一株产G418单组分工程菌的构建

Construction of an engineering strain to produce G418 single component

【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.