Jia Bin, Liu Wenshan, Yang Jiangke, Ye Caiwei, Xu Li, Yan Yunjun
Key Laboratory of Molecular Biophysics, Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
Wei Sheng Wu Xue Bao. 2010 Sep;50(9):1194-201.
To achieve fast, safe and stable expression of Burkholderia cepacia lipase in Pichia pastoris.
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 pGAPZalpha 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.
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 degrees C and 9.0, respectively. The lipase was rather stable at 40 degrees C - 65 degrees C and pH 6.0-pH10.0.
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.
在毕赤酵母中实现洋葱伯克霍尔德菌脂肪酶的快速、安全和稳定表达。
首先扩增洋葱伯克霍尔德菌脂肪酶基因，然后运用生物信息学方法分析洋葱伯克霍尔德菌和毕赤酵母的密码子使用情况以及脂肪酶基因信号肽。在此基础上，应用重叠PCR对脂肪酶基因进行修饰，最终获得具有毕赤酵母密码子使用且G + C含量较低的优化基因。随后，分别将优化后的脂肪酶基因和野生型脂肪酶基因克隆至载体pGAPZalpha和pPIC9K中。结果，获得了组成型表达载体pGAPlipW、pGAPlipO以及诱导型表达载体pPIClipW、pPIClipO。最后，将这些表达载体电穿孔导入GS115，从而获得一系列工程菌株。经发酵和NTA树脂纯化后，对脂肪酶的酶学性质进行了研究。
pPIClipW、pPIClipO、pGAPlipW和pGAPlipO的脂肪酶活性分别为37.8 U/mL、129.5 U/mL、40.2 U/mL和184.3 U/mL。优化后的脂肪酶活性提高了4.6倍。酶学性质研究表明，其最适温度和pH分别为60℃和9.0。该脂肪酶在40℃ - 65℃和pH 6.0 - pH10.0条件下相当稳定。
经重叠PCR修饰后，毕赤酵母中脂肪酶的表达效率显著提高，这表明重叠PCR修饰是脂肪酶过表达的一种潜在策略。对于洋葱伯克霍尔德菌脂肪酶的表达，GAP启动子比AOX1启动子更合适。此外，重组脂肪酶的酶学性质与野生型相同，满足工业应用需求。
