中国红豆杉羟化酶基因TcCYP725A22的克隆与功能研究
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摘要
基于中国红豆杉(Taxus wallichiana var. chinensis(Pilger) Florin)转录组序列,从中扩增到1个新的P450羟化酶基因TcCYP725A22,并对其进行了克隆、表达及功能分析。生物信息学分析结果显示:该基因编码区长1500 bp,共编码499个氨基酸;编码蛋白无信号肽,包含1个跨膜区,具有催化活性中心结构域。进一步将该基因在酿酒酵母WAT11中进行异源表达功能研究,蛋白质免疫印迹(Western blot)实验结果表明,目的蛋白TcCYP725A22可在WAT11中成功表达;液相色谱-质谱联用(LC-MS)分析结果显示,羟化酶TcCYP725A22对底物紫杉素表现出催化活性。依据质谱结果及反应底物紫杉素的分子结构,判断其催化产物可能为添加了2个羟基的紫杉烷新衍生物。
Based on our previous transcriptome analysis,a new P450 hydroxylase gene was identified and named TcCYP725 A22 according to homological analysis. The TcCYP725A22 cDNA was 1500 bp in length and encoded 499 amino acids. Bioinformatics predicted that the TcCYP725A22 protein contained a transmembrane region and catalytically active domains but no signal peptide. Functional analysis of TcCYP725A22 was carried out in Saccharomyces cerevisiae strain WAT11. Western blotting results indicated that the target protein was expressed successfully in WAT11. Furthermore,LC-MS analysis showed that TcCYP725A22 expressed in yeast was able to transform taxusin to its hydroxyl derivative,indicating that it had catalytic activity.
Based on our previous transcriptome analysis,a new P450 hydroxylase gene was identified and named TcCYP725 A22 according to homological analysis. The TcCYP725A22 cDNA was 1500 bp in length and encoded 499 amino acids. Bioinformatics predicted that the TcCYP725A22 protein contained a transmembrane region and catalytically active domains but no signal peptide. Functional analysis of TcCYP725A22 was carried out in Saccharomyces cerevisiae strain WAT11. Western blotting results indicated that the target protein was expressed successfully in WAT11. Furthermore,LC-MS analysis showed that TcCYP725A22 expressed in yeast was able to transform taxusin to its hydroxyl derivative,indicating that it had catalytic activity.
引文
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[2] Ketchum RE,Wherland L,Croteau R. Stable transformation and long-term maintenance of transgenic Taxus cell suspension cultures[J]. Plant Cell Rep,2007,26(7):1025-1033.
[3] Julsing MK,Koulman A,Woerdenbag HJ,Quax WJ,Kayser O. Combinatorial biosynthesis of medicinal plant secondary metabolites[J]. Biomol Eng,2006,23(6):265-279.
[4] Malik S,Cusido RM,Mirjalili MH,Moyano E,Palazon J,et al. Production of the anticancer drug taxol in Taxus baccata suspension cultures:a review[J]. Process Biochem,2011,46(1):23-34.
[5] Mirjalili MH,Farzaneh M,Bonfill M,Rezadoost H,Ghassempour A. Isolation and characterization of Stemphylium sedicola SBU-16 as a new endophytic taxol-producing fungus from Taxus baccata grown in Iran[J]. FEMS Microbiol Lett,2012,328(2):122-129.
[6] Yang N,Pan X,Chen G,Sarsaiya S,Yu J,et al. Fermentation engineering for enhanced paclitaxel production by Taxus media endophytic fungus MF-5(Alternaria sp.)[J].J Biobased Mater Bio,2018,12(6):545-550.
[7] Tije AJ,Verweij J,Loos WJ,Sparreboom A. Pharmacological effects of formulation vehicles:implications for cancer chemotherapy[J]. Clin Pharmac Okinet,2003,42(7):665-685.
[8] Matoba H,Watanabe T,Nagatomo M,Inoue M. Convergent synthesis of taxol skeleton via decarbonylative radical coupling reaction[J]. Org Lett,2018,20(23):7554-7557.
[9] Ro DK,Paradise EM,Ouellet M,Fisher KJ,Newman KL,et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast[J]. Nature,2006,440(7086):940-943.
[10] Yan X,Fan Y,Wei W,Wang P,Liu QF,et al. Production of bioactive ginsenoside compound K in metabolically engineered yeast[J]. Cell Res,2014,24(6):770-773.
[11] Awasthi D,Wang L,Rhee MS,Wang Q,Chauliac D,et al. Metabolic engineering of Bacillus subtilis for production of D-lactic acid[J]. Biotech Bioeng,2018,115(2):453-463.
[12] Ajikumar PK,Xiao WH,Tyo KE,Wang Y,Simeon F,et al.Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli[J]. Science,2010,330(6000):70-74.
[13] Zhou K,Qiao K,Edgar S,Stephanopoulos G. Distributing a metabolic pathway among a microbial consortium enhances production of natural products[J]. Nature Biotech,2015,33(4):377-383.
[14] Biggs BW,Lim CG,Sagliani K,Shankar S,Stephanopoulos G,et al. Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli[J]. Proc Natl Acad Sci USA,2016,113(12):3209-3214.
[15] Liao WF,Zhao SY,Zhang M,Dong KG,Chen Y,et al.Transcriptome assembly and systematic identification of novel cytochrome P450s in Taxus chinensis[J]. Front Plant Sci,2017,8:1468.
[16] Zhang CH,Mei XG,Liu L,Yu LJ. Enhanced paclitaxel production induced by the combination of elicitors in cell suspension cultures of Taxus chinensis[J]. Biotechnol Lett,2000,22(19):1561-1564.
[17] Roberts SC. Production and engineering of terpenoids in plant cell culture[J]. Nat Chem Biol,2007,3(7):387-395.
[18] Howat S,Park B,Oh IS,Jin YW,Lee EK,et al. Paclitaxel:biosynthesis,production and future prospects[J].New Biotechnol,2014,31(3):242-245.
[19] Jennewein S,Long RM,Williams RM,Croteau R. Cytochrome P450 taxadiene 5α-hydroxylase,a mechanistically unusual monooxygenase catalyzing the first oxygenation step of taxol biosynthesis[J]. Chem Biol,2004,11(3):379-387.
[20] Schoendorf A,Rithner CD,Williams RM,Croteau R. Molecular cloning of a cytochrome P450 taxane 10β-hydroxylase c DNA from Taxus and functional expression in yeast[J]. Proc Natl Acad Sci USA,2001,98(4):1501-1506.
[21] Jennewein S,Rithner CD,Williams RM,Croteau R. Taxol biosynthesis:taxane 13α-hydroxylase is a cytochrome P450-dependent monooxygenase[J]. Proc Natl Acad Sci USA,2001,98(24):13595-13600.
[22] Chau M,Croteau R. Molecular cloning and characterization of a cytochrome P450 taxoid 2α-hydroxylase involved in taxol biosynthesis[J]. Arch Biochem Biophys,2004,427(1):48-57.
[23] Chau M,Jennewein S,Walker K,Croteau R. Taxol biosynthesis:molecular cloning and characterization of a cytochrome P450 taxoid 7β-hydroxylase[J]. Chem Biol,2004,11(5):663-672.
[24]陈清浦,廖卫芳,付春华,赵春芳,余龙江.紫杉醇生物合成途径中羟化酶的研究进展[J].生物工程学报,2016,32(5):554-564.Chen QP,Liao WF,Fu CH,Zhao CF,Yu LJ. Research progress in hydroxylase in taxol biosynthetic pathway[J].Chinese Journal of Biotechnology,2016,32(5):554-564.
[25] Eisenreich W,Menhard B,Lee MS. Multiple oxygenase reactions in the biosynthesis of taxoids[J]. J Am Chem Soc,1998,120(37):9694-9695.
[26] Walker K,Schoendorf A,Croteau R. Molecular cloning of a taxa-4(20),11(12)-dien-5alpha-ol-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli[J]. Arch Biochem Biophys,2000,374(2):371-380.
[27] Walker K,Croteau R. Taxol biosynthesis:molecular cloning of a benzoyl-CoA:taxane 2alpha-O-benzoyltransferase cDNA from Taxus and functional expression in Escherichia coli[J]. Proc Natl Acad Sci USA,2000,97(25):13591-13596.