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目的:以Streptomyces lividans TK24为表达宿主,构建高效表达棘白霉素脱酰酶(Ech DA)的基因工程菌。方法:从Actinoplanesutahensis NRRL 12052中获取Ech DA的基因片段,连接到链霉菌表达载体p NW-S1,采用诱导型启动子Plac与组成型启动子Perm E*和PSau3A,依次构建基因工程菌株ZNW-S11、ZNW-S12和ZNW-S13。通过分析启动子对Ech DA分泌表达的影响,确定工程菌的表达能力,随后完善发酵放大的工艺过程,将工程菌株发酵放大到300 L发酵罐水平。结果:成功实现了Ech DA在S.lividans TK24中的诱导分泌表达;在摇瓶水平,Ech DA的酶活可达到125U/L,在24h内可实现对15g/L米卡芬净前体FR901379的完全转化;在300L发酵罐水平,Ech DA的酶活可达到80U/L,可用于10g/LFR901379的完全转化。当前,国内在工业生产中仍使用原始菌A.utahensis来表达Ech DA,而开发的Ech DA基因工程菌,与之相比在生产效率上有了大幅度提升,有助于改良当前棘白霉素类抗生素的生产工艺。
OBJECTIVE: To construct a genetically engineered bacterium expressing Ech DA efficiently by using Streptomyces lividans TK24 as an expression host. Methods: The gene fragment of Ech DA was obtained from Actinoplanesutahensis NRRL 12052 and ligated into the Streptomyces expression vector p NW-S1. The inducible promoter Plac and the constitutive promoters Perm E * and PSau3A were used to construct the engineered strain ZNW-S11 , ZNW-S12 and ZNW-S13. By analyzing the effect of the promoter on the secretion of Ech DA, the expression capacity of the engineered bacteria was determined, and then the process of fermentation amplification was perfected, and the engineered strain was expanded to 300 L fermentor level. Results: Ech DA was successfully induced in S.lividans TK24. At the shake flask level, the enzyme activity of Ech DA reached 125U / L, and the effect of 15g / L micafafen net precursor FR901379 Of the complete conversion; 300L fermentor level, Ech DA enzyme activity up to 80U / L, can be used for 10g / LFR901379 complete conversion. At present, the domestic industrial production still use the original bacteria A.utahensis to express Ech DA, compared with the development of Ech DA genetically engineered bacteria, in terms of production efficiency has greatly improved, help to improve the current Echinococcus Class antibiotic production process.