以糖蜜为碳源在真菌灰盖鬼伞中高效表达碱性漆酶PIE5

Efficient expression of the alkaline laccase PIE5 in Coprinopsis cinerea with molasses as a carbon source

摘要:

目的 利用廉价糖蜜替代葡萄糖作为碳源，在真菌灰盖鬼伞(Coprinopsis cinerea)中高效表达碱性真菌漆酶PIE5。方法 通过外源添加蔗糖酶GspInv或在C. cinerea中共表达GspInv分解糖蜜中的蔗糖；以漆酶活力为参考，优化菌株发酵条件。结果 以40 g/L糖蜜为碳源时，菌株CcPIE5-14表达漆酶活力为(11.9±1.2) U/mL，发酵液中蔗糖未被利用。添加外源蔗糖酶GspInv后，发酵液中的蔗糖被水解为果糖与葡萄糖，菌株CcPIE5-14在30 g/L糖蜜条件下漆酶活力最高，达到(14.8±0.7) U/mL。在CcPIE5-14中共表达GspInv获得菌株CcPIE5-14-GspInv-12，在mKjalke培养基中最高漆酶活性为(28.1±2.4) U/mL。利用糖蜜为碳源发酵制备漆酶时，共表达菌株CcPIE5-14-GspInv-12的最高漆酶活性为(20.1±2.7) U/mL，较出发菌株CcPIE5-14提高了2.24倍。发酵条件优化后，菌株CcPIE5-14-GspInv-12的最高漆酶活性为(44.6±2.6) U/mL，是优化前的2.22倍。结论 本研究成功构建了漆酶和蔗糖酶共表达菌株，菌株CcPIE5-14-GspInv-12能以廉价糖蜜为碳源高效表达碱性真菌漆酶PIE5。

Abstract:

Objective To achieve efficient expression of the alkaline laccase PIE5 in Coprinopsis cinerea with molasses as a substitute for glucose as the carbon source.Methods We enhanced the laccase production in C. cinerea by either exogenous addition of the invertase GspInv or endogenous co-expression of GspInv to hydrolyze sucrose in molasses. The fermentation conditions were optimized based on the laccase activity.Results With 40 g/L molasses as the carbon source, strain CcPIE5-14 achieved the laccase activity of (11.9±1.2) U/mL, while sucrose remained unutilized in the fermentation liquid. Upon addition of exogenous GspInv, sucrose in the fermentation liquid was hydrolyzed into fructose and glucose, and strain CcPIE5-14 exhibited the peak laccase activity of (14.8±0.7) U/mL in the medium with 30 g/L molasses. Co-expression of GspInv in CcPIE5-14 generated the engineered strain CcPIE5-14-GspInv-12, which demonstrated the maximum laccase activity of (28.1±2.4) U/mL in the mKjalke medium. When using molasses as the carbon source for laccase production, strain CcPIE5-14-GspInv-12 achieved the peak laccase activity of (20.1±2.7) U/mL, which represented a 2.24-fold increase over that of the parental strain CcPIE5-14. Following fermentation condition optimization, strain CcPIE5-14-GspInv-12 attained the maximum laccase activity of (44.6±2.6) U/mL, which marked a 2.22-fold enhancement over the pre-optimization level.Conclusion The engineered strain CcPIE5-14-GspInv-12, co-expressing laccase and invertase, demonstrates efficient production of the alkaline laccase PIE5 in C. cinerea with cost-effective molasses as the carbon source.