Original articles
Jing-Jing Chen, Xiao Liang, Fen Wang, Yan-Hua Wen, Tian-Jiao Chen, Wan-Cang Liu, Ting Gong, Jin-Ling Yang, Ping Zhu. Combinatorial mutation on the β-glycosidase specific to 7-β-xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast[J]. Acta Pharmaceutica Sinica B, 2019, 9(3): 626-638

Combinatorial mutation on the β-glycosidase specific to 7-β-xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast
Jing-Jing Chen, Xiao Liang, Fen Wang, Yan-Hua Wen, Tian-Jiao Chen, Wan-Cang Liu, Ting Gong, Jin-Ling Yang, Ping Zhu
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, Beijing 100050, China
Abstract:
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-P1-1 and LXYL-P1-2 can convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol for Taxol semi-synthesis. Of them, LXYL-P1-2 is twice more active than LXYLP1-1, 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-P1-1 to LXYL-P1-2 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 (A72T/V91S) was the most active and even displayed 2.8-and 3-fold higher than LXYL-P1-2 on β-xylosidase and β-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-P1E12-7 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 GS115-3.5K-P1-2 that had been used for demo-scale fermentation. Thus, GS115-P1E12-7 becomes a promising candidate to replace GS115-3.5K-P1-2 for industrial purpose.
Key words:    β-Glycosidases    Combinatorial mutation    Improved catalytic property    Molecular docking    Engineered yeast    Taxol   
Received: 2018-09-05     Revised:
DOI: 10.1016/j.apsb.2018.11.003
Funds: This work was 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), and CAMS Innovation Fund for Medical Sciences (Grant no. CIFMS-2017-I2M-4-004, China).
Corresponding author: Ping Zhu     Email:zhuping@imm.ac.cn
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Jing-Jing Chen
Xiao Liang
Fen Wang
Yan-Hua Wen
Tian-Jiao Chen
Wan-Cang Liu
Ting Gong
Jin-Ling Yang
Ping Zhu

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