Role of a Conserved Arginine Residue in Linkers between the Ketosynthase and Acyltransferase Domains of Multimodular Polyketide Synthases

The role of interdomain linkers in modular polyketide synthases is poorly understood. Analysis of the 6-deoxyerythronolide B synthase (DEBS) has yielded a model in which chain elongation is governed by interactions between the acyl carrier protein domain and the ketosynthase domain plus an adjacent linker. Alanine scanning mutagenesis of the conserved residues of this linker in DEBS module 3 led to the identification of the R513A mutant with a markedly reduced rate of chain elongation. Limited proteolysis supported a structural role for this Arg. Our findings highlight the importance of domain–linker interactions in assembly line polyketide biosynthesis

Broad Substrate Specificity of the Loading Didomain of the Lipomycin Polyketide Synthase

LipPks1, a polyketide synthase subunit of the lipomycin synthase, is believed to catalyze the polyketide chain initiation reaction using isobutyryl-CoA as a substrate, followed by an elongation reaction with methylmalonyl-CoA to start the biosynthesis of antibiotic α-lipomycin in <i>Streptomyces aureofaciens</i> Tü117. Recombinant LipPks1, containing the thioesterase domain from the 6-deoxyerythronolide B synthase, was produced in <i>Escherichia coli</i>, and its substrate specificity was investigated <i>in vitro</i>. Surprisingly, several different acyl-CoAs, including isobutyryl-CoA, were accepted as the starter substrates, while no product was observed with acetyl-CoA. These results demonstrate the broad substrate specificity of LipPks1 and may be applied to producing new antibiotics

Alteration of Polyketide Stereochemistry from <i>anti</i> to <i>syn</i> by a Ketoreductase Domain Exchange in a Type I Modular Polyketide Synthase Subunit

Polyketide natural products have broad applications in medicine. Exploiting the modular nature of polyketide synthases to alter stereospecificity is an attractive strategy for obtaining natural product analogues with altered pharmaceutical properties. We demonstrate that by retaining a dimerization element present in LipPks1+TE, we are able to use a ketoreductase domain exchange to alter α-methyl group stereochemistry with unprecedented retention of activity and simultaneously achieve a novel alteration of polyketide product stereochemistry from <i>anti</i> to <i>syn</i>. The substrate promiscuity of LipPks1+TE further provided a unique opportunity to investigate the substrate dependence of ketoreductase activity in a polyketide synthase module context

<i>In Vitro</i> Analysis of Carboxyacyl Substrate Tolerance in the Loading and First Extension Modules of Borrelidin Polyketide Synthase

The borrelidin polyketide synthase (PKS) begins with a carboxylated substrate and, unlike typical decarboxylative loading PKSs, retains the carboxy group in the final product. The specificity and tolerance of incorporation of carboxyacyl substrate into type I PKSs have not been explored. Here, we show that the first extension module is promiscuous in its ability to extend both carboxyacyl and non-carboxyacyl substrates. However, the loading module has a requirement for substrates containing a carboxy moiety, which are not decarboxylated <i>in situ</i>. Thus, the loading module is the basis for the observed specific incorporation of carboxylated starter units by the borelidin PKS

Comprehensive <i>in Vitro</i> Analysis of Acyltransferase Domain Exchanges in Modular Polyketide Synthases and Its Application for Short-Chain Ketone Production

Type I modular polyketide synthases (PKSs) are polymerases that utilize acyl-CoAs as substrates. Each polyketide elongation reaction is catalyzed by a set of protein domains called a module. Each module usually contains an acyltransferase (AT) domain, which determines the specific acyl-CoA incorporated into each condensation reaction. Although a successful exchange of individual AT domains can lead to the biosynthesis of a large variety of novel compounds, hybrid PKS modules often show significantly decreased activities. Using monomodular PKSs as models, we have systematically analyzed the segments of AT domains and associated linkers in AT exchanges <i>in vitro</i> and have identified the boundaries within a module that can be used to exchange AT domains while maintaining protein stability and enzyme activity. Importantly, the optimized domain boundary is highly conserved, which facilitates AT domain replacements in most type I PKS modules. To further demonstrate the utility of the optimized AT domain boundary, we have constructed hybrid PKSs to produce industrially important short-chain ketones. Our <i>in vitro</i> and <i>in vivo</i> analysis demonstrated production of predicted ketones without significant loss of activities of the hybrid enzymes. These results greatly enhance the mechanistic understanding of PKS modules and prove the benefit of using engineered PKSs as a synthetic biology tool for chemical production

Heterologous Gene Expression of <i>N</i>‑Terminally Truncated Variants of LipPks1 Suggests a Functionally Critical Structural Motif in the <i>N</i>‑terminus of Modular Polyketide Synthase

<i>Streptomyces</i> genomes have a high G + C content and typically use an ATG or GTG codon to initiate protein synthesis. Although gene-finding tools perform well in low GC genomes, it is known that the accuracy in predicting a translational start site (TSS) is much less for high GC genomes. LipPks1 is a <i>Streptomyces</i>-derived, well-characterized modular polyketide synthase (PKS). Using this enzyme as a model, we experimentally investigated the effects of alternative TSSs using a heterologous host, <i>Streptomyces venezuelae</i>. One of the TSSs employed boosted the protein level by 59-fold and the product yield by 23-fold compared to the originally annotated start codon. Interestingly, a structural model of the PKS indicated the presence of a structural motif in the <i>N</i>-terminus, which may explain the observed different protein levels together with a proline and arginine-rich sequence that may inhibit translational initiation. This structure was also found in six other modular PKSs that utilize noncarboxylated starter substrates, which may guide the selection of optimal TSSs in conjunction with start-codon prediction software

Engineered Production of Short-Chain Acyl-Coenzyme A Esters in <i>Saccharomyces cerevisiae</i>

Short-chain acyl-coenzyme A esters serve as intermediate compounds in fatty acid biosynthesis, and the production of polyketides, biopolymers and other value-added chemicals. <i>S. cerevisiae</i> is a model organism that has been utilized for the biosynthesis of such biologically and economically valuable compounds. However, its limited repertoire of short-chain acyl-CoAs effectively prevents its application as a production host for a plethora of natural products. Therefore, we introduced biosynthetic metabolic pathways to five different acyl-CoA esters into <i>S. cerevisiae</i>. Our engineered strains provide the following acyl-CoAs: propionyl-CoA, methylmalonyl-CoA, <i>n</i>-butyryl-CoA, isovaleryl-CoA and <i>n</i>-hexanoyl-CoA. We established a yeast-specific metabolite extraction protocol to determine the intracellular acyl-CoA concentrations in the engineered strains. Propionyl-CoA was produced at 4–9 μM; methylmalonyl-CoA at 0.5 μM; and isovaleryl-CoA, <i>n</i>-butyryl-CoA, and <i>n</i>-hexanoyl-CoA at 6 μM each. The acyl-CoAs produced in this study are common building blocks of secondary metabolites and will enable the engineered production of a variety of natural products in <i>S. cerevisiae</i>. By providing this toolbox of acyl-CoA producing strains, we have laid the foundation to explore <i>S. cerevisiae</i> as a heterologous production host for novel secondary metabolites

Probing the Flexibility of an Iterative Modular Polyketide Synthase with Non-Native Substrates <i>in Vitro</i>

In the search for molecular machinery for custom biosynthesis of valuable compounds, the modular type I polyketide synthases (PKSs) offer great potential. In this study, we investigate the flexibility of BorM5, the iterative fifth module of the borrelidin synthase, with a panel of non-native priming substrates <i>in vitro</i>. BorM5 differentially extends various aliphatic and substituted substrates. Depending on substrate size and substitution BorM5 can exceed the three iterations it natively performs. To probe the effect of methyl branching on chain length regulation, we engineered a BorM5 variant capable of incorporating methylmalonyl- and malonyl-CoA into its intermediates. Intermediate methylation did not affect overall chain length, indicating that the enzyme does not to count methyl branches to specify the number of iterations. In addition to providing regulatory insight about BorM5, we produced dozens of novel methylated intermediates that might be used for production of various hydrocarbons or pharmaceuticals. These findings enable rational engineering and recombination of BorM5 and inform the study of other iterative modules