Combinatorial mutation on the β-glycosidase specific to 7-β-xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast

作     者：Jing-Jing Chen Xiao Liang Fen Wang Yan-Hua Wen Tian-Jiao Chen Wan-Cang Liu Ting Gong Jin-Ling Yang Ping Zhu 

作者机构：State Key Laboratory of Bioactive Substance and Function of Natural Medicines & NHC Key Laboratory of Biosynthesis of Natural Products Institute of Materia Medica Chinese Academy of Medical Sciences & Peking Union Medical College 

出 版 物：《Acta Pharmaceutica Sinica B》 (药学学报（英文版）)

年 卷 期：2019年第9卷第3期

页      面：626-638页

核心收录：

学科分类：1007[医学-药学(可授医学、理学学位)] 10[医学] 

基　　金：supported by the National Natural Science Foundation of China (Grants nos. 81573325 and 31270796) the National Mega-project for Innovative Drugs (Grants nos. 2018ZX09711001-006-001 and 2012ZX09301002-001-005, China) the fundamental Research Funds for the Central Universities (Grant no. 2017PT35001, China) CAMS Innovation Fund for Medical Sciences (Grant no. CIFMS-2017-I2M-4-004, China) 

主　　题：β-Glycosidases Combinatorial mutation Improved catalytic property Molecular docking Engineered yeast Taxol 

摘      要：Taxol is a blockbuster antitumor drug produced by Taxus species with extremely low amount, while its analogue 7-β-xylosyl-10-deacetyltaxol is generally much higher in the plants. Both the fungal enzymes LXYL-P1à1 and LXYL-P1à2 can convert 7-β-xylosyl-10-deacetyltaxol into10-deacetyltaxol for Taxol semi-synthesis. Of them, LXYL-P1à2 is twice more active than LXYLP1à1, but there are only 11 significantly different amino acids in terms of the polarity and acidic-basic properties between them. In this study, single and multiple site-directed mutations at the 11 sites from LXYL-P1à1 to LXYL-P1à2 were performed to define the amino acids with upward bias in activities and to acquire variants with improved catalytic properties. Among all the 17 mutants, E12(A72 T/V91 S) was the most active and even displayed 2.8-and 3-fold higher than LXYL-P1à2 on β-xylosidase andβ-glucosidase activities. The possible mechanism for such improvement was proposed by homology modeling and molecular docking between E12 and 7-β-xylosyl-10-deacetyltaxol. The recombinant yeast GS115-P1 E12-7 was constructed by introducing variant E12, the molecular chaperone gene pdi and the bacterial hemoglobin gene vhb. This engineered yeast rendered 4 times higher biomass enzyme activity than GS115-3.5 K-P1à2 that had been used for demo-scale fermentation. Thus, GS115-P1 E12-7 becomes a promising candidate to replace GS115-3.5 K-P1à2 for industrial purpose.