Poly Specific trans-Acyltransferase Machinery Revealed via Engineered Acyl-CoA Synthetases

Abstract

Polyketide synthases construct polyketides with diverse structures and biological activities via the condensation of extender units and acyl thioesters. Although a growing body of evidence suggests that polyketide synthases might be tolerant to non-natural extender units, in vitro and in vivo studies aimed at probing and utilizing polyketide synthase specificity are severely limited to only a small number of extender units, owing to the lack of synthetic routes to a broad variety of acyl-CoA extender units. Here, we report the construction of promiscuous malonyl-CoA synthetase variants that can be used to synthesize a broad range of malonyl-CoA extender units substituted at the C2-position, several of which contain handles for chemoselective ligation and are not found in natural biosynthetic systems. We highlighted utility of these enzymes by probing the acyl-CoA specificity of several trans-acyltransferases, leading to the unprecedented discovery of poly specificity toward non-natural extender units, several of which are not found in naturally occurring biosynthetic pathways. These results reveal that polyketide biosynthetic machinery might be more tolerant to non-natural substrates than previously established, and that mutant synthetases are valuable tools for probing the specificity of biosynthetic machinery. Our data suggest new synthetic biology strategies for harnessing this promiscuity and enabling the regioselective modification of polyketides.

A Chemo-Enzymatic Road Map to the Synthesis of CoA Esters

“…Judging by the yield and ease of use of our model syntheses we provide a roadmap of which method to use for each individual acyl-CoA class (Figure 3). [8]; Route 6, this study and [12,13]). These routes give access to all three classes of acyl-CoA esters with high yield and stereoselectivity.…”

“…Additionally, this method offers the possibility to generate reactive desaturated acyl-CoAs (e.g., acrylyl-CoA (8)) in reaction mixtures in situ. As an alternative enzymatic route many [8]; Route 6, this study and [12,13]). These routes give access to all three classes of acyl-CoA esters with high yield and stereoselectivity.…”

“…Through active site mutagenesis, a more promiscuous MatB variant was created recently. This variant was used to generate other α-carboxylated (R)-malonyl-CoA derivatives in situ for in vitro polyketide synthesis assays [12,16]. Note however, that MatB synthesis is limited by commercial availablility of free malonic acid starting precursors.…”

The Combinatorial Biosynthesis of “Unnatural” Products with Polyketides

“…Compared to cis-AT, the trans-AT may have a wider substrate library, thus, it can be used to obtain "unnatural" analogs [33]. A promiscuous malonyl-CoA synthase variant was constructed by Koryakina et al [34], and it can be applied to synthesize a broad range of malonyl-CoA extender units, some of these extender units are not found in natural biosynthetic systems. The utility of the variant in probing the acyl-CoA specificity of several trans-ATs has led to the discovery of poly-specificity toward non-natural extender units.…”

Engineering the acyltransferase substrate specificity of assembly line polyketide synthases

“…The majority of trans-AT domains are thought to be specific for malonyl-CoA, but domains with varying specificities have been proposed recently [80]. The ethylmalonyl-CoA specific kirromycin trans-AT domain was shown to be promiscuous, capable of catalysing the transacylation of several a-carboxyacyl-CoA substrates not found in its native host [37]. Further, several trans-AT domains have shown promiscuity towards their ACP substrates as well, with activities towards heterologous ACP domains that meet or surpass the activities of native cis-AT domains [42,83].…”