玉米赤霉烯酮降解酶多拷贝毕赤酵母菌株的构建及高效表达

High expression of zearalenone degrading enzyme in Pichia pastoris

摘要:

通过密码子优化、体外多拷贝构建实现玉米赤霉烯酮 (Zearalenone，ZEN) 降解酶基因 (zlhy-6) 在毕赤酵母GS115菌株中的高效表达。按酵母密码子偏好性优化zlhy-6基因的密码子，与α因子信号肽编码序列一起合成，插入到pAO815质粒中，通过酶切酶连构建含1–6个表达盒的表达质粒，将其转入毕赤酵母 GS115菌中，获得ZEN降解酶重组菌株。重组蛋白分子量为28.9 kDa，与预期一致。重组菌用甲醇诱导3 d，蛋白浓度达最高，之后下降；在pH 5.0、4.5条件下诱导培养，表达量最高；每天添加0.8%的甲醇、接种量10%表达水平最高；4拷贝的转化子表达水平最高，三角瓶发酵3 d，酶活性达到10 U/mL。在1 g玉米渣中添加0.1–0.5 mL发酵上清液，水解24 h，玉米渣中ZEN的降解率为44.08%–75.51%。研究结果为ZEN降解酶工业生产及在食品饲料中的应用奠定了基础。

Abstract:

High expression of zearalenone (ZEN) degrading enzyme gene (zlhy-6) in Pichia pastoris strain GS115 was achieved by codon optimization and multi-copy construction in vitro. The codon-optimized zlhy-6 gene sequence was synthesized with the alpha factor signal peptide coding sequence and inserted into the pAO815 plasmid. The expression plasmid containing 1–6 expression cassettes was constructed by enzyme digestion and transferred into P. pastoris GS115 strain to obtain the ZEN degrading enzyme recombinant strain. The molecular weight of the recombinant protein was 28.9 kDa, which was consistent with the theoretical value. After 3 days of induction fermentation, the protein concentration reached the highest level and then decreased; the expression level was the highest in the induction culture at pH 5.0 and 4.5, while the expression level at other pH was very low; the expression level was the highest when 0.8% methanol was added every day and 10% inoculation was added; the expression level of four-copy transformants was the highest, and the enzyme activity reached 10 U/mL after 3 days of flask fermentation, The degradation rate of ZEN in 1 g corn ballast was 44.08%–75.51% when 0.1–0.5 mL fermentation supernatant added and hydrolyzed for 24 hours. The results of this study laid a foundation for improving the industrial fermentation level of ZEN degrading enzyme and its application in eliminating ZEN in food and feed.