Study Of The Gene Regulation And Reconstrution Of Vancomycin Biosynthesis In Amycolatopsis Orientalis HCCB10007

Amycolatopsis orientalis HCCB10007 is a high-yield strain for industrial production of vancomycin.The complete genome of this strain has been sequenced in 2014.It was predicted that there were 10% of regulatory genes in the genome of HCCB10007 according to comparative genomic analyses,and there were at least 25 kinds of secondary metabolite synthesis pathways,which were largely unknown,other than the vancomycin synthetic pathway.It was studied in the strain of HCCB 10007 that the activity of the metabolite from a complete gene cluster,the regulatory genes that played an important in the biosynthesis of vancomycin,and obtaining the important precursor of a marketed drug oritavacin by using of gene reconstration.1.The biological activity of LYXLF2 produced by gene cluster n?p2It was predicted that the gene cluster n?p2 was a more complete gene cluster in A.orientalis HCCB10007,and a new natural product LYXLF2 was unearthed.Based on LYXLF2 belonging to the structure of indole this work investigated its effects on the plant growth.The seeds of model plant Arabidopsis thaliana and economic crop wheat were chosed.The results showed that LYXLF2 could promote the seeding growth of Arabidopsis thaliana and wheat.LYXLF2 increased the root length of Arabidopsis thaliana by 23% at the concentration of 0.05mg/l and wheat seedlings by 37% at the concentration of 0.5mg/l.LYXLF2 was a new plant growth promoter.2.The effects of regulatory genes on the biosynthesis of vancomycinIn this study,six genes with potential regulatory effects on vancomycin biosynthesis were selected,one of which was pathway-specific regulatory gene and five of which were global regulatory genes.After over-expression of six gene in the strsin HCCB10007,the binsynthesis of vancomycin was changed.The results showed that AORI?1475,AORI?1832,AORI?2956 and AORI?3531 had positive effects on the biosynthesis of vancomycin,while AORI?3398 and AORI?7412 had negative regulations.AORI?1475 and AORI?1832 increased the synthesis of vancomycin by 55% and 51% respectively,and the aglycovancomycin was increased by 4 times and 6 times respectively too.By transcription analysis of AORI?1475 and AORI?1832,it was found that the two genes upregulated the synthesis of Hpg and Dpg amino acids and the cyclization of heptapeptide backbone in the biosynthesis pathway of vancomycin.This had a meaningful guidianc e for increasing the production of vancomycin by gene regulation.3.The study on the remodeling of secondary metabolites producing by Amycolatopsis orientalisIn the previous study,LYV07WW01,a vancomycin analogue was biosynthed in our laboratory by glycosyltransferase genetic engineering.In this study,the epi-vancosamine synthase gene of another strain was recombined into LYV07WW01 producing bacteria to obtain chloroeremomycin,a precursor substance of the marketed drug Oritavancin.After genetic recombination,fermentation of engineering strain and identification of the product,it was confirmed the success of the reconstruction.This application laid the foundation for obtaining chloroeremomycin producing strain with high yield.