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

摘要:

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

Abstract:

Abstract: [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.