Identification And Analysis Of Regulatory Gene LmbU In Streptomyces Lincolnensis

Lincomycin is a lincosamide antibiotic which derives from Streptomyces lincolnensis.The biosynthetic gene cluster of lincomycin was firstly obtained by chromosome walking in 1992.Extensive researches focused on each gene of the cluster shows profound significance on surveying the biosynthetic pathway.lmbU,flanking lmrC,is a gene in S.lincolnensis NRRL 2936.According to the previous findings which mainly include lmbU-deletion and lmbU-complementation,we confirm lmbU is the positive gene related to the lincomycin biosynthesis.In the present research,firstly,comparing wild-type strain with lmbU-deletion strain in primary and secondary metabolism,we find that the absence of lmbU has no significant effect on the growth of strain and sporification.However,the result of HPLC-MS denotes that the absence of lmbU leads to incapable of producing lincomycin.It is noteworthy that there is a new kind of pigment appeared in fermentation supernatant of lmbU mutant,suggesting ImbU is a multi-functional gene.Then we presume that LmbU is a potential DNA-binding protein by modeling.Afterwards,we screen 18 promoter regions inside gene cluster by EMSA and 10 could be combined by LmbU.According to the analysis of lmbWp,lmbKp and lmbCp by DNase I footprinting assay and sequence alignment,we demonstrate that the three regions contain a relatively conservative domain "...GNNNNCCCNNNNC...".In addition,we analyze the effect of lmbU gene on transcription of other genes in cluster by qRT-PCR,revealing that most of the genes are up-regulation and a part of genes are down-regulation by lmbU.The absence of lmbU dramatically promotes the transcription of lmbBl which encodes a tyrosine hydroxylase.According to the result of the new kind of pigment,it indicates that other secondary metabolic pathways are switched on.Finally,in vivo experiments provide another proof of binding of LmbU to the lmbV-lmbW intergenic spacer,and reveal differentiation of LmbU on lmbW and lmbV in transcriptional regulation.The result of dual-plasmids system in E.coli proves that one or more partners in Streptomyces are necessary for normal functioning of LmbU.