Polyketides are a large class of pharmaceutically relevant natural products with wide-ranging biological activities. Polyketides are biosynthesized by enzymatic assembly lines called polyketide synthases (PKSs) which catalyze the condensation of building block acyl-CoA extender units and other acyl thioesters. Typically, polyketide producing organisms possess biosynthetic routes to only 2-3 extender units. Accordingly, most PKS in vitro and in vivo studies have probed PKS specificity using a very small number of extender units, and the acyltransferase (AT) domains of PKSs have been subsequently described as 'gatekeeper' domains. Our central hypothesis is that PKS machinery is significantly more promiscuous toward extender units not normally provided to the PKS within the native host, and this promiscuity could be harnessed to produce polyketide analogues. Firstly, we have used structure-guided mutagenesis to alter the specificity of the malonyl-CoA synthetase MatB, and produced variants capable of synthesizing a broad range of natural and non-natural acyl-CoA extender units. Secondly, these promiscuous synthetases were used to probe the specificity of several polyketide biosynthetic systems. These studies revealed unprecedented promiscuity toward several non-natural extender units, including those that contained chemical handles for bioorthogonal ligation chemistry. Thirdly, we have created mutant AT's with specificities shifted toward specific acyl-CoA substrates, including methylmalonyl- and propargylmalonyl-CoA. These mutant AT's will be used for the in vitro and in vivo regioselective modification of polyketides. Access to a broad variety of polyketide building blocks and substrate-specific AT's provides a synthetic biology platform for regioselective modification of polyketides. In addition, this work has enabled development of high-throughput screens and selections for directed evolution of polyketide biosynthesis.

Synthetic biology approaches for combinatorial biosynthesis of polyketide natural products

247th National Meeting (2014)

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