S28: Engineering acyl-CoA and carrier protein specificity of trans-acyltransferases for polyketide diversification

Acyltransferase (AT) domains of polyketide synthases (PKS) are responsible for selecting extender units for incorporation into polyketides. Typically, ATs are an integral part of each PKS module, and operate in cis to load the requisite acyl-CoA substrate onto the cognate acyl carrier protein. A less common variant of this organization involves a freestanding AT enzyme that is physically separated from the PKS and operates in trans. Such trans-ATs offer interesting possibilities for combinatorial biosynthesis of regioselectively modified polyketide analogues, but critically usually display limited specificity towards both the acyl-CoA extender unit and carrier protein substrate. Here, we reveal trans-AT promiscuity towards natural and non-natural acyl-CoA extender units, and report the use of novel high-throughput screens for identifying mutants which display altered acyl-CoA specificity and orthogonal carrier protein interactions. Using a synthetic biology philosophy, our mutant enzymes will be used to construct artificial biosynthetic pathways for polyketide diversification.