Functional Analysis Of Lincomycin Biosynthetic Resistance Genes In Streptomyces Lincolnensis

Lincomycin and its derivatives are a class of lincosamide antibiotics, which are widely used for the treatment of infections caused by Gram-positive bacteria in clinic. It is always the focus of attention of researchers and entrepreneurs to improve the production of linomycin for its high medical and economic value. In recent years great progress has been made in research from two aspects, of which one is optimization of fermentation conditions, the other is to obtain good strains through genetic engineering. Now optimization of fermentation conditions has reached its ultimate but there is great potential in improving strains through genetic engineering.So the latter is always the focus of attention. In lincomycin biosynthetic gene cluster there are threelinc omycin resistance genes, lmrA, lmrB and lmrC, of which lmrA and lmrC are putative transporter genes. According to bioinformatic analysis, LmrA is a proton dependent transporter with14transmembrane most likely related with lincomycin export,and LmrC belongs to ABC transporter family, however, whose structure contains no transmembrane but ATP binding domain of ABC transporter. LmrB is significantly similar to the members of AdoMet_MTases superfamily, which can catalyse the N6-mono-or dimethylation of an adenosine in position2058of the23S rRNA. We speculated LmrB could make host strains resistant to lincomycin through modificating its23S rRNA. So in Steptomyces lincolensis there are two kinds of lincomycin resistance mechanism, of which one is to reduce lincomycin affinity through modificating rRNA, and the other is to transport lincomycin to extracellular space. However, it has not been studied further so far. In this project we set the stage for obtaining high resistance and high yield strains through studying the function of lincomycin biosynthetic resistance genes.First, we constructed lmrA deletion mutant XJJ4by homologous recombination. In liquid industrial fermentation medium, the productivity of lincomycin in XJ J4decreased by75%compared with that in LC-G. Through resistance assay self-resistance in XJJ4decreased by90%.Quantitative real-time PCR analysis showed that the transcriptional levels of structure genes lmbA, lmbR and regulatory gene lmbU were drastically reduced in XJJ4. We also constructed lmrA complementation strain XJJ5. Its productivity of lincomycin returned to42%of LC-G. Furthermore we constructed lmrA-overexpressed strain XJJ6. Then we analyzed its productivty, resistance and transcriptional level. The productivity of lincomycin in XJJ6increased by29%, while self-resistance increased by800%, compared with those in LC-G. Overexpression of lmrA increased the concentration of lmrA mRNA20-30folds, and also caused increased expression of the lincomycin biosynthetic genes detected by real-time RT-PCR analysis. These results suggest that LmrA was major transporter for exporting lincomycin. LmrA was proved to be involved in lincomycin biosynthesis by affecting the transcription of structure genes and conferred lincomycin resistance to Streptomyces lincolnensis.Second, we also constructed lmrC deletion mutant XJJ7. Through HPLC analysis and resistance assay, lincomycin production and self-resistance in XJJ7were decreased by55%and50%, respectively. Quantitative real-time PCR analysis showed that the transcriptional levels of lmbA and lmbR were drastically reduced, whereas the expression of the regulatory gene lmbU did not change in XJJ7. The productivity of lmrC complementation strain, XJJ8, returned to55%of LC-G. Then we constructed lmrC-overexpression strain XJJ9and made analysis of lincomycin yield, self-resistance and transcriptional level of lincomycin biosynthetic genes. Overexpression of ImrC in LC-G couldn’t increase the lincomycin yield and the expression of lmbA, lmbR and lmbU as well. However, the resistance to lincomycin was improved in the lmrC-overexpressed strain. According to these results, we concluded the ATP binding domain of ABC transporter encoded by lmrC was a non specific transporter for exporting antibiotics and might function with transmembrane subunit of ABC transporter encoded by some gene in S. lincolnensis genome. So that means LmrC can export lincomycin well in high concentration of lincomycin in vitro, but it’s hard to increase lincomycin yield by overexpression of lmrC in low concentration of lincomycin in vivo.Third, we found that lmrB gene in LC-G missed156bp nucleotides through comparative genomics analysis of lmrB gene in wild type strain and that in high yield LC-G. Then we succeeded in disrupting the gene ImrB in wild type strain by Redirect technology. Furthermore we overexpressed wildtype lmrB gene in LC-G to investigate if lincomycin yield can be increased. Now experiments on yield analysis of these strains are on progress. In our study on the one hand we inactivated lmrA and lmrC in LC-G by homologous recombination, and then overexpressed these two genes. Later we made analysis of lincomycin yield, resistance and transcriptional level of lincomycin biosynthetic genes. According to the results, we concluded the function of these two genes and the relations between the genes and the yields. On the other hand we disrupted lmrB in wild type strain and overexpressed wildtype lmrB in high yield strain. Further study on lmrB gene is under way. Our project not only broadens a horizon for improving lincomycin yield, but also provides important theoretical base for the industrial production.