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 describe 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 that 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 activates L-ornithine and that the four-domain NRPS is 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.

Figure 1

(A) Structure of HSAF and related tetramic acid-containing macrolactams. The compounds are divided into two groups based on their possible biosynthetic mechanisms: the hexaketide-ornithine-hexaketide group and the pentaketide-ornithine-hexaketide group. Both groups can have a 17-membered macrolactam fused to a tricyclic system or a 21-membered macrolactam fused to a bicyclic system. (B) Map of the HSAF gene cluster (EF028635.2). The ORFs in the blocked region are annotated here; the annotation of the rest of the ORFs is included in Figure S6.

Figure 2

In vitro synthesis of a tetramic acid-containing product (8) using the purified HSAF NRPS. (A) Schematic illustration of the reactions. The acyl-S-ACP substrate was prepared by incubating ACP and stearoyl-CoA with Svp, and Orn-S-NRPS was prepared by incubating NRPS and CoA with Svp, followed by addition of L-ornithine and ATP. (B) SDS-PAGE showing the expression and purification of the 4-domain NRPS. The expected size of the NRPS is 148.6 kDa. Lane-1, markers; lane-2, purified NRPS; lane-3, soluble fraction of protein extracts; lane-4, total protein extracts upon IPTG induction; lane-5, total protein extracts before IPTG induction. (C) LC-MS detection of the expected product 8. The first is a control reaction without the NRPS, the second is a complete reaction to show the production of the expected product with a [M+H]⁺ of 647.6080, and the third is the chemically synthesized standard 8 (mixture of diastereomers).

Scheme 1

Preparation of tetramic acid 8.