克氏梭菌硫解酶的鉴定及其功能研究
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摘要
【目的】硫解酶是梭菌属微生物合成短中链脂肪酸的关键酶。克氏梭菌(Clostridium kluyveri)具有3个高度同源的硫解酶编码基因,对这3个基因的功能鉴定是解析克氏梭菌高己酸合成能力的关键。【方法】通过发酵动力学分析确定克氏梭菌的己酸和丁酸生成动力学特征;转录组测序结合反转录-荧光定量RCR分析克氏梭菌3个硫解酶编码基因的表达水平和时序表达特征;在大肠杆菌中异源表达这3个硫解酶,并对其硫解酶动力学参数进行测定。【结果】克氏梭菌生成丁酸、己酸、辛酸,其中己酸为主要代谢产物;转录组数据显示,在乙酸消耗完全之前,thlA1基因维持恒定表达,thlA2基因表达时序上调,thlA3基因表达时序下调,转录组测序表明3个硫解酶编码基因均具有较高水平的转录活性,thlA2和thlA3的最高表达量分别约为thlA1的29%和43%;硫解酶动力学参数测定结果表明,克氏梭菌3个硫解酶对于四碳底物均显示出相似的底物亲和力(K_m),但ThlA1对四碳底物的催化效率(k_(cat)/K_m)略低于ThlA2和ThlA3。【结论】克氏梭菌的3个硫解酶均具有催化活性,在克氏梭菌体内均呈活跃表达,表明克氏梭菌拥有3个具有催化活性的硫解酶,这为后续深入研究克氏梭菌己酸合成机理奠定了基础。
[Objective] Clostridium kluyveri genome encodes for three highly homologous thiolases. To identify the function of these three thiolases will help us to understand how Clostridium kluyveri can efficiently produce hexanoate. [Methods] The characteristics of hexanoate and butyrate production were examined via fermentation kinetics analysis. The transcriptome and reverse transcription-quantitative RCR were used to analyze the expression profiles of thiolase-encoding genes during fermentation. Thiolases from Clostridium kluyveri were heterologously expressed in Escherichia coli and their enzyme kinetic parameters were examined. [Results] Clostridium kluyveriproduced butyrate, hexanoate and octanoate, of which hexanoate was the major product. Transcription analysis showed that thlA1 gene was constitutively expressed, thlA2 gene was up-regulated and thlA3 gene was down-regulated before the depletion of acetate. The three thiolase-encoding genes all had higher transcription levels, and the highest expression levels of thlA2 and thlA3 were approximately 29% and 43% that of thlA1, respectively. The enzyme kinetic parameters with four-carbon substrate demonstrated that the three thiolases from Clostridium kluyveri had similar affinity. However, the catalytic efficiency(k_(cat)/K_m) of ThlA1 for four-carbon substrates was lower than that of ThlA2 and ThlA3. [Conclusion] All of the three thiolases from Clostridium kluyveri had catalytic activities, and also actively expressed in vivo.
[Objective] Clostridium kluyveri genome encodes for three highly homologous thiolases. To identify the function of these three thiolases will help us to understand how Clostridium kluyveri can efficiently produce hexanoate. [Methods] The characteristics of hexanoate and butyrate production were examined via fermentation kinetics analysis. The transcriptome and reverse transcription-quantitative RCR were used to analyze the expression profiles of thiolase-encoding genes during fermentation. Thiolases from Clostridium kluyveri were heterologously expressed in Escherichia coli and their enzyme kinetic parameters were examined. [Results] Clostridium kluyveriproduced butyrate, hexanoate and octanoate, of which hexanoate was the major product. Transcription analysis showed that thlA1 gene was constitutively expressed, thlA2 gene was up-regulated and thlA3 gene was down-regulated before the depletion of acetate. The three thiolase-encoding genes all had higher transcription levels, and the highest expression levels of thlA2 and thlA3 were approximately 29% and 43% that of thlA1, respectively. The enzyme kinetic parameters with four-carbon substrate demonstrated that the three thiolases from Clostridium kluyveri had similar affinity. However, the catalytic efficiency(k_(cat)/K_m) of ThlA1 for four-carbon substrates was lower than that of ThlA2 and ThlA3. [Conclusion] All of the three thiolases from Clostridium kluyveri had catalytic activities, and also actively expressed in vivo.
引文
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[11]Weimer PJ,Stevenson DM.Isolation,characterization,and quantification of Clostridium kluyveri from the bovine rumen.Applied Microbiology and Biotechnology,2012,94(2):461-466.
[12]Jeon BS,Kim BC,Um Y,Sang BI.Production of hexanoic acid from D-galactitol by a newly isolated Clostridium sp.BS-1.Applied Microbiology and Biotechnology,2010,88(5):1161-1167.
[13]Jiang H,Wong WH.Statistical inferences for isoform expression in RNA-Seq.Bioinformatics,2009,25(8):1026-1032.
[14]Hartmanis MG,Gatenbeck S.Intermediary metabolism in Clostridium acetobutylicum:levels of enzymes involved in the formation of acetate and butyrate.Applied and Environmental Microbiology,1984,47(6):1277-1283.
[15]Cho C,Choe D,Jang YS,Kim KJ,Kim WJ,Cho BK,Papoutsakis ET,Bennett GN,Seung DY,Lee SY.Genome analysis of a hyper acetone-butanol-ethanol(ABE)producing Clostridium acetobutylicum BKM19.Journal of Biotechnology,2017,12(2):1600457.
[16]Flamholz A,Noor E,Bar-Even A,Milo R.eQuilibrator-the biochemical thermodynamics calculator.Nucleic Acids Research,2012,40(D1):D770-D775.
[17]Mann MS,Lütke-Eversloh T.Thiolase engineering for enhanced butanol production in Clostridium acetobutylicum.Biotechnology and Bioengineering,2013,110(3):887-897.
[18]Meng YH,Li JL.Cloning,expression and characterization of a thiolase gene from Clostridium pasteurianum.Biotechnology Letters,2006,28:1227.
[19]Berndt H,Schlegel HG.Kinetics and properties ofβ-ketothiolase from Clostridium pasteurianum.Archives of Microbiology,1975,103(1):21-30.
[20]Klein M,Wenk P,Ansorge-Schumacher MB,Fritsch M,Hartmeier W.Heterologous expression and characterisation of a biosynthetic thiolase from Clostridium butyricum DSM10702.Enzyme and Microbial Technology,2009,45(5):361-366.
[21]Wiesenborn DP,Rudolph FB,Papoutsakis ET.Thiolase from Clostridium acetobutylicum ATCC 824 and its role in the synthesis of acids and solvents.Applied and Environmental Microbiology,1988,54(11):2717-2722.
[22]Dellomonaco C,Clomburg JM,Miller EN,Gonzalez R.Engineered reversal of theβ-oxidation cycle for the synthesis of fuels and chemicals.Nature,2011,476(7360):355-359.
[23]Yu MR,Zhang YL,Tang IC,Yang ST.Metabolic engineering of Clostridium tyrobutyricum for n-butanol production.Metabolic Engineering,2011,13(4):373-382.
[24]Kim S,Jang YS,Ha SC,Ahn JW,Kim EJ,Lim JH,Cho C,Ryu YS,Lee SK,Lee SY,Kim KJ.Redox-switch regulatory mechanism of thiolase from Clostridium acetobutylicum.Nature Communications,2015,6:8410.
[25]Ren C,Gu Y,Hu SY,Wu Y,Wang P,Yang YL,Yang C,Yang S,Jiang WH.Identification and inactivation of pleiotropic regulator CcpA to eliminate glucose repression of xylose utilization in Clostridium acetobutylicum.Metabolic Engineering,2010,12(5):446-454.
[26]Diender M,Stams AJM,Sousa DZ.Production of medium-chain fatty acids and higher alcohols by a synthetic co-culture grown on carbon monoxide or syngas.Biotechnology for Biofuels,2016,9:82.
[27]Weimer PJ,Nerdahl M,Brandl DJ.Production of medium-chain volatile fatty acids by mixed ruminal microorganisms is enhanced by ethanol in co-culture with Clostridium kluyveri.Bioresource Technology,2015,175:97-101.
[28]Leng L,Yang PX,Mao YP,Wu ZY,Zhang T,Lee PH.Thermodynamic and physiological study of caproate and1,3-propanediol co-production through glycerol fermentation and fatty acids chain elongation.Water Research,2017,114:200-209.
[2]Agler MT,Spirito CM,Usack JG,Werner JJ,Angenent LT.Chain elongation with reactor microbiomes:upgrading dilute ethanol to medium-chain carboxylates.Energy&Environmental Science,2012,5(8):8189-8192.
[3]Ren ZZ,Gu CA,Wang ZG,Hu CQ,Niu JY.Catalysis of polybasic heteropolyacid in preparation of hexanoic acid from2-octanol.Fine Chemicals,2003,20(8):463-465.(in Chinese)任知忠,谷长安,王战国,胡长青,牛景扬.多元杂多酸在仲辛醇制己酸中的催化作用.精细化工,2003,20(8):463-465.
[4]Barker HA,Taha SM.Clostridium kluyverii,an organism concerned in the formation of caproic acid from ethyl alcohol.Journal of Bacteriology,1942,43(3):347-363.
[5]Weimer PJ,Moen GN.Quantitative analysis of growth and volatile fatty acid production by the anaerobic ruminal bacterium Megasphaera elsdenii T81.Applied Microbiology and Biotechnology,2013,97(9):4075-4081.
[6]Kim BC,Jeon BS,Kim S,Kim H,Um Y,Sang BI.Caproiciproducens galactitolivorans gen.nov.,sp.nov.,a bacterium capable of producing caproic acid from galactitol,isolated from a wastewater treatment plant.International Journal of Systematic and Evolutionary Microbiology,2015,65(12):4902-4908.
[7]Zhu XY,Zhou Y,Wang Y,Wu TT,Li XZ,Li DP,Tao Y.Production of high-concentration n-caproic acid from lactate through fermentation using a newly isolated Ruminococcaceae bacterium CPB6.Biotechnology for Biofuels,2017,10:102.
[8]Seedorf H,Fricke WF,Veith B,Brüggemann H,Liesegang H,Strittmatter A,Miethke M,Buckel W,Hinderberger J,Li FL,Hagemeier C,Thauer RK,Gottschalk G.The genome of Clostridium kluyveri,a strict anaerobe with unique metabolic features.Proceedings of the National Academy of Sciences of the United States of America,2008,105(6):2128-2133.
[9]Yin YN,Zhang YF,Karakashev DB,Wang JL,Angelidaki I.Biological caproate production by Clostridium kluyveri from ethanol and acetate as carbon sources.Bioresource Technology,2017,241:638-644.
[10]Hu XL,Du H,Xu Y.Identification and quantification of the caproic acid-producing bacterium Clostridium kluyveri in the fermentation of pit mud used for Chinese strong-aroma type liquor production.International Journal of Food Microbiology,2015,214:116-122.
[11]Weimer PJ,Stevenson DM.Isolation,characterization,and quantification of Clostridium kluyveri from the bovine rumen.Applied Microbiology and Biotechnology,2012,94(2):461-466.
[12]Jeon BS,Kim BC,Um Y,Sang BI.Production of hexanoic acid from D-galactitol by a newly isolated Clostridium sp.BS-1.Applied Microbiology and Biotechnology,2010,88(5):1161-1167.
[13]Jiang H,Wong WH.Statistical inferences for isoform expression in RNA-Seq.Bioinformatics,2009,25(8):1026-1032.
[14]Hartmanis MG,Gatenbeck S.Intermediary metabolism in Clostridium acetobutylicum:levels of enzymes involved in the formation of acetate and butyrate.Applied and Environmental Microbiology,1984,47(6):1277-1283.
[15]Cho C,Choe D,Jang YS,Kim KJ,Kim WJ,Cho BK,Papoutsakis ET,Bennett GN,Seung DY,Lee SY.Genome analysis of a hyper acetone-butanol-ethanol(ABE)producing Clostridium acetobutylicum BKM19.Journal of Biotechnology,2017,12(2):1600457.
[16]Flamholz A,Noor E,Bar-Even A,Milo R.eQuilibrator-the biochemical thermodynamics calculator.Nucleic Acids Research,2012,40(D1):D770-D775.
[17]Mann MS,Lütke-Eversloh T.Thiolase engineering for enhanced butanol production in Clostridium acetobutylicum.Biotechnology and Bioengineering,2013,110(3):887-897.
[18]Meng YH,Li JL.Cloning,expression and characterization of a thiolase gene from Clostridium pasteurianum.Biotechnology Letters,2006,28:1227.
[19]Berndt H,Schlegel HG.Kinetics and properties ofβ-ketothiolase from Clostridium pasteurianum.Archives of Microbiology,1975,103(1):21-30.
[20]Klein M,Wenk P,Ansorge-Schumacher MB,Fritsch M,Hartmeier W.Heterologous expression and characterisation of a biosynthetic thiolase from Clostridium butyricum DSM10702.Enzyme and Microbial Technology,2009,45(5):361-366.
[21]Wiesenborn DP,Rudolph FB,Papoutsakis ET.Thiolase from Clostridium acetobutylicum ATCC 824 and its role in the synthesis of acids and solvents.Applied and Environmental Microbiology,1988,54(11):2717-2722.
[22]Dellomonaco C,Clomburg JM,Miller EN,Gonzalez R.Engineered reversal of theβ-oxidation cycle for the synthesis of fuels and chemicals.Nature,2011,476(7360):355-359.
[23]Yu MR,Zhang YL,Tang IC,Yang ST.Metabolic engineering of Clostridium tyrobutyricum for n-butanol production.Metabolic Engineering,2011,13(4):373-382.
[24]Kim S,Jang YS,Ha SC,Ahn JW,Kim EJ,Lim JH,Cho C,Ryu YS,Lee SK,Lee SY,Kim KJ.Redox-switch regulatory mechanism of thiolase from Clostridium acetobutylicum.Nature Communications,2015,6:8410.
[25]Ren C,Gu Y,Hu SY,Wu Y,Wang P,Yang YL,Yang C,Yang S,Jiang WH.Identification and inactivation of pleiotropic regulator CcpA to eliminate glucose repression of xylose utilization in Clostridium acetobutylicum.Metabolic Engineering,2010,12(5):446-454.
[26]Diender M,Stams AJM,Sousa DZ.Production of medium-chain fatty acids and higher alcohols by a synthetic co-culture grown on carbon monoxide or syngas.Biotechnology for Biofuels,2016,9:82.
[27]Weimer PJ,Nerdahl M,Brandl DJ.Production of medium-chain volatile fatty acids by mixed ruminal microorganisms is enhanced by ethanol in co-culture with Clostridium kluyveri.Bioresource Technology,2015,175:97-101.
[28]Leng L,Yang PX,Mao YP,Wu ZY,Zhang T,Lee PH.Thermodynamic and physiological study of caproate and1,3-propanediol co-production through glycerol fermentation and fatty acids chain elongation.Water Research,2017,114:200-209.