Pectate lyase is widely applied in ramie degumming and fabric bioscouring in the textile industry. Compared to conventional processes that involve high alkaline and high temperature treatment, enzyme based treatments have significant advantages in fibers protectiveness, improved efficiency of refining, reduced energy consumption and pollution. Hence, it would be highly desirable to construct high-yield alkaline pectate lyase engineered strains and reduce the pectate lyase production cost. In the previous study, pectate lyase gene pel from Bacillus subtilis168 was expressed in Pichia pastoris GS115 after codon usage optimization based on the vector pHBM905A. To improve the expression level, the vector pHBM905BDM with optimized promoter and signal peptide was used to express the optimized gene pels in GS115. The transformant had increased activity from 68 U/mL to 100 U/mL with the improvement in the transcription level by 27% measured by qPCR. The transformants were further screened on pectin plates, where higher halo forming strains were picked for shake-flask fermentation and strain GS115-pHBM905BDM-pels4 showed the highest activity of 536 U/mL. Then plasmid pPIC9K-pels was constructed and electroporated into the GS115-pHBM905BDM-pels4 cells. Subsequently, high-copy transformant was screened by using the medium containing antibiotics G418, strain GS115-pHBM905BDMpPIC9K- pels1 was identified with increased activity of 770 U/mL and the copy number of pels was 7 confirmed by qPCR. Finally, the activity of pectate lyase produced by GS115-pHBM905BDM-pPIC9K-pels1reached to 2 271 U/mL in a 5-L fermentor. The activity of pectate lyase in our study reached the highest level of expression in P. pastoris, showing good application potential in the textile industry.
碱性果胶酶可用于苎麻脱胶和棉织物前处理的精练工艺，与传统的高温碱煮相比，具有保护纤维、降低能耗和化学污染的优势，因此获得高表达的碱性果胶酶基因工程菌，低成本生产碱性果胶酶对于纺织工业节能减排具有重要的意义。前期研究工作已经将来源于枯草芽孢杆菌Bacillus subtilis 168 的碱性果胶酶基因pel 经过密码子优化后在毕赤酵母Pichia pastoris GS115 中成功表达。本研究为了提高其表达量，首先利用启动子和信号肽都优化的载体pHBM905BDM 进行表达，摇瓶酶活从68 U/mL 增加到100 U/mL，qPCR 检测转录水平提高了27%。再利用果胶底物平板筛选水解圈大的转化子进行摇瓶发酵获得菌株GS115-pHBM905BDM-pels4，摇瓶酶活为536 U/mL。随后构建重组质粒pPIC9K-pels，电转化菌株GS115-pHBM905BDM-pels4，利用抗生素G418 平板进行筛选，在含4 mg/mL 的G418 抗性平板上得到菌株GS115-pHBM905BDM-pPIC9K-pels1，摇瓶酶活为770 U/mL，qPCR 测定含7 个拷贝目的基因。最后将该菌株在5 L 的发酵罐中进行高密度发酵，果胶酶酶活提高至2 271 U/mL。该碱性果胶酶酶活已达到目前酵母表达的最高水平，说明其具有很好的应用于纺织工业的潜力。.
Keywords: G418 marker; HIS4 selection marker; Pichia pastoris; alkaline pectate lyase; high-density fermentation.