甘露醇磷酸化酶基因的敲除对D-甘露醇合成的影响
|
摘要
为了探究甘露醇分解利用途径对甘露醇合成的影响,在重组菌株R_1(K-12/pTrc99a-mdh)的基础上,通过CRISPR/Cas9敲除E.coli PTS系统中的cmtA、cmtB、mtlA基因,阻断了E. coli K-12中甘露醇的分解途径,获得了重组菌株R_3(K-12/?cmtA?cmtBmdh~+)和R_5(K-12/?cmtA?cmtB?mtlAmdh~+)。与出发菌株R_1相比,R_3的生长速率没有降低,而R_5的生长速率明显下降。并且R_5在以甘露醇为唯一碳源的培养基上已无法生长,说明cmtA、cmtB、mtlA三个基因全部敲除后,菌株已无法再利用甘露醇作为碳源进行生长。最后,构建的重组菌株R_5,测得MDH酶活力为258 U/mL,用高效液相色谱对胞外产物进行检测,可检测到少量的甘露醇,为进一步探究大肠杆菌合成甘露醇的调控机制奠定基础。
In order to explore the effect of mannitol decomposition and utilization pathways on mannitol synthesis,the cmtA,cmtB and mtlA genes in Escherichia. coli PTS system were knocked out by CRISPR/Cas9 on the basis of recombinant strain R_1(K-12/pTrc99 amdh),which blocked the decomposition pathway of mannitol in E. coli K-12,and the recombinant strain R_3(K-12/?cmtA?cmtBmdh~+)and R_5(K-12/?cmtA?cmtB?mtlAmdh~+)were obtained. Compared with the original strain R_1,the growth rate of R_3 did not decrease,but that of R_5 decreased significantly. Moreover,R_5 did not grow on the medium with mannitol as the sole carbon source,indicating that the strain could no longer use mannitol as the carbon source after all the three genes cmtA,cmtB and mtlA were knocked out. Finally,the recombinant strain R_5 was constructed,the activity of MDH enzyme was 258 U/mL,and a small amount of mannitol was detected by high performance liquid chromatography,which laid a foundation for further exploring the regulation mechanism of mannitol synthesis in E. coli.
In order to explore the effect of mannitol decomposition and utilization pathways on mannitol synthesis,the cmtA,cmtB and mtlA genes in Escherichia. coli PTS system were knocked out by CRISPR/Cas9 on the basis of recombinant strain R_1(K-12/pTrc99 amdh),which blocked the decomposition pathway of mannitol in E. coli K-12,and the recombinant strain R_3(K-12/?cmtA?cmtBmdh~+)and R_5(K-12/?cmtA?cmtB?mtlAmdh~+)were obtained. Compared with the original strain R_1,the growth rate of R_3 did not decrease,but that of R_5 decreased significantly. Moreover,R_5 did not grow on the medium with mannitol as the sole carbon source,indicating that the strain could no longer use mannitol as the carbon source after all the three genes cmtA,cmtB and mtlA were knocked out. Finally,the recombinant strain R_5 was constructed,the activity of MDH enzyme was 258 U/mL,and a small amount of mannitol was detected by high performance liquid chromatography,which laid a foundation for further exploring the regulation mechanism of mannitol synthesis in E. coli.
引文
[1]Dai Y, Meng Q, Mu W, et al. Recent advances in the applications and biotechnological production of mannitol[J]. Journal of Functional Foods, 2017, 36:404-409.
[2]Park YC, Oh EJ, Jo JH, et al. Recent advances in biological productionofsugaralcohols[J].CurrentOpinionin Biotechnology, 2016, 37:105.
[3]杨海军,袁卫涛.甘露醇的工业化生产及市场应用[J].山东食品发酵, 2008, 36(1):27-29.
[4]Reshamwala SM, Pagar SK, Velhal VS, et al. Construction of an efficient Escherichia coli whole-cell biocatalyst for d-mannitol production[J]. Journal of Bioscience&Bioengineering, 2014, 118(6):628-631.
[5]Gaspar P, Neves AR, Ramos A, et al. Engineering Lactococcus lactis for production of mannitol:high yields from food-grade strains deficient in lactate dehydrogenase and the mannitol transport system[J]. Applied&Environmental Microbiology, 2004, 70(3):1466.
[6]Kaup B, Bringer-Meyer S, Sahm H. D-Mannitol formation from d-glucose in a whole-cell biotransformation with recombinant Escherichia coli[J]. Applied Microbiology and Biotechnology,2005, 69(4):397.
[7]Kaup B, Bringer-Meyer S, Sahm H. Metabolic engineering of Escherichia coli:construction of an efficient biocatalyst for D-mannitol formation in a whole-cell biotransformation[J].Applied Microbiology and Biotechnology, 2016, 68(2):235-240.
[8]Kaup B, Bringer-Meyer S, Sahm H. Metabolic engineering of Escherichia coli:construction of an efficient biocatalyst for D-mannitol formation in a whole-cell biotransformation[J].Communications in Agricultural&Applied Biological Sciences,2004, 64(3):333-339.
[9]陈艳,田康明,李玉,等.以蔗糖为底物利用重组大肠杆菌合成甘露醇[J].微生物学通报, 2014, 41(11):2182-2190.
[10]Ortiz ME, Fornaguera MJ, Raya RR, et al. Lactobacillus reuteri CRL1101 highly produces mannitol from sugarcane molasses as carbon source[J]. Applied Microbiology and Biotechnology,2012, 95(1):991-999.
[11]Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing.[J]. Metabolic Engineering, 2015, 31:13-21.
[12]蔡友华,郑明英,严杰能,等.一种嘌呤核苷磷酸化酶基因敲除的大肠杆菌Ecoli-SL26的构建方法:中国102994537A[P], 2013.
[13]邱磊.球孢白僵菌组氨酸激酶家族、核激酶Weel和磷酸化酶Cdc25的功能解析[D].杭州:浙江大学, 2014.
[14]程雅韵,王新,郑琳,等.假肠膜明串珠菌甘露醇脱氢酶基因的克隆及表达[J].食品科学, 2016, 37(13):153-156.
[15]Liu S, Saha B, Cotta M. Cloning, expression, purification, and analysis of mannitol dehydrogenase gene mtlK from Lactobacillus brevis[J]. Applied Biochemistry&Biotechnology Part A Enzyme Engineering&Biotechnology, 2005, 121-124(1-3):391.
[16]Wang ZL, Ying SH, Feng MG. Gene cloning and catalysis features of a new mannitol-1-phosphate dehydrogenase(BbMPD)from Beauveria bassiana[J]. Carbohydrate Research, 2010, 345(1):50.
[17]王芳.甘露醇发酵及甘露醇脱氢酶的克隆表达[D].石家庄:河北科技大学, 2009.
[18]封志媚,赵雅童,刘业学,等. NADPH依赖的甘露醇脱氢酶的重组表达及甘露醇的转化条件[J].生物技术通报, 2017, 33(8):186-191.
[19]Kaup B, Bringer-Meyer S, Sahm H. D-Mannitol formation from d-glucose in a whole-cell biotransformation with recombinant Escherichia coli[J]. Applied Microbiology and Biotechnology,2005, 69(4):397.
[20]Song SH, Vieille C. Recent advances in the biological production of mannitol[J]. Applied Microbiology&Biotechnology, 2009, 84(1):55.
[2]Park YC, Oh EJ, Jo JH, et al. Recent advances in biological productionofsugaralcohols[J].CurrentOpinionin Biotechnology, 2016, 37:105.
[3]杨海军,袁卫涛.甘露醇的工业化生产及市场应用[J].山东食品发酵, 2008, 36(1):27-29.
[4]Reshamwala SM, Pagar SK, Velhal VS, et al. Construction of an efficient Escherichia coli whole-cell biocatalyst for d-mannitol production[J]. Journal of Bioscience&Bioengineering, 2014, 118(6):628-631.
[5]Gaspar P, Neves AR, Ramos A, et al. Engineering Lactococcus lactis for production of mannitol:high yields from food-grade strains deficient in lactate dehydrogenase and the mannitol transport system[J]. Applied&Environmental Microbiology, 2004, 70(3):1466.
[6]Kaup B, Bringer-Meyer S, Sahm H. D-Mannitol formation from d-glucose in a whole-cell biotransformation with recombinant Escherichia coli[J]. Applied Microbiology and Biotechnology,2005, 69(4):397.
[7]Kaup B, Bringer-Meyer S, Sahm H. Metabolic engineering of Escherichia coli:construction of an efficient biocatalyst for D-mannitol formation in a whole-cell biotransformation[J].Applied Microbiology and Biotechnology, 2016, 68(2):235-240.
[8]Kaup B, Bringer-Meyer S, Sahm H. Metabolic engineering of Escherichia coli:construction of an efficient biocatalyst for D-mannitol formation in a whole-cell biotransformation[J].Communications in Agricultural&Applied Biological Sciences,2004, 64(3):333-339.
[9]陈艳,田康明,李玉,等.以蔗糖为底物利用重组大肠杆菌合成甘露醇[J].微生物学通报, 2014, 41(11):2182-2190.
[10]Ortiz ME, Fornaguera MJ, Raya RR, et al. Lactobacillus reuteri CRL1101 highly produces mannitol from sugarcane molasses as carbon source[J]. Applied Microbiology and Biotechnology,2012, 95(1):991-999.
[11]Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing.[J]. Metabolic Engineering, 2015, 31:13-21.
[12]蔡友华,郑明英,严杰能,等.一种嘌呤核苷磷酸化酶基因敲除的大肠杆菌Ecoli-SL26的构建方法:中国102994537A[P], 2013.
[13]邱磊.球孢白僵菌组氨酸激酶家族、核激酶Weel和磷酸化酶Cdc25的功能解析[D].杭州:浙江大学, 2014.
[14]程雅韵,王新,郑琳,等.假肠膜明串珠菌甘露醇脱氢酶基因的克隆及表达[J].食品科学, 2016, 37(13):153-156.
[15]Liu S, Saha B, Cotta M. Cloning, expression, purification, and analysis of mannitol dehydrogenase gene mtlK from Lactobacillus brevis[J]. Applied Biochemistry&Biotechnology Part A Enzyme Engineering&Biotechnology, 2005, 121-124(1-3):391.
[16]Wang ZL, Ying SH, Feng MG. Gene cloning and catalysis features of a new mannitol-1-phosphate dehydrogenase(BbMPD)from Beauveria bassiana[J]. Carbohydrate Research, 2010, 345(1):50.
[17]王芳.甘露醇发酵及甘露醇脱氢酶的克隆表达[D].石家庄:河北科技大学, 2009.
[18]封志媚,赵雅童,刘业学,等. NADPH依赖的甘露醇脱氢酶的重组表达及甘露醇的转化条件[J].生物技术通报, 2017, 33(8):186-191.
[19]Kaup B, Bringer-Meyer S, Sahm H. D-Mannitol formation from d-glucose in a whole-cell biotransformation with recombinant Escherichia coli[J]. Applied Microbiology and Biotechnology,2005, 69(4):397.
[20]Song SH, Vieille C. Recent advances in the biological production of mannitol[J]. Applied Microbiology&Biotechnology, 2009, 84(1):55.