Combinatorial mutation on the -glycosidase specific to 7--xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast.
10.1016/j.apsb.2018.11.003
- Author:
Jing-Jing CHEN
1
;
Xiao LIANG
1
;
Fen WANG
1
;
Yan-Hua WEN
1
;
Tian-Jiao CHEN
1
;
Wan-Cang LIU
1
;
Ting GONG
1
;
Jin-Ling YANG
1
;
Ping ZHU
1
Author Information
1. 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.
- Publication Type:Journal Article
- Keywords:
Combinatorial mutation;
Engineered yeast;
Improved catalytic property;
Molecular docking;
Taxol;
β-Glycosidases
- From:
Acta Pharmaceutica Sinica B
2019;9(3):626-638
- CountryChina
- Language:English
-
Abstract:
Taxol is a "blockbuster" antitumor drug produced by species with extremely low amount, while its analogue 7--xylosyl-10-deacetyltaxol is generally much higher in the plants. Both the fungal enzymes LXYL-P1-1 and LXYL-P1-2 can convert 7--xylosyl-10-deacetyltaxol into 10-deacetyltaxol for Taxol semi-synthesis. Of them, LXYL-P1-2 is twice more active than LXYL-P1-1, 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 -xylosidase and -glucosidase activities. The possible mechanism for such improvement was proposed by homology modeling and molecular docking between E12 and 7--xylosyl-10-deacetyltaxol. The recombinant yeast GS115-P1E12-7 was constructed by introducing variant , the molecular chaperone gene and the bacterial hemoglobin gene . 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.
