大肠杆菌产L-苯丙氨酸发酵调控及代谢通量分析
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摘要
考察了外源添加中间代谢产物、维生素B1和硫酸镁对大肠杆菌发酵产L-苯丙氨酸的影响,结果表明,添加1g/L柠檬酸三钠、1g/Lα-酮戊二酸、150mg/L维生素B1及3g/L硫酸镁均对L-苯丙氨酸的合成有利。根据构建的大肠杆菌合成L-苯丙氨酸的生化反应网络,利用代谢通量分析其原因。结果表明,这些物质的添加均可以调节G6P和PEP节点处的代谢通量分布,为L-苯丙氨酸的合成提供更多的前体物质赤藓糖四磷酸(E4P)、磷酸烯醇式丙酮酸(PEP)和还原力NADPH。通过补料分批发酵实验得出,优化后菌体对总葡萄糖的消耗提高了24.49%,菌体终浓度提高了23.50%,L-苯丙氨酸的终浓度提高了62.87%,L-苯丙氨酸的收率提高了30.88%,乙酸的合成降低了56.51%。
The effect of adding intermediate metabolites,thiamine,magnesium on cell growth and Lphenylalanine production were investigated. The yield of L-phenylalanine( L-phe) improved when 1g / L sodium citrate,1g / L α-ketoglutarate,150 mg / L thiamine or 3g / L magnesium was added. According to the metabolic network of E. coli YP1617,the reason was acquired by metabolic flux analysis. The addition of them can adjust the metabolic flux distribution of G6 Pand PEPnode, which provide erythrose-4-phosphate( E4P),phosphoenolpyruvate( PEP) and NADPH for L-Phe production. In the optimal fed-batch fermentation,glucose consumption,cell and L-Phe concentrations,the yield of L-Phe was 24. 49%,23. 50%,62. 87% and 30. 88%higher than the control,respectively. Moreover,acetate production decreased 56. 51%.
The effect of adding intermediate metabolites,thiamine,magnesium on cell growth and Lphenylalanine production were investigated. The yield of L-phenylalanine( L-phe) improved when 1g / L sodium citrate,1g / L α-ketoglutarate,150 mg / L thiamine or 3g / L magnesium was added. According to the metabolic network of E. coli YP1617,the reason was acquired by metabolic flux analysis. The addition of them can adjust the metabolic flux distribution of G6 Pand PEPnode, which provide erythrose-4-phosphate( E4P),phosphoenolpyruvate( PEP) and NADPH for L-Phe production. In the optimal fed-batch fermentation,glucose consumption,cell and L-Phe concentrations,the yield of L-Phe was 24. 49%,23. 50%,62. 87% and 30. 88%higher than the control,respectively. Moreover,acetate production decreased 56. 51%.
引文
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[18]陈宁,刘辉.柠檬酸钠对L-亮氨酸发酵代谢流分布的影响.高校化学工程学报,2008,22(3):478-483.Chen N,Liu H.Effects of sodium citrate on metabolic flux distributions of L-Leucine production by Brevibacterium flavum TK0303.J Chem Eng Chin Uni,2008,22(3):478-483.
[19]Muhammad Akram.Citric acid cycle and role of its intermediates in metabolism.Cell Biochem Biophys,2014,68(3):475-478.
[20]Ren L J,Huang H,Xiao A H,et al.Enhanced docosahexaenoic acid production by reinforcing acetyl-Co A and NADPH supply in Schizochytrium sp.HX-308.Bioprocess Biosyst Eng,2009,32(6):837-843.
[21]Yoichiro S,Eric M P,James K,et al.Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae for high-level production of isoprenoids.Metab Eng,2007,9(2):160-168.
[22]Guo H W,Catherine M,Du G C,et al.Effects of pyruvate dehydrogenase subunits overexpression on theα-ketoglutarate production in Yarrowia lipolytica WSH-Z06.Appl Microbiol Biotechnol,2014,98(16):7003-7012.
[23]Waseem R,Yang X M,Wu H S,et al.Evaluation of metal ions(Zn2+,Fe3+and Mg2+)effect on the production of fusaricidintype antifungal compounds by Paenibacillus polymyxa SQR-21.Bioresour Technol,2010,101(2):9264-9271.
[24]Jia Y L,Zhong J J.Enhanced production of ansamitocin P-3 by addition of Mg2+in fermentation of Actinosynnema pretiosum.Bioresour Technol,2011,102(2):10147-10150.
[25]Liu B F,Ren N Q,Ding J,et al.The effect of Ni2+,Fe2+and Mg2+concentration on photo-hydrogen production by Rhodopseudomonas faecalis RLD-53.Int J hydrogen Energ,2009,34:721-726.
[2]Park S H,Hong K T,You S J,et al.L-phenylalanine production by auxotrophic regulatory mutants of Escherichia coli—Lphenylalanine production by mutants of E.coli.Korean J Chem Eng,1984,1(1):65-69.
[3]Hwang S O,Gil G H,Cho Y J,et al.The fermentation process for L-phenylalanine production using an auxotrophic regulatory mutant of Escherichia coli.Appl Microbiol Biotechnol,1985,22(2):108-113.
[4]周海岩.L-苯丙氨酸生产菌株的构建、代谢调控和发酵条件优化.无锡:江南大学,生物工程学院,2011.Zhou H Y.Strain Construction,Metabolic Regulation and Process Optimization for L-Phenylalanine Production.Wuxi:Jiangnan University,College of Biological Engineering,2011.
[5]Wu Y Q,Jiang P H,Fan C S,et al.Co-expression of five genes in E.coli for L-phenylalanine in Brevibacterium flavum.World J Gastroenterol,2003,9(2):342-346.
[6]Mutsumi T,Yoshinori N,Gyuseop O,et al.Control of Lphenylalanine production by dual feeding of glucose and Ltyrosine.Biotechnol Bioeng,1996,52(6):653-660.
[7]Yakandawala N,Romeo T,Friesen A D,et al.Metabolic engineering of Escherichia coli to enhance phenylalanine production.Appl Microbiol Biotechnol,2008,78(2):283-291.
[8]姜岷,黄秀梅,李建,等.氧化还原电位调控对产琥珀酸放线杆菌代谢通量分布的影响.化工学报,2009,60(10):2555-2561.Jiang M,Huang X M,Li J,et al.Effect of redox potential regulation on metabolic flux distribution of succinate production by Actinobacillus succinogenes.J Chem Ind Eng,2009,60(10):2555-2561.
[9]潘军华,潘中明,曾嵋涓,等.营养因子对乳酸发酵短杆菌合成赖氨酸的影响.无锡轻工大学学报,2002,21(2):130-134.Pan J H,Pan Z M,Zeng M J,et al.Effects of nutrition factors on the biosynthesis of lysine in a lysine producer Brevibacterium lactofermentum FP094.J Wuxi Univ Light Ind,2002,21(2):130-134.
[10]Underwood S A,Buszko M L,Shanmugam K T,et al.Flux through citrate synthase limits the growth of ethanologenic Escherichia coli KO11 during xylose fermentation.Appl Environ Microbiol,2002,68(3):1071-1081.
[11]Akshay G,Jinwoon L,Michael M D,et al.Metabolic fluxes,pools,and enzyme measurements suggest a tighter coupling of energetics and biosynthetic reactions associated with reduced pyruvate kinase flux.Biotechnol Bioeng,1999,64(2):129-134.
[12]Bruce E Waygood,Sanwa B D.The control of pyruvate kinases of Escherichia coli.J Biol Chem,1974,249(1):265-274.
[13]Sanwal B D.Regulatory mechanisms involving nicotinamide adenine nucleotides as allosteric effectors.J Biol Chem,1970,254(7):1626-1631.
[14]黄秀梅,姜岷,李建,等.外源添加代谢中间体对产L-琥珀酸放线杆菌厌氧发酵制备丁二酸的影响.生物工程学报,2010,26(9):1249-1256.Huang X M,Jiang M,Li J,et al.Effect of adding intermediate metabolites on succinate production by A.succinogenes.Chin J Biotech,2010,26(9):1249-1256.
[15]Maciek R A,David F K,Lisa A L,et al.Metabolic flux analysis in a nonstationary system:Fed-batch fermentation of a high yielding strain of E.coli producing 1,3-propanediol.Metab Eng,2007,9(3):277-292.
[16]Jens K,Christoph W,Hartwig S,et al.Metabolic pathway analysis for rational design of L-methionine production by E.coli and C.glutamicum.Metab Eng,2006,8(4):353-369.
[17]陈飞,冯小海,吴波,等.丙酸杆菌的两种固定化细胞反应器发酵生产丙酸及其代谢通量分析.化工学报,2011,62(4):1034-1041.Chen F,Feng X H,Wu B,et al.Metabolic flux analysis of proponic acid biosysbthesis with two immobilized cell reactor fermentation by Propionibacterium.J Chem Ind Eng,2011,62(4):1034-1041.
[18]陈宁,刘辉.柠檬酸钠对L-亮氨酸发酵代谢流分布的影响.高校化学工程学报,2008,22(3):478-483.Chen N,Liu H.Effects of sodium citrate on metabolic flux distributions of L-Leucine production by Brevibacterium flavum TK0303.J Chem Eng Chin Uni,2008,22(3):478-483.
[19]Muhammad Akram.Citric acid cycle and role of its intermediates in metabolism.Cell Biochem Biophys,2014,68(3):475-478.
[20]Ren L J,Huang H,Xiao A H,et al.Enhanced docosahexaenoic acid production by reinforcing acetyl-Co A and NADPH supply in Schizochytrium sp.HX-308.Bioprocess Biosyst Eng,2009,32(6):837-843.
[21]Yoichiro S,Eric M P,James K,et al.Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae for high-level production of isoprenoids.Metab Eng,2007,9(2):160-168.
[22]Guo H W,Catherine M,Du G C,et al.Effects of pyruvate dehydrogenase subunits overexpression on theα-ketoglutarate production in Yarrowia lipolytica WSH-Z06.Appl Microbiol Biotechnol,2014,98(16):7003-7012.
[23]Waseem R,Yang X M,Wu H S,et al.Evaluation of metal ions(Zn2+,Fe3+and Mg2+)effect on the production of fusaricidintype antifungal compounds by Paenibacillus polymyxa SQR-21.Bioresour Technol,2010,101(2):9264-9271.
[24]Jia Y L,Zhong J J.Enhanced production of ansamitocin P-3 by addition of Mg2+in fermentation of Actinosynnema pretiosum.Bioresour Technol,2011,102(2):10147-10150.
[25]Liu B F,Ren N Q,Ding J,et al.The effect of Ni2+,Fe2+and Mg2+concentration on photo-hydrogen production by Rhodopseudomonas faecalis RLD-53.Int J hydrogen Energ,2009,34:721-726.