Mining Secondary Metabolites From Deep-sea Derived Streptomyces Somaliensis SCSIO ZH66 By Genetic Manipulation

The deep-sea drived bacterium S. somaliensis SCSIO ZH66 was isolated from the sediments of the South China Sea (a depth of 3536 m). The extreme living environment such as high salt, high pressure, low temperature, low oxygen, low light and oligotrophic conditions endow the S. somaliensis SCSIO ZH66 with unique secondary metabolic pathways, which are able to produce diverse secondary metabolites with novel chemical structures and biological activities. The genome size of S. somaliensis SCSIO ZH66 is about 7.1 Mb, and is composed of 124 contigs, harboring 6312 putative open reading frames (ORFs). Bioinformatic analysis revealed the presence of at least 16 secondary metabolites biosynthetic gene clusters. In this study, gene inactivation and heterologous expression were performed to do genome mining of S. somaliensis SCSIO ZH66.Firstly, we studied the nonribosomal peptide biosynthetic gene cluster nrps-3 located in contig67. Bioinformatic analysis showed that nrps-3 probably encodes a compound with novel structure, which is built from 6 amino acids and contains modifications of glycosylation and methylation. To identify the product encoded by nrps-3, the nonpeptide synthetase gene orf8 was inactivated. The fermentation broth of the △orf8 mutant was extracted with EtOAc, and then was subjected to the HPLC analysis. According to the result, the secondary metabolites of mutant strain △orf8 showed significant difference compared to those of the wild type strain. Then, the EtOAc extract of △orf8 mutant was fractionated by C18 open column chromatography to obtain 15 fractions. In the following antibacterial test, Fr.6 exhibited significant activity against methicillin-resistant Staphylococcus aureus (MRSA). After further separation of Fr.6 using semi-preparative HPLC, we obtained 5 compounds, which were identified to be violapyrones A-C, H and I by combination of MS and NMR analysis. Violapyrone B (100 μg) exhibited strong antibacterial activity against MRS A with the inhibition zone of 25 mm.Secondly, the pleiotropic regulatory genes regP12, which are from Streplomyces sp. OUC6819, were heterologously expressed in S. somaliensis SCSIO ZH66. The regP12 gene was expressed under the control of the constitutive promoter gapDHp. The recombinant strain was cultured under the same conditions with those of the wild type strain. HPLC analysis of the fermentation products showed that the profiles of secondary metabolites are quite different between them. Further study is going on to isolate the new compounds accumulated in the recombinant strain.In summary, production of 5 pyranoid ketone compounds (violapyrones A-C, H and I) was significantly increased in the △orf8 mutant strain, one of which (violapyrone B) exhibited strong inhibition against MRSA. The results showed that inactivation of orf8 gene impacted on the production of violapyrone compounds in S. somaliensis SCSIO ZH66. The heterologous expression of the pleiotropic regulatory genes regP12 in S. somaliensis SCSIO ZH66 revealed that the production of secondary metabolites in the recombinant strain changed a lot compared to that of the wild type strain. Thus, it is speculated that regP12 have regulate the production of secondary metabolites in S. somaliensis SCSIO ZH66 as well.