光滑球拟酵母中AMP代谢对其生理功能的影响
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摘要
【目的】研究光滑球拟酵母(Candida glabrata)中AMP代谢影响其碳流代谢和耐酸胁迫的生理机制。【方法】同源重组法敲除基因cgade12(ORF CAGL0K05027g)和cgade13(ORF CAGL0B02794g)构建双缺菌株cgade12Δade13Δ,与出发菌株(ATCC55)比较ATP水平、碳流代谢酶活性和中间代谢物含量变化分析和AMP代谢对其碳流代谢的影响,对比菌株有机酸胁迫下生长情况及胞内环境变化,研究AMP代谢变化对光滑球拟酵母酸胁迫耐受性的影响。【结果】与出发菌株ATCC55相比,cgade12Δade13Δ的ATP水平下降了12.50%。与ATCC55相比,cgade12Δade13Δ中柠檬酸合成酶、苹果酸脱氢酶、异柠檬酸脱氢酶、α-酮戊二酸脱氢酶的活性分别上升了31.26%、19.45%、28.96%、18.36%,柠檬酸、α-酮戊二酸、苹果酸、琥珀酸含量分别提高了44.11%、73.60%、50.00%、65.68%。胞内丙酮酸浓度下降20.00%,丙酮酸产量下降73.11%。与ATCC55相比,cgade12Δade13Δ在0.4%丙酮酸、0.6%苹果酸和0.2%乙酸胁迫下的菌体浓度分别提高了8.71%、11.21%和12.71%。在0.2%乙酸胁迫下,菌株cgade12Δade13Δ的H~+-ATPase活性、细胞膜完整度、细胞膜电势分别比出发菌株ATCC55上升了7.04%、8.71%、25.14%,ROS水平下降了19.51%。【结论】基因cgade12、cgade13的缺失导致菌株的ATP水平下降,TCA循环活性和有机酸耐受性上升。
[Objective] The aim of this study is to investigate the effects of AMP metabolism on the physiological function of Torulopsis glabrata.[Methods] Strain cgade12Δade13Δ was constructed by deleting cgade12 and cgade13 with homologous recombination,and was used to study the effects of AMP metabolism on carbon metabolism by comparing the ATP levels,enzymes activity and inter-metabolite concentrations of carbon metabolism to that of ATCC55.And the effects of AMP on metabolisms on organic acid tolerance were studied by compared the cell growth and intracellular environment of cgade12Δade13Δ to that of ATCC55 under organic acid stress.[Results]The ATP levels of mutant cgade12Δade13Δ was decreased by 12.50% when compared with that of strain ATCC55.The enzymes activity of citrate synthetase,malate dehydrogenase,isocitrate dehydrogenase and α-ketoglutarate dehydrogenase was increased by 31.26%,19.45%,28.96%,18.36% and the intracellular citric acid,α-ketoglutarate,malic acid,succinic acid contents were increased by 44.11%,73.60%,50.00%,65.68%,respectively,compared with the corresponding value of strain ATCC55.However,the intracellular concentration of pyruvic acid in mutant cgade12Δade13Δ was decreased by 20.00% which led to a 73.11% reduction of pyruvic production in fermentation broth.Compared with strain ATCC55,the cell concentrations of cgade12Δade13Δ were increased by 8.71%,11.21%and 12.71% grown in YNB with 0.4% pyruvic,0.6% malic acid and 0.2% acetic acid,respectively.Grown in YNB with 0.2% acetic acid the H~+-ATPase activity,cell membrane integrity,cell membrane electric potential of mutant cgade12Δade13Δ was increased by 7.04%,8.71%,25.14% than that of strain ATCC55,respectively,while the ROS concentration was decreased by 19.51%.[Conclusion] The deletion of genes cgade12 and cgade13 resulted in a reduction in ATP level but led to an increase in activity of TCA cycle and organic acid tolerance.
[Objective] The aim of this study is to investigate the effects of AMP metabolism on the physiological function of Torulopsis glabrata.[Methods] Strain cgade12Δade13Δ was constructed by deleting cgade12 and cgade13 with homologous recombination,and was used to study the effects of AMP metabolism on carbon metabolism by comparing the ATP levels,enzymes activity and inter-metabolite concentrations of carbon metabolism to that of ATCC55.And the effects of AMP on metabolisms on organic acid tolerance were studied by compared the cell growth and intracellular environment of cgade12Δade13Δ to that of ATCC55 under organic acid stress.[Results]The ATP levels of mutant cgade12Δade13Δ was decreased by 12.50% when compared with that of strain ATCC55.The enzymes activity of citrate synthetase,malate dehydrogenase,isocitrate dehydrogenase and α-ketoglutarate dehydrogenase was increased by 31.26%,19.45%,28.96%,18.36% and the intracellular citric acid,α-ketoglutarate,malic acid,succinic acid contents were increased by 44.11%,73.60%,50.00%,65.68%,respectively,compared with the corresponding value of strain ATCC55.However,the intracellular concentration of pyruvic acid in mutant cgade12Δade13Δ was decreased by 20.00% which led to a 73.11% reduction of pyruvic production in fermentation broth.Compared with strain ATCC55,the cell concentrations of cgade12Δade13Δ were increased by 8.71%,11.21%and 12.71% grown in YNB with 0.4% pyruvic,0.6% malic acid and 0.2% acetic acid,respectively.Grown in YNB with 0.2% acetic acid the H~+-ATPase activity,cell membrane integrity,cell membrane electric potential of mutant cgade12Δade13Δ was increased by 7.04%,8.71%,25.14% than that of strain ATCC55,respectively,while the ROS concentration was decreased by 19.51%.[Conclusion] The deletion of genes cgade12 and cgade13 resulted in a reduction in ATP level but led to an increase in activity of TCA cycle and organic acid tolerance.
引文
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[14]Lopes AM,Toralles RP,Rombaldi CV.Thermal inactivation of polyphenoloxidase and peroxidase in Jubileu clingstone peach and yeast isolated from its spoiled puree.Food Science and Technology(Campinas),2014,34(1):150-156.
[15]Giannattasio S,Guaragnella N,?dralevi?M,Marra E.Molecular mechanisms of Saccharomyces cerevisiae stress adaptation and programmed cell death in response to acetic acid.Frontiers in Microbiology,2013,4:33.
[2]Lin SJ,Ford E,Haigis M,Liszt G,Guarente L.Calorie restriction extends yeast life span by lowering the level of NADH.Genes&Development,2004,18(1):12-16.
[3]Zhou JW,Liu LM,Shi ZP,Du GC,Chen J.ATP in current biotechnology:regulation,applications and perspectives.Biotechnology Advances,2009,27(1):94-101.
[4]Kowalski D,Pendyala L,Daignan-Fornier B,Howell SB,Huang RY.Dysregulation of purine nucleotide biosynthesis pathways modulates cisplatin cytotoxicity in Saccharomyces cerevisiae.Molecular Pharmacology,2008,74(4):1092-1100.
[5]You CH,Okano H,Hui S,Zhang ZG,Kim M,Gunderson CW,Wang YP,Lenz P,Yan DL,Hwa T.Coordination of bacterial proteome with metabolism by cyclic AMP signalling.Nature,2013,500(7462):301-306.
[6]Saint-Marc C,Pinson B,Coulpier F,Jourdren L,Lisova O,Daignan-Fornier B.Phenotypic consequences of purine nucleotide imbalance in Saccharomyces cerevisiae.Genetics,2009,183(2):529-538.
[7]Wu J,Chen XL,Cai LJ,Tang L,Liu LM.Transcription factors Asg1p and Hal9p regulate p H homeostasis in Candida glabrata.Frontiers in Microbiology,2015,6:843.
[8]Hou ZL,Wang WY,Fromm HJ,Honzatko RB.IMP alone organizes the active site of adenylosuccinate synthetase from Escherichia coli.The Journal of Biological Chemistry,2002,277(8):5970-5976.
[9]Woodward DO.Adenylosuccinate AMP-lyase(Neurospora crassa).Methods in Enzymology,1978,51:202-207.
[10]Zhou JW,Liu LM,Chen J.Improved ATP supply enhances acid tolerance of Candida glabrata during pyruvic acid production.Journal of Applied Microbiology,2011,110(1):44-53.
[11]Machida K,Tanaka T.Farnesol-induced generation of reactive oxygen species dependent on mitochondrial transmembrane potential hyperpolarization mediated by F0F1-ATPase in yeast.FEBS Letters,1999,462(1/2):108-112.
[12]Bracey D,Holyoak CD,Nebe-von Caron G,Coote PJ.Determination of the intracellular p H(p Hi)of growing cells of Saccharomyces cerevisiae:the effect of reduced-expression of the membrane H+-ATPase.Journal of Microbiological Methods,1998,31(3):113-125.
[13]Nakamura K,Niimi M,Niimi K,Holmes AR,Yates JE,Decottignies A,Monk BC,Goffeau A,Cannon RD.Functional expression of Candida albicans drug efflux pump Cdr1p in a Saccharomyces cerevisiae strain deficient in membrane transporters.Antimicrobial Agents and Chemotherapy,2001,45(12):3366-3374.
[14]Lopes AM,Toralles RP,Rombaldi CV.Thermal inactivation of polyphenoloxidase and peroxidase in Jubileu clingstone peach and yeast isolated from its spoiled puree.Food Science and Technology(Campinas),2014,34(1):150-156.
[15]Giannattasio S,Guaragnella N,?dralevi?M,Marra E.Molecular mechanisms of Saccharomyces cerevisiae stress adaptation and programmed cell death in response to acetic acid.Frontiers in Microbiology,2013,4:33.