P124: FK228 biosynthetic pathway elucidation and reconstitution reveals cross talk between modular polyketide synthases and fatty acid synthase

The biosynthetic gene cluster of FK228, an FDA-approved anticancer depsipeptide natural product, was previously reported to encode a hybrid modular nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) biosynthetic pathway in Chromobacterium violaceum No. 968. This pathway contains two PKS modules on the DepB and DepC enzymes that lack a functional acyltransferase (AT) domain, and no apparent AT-encoding gene exists within the gene cluster or its vicinity. Through gene deletion, transcriptional analysis and pathway reconstitution of the FK228 biosynthesis in Escherchia coli cells, we have redefined the gene cluster to contain 12 genes, identified a new pathway regulatory gene, depR, and revealed the trans-acting participation of two essential genes, fabD1 and fabD2, both encoding a putative malonyl coenzyme A (CoA) acyltransferase component of the fatty acid synthase complex. Either fabD gene product appears sufficient to complement the AT-less PKS modules on DepB and DepC for polyketide chain elongation. Concurrently, another gene outside the gene cluster encoding a putative Sfp-type phosphopantetheinyltransferase (sfp) was identified to be necessary for FK228 biosynthesis.  Most interestingly, engineered E. coli strains carrying variable genetic components produced significant levels of FK228 under both aerobic and anaerobic cultivation conditions.  Discovery of the trans complementation of modular PSKs by housekeeping ATs reveals natural product biosynthesis diversity.  Moreover, the elucidation of the complete FK228 biosynthetic pathway and anaerobic production by an engineered facultative bacterial strain validates the research goals toward genetic engineering of new FK228 analogs and the construction of novel tumor-targeting bioagents.