Draft Genome Sequence of Marine Actinobacterium Streptomyces spinoverrucosus SNB-032.

Actinobacteria represent a large source of diverse bioactive compounds of medical and economic importance. Here, we report the 8.8-Mb draft genome of the marine bacterium Streptomyces spinoverrucosus SNB-032. Bioinformatic sequence analysis proved similarities to known Streptomyces strains and revealed the capacity for the production of various secondary metabolites.

Biosynthesis of the Tricyclic Aromatic Type II Polyketide Rishirilide: New Potential Third Ring Oxygenation after Three Cyclization Steps.

Rishirilides are a group of PKS II secondary metabolites produced by Streptomyces bottropensis Gö C4/4. Biosynthetic studies in the past have elucidated early and late steps of rishirilide biosynthesis. This work is aiming to solve the remaining steps in the rishirilide biosynthesis. Inactivation of the cyclase gene rslC3 in Streptomyces bottropensis resulted in an interruption of rishirilide production. Instead, accumulation of the tricyclic aromatic galvaquinones was observed. Similar results were observed after deletion of rslO4. Closer inspection into RslO4 crystal structure in addition to site-directed mutagenesis and molecular dynamic simulations revealed that RslO4 might be responsible for quinone formation on the third ring. The RslO1 three-dimensional structure shows a high similarity to FMN-dependent luciferase-like monooxygenases such as the epoxy-forming MsnO8 which acts with the flavin reductase MsnO3 in mensacarcin biosynthesis in the same strain. The high sequence similarity between RslO2 and MsnO3 suggests that RslO2 provides RslO1 with reduced FMN to form an epoxide that serves as substrate for RslO5.

Early Steps in the Biosynthetic Pathway of Rishirilide B.

The biological active compound rishirilide B is produced by Streptomyces bottropensis. The cosmid cos4 contains the complete rishirilide B biosynthesis gene cluster. Its heterologous expression in the host Streptomyces albus J1074 led to the production of rishirilide B as a major compound and to small amounts of rishirilide A, rishirilide D and lupinacidin A. In order to gain more insights into the biosynthesis, gene inactivation experiments and gene expression experiments were carried out. This study lays the focus on the functional elucidation of the genes involved in the early biosynthetic pathway. A total of eight genes were deleted and six gene cassettes were generated. Rishirilide production was not strongly affected by mutations in rslO2, rslO6 and rslH. The deletion of rslK4 and rslO3 led to the formation of polyketides with novel structures. These results indicated that RslK4 and RslO3 are involved in the generation or selection of the starter unit for rishirilide biosynthesis. In the rslO10 mutant strain, two novel compounds were detected, which were also produced by a strain containing solely the genes rslK1, rslK2, rslK3, rslK4, and rslA. rslO1 and rslO4 mutants predominately produce galvaquinones. Therefore, the ketoreductase RslO10 is involved in an early step of rishirilide biosynthesis and the oxygenases RslO1 and RslO4 are most probably acting on an anthracene moiety. This study led to the functional elucidation of several genes of the rishirilide pathway, including rslK4, which is involved in selecting the unusual starter unit for polyketide synthesis.

Biosynthesis of Rishirilide B.

Rishirilide B was isolated from Streptomyces rishiriensis and Streptomyces bottropensis on the basis of its inhibitory activity towards alpha-2-macroglobulin. The biosynthesis of rishirilide B was investigated by feeding experiments with different 13C labelled precursors using the heterologous host Streptomyces albus J1074::cos4 containing a cosmid encoding of the gene cluster responsible for rishirilide B production. NMR spectroscopic analysis of labelled compounds demonstrate that the tricyclic backbone of rishirilide B is a polyketide synthesized from nine acetate units. One of the acetate units is decarboxylated to give a methyl group. The origin of the starter unit was determined to be isobutyrate.