similar to example 13, the lactone formation can be analyzed by HPLC-MS analysis. In a competitive reaction set-up using MM-CoA and non-CoA acyl-thioesters, the introduction of either the methyl moiety from MM-CoA or an acyl moiety from the non-CoA substrate 15 can be determined by the mass information. A possible assay could look like the following: Briefly, 1-6 µM of the DEBS3 mutants were combined with 4-20 mM of the priming substrate (NDK-SNAC or the active thioester of a pentaketide), 60 µM NADPH, 100-300 µM MM-CoA and optionally 100–300 µM of a non- CoA acyl-thioester like S-Fluoromethylmalonyl-N-hexanoylcysteamine or S-Ethylmalonyl- 20 N-hexanoylcysteamine. The reactions containing the same material as in example 13, but the concentrations can be adjusted to the following concentration to produce more lactone derivatives: 4-15 µM of DEBS3 mutants, 10-50 µM of the priming substrate (NDK-SNAC or the active thioester of a pentaketide), 500-4000 µM NADPH, 1500-20000 µM MM-CoA and 1500–20000 µM of a25 non-CoA thioester like S-Fluoromethylmalonyl-N-hexanoylcysteamine or S-Ethylmalonyl- N-hexanoylcysteamine. The reaction mixtures were acidified with 3 M HCl to pH 3 and extracted two to three times with the equal amount of ethyl acetate (EE). The organic solvent was evaporated under vacuum and then analyzed by HPLC-MS. The extract was analyzed by HPLC-MS for the masses of the triketide lactone corresponding to the 30 introduction of methyl, fluoromethyl and / or ethyl moieties. - 72 - Example 15: Chemobiosynthetic derivatization of triketide lactones in-vivo The same concept as in examples 8-12 was applied for the production of lactones in-vivo, which allows a much higher scaling of the product. To do so, the two proteins from examples 8-12 including the loading domain fused to the first elongation module and the 5 second elongation module containing the thioesterase were expressed in different E. coli strains like Rosetta, BL21(DE3), NiCo21 (DE3) or K207-3 (Zhang 2023, Menzella 2005, Lau 2004). At least one of the two proteins must contain a mutated acyltransferase, accordingly to the present invention. After induction, the non-CoA substrates were fed. If the strain cannot produce MM-CoA in sufficient amount, S-Methylmalonyl-N- 10 hexanoylcysteamine was additionally fed. Briefly, the cells were transformed with one or to plasmids containing the two proteins by electroporation or by using chemical competent cells. If the used strain does not contain a phosphopantetheinyl transferase that is able to activate the ACPs of PKSs, a Sfp (Lambalot 1996) or Npt (Geyer 2020) gene was additionally provided on one of the two plasmids or 15 separately on a third plasmid. The transformed cells were recovered in 10 times the volume of SOC-medium for at least 1 h at 37°C and 250-350 rpm, then transferred to LB-media containing the appropriate antibiotics and incubated over night at 37 °C, 250-300 rpm. The pre-culture was used to inoculate a Main culture of 100-2000 L of Media, for example TB- medium or KC-Medium (5 g / L yeast extract, 10 g / L casein, 15 g / L glycerol, 10 g / L NaCl, 20 100 mM potassium phosphate, pH 7.6 Zhang et al.2023), with appropriate antibiotics. The main cultures were grown for 1-2 h at 37 °C and 250-300 rpm to reach an OD of 0.3-0.6. The main culture was cooled down to 20 °C and then induced with 100-300 µM IPTG. The Bap1 und K207-3 strains were fed with 80 mM sodium propionate and optionally 5 mM hydroxocobalamin hydrochloride. Additionally, all cultures were fed with 5000–40000 µM25 non-CoA acyl-thioesters like e.g. S-Ethylmalonyl-N-hexanoylcysteamine or S- Fluoromethylmalonyl-N-hexanoylcysteamine. If the strain cannot produce MM-CoA in sufficient amount, 5000–40000 µM of S-Methylmalonyl-N-hexanoylcysteamine was additionally fed. The cultures were grown for 5-10 days at 20 °C and 250-300 rpm. The cultures were acidified to pH 2-3 with concentrated HCl and then extracted two to three 30 times with the equal volume of ethyl acetate (EE). Phases were separated by centrifugation. All organic phases were combined and dried with MgSO4. The solvent was removed in vacuum. The crude product was analyzed for product formation by HPLC-MS and optionally purified using HPLC or other chromatography strategies like normal phase chromatography using silica gel. - 73 - Example 16: Chemobiosynthetic derivatization of 6-deoxyerythronolide B in a heterologous host It is well established, that 6-deoxyerythronolide B (6-dEB) can be produced in specialized Escherichia coli (E. coli) cells when the three genes of the polyketide synthase DEBS 5 (eryAI-eryAIII) and additional genes are heterologously expressed and a propionyl- thioester is fed to the culture (Pfeifer et al.2001, Murli et al.2005). This way, several 15- R-6-dEB analogues were produced by mutation of the loading module and feeding of different starter molecules as thioesters. The herein presented invention allows a general regioselective chemobiosynthetic derivatization of polyketides when the respective 10 acyltransferase(s) are altered, and the respective function is fed as non-CoA thioester during cultivation. Methods of the present invention are performed, for example, by production of 2-R-6-dEB analogues in specialized E. coli cells by alteration of the DEBS AT6 and feeding of various elongation substrates during cultivation. For producing a specific derivative, e.g.2-Fluoro- 15 2-methyl-6-dEB, the native acyl specificity of the DEBS AT6 can be utilized and hence, for the reason of minimally engineering the polyketide synthase, it is only required to introduce point mutations into the DEBS AT6 as given in Examples 3 and 4, and to feed the culture with a 2-fluoro-2-methylmalonyl-thioester, linked via the thioester to a non-CoA coenzyme of the present invention. For producing a library of 2-R-6-dEB analogues, one would 20 introduce the selectase by exchanging the native DEBS AT6, for example by exchanging the acyltransferase as in Rittner et al. (2022) and feed the culture(s) with various different elongation substrates as non-CoA substrates during cultivation. Briefly, the encoding DNA of DEBS AT6 is exchanged with a DEBS AT6 variant DNA or a MAT variant DNA from example 2, as e.g. in Rittner et al.2022, using the in-Fusion Snap 25 Assembly Master Mix (Takara). The two plasmids encoding for DEBS1, DEBS2 and the altered DEBS3 together with additional genes are transformed into specialized E. coli cells, which are used to inoculate pre-cultures. These are used to inoculate production cultures, which are grown for 6-dEB production as described previously (Murli et al.2003). Non-CoA substrates for chemobiosynthesis are added upon induction of the expression of T7 RNA 30 polymerase at final concentrations around 4 mM unless otherwise stated. Sample broths for 6-dEB analysis were extracted with ethyl acetate and are analyzed by thin layer chromatography and staining with Vanilin or for deeper analysis with high-performance liquid chromatography (HPLC) / mass spectrometry (MS). For determination of the toxicity - 74 - of non-CoA substrates a titration in the final concentration range between 0.5 mM-8 mM is performed (Murli et al.2005). Example 17: In-vivo polyketide derivatives production by using exchanged acyltransferase mutants 5 Instead of manipulating the polyketide production in a heterologous host, the methods of the present invention can be used for producing polyketide derivatives directly in the native or industrial producing strain. This is achieved, for example, by production of 2-R- Erythromycin analogues in Saccharopolyspora erythraea (S. erythraea) NRRL2338 or an industrial strain variants by modifying the encoding DNA for the DEBS AT6 with a DEBS 10 AT6 variant, like e.g. described in examples 3 and 4, or swapped-in selectase domains, as described in example 16, and feeding of various non-CoA elongation substrates as thioesters during cultivation. Additionally, the DEBS AT6 can also be exchanged by Nid- AT5, Ans-AT8 or Raps AT14 variants, as e.g. described in examples 5 and 6. All acyltransferases were exchanged with or without the adjacent linker domain. The genetic 15 manipulation of the producing actinomycetes strains can be accomplished by several well described methods, essentially by intergeneric conjugation with e.g. E. coli ET12567 / pUZ8002 and subsequent induced homology-directed repair (HDR) (Tong et al. 2020). The cultivation of S. erythraea for production of erythromycin and derivatives thereof is well 20 described in literature (Minas et al.1998, Wu et al.2011) and established protocols can be followed Example 18: In-vivo polyketide derivatives production by exchanging DEBS AT1 The same concept as in example 17 can be applied to the module 1 of DEBS to achieve modification of a different position in the polyketide product and generate 12-R-6- 25 Erythromycin analogues. Instead of altering the acyltransferase of module 6 of DEBS, the DEBS AT1 is exchanged with DEBS AT1 variants, swapped-in selectase domains or swapped-in Nid-AT5, Ans-AT8 or Raps AT14 variant domains. The acyltransferases were exchanged with or without the adjacent linker domain. This approach allows a general regiolselective chemobiosynthetic synthesis of 12-R-6-Erythromycin analogues, when the 30 culture is fed with different elongation substrates as non-CoA substrates during cultivation. - 75 - Additionally, it could make sense, if the Nid-AT5, Ans-AT8 and Raps AT14 variants are swapped-in and cultures are fed with rather bulky non-CoA extender substrates, to simultaneously exchange the respective downstream domains ACP and KS to match the required specificity for efficient biocatalysis. 5 Example 19: In-vivo polyketide derivatives production by exchanging DEBS AT0 (acyltransferase of the loading domain) The same concept as in example 17, can be applied to the loading domain of DEBS. Instead of altering the DEBS AT6, the DEBS AT0 (the acyltransferase of the loading domain) is exchanged with DEBS AT0 variants, similar to the AVES loading domain 10 variants described in examples 5 and 6, swapped-in selectase domains or swapped-in AVES AT0 variants. This approach allows a general regiolselective chemobiosynthetic synthesis of 14-R-6-Erythromycin analogues, when the culture is fed with different starter substrates as non-CoA substrates during cultivation. Example 20: In-vivo FK506 derivatives production by using fkb AT4 mutants 15 The same concept of examples 17-19 can be transferred to a FK506 producing strain. FK506 (tacrolimus) is an important drug, which is produced by several streptomyces strains including Streptomyces tsukubaensis or Streptomyces sp. KCTC 11604BP (Yeon 2016, Beom 2019). Applying the herein presented invention to these strains or industrial strains allows a general regioselective chemobiosynthetic derivatization of FK506. 20 In this example the encoding DNA sequence for the acyltransferase of module 4 (Fkb AT4) is exchanged with Fkb AT4 variants, according to the present invention, to prevent binding of the native allylmalonyl-CoA substrate present in the cells. This acyltransferase domain was chosen as it is known for high substrate flexibility for various acyl moieties (Jiang 2015). Non-CoA acyl-thioesters as elongation substrates are fed during the cultivation of the 25 strains. This allows the regioselective chemobiosynthetic synthesis of 21-R-FK506. The same concept for genomic manipulation and feeding strategies as described in example 17 can be applied. - 76 - Example 21: In-vivo FK506 derivatives production by exchanging fkb AT9 with acyltransferase mutants. The same strains as in example 20 can be used for the regioselective chemobiosynthetic synthesis of 12-R-FK506 analogues. In this example the acyltransferase of module 9 (Fkb 5 AT9) was exchanged with swapped-in selectase domains or swapped-in Nid-AT5, Ans- AT8 or Raps AT14 variants, according to the present invention. The culture was fed with different elongation substrates as non-CoA substrates during cultivation. Additionally, it could make sense, if the Nid-AT5, Ans-AT8 and Raps AT14 variants are swapped-in and cultures are fed with rather bulky non-CoA extender substrates, to 10 simultaneously exchange the respective downstream domains ACP and KS to match the required specificity for efficient biocatalysis. The same concept for genomic manipulation and feeding strategies as described in example 17 can be applied. Example 22: In-vivo kirromycin derivatives production by using KirCII variants Similarly to example 20, the principle of this invention can be also applied to a trans-AT 15 enzyme. We chose KirCII from the kirromycin biosynthesis as a well-established example for the introduction of ethylmalonyl moieties from ethylmalonyl-CoA and having a relaxed specificity for different acyl moieties (Musiol 2011). Kirromycin is an antibiotic which is produced by the strain Streptomyces collinus Tü 365. Exchanging the encoding DNA sequence for KirCII with KirCII variants, according to the present invention, allows the 20 discrimination against the ethylmalonyl-CoA substrate in the cell. By feeding other non-CoA acyl-thioester to Streptomyces collinus Tü 365 strains during cultivation, 27-R kirromycin derivatives can be generated in-vivo by regioselective chemobiosynthesis. The same concept for genomic manipulation and feeding strategies as described in example 17 can be applied. 25 Example 23: Transfer of different KEZ1-esters to the ACP To validate that the altered acyltransferase can transfer different non-CoA substrates, the mutant Selectase-A34 (T161L F184L R286L K186D; SEQ ID NO: 122) was used as an example. We used a thiol-sensitive fluorophore (ThioGlo4; Yi et al.2009) to quantify the released free thiol groups from the substrate Acyl-N-hexanoylcysteamine after the enzyme- 30 specific transfer of the acyl moiety to the FAS ACP (KEZ0B03; SEQ ID NO: 170). Three solutions were prepared in either a two-fold or as a 4x concentrated stock in the assay - 77 - buffer (50 mM sodium phosphate buffer, 10 % (v / v) glycerol, 1 mM EDTA, pH 7.6). Solution 1 contained the Acyl-KEZ1 substrates at 200 µM (KEZ1 being the non-CoA coenzyme according to formula (1)). Solution 2 contained the ACP at 224 µM which also contained TCEP at a final concentration of 400 µM. Solution 3 contained the Selectase-A34 enzyme 5 at 44 µM or 355 µM depending on the transfer rate for the used non-CoA acetyltransferase substrate. The enzyme solution contained also 0.1 mg / mL BSA. 12.5 µL solution 2 and 12.5 µL solution 3 were combined in a 96-well PCR plate. Then 25 µL of Acyl-KEZ1 solutions were added and the solution was mixed. The reaction mixture was incubated for 90 s at room temperature and then stopped by adding 70 µL acetonitrile and 20 µL Brine. 10 The mixture was mixed and after phase separation the lower phase was discarded (90 µL). 10 µL of the upper ACN-phase was transferred from the bottom into a 384-well Small Volume HiBase Microplates (Greiner Bio-one). 10 µL of a 240 µM ThioGlo4 (in 100 mM NaPi, pH 7.0) was added and the reaction mixture was mixed. All reactions were measured twice with the plate reader (ClarioStar, BMG Labtech) directly and after 5 min using the 15 following settings: 400-20 nm; emission: 465-20 nm; gain: 700; focal height: 12.5 mm; flashes: 20; orbital averaging: off. Background was measured without the enzyme selectase-A34 (solution contained 0.1 mg / mL BSA), but with the respective substrate and acceptor in the mentioned concentrations and was subtracted from the measurements. Exemplary results for acyl groups according to formulas (4) to (12) are shown in Figure 7. 20 Data show that the Selectase-A34 has excellent turnover rates for various acyl-moieties from synthetic non-CoA substrates and is therefore a promising tool for the derivatization of polyketides. Example 24: NADPH consumption assay to monitor the synthesis of lactones A NAPDH consumption was performed to track the production rate of lactones, by 25 measuring the NAPDH consumption fluorometrically. Three solutions were prepared as 4x and 2x concentrated stocks in the assay buffer (400 mM phosphate buffer, 20 % (v / v) glycerol, 1 mM EDTA, pH 7.2). Solution 1 contained all substrates, including 240 µM NADPH, 1 mM MM-CoA and optionally 400 µM Bu-CoA or 400 µM Bu-KEZ1 (with an acyl group according to formula (10) and KEZ1 being the non-CoA coenzyme according to 30 formula (1)). Solution 2 contained the Enzyme KEZ0F10 (pikAIV, SEQ ID NO: 166; Zhang et al 2023) at 8 µM. Solution 3 contained either the protein KEZ0F16 (pik167, SEQ ID NO: 167; Zhang et al 2023), KEZ0F19 (StrI (Strep Tag sequences I (1)-LD-MAT-pik167), KEZ0F22 (StrI-LD-MATKO(used as control)-pik167; SEQ ID NO: 168) or KEZ0F25 (StrI- LD-SelectaseA34-pik167; SEQ ID NO: 169) at around 4 µM.5 µL of solution 1 and 5 µL of 35 solution 2 were combined and mixed with 10 µL of solution 3. The assay was performed in - 78 - 384-well Small Volume HiBase Microplates (Greiner Bio-one) and mixed manually. The reaction was recorded using following settings: (ClarioStar, BMG labtech): 348-20 nm; emission: 476-20 nm; gain: 1250; focal height: 12.5 mm. Calibration was performed with NADPH. A scheme of pik167, pikAIV, StrI-LD-SelectaseA34-pik167 and the reaction is 5 shown in Figure 8A. Turnover results comparing StrI-LD-MAT-pik167 and StrI-LD- SelectaseA34-pik167 are shown in Figure 8B. Data show the success of the engineering approach, as the SelectaseA34 variant does not produce products with Bu-CoA, but with Bu-KEZ1. Example 25: Transfer of acyl moieties from CoA- and non-CoA substrates by the FAS 10 AT of Caenorhabditis elegans The same assay as described in example 3 was used for Caenorhabditis elegans (C. elegans) FAS AT and its respective variants. For solution 1, 24 nM of the WT and 2400 nM of the mutants were used. For solution 3, 200 µM Ac-CoA or 200 µM Mal-CoA or 200 µM MM-CoA were used. For solution 4, 240 µM mouse FAS ACP was used. 15 To validate the shifted specificity of the altered acyltransferases towards non-CoA substrates, the assay described in example 4 (second part) was used to demonstrate the transfer of acyl moieties from non-CoA substrates to ACPs. For solution 1, 500 nM of the wildtype AT was used and 500-1200 nM of the variants. For solution 2, 240 µM mouse FAS 20 ACP was used. For solution 3, 200 µM S-Malonyl-N-hexanoylcysteamine was used. Results for the transfer of acyl moieties from CoA- and non-CoA substrates to ACP as described are shown in Figure 17. The results show that the mutation within the AT domain led to a full knockout of the enzyme, which, more specifically, was caused most likely by the three alanine mutations 25 within positions 1, 2 and 3. It is well known that alanine mutations can lead sometimes to a destabilized fold due to a disruption of alpha or beta-folds or hydrogen bond networks. Therefore, although the approach of the present invention is generally applicable, one has to be careful in some cases with the accumulation of destabilizing residues, which may impair the protein fold. The skilled person can easily change each position simultaneously 30 or successively with various amino acids to find variants with excellent ratios between slow turnover rates for acyl moieties from CoA esters and fast turnover rates for acyl moieties from synthetic non-CoA esters without affecting the protein fold. - 79 - References Beom, Ji Yoon, et al. "Biosynthesis of nonimmunosuppressive FK506 analogues with antifungal activity." Journal of natural products 82.8 (2019): 2078-2086. Chen AY, Schnarr NA, Kim CY, Cane DE, Khosla C. Extender unit and acyl carrier protein 5 specificity of ketosynthase domains of the 6-deoxyerythronolide B synthase. J Am Chem Soc.2006 Mar 8;128(9):3067-74. doi: 10.1021 / ja058093d. Englund, E. et al. Expanding Extender Substrate Selection for Unnatural Polyketide Biosynthesis by Acyltransferase Domain Exchange within a Modular Polyketide Synthase. J. Am. Chem. Soc. 145, 8822–8832 (2023). Hertweck C. The biosynthetic logic of 10 polyketide diversity. Angew Chem Int Ed Engl. 2009;48(26):4688-716. doi: 10.1002 / anie.200806121. Jiang, Hui, et al. "An acyltransferase domain of FK 506 polyketide synthase recognizing both an acyl carrier protein and coenzyme A as acyl donors to transfer allylmalonyl and ethylmalonyl units." The FEBS Journal 282.13 (2015): 2527-2539. 15 Le, Q et al. Extraction of erythromycin from fermentation broth using salt-induced phase separation processes. Separation and Purification Technology,24 (1–2): 85-91, doi: 10.1016 / S1383-5866(00)00217-3. Minas W, Brünker P, Kallio PT, Bailey JE. Improved erythromycin production in a genetically engineered industrial strain of Saccharopolyspora erythraea. Biotechnol Prog. 20 1998 Jul-Aug;14(4):561-6. doi: 10.1021 / bp980055t. Miyazawa, T., Fitzgerald, B. J. & Keatinge-Clay, A. T. Preparative production of an enantiomeric pair by engineered polyketide synthases. Chem. Commun.57, 8762–8765 (2021). Murli S, Kennedy J, Dayem LC, Carney JR, Kealey JT. Metabolic engineering of 25 Escherichia coli for improved 6-deoxyerythronolide B production. J Ind Microbiol Biotechnol.2003 Aug;30(8):500-9. doi: 10.1007 / s10295-003-0073-x. Murli S, MacMillan KS, Hu Z, Ashley GW, Dong SD, Kealey JT, Reeves CD, Kennedy J. Chemobiosynthesis of novel 6-deoxyerythronolide B analogues by mutation of the loading - 80 - module of 6-deoxyerythronolide B synthase 1. Appl Environ Microbiol. 2005 Aug;71(8):4503-9. doi: 10.1128 / AEM.71.8.4503-4509.2005. Musiol, E. M. et al. Supramolecular Templating in Kirromycin Biosynthesis: The Acyltransferase KirCII Loads Ethylmalonyl-CoA Extender onto a Specific ACP of the trans- 5 AT PKS. Chem. Biol.18, 438–444 (2011). Pfeifer BA, Admiraal SJ, Gramajo H, Cane DE, Khosla C. Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli. Science. 2001 Mar 2;291(5509):1790-2. doi: 10.1126 / science.1058092. Rittner A, Paithankar KS, Huu KV, Grininger M. Characterization of the Polyspecific 10 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. 15 Rittner, A., Joppe, M., Schmidt, J.J. et al. Chemoenzymatic synthesis of fluorinated polyketides. Nat. Chem. 14, 1000–1006 (2022). https: / / doi.org / 10.1038 / s41557-022- 00996-z. 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. 20 Tong Y, Whitford CM, Blin K, Jørgensen TS, Weber T, Lee SY. CRISPR-Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes. Nat Protoc. 2020 Aug;15(8):2470-2502. doi: 10.1038 / s41596-020-0339-z. Wang, Fen, et al. "Structural and functional analysis of the loading acyltransferase from avermectin modular polyketide synthase." ACS chemical biology 10.4 (2015): 1017-1025. 25 Wu J, Zhang Q, Deng W, Qian J, Zhang S, Liu W. Toward improvement of erythromycin A production in an industrial Saccharopolyspora erythraea strain via facilitation of genetic manipulation with an artificial attB site for specific recombination. Appl Environ Microbiol. 2011 Nov;77(21):7508-16. doi: 10.1128 / AEM.06034-11. - 81 - Yeon Hee Ban, Sung Ryeol Park, Yeo Joon Yoon, The biosynthetic pathway of FK506 and its engineering: from past achievements to future prospects, Journal of Industrial Microbiology and Biotechnology, Volume 43, Issue 2-3, 1 March 2016, Pages 389–400, https: / / doi.org / 10.1007 / s10295-015-1677-7 5 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. 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Claims

- 82 - Claims 1. A method comprising: (a) providing (i) at least one non-CoA acyltransferase substrate; and 5 (ii) at least one altered acyltransferase; and (iii) at least one acyl carrier protein (ACP); (b) allowing the at least one acyltransferase to transfer the acyl group of the at least one non-CoA acyltransferase substrate to the at least one ACP; (c) obtaining at least one acyl-ACP; 10 wherein the at least one altered acyltransferase has a CoA-binding pocket that is altered to reduce CoA acyltransferase substrate transfer activity.

2. The method of claim 1, wherein step (b) is performed in at least one cell comprising the components (i), (ii), and (iii) of step (a).

3. The method of claim 1, wherein step (b) is performed outside of a cell, optionally, 15 wherein the at least one altered acyltransferase is provided in a cell-free system and / or as a purified or partially purified protein or as a lysate comprising the altered acyltransferase, optionally wherein the at least one altered acyltransferase is provided as a fusion protein further comprising at least one linker domain and optionally at least one ketoacyl synthase domain. 20 4. A method for polyketide synthesis, the method comprising: (a) providing (i) at least one non-CoA acyltransferase substrate; and (ii) optionally at least one CoA acyltransferase substrate; and (iii) a polyketide synthase (PKS) system, wherein the PKS system 25 comprises at least one altered acyltransferase; (b) allowing polyketide synthesis, wherein the at least one non-CoA acyltransferase substrate, and optionally the at least one CoA acyltransferase substrate, is / are used as a substrate in the polyketide synthesis;- 83 - (c) obtaining at least one polyketide or derivative thereof, and optionally purifying said at least one polyketide or derivative thereof; wherein the at least one altered acyltransferase has a CoA-binding pocket that is altered to reduce CoA acyltransferase substrate transfer activity. 5 5. The method of claim 4, wherein the PKS system is a PKS type I system or a PKS type II system, wherein one, two, three or more PKS modules comprise and / or interact with at least one altered acyltransferase.

6. The method of claim 4 or 5, wherein the loading domain, the first, second, third, fourth, fifth, six, seventh, eighth, ninth, tenth, eleventh, thirteenth, fourteenths, fifteenth, 10 and / or sixteenth PKS module and / or one or more subsequent PKS modules comprise(s) and / or interact(s) with at least one altered acyltransferase.

7. The method of any one of claims 4 to 6, wherein PKS system is a PKS type I system and the at least one altered acyltransferase is part of at least one multifunctional PKS type I polypeptide, optionally through replacement of at least one of the PKS acyltransferases. 15 8. The method of any one of claims 4 to 7, wherein the at least one altered acyltransferase is provided as a separate polypeptide, optionally as a fusion protein further comprising at least one linker domain and optionally at least one ketoacyl synthase domain.

9. The method of any one of claims 4 to 8, wherein the polyketide synthesis, or part of the polyketide synthesis, is performed in at least one cell comprising the PKS system or 20 parts thereof.

10. The method of any one of claims 4 to 9, wherein the polyketide synthesis, or part of the polyketide synthesis, is performed outside of a cell, optionally wherein the at least one altered acyltransferase is provided in a cell-free system and / or as a purified or partially purified protein or as a lysate comprising the altered acyltransferase, optionally wherein the 25 at least one altered acyltransferase is provided as a fusion protein further comprising at least one linker domain and optionally at least one ketoacyl synthase domain.

11. The method of any of the previous claims, wherein the at least one altered acyltransferase has a CoA-binding pocket that is altered to transfer the at least one non- CoA acyltransferase substrate at a higher rate than the equivalent CoA acyltransferase 30 substrate with the same acyl group and / or, in case at least one CoA acyltransferase- 84 - substrate is provided in step (a), the at least one CoA acyltransferase substrate provided in step (a).

12. The method of any of the previous claims, wherein the at least one non-CoA substrate comprises or consists of 5 (i) a non-CoA coenzyme; and (ii) an acyl group; optionally wherein part (i) and part (ii) are linked via a thioester, optionally wherein a) the non-CoA coenzyme has a neutral net charge or a positive net charge, further optionally, wherein the positive net charge is a single, two-fold, three-fold or 10 four-fold positive net charge; and / or b) wherein the non-CoA coenzyme does not comprise a negative charge.

13. The method of claim 12, wherein the acyl group comprises or consist of a malonyl group, an acetyl group, a methylmalonyl group, a propionyl group, an ethymalonyl group, or a derivative thereof. 15 14. A non-CoA polyketide substrate comprising or consisting of (i) a non-CoA coenzyme; and (ii) an acyl group; wherein part (i) and part (ii) are linked via a thioester, optionally wherein (a) the non-CoA coenzyme has a neutral net charge or a positive net charge, 20 further optionally, wherein the positive net charge is a single, two-fold, three-fold or four-fold positive net charge; and / or (b) wherein the non-CoA coenzyme does not comprise a negative charge.

15. The non-CoA polyketide substrate of claim 14, wherein the non-CoA coenzyme comprises or consist of a compound according to formula (1):- 85 -(1), wherein R1 represents the linked acyl group, wherein the acyl group is not a malonyl group.

16. The non-CoA polyketide substrate of claim 14 or 15, wherein the acyl group comprises or consist of an acetyl group, a methylmalonyl group, a propionyl group, an ethymalonyl group, or a derivative thereof.

17. The non-CoA polyketide substrate of claim 14 or 15, wherein the acyl group comprises or consist of the acyl group according to any one of formulas 5 to 10 or 12 to 19, wherein the asterisk denotes the non-CoA coenzyme:- 87 - 18. A compound according to formula (1):(1), wherein R1 is hydrogen. 5 19. Use of at least altered acyltransferase as defined in any one of claims 1 to 11, wherein the at least one altered acyltransferase originates from a PKS; and / or at least one non-CoA acyltransferase substrate as defined in any one of claims 12 to 17, and / or at least one compound as defined in claim 18, and optionally at least one CoA acyltransferase substrate for polyketide synthesis, fatty acid synthesis, and / or nonribosomal peptide 10 synthesis.

20. A kit comprising at least altered acyltransferase as defined in any one of claims 1 to 11; and / or at least one non-CoA acyltransferase substrate as defined in any one of claims 12 to 17; and / or at least one compound as defined in claim 18, optionally further comprising at least one CoA acyltransferase substrate, and / or at least one container, and / or a set of 15 reagents including buffer and / or medium, and / or means of transfection.

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