The Function Research Of Lincomycin Biosynthetic Gene

Lincomycin and its derivatives are antibiotics exhibiting biological activity against Gram-positive bacteria. We constructed and optimized the Escherichia coli-Streptomyces lincolnensis inter-species conjugation system. Intergeneric transfer of temperature-sensitive plasmid vectors pKC1139 from donor strain E. coli ET12567 to S. lincolnensis. The genomic library of S. lincolnensis was constructed with pJTU2554 as a vector. We successfully identified a Cosmid 1E8 that harbors the entire lincomycin biosynthetic gene cluster from the S. lincolnensis genomic library.The 35 kb-long chromosomal region bearing the lincomycin biosynthetic gene cluster. There are 27 open reading frames with putative biosynthetic or regulatory functions (Imb genes) and three resistance (lmr) genes in the region. Three putative regulatory gene lmbIH、lmbQ�'�lmbU were knockout through in-frame deletion. The fermentation at the flask level proved that three genes are positive regulation gene. Three putative methylase gene lmbM1、1mbM2�'�lmbM3 were knockout used the same method. We separated the intermediate product generated by the lmbMl gene knockout mutant, its structure was identified to lincomycin B by NMR. It provided a very important evidence to lmbM1 gene function verification.The fermentation broth of the lmbM3 interruption mutant was separated initially. The lmbM2 knockout mutants lost the ability of producing lincomycin, indicating that lmbM2 is a key gene in lincomysin biosynthesis.