Construction Of A Genetic Manipulation System For Micromonospora Sp.A29and The Activation Of Secondary Metabolic Pathways Containing AHBA Synthase Gene

In order to discover new ansamycins antibiotics, we found three different AHBA synthase gene postive strains by chosing AHBA synthase gene as the anchor gene:1) A representative ansa-synthetic gene cluste of Streptomyces. sp xzqhl2(not only has AHBA synthase gene but also has PKS);2) A Noansa-synthetic gene cluster of Streptomyce xzqhl2(only has AHBA synthase gene);3) A NRPS-PKS heterozygous synthetic gene cluster of Micromonospora sp.A29(based on the analysis of Micromonospora sp.L5which has finished the whole-genome sequencing).In order to activat the secondary metabolic pathways in Micromonospora sp.A29, we should find an efficient genetic manipulation system for Micromonospora sp.A29. A29mycelium has be chosed as the genetic operation object. pSET152has be chosed as the recombinant plasmid. Finally we successfully have constructed an efficient,universal conjugational transfer system for Micromonospora.Through the comparison of experimental results, we found an efficient culture method of Micromonospora mycelium by shaking culture flask at a high-speed in a small shaker. This method can cultivate a large number of dispersive and vimineous mycelium.With mycelium as action object we respectively trid two methods of genetic operations:electrotransformation and conjugational transfer.The electrotransformation of Micromonospora mycelium has been failed. May be it is difficult to make the recombinant plasmid get through the thick and hard cell wall of Micromonospora only by electric shocking.At the same time, we found that conjugational transfer. between E.coli and Micromonospora can successfully make the recombinant plasmid into Micromonospora mycelium.we have increased the conjugal transfer efficiency to more than10%by optimizing operating conditions, This efficiency is far more than the literature reported efficiency of Micromonospora spores.We have compared the metabolic profiling between wild type strain and mutant strains Knockouted synthetic gene of the three types of AHBA synthase gene postive gene clusters in Streptomyces.sp xzqhl2and Micromonospora sp.A29to detect the expression level of the three types of secondary metabolic pathways. We found that these three synthetic gene clusters are silent, therefore, a series of gene regulation has been exercised for the three types of secondary metabolic pathways.Increasing a set of LZ35RNA polymerase gene in xzqhl2did not activate the synthetic gene clusters. Ansa in xzqh12LuxR expression have been done on the kind of synthetic gene cluster.The discrete compounds from the mutant strain which has exceedingly expressed the LuxR gene in xzqh12do not meet expectations. Speculated that possibility is LuxR family proteins may not only refers to the way to play a specific regulation,but also a global regulation for the secondary metabolism of bacteria.TetR gene and PadR gene have been knockout, but it did not activate the NRPS-PKS secondary metabolic pathways in A29. Possibly the reasons is the TetR gene cluster and PadR do not express, TetR family and PadR family are not negative regulatory factors. Compared the metabolic profiling between wild type strain and mutant strains which have exceedingly expressed the LuxR gene in A29,we found that there are different peak.We should separate the chemical compound of the different peak and determine its structure in the future.