Biosynthesis of HSAF, a Tetramic Acid-Containing Macrolactam from Lysobacter enzymogenes

Abstract

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.

Lysobacter enzymogenes Uses Two Distinct Cell-Cell Signaling Systems for Differential Regulation of Secondary-Metabolite Biosynthesis and Colony Morphology

“…The extraction of HSAF from L. enzymogenes was performed as described previously (31,33). We cultured wild-type strain OH11 and its derivative strains in 50 ml of 10% TSB supplemented with 100 g/ml Km at 28°C in a rotary shaker at 200 rpm for 3 days.…”

“…The production of lytic enzymes, such as chitinases, proteases, and glucanases, is tightly linked to L. enzymogenes biocontrol activity. Recently, two antimicrobial secondary metabolites, including heat-stable antifungal factor (HSAF) and WAP-8294A2, have also been shown to be important in L. enzymogenes for growth inhibition of filamentous fungi and Gram-positive bacteria, respectively (31)(32)(33)(34).…”

“…HSAF is a tetramic acid (2,4-pyrrolidinedione)-containing macrolactam and contains a 5,5,6-tricyclic system fused to the macrolactam (31,33). This antibiotic exhibits remarkably inhibitory activities against a wide range of plant and animal fungal pathogens (32).…”

“…These results indicate that HSAF potentially represents a novel and important source of environmentally friendly pesticides and antifungal drugs as the sphingolipid structure in fungal cells is distinct from that in plants and mammalian cells (35). Recently, we cloned the gene clusters responsible for HSAF biosynthesis and investigated the unique biosynthetic mechanism in L. enzymogenes (33). Collectively, the novelty in chemical structure, mode of action, and biosynthetic mechanism of HSAF has encouraged us to understand the regulatory mechanism for its biosynthesis.…”

Volatile communication between fungi and bacteria

“…S4D) resembles clusters encoding the Heat-stable antifungal factor (HSAF), also referred to as dihydromaltophilin, produced by Lysobacter species (Yu et al, 2007, Lou et al, 2011. HSAF exhibits inhibitory activities against a wide range of fungal species by disrupting the polarized growth or the biosynthesis of a distinct group of sphingolipids of fungi (Yu et al, 2007, Lou et al, 2011.…”