Structure and biosynthesis of heat-stable antifungal factor (HSAF), a broad-spectrum antimycotic with a novel mode of action

Abstract

A screen for antifungal compounds from Lysobacter enzymogenes strain C3, a bacterial biological control agent of fungal diseases, has previously led to the isolation of heat-stable antifungal factor (HSAF). HSAF exhibits inhibitory activities against a wide range of fungal species and shows a novel mode of antifungal action by disrupting the biosynthesis of a distinct group of sphingolipids. We have now determined the chemical structure of HSAF, which is identical to that of dihydromaltophilin, an antifungal metabolite with a unique macrocyclic lactam system containing a tetramic acid moiety and a 5,5,6-tricyclic skeleton. We have also identified the genetic locus responsible for the biosynthesis of HSAF in strain C3. DNA sequencing of this locus revealed genes for a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), a sterol desaturase, a ferredoxin reductase, and an arginase. The disruption of the PKS-NRPS gene generated C3 mutants that lost the ability to produce HSAF and to inhibit fungal growth, demonstrating a hybrid PKS-NRPS that catalyzed the biosynthesis of the unique macrolactam system that is found in many biologically active natural products isolated from marine organisms. In addition, we have generated mutants with disrupted sterol desaturase, ferredoxin reductase, and arginase and examined the metabolites produced in these mutants. The work represents the first study of the genetic basis for the biosynthesis of the tetramic acid-containing macrolactams. The elucidation of the chemical structure of HSAF and the identification of the genetic locus for its biosynthesis establish the foundation for future exploitation of this group of compounds as new fungicides or antifungal drugs.

FIG. 1.

Structures of HSAF and several tetramic acid-containing macrolactams.

FIG. 2.

Scheme for PKS gene disruption in L. enzymogenes strain C3. A 407-bp fragment of the gene (shaded bar) was amplified from the genomic DNA and cloned into a gene disruption vector to produce pJQ200SK-PKS. PCR primers (primers P1, P2, P3, P4, T3, and T7) and their relative positions are shown as small arrows. B, BamHI; E, EcoRI; K, KpnI; P, PstI; S, SacI; Gm^r, gentamicin resistance gene.

FIG. 3.

Screening of the PKS gene-disrupted mutants by PCR assays. Genomic DNA was prepared from the gentamicin-resistant colonies and used as the template for PCR with primers P4 and T7 (Fig. 2). The expected size of the PCR product from homologous recombinants is 522 bp. Lane 1, control reaction without template; lane 2, wild type; middle unnumbered lanes, gentamicin (Gm)-resistant colonies; lane 4, 100-bp DNA ladder.

FIG. 4.

HPLC analysis and antifungal activity assay for the PKS-disrupted mutants. (A and B) HPLC traces of culture broths from mutant strain K19 (A) and wild type (B), with the peak of HSAF indicated; (C and D) activity assay for the culture broth from mutant strain K19 (C) and the wild type (D) by using Bipolaris sorokiniana as the test organism.

FIG. 5.

Partial map of the 13.5-kb region of L. enzymogenes strain C3 hosting the HSAF biosynthetic genes. The predicted functions of the open reading frames are indicated. Restriction enzyme sites: A, ApaI; B, BamHI; E, EcoRI; K, KpnI; S, SacI. Domains of the hybrid PKS-NRPS: KS, ketosynthase; AT, acyltransferase; DH, dehydratase; KR, ketoreductase; ACP, acyl carrier protein; C, condensation; A, adenylation; PCP, peptidyl carrier protein; TE, thioesterase. The highly conserved signature motifs within the domains are shown, with the active-site residues highlighted in boldface letters. MxaD, PKS from Stigmatella aurantiaca (GenBank accession number AAK57188); MxaE, PKS from S. aurantiaca (GenBank accession number AAK57189); HSAF, the PKS-NRPS from L. enzymogenes C3; GenBank accession number YP_529192, PKS-NRPS sequence from Saccharophagus degradans 2-40 (GenBank accession number YP_529192).

FIG. 6.

HPLC analysis of metabolites from mutants with disrupted gene for the NRPS module, ferredoxin reductase, arginase, and sterol desaturase. The results for crude extracts from wild-type strain C3 (A), NRPS-disrupted mutants (B), ferredoxin reductase-disrupted mutants (C), arginase-disrupted mutants (D) and sterol desaturase-disrupted mutants (E) are shown.

FIG. 7.

Proposed biosynthetic pathway for HSAF involving both polyketide and nonribosomal peptide mechanisms. SEnz, the thiol group of the phosphopantetheinyl cofactor of the carrier proteins (acyl carrier protein or peptidyl carrier protein) within the polyketide synthase or nonribosomal pepetide synthetase; OH-Orn, β-hydroxyornithine. Note that the sequence of the biosynthetic steps illustrated here is just one of several possibilities.

FIG. 8.

Proposed mechanism for the formation of the two lactam moieties in HSAF. Note that the NRPS may use ornithine as a substrate and that the β-hydroxyl group may be added after the incorporation of the amino acid; alternatively, NRPS may use β-hydroxyornithine (OH-Orn) directly as substrate. SEnz, the thiol group of the phosphopantetheinyl cofactor of the carrier proteins (acyl carrier protein or peptidyl carrier protein) within the polyketide synthase or nonribosomal pepetide synthetase.