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

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(AnfaeA),并对AnfaeA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-AnfaeA、pPICZαA-opAnfaeA Ⅰ和pPICZαA-opAnfaeA 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4.5d后,测定并比较重组阿魏酸酯酶(reAnfaeA)酶活.[结果]密码子优化前阿魏酸酯酶酶活为6.8士0.1 U/mL,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/mL和39.9±0.1 U/mL,“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pH为5.5,且在pH 4.5-7.0稳定性较好;最适反应温度50℃,在45-50℃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 pPICZαA,respectively,obtaining the recombinant expression vectors pPICZαA-AnfaeA,pPICZαA-opAnfaeA Ⅰ and pPICZαA-opAnfFaeA Ⅱ.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 6.8±0.1 U/mL,5.2±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,reAnfaeA showed stability when incubated in 45-50 ℃ 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.