Increasing production of spinosad in Saccharopolyspora spinosa by metabolic engineering.

Spinosad, a combination of spinosyn A and D produced by Saccharopolyspora spinosa, is a highly efficient pesticide. There has been a considerable interest in the improvement of spinosad production because of a low yield achieved by wild-type S. spinosa. In this study, we designed and constructed a pIBR-SPN vector. pIBR-SPN is an integrative vector that can be used to introduce foreign genes into the chromosome of S. spinosa. Different combinations of genes encoding forasamine and rhamnose were synthesized and used for the construction of different recombinant plasmids. The following recombinant strains were developed: S. spinosa pIBR-SPN (only the vector), S. spinosa pIBR-SPN F (forosamine genes), S. spinosa pIBR-SPN R (rhamnose genes), S. spinosa pIBR-SPN FR (forosamine and rhamnose genes), S. spinosa pIBR-SPN FRS (forosamine, rhamnose, and SAM [S-adenosyl-L-methionine synthetase] genes), and S. spinosa MUV pIBR-SPN FR. Among these recombinant strains, S. spinosa pIBR-SPN FR produced 1394 ± 163 mg/L spinosad, which was 13-fold higher than the wild-type. S. spinosa MUV pIBR-SPN FR produced 1897 (±129) mg/L spinosad, which was seven-fold higher than S. spinosa MUV and 17-fold higher than the wild-type strain.

Identification and enhancing production of a novel macrolide compound in engineered Streptomyces peucetius.

Streptomyces peucetius produces doxorubicin and daunorubicin, which are important anticancer drugs. In this study, we activate peucemycin, a new antibacterial compound, using an OSMAC strategy. In general, bioactive compounds are produced in a higher amount at room temperature; however, in this study, we have demonstrated that a bioactive novel compound was successfully activated at a low temperature (18 °C) in S. peucetius DM07. Through LC-MS/MS, IR spectroscopy, and NMR analysis, we identified the structure of this compound as a γ-pyrone macrolide. This compound was found to be novel, thus named peucemycin. It is an unusual 14-membered macrocyclic γ-pyrone ring with cyclization. Also, peucemycin exhibits potential antibacterial activity and a suppressive effect on the viability of various cancer cell lines.

Recent Advances in Strategies for Activation and Discovery/Characterization of Cryptic Biosynthetic Gene Clusters in Streptomyces.

Streptomyces spp. are prolific sources of valuable natural products (NPs) that are of great interest in pharmaceutical industries such as antibiotics, anticancer chemotherapeutics, immunosuppressants, etc. Approximately two-thirds of all known antibiotics are produced by actinomycetes, most predominantly by Streptomyces. Nevertheless, in recent years, the chances of the discovery of novel and bioactive compounds from Streptomyces have significantly declined. The major hindrance for obtaining such bioactive compounds from Streptomyces is that most of the compounds are not produced in significant titers, or the biosynthetic gene clusters (BGCs) are cryptic. The rapid development of genome sequencing has provided access to a tremendous number of NP-BGCs embedded in the microbial genomes. In addition, the studies of metabolomics provide a portfolio of entire metabolites produced from the strain of interest. Therefore, through the integrated approaches of different-omics techniques, the connection between gene expression and metabolism can be established. Hence, in this review we summarized recent advancements in strategies for activating cryptic BGCs in Streptomyces by utilizing diverse state-of-the-art techniques.

Streptomyces sp. VN1, a producer of diverse metabolites including non-natural furan-type anticancer compound.

Streptomyces sp. VN1 was isolated from the coastal region of Phu Yen Province (central Viet Nam). Morphological, physiological, and whole genome phylogenetic analyses suggested that strain Streptomyces sp. VN1 belonged to genus Streptomyces. Whole genome sequencing analysis showed its genome was 8,341,703 base pairs in length with GC content of 72.5%. Diverse metabolites, including cinnamamide, spirotetronate antibiotic lobophorin A, diketopiperazines cyclo-L-proline-L-tyrosine, and a unique furan-type compound were isolated from Streptomyces sp. VN1. Structures of these compounds were studied by HR-Q-TOF ESI/MS/MS and 2D NMR analyses. Bioassay-guided purification yielded a furan-type compound which exhibited in vitro anticancer activity against AGS, HCT116, A375M, U87MG, and A549 cell lines with IC50 values of 40.5, 123.7, 84.67, 50, and 58.64 µM, respectively. In silico genome analysis of the isolated Streptomyces sp. VN1 contained 34 gene clusters responsible for the biosynthesis of known and/or novel secondary metabolites, including different types of terpene, T1PKS, T2PKS, T3PKS, NRPS, and hybrid PKS-NRPS. Genome mining with HR-Q-TOF ESI/MS/MS analysis of the crude extract confirmed the biosynthesis of lobophorin analogs. This study indicates that Streptomyces sp. VN1 is a promising strain for biosynthesis of novel natural products.

Exploration of cryptic organic photosensitive compound as Zincphyrin IV in Streptomyces venezuelae ATCC 15439.

Zincphyrin IV is a potential organic photosensitizer which is of significant interest for applications in biomedicine, materials science, agriculture (as insecticide), and chemistry. Most studies on Zincphyrin are focused on Zincphyrin III while biosynthesis and application of Zincphyrin IV is comparatively less explored. In this study, we explored Zincphyrin IV production in Streptomyces venezuelae ATCC 15439 through combination of morphology engineering and "One strain many compounds" approach. The morphology engineering followed by change in culture medium led to activation of cryptic Zincphyrin IV biosynthetic pathway in S. venezuelae with subsequent detection of Zincphyrin IV. Morphology engineering applied in S. venezuelae increased the biomass from 7.17 to 10.5 mg/mL after 48 h of culture. Moreover, morphology of engineered strain examined by SEM showed reduced branching and fragmentation of mycelia. The distinct change in color of culture broth visually demonstrated the activation of the cryptic biosynthetic pathway in S. venezuelae. The production of Zincphyrin IV was found to be initiated after overexpression ssgA, resulting in the increase in titer from 4.21 to 7.54 µg/mL. Furthermore, Zincphyrin IV demonstrated photodynamic antibacterial activity against Bacillus subtilis and photodynamic anticancer activity against human ovarian carcinoma cell lines.