Abstract
The natural concentration of the anticancer drug Taxol is about 0.02% in yew trees, whereas that of its analogue 7-β-xylosyl-10-deacetyltaxol is up to 0.5%. While this compound is not an intermediate in Taxol biosynthetic route, it can be converted into Taxol by de-glycosylation and acetylation. Here, we improve the catalytic efficiency of 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT) of Taxus towards 10-deacetyltaxol, a de-glycosylated derivative of 7-β-xylosyl-10-deacetyltaxol to generate Taxol using mutagenesis. We generate a three-dimensional structure of DBAT and identify its active site using alanine scanning and design a double DBAT mutant (DBATG38R/F301V) with a catalytic efficiency approximately six times higher than that of the wild-type. We combine this mutant with a β-xylosidase to obtain an in vitro one-pot conversion of 7-β-xylosyl-10-deacetyltaxol to Taxol yielding 0.64 mg ml−1 Taxol in 50 ml at 15 h. This approach represents a promising environmentally friendly alternative for Taxol production from an abundant analogue.
Highlights
The natural concentration of the anticancer drug Taxol is about 0.02% in yew trees, whereas that of its analogue 7-b-xylosyl-10-deacetyltaxol is up to 0.5%
We improve the catalytic efficiency of 10-deacetylbaccatin III-10-Oacetyltransferase (DBAT) of Taxus towards 10-deacetyltaxol, a de-glycosylated derivative of 7-b-xylosyl-10-deacetyltaxol to generate Taxol using mutagenesis
Taxol produced from seedling culture, forestation of yew trees and chemical semi-synthesis of Taxol from the precursor 10-deacetylbaccatin III (10-DAB) have become the major Taxol sources for clinical supply15,16. 10-DAB is one of the key intermediates in the biosynthetic pathway of Taxol and is converted into baccatin III by the enzyme 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT)[17]
Summary
Activities of the recombinant DBATs against 10-DAB and DT. Six full-length DBAT cDNAs derived from Taxus cuspidata (GenBank accession: Q9M6E2.1), T. brevifolia (GenBank accession: EU107143.1), T. baccata (GenBank accession: AF456342.1), T. canadensis (GenBank accession: EU107134.1), T. wallichiana var (GenBank accession: EU107140.1) and T. x media (GenBank accession: AY452666.1) were synthesized and expressed in E. coli. The DBATs of T. wallichiana var., T. brevifolia, T. cuspidata and T. x media origins showed similar conversion rates for 10-DAB (Fig. 2a,b), HO O HO. Regarding the substrate DT, the DBATs of T. cuspidata and T. brevifolia showed the highest specific activities (B0.26 U mg À 1), again over 80 times more than that of T. baccata (0.003 U mg À 1) (Fig. 2f; Supplementary Table 2). Enzyme activity generally declines if the amino acids involved in the substrate binding or catalytic sites are subjected to L-alanine scanning mutagenesis (mutated to Ala). DBATG38R and DBATF301V, were combined to form the double mutant DBATG38R/F301V This combination did not change the optimum temperature and optimum pH when compared to those of the wild-type DBAT (Fig. 6c,d), but further increased enzyme activity 3.7 times against DT (Table 1). The product was purified by HPLC (Fig. 7h) for structure elucidation and was confirmed to be Taxol by MS, 1H-NMR and 13C-NMR analysis (Supplementary Fig. 1, Supplementary Table 3)
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