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 × 104 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.
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References
Celik E, Calik P, Oliver SG (2009) Fed-batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co-substrate sorbitol. Yeast 26:473–484
Chattopadhyay SK, Sharma RP, Kumar S (2000) Process for the production of important taxol analogues 10-deacetyl taxol A, B, and C. US Patent 6028206
Chattopadhyay SK, Sharma RP, Kumar S, Madhusudanan KP (2002) A process for the production of taxol. European Patent 0905130 B1
Cregg JM, Cereghino JL, Shi JY, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16:23–52
Frense D (2007) Taxanes: perspectives for biotechnological production. Appl Microbiol Biotechnol 73:1233–1240
Gao MJ, Zheng ZY, Wu JR, Dong SJ, Li Z, Jin H, Zhan XB, Lin CC (2012) Improvement of specific growth rate of Pichia pastoris for effective porcine interferon- α production with an on-line model-based glycerol feeding strategy. Appl Microbiol Biotechnol 93:1437–1445
Hanson RL, Howell JM, Brzozowski DB, Sullivan SA, Patel RN, Szarka LJ (1997) Enzymatic hydrolysis of 7-xylosyltaxanes by xylosidase from Moraxella sp. Biotechnol Appl Biochem 26:153–158
Hanson RL, Patel RN, Szarka LJ (1997) Enzymatic hydrolysis method for the conversion of C-7 sugar to C-7 hydroxyl taxanes. US Patent 5700669
Holmel WJ, Darby RAJ, Wilks MDB, Smith R, Bill MB (2009) Developing a scalable model of recombinant protein yield from Pichia pastoris: the influence of culture conditions, biomass and induction regime. Microb Cell Fact 8:35
Huang HQ, Yang PL, Luo HY, Tang HG, Shao N, Yuan TZ, Wang YR, Bai YG, Yao B (2008) High-level expression of a truncated 1,3-1,4-β-d-glucanase from Fibrobacter succinogenes in Pichia pastoris by optimization of codons and fermentation. Appl Microbiol Biotechnol 78:95–103
Invitrogen Life Technologies Version B. Pichia fermentation process guidelines 1–11
Khatri NK, Hoffmann F (2005) Impact of methanol concentration on secreted protein production in oxygen-limited cultures of recombinant Pichia pastoris. Biotechnol Bioeng 93(5):871–879
Lee CY, Nakano A, Shiomi N, Lee EK, Katoh S (2003) Effects of substrate feed rates on heterologous protein expression by Pichia pastoris in DO-stat fed-batch fermentation. Enzyme Microb Tech 33:358–365
Naidu R (2011) Semi-synthesis of taxane intermediates and their conversion to paclitaxel and docetaxel. US Patent 7893283 B2
Patrick SM, Fazenda ML, Mcneil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22(4):249–270
Rao KV (1993) Process for the preparation of taxol and 10-deacetyltaxol. US Patent 520053417
Rowenski EK, Donehower RC (1995) Paclitaxel (Taxol). N Engl J Med 332:1004–1014
Schipperus R, Teeuwen RLM, Werten MWT, Eggink G, Wolf FA (2009) Secreted production of an elastin-like polypeptide by Pichia pastoris. Appl Microbiol Biotechnol 85:293–301
Soyaslan ES, Calik P (2011) Enhanced recombinant human erythropoietin production by Pichia pastoris in methanol fed-batch/sorbitol batch fermentation through pH optimization. Biochem Eng J 55:59–65
Surribas A, Stahn R, Montesinos JL, Enfors SO, Valero F, Jahic M (2007) Production of a Rhizopus oryzae lipase from Pichia pastoris using alternative operational strategies. J Biotechnol 130:291–299
Trentmann O, Khatri NK, Hoffmann F (2004) Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris. Biotechnol Prog 20(6):1766–1775
Wang J, Nguyen V, Glen J, Henderson B, Saul A, Miller LH (2005) Improved yield of recombinant merozoite surface protein 3 (MSP3) from Pichia pastoris using chemically defined media. Biotechnol Bioeng 90(7):838–847
Wang K, Wang TT, Li JH, Zou JH, Chen YQ, Dai JG (2011) Microbial hydrolysis of 7-xylosyl-10-deacetyltaxol to 10-deacetyltaxol. J Mol Catal B-Enzym 68:250–255
Wang Y, Wang ZH, Xu QL, Du GH, Hua ZZ, Liu LM, Li JH, Chen J (2009) Lowering induction temperature for enhanced production of polygalacturonate lyase in recombinant Pichia pastoris. Process Biochem 44:949–954
Wang ZH, Wang Y, Zhang DX, Li JH, Hua ZZ, Du GC, Chen J (2010) Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation by sorbitol co-feeding with methanol in Pichia pastoris fermentation. Bioresource Technol 101:1318–1323
Wu D, Hao YY, Chu J, Zhuang YP, Zhang SL (2008) Inhibition of degradation and aggregation of recombinant human consensus interferon-α mutant expressed in Pichia pastoris with complex medium in bioreactor. Appl Microbiol Biotechnol 80:1063–1071
Yang L, Luan H, Liu X (2008) A Cellulosimicrobium cellulans, its hydrolase and in the use of transformation of taxanes. Patent International Application No. PCT/CN2008/000618
Zhao W, Wang JW, Deng RQ, Wang XZ (2008) Scale-up fermentation of recombinant Candida rugosa lipase. J Ind Microbiol Biotechnol 35:189–195
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This work was supported by the National Natural Science Foundation (No. 30770229) and the National High-Tech Research and Development Program of China (No. 2007AA021501).
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Yu, WB., Liang, X. & Zhu, P. High-cell-density fermentation and pilot-scale biocatalytic studies of an engineered yeast expressing the heterologous glycoside hydrolase of 7-β-xylosyltaxanes. J Ind Microbiol Biotechnol 40, 133–140 (2013). https://doi.org/10.1007/s10295-012-1212-z
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DOI: https://doi.org/10.1007/s10295-012-1212-z