title: Iterative assembly of two separate polyketide chains by the same single-module bacterial polyketide synthase in the biosynthesis of HSAF.
abstract: Antifungal HSAF (heat-stable antifungal factor, dihydromaltophilin) is a polycyclic tetramate macrolactam from the biocontrol agent Lysobacter enzymogenes. 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 skeleton. To address the unusual biosynthetic mechanism, we expressed the biosynthetic genes in two "clean" strains of Streptomyces and showed the production of HSAF analogues and a polyene tetramate intermediate. We then expressed the PKS module in Escherichia coli and purified the enzyme. Upon incubation of the enzyme with acyl-coenzyme A and reduced nicotinamide adenine dinucleotide phosphate (NADPH), a polyene was detected in the tryptic acyl carrier protein (ACP). Finally, we incubated the polyene-PKS with the NRPS module in the presence of ornithine and adenosine triphosphate (ATP), and we detected the same polyene tetramate as that in Streptomyces transformed with the PKS-NRPS alone. Together, our results provide evidence for an unusual iterative biosynthetic mechanism for bacterial polyketide-peptide natural products.
authors.affiliation: Key Laboratory of Chemical Biology, School of Pharmaceutical Sciences, Shandong University, Jinan 250100, China.
authors.forename: Yaoyao
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
authors.forename: Haotong
authors.initials: H
authors.lastname: Chen
authors.affiliation: Key Laboratory of Chemical Biology, School of Pharmaceutical Sciences, Shandong University, Jinan 250100, China.
authors.forename: Yanjiao
authors.initials: Y
authors.lastname: Ding
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
authors.forename: Yunxuan
authors.initials: Y
authors.lastname: Xie
authors.affiliation: Key Laboratory of Chemical Biology, School of Pharmaceutical Sciences, Shandong University, Jinan 250100, China; State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan 250100, China.
authors.forename: Haoxin
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Cerny
authors.affiliation: Key Laboratory of Chemical Biology, School of Pharmaceutical Sciences, Shandong University, Jinan 250100, China; State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan 250100, China.
authors.forename: Yuemao
authors.initials: Y
authors.lastname: Shen
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
authors.forename: Liangcheng
authors.initials: L
authors.lastname: Du
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D053528:Lysobacter / Q000201:enzymology*; D008969:Molecular Sequence Data; D048630:Polyketide Synthases / Q000737:chemistry / Q000378:metabolism*; D013057:Spectrum Analysis
chemical_list: D048630:Polyketide Synthases
keywords: biosynthesis; enzymes; macrocycles; natural products; polyketides