On May 10, 2013, Professor Zhu's group of the State Key Laboratory of Bioactive Substance and Function of Natural Medicines published (online) the article: Cloning and characterization of the glycoside hydrolases that remove xylosyl group from 7-β-xylosyl-10-deacetyltaxol and its analogues (doi:10.1074/mcp.M113.030619) on Molecular & Cellular Proteomics (MCP), an international top journal in the field of proteomics (http://www.mcponline.org/content/early/recent) (the impact factor was 7.398 published in 2012, with the 5-year impact factor of 8.633). Firstly, they purified an unknown glycoside hydrolase from the mycelium of the mushroomLentinula edodes by active tracking, and confirmed that the natural enzyme was active against 7-xylosyltaxanes and was a bi-functional β-xylosidase/β-glucosidase. Then, they applied the strategy of combination of the protein de novo sequencing with RT-PCR/RACE for mining and obtained the targeted gene from this fungus conducted by the bioinformatics. Blast results showed that the targeted gene had very low identity with the other DNA sequences accessed in NCBI and other databases, including JGI. The encoded protein showed the maximum identities of 43-59% with the hypothetical proteins accessed in the databases. The heterologous gene was highly expressed in Pichia pastoris . The recombinant enzyme was in accordance with the natural enzyme on activity and other characteristics. Importantly, at the substrate concentrations up to 10 mg/ml (over-saturated), the engineered yeast could still robustly convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol with over 85% conversion rate and the highest yield of 8.42 mg/ml within 24 h, which is much higher than those of previous reports. The following schematic diagram exhibits the process of the research:
Paclitaxel (Taxol®) is a natural antitumor compound and is produced by yew trees at very low concentrations (~0.02%), causing a worldwide shortage of this important anticancer medicine. These plants also produce significant amounts (up to 0.5%) of the by-product 7-β-xylosyl-10-deacetyltaxol, which can be bio-converted into 10-deacetyltaxol for semi-synthesis of paclitaxel. This work may bring significant progress in the development of new 7-β-xylosyl-10-deacetyltaxol converting enzymes for more efficient use of 7-β-xylosyltaxanes to semi-synthesize paclitaxel and its analogues (the gene and the encoded enzyme have been patented with the international application and the international publication). The work may also lead to further studies on detailed mechanisms of how these enzymes act on 7-β-xylosyltaxanes and contribute to the growing database of glycoside hydrolases.
