Activation And Enhancement Of Cryptic Secondary Metabolite From Deep Sea-derived Streptomyces Somaliensis SCSIO ZH66

Marine Streptomyces strains are an important source of new drug leads. However, the discovery and sustainable production of these compounds are often hampered due to the unavailable expression of cryptic biosynthetic gene clusters or limited titer.Deepsea-derived S. somaliensis SCSIO ZH66 was studied in this paper, ribosome engineering was applied to activate the anti-cancer lead compound FDM A cryptic gene cluster, and then FDM A production was effectively increased by fermentation engineering. The main results are as follows:7 rifampin-resistant strains and 4 streptomycin-resistant strains were obtained by ribosome engineering. Among them, RIF1 mutant strain, which is resistant to 300 μg/ mL of rifampicind, accumulated a brown pigment with antitumor bioactivity on MS plate while absent in the other mutants and wide type strains. After screening of fermentation conditions, the compound with pigment was purified and identified to be FDM A according to NMR data, indicating that the activation of cryptic FDM A biosynthetic gene cluster was taken place in strain RIF1.Further, the ropB gene sequence of RIF1 mutant was analysed, revealing the mut ation of R444H in the β sub unit of RNA polymerase. To further clarify the impact of RNAP (3-subunit change on the expression of FDM A gene cluster, semi-quantitative RT-PCR and quantitative real time RT-PCR were performed. The wild-type strain and RIF1 mutant were cultured on MS plate for 3d,5d and 7d. The result showed that the expression of fdmD and fdmR1 genes were actiated in RIF1 mutant after incubation fo r 5d and 7d, and the transcriptional levels of fdmD and fdmRl genes in the RIF1 muta nt were significantly increased by 15-fold compared with those in the wild-type strain when cultured on MS plate for 7d.To improve the yield efficiently, culture conditions and medium components wer e optimized for FDM A production by single factor experiment, Plackett-Burman factorial design and Box-Behnken design. The optimum medium compositio n was achieved under which the titer of FDM A reached 679.5 mg/L, representing a 3-fold increase compared to the original medium. In terms of short fermentation time a nd low-cost fermentation medium, strain RIF1 would be an ideal alternative source fo r FDM A production.In summary, ribosome engineering was applied to generate resistant strains of S. somaliensis SCSIO ZH66. RIF1 mutant was obtained, in which anticancer activity co mpound FDM A was accumulated. Our results would hasten the efforts for further de velopment of FDM A as a drug candidate. Moreover, this ribosomal engineering and f ermentation engineering integrated methodology is effective, fast and efficient; it wou Id be applicable to genome mining for novel natural products from other strains.