bkdFGH和aveD基因缺失多拉菌素工程菌株的构建  

Construction of bkdFGH and ave D-deleted Doramectin-producing Strains

作　　者：刘振[1] 潘媛媛[2] 杨在清[1] 雷霆[2,1] 

机构地区：[1]华中农业大学生命科学技术学院,武汉430070 [2]南阳师范学院生命科学与技术学院,南阳473061

出　　处：《农业生物技术学报》2016年第3期454-460,共7页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育科技重大专项(No.2009ZX08009-151B)

摘　　要：多拉菌素(doramectin)是一种新型大环内酯类抗寄生虫药物,为阿维菌素的衍生物,可由基因重组阿维链霉菌(Streptomyces avermitilis)发酵产生,在畜牧业生产中应用广泛。为获得可用于生产的能够稳定制造多拉菌素的阿维链霉菌工业菌株,本研究以阿维菌素高产菌株SAV939为基础,通过对阿维菌素生物合成基因簇进行遗传改造,构建获得阿维链霉菌突变株S.avermitilis LZ-01和LZ-02。LZ-01是通过同源双交换法构建的支链α-酮酸脱氢酶基因簇(branched-chain alpha-keto acid dehydrogenase gene cluster,bkd FGH)缺失突变株,自身无法合成多拉菌素,当在发酵过程中添加环己甲酸时即可产生多拉菌素(CHCB1)。LZ-01缺失avermectin D(ave D)基因即获得突变株LZ-02,对其发酵仍可获得多拉菌素,但不再产生无效的CHC-A组分,为分离纯化提供了便利。突变菌株LZ-01和LZ-02的构建为进一步优化发酵条件,提高多拉菌素产率奠定了基础。Doramectin is a novel macrolide antibiotics derived from avermectin and widely used as an antiparasitic drug in animal husbandry. Doramectin can be produced by genetically modified Streptomyces avermitilis with altered avermectin synthesis pathways. Disruption of branched- chain alpha- keto acid dehydrogenase gene cluster（bkd FGH）, which encodes the components of the branched-chain alpha-keto acid dehydrogenase（BCDH） complex multi- enzyme system, can direct S. avermitilis to produce effective doramectin（CHC- B1）, along with other undesired analogues such as CHC- A1 and- A2, when cyclohexanecarboxylic acid（CHC） is added to the fermentation medium as a precursor. ave D, which encodes avermectin B 5-O-methyltransferase, is reported to be responsible for transforming avermectin ＂B＂ component to ＂A＂. Deletion of ave D may reduce the amount of CHC-A component in doramectin produced by bkd FGHdeleted S. avermitilis. Here we reported the construction of 2 mutant S. avermitilis strains capable of doramectin biosynthesis. S. avermitilis strain SAV939, an avermectin high-producing strain, was deleted for bkd FGH gene by homologous double crossover to generate mutant strain LZ- 01. High performance liquid chromatography（HPLC） analysis demonstrated that LZ- 01 could produce effective doramectin（CHC- B1） when CHC was added as a precursor in the fermentation process. Based on that, we further deleted avermectin D（ave D） in LZ-01 and obtained mutant strain LZ-02. Like LZ-01, LZ-02 could also produce CHC-B1 through fermentation,but without the ineffective component CHC- A. This work provides 2 novel doramectin producer mutants from an avermectin- overproducing industrial strain of S. avermitilis, which could potentially become industrial doramectin-producing strains after further improvement.

关 键 词：阿维链霉菌 多拉菌素 支链α-酮酸脱氢酶基因簇(bkd FGH) AVERMECTIN D(ave D) 

分 类 号：Q784[生物学—分子生物学]