朱黄青霉α-葡聚糖酶在毕赤酵母中的高效表达

摘要

【目的】提高朱黄青霉（Penicillium minioluteum ）α-葡聚糖酶（Dextranase）基因在毕赤酵母（Pichia pastoris）中的表达水平。【方法】根据毕赤酵母的密码子偏爱对酶基因进行优化与合成。优化后的基因片段与载体 pGAPZαA 连接，转化毕赤酵母 KM71 H。【结果】获得组成型分泌表达α-葡聚糖酶的工程菌 KM71 H／pGAPZαA-dex。发酵工艺试验中，摇瓶培养144 h，酶活为153 U／mL。6．8 L发酵罐补料分批培养92 h，酶活达到1218 U／mL。【结论】该工程菌以甘油作为碳源，发酵调控简单，产酶水平较高，具有适用于大规模生产的潜力。%Objective]The aim of this paper is to enhance the expression level of a dextranase gene dex of Penicillium minioluteum in Pichia pastoris.[Methods]Codon optimization was ap-plied and the dextranase gene was synthesized using the preferential condon of pichia pastoris . The codon-optimized gene was cloned into the vector pGAPZαA and transformed into host strain KM7 1 H to achieve constitutive expression and secretion of dextranase.[Results]The ac-tivity of dextranase reached 153 U/mL after 144 h when the engineering strain KM71H/pGAPZαA-dex was cultured in shake flask.Using glycerol as the sole carbon source,a simple fermentation control strategy has been developed and expression level of 1218 U/mL after 92 h has been demonstrated in 6.8 L scale fed-batch fermentation.[Conclusion]The high expression level makes the engineering strain a good candidate for industrial production.