HSAF was isolated from Lysobacter enzymogenes, a bacterium used in the biological control of fungal diseases of plants. Structurally, it is a tetramic acid-containing macrolactam fused to a tricyclic system. HSAF exhibits a novel mode of action by disrupting sphingolipids important to the polarized growth of filamentous fungi. Here, we described the HSAF biosynthetic gene cluster which contains only a single-module polyketide synthase-nonribosomal peptide synthetase (PKS/NRPS), although the biosynthesis of HSAF apparently requires two separate polyketide chains that are linked together by one amino acid (ornithine) via two amide bonds. Flanking the PKS/NRPS are six genes, encoding a cascade of four tightly clustered redox enzymes on one side and a sterol desaturase/fatty acid hydroxylase and a ferredoxin reductase on the other side. The genetic data demonstrate that the four redox genes, in addition to the PKS/NRPS gene and the sterol desaturase/fatty acid hydroxylase gene, are required for HSAF production. The biochemical data show that the adenylation domain of the NRPS specifically activated L-ornithine and the four-domain NRPS was able to catalyze the formation of a tetramic acid-containing product from acyl-S-ACP and ornithinyl-S-NRPS. These results reveal a previously unrecognized biosynthetic mechanism for hybrid PK/NRP in prokaryotic organisms.

Lysobactor enzymogenes strain OH11 is an emerging biological control agent of fungal and bacterial diseases. We recently completed its genome sequence and found it contains a large number of gene clusters putatively responsible for the biosynthesis of nonribosomal peptides and polyketides, including the previously identified antifungal dihydromaltophilin (HSAF). One of the gene clusters contains two huge open reading frames, together encoding 12 modules of nonribosomal peptide synthetases (NRPS). Gene disruption of one of the NRPS led to the disappearance of a metabolite produced in the wild type and the elimination of its antibacterial activity. The metabolite and antibacterial activity were also affected by the disruption of some of the flanking genes. We subsequently isolated this metabolite and subjected it to spectroscopic analysis. The mass spectrometry and nuclear magnetic resonance data showed that its chemical structure is identical to WAP-8294A2, a cyclic lipodepsipeptide with potent antimethicillin-resistant Staphylococcus aureus (MRSA) activity and currently in phase I/II clinical trials. The WAP-8294A2 biosynthetic genes had not been described previously. So far, the Gram-positive Streptomyces have been the primary source of anti-infectives. Lysobacter are Gram-negative soil/water bacteria that are genetically amendable and have not been well exploited. The WAP-8294A2 synthetase represents one of the largest NRPS complexes, consisting of 45 functional domains. The identification of these genes sets the foundation for the study of the WAP-8294A2 biosynthetic mechanism and opens the door for producing new anti-MRSA antibiotics through biosynthetic engineering in this new source of Lysobacter.

Heterocyclic aromatic N-oxides often have potent biological activities, but the mechanism for aromatic N-oxidation is unclear. Six phenazine antibiotics were isolated from Lysobacter antibioticus OH13. A 10 gene cluster was identified for phenazine biosynthesis. Mutation of LaPhzNO1 abolished all N-oxides, while non-oxides markedly increased. LaPhzNO1 is homologous to Baeyer-Villiger flavoproteins but was shown to catazlye phenazine N-oxidation. LaPhzNO1 and LaPhzS together converted phenazine 1,6-dicarboxylic acid to 1,6-dihydroxyphenazine N5,N10-dioxide. LaPhzNO1 also catalyzed N-oxidation of 8-hydroxyquinoline.

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.

Modular polyketide synthases (type I PKSs) in bacteria are responsible for synthesizing a significant percentage of bioactive natural products. This group of synthases has a characteristic modular organization, and each module within a PKS carries out one cycle of polyketide chain elongation; thus each module is “non-iterative” in function. It was possible to predict the basic structure of a polyketide product from the module organization of the PKSs, since there generally existed a co-linearity between the number of modules and the number of chain elongations. However, more and more bacterial modular PKSs fail to conform to the “canonical rules”, and a particularly noteworthy group of non-canonical PKSs is the bacterial iterative type I PKSs. This review covers recent examples of iteratively-used modular PKSs in bacteria. These non-canonical PKSs give rise to a large array of natural products with impressive structural diversity. The molecular mechanism behind the iterations is often unclear, presenting a new challenge to the rational engineering of these PKSs with the goal of generating new natural products. Structural elucidation of these synthase complexes and better understanding of potential PKS-PKS interactions as well as PKS-substrate recognition may provide new prospects and inspirations for the discovery and engineering of new bioactive polyketides.