双组份转运系统BrnFE的过表达和表面活性剂的添加对谷氨酸棒杆菌发酵生产L-异亮氨酸的影响
|
摘要
为了提高L-异亮氨酸生产菌株Corynebacterium glutamicum LD320的产酸水平,通过改善其分泌系统,在C.glutamicum LD320中分别过表达突变型和野生型的双组份转运系统BrnFE操纵子,构建了重组菌LD320/p XMJ19-brn FE和LD320/p XMJ19-brnFE1。通过对两株重组菌的L-异亮氨酸生产分析比较,发现突变型比野生型能更有效地提高L-异亮氨酸产量。同时对LD320/p XMJ19-brnFE1进行表面活性剂添加实验,发现Tween-80为最佳选择,其最佳添加量为0.5 g/L,最佳添加时间为对数期的16 h。最后通过7 L发酵罐放大实验,LD320/pXMJ19-brnFE1的L-异亮氨酸产量由18.53 g/L提高到25.45 g/L,比对照组提高了37%。
In order to increase L-isoleucine productivity in Corynebacterium glutamicum LD320, the L-isoleucine secretion system was improved. Both the mutated brn FE operon and the wild type brn FE operon were cloned into the shutter expression vector p XMJ19 individually,and the resultant plasmids pXMJ19-brn FE and p XMJ19-brnFE1 were transformed into LD320. Then the L-isoleucine production by these two strains was compared and analyzed. More Lisoleucine was produced by LD320 / p XMJ19-brn FE than that by LD320 / pXMJ19-brn FE1. In addition,the effects of several surfactants on growth and L-isoleucine production by LD320 / p XMJ19-brn FE1 were investigated. The shaking flask results revealed that Tween-80 played a key role in cell growth and L-isoleucine production. The suitable concentration of Tween-80 was 0. 5 g / L and the optimal adding time was 16 h. Furthermore,the effects of Tween-80 on cell growth and Lisoleucine production by LD320 / pXMJ19-brn FE1 was also investigated in a 7 L fermentor. The concentration of Lisoleucine increased from 18. 53 g / L to 25. 45 g / L,which was 37% higher than that of the control strain.
In order to increase L-isoleucine productivity in Corynebacterium glutamicum LD320, the L-isoleucine secretion system was improved. Both the mutated brn FE operon and the wild type brn FE operon were cloned into the shutter expression vector p XMJ19 individually,and the resultant plasmids pXMJ19-brn FE and p XMJ19-brnFE1 were transformed into LD320. Then the L-isoleucine production by these two strains was compared and analyzed. More Lisoleucine was produced by LD320 / p XMJ19-brn FE than that by LD320 / pXMJ19-brn FE1. In addition,the effects of several surfactants on growth and L-isoleucine production by LD320 / p XMJ19-brn FE1 were investigated. The shaking flask results revealed that Tween-80 played a key role in cell growth and L-isoleucine production. The suitable concentration of Tween-80 was 0. 5 g / L and the optimal adding time was 16 h. Furthermore,the effects of Tween-80 on cell growth and Lisoleucine production by LD320 / pXMJ19-brn FE1 was also investigated in a 7 L fermentor. The concentration of Lisoleucine increased from 18. 53 g / L to 25. 45 g / L,which was 37% higher than that of the control strain.
引文
[1]Ikeda S,Fujita I,Yoshinaga F.Screening of L-isoleucine producers among ethionine resistant mutants of L-threonine producing Bacteria[J].Agricultural and Biological Chemistry,1976,40(3):511-516.
[2]Rose WC,Haines WJ,Johnson JE.The role of the amino acids in human nutrition[J].Journal of Biological Chemistry,1942,146(2):683-684.
[3]Blomstrand E.A role for branched-chain amino acids in reducing central fatigue[J].Journal of Nutrition[J].Journal of Nutrition,2006,136(2):544-547.
[4]Park JH,Lee SY.Metabolic pathways and fermentative production of L-aspartate family amino acids[J].Biotechnology Journal,2010,5(6):560-577.
[5]Kind S,Jeong WK,Schrder H,et al.Systems-wide metabolic pathway engineering in Corynebacterium glutamicum for bio-based production of diaminopentane[J].Metabolic Engineering,2010,12(4):341-351.
[6]Kind S,Kreye S,Wittmann C.Metabolic engineering of cellular transport for overproduction of the platform chemical 1,5-diaminopentane in Corynebacterium glutamicum[J].Metabolic Engineering,2011,13(5):617-627.
[7]Park SD,Lee JY,Sim SY,et al.Characteristics of methionine production by an engineered Corynebacterium glutamicum strain[J].Metabolic Engineering,2007,9(4):327-336.
[8]Morbach S,Sahm H,Eggeling L.Use of feedback-resistant threonine dehydratases of Corynebacterium glutamicum to increase carbon flux towards L-isoleucine[J].Appl Environ Microbiol,1995,61(12):4315-4320.
[9]Kennerknecht N,Sahm H,Yen MR,et al.Export of L-Isoleucine from Corynebacterium glutamicum:a two-gene-encoded member of a new translocator family[J].Journal of Bacteriology,2002,184(14):3947-3956.
[10]Trotschel C,Deutenberg D,Bathe B,et al.Characterization of methionine export in Corynebacterium glutamicum[J].Journal of Bacteriology,2005,187(11):3786-3794.
[11]Yin L,Shi F,Hu X,et al.Increasing L-isoleucine production in Corynebacterium glutamicum by overexpressing global regulator Lrp and two-component export system Brn FE[J].Journal of Applied Microbiology,2013,114(5):1369-1377.
[12]梅建凤,航闵.生物表面活性剂及其应用[J].工业微生物,2001,31(1):54-57.
[13]Nemec T,Jernejc K.Influence of Tween 80 on lipid metabolism of an Aspergillus niger strain[J].Applied Biochemistry and Biotechnology,2002,101(3):229-238.
[14]朱艳,袁其朋,王航.添加氧载体及表面活性剂对番茄红素发酵的影响[J].微生物学通报,2006,33(1):90-94.
[15]韦祎,张淑荣,刘春巧等.添加表面活性剂对α-熊果苷发酵的影响[J].化工学报,2007,58(9):2352-2356.
[16]余秉琦,沈微,诸葛健.适于异源DNA高效率整合转化的谷氨酸棒杆菌电转化法[J].中国生物工程杂志,2005,25(2):78-81.
[17]van der Rest ME,Lange C,Molenaar D.A heat shock following electroporation induces highly effcient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA[J].Appl Microbiol Biotechnol,1999,52(4):541-545.
[18]许彦芳,许新民,王永利.高效液相色谱柱前自动衍生法测定氨基酸含量[J].河北医科大学学报,1996,17(3):132-134.
[19]何晨光,马雷,徐庆阳等.用高效液相色谱定量分析分支链氨基酸[[J].生物技术通讯,2009,20(4):556-558.
[20]卫云路,宁正祥,郑成.新型表面活性剂在谷氨酸发酵中的应用[J].现代食品科技,2009,25(3):289-295.
[2]Rose WC,Haines WJ,Johnson JE.The role of the amino acids in human nutrition[J].Journal of Biological Chemistry,1942,146(2):683-684.
[3]Blomstrand E.A role for branched-chain amino acids in reducing central fatigue[J].Journal of Nutrition[J].Journal of Nutrition,2006,136(2):544-547.
[4]Park JH,Lee SY.Metabolic pathways and fermentative production of L-aspartate family amino acids[J].Biotechnology Journal,2010,5(6):560-577.
[5]Kind S,Jeong WK,Schrder H,et al.Systems-wide metabolic pathway engineering in Corynebacterium glutamicum for bio-based production of diaminopentane[J].Metabolic Engineering,2010,12(4):341-351.
[6]Kind S,Kreye S,Wittmann C.Metabolic engineering of cellular transport for overproduction of the platform chemical 1,5-diaminopentane in Corynebacterium glutamicum[J].Metabolic Engineering,2011,13(5):617-627.
[7]Park SD,Lee JY,Sim SY,et al.Characteristics of methionine production by an engineered Corynebacterium glutamicum strain[J].Metabolic Engineering,2007,9(4):327-336.
[8]Morbach S,Sahm H,Eggeling L.Use of feedback-resistant threonine dehydratases of Corynebacterium glutamicum to increase carbon flux towards L-isoleucine[J].Appl Environ Microbiol,1995,61(12):4315-4320.
[9]Kennerknecht N,Sahm H,Yen MR,et al.Export of L-Isoleucine from Corynebacterium glutamicum:a two-gene-encoded member of a new translocator family[J].Journal of Bacteriology,2002,184(14):3947-3956.
[10]Trotschel C,Deutenberg D,Bathe B,et al.Characterization of methionine export in Corynebacterium glutamicum[J].Journal of Bacteriology,2005,187(11):3786-3794.
[11]Yin L,Shi F,Hu X,et al.Increasing L-isoleucine production in Corynebacterium glutamicum by overexpressing global regulator Lrp and two-component export system Brn FE[J].Journal of Applied Microbiology,2013,114(5):1369-1377.
[12]梅建凤,航闵.生物表面活性剂及其应用[J].工业微生物,2001,31(1):54-57.
[13]Nemec T,Jernejc K.Influence of Tween 80 on lipid metabolism of an Aspergillus niger strain[J].Applied Biochemistry and Biotechnology,2002,101(3):229-238.
[14]朱艳,袁其朋,王航.添加氧载体及表面活性剂对番茄红素发酵的影响[J].微生物学通报,2006,33(1):90-94.
[15]韦祎,张淑荣,刘春巧等.添加表面活性剂对α-熊果苷发酵的影响[J].化工学报,2007,58(9):2352-2356.
[16]余秉琦,沈微,诸葛健.适于异源DNA高效率整合转化的谷氨酸棒杆菌电转化法[J].中国生物工程杂志,2005,25(2):78-81.
[17]van der Rest ME,Lange C,Molenaar D.A heat shock following electroporation induces highly effcient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA[J].Appl Microbiol Biotechnol,1999,52(4):541-545.
[18]许彦芳,许新民,王永利.高效液相色谱柱前自动衍生法测定氨基酸含量[J].河北医科大学学报,1996,17(3):132-134.
[19]何晨光,马雷,徐庆阳等.用高效液相色谱定量分析分支链氨基酸[[J].生物技术通讯,2009,20(4):556-558.
[20]卫云路,宁正祥,郑成.新型表面活性剂在谷氨酸发酵中的应用[J].现代食品科技,2009,25(3):289-295.