黑曲霉阿魏酸酯酶基因密码子优化及在毕赤酵母中的高效表达  

Codon optimization of feruloyl esterase gene of Aspergillus niger and its high expression in Pichia pastoris

作　　者：李兵[1] 蔡国林[1] 朱德伟[1] 陆健[1] 

机构地区：[1]江南大学工业生物技术教育部重点实验室粮食发酵工艺与技术国家工程实验室生物工程学院,江苏无锡214122

出　　处：《微生物学通报》2017年第5期1065-1073,共9页Microbiology China

基　　金：国家高技术研究发展计划(863计划)项目(No.2013AA102109);江苏高校优势学科建设工程资助项目~~

摘　　要：【目的】对黑曲霉(Aspergillus niger)阿魏酸酯酶基因进行克隆和密码子优化,使其在毕赤酵母(Pichia pastoris X-33)中高效表达。【方法】以黑曲霉基因组为模板,经重叠延伸PCR扩增得到阿魏酸酯酶基因(Anfae A),并对Anfae A基因进行毕赤酵母密码子偏好性"随机优化"和"一对一优化",全基因合成后分别与表达载体pPICZαA连接,构建表达载体pPICZαA-Anfae A、pPICZαA-op Anfae A I和pPICZαA-op Anfae A II。经Sac I线性化后电转化至P.pastoris X-33中,筛选阳性转化子。摇瓶发酵4.5 d后,测定并比较重组阿魏酸酯酶(re Anfae A)酶活。【结果】密码子优化前阿魏酸酯酶酶活为6.8±0.1 U/m L,基因"一对一优化"和"随机优化"后的重组酶酶活分别为5.2±0.1 U/m L和39.9±0.1 U/m L,"随机优化"后酶活比优化前提高了近6倍,而"一对一优化"后酶活仅为优化前酶活的76.5%。重组阿魏酸酯酶的最适p H为5.5,且在pH 4.5-7.0稳定性较好;最适反应温度50°C,在45-50°C较稳定。【结论】阿魏酸酯酶基因经密码子"随机优化"后进行重组表达,酶活显著提高,对研究阿魏酸酯酶在毕赤酵母及其它宿主中的高效表达具有一定的借鉴意义,也为大规模工业化应用奠定了基础。[Objective] Gene cloning and codon optimization of the feruloyl esterase from Aspergillus niger （AnfaeA） for its inducible expression in Pichia pastoris X-33. [Methods] The AnfaeA gene was amplified by overlap extension PCR using the genome of A. niger as template. At the same time, the AnfaeA gene was optimized by ＇one amino acid one codon＇ and ＇codon randomization＇ and then synthesized. The three kinds of ferulic acid esterase gene were cloned into the expression vector pPICZaA, respectively, obtaining the recombinant expression vectors pPICZaA-AnfaeA, pPICZaA-opAnfaeA I and pPICZaA-opAnf FaeA lI. The plasmids were then linearized by Sac I, and transformed into P. pastoris X-33. The positive transformants of each gene type were identified by PCR, and further screened by determination of feruloyl esterase activity in fermentations using high performance liquid chromatography （HPLC）. [Results] The feruloyl esterase activity of FaeA-ori, FaeA-opt I and FaeA-opt II were 6.84-0.1 U/mL, 5.24-0.1 U/mL, and 39.9±0.1 U/mL, respectively. By ＇codon randomization＇ optimization, the activity of recombinant enzyme was 6 times higher than that coded by original AnfaeA gene. However, the feruloyl esterase coded by ＇one amino acid one codon＇ optimized gene was only 76.5% of the original enzyme. The optimal temperature and pH for recombinant AnfaeA （reAnfaeA） was 50℃and 5.5, respectively. In addition, reAnPaeA showed stability when incubated in 45-50 ~C and pH 4.5-7.0. [Conclusions] By ＇codon randomization＇ optimization, the resultant recombinant feruloyl esterase expressed in P. pastoris X-33 was 6 times higher than that coded by original AnfaeA, reaching the highest activity among existed recombinant feruloyl esterase until now. The results provided large-scale application potential of feruloyl esterase in industrial, and laid the experimental foundation for the further improvement the enzyme activity.

关 键 词：黑曲霉 毕赤酵母 阿魏酸酯酶 密码子优化 

分 类 号：Q78[生物学—分子生物学] Q936