【摘 要】
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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 h
【机 构】
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State Key Laboratory of Bioactive Substance and Function of Natural Medicines & NHC Key Laboratory o
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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 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,El2 (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 El2 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.
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