大肠杆菌酪氨酸转运系统基因敲除对酪氨酸生产的影响
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摘要
酪氨酸是三大芳香族氨基酸之一,广泛用于食品、医药和化工等领域。转运系统工程为代谢工程改造大肠杆菌选育酪氨酸生产菌株提供了一种重要的研究策略。大肠杆菌中酪氨酸胞内转运主要通过aroP和tyrP基因编码的通透酶进行调控。以酪氨酸生产菌株HGXP为出发菌株,利用CRISPR-Cas9技术成功构建了aroP和tyrP基因敲除菌,并通过发酵试验考察了调节转运系统对酪氨酸生产的影响。发酵结果表明,aroP和tyrP基因敲除菌酪氨酸产量分别达到3.74 g/L和3.45 g/L,较出发菌株酪氨酸产量分别提高了19%和10%。对诱导温度进行了优化,结果表明38℃为最佳诱导温度。在3 L发酵罐上进行了补料分批发酵,aroP和tyrP基因敲除菌酪氨酸产量进一步提高至44.5 g/L和35.1 g/L,较出发菌株酪氨酸产量分别提高了57%和24%。研究结果对代谢工程强化大肠杆菌生产酪氨酸具有重要的参考价值。
L-tyrosine is one of three aromatic amino acids that are widely used in food,pharmaceutical and chemical industries.The transport system engineering provides an important research strategy for the metabolic engineering of E.scherichia coli to breed L-tyrosine producing strain.The intracellular transport of L-tyrosine in E.coli is mainly regulated by two distinct permeases encoded by aroP and tyrP genes.The aroP and tyrP gene knockout mutants were constructed by CRISPR-Cas technique on the basis of L-tyrosine producing strain HGXP,and the effects of regulating transport system on L-tyrosine production were investigated by fermentation experiments.The fermentation results showed that the aroP and tyrP knockout mutants produced 3.74 and 3.45 g/L L-tyrosine,respectively,which were 19% and 10% higher than that of the original strain.The optimum induction temperature was determined to be 38 ℃.Fed-batch fermentation was carried out on a3-L fermentor.The L-tyrosine yields of aroP and tyrP knockout mutants were further increased to 44.5 and 35.1 g/L,respectively,which were 57% and 24% higher than that of the original strain.The research results are of great reference value for metabolic engineering of E.coli to produce L-tyrosine.
L-tyrosine is one of three aromatic amino acids that are widely used in food,pharmaceutical and chemical industries.The transport system engineering provides an important research strategy for the metabolic engineering of E.scherichia coli to breed L-tyrosine producing strain.The intracellular transport of L-tyrosine in E.coli is mainly regulated by two distinct permeases encoded by aroP and tyrP genes.The aroP and tyrP gene knockout mutants were constructed by CRISPR-Cas technique on the basis of L-tyrosine producing strain HGXP,and the effects of regulating transport system on L-tyrosine production were investigated by fermentation experiments.The fermentation results showed that the aroP and tyrP knockout mutants produced 3.74 and 3.45 g/L L-tyrosine,respectively,which were 19% and 10% higher than that of the original strain.The optimum induction temperature was determined to be 38 ℃.Fed-batch fermentation was carried out on a3-L fermentor.The L-tyrosine yields of aroP and tyrP knockout mutants were further increased to 44.5 and 35.1 g/L,respectively,which were 57% and 24% higher than that of the original strain.The research results are of great reference value for metabolic engineering of E.coli to produce L-tyrosine.
引文
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[16]Ikeda M,Katsumata R.Transport of aromatic amino acids and its influence on overproduction of the amino acids in Corynebacterium glutamicum.JFerment Bioeng,1994,78(6):420-425.
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[18]Zhou HY,Liao XY,Wang TW,et al.Enhanced L-phenylalanine biosynthesis by co-expression of pheAfbr and aroFwt.Bioresour Technol,2010,101(11):4151-4156.
[19]Jiang Y,Chen B,Duan CL,et al.Multigene editing in the Escherichia coli genome via the CRISPR-Cas9system.Appl Environ Microbiol,2015,81(7):2506-2514.
[20]Ji CX.Determination of tryptophan and tyrosine of fermentation liquid by HPLC.Anhui Chem Ind,2014,40(1):68-69,74(in Chinese).纪传侠.高效液相色谱法测定发酵液中色氨酸和酪氨酸含量的研究.安徽化工,2014,40(1):68-69,74.
[21]Juminaga D,Baidoo EEK,Redding-Johanson AM,et al.Modular engineering of L-tyrosine production in Escherichia coli.Appl Environ Microbiol,2012,78(1):89-98.
[22]Burkovski A,Kr?mer R.Bacterial amino acid transport proteins:occurrence,functions,and significance for biotechnological applications.Appl Microbiol Biotechnol,2002,58(3):265-274.
[23]Ikeda M,Katsumata R.Tryptophan production by transport mutants of Corynebacterium glutamicum.Biosci Biotechnol Biochem,1995,59(8):1600-1602.
[24]Vrljic M,Sahm H,Eggeling L.A new type of transporter with a new type of cellular function:L-lysine export from Corynebacterium glutamicum.Mol Microbiol,1996,22(5):815-826.
[25]Okamoto K,Kino K,Ikeda M.Hyperproduction of L-threonine by an Escherichia coli mutant with impaired L-threonine uptake.Biosci Biotechnol Biochem,1997,61(11):1877-1882.
[26]Kim B,Binkley R,Kim HU,et al.Metabolic engineering of Escherichia coli for the enhanced production of L-tyrosine.Biotechnol Bioeng,2018,115(10):2554-2564.
[2]Seetharam G,Saville BA.L-DOPA production from tyrosinase immobilized on zeolite.Enzyme Microb Tech,2002,31(6):747-753.
[3]Katsuyama Y,Funa N,Miyahisa I,et al.Synthesis of unnatural flavonoids and stilbenes by exploiting the plant biosynthetic pathway in Escherichia coli.Chem Biol,2007,14(6):613-621.
[4]Lütke-Eversloh T,Santos CNS,Stephanopoulos G.Perspectives of biotechnological production of L-tyrosine and its applications.Appl Microbiol Biot,2007,77(4):751-762.
[5]Wu JJ,Liu PR,Fan YM,et al.Multivariate modular metabolic engineering of Escherichia coli to produce resveratrol from L-tyrosine.J Biotechnol,2013,167(4):404-411.
[6]Kim DY,Rha E,Choi SL,et al.Development of bioreactor system for L-tyrosine synthesis using thermostable tyrosine phenol-lyase.J Microbiol Biotechnol,2007,17(1):116-122.
[7]Kim JH,Song JJ,Kim BG,et al.Enhanced stability of tyrosine phenol-lyase from Symbiobacterium toebii by DNA shuffling.J Microbiol Biotechnol,2004,14(1):153-157.
[8]Rha E,Kim S,Choi SL,et al.Simultaneous improvement of catalytic activity and thermal stability of tyrosine phenol-lyase by directed evolution.FEBS J,2009,276(21):6187-6194.
[9]Santos CNS,Xiao WH,Stephanopoulos G.Rational,combinatorial,and genomic approaches for engineering L-tyrosine production in Escherichia coli.Proc Natl Acad Sci USA,2012,109(34):13538-13543.
[10]Kim SC,Min BE,Hwang HG,et al.Pathway optimization by re-design of untranslated regions for L-tyrosine production in Escherichia coli.Sci Rep,2015,5:13853.
[11]Patnaik R,Zolandz RR,Green DA,et al.L-tyrosine production by recombinant Escherichia coli:fermentation optimization and recovery.Biotechnol Bioeng,2008,99(4):741-752.
[12]Olson MM,Templeton LJ,Suh W,et al.Production of tyrosine from sucrose or glucose achieved by rapid genetic changes to phenylalanine-producing Escherichia coli strains.Appl Microbiol Biot,2007,74(5):1031-1040.
[13]Gosset G,Yong-Xiao J,Berry A.A direct comparison of approaches for increasing carbon flow to aromatic biosynthesis in Escherichia coli.J Ind Microbiol,1996,17(1):47-52.
[14]Lütke-Eversloh T,Stephanopoulos G.Feedback inhibition of chorismate mutase/prephenate dehydrogenase(TyrA)of Escherichia coli:generation and characterization of tyrosine-insensitive mutants.Appl Environ Microbiol,2005,71(11):7224-7228.
[15]Ikeda M,Katsumata R.Metabolic engineering to produce tyrosine or phenylalanine in a tryptophan-producing Corynebacterium glutamicum strain.Appl Environ Microbiol,1992,58(3):781-785.
[16]Ikeda M,Katsumata R.Transport of aromatic amino acids and its influence on overproduction of the amino acids in Corynebacterium glutamicum.JFerment Bioeng,1994,78(6):420-425.
[17]Hu CY,Jiang PH,Xu JF,et al.Mutation analysis of the feedback inhibition site of phenylalaninesensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Escherichia coli.J Basic Microbiol,2003,43(5):399-406.
[18]Zhou HY,Liao XY,Wang TW,et al.Enhanced L-phenylalanine biosynthesis by co-expression of pheAfbr and aroFwt.Bioresour Technol,2010,101(11):4151-4156.
[19]Jiang Y,Chen B,Duan CL,et al.Multigene editing in the Escherichia coli genome via the CRISPR-Cas9system.Appl Environ Microbiol,2015,81(7):2506-2514.
[20]Ji CX.Determination of tryptophan and tyrosine of fermentation liquid by HPLC.Anhui Chem Ind,2014,40(1):68-69,74(in Chinese).纪传侠.高效液相色谱法测定发酵液中色氨酸和酪氨酸含量的研究.安徽化工,2014,40(1):68-69,74.
[21]Juminaga D,Baidoo EEK,Redding-Johanson AM,et al.Modular engineering of L-tyrosine production in Escherichia coli.Appl Environ Microbiol,2012,78(1):89-98.
[22]Burkovski A,Kr?mer R.Bacterial amino acid transport proteins:occurrence,functions,and significance for biotechnological applications.Appl Microbiol Biotechnol,2002,58(3):265-274.
[23]Ikeda M,Katsumata R.Tryptophan production by transport mutants of Corynebacterium glutamicum.Biosci Biotechnol Biochem,1995,59(8):1600-1602.
[24]Vrljic M,Sahm H,Eggeling L.A new type of transporter with a new type of cellular function:L-lysine export from Corynebacterium glutamicum.Mol Microbiol,1996,22(5):815-826.
[25]Okamoto K,Kino K,Ikeda M.Hyperproduction of L-threonine by an Escherichia coli mutant with impaired L-threonine uptake.Biosci Biotechnol Biochem,1997,61(11):1877-1882.
[26]Kim B,Binkley R,Kim HU,et al.Metabolic engineering of Escherichia coli for the enhanced production of L-tyrosine.Biotechnol Bioeng,2018,115(10):2554-2564.