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DO-stat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(OD600=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 (OD600=7), the maximal cell density, TPL activity and L-DOPA titer raised to 51.2 g/L, 2.43 U/mL, 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.
[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] VALD S 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. Ground-state 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 phenol-lyase 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 CoQ10 production 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.
