Molecular Mechanism for the Biosynthesis of Antifungal HSAF and Antibacterial WAP-8294A2

Bioactive natural products are a major source of anti-infectives. Many ubiquitous inhabitants of soil and water, such as the Gram-negative, prolific producers of natural products, Lysobacter, remain largely unexplored. This PhD thesis reports our studies of the biosynthetic mechanism for a novel antifungal natural product HSAF and a potent anti-MRSA natural product WAP-8294A2. In the first chapter, we give a short review of the biosynthesis of polyketides, focusing on those which exhibit new biosynthetic features. In subsequent chapters, we present original research on the biosynthetic mechanisms of dihydromaltophilin, a heat stable antifungal factor (HSAF). HSAF is the main antifungal factor that the biocontrol agent Lysobacter enzymogenes produces to fight against fungal pathogens. Most interestingly, the genetic feature of HSAF biosynthesis suggests that the same polyketide synthase (PKS) module acts not only iteratively, but also separately. In the thesis, we demonstrated the in vitro and in vivo production of the polyene tetramate, providing direct evidence for this highly unusual iterative polyketide biosynthetic mechanism that is likely general for this type of hybrid polyketide-peptides. We also investigated four oxidoreductase (OX1-4) genes in the HSAF biosynthetic gene cluster, to define the minimal gene cluster required for HSAF biosynthesis. Together, the results support a new biosynthetic mechanism: a single set of domains of an iterative PKS-NRPS, in cooperating with a cascade of redox enzymes, to synthesize a complex and highly modified polycyclic tetramate macrolactam. The final part of the thesis presents research on WAP-8294A, a complex of at least 20 cyclic lipodepsipeptides exhibiting remarkable activity against methicillin-resistant Staphylococcus aureus (MRSA). All WAP-8294A compounds contain a 3-hydroxy fatty acyl chain, varied only in the chain length and branching pattern. The mechanism for activating and introducing the 3-hydroxy fatty acid chain into the peptide is unclear. We have identified seven putative acyl CoA ligase (ACL) genes and generated gene-disruption mutants. We also expressed the genes in E. coli to obtain the pure enzymes. Both the in vivo and in vitro results showed that ACL-6 is the dominant enzyme responsible for the activation of 3-hydroxyl-7-methyloctanoic acid and the subsequent initiation of WAP core structure formation. Advisor: Liangcheng D