An industrial microorganism Streptomyces avermitilis, which is a producer of anthelmintic macrocyclic lactones, avermectins, has been constructed as a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis. Twenty of the entire biosynthetic gene clusters for secondary metabolites were successively cloned and introduced into a versatile model host S. avermitilis SUKA17 or 22. Almost all S. avermitilis transformants carrying the entire gene cluster produced metabolites as a result of the expression of biosynthetic gene clusters introduced. A few transformants were unable to produce metabolites but their production was restored by the expression of biosynthetic genes using an alternative promoter or the expression of a regulatory gene in the gene cluster that controls the expression of biosynthetic genes in the cluster using an alternative promoter. Production of metabolites in some transformants of the versatile host was higher than that of the original producers and cryptic biosynthetic gene clusters in the original producer were also expressed in a versatile host.

Polyketides form many clinically valuable compounds. However, manipulation of their biosynthesis remains highly challenging. An understanding of gene cluster evolution provides a rationale for reprogramming of the biosynthetic machinery. Herein, we report characterization of giant modular polyketide synthases (PKSs) responsible for the production of aminopolyol polyketides. Heterologous expression of over 150 kbp polyketide gene clusters successfully afforded their products, whose stereochemistry was established by taking advantage of bioinformatic analysis. Furthermore, phylogenetic analysis of highly homologous but functionally diverse domains from the giant PKSs demonstrated the evolutionary mechanism for structural diversification of polyketides. The gene clusters characterized herein, together with their evolutionary insights, are promising genetic building blocks for de novo production of unnatural polyketides.

Versipelostatin (VST) is an unusual 17-membered macrocyclic polyketide product that contains a spirotetronate skeleton. In this study, the entire VST biosynthetic gene cluster (vst) spanning 108 kb from Streptomyces versipellis 4083-SVS6 was identified by heterologous expression using a bacterial artificial chromosome vector. Here, we demonstrate that an enzyme, VstJ, catalyzes the stereoselective [4+2]-cycloaddition between the conjugated diene and the exocyclic olefin of a newly identified tetronate-containing intermediate to form the spirotetronate skeleton during VST biosynthesis.

JBIR-34 and -35 produced by Streptomyces sp. Sp080513GE-23 are nonribosomal peptides that possess an unusual 4-methyloxazoline moiety. Through draft genome sequencing, cosmid cloning, and gene disruption, the JBIR-34 and -35 biosynthesis gene cluster (fmo cluster) was identified; it encodes 20 proteins including five nonribosomal peptide synthetases (NRPSs). Disruption of one of these NRPS genes (fmoA3) resulted in no JBIR-34 and -35 production and accumulation of 6-chloro-4-hydroxyindole-3-carboxylic acid. Stable isotope-feeding experiments indicated that the methyl group of the methyloxazoline ring is derived from alanine rather than methionine. A recombinant FmoH protein, a glycine/serine hydroxymethyltransferase homolog, catalyzed conversion of α-methyl-l-serine into d-alanine (the reverse reaction of α-methyl-l-serine synthesis catalyzed by FmoH in vivo). Taken together, we concluded that α-methyl-l-serine synthesized from d-alanine is incorporated into JBIR-34 and -35 to form the 4-methyloxazoline moiety. We also propose the biosynthesis pathway of JBIR-34 and -35.

Thioviridamide (1) is an N-acylated undecapeptide antibiotic that induces apoptosis selectively in E1A-transformed cells. 1 The most unique structural feature of thioviridamide is the presence of five thioamide bonds between the amino acids. 2 Recently, the thioviridamide biosynthesis gene cluster of Streptomyces olivoviridis NA05001 was identified, and the heterologous expression of thioviridamide in Streptomyces lividans TK23 was demonstrated. 3 Thioviridamide is synthesized by the posttranslational modification of a ribosomal precursor peptide containing a VMAAAASIALHC sequence. 3 Heterologous expression of a bacterial artificial chromosome (BAC) clone prepared from S. olivoviridis OM13 containing the entire gene cluster for thioviridamide biosynthesis in S. avermitilis SUKA17 strain, 4,5 which is the suitable host for heterologous expression, resulted in the expression of a novel thioviridamide derivative, JBIR-140 (2), together with thioviridamide. Here, we report the isolation, structure determination and biological activity of 2.The BAC genomic library of S. olivoviridis OM13 was prepared according to a previously reported method. 4 S. olivoviridis OM13 genome was partially digested with BamHI in an agarose gel and then subjected to contour-clamped homogeneous electric field electrophoresis. Fragments (size, 100-130 kb) were then excised from the gel. The purified DNA fragments were ligated to the BamHI fragment of the integrating BAC vector pKU503. 5 The ligated DNAs were transformed into Escherichia coli NEB 10-β cells (New England Biolabs, Ipswich, MA, USA) by electroporation. 4,5 Each BAC clone was stored in five 384-well plates containing Plusgrow II (100 μg ml − 1 ampicillin and 20% glycerol) at − 80°C. Clones carrying the entire gene cluster for thioviridamide biosynthesis were screened by PCR amplification using two pairs of primers corresponding to the upstream and downstream regions of the gene cluster (upstream primer pair, forward: 5′-CA GATGGACACGTACACGCAGAC-3′ (corresponding to the region from nucleotides 5539 to 5561 of the thioviridamide biosynthesis gene cluster 3 ) and reverse: 5′-CACGTTCGTAGTAGCTGTCGGA GA-3′ (6187-6164 3 ); downstream primer pair, forward: 5′-AGT GGAGCGGTGCTACGACATC-3′ (28 124-28 145 3 )) and reverse: 5′-T CCGGTACAGCTTCTTGTACTCCA-3′(27 567-27 544 3 )). The amplification was performed under the following conditions: denaturation at 95°C for 5 min, 35 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 60 s, and a final incubation at 72°C for 5 min, and then soaked at 12°C. One of the BAC clones, pKU503thvP3-F8 (inset size: 71 900 bp), contained the entire gene cluster for thioviridamide biosynthesis. Unmethylated pKU503thvP3-F8 was prepared by transforming the plasmid into E. coli GM2929 hsdS::Tn10 cells. 5 The resultant DNA preparation was introduced into S. avermitilis SUKA17 by polyethylene glycol-assisted protoplast transformation. 4,5 Integration of the plasmids was confirmed by neomycin resistance and PCR analysis using the above-mentioned primer pairs. SUKA17 carry...