title: High-cell-density fermentation and pilot-scale biocatalytic studies of an engineered yeast expressing the heterologous glycoside hydrolase of 7-β-xylosyltaxanes.
abstract: The glycoside hydrolase of 7-β-xylosyltaxanes (designated as LXYL-P1-2) is encoded by Lxyl-p1-2 isolated from Lentinula edodes. This hydrolase specifically removes C-7 xylose from 7-β-xylosyltaxanes to form 7-β-hydroxyltaxanes, which can be used for the semi-synthesis of paclitaxel or its analogues. In our present study, we established a high-cell-density fermentation of the recombinant Pichia pastoris harboring the Lxyl-p1-2 gene. Moreover, we further optimized the fermentation conditions, including the initial cell density and the dissolved oxygen level in the induction phase. Under optimized conditions, the biomass of 312.3 g/l (wet cell weight, WCW) was obtained, and the biomass activity of the recombinant enzyme reached 6.55 × 10(4) U/g (WCW). The freeze-dried cells (32 g/l) were used to convert 7-β-xylosyltaxanes (10 g/l, 7-β-xylosyl-10-deacetyltaxol = 62.12 %) in a 5-l reaction volume, and a bioconversion rate about 80 % was achieved. The product purification was performed by ethyl acetate, silica gel chromatography, and preparative HPLC (prep-HPLC), yielding 15.13 g of 10-deacetyltaxol, 3.07 g of 10-deacetylcephalomanine, and 3.47 g of 10-deacetyltaxol C, respectively. In addition, the average recovery rate was around 70 %. Our work provided a foundation for the industrial utilization of the recombinant enzyme on the semi-synthesis of paclitaxel using 7-β-xylosyltaxanes.
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines and Ministry of Health Key Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, People's Republic of China.
authors.forename: Wen-Bo
authors.initials: WB
authors.lastname: Yu
authors.forename: Xiao
authors.initials: X
authors.lastname: Liang
authors.forename: Ping
authors.initials: P
authors.lastname: Zhu
mesh_terms: D055162:Biocatalysis; D018533:Biomass; D005285:Fermentation; D005818:Genetic Engineering; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D010865:Pilot Projects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D043823:Taxoids / Q000378:metabolism*; D014994:Xylose / Q000737:chemistry / Q000378:metabolism
chemical_list: C100478:7-xylosyl-10-deacetyltaxol; D011994:Recombinant Proteins; D043823:Taxoids; C031494:10-deacetyltaxol; D014994:Xylose; D006026:Glycoside Hydrolases