Constructing Combinatorial Synthases Using Acyl Carrier Protein Chimeras

Abstract

Synthases encoded by biosynthetic gene clusters in microorganisms are capable of producing structurally complex molecules of diverse function. The ability to mix‐and‐match enzymes from different synthases to create “unnatural natural products” could enable the environmentally‐responsible construction of new pharmaceutically relevant molecules. Acyl carrier proteins (ACPs) are central hubs within biosynthetic pathways as they mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural product. Past combinatorial biosynthesis efforts have failed because of incompatible ACP‐enzyme interactions. To overcome this obstacle, chimeric ACPs capable of cross‐talk between different biosynthetic pathways were designed to serve as a lynchpin in functional hybrid synthases. Chimeric ACPs were constructed by combining features of the actinorhodin polyketide synthase ACP (ACT ACP) and of E. coli fatty acid synthase ACP (AcpP). Given the premise that AcpP interacts with its cognate ketosynthase, FabF, whereas ACT ACP does not, the ability of chimeric ACPs to form a mechanistically‐relevant crosslink with FabF was evaluated. A colorimetric assay was used to assess ACP interactions and revealed the role of Helix II of AcpP in driving ACP‐FabF binding. Results suggest that the residues guiding ACP‐FabF compatibility may reside in Helix II. Future directions will include using site directed mutagenesis to investigate the specific residues responsible for ACP‐FabF recognition.Support or Funding InformationNational Institutes of Health (R15GM120704 to L.K.C)National Science Foundation CAREER Award (CHE‐1652424 to L.K.C.)Cottrell Scholars Award (24350 to L.K.C.)