高密度发酵产酪氨酸酚裂解酶及催化合成L-DOPA
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摘要
为了提高酪氨酸酚裂解酶(tyrosine phenol-lyase,TPL)的表达量和酶活,使之更高效地催化合成左旋多巴(3,4-2-dihydroxylphenylalanine,L-DOPA),在摇瓶优化的基础上,研究了溶氧控制策略、补料策略、诱导温度和诱导策略对菌体生长、TPL酶活和L-DOPA产量的影响。结果表明,在5 L发酵罐中25℃诱导条件下,采用DOstat补料策略控制罐内溶氧的体积分数为20%,菌体浓度、TPL酶活和催化合成L-DOPA产量较分批培养分别提高了17. 9%、57. 7%和27. 8%。诱导后控制溶氧体积分数在40%相比于20%更利于TPL的表达,酶活相比于20%的DO的提高了33. 8%。通过优化起始诱导时间,在菌体生长的对数前期(OD_(600)=7)诱导,细胞浓度提高到51. 2 g/L,酶活提高到2. 43 U/mL,L-DOPA产量为56. 58 g/L,较分批培养分别提高了64. 1%、170%和209. 2%。初步实现了重组大肠杆菌高密度培养生产TPL,为L-DOPA产业化研究奠定了基础。
In order to improve the expression level of tyrosine phenol lyase(TPL) and to make it more efficient to catalyze L-DOPA synthesis,influences of dissolved oxygen control strategy,feeding strategy and induction strategy on cell growth,TPL activity and L-DOPA production were studied. By using DO-stat feeding strategy to maintain the volume fraction of dissolved oxygen at 20% in a 5 L fermenter at 25℃,the cell density,TPL activity and the titer of L-DOPA increased by 17. 9%,57. 7%,and 27. 8%,respectively. The enzyme activity further increased by 33. 8% with the volume fraction of the dissolved oxygen changed from 20% to 40% after induction. When the cells were inducted during the logarithmic phase of cell growth(OD_(600)= 7),the maximal cell density,TPL activity and L-DOPA titer raised to 51. 2 g/L,2. 43 U/m L,and 56. 58 g/L,respectively,which was 64. 1%,170%,and 209. 2%,respectively,higher than that in batch cultures. This study preliminarily realized high density culture of recombinant Escherichia coli to produce TPL,which provides technical supports for the industrialization of L-DOPA.
In order to improve the expression level of tyrosine phenol lyase(TPL) and to make it more efficient to catalyze L-DOPA synthesis,influences of dissolved oxygen control strategy,feeding strategy and induction strategy on cell growth,TPL activity and L-DOPA production were studied. By using DO-stat feeding strategy to maintain the volume fraction of dissolved oxygen at 20% in a 5 L fermenter at 25℃,the cell density,TPL activity and the titer of L-DOPA increased by 17. 9%,57. 7%,and 27. 8%,respectively. The enzyme activity further increased by 33. 8% with the volume fraction of the dissolved oxygen changed from 20% to 40% after induction. When the cells were inducted during the logarithmic phase of cell growth(OD_(600)= 7),the maximal cell density,TPL activity and L-DOPA titer raised to 51. 2 g/L,2. 43 U/m L,and 56. 58 g/L,respectively,which was 64. 1%,170%,and 209. 2%,respectively,higher than that in batch cultures. This study preliminarily realized high density culture of recombinant Escherichia coli to produce TPL,which provides technical supports for the industrialization of L-DOPA.
引文
[1]ONDO W,COSS P,CHRISTIE M,et al.Conversion of L-dopa to extended release L-dopa(Rytary(R))in patients with fluctuating parkinson's disease:Predictor's of dose[J].Journal of Parkinson's disease,2019,1(9):153-156.
[2]KATZENSCHLAGER R,LEES A J.Treatment of Parkinson's disease:Levodopa as the first choice[J].Journal of neurology,2002,249(Suppl 2):ii19-ii24.
[3]VALDS R H,PUZER L,GOMES J M,et al.Production of L-DOPA under heterogeneous asymmetric catalysis[J].Catalysis Communications,2004,5(10):631-634.
[4]TANG Xiaoling,LIU Xiao,SUO Hui,et al.Process development for efficient biosynthesis of L-DOPA with recombinant Escherichia coli harboring tyrosine phenol lyase from Fusobacterium nucleatum[J].Bioprocess and Biosystems Engineering,2018,41(9):1 347-1 354.
[5]FOOR F,MORIN N,BOSTIAN K A.Production of L-dihydroxyphenylalanine in Escherichia-coli with the tyrosine phenol-lyase gene cloned from Erwinia-herbicola[J].Applied and Environmental Microbiology,1993,59(9):3 070-3 075.
[6]KOYANAGI T,KATAYAMA T,SUZUKI H,et al.Hyperproduction of 3,4-dihydroxyphenyl-L-alanine(L-Dopa)using Erwinia herbicola cells carrying a mutant transcriptional regulator TyrR[J].Bioscience Biotechnology and Biochemistry,2009,73(5):1 221-1 223.
[7]PHILLIPS R S.Chemistry and diversity of pyridoxal-5`-phosphate dependent enzymes[J].Biochim Biophys Acta,2015,1 854(9):1 167-1 174.
[8]PHILLIPS R S,VITA A,SPIVEY J B,et al.Groundstate destabilization by Phe-448 and Phe-449 contributes to tyrosine phenol-lyase catalysis[J].ACS Catalysis,2016,6(10):6 770-6 779.
[9]CHANDEL M,MAZMI W.Purification and characterization of tyrosine phenol lyase from Citrobacter freundii[J].Appl Biochem Biotech,2013,171(8):2 040-2 052.
[10]KIM D Y,RHA E,CHOI S L,et al.Development of bioreactor system for L-tyrosine synthesis using thermostable tyrosine phenol-lyase[J].Journal of Microbiology and Biotechnology,2007,17(1):116-122.
[11]RHA E,KIM S,CHOI S L,et al.Simultaneous improvement of catalytic activity and thermal stability of tyrosine phenol-lyase by directed evolution[J].Febs Journal,2009,276(21):6 187-6 194.
[12]LUNA-VELASCO A,FIELD J A,COBO-CURIEL A,et al.Inorganic nanoparticles enhance the production of reactive oxygen species(ROS)during the autoxidation of L-3,4-dihydroxyphenylalanine(L-dopa)[J].Chemosphere,2011,85(1):19-25.
[13]ZHENG Renchao,TANG Xiaoling,SUO Hui,et al.Biochemical characterization of a novel tyrosine phenol-lyase from Fusobacterium nucleatum for highly efficient biosynthesis of L-DOPA[J].Enzyme and Microbial Technology,2018,112:88-93.
[14]YAMADA H,KUMAGAI H,KASHIMA N,et al.Synthesis of L-tyrosine from pyruvate,ammonia and phenol by crystalline tyrosine phenol lyase[J].Biochemical and biophysical Research Communications,1972,46(2):370-374.
[15]李华钟,孙伟,刘吉泉,等.弗氏柠檬酸细菌酪氨酸酚解酶基因在大肠杆菌中的克隆与表达[J].工业微生物,2001,31(3):9-12.
[16]PHILLIPS R S,CRAIG S.Crystal structures of wild-type and F448A mutant Citrobacter freundii tyrosine phenollyase complexed with a substrate and inhibitors:Implications for the reaction mechanism[J].Biochemistry,2018,57(43):6 166-6 179.
[17]刘潇,汤晓玲,郑仁朝.生物法合成左旋多巴的研究进展[J].发酵科技通讯,2018,47(3):145-150.
[18]WACHTMEISTER J,ROTHER D.Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale[J].Current Opinion in Bitechnology,2016,42:169-177.
[19]TANG Xiaoling,LU Xiafeng,WU Zheming,et al.Biocatalytic production of(S)-2-aminobutanamide by a novel d-aminopeptidase from Brucella sp.with high activity and enantioselectivity[J].Journal of Biotechnology,2018,266(20):20-26.
[20]WU Zufang,DU Guocheng,CHEN Jian.Effects of dissolved oxygen concentration and DO-stat feeding strategy on Co Q10production with Rhizobium radiobacter[J].World Journal of Microbiology and Biotechnology,2003,19(9):925-928.
[21]PICOTTO L D,SGUAZZA G H,TIZZANO M A,et al.An effective and simplified DO-stat control strategy for production of rabies glycoprotein in Pichia pastoris[J].Protein Expression and Purification,2017,132:124-130.
[22]张俊丽,刘龙,房峻,等.基于DO-stat流加培养控制的L-异亮氨酸发酵条件优化[J].应用与环境生物学报,2011,17(3):317-320.
[23]杨军,梁凌宇,高雪峰,等.基因工程菌发酵液中多种成分快速检测方法的建立[J].中国生物制品学杂志,2013,26(12):1 831-1 834.
[24]AMIRZADA M I,YU M,GONG X,et al.Cost-effective production of recombinant human interleukin 24 by lactose induction and a two-step denaturing and one-step refolding method[J].Journal of industrial microbiology and biotechnology,2014,41(1):135-142.
[25]DEMIDKINA T V,FALEEV N G,PAPISOVA A I,et al.Aspartic acid 214 in citrobacter freundii tyrosine phenol-lyase ensures sufficient C-H-acidity of the external aldimine intermediate and proper orientation of the cofactor at the active site[J].Biochimica Et Biophysica Acta Proteins and Proteomics,2006,1 764(7):1 268-1 276.
[26]SURWASE S N,PATIL S A,APINE O A,et al.Efficient microbial conversion of tyrosine to L-DOPA by Brevundimonas sp.SGJ[J].Applied Biochemistry and Biotechnology,2012,167(5):1 015-1 028.
[2]KATZENSCHLAGER R,LEES A J.Treatment of Parkinson's disease:Levodopa as the first choice[J].Journal of neurology,2002,249(Suppl 2):ii19-ii24.
[3]VALDS R H,PUZER L,GOMES J M,et al.Production of L-DOPA under heterogeneous asymmetric catalysis[J].Catalysis Communications,2004,5(10):631-634.
[4]TANG Xiaoling,LIU Xiao,SUO Hui,et al.Process development for efficient biosynthesis of L-DOPA with recombinant Escherichia coli harboring tyrosine phenol lyase from Fusobacterium nucleatum[J].Bioprocess and Biosystems Engineering,2018,41(9):1 347-1 354.
[5]FOOR F,MORIN N,BOSTIAN K A.Production of L-dihydroxyphenylalanine in Escherichia-coli with the tyrosine phenol-lyase gene cloned from Erwinia-herbicola[J].Applied and Environmental Microbiology,1993,59(9):3 070-3 075.
[6]KOYANAGI T,KATAYAMA T,SUZUKI H,et al.Hyperproduction of 3,4-dihydroxyphenyl-L-alanine(L-Dopa)using Erwinia herbicola cells carrying a mutant transcriptional regulator TyrR[J].Bioscience Biotechnology and Biochemistry,2009,73(5):1 221-1 223.
[7]PHILLIPS R S.Chemistry and diversity of pyridoxal-5`-phosphate dependent enzymes[J].Biochim Biophys Acta,2015,1 854(9):1 167-1 174.
[8]PHILLIPS R S,VITA A,SPIVEY J B,et al.Groundstate destabilization by Phe-448 and Phe-449 contributes to tyrosine phenol-lyase catalysis[J].ACS Catalysis,2016,6(10):6 770-6 779.
[9]CHANDEL M,MAZMI W.Purification and characterization of tyrosine phenol lyase from Citrobacter freundii[J].Appl Biochem Biotech,2013,171(8):2 040-2 052.
[10]KIM D Y,RHA E,CHOI S L,et al.Development of bioreactor system for L-tyrosine synthesis using thermostable tyrosine phenol-lyase[J].Journal of Microbiology and Biotechnology,2007,17(1):116-122.
[11]RHA E,KIM S,CHOI S L,et al.Simultaneous improvement of catalytic activity and thermal stability of tyrosine phenol-lyase by directed evolution[J].Febs Journal,2009,276(21):6 187-6 194.
[12]LUNA-VELASCO A,FIELD J A,COBO-CURIEL A,et al.Inorganic nanoparticles enhance the production of reactive oxygen species(ROS)during the autoxidation of L-3,4-dihydroxyphenylalanine(L-dopa)[J].Chemosphere,2011,85(1):19-25.
[13]ZHENG Renchao,TANG Xiaoling,SUO Hui,et al.Biochemical characterization of a novel tyrosine phenol-lyase from Fusobacterium nucleatum for highly efficient biosynthesis of L-DOPA[J].Enzyme and Microbial Technology,2018,112:88-93.
[14]YAMADA H,KUMAGAI H,KASHIMA N,et al.Synthesis of L-tyrosine from pyruvate,ammonia and phenol by crystalline tyrosine phenol lyase[J].Biochemical and biophysical Research Communications,1972,46(2):370-374.
[15]李华钟,孙伟,刘吉泉,等.弗氏柠檬酸细菌酪氨酸酚解酶基因在大肠杆菌中的克隆与表达[J].工业微生物,2001,31(3):9-12.
[16]PHILLIPS R S,CRAIG S.Crystal structures of wild-type and F448A mutant Citrobacter freundii tyrosine phenollyase complexed with a substrate and inhibitors:Implications for the reaction mechanism[J].Biochemistry,2018,57(43):6 166-6 179.
[17]刘潇,汤晓玲,郑仁朝.生物法合成左旋多巴的研究进展[J].发酵科技通讯,2018,47(3):145-150.
[18]WACHTMEISTER J,ROTHER D.Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale[J].Current Opinion in Bitechnology,2016,42:169-177.
[19]TANG Xiaoling,LU Xiafeng,WU Zheming,et al.Biocatalytic production of(S)-2-aminobutanamide by a novel d-aminopeptidase from Brucella sp.with high activity and enantioselectivity[J].Journal of Biotechnology,2018,266(20):20-26.
[20]WU Zufang,DU Guocheng,CHEN Jian.Effects of dissolved oxygen concentration and DO-stat feeding strategy on Co Q10production with Rhizobium radiobacter[J].World Journal of Microbiology and Biotechnology,2003,19(9):925-928.
[21]PICOTTO L D,SGUAZZA G H,TIZZANO M A,et al.An effective and simplified DO-stat control strategy for production of rabies glycoprotein in Pichia pastoris[J].Protein Expression and Purification,2017,132:124-130.
[22]张俊丽,刘龙,房峻,等.基于DO-stat流加培养控制的L-异亮氨酸发酵条件优化[J].应用与环境生物学报,2011,17(3):317-320.
[23]杨军,梁凌宇,高雪峰,等.基因工程菌发酵液中多种成分快速检测方法的建立[J].中国生物制品学杂志,2013,26(12):1 831-1 834.
[24]AMIRZADA M I,YU M,GONG X,et al.Cost-effective production of recombinant human interleukin 24 by lactose induction and a two-step denaturing and one-step refolding method[J].Journal of industrial microbiology and biotechnology,2014,41(1):135-142.
[25]DEMIDKINA T V,FALEEV N G,PAPISOVA A I,et al.Aspartic acid 214 in citrobacter freundii tyrosine phenol-lyase ensures sufficient C-H-acidity of the external aldimine intermediate and proper orientation of the cofactor at the active site[J].Biochimica Et Biophysica Acta Proteins and Proteomics,2006,1 764(7):1 268-1 276.
[26]SURWASE S N,PATIL S A,APINE O A,et al.Efficient microbial conversion of tyrosine to L-DOPA by Brevundimonas sp.SGJ[J].Applied Biochemistry and Biotechnology,2012,167(5):1 015-1 028.
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