HSAF is a polycyclic tetramate macrolactam we previously isolated from the biocontrol agent Lysobacter enzymogenes. HSAF exhibits activity against a wide range of fungi and shows a novel mode of action. Its biosynthetic gene cluster contains only a single-module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), although two separate hexaketide chains are required to assemble the HSAF skeleton. To address the molecular logic behind HSAF biosynthesis, we generated expression constructs, in which the HSAF biosynthetic genes were placed under the control of ermE* promoter. The constructs were then transferred into two �clean� strains of Streptomyces. The results showed that HSAF analogs were produced in the heterologous hosts, demonstrating that the bacterial modular PKS-NRPS acts iteratively to assemble the two separate polyketide chains and to form the tetramate macrolactam. To obtain further evidence, we expressed the PKS module (KS-AT-DH-KR-ACP) in E. coli and purified the 199.8 kDa protein. This PKS was converted to its holo form in vitro by 4'-phosphopantetheinylation and, upon incubating the holo PKS with acyl-CoA and NADPH, a hexaketide was detected in the tryptic fragment containing the ACP domain. Finally, we incubated the hexaketide-attached PKS with the separately expressed and purified NRPS module (C-A-PCP-TE, 148.6 kDa) in the presence of ornithine and ATP. The data indicated the production of a tetramate-containing polyene product resulted from condensations between the two hexaketide chains and ornithine. Together, our results provide evidence for an unusual iterative biosynthetic mechanism for bacterial polyketide-peptide natural products.

Iterative assembly of two separate polyketide chains by the same single-module bacterial PKS-NRPS in the biosynthesis of antifungal HASF

248th National Meeting (2014)

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