组合策略促进碱性果胶酶在毕赤酵母中高效表达
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摘要
本研究组合了基因密码子优化及分子伴侣共表达策略提高碱性果胶酶(PGL)在毕赤酵母中的胞外产量。基于Pichia pastoris密码子偏好性,优化了来源于Bacillus sp.WSHB04-02 PGL高热稳定性突变体K314M基因,并克隆至pPIC9K的Eco R I-Not I得到PGL表达载体p PIC9K-PGL。p PIC9K-PGL转化Pichia pastoris GS115(his-)得到重组菌GS115/PGL 14#,胞外PGL酶活达到301.32 U/mL,较优化前提高25.1%。在此基础上,分别及同时共表达分子伴侣内质网蛋白折叠氧化还原辅助因子(ERO1)和泛素共轭酶(UBC1)。结果显示,同时表达ERO1和UBC1(GS115/PGLl-ERO1-UBC1 2#)使重组菌胞外PGL较共表达前提高49.3%,达到450.12 U/mL。应用指数流加策略对重组菌株GS115/PGLl-ERO1-UBC1 2#进行3 L罐发酵,诱导发酵96h胞外PGL可达1 362.31 U/mL。研究结果对促进PGL产量的提升及其应用具有重要现实意义。
In this study,codon usage optimization and co-expression of molecular chaperone are used to improve the extracellular production of alkaline polygalacturonate lyase(PGL) in Pichia pastoris. According to the codon bias of P. pastoris,the gene of the stabilized mutant(K314 M) of Bacillus sp. WSHB04-02 PGL is optimized and cloned into the Eco R I-Not I sites of pPIC9 K-PGL,yielding the expression plasmid pPIC9 K-PGL. pPIC9 K-PGL is transformed into the P. pastoris GS115(his-),yielding the strain GS115/PGL 14#. GS115/PGL 14# produced 301.32 U/mL of extracellular PGL which is 25.1% higher than that of the strain with native PGL gene. On this basis,two molecular chaperones the protein folding factors endoplasmic reticulum oxidoreduction 1(ERO1) and ubiquitin-conjugating enzyme(UBC1) are co-expressed with the optimized K314 M gene individually and simultaneously. It is showed that co-expression of ERO1 and UBC1 at the same time(GS115/PGLl-ERO1-UBC1 2#) increased the extracellular PGL by 49.3%,which is up to450.12 U/mL. By using an exponential fed-batch strategy in 3 L-fermenter,GS115/PGLl-ERO1-UBC1 2# produces 1 362.31 U/mL after the 96 h induction by methanol. The results here may benefit the industrial production and application of the PGL.
In this study,codon usage optimization and co-expression of molecular chaperone are used to improve the extracellular production of alkaline polygalacturonate lyase(PGL) in Pichia pastoris. According to the codon bias of P. pastoris,the gene of the stabilized mutant(K314 M) of Bacillus sp. WSHB04-02 PGL is optimized and cloned into the Eco R I-Not I sites of pPIC9 K-PGL,yielding the expression plasmid pPIC9 K-PGL. pPIC9 K-PGL is transformed into the P. pastoris GS115(his-),yielding the strain GS115/PGL 14#. GS115/PGL 14# produced 301.32 U/mL of extracellular PGL which is 25.1% higher than that of the strain with native PGL gene. On this basis,two molecular chaperones the protein folding factors endoplasmic reticulum oxidoreduction 1(ERO1) and ubiquitin-conjugating enzyme(UBC1) are co-expressed with the optimized K314 M gene individually and simultaneously. It is showed that co-expression of ERO1 and UBC1 at the same time(GS115/PGLl-ERO1-UBC1 2#) increased the extracellular PGL by 49.3%,which is up to450.12 U/mL. By using an exponential fed-batch strategy in 3 L-fermenter,GS115/PGLl-ERO1-UBC1 2# produces 1 362.31 U/mL after the 96 h induction by methanol. The results here may benefit the industrial production and application of the PGL.
引文
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[21]WANG Mingxing,LIU Song,LIU Long,et al. Fusions of amphipathic peptide to the alkaline polygalacturonate lyase from Bacillus sp. WSHB04-02 improves the production[J]. Journal of Food Science and Biotechnology,2016,35(5):504-509.(in Chinese)
[22]HAGGLUND P,BUNKENBORG J,ELORTZA F,et al. A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation[J]. Journal of Proteome Research,2004,3(3):556-566.
[23]XU Z,LIU S,LU X,et al. Thermal inactivation of a recombinant lipoxygenase from Pseudomonas aeruginosa BBE in the absence and presence of additives[J]. Journal of the Science of Food and Agriculture,2014,94(9):1753-1757.
[24]ATA O,BOY E,GUNES H,et al. Codon optimization of xylA gene for recombinant glucose isomerase production in Pichia pastoris and fed-batch feeding strategies to fine-tune bioreactor performance[J]. Bioprocess and Biosystems Engineering,2015,38(5):889-903.
[25]PAUL M S,LI W H. The codon adaptation index-a measure of directional synonymous codon usage bias,and its potential applications[J]. Nucleic Acids Research,1987,15:1281-1296.
[26]ADIVITIY A,DAGAR V K,DEVI N,et al. High level production of active streptokinase in Pichia pastoris fed-batch culture[J].International Journal of Biological Macromolecules,2015,83:50-60.
[27]HONG F,MEINANDER N Q,JONSSON L J. Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris[J]. Biotechnology and Bioengineering,2002,79(4):438-449.
[28]WEI W,CHEN L,ZOU G,et al. N-glycosylation affects the proper folding,enzymatic characteristics and production of a fungalβ-glucosidase[J]. Biotechnology and Bioengineering,2013,110:3075-3084.
[2]HOONDAL G S,TIWARI R P,TEWARI R,et al. Microbial alkaline pectinases and their industrial applications:a review[J].Applied Microbiology and Biotechnology,2002,59(4/5):409-418.
[3]ZOU M Y,LI X Z,SHI W J,et al. Improved production of alkaline polygalacturonate lyase by homologous overexpression pelA in Bacillus subtilis[J]. Process Biochemistry,2013,48(8):1143-1150.
[4]WANG H L,LI X M,MA Y H,et al. Process optimization of high-level extracellular production of alkaline pectate lyase in recombinant Escherichia coli BL21(DE3)[J]. Biochemical Engineering Journal,2015,93:38-46.
[5]WANG Y,WANG Z H,DU G C,et al. Enhancement of alkaline polygalacturonate lyase production in recombinant Pichia pastoris according to the ratio of methanol to cell concentration[J]. Bioresource Technology,2009,100(3):1343-1349.
[6]CEREGHINO J L,CREGG J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J]. FEMS Microbiology Reviews,2000,24(1):45-66.
[7]WANG Z H,WANG Y,ZHANG D X,et al. Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation[J]. Bioresource Technology,2010,101(4):1318-1323.
[8]AHMAD M,HIRZ M,PICHLER H,et al. Protein expression in Pichia pastoris:recent achievements and perspectives for heterologous protein production[J]. Applied Microbiology and Biotechnology,2014,98(12):5301-5317.
[9]HOU J,TYO K E,LIU Z,et al. Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae[J]. FEMS Yeast Res,2012,12(5):491-510.
[10]HOHENBLUM H,GASSER B,MAURER M,et al. Effects of gene dosage,promoters,and substrates on unfolded protein stress of recombinant Pichia pastoris[J]. Biotechnology and Bioengineering,2004,85(4):367-375.
[11]VASSILEVA A,CHUGH D A,SWAMINATHAN S,et al. Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris[J]. Protein Expression and Purification,2001,21(1):71-80.
[12]DEML L,BOJAK A,STECK S,et al. Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein[J]. Journal of Virology,2001,75(22):10991-11001.
[13]AKCAPINAR G B,GUL O,SEZERMAN U. Effect of Codon optimization on the expression of Trichoderma reesei Endoglucanase 1 in Pichia pastoris[J]. Biotechnology Progress,2011,27(5):1257-1263.
[14]GASSER B,MAURER M,RAUTIO J,et al. Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions[J]. BMC Genomics,2007,8:179.
[15]SHA C,YU X W,LIN N X,et al. Enhancement of lipase r27RCL production in Pichia pastoris by regulating gene dosage and co-expression with chaperone protein disulfide isomerase[J]. Enzyme and Microbial Technology,2013,53:438-443.
[16]YU P,ZHU Q,CHEN K,et al. Improving the secretory production of the heterologous protein in Pichia pastoris by focusing on protein folding[J]. Applied Biochemistry and Biotechnology,2015,175(1):535-548.
[17]LIU Song,WANG Mingxing,DU Guocheng,et al. Improvement of thermal stability of alkaline polygalacturonate lyase through site-directed mutagenesis[J]. Food and Fermentation Industries,2015,41(9):333-339.(in Chinese)
[18]XUAN Yaoji,ZHOU Xiangshan,ZHANG Yuanxing. Determination of copy number of foreign gene in genome of Pichia pastoris by real-time fluorescent quantitative PCR[J]. Chinese Journal of Biologicals,2009,22(19):1236-1239.(in Chinese)
[19]ABAD S,KITZ K,HORMANN A,et al. Real-time PCR-based determination of gene copy numbers in Pichia pastoris[J].Biotechnology Journal,2010,5(4):413-420.
[20]WANG Yun,HUA Zhaozhe,LIU Liming,et al. High-level production of alkaline polygalacturonate lyase in recombinant Pichia pastoris[J]. Chinese Journal of Biotechnology,2008,24:835-839.(in Chinese)
[21]WANG Mingxing,LIU Song,LIU Long,et al. Fusions of amphipathic peptide to the alkaline polygalacturonate lyase from Bacillus sp. WSHB04-02 improves the production[J]. Journal of Food Science and Biotechnology,2016,35(5):504-509.(in Chinese)
[22]HAGGLUND P,BUNKENBORG J,ELORTZA F,et al. A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation[J]. Journal of Proteome Research,2004,3(3):556-566.
[23]XU Z,LIU S,LU X,et al. Thermal inactivation of a recombinant lipoxygenase from Pseudomonas aeruginosa BBE in the absence and presence of additives[J]. Journal of the Science of Food and Agriculture,2014,94(9):1753-1757.
[24]ATA O,BOY E,GUNES H,et al. Codon optimization of xylA gene for recombinant glucose isomerase production in Pichia pastoris and fed-batch feeding strategies to fine-tune bioreactor performance[J]. Bioprocess and Biosystems Engineering,2015,38(5):889-903.
[25]PAUL M S,LI W H. The codon adaptation index-a measure of directional synonymous codon usage bias,and its potential applications[J]. Nucleic Acids Research,1987,15:1281-1296.
[26]ADIVITIY A,DAGAR V K,DEVI N,et al. High level production of active streptokinase in Pichia pastoris fed-batch culture[J].International Journal of Biological Macromolecules,2015,83:50-60.
[27]HONG F,MEINANDER N Q,JONSSON L J. Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris[J]. Biotechnology and Bioengineering,2002,79(4):438-449.
[28]WEI W,CHEN L,ZOU G,et al. N-glycosylation affects the proper folding,enzymatic characteristics and production of a fungalβ-glucosidase[J]. Biotechnology and Bioengineering,2013,110:3075-3084.