Identification of a cyclohexylcarbonyl CoA biosynthetic gene cluster and application in the production of doramectin

Abstract

The side chain of the antifungal antibiotic ansatrienin A from Streptomyces collinus contains a cyclohexanecarboxylic acid (CHC)-derived moiety. This moiety is also observed in trace amounts of omega-cyclohexyl fatty acids (typically less than 1% of total fatty acids) produced by S. collinus. Coenzyme A-activated CHC (CHC-CoA) is derived from shikimic acid through a reductive pathway involving a minimum of nine catalytic steps. Five putative CHC-CoA biosynthetic genes in the ansatrienin biosynthetic gene cluster of S. collinus have been identified. Plasmid-based heterologous expression of these five genes in Streptomyces avermitilis or Streptomyces lividans allows for production of significant amounts of omega-cyclohexyl fatty acids (as high as 49% of total fatty acids). In the absence of the plasmid these organisms are dependent on exogenously supplied CHC for omega-cyclohexyl fatty acid production. Doramectin is a commercial antiparasitic avermectin analog produced by fermenting a bkd mutant of S. avermitilis in the presence of CHC. Introduction of the S. collinus CHC-CoA biosynthetic gene cassette into this organism resulted in an engineered strain able to produce doramectin without CHC supplementation. The CHC-CoA biosynthetic gene cluster represents an important genetic tool for precursor-directed biosynthesis of doramectin and has potential for directed biosynthesis in other important polyketide-producing organisms.

Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate

“…2). In light of the present findings, it would be worthwhile to investigate the in vitro activity of those enzymes thought to catalyze the latter reaction (20)(21)(22)(23)(24)(25) to obtain definitive evidence for or against their acting as chorismatases, rather than shikimate dehydratases.…”

“…2), which is activated by an ATPdependent adenylation domain in the loading module of the polyketide synthase, transferred to the acyl carrier protein domain of this module, and then reduced in situ by an adjacent enoylreductase domain (20). The route to DHCHC from shikimate remains unknown, but it has been suggested, based on extensive isotope labeling and heterologous gene expression analysis of related pathways in other bacteria (21)(22)(23)(24)(25), to involve the pathway shown in Fig. 2.…”

Biochemical and Genetic Insights into Asukamycin Biosynthesis

“…asukaensis was constructed and screened with two 32 Plabeled DNA probes. ChcA, encoding the 1-cyclohexenylcarbonyl CoA reductase of ansatrienin biosynthesis in S. collinus (12), identified two cosmids, 2B9 and 10D6, which revealed a 1.8-kb overlapping region of the cloned DNA inserts. AsuD2, encoding a 2-oxoamine synthase, hybridized only with cosmid 10D6 (15).…”

“…The identical lower and upper triene polyketide chains are built up from 3,4-AHBA and cyclohexylcarbonyl CoA (CHC-CoA), respectively, by three steps of classical polyketide condensations. A gene set involved in the CHCCoA biosynthesis of ansatrienin has been well characterized in Streptomyces collinus (12), but the genes involved in 3,4-AHBA and polyketide chain assembly of asukamycin have not been identified. The C 5 N ring moiety, found in several natural products including reductiomycin, moenomycin A, and ECO-02301, was suggested to be derived from a 5-aminolevulinate (5-ALA) intermediate (13)(14)(15)(16).…”

Hyper-inducible expression system for streptomycetes

“…These organisms have been very well studied, especially S. coelicolor (28,29) and S. avermitilis (30,31), for each of which the whole genome sequence has already been determined and extensive genomic information involving biosynthetic pathways for useful products or other metabolic pathways has been revealed. The combined application of the genomic information and regulated expression systems such as that described here could contribute to the systematic improvement of productivity in streptomycetes, for example, by enhancing the predicted rate-limiting steps of biosynthetic pathways (32)(33)(34)(35)(36)(37)(38). In the cases that the tsr gene in pSH19 is not ideal as a selection marker [e.g., the host strains are resistant to thiostrepton (3) or the addition of thiostrepton can induce a regulon of genes via the tipA system (3, 7)], an alternative selection marker that could be substituted for tsr would be preferable.…”