有机氮源对L-色氨酸发酵的影响
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摘要
以L-色氨酸生产菌Escherichia coli TRP03为供试菌株,研究了多种有机氮源对L-色氨酸发酵的影响。首先对不同来源的酵母粉进行了优化试验,确定一种最优酵母粉,摇瓶发酵时L-色氨酸可积累10.21 g/L;利用5 L发酵罐发酵1.5～3.0 h时,细胞出现二次生长现象,选择添加氨基酸粉和氯化胆碱促进细胞生长,经优化实验,确定同时添加氨基酸粉2 g/L、氯化胆碱0.5 g/L可在很大程度上解决细胞的二次生长问题并提高L-色氨酸产量至44.21 g/L;为提高中后期菌体活力及产酸能力,选择在不同发酵时期流加质量浓度为1 g/L的酵母粉、蛋氨酸及谷氨酰胺混合液,确定10 h流加时,中后期的活细胞数提高了30.18%,保证了菌体活力。菌株E. coli TRP03经36 h发酵,可积累L-色氨酸51.23 g/L,较未经任何优化的菌株提高41.91%。
The effect of various organic nitrogen sources on L-tryptophan fermentation was studied by using L-tryptophan producing strain Escherichia coli TRP03 as test strain. Different sources of yeast powders were optimized to determine an optimal yeast powder, and the L-tryptophan accumulation was 10.21 g/L using shaking flask fermentation. After fermented for 1.5-3.0 h in 5 L fermenter, the secondary growth phenomenon of cell was occurred. To solve the problem, amino acid powder and choline chloride were added to medium to promote the growth of cell, and the addition of amino acid powder and choline chloride was determined as 2 g/L and 0.5 g/L, respectively, which could solve the problem of secondary growth of cell in great extent, and increase the production of L-tryptophan to 44.21 g/L. In order to improve activity and acid production ability of cell in the middle and late stages, the yeast powder, methionine and glutamine mixture with a concentration of 1 g/L were fed in different fermentation periods.When the mixture was fed at 10 h, the viable cell count increased 30.18% in the middle and late stages, and the cell activity was ensured. The L-tryptophan accumulation was 51.23 g/L with fermentation by E. coli TRP03 for 36 h, which was 41.91% higher than that before optimization.
The effect of various organic nitrogen sources on L-tryptophan fermentation was studied by using L-tryptophan producing strain Escherichia coli TRP03 as test strain. Different sources of yeast powders were optimized to determine an optimal yeast powder, and the L-tryptophan accumulation was 10.21 g/L using shaking flask fermentation. After fermented for 1.5-3.0 h in 5 L fermenter, the secondary growth phenomenon of cell was occurred. To solve the problem, amino acid powder and choline chloride were added to medium to promote the growth of cell, and the addition of amino acid powder and choline chloride was determined as 2 g/L and 0.5 g/L, respectively, which could solve the problem of secondary growth of cell in great extent, and increase the production of L-tryptophan to 44.21 g/L. In order to improve activity and acid production ability of cell in the middle and late stages, the yeast powder, methionine and glutamine mixture with a concentration of 1 g/L were fed in different fermentation periods.When the mixture was fed at 10 h, the viable cell count increased 30.18% in the middle and late stages, and the cell activity was ensured. The L-tryptophan accumulation was 51.23 g/L with fermentation by E. coli TRP03 for 36 h, which was 41.91% higher than that before optimization.
引文
[1]杨梦晨,户红通,刘子强,等.响应面分析优化L-色氨酸清液发酵培养基[J].生物资源,2017(6):78-85.
[2]GU P,YANG F,KANG J,et al.One-step of tryptophan attenuator inactivation and promoter swapping to improve the production of L-tryptophan in Escherichia coli[J].Microb Cell Factor,2012,11(1):30.
[3]吴涛,赵津津,毛贤军.大肠杆菌磷酸烯醇式丙酮酸-糖磷酸转移酶系统改造对产L-色氨酸的影响[J].生物工程学报,2017,33(11):1877-1882.
[4]大肠杆菌芳香族氨基酸代谢工程研究进展[J].生物加工过程,2017,15(5):32-39.
[5]张涛,董延,张志博.猪饲粮中色氨酸的营养研究进展[J].饲料广角,2017(8):38-41.
[6]LIU L N,DUAN X G,WU J,et al.L-tryptophan production in Escherichia coli improved by weakening the Pta-AckA pathway[J].Plos One,2016,11(6):e0158200.
[7]CHEN L,CHEN M,MA C,et al.Discovery of feed-forward regulation in L-tryptophan biosynthesis and its use in metabolic engineering of E.coli for efficient tryptophan bioproduction[J].Metab Eng,2018,47:434-444.
[8]赵春光,程立坤,徐庆阳,等.微生物法生产L-色氨酸的研究进展[J].发酵科技通讯,2008,37(4):34-36.
[9]CHEN L,CHEN M,MA C,et al.Discovery of feed-forward regulation in L-tryptophan biosynthesis and its use in metabolic engineering of E.coli,for efficient tryptophan bioproduction[J].Metabol Eng,2018,47:434.
[10]CHEN L,ZENG A P.Rational design and metabolic analysis of Escherichia coli for effective production of L-tryptophan at high concentration[J].Appl Microbiol Biot,2017,101(2):559-568.
[11]IKEDA M,KATSUMATA R.Transport of aromatic amino acids and its in覲uence on overproduction of the amino acids in Corynebacterium glutamicum[J].Ferment Bioeng,1994,78:420-425.
[12]NEUBAUER P,LIN H Y,MATHISZIK B.Metabolic load of recombinant protein production:inhibition of cellular capacities for glucose uptake and respiration after induction of a heterologous gene in Escherichia coli[J].Biotechnol Bioeng,2003,83:53-64.
[13]黄静,史建明,霍文婷,等.氮源对L-色氨酸发酵的影响[J].食品与发酵工业,2011,37(5):21-25.
[14]刘子强,张震,蔡萌萌,等.稀糖分罐发酵对L-色氨酸发酵的影响[J].中国酿造,2018,37(4):132-136.
[15]王宁宁,吴振,江建梅,等.酵母类有机氮源及其在发酵行业的应用[J].产业与科技论坛,2014,13(2):69-71.
[16]郭翠.有机氮源对L-赖氨酸发酵的影响[J].安徽科技,2013(7):42-44.
[17]陈建华,殷云飞,赵彦光,等.发酵豆粕与合成氨基酸替代鱼粉仔猪饲料饲养对比试验[J].云南畜牧兽医,2017(3):4-6.
[18]罗俊,高淑红,陈长华,等.甲基化试剂对螺旋霉素发酵的影响[J].化学与生物工程,2011,28(5):46-50.
[19]刘敏,齐继红,汪之顼.059胆碱营养研究进展[J].环境卫生学杂志,2007,34(4):254-256.
[20]佚名.一种通过添加胆碱提高L-异亮氨酸发酵产量的方法:CN104878051A[P].2015-05-25.
[2]GU P,YANG F,KANG J,et al.One-step of tryptophan attenuator inactivation and promoter swapping to improve the production of L-tryptophan in Escherichia coli[J].Microb Cell Factor,2012,11(1):30.
[3]吴涛,赵津津,毛贤军.大肠杆菌磷酸烯醇式丙酮酸-糖磷酸转移酶系统改造对产L-色氨酸的影响[J].生物工程学报,2017,33(11):1877-1882.
[4]大肠杆菌芳香族氨基酸代谢工程研究进展[J].生物加工过程,2017,15(5):32-39.
[5]张涛,董延,张志博.猪饲粮中色氨酸的营养研究进展[J].饲料广角,2017(8):38-41.
[6]LIU L N,DUAN X G,WU J,et al.L-tryptophan production in Escherichia coli improved by weakening the Pta-AckA pathway[J].Plos One,2016,11(6):e0158200.
[7]CHEN L,CHEN M,MA C,et al.Discovery of feed-forward regulation in L-tryptophan biosynthesis and its use in metabolic engineering of E.coli for efficient tryptophan bioproduction[J].Metab Eng,2018,47:434-444.
[8]赵春光,程立坤,徐庆阳,等.微生物法生产L-色氨酸的研究进展[J].发酵科技通讯,2008,37(4):34-36.
[9]CHEN L,CHEN M,MA C,et al.Discovery of feed-forward regulation in L-tryptophan biosynthesis and its use in metabolic engineering of E.coli,for efficient tryptophan bioproduction[J].Metabol Eng,2018,47:434.
[10]CHEN L,ZENG A P.Rational design and metabolic analysis of Escherichia coli for effective production of L-tryptophan at high concentration[J].Appl Microbiol Biot,2017,101(2):559-568.
[11]IKEDA M,KATSUMATA R.Transport of aromatic amino acids and its in覲uence on overproduction of the amino acids in Corynebacterium glutamicum[J].Ferment Bioeng,1994,78:420-425.
[12]NEUBAUER P,LIN H Y,MATHISZIK B.Metabolic load of recombinant protein production:inhibition of cellular capacities for glucose uptake and respiration after induction of a heterologous gene in Escherichia coli[J].Biotechnol Bioeng,2003,83:53-64.
[13]黄静,史建明,霍文婷,等.氮源对L-色氨酸发酵的影响[J].食品与发酵工业,2011,37(5):21-25.
[14]刘子强,张震,蔡萌萌,等.稀糖分罐发酵对L-色氨酸发酵的影响[J].中国酿造,2018,37(4):132-136.
[15]王宁宁,吴振,江建梅,等.酵母类有机氮源及其在发酵行业的应用[J].产业与科技论坛,2014,13(2):69-71.
[16]郭翠.有机氮源对L-赖氨酸发酵的影响[J].安徽科技,2013(7):42-44.
[17]陈建华,殷云飞,赵彦光,等.发酵豆粕与合成氨基酸替代鱼粉仔猪饲料饲养对比试验[J].云南畜牧兽医,2017(3):4-6.
[18]罗俊,高淑红,陈长华,等.甲基化试剂对螺旋霉素发酵的影响[J].化学与生物工程,2011,28(5):46-50.
[19]刘敏,齐继红,汪之顼.059胆碱营养研究进展[J].环境卫生学杂志,2007,34(4):254-256.
[20]佚名.一种通过添加胆碱提高L-异亮氨酸发酵产量的方法:CN104878051A[P].2015-05-25.