一株产G418单组分工程菌的构建  

Construction of an engineering strain to produce G418 single component

作　　者：阙新桥 林艺辉[1] 胡育龙 肖剑萍[1] 洪文荣[1] 

机构地区：[1]福州大学生物科学与工程学院,福建福州350116

出　　处：《微生物学通报》2015年第2期307-314,共8页Microbiology China

基　　金：国家自然科学基金项目(No.31070093);国家"重大新药创制"科技重大专项项目(No.2012ZX09201101-008)

摘　　要：【目的】定向改造庆大霉素产生菌绛红小单孢菌G1008,获得产G418单组分工程菌。【方法】以温敏型穿梭质粒p KC1139为载体,构建敲除基因genQ*重组质粒p QB303,通过接合转移,导入绛红小单孢菌G1008中,影印筛选和PCR鉴定genQ*框内缺失突变菌,利用TLC和MS分析其代谢产物组分。【结果】获得一株genQ*缺失工程菌Micromonospora purpurea GQ175,其代谢产物为G418单组分,生物效价达828 mg/L,与出发菌G1008的产抗能力相当。【结论】工程菌GQ175产G418单组分,具有很好的工业开发价值。同时,证明绛红小单孢菌G1008中,庆大霉素C-6′脱氢酶基因为genQ*,且无其他同功酶基因。[Objective] To obtain an engineering strain producing G418 single component by directional reconstruction of Micromonospora purpurea G1008 producing gentamicins. [Methods] Using the temperature-sensitive plasmid pKCl139 as vector, the recombinant plasmid pQB303 was constructed and introduced into the M. purpurea G1008 by conjugation. The genQ＊-disruption mutant was screened out by replica plating and PCR analysis. Use MS and TLC for analysis of metabolites. [Results[ A genQ＊-disruption strain, named as M. purpurea GQ175, was obtained to produce G418 single component. The level of antibiotic production was 828 mg/L, similar to the level of the parent strain. [Conclusion] The engineering strain GQ175 produced G418 single component, which had great value for industrial development. Collectively, these results demonstrated that the C-6＇ dehydrogenase gene for gentamicin biosynthesis is genQ＊ and there is no other isoenzyme genes in M. purpurea G1008.

关 键 词：绛红小单孢菌 G418 基因敲除 genQ+ 

分 类 号：Q93[生物学—微生物学]