A gene designated jadI from the jadomycin B producer Streptomyces venezuelae has homology to the tetracenomycin pathway tcmI gene and encodes a putative cyclase for angucyclinone biosynthesis. Expression in Streptomyces lividans of a jadomycin (jad) gene cassette composed of the minimal polyketide synthase (PKS, jadABC), a cyclase (jadD), a ketoreductase (jadE), and jadI leads to production of several yellow compounds in liquid culture. Rabelomycin (1), a known angucyclinone, results from dehydration and oxidation of a new product, UWM6, isolated from the culture extracts. Characterization of UWM6 by UV, MS, and NMR analyses revealed a new angucyclinone structure, 4-hydroxy-12bH-12-deoxyrabelomycin. Production of these angucyclinones occurs only when jadI is present in the cassette, suggesting an essential cyclase phenotype for this gene. Engineered replacement of jadI in the jad cassette with the tcmI cyclase gene, and similar replacement of tcmI in a functional tcm PKS cassette with jadI, leads to the same set of aromatic decapolyketides produced by either cassette in the absence of jadI. From these results we conclude that both cyclases are nonfunctional out of their normal context.

The lomaiviticins are a family of cytotoxic marine natural products that have captured the attention of both synthetic and biological chemists due to their intricate molecular scaffolds and potent biological activities. Here we describe the identification of the gene cluster responsible for lomaiviticin biosynthesis in Salinispora pacifica strains DPJ-0016 and DPJ-0019 using a combination of molecular approaches and genome sequencing. The link between the lom gene cluster and lomaiviticin production was confirmed using bacterial genetics, and subsequent analysis and annotation of this cluster revealed the biosynthetic basis for the core polyketide scaffold. Additionally, we have used comparative genomics to identify candidate enzymes for several unusual tailoring events, including diazo formation and oxidative dimerization. These findings will allow further elucidation of the biosynthetic logic of lomaiviticin assembly and provide useful molecular tools for application in biocatalysis and synthetic biology.

Expression of biosynthetic pathways in heterologous hosts is an emerging approach to expedite production improvement and biosynthetic modification of natural products derived from microbial secondary metabolites. Herein we describe the development of a versatile Escherichia coli-Streptomyces shuttle Bacterial Artificial Chromosomal (BAC) conjugation vector, pSBAC, to facilitate the cloning, genetic manipulation, and heterologous expression of actinomycetes secondary metabolite biosynthetic gene clusters. The utility of pSBAC was demonstrated through the rapid cloning and heterologous expression of one of the largest polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic pathways: the meridamycin biosynthesis gene cluster (mer). The entire mer gene cluster ( approximately 90 kb) was captured in a single pSBAC clone through a straightforward restriction enzyme digestion and cloning approach and transferred into Streptomyces lividans. The production of meridamycin (1) in the heterologous host was achieved after replacement of the original promoter with an ermE* promoter and was enhanced by feeding with a biosynthetic precursor. The success of heterologous expression of such a giant gene cluster demonstrates the versatility of BAC cloning technology and paves the road for future exploration of expression of the meridamycin biosynthetic pathway in various hosts, including strains that have been optimized for polyketide production.

The plasmid-based reconstruction of the avr deoxysugar genes for expression in a heterologous system combined with biotransformation has led to new information about the mechanism of 2,6-deoxysugar biosynthesis. The structures of the di-demethyldeoxysugar avermectins accumulated indicate that in the oleandrose pathway the stereochemistry at C-3 is ultimately determined by the 3-O-methyltransferase and not by the 3-ketoreductase or a possible 3,5-epimerase. The AvrF protein is therefore a 5-epimerase and not a 3,5-epimerase. The ability of the AvrB (mono-)glycosyltransferase to accommodate different deoxysugar intermediates is evident from the structures of the novel avermectins produced.

Analysis of a region of chromosomal DNA lying between jadR 1 and jadI in the gene cluster for jadomycin biosynthesis in Streptomyces venezuelae ISP5230 detected an ORF encoding 584 amino acids similar in sequence to the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) components of acyl-coenzyme A carboxylases. Multiple sequence alignments of the deduced Jad protein with acyl-coenzyme A carboxylases from various sources located the BC and BCCP components in the N-and C-terminal regions, respectively, of the deduced polypeptides. The organization and amino acid sequence of the deduced polypeptide most closely resembled those in other Gram-positive bacteria broadly classified as actinomycetes. Disrupting the gene, designated jadJ, severely reduced but did not eliminate jadomycin production. The disruption had no effect on growth or morphology of the organism, implying that the product of jadJ is not essential for fatty acid biosynthesis. It is concluded that jadJ supplies malonyl-coenzyme A for biosynthesis of the polyketide intermediate that is eventually processed to form the antibiotic jadomycin B.

Microbial transformations were conducted with prosulfuron {1-(4‘-methoxy-6‘-methyltriazin-2‘-yl)-3-[[2‘‘-(3‘‘‘,3‘‘‘,3‘‘‘-trifluoropropyl)phenyl]sulfonyl]urea} to identify pathways by which the sulfonylurea herbicide is metabolized by microorganisms. Streptomyces griseolus produced four new hydroxylated metabolites which were isolated, chromatographically purified, and characterized by mass spectrometry and NMR spectroscopy as the following:  a benzyl alcohol from methyl hydroxylation on the triazine ring, phenolic and catechol metabolites by 3‘‘ and subsequent 4‘‘ hydroxylation on the carbocyclic aromatic ring, and a second benzylic alcohol obtained by microbial hydroxylation of the trifluoropropyl side chain. The new metabolites were used as analytical standards for soil incubation studies. Keywords: Prosulfuron; sulfonylurea; oxidative biotransformations; microbial metabolites; benzylic and aromatic hydroxylations; O-demethylation; hydrolysis

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