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“Trash to Treasure”: Producing Taxol by Enzymatic One-pot Reaction

Time:2017-5-22 Source:State Key Laboratory of Bioactive Substance and Function of Natural Medicines Read:1243

 

On May 18th, 2017, the Series Journal of Nature Publishing Group: Nature Communications published online the full article of “Improving 10-deacetylbaccatin III-10-β-O-acetyltransferase fitness for Taxol production”, which was achieved by Professor Ping Zhu’s research group affiliated to the State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College. The first author is Dr. Bing-Juan Li graduated from Peking Union Medical College and the corresponding author is Professor Zhu. This work was supported by the National Natural Science Foundation of China (Grant Nos. 31270796 and 30770229), the National Mega-project for Innovative Drugs (Grant No. 2012ZX09301002-001-005), and the Youth Fund of Peking Union Medical College (Grant No. 3332015137). The article described the process for improving the catalytic tness of 10-deacetylbaccatin III-10-β-O-acetyltransferase (DBAT) of Taxus towards the unnatural substrate 10-deacetyltaxol (DT) using protein engineering technique, and realizing the production of Taxol, a “blockbuster” antitumor drug, from the ample analogue 7-β-Xylosyl-10-deacetyltaxol (XDT) through DT by enzymatic one-pot reaction.

Taxol (generic name: paclitaxel) is the biggest-selling drug among the botanical antitumor drugs up to date and its indications are still expanding. However, the natural content in yew trees is extremely low (average amount: 0.02%). In the biosynthetic pathway of Taxol, DBAT is one of the key enzymes which acetylates the natural substrate 10-deacetyl baccatin III  (10-DAB) into baccatin III, an important intermediate for Taxol biosynthesis. 10-Deacetyltaxol (DT) is not involved in the Taxol biosynthetic pathway from 10-DAB to Taxol, but can be converted into Taxol through acetylation at the C10 hydroxyl position of DT. Compared with DT, another analogue 7-β-xylosyl-10-deacetyltaxol (XDT) harbors an additional β-xylosyl group at the C7 position (Figure 1). XDT is an ample analogue of Taxol and its content in yew trees, especially in Taxus chinensis (Chinese yew) and Taxus wallichiana (Himalayan yew), is as much as 0.5% (dry weight of the bark). This analogue is often regarded as the waste during Taxol extraction process, causing both resource loss and potential environmental pollution.

In this study, they confirmed the enzymatic acetylation of DT at its C10 hydroxyl position via the recombinant DBAT, but the catalytic efficiency was much lower than that on the natural substrate 10-DAB. Thus, they used protein engineering of DBAT to improve its enzymatic activity against DT, generating a double mutant DBATG38R/F301V with a catalytic efficiency approximately six times higher than that of the wild-type.  In the previous study, they had cloned a new β-xylosidase (designated as LXYL-P1-2) from Lentinula edodes for specically removing the β-xylosyl group from XDT and efficiently converting XDT into DT. In the present study, they constructed an in vitro one-pot reaction system harboring LXYL-P1-2 and the improved acetyltransferase (DBATG38R/F301V) to enzymatically convert XDT into Taxol, realizing the gorgeous turn of “trash to treasure” ((Figure 1).

Briefly, this study provides another choice for environmentally friendly production of Taxol from the ample analogue, which may also reduce resource waste and alleviate the shortage for the clinical supply of Taxol.

 

Figure 1.  One-pot reaction system for the bioconversion of XDT to Taxol and partial results.

(a) The in vitro one-pot reaction system contained a specific 7-β-xlyosidase (LXYL-P1-2), the improved 10-β-acetyltransferase (DBATG38R/F301V), the substrate 7-β-xylosyl-10-deacetyltaxol (XDT) and the acetyl group donor acetyl-CoA, respectively. LXYL-P1-2 deglycosylates XDT into the intermediate product 10-deacetyltaxol (DT) and then DBATG38R/F301V acetylates 10-deacetyltaxol into the final product Taxol. (b) Taxol yields detected by HPLC after the one-pot reaction (reaction volume: 1 mL, 10 mL and 50 mL). (c) HPLC chromatogram of the 50 mL reaction.

 

Reference NATURE COMMUNICATIONS | 8:15544 |DOI: 10.1038/ncomms15544

 

 

 

 

 

 

 

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