Preliminary Exploration Of Discovering New Ansamycins By Heterologous Expression

Actinomycetes is a kind of Gram-positive bacterial with complex life style, which produces more than half of the currently known antibiotics. It mainly distributed in terrestrial and marine while extreme environment and plant tissue became the new source of actinomycetes.Ansamycins is a family of antibiotics, which often possess strong antimicrobial, antifungal, anticancer and antiviral activity. Most members of this family were isolated from actinomycetes. A subfamily named maytansinoids was found in higher plants and mosses. Ansamycins is Type Ⅰ PKS compound. It has a common biosynthetic starting unit AHBA. By using the DNA probe of AHB A, we can screen for ansamycin biosynthetic gene cluster. The biosynthesis of ansamycin is not only under global regulation of the cell but the pathway-specific regulation. Many of the ansamycin biosynthetic regulators belong to the LuxR family, by overexpression of which we can activate the silent ansamycin biosynthetic gene cluster or improve the production.Natural products was the most important source of drugs, but also a great treasure of new ansamycins. Megagenomic technology provides a strong support for the extraction of drugs from natural products.This thesis discussed two main ways of extracting new ansamycins.Approach Ⅰ:construct BAC library and expect to capture large integrated ansamycin biosynthetic gene cluster. First, a serials of E.coli-Streptomycets shuttle vector were constructed. pCCESAC82has advantages such as inducible replicon, PermE promoter, BstⅪ adaptor supported ligation, capability of conjugation and integration based on CIS element. Methods of constructing BAC library including extraction of high quality HMW DNA, preparation of high-efficient competent cells and improvement of ligation and transformation efficiency was investigated. Then a BAC library of Actinosynnema pretiosum31565was constructed and clones with an insertion of up to100kb was obtained. Approach Ⅱ:reassemble of overlapped DNA fragments by using Red recombination and heterologous expression of the biosynthetic gene cluster in suitable host. Utilizing the homologous arms between pCCESAC2and pCC1FOS, and the overlap fragment between the two fosmid insertion, two rounds of Red recombination was carried out resulting in the capture of the whole biosynthetic gene cluster in pCCESAC2. So, we developed a simple method for reassembling large DNA fragments into one vector. This method was much simple than the reported ones. It doesn’t need any additional modifications of donor fosmids and in vitro preparation of the homologous arms and selected marker. The plasmid can be directly conjugated to host for expression.We successfully constructed tam gene cluster obtained from metagenomic library of Trewia nudiflora and the unknown nas gene cluster of Streptomyces sp. LZ35. The obtained clusters were transformed into Streptomyces coelicolor ZM12, Amycolatopsis mediterranei s699△rifA and Thermophilic Streptomyces sp.4F for heterologous expression and product detection.