洋葱伯克霍尔德菌脂肪酶的基因改造及其在毕赤酵母中组成型和诱导型的表达  

Burkholderia cepacia lipase gene modification and its constitutive and inducible expression in Pichia pastoris

作　　者：贾彬[1] 刘文山[1] 杨江科[1] 叶才伟[1] 徐莉[1] 闫云君[1] 

机构地区：[1]分子生物物理教育部重点实验室,华中科技大学生命科学与技术学院

出　　处：《微生物学报》2010年第9期1194-1201,共8页Acta Microbiologica Sinica

基　　金：国家“863计划”(2006AA020203,2007AA05Z417,2007AA100703,2009AA03Z232);教育部新世纪人才基金(NCET070336);湖北省自然科学基金(2008CDB359,2009CDA046)~~

摘　　要：【目的】克隆洋葱伯克霍尔德菌（Burkholderia cepacia）脂肪酶基因,实现其在毕赤酵母中快速、安全和稳定性的大量表达。【方法】首先设计引物扩增B.cepacia脂肪酶基因,然后应用生物信息学方法分析B.cepacia和毕赤酵母整体密码子使用情况、脂肪酶基因信号肽及密码子偏好性。在此基础上,运用overlap PCR对脂肪酶基因中低使用频率密码子进行改造并同时降低基因的G＋C含量,获得优化的脂肪酶基因。再分别把原始和优化的脂肪酶基因导入载体pGAPZα和pPIC9K中,获得组成型表达载体pGAPlipW、pGAPlipO和诱导型表达载体pPIClipW、pPIClipO。分别将所得4种载体转入GS115中,得到一系列工程菌。经发酵和NTA树脂纯化后,对脂肪酶的酶学性质进行了初步研究。【结果】4种工程菌的脂肪酶活力分别为pPIClipW37.8U/mL,pPIClipO129.5U/mL,pGAPlipW40.2U/mL和pGAPlipO184.3U/mL。改造后脂肪酶活力比原始脂肪酶提高了4.6倍。酶学性质研究表明,脂肪酶在60℃时活力最高,在40℃-65℃范围内非常稳定;脂肪酶最适pH值为9.0,在pH6.0-pH10.0范围均表现很好的稳定性。【结论】通过overlap PCR改造后的脂肪酶显著提高了其在毕赤酵母中的表达效率,且GAP启动子比AOX1启动子更适合于B.cepacia脂肪酶的表达。大量表达的重组脂肪酶的性质与野生脂肪酶的性质相同,符合生产要求。[Objective]To achieve fast,safe and stable expression of Burkholderia cepacia lipase in Pichia pastoris.[Methods]We first amplified B.cepacia lipase gene,and then analyzed the codon usage of B.cepacia and Pichia,lipase gene signal peptide with bioinformatics methods.On this basis,we applied the overlap PCR to modify the lipase gene and finally got the optimized gene with Pichia codon usage and lower G ＋ C content.Subsequently,we cloned the optimized and wild lipase gene into vector pGAPZα and pPIC9K,respectively.As a result,constitutive expression vector pGAPlipW,pGAPlipO and inducible expression vector pPIClipW,pPIClipO were obtained.Finally,we electroporated these expression vectors into GS115,and therefore,got a series of engineering strains.After fermentation and NTA resin purification,the enzymatic properties of lipase were studied.[Results]The lipase activities of pPIClipW,pPIClipO,pGAPlipW and pGAPlipO were 37.8 U/mL,129.5 U/mL,40.2 U/mL,and 184.3 U/mL,respectively.The optimized lipase activity increased 4.6-fold.Enzymatic properties study showed that the optimal temperature and pH was 60℃ and 9.0,respectively.The lipase was rather stable at 40℃-65℃ and pH 6.0-pH10.0.[Conclusion]After overlap PCR modification,the lipase expression efficiency in Pichia was significantly increased,which indicates that the overlap PCR modification is a potential strategy for lipase overexpression.The GAP promoter is more appropriate than the AOX1 promoter for the B.cepacia lipase expression.Additionally,the recombinant lipase whose enzymatic properties were identical to the wild type satisfies the needs of industrial application.

关 键 词：洋葱伯克霍尔德菌 脂肪酶 OVERLAP PCR GAP启动子 AOX1启动子 

分 类 号：Q78[生物学—分子生物学]