Genetic Localization and Molecular Characterization of the nonS Gene Required for Macrotetrolide Biosynthesis in Streptomyces griseus DSM40695

Abstract

The macrotetrolides are a family of cyclic polyethers derived from tetramerization, in a stereospecific fashion, of the enantiomeric nonactic acid (NA) and its homologs. Isotope labeling experiments established that NA is of polyketide origin, and biochemical investigations demonstrated that 2-methyl-6,8-dihydroxynon-2E-enoic acid can be converted into NA by a cell-free preparation from Streptomyces lividans that expresses nonS. These results lead to the hypothesis that macrotetrolide biosynthesis involves a pair of enantiospecific polyketide pathways. In this work, a 55-kb contiguous DNA region was cloned from Streptomyces griseus DSM40695, a 6.3-kb fragment of which was sequenced to reveal five open reading frames, including the previously reported nonR and nonS genes. Inactivation of nonS in vivo completely abolished macrotetrolide production. Complementation of the nonS mutant by the expression of nonS in trans fully restored its macrotetrolide production ability, with a distribution of individual macrotetrolides similar to that for the wild-type producer. In contrast, fermentation of the nonS mutant in the presence of exogenous (؎)-NA resulted in the production of nonactin, monactin, and dinactin but not in the production of trinactin and tetranactin. These results prove the direct involvement of nonS in macrotetrolide biosynthesis. The difference in macrotetrolide production between in vivo complementation of the nonS mutant by the plasmid-borne nonS gene and fermentation of the nonS mutant in the presence of exogenously added (؎)-NA suggests that NonS catalyzes the formation of (؊)-NA and its homologs, supporting the existence of a pair of enantiospecific polyketide pathways for macrotetrolide biosynthesis in S. griseus. The latter should provide a model that can be used to study the mechanism by which polyketide synthase controls stereochemistry during polyketide biosynthesis.

Comment on “Asymmetric syntheses of sceptrin and massadine and evidence for biosynthetic enantiodivergence”

“…(3-14) Little is known about the intricate genetic, biocatalytic and mechanistic details of how enantiomeric, or pseudo-enantiomeric metabolites, are constructed. Co-isolated congeners in the same, as well as distinct oxidation states, presumably derived from the same pathway or, via parallel pathways are evident, including the biogenesis postulated by Ma.…”

Nitrogen Incorporation in the Biosynthetic Pathway of the Nitrogen-containing Polyketide, Pamamycin in Streptomyces alboniger

“…Molecular biological research of nonactin which consists of stereoisomeric homologues of hydroxy acid S in pamamycin, was carried by Priestley et al [18] and Shen et al [19]. We postulate that the homologue of nonS gene, which encodes the enzyme catalyzing the formation of a tetrahydrofuran ring of the constituent hydroxy acid of nonactin, exists in the pamamycin biosynthetic gene cluster.…”

Polyketide Biosynthesis beyond the Type I, II, and III Polyketide Synthase Paradigms: A Progress Report

“…[15][16][17][18] On the basis of these results we concluded that NonJK represented C-0 bond-forming type II PKSs that act directly on (±)-8 to catalyze their tetramerization and cyclization into 7, demonstrating the first time that PKS has the intrinsic propensity to evole catalytic activities catalyzing other than C-C bond formation ( Figure 3A). We have now co-expressed nonJK in E. coli and showed that cell-free extract made from E. coli overexpressing nonJK can efficiently catalyze the synthesis of 7 from (±)-8; no activity was detected in cell-free extracts made from E. coli carrying the expression vector as a negative control or constructs expressing nonJ or nonK alone.…”