基于分子改造和宿主优化促地衣芽胞杆菌高效表达γ-谷氨酰转肽酶

Molecular modification and host optimization enhance the expression of γ-glutamyltranspeptidase in Bacillus licheniformis

摘要:

γ-谷氨酰转肽酶(γ-glutamyltranspeptidase, GGT)在临床诊断、食品加工和医药制造等领域应用广泛，然而较低的表达量限制了其广泛应用。为提高GGT表达水平，本研究首先使用MūtCompute软件对来源于地衣芽胞杆菌的GGT进行蛋白质工程改造，获得了催化效率提高的突变体GGT^A339C。通过对底物结合口袋进行分析，发现突变体GGT^A339C的隧道结构更直，底物结合区域更开放，酶活较对照提高255%。随后，以地衣芽胞杆菌BL11为出发菌株，通过整合表达分子伴侣PrsA，构建了高效分泌GGT的地衣芽胞杆菌BL11::prsA/pP_ykzA+rbs6-SP_SacC-GGT^A339C，其GGT酶活达到22.1 U/mL。最后，通过5 L发酵罐进行放大，GGT酶活达180.14 U/mL。本研究获得了高催化性能的突变体GGT^A339C，并构建了一株高产GGT的地衣芽胞杆菌BL11::prsA/pP_ykzA+rbs6-SP_SacC-GGT^A339C，为GGT工业生产及催化应用奠定了基础。

Abstract:

γ-glutamyltranspeptidase (GGT) is widely used in clinical diagnosis, food processing, and pharmaceutical manufacturing, whereas the low expression limits its application expansion. To improve the expression of GGT, we employed MūtCompute to engineer the GGT derived from Bacillus licheniformis and obtained the mutant GGT^A339C with improved catalytic efficiency. By analyzing the substrate-binding pocket, we found that the mutant GGT^A339C had a more open substrate-binding region and a substantially straighter tunnel than the control, with a 255% increase in activity. Subsequently, with B. licheniformis BL11 as the starting strain, we constructed the strain BL11::prsA/pP_ykzA+rbs6-SP_SacC-GGT^A339C with efficient GGT secretion by integrating the expression of chaperone PrsA, and the enzyme activity reached 22.1 U/mL. Finally, after optimization of the fermentation process in a 5 L fermenter, the GGT activity reached 180.14 U/mL, marking the highest level of GGT activity reported to date. In conclusion, the mutant GGT^A339C with high catalytic performance was successfully obtained, and the strain B. licheniformis BL11::prsA/pP_ykzA+rbs6-SP_SacC-GGT^A339C was attained with high production of GGT, which provided an excellent strain for the industrial production and catalytic application of GGT.