Study On The Biosynthesis Of Dirmentines From Mangrove Conservation Derived Streptomyces Sp.OUC6819

Streptomyces sp.OUC6819was isolated from the mangrove sediment of South China Sea. It produces terpenoid-diketopiperazine hybrid compounds Drimentines, which exhibit antibacterial, antifungal, anticancer, and anthelmintic activities. Due to their unique heterozygosity structure and significant bioactivities, Drimentines have attracted much attention. To reveal the biosynthetic mechanism of Drimentines in Streptomyces sp.OUC6819, we have carried out the following studies:1.The genomic library of Streptomyces sp.OUC6819was constructed. Total DNA of Streptomyces sp.OUC6819was extracted and partially digested by restriction endonuclease Sau3AI, and then DNA fragments at the size of30-50kb were recovered and ligated with SuperCos1, which was digested by XbaI,dephosphorylated and redigested by BamHI. The ligation product was packaged into phage particles with Lambda Packaging Extracts, which was then transfected E. coli Trans10to yield the genomic library of Streptomyces sp.OUC6819. The titer of the library was6.0×106cfu/mL, meeting the requirements as a standard genomic library. The clones were stored at-80℃. Construction of the genomic library of Streptomyces sp.OUC6819laid the foundation for cloning of biosynthesis gene cluster of secondary metabolites and their heterologous expression.2. By using strategies of chemical informatics and bioinformatics analysis, the biosynthetic gene cluster of Drimentines was identified and isolated from the genome of Streptomyces sp.OUC6819. The gene cluster is about33kb, consisting of23open reading frames (ORFs):driB, C, D, E, F are proposed to be associated with the biosynthesis of the sesquiterpene structure; driK, M, N, O, P, V are proposed to be involved in the biosynthesis of diketopiperazine skeleton; driJ and driL are responsible for the synthesis of tryptophan; driA,Q,T,W are regulatory genes; driS is proposed to be related to resistance.3.By λ-RED combination strategy, the recombinant cosmid for driB (encoding polyprenyl diphosphate synthetase) inactivation was constructed and transferred to Streptomyces sp.OUC6819by conjugation. Through double crossover,△driB mutant was selected and confirmed by antibiotic resistance and PCR analysis. Both the wide type and mutant strain were fermented under the same condition, and the fermentation products were analyzed by using HPLC-ESI-MS analysis. The result showed that the△driB mutant lost the ability to produce Drimentines, which suggests that driB is essential for Drimentines biosynthesis.4.Heterologous expression of the Drimentines biosynthetic gene cluster was investigated. Cosmids containing different version of the Drimentines biosynthetic gene cluster were constructed, and then introducted into Streptomyces coelicolor M1152by conjugation. The exconjugants were fermentated using different growth media, however, no Drimentines could be detectable by using HPLC-MS analysis, indicating the cluster may be incomplete or unexpressed in S. coelicolor M1152.5. The catalytic activity of DriM was studied in vitro. By bioinformatics analysis, driM is proposed to be involved in the biosynthesis of diketopiperazine skeleton of Drimentines. The recombinant plasmid for driM expression was constructed, and was successfully expressed in E. coli CondonPlus(DE3). The catalytic activity of DriM was characterized, and the result revealed DriM showed wide substrate flexibility and is able to catalyze the adenylylation of all the tested amino acids. Futher kinetics studies are currently going on.The above studies laid the foundation for deciphering the molecular mechanism of the biosynthetic pathway of Drimentines, and furthermore carrying out combinatorial biosynthesis studies to obtain Drimentines analogues or high-yield producing strains.