代谢工程改造酿酒酵母生产L-苹果酸
|
摘要
为了研究胞质还原路径对酿酒酵母积累L-苹果酸的影响,通过在酿酒酵母中过量表达源于黄曲霉的丙酮酸羧化酶(Afpyc)、苹果酸脱氢酶(Afmdh)及C4-二羧酸转运蛋白(Afmae),成功构建了L-苹果酸合成的胞质还原路径。结果表明:(1)当低水平表达Afpyc时,其丙酮酸浓度降低了42%,但不能积累L-苹果酸;(2)当共表达Afpyc和Afmdh时,菌株W005积累了1.93 g/L的L-苹果酸,与对照菌株W004相比细胞干重提高了350%,丙酮酸降低了65.9%;(3)当共表达Afpyc、Afmdh和Afmae时,菌株W006的L-苹果酸产量提高了21.2%,达到2.34 g/L;4)通过提高接种量至初始OD_(600)=2,L-苹果酸的产量提高到3.28 g/L。通过在酿酒酵母中过量表达黄曲霉胞质还原路径的关键基因,使得工程菌能够积累L-苹果酸,为目标产物的高效积累提供了一种可借鉴的思路。
With the purpose of elucidating the introduction of cytoplasmic reductive pathway in Saccharomyces cerevisiae on the production of L-malic acid, the reductive pathway for L-malic acid biosynthesis is constructed in S. cerevisiae by overexpressing genes Afpyc,Afmdh and Afmae from Aspergillus flavus. When gene Afpyc is kept in low level,the pyruvate titers of W003 are decreased42% compared with control strain W001,respectively. To further introduced gene Afmdh in strain W003,the concentration of L-malic acid in W005 is up to 1.93 g/L,the DCW is increased by 350%and the pyruvate titers is decreased by 65.9% than that of the strain W004. Moreover,the malate efflux system is enhanced by introducting the tansporter gene Afmae and the L-malic acid titers of strain W006 is increased by 21.2%(2.34 g/L). The concentrations of L-malic acid is increased from2.34 g/L to 3.28 g/L with the initial OD_(600)=2. It could be summarized that introducting the reductive pathway of overproducing L-malic acid strain A. flavus in S. cerevisiae is indeed producted L-malic acid,and this successful method provides a strategy to the metabolic engineering.
With the purpose of elucidating the introduction of cytoplasmic reductive pathway in Saccharomyces cerevisiae on the production of L-malic acid, the reductive pathway for L-malic acid biosynthesis is constructed in S. cerevisiae by overexpressing genes Afpyc,Afmdh and Afmae from Aspergillus flavus. When gene Afpyc is kept in low level,the pyruvate titers of W003 are decreased42% compared with control strain W001,respectively. To further introduced gene Afmdh in strain W003,the concentration of L-malic acid in W005 is up to 1.93 g/L,the DCW is increased by 350%and the pyruvate titers is decreased by 65.9% than that of the strain W004. Moreover,the malate efflux system is enhanced by introducting the tansporter gene Afmae and the L-malic acid titers of strain W006 is increased by 21.2%(2.34 g/L). The concentrations of L-malic acid is increased from2.34 g/L to 3.28 g/L with the initial OD_(600)=2. It could be summarized that introducting the reductive pathway of overproducing L-malic acid strain A. flavus in S. cerevisiae is indeed producted L-malic acid,and this successful method provides a strategy to the metabolic engineering.
引文
[1]BATTAT E,PELEG Y,BERCOVITZ A,et al. Optimization of L-malic acid production by Aspergillus flavus in a stirred fermentor[J]. Biotechnology and Bioengineering,1991,37:1108-1116.
[2]TAING O,TAING K. Production of malic and succinic acids by sugar-tolerant yeast Zygosaccharomyces rouxii[J]. European Food Research and Technology,2006,224(3):343-347.
[3]BERCOVITZ A,PELEG Y,BATTAT E,et al. Localization of pyruvate carboxylase in organic acid-producing[J]. Applied and Environment Microbiology,1990,56(6):1594.
[4]KNUF C,NOOKAEW I,REMMERS I,et al. Physiological characterization of the high malic acid-producing Aspergillus oryzae strain 2103a-68[J]. Applied Microbiology and Biotechnology,2014,98(8):3517-3527.
[5]ZELLE R M,DE HULSTER E,VAN W W A,et al. Malic acid production by Saccharomyces cerevisiae:engineering of pyruvate carboxylation,oxaloacetate reduction,and malate export[J]. Applied and Environment Microbiology,2008,74(9):2766-2777.
[6]CHEN X,XU G,XU N,et al. Metabolic engineering of Torulopsis glabrata for malate production[J]. Metabolic Engineering,2013,19:10-16.
[7]MOON S Y,HONG S H,KIM T Y,et al. Metabolic engineering of Escherichia coli for the production of malic acid[J].Biochemical Engineering Journal,2008,40(2):312-320.
[8]ZHANG X,WANG X,SHANMUGAM K T,et al. L-malate production by metabolically engineered Escherichia coli[J]. Applied and Environment Microbiology,2011,77(2):427-434.
[9]ABBOTT D A,ZELLE R M,PRONK J T,et al. Metabolic engineering of Saccharomyces cerevisiae for production of carboxylic acids:current status and challenges[J]. FEMS Yeast Research,2009,9(8):1123-1136.
[10]XU G,LIU L,CHEN J. Reconstruction of cytosolic fumaric acid biosynthetic pathways in Saccharomyces cerevisiae[J].Microbial Cell Factories,2012,11:24-34.
[11]CHEN X,XU G,XU N,et al. Metabolic engineering of Torulopsis glabrata for malate production[J]. Metabolic Engineering,2013,19:10-16.
[12]BROWN S H,BASHKIROVA L,BERKA R,et al. Metabolic engineering of Aspergillus oryzae NRRL 3488 for increased production of L-malic acid[J]. Applied Microbiology and Biotechnology,2013,97(20):8903-8912.
[13]PINES O,SHEMESH S,BATTAT E,et al. Overexpression of cytosolic malate dehydrogenase(MDH2)causes overproduction of specific organic acids in Saccharomyces cerevisiae[J]. Applied Microbiology and Biotechnology,1997,48(2):248-255.
[14]CANELAS A B,PIERICK A,RAS C,et al. Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics[J]. Analytical Chemistry,2009,81(17):7379-7389.
[15]CANELAS A B,RAS C,TEN PIERICK A,et al. Leakage-free rapid quenching technique for yeast metabolomics[J].Metabolomics,2008,4(3):226-239.
[16]JONG-GUBBELS P,VANROLLEGHEMI P,HEIJNENI S,et al. Regulation of carbon metabolism in chemostat cultures of Saccharomyces cerevisiae grown on mixtures of glucose and ethanol[J]. Yeast,1995,11(5):407-418.
[17]YAN D,WANG C,ZHOU J,et al. Construction of reductive pathway in Saccharomyces cerevisiae for effective succinic acid fermentation at low pH value[J]. Bioresource Technology,2014,156:232-239.
[18]LIU L,LI Y,ZHU Y,et al. Redistribution of carbon flux in Torulopsis glabrata by altering vitamin and calcium level[J].Metabolic Engineering,2007,9(1):21-29.
[19]PETERS-WENDISCH P G,SCHIEL B,WENDISCH V F,et al. Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum[J]. Journal of Molecular Microbiology and Biotechnology,2001,3(2):295-300.
[20]BLANKSCHIEN M D,CLOMBURG J M,GONZALEZ R. Metabolic engineering of Escherichia coli for the production of succinate from glycerol[J]. Metabolic Engineering,2010,12(5):409-419.
[21]QIN Jingru,XU Meijuan,ZHANG Xian,et al. Co-production of L-arginine and polyhydroxybutyrate in recombinant Corynebacterium crenatum[J]. Journal of Food Science and Biotechnology,2016,35(3):240-246.(in Chinese)
[22]KOFFAS M A,JUNG G Y,STEPHANOPOULOS G. Engineering metabolism and product formation in Corynebacterium glutamicum by coordinated gene overexpression[J]. Metabolic Engineering,2003,5(1):32-41.
[23]PINES O,EVEN-RAM S,ELNATHAN N,et al. The cytosolic pathway of L-malic acid synthesis in Saccharomyces cerevisiae:the role of fumarase[J]. Applied Microbiology and Biotechnology,1996,46(4):393-399.
[24]HOU J,LAGES N F,OLDIGES M,et al. Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae[J].Metabolic Engineering,2009,11(4/5):253-261.
[25]MOLENAAR D,VAN DER REST M E,DRYSCH A,et al. Functions of the membrane-associated and cytoplasmic malate dehydrogenases in the citric acid cycle of Corynebacterium glutamicum[J]. Journal of Bacteriology,2000,182(24):6884-6891.
[26]GROBLER J,BAUER F,SUBDEN R E,et al. The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids[J]. Yeast,1995,11(15):1485-1491.
[27]LEE J W,NA D,PARK J M,et al. Systems metabolic engineering of microorganisms for natural and non-natural chemicals[J].Nature Chemical Biology,2012,8(6):536-546.
[28]JUMINAGA D,BAIDOO E E,REDDING-JOHANSON A M,et al. Modular engineering of L-tyrosine production in Escherichia coli[J]. Applied and Environment Microbiology,2012,78(1):89-98.
[2]TAING O,TAING K. Production of malic and succinic acids by sugar-tolerant yeast Zygosaccharomyces rouxii[J]. European Food Research and Technology,2006,224(3):343-347.
[3]BERCOVITZ A,PELEG Y,BATTAT E,et al. Localization of pyruvate carboxylase in organic acid-producing[J]. Applied and Environment Microbiology,1990,56(6):1594.
[4]KNUF C,NOOKAEW I,REMMERS I,et al. Physiological characterization of the high malic acid-producing Aspergillus oryzae strain 2103a-68[J]. Applied Microbiology and Biotechnology,2014,98(8):3517-3527.
[5]ZELLE R M,DE HULSTER E,VAN W W A,et al. Malic acid production by Saccharomyces cerevisiae:engineering of pyruvate carboxylation,oxaloacetate reduction,and malate export[J]. Applied and Environment Microbiology,2008,74(9):2766-2777.
[6]CHEN X,XU G,XU N,et al. Metabolic engineering of Torulopsis glabrata for malate production[J]. Metabolic Engineering,2013,19:10-16.
[7]MOON S Y,HONG S H,KIM T Y,et al. Metabolic engineering of Escherichia coli for the production of malic acid[J].Biochemical Engineering Journal,2008,40(2):312-320.
[8]ZHANG X,WANG X,SHANMUGAM K T,et al. L-malate production by metabolically engineered Escherichia coli[J]. Applied and Environment Microbiology,2011,77(2):427-434.
[9]ABBOTT D A,ZELLE R M,PRONK J T,et al. Metabolic engineering of Saccharomyces cerevisiae for production of carboxylic acids:current status and challenges[J]. FEMS Yeast Research,2009,9(8):1123-1136.
[10]XU G,LIU L,CHEN J. Reconstruction of cytosolic fumaric acid biosynthetic pathways in Saccharomyces cerevisiae[J].Microbial Cell Factories,2012,11:24-34.
[11]CHEN X,XU G,XU N,et al. Metabolic engineering of Torulopsis glabrata for malate production[J]. Metabolic Engineering,2013,19:10-16.
[12]BROWN S H,BASHKIROVA L,BERKA R,et al. Metabolic engineering of Aspergillus oryzae NRRL 3488 for increased production of L-malic acid[J]. Applied Microbiology and Biotechnology,2013,97(20):8903-8912.
[13]PINES O,SHEMESH S,BATTAT E,et al. Overexpression of cytosolic malate dehydrogenase(MDH2)causes overproduction of specific organic acids in Saccharomyces cerevisiae[J]. Applied Microbiology and Biotechnology,1997,48(2):248-255.
[14]CANELAS A B,PIERICK A,RAS C,et al. Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics[J]. Analytical Chemistry,2009,81(17):7379-7389.
[15]CANELAS A B,RAS C,TEN PIERICK A,et al. Leakage-free rapid quenching technique for yeast metabolomics[J].Metabolomics,2008,4(3):226-239.
[16]JONG-GUBBELS P,VANROLLEGHEMI P,HEIJNENI S,et al. Regulation of carbon metabolism in chemostat cultures of Saccharomyces cerevisiae grown on mixtures of glucose and ethanol[J]. Yeast,1995,11(5):407-418.
[17]YAN D,WANG C,ZHOU J,et al. Construction of reductive pathway in Saccharomyces cerevisiae for effective succinic acid fermentation at low pH value[J]. Bioresource Technology,2014,156:232-239.
[18]LIU L,LI Y,ZHU Y,et al. Redistribution of carbon flux in Torulopsis glabrata by altering vitamin and calcium level[J].Metabolic Engineering,2007,9(1):21-29.
[19]PETERS-WENDISCH P G,SCHIEL B,WENDISCH V F,et al. Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum[J]. Journal of Molecular Microbiology and Biotechnology,2001,3(2):295-300.
[20]BLANKSCHIEN M D,CLOMBURG J M,GONZALEZ R. Metabolic engineering of Escherichia coli for the production of succinate from glycerol[J]. Metabolic Engineering,2010,12(5):409-419.
[21]QIN Jingru,XU Meijuan,ZHANG Xian,et al. Co-production of L-arginine and polyhydroxybutyrate in recombinant Corynebacterium crenatum[J]. Journal of Food Science and Biotechnology,2016,35(3):240-246.(in Chinese)
[22]KOFFAS M A,JUNG G Y,STEPHANOPOULOS G. Engineering metabolism and product formation in Corynebacterium glutamicum by coordinated gene overexpression[J]. Metabolic Engineering,2003,5(1):32-41.
[23]PINES O,EVEN-RAM S,ELNATHAN N,et al. The cytosolic pathway of L-malic acid synthesis in Saccharomyces cerevisiae:the role of fumarase[J]. Applied Microbiology and Biotechnology,1996,46(4):393-399.
[24]HOU J,LAGES N F,OLDIGES M,et al. Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae[J].Metabolic Engineering,2009,11(4/5):253-261.
[25]MOLENAAR D,VAN DER REST M E,DRYSCH A,et al. Functions of the membrane-associated and cytoplasmic malate dehydrogenases in the citric acid cycle of Corynebacterium glutamicum[J]. Journal of Bacteriology,2000,182(24):6884-6891.
[26]GROBLER J,BAUER F,SUBDEN R E,et al. The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids[J]. Yeast,1995,11(15):1485-1491.
[27]LEE J W,NA D,PARK J M,et al. Systems metabolic engineering of microorganisms for natural and non-natural chemicals[J].Nature Chemical Biology,2012,8(6):536-546.
[28]JUMINAGA D,BAIDOO E E,REDDING-JOHANSON A M,et al. Modular engineering of L-tyrosine production in Escherichia coli[J]. Applied and Environment Microbiology,2012,78(1):89-98.