Regulation And Modification On The Biosynthesis Of Lincomycin

Lincomycin, which is produced by Streptomyces lincolnensis, is a lincosamideantibiotic for the treatment of infective diseases caused by Gram-positive bacteria.Lincomycin biosynthesis occurs via a biphasic pathway producing propylproline （PPL）and methylthiolincosamide （MTL） followed by condensation of these subunits toN-demethyllincomycin and methyllation by S-adenosylmethionine to produce theantibiotic lincomycin.30genes are involved in the biosynthesis of lincomycin,however, most of the genes’ function is largely confined to the sequence alignmentand bioinformatics analysis. To determine the genes’ function by experimental studiesis of great significance for genetically modification of S. lincolnensis andimprovement of the fermentation yield of lincomycin.In this study, a simple and efficient method of transferring plasmids into S.lincolnensis through the intergeneric Escherichia coli-mycelia conjugation wasestablished and optimized for the first time. The recipient mycelia of S. lincolnensiswere prepared in liquid SM medium containing10.3%sucrose for three days. Thedispersed mycelia were conjugated with competent E. coli donor cells. Theexconjugants were regenerated efficiently on solid mannitol soya flour （MS） mediumcontaining20mM MgCl₂. The efficient and convenient method of intergeneric E.coli-mycelia conjugation in this study provides the promising procedure to introduceplasmid DNA into S. lincolnensis and other refractory streptomycetes with poorsporulation.The lmbU, lmbD, lmbV, lmbW deleted strains were constructed in this study.Researches on the transcription level of lincomycin biosynthetic gene clusters and theyield of lincomycin fermentation showed that lmbU, lmbD and lmbV were globalpositive regulatory genes, affecting the transcription level of PPL synthetic genes,MTL synthetic genes, resistance genes and other genes. The strain with lmbW deletedonly produced lincomycin B without lincomycin A component. The function of lmbW,catalyzing vinyl to propyl by methylation in the PPL synthesis branch, is confirmedthrough experiment for the first time.The lmrABC, lmbGHIJ, lmbW, lmbUYX over-expression strains were constructedin this study. Fermentation researches showed that increasing copy number of these genes have great significance for improving lincomycin yield. Over-expression ofS-adenosyl-L-methionine synthetase gene （metK） or Vitreoscilla hemoglobin gene（vgb） also promoted bacterial biomass and lincomycin fermentation titer.In conclusion, an efficient intergeneric conjugation of E. coli-S. lincolnensismycelia was first developed. The three regulatory genes in lincomycin biosyntheticgene cluster and their target genes were confirmed and demonstrated. A preliminarygenetic engineering study of lincomycin biosynthesis were carried out through geneover-expression and exogenous gene expression. This study laid the foundation offurther researches into lincomycin biosynthetic mechanism and pathway engineering,and improvement of S. lincolnensis fermentation yield and quality.