Heterologous Expression Of Methoxymalonyl-ACP Biosynthetic Genes Involved In The Biosynthesis Of FK506

Polyketides are a large family of natural products produced by bacteria, fungi and plants, and include many clinically and agriculturally important antibiotics. Polyketides are biosynthesized from many low-level fatty acids, such as acyl CoA, malonyl CoA, propionyl CoA and so on. The immunosuppressant FK506is a polyketide compound which is regarded as one of the most potential clinically immunosuppressant due to its high efficiency and low toxicity. However, the natural producer of FK506has slow growth rate and low yield, to achieve large-scale fermentation and production of FK506, choosing host strains with faster growth rate or clear genetic background for heterologous production is a new alternative strategy.The size of the biosynthetic gene cluster of FK506is about90kb, and approximately10different kinds of small molecules participate in the biosynthetic process. Among them, MM-ACP (methoxymalonyl-ACP) is the most special one, and the biosynthetic genes of MM-ACP include fkbG,fkbH, fkbI, fkbJ and fkbK with the respective size667bp,1088bp,1100bp,260bp and875bp, respectively. In this study, the biosynthetic genes of MM-ACP from Streptomyces tsukubaensis were cloned into the expression plasmid pET28a either separately or as a whole expression unit to achieve heterologous expression. Meanwhile an integrative vector based on the ribosome DNA sequence as homologous arms is constructed. The protein expression of the constructed strains was monitored by SDS-PAGE. All the five genes were heterologously expressed in E. coli BAP1with the right size. Further research was carried out to realize soluble expression through coexpression with plasmid pL1SL2which contains Streptomyces chaperonins, and soluable protein of FkbH was successfully obtained.Marine Streptomyces is the main source of novel natural products. Genomic sequencing of a novel species, S. xinghaiensis S187, which is one of the marine actinobacteria previously isolated in our lab, indicated that it has the potential to produce polyketides, and can be developed as alternative host for FK506production. Therefore, genetic transformation system of S187was attempted. S187was found to be sensitive to apramycin, kanamycin, thiostrepton and tetracycline, and was easily transformed with pSET152plasmid using Gaus No.1medium for conjugation.The results in this study indicated that modulation of MM-ACP biosynthetic genes to achieve soluble protein expression is important for the heterologous production of FK506. The establishment of genetic manipulation systems of S. xinghaiensis provided basis for heterologous production of FK506using this marine streptomycete as host strain.