短乳杆菌和植物乳杆菌产γ-氨基丁酸及其关键基因的研究
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摘要
为研究乳杆菌γ-氨基丁酸(γ-aminobutyric acid,GABA)的产量以及其与谷氨酸脱羧酶系统关键基因表达的相关性,实验结合GABA产量、基因组学调查和谷氨酸脱羧酶(glutamate decarboxylase,GAD)系统基因的扩增结果,发现产GABA的菌株在短乳杆菌、植物乳杆菌中分布较多,这2个种属可作为产GABA菌株的主要筛选对象。通过qRT-PCR研究短乳杆菌和植物乳杆菌不同生长时期GAD相关基因转录情况,结果表明,短乳杆菌的gad B和gad C的表达量变化情况相似,且在对数后期或稳定前期达到高峰。文中的短乳杆菌高产GABA可能与其gad C和gad B的高表达有关。植物乳杆菌的gad B表达变化与短乳杆菌类似,但是与GABA的产量之间相关性较弱,可能存在其他因素的影响。该研究为产GABA菌株的筛选,乳杆菌中GAD基因的表达调控,GABA发酵工艺的优化提供了理论基础。
This study was conducted to study differences in γ-aminobutyric acid( GABA) production in Lactobacillus and the correlation between its yield and key genes expressions in the glutamate decarboxylase( GAD) system.The results showed that most GABA-producing strains belonged to L. brevis and L. plantarum. The transcriptional activities of GAD-related genes at different growth stages of L. brevis and L. plantarum were studied by qRT-PCR. It was found that the expression levels of gadB and gadC in L. brevis were similar and peaked at late logarithm or early stable phase. Therefore,the high-yield of GABA in L. brevis may be associated with high expressions of gadC and gadB. Moreover,the expression of gadB in L. plantarum was similar to that of L. brevis,but its GABA production had little correlation with gadB expression,which indicated that other factors might affect L. plantarum producing GABA. Overall,this study provides a theoretical basis for screening GABA-producing strains and regulating GAD gene expression in Lactobacillus as well as for optimizing GABA fermentation process.
This study was conducted to study differences in γ-aminobutyric acid( GABA) production in Lactobacillus and the correlation between its yield and key genes expressions in the glutamate decarboxylase( GAD) system.The results showed that most GABA-producing strains belonged to L. brevis and L. plantarum. The transcriptional activities of GAD-related genes at different growth stages of L. brevis and L. plantarum were studied by qRT-PCR. It was found that the expression levels of gadB and gadC in L. brevis were similar and peaked at late logarithm or early stable phase. Therefore,the high-yield of GABA in L. brevis may be associated with high expressions of gadC and gadB. Moreover,the expression of gadB in L. plantarum was similar to that of L. brevis,but its GABA production had little correlation with gadB expression,which indicated that other factors might affect L. plantarum producing GABA. Overall,this study provides a theoretical basis for screening GABA-producing strains and regulating GAD gene expression in Lactobacillus as well as for optimizing GABA fermentation process.
引文
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[15] RADHIKA D,BAJPAI V K,KWANG-HYUN B. Production of gaba(γ-aminobutyric acid)by microorganisms:A review[J]. Brazilian Journal of Microbiology,2012,43(4):1 230-1 241.
[16] KRULWICH T A,SACHS G,PADAN E. Molecular aspects of bacterial p H sensing and homeostasis[J]. Nat Rev Microbiol,2011,9(5):330-343.
[17] LYU Changjiang,ZHAO Weirui,HU Sheng,et al. Physiology-oriented engineering strategy to improve gammaaminobutyrate production in Lactobacillus brevis[J]. J Agric Food Chem,2017,65(4):858-866.
[18]彭春龙,黄俊,赵伟睿,等.酸胁迫下短乳杆菌谷氨酸脱羧酶系统关键基因的表达及酶活性响应[J].高校化学工程学报,2015(2):359-365.
[19] WU Qinglong,TUN Heinming,LAW Yeesong,et al.Common distribution of gad operon in Lactobacillus brevis and its Gad A contributes to efficient GABA synthesis toward cytosolic near-neutral p H[J]. Front Microbiol,2017,8:206.
[20] SU M S,SCHLICHT S,GANZLE M G. Contribution of glutamate decarboxylase in Lactobacillus reuteri to acid resistance and persistence in sourdough fermentation[J].Microb Cell Fact,2011,10 Suppl 1:S8.
[21] YUNES R A,POLUEKTOVA E U,DYACHKOVA M S,et al. GABA production and structure of gad B/gad C genes in Lactobacillus and Bifidobacterium strains from human microbiota[J]. Anaerobe,2016,42:197-204.
[22] KOMATSUZAKI N,NAKAMURA T,KIMURA T,et al.Characterization of glutamate decarboxylase from a high gamma-aminobutyric acid(GABA)-producer,Lactobacillus paracasei[J]. Biosci Biotechnol Biochem,2008,72(2):278-285.
[23] SA H D,PARK J Y,JEONG S J,et al. Characterization of glutamate decarboxylase(GAD)from Lactobacillus sakei A156 isolated from jeot-gal[J]. J Microbiol Biotechnol,2015,25(5):696-703.
[24] DE BIASE D and PENNACCHIETTI E. Glutamate decarboxylase-dependent acid resistance in orally acquired bacteria:function,distribution and biomedical implications of the gad BC operon[J]. Mol Microbiol,2012,86(4):770-786.
[25]李理.植物乳杆菌γ物氨基丁酸产量影响因素及代谢途径的研究[D].哈尔滨:东北农业大学,2015.
[2] AKAMA K,KANETOU J,SHIMOSAKI S,et al. Seedspecific expression of truncated Os GAD2 produces GABAenriched rice grains that influence a decrease in blood pressure in spontaneously hypertensive rats[J]. Transgenic Res,2009,18(6):865-876.
[3] JUNG W Y,KIM S G,LEE J S,et al. Effect of feeding high gamma-aminobutyric acid-containing giant embryo black sticky rice(Oryza sativa L.)on anxiety-related behavior of C57BL/6 Mice[J]. J Med Food,2017,20(8):777-781.
[4] KOBUCHI S,TANAKA R,SHINTANI T,et al. Mechanisms underlying the renoprotective effect of GABA against ischaemia/reperfusion-induced renal injury in rats[J].Clin Exp Pharmacol Physiol,2015,42(3):278-286.
[5] PINILLA L,GONZALEZ L C,TENA-SEMPERE M,et al. Cross-talk between excitatory and inhibitory amino acids in the regulation of growth hormone secretion in neonatal rats[J]. Neuroendocrinology,2001,73(1):62-67.
[6] ABDOU A M,HIGASHIGUCHI S,HORIE K,et al. Relaxation and immunity enhancement effects of gamma-aminobutyric acid(GABA)administration in humans[J].Biofactors,2010,26(3):201-208.
[7] LIM H S,CHA I T,ROH S W,et al. Enhanced production of gamma-aminobutyric acid by optimizing culture conditions of Lactobacillus brevis HYE1 isolated from Kimchi,a Korean fermented food[J]. J Microbiol Biotechnol,2017,27(3):450-459.
[8] LI Haixing,LI Wenming,LIU Xiaohua,et al. gad A gene locus in Lactobacillus brevis NCL912 and its expression during fed-batch fermentation[J]. FEMS Microbiol Lett,2013,349(2):108-116.
[9] LYU Changjiang,ZHAO Weirui,PENG Chunlong,et al.Exploring the contributions of two glutamate decarboxylase isozymes in Lactobacillus brevis to acid resistance and gamma-aminobutyric acid production[J]. Microb Cell Fact,2018,17(1):180.
[10] SHANY,MAN C X,HAN X,et al. Evaluation of improved gamma-aminobutyric acid production in yogurt using Lactobacillus plantarum NDC75017[J]. J Dairy Sci,2015,98(4):2 138-2 149.
[11] YANG Taowei,RAO Zhiming,KIMANI B G,et al. Twostep production of gamma-aminobutyric acid from cassava powder using Corynebacterium glutamicum and Lactobacillus plantarum[J]. J Ind Microbiol Biotechnol,2015,42(8):1 157-1 165.
[12] TAJABADI N,BARADARAN A,EBRAHIMPOUR A,et al. Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj-Apis362 for high gamma-aminobutyric acid production[J]. Microb Biotechnol,2015,8(4):623-632.
[13] SIRAGUSA S,DE ANGELIS M,DI CAGNO R,et al.Synthesis of gamma-aminobutyric acid by lactic acid bacteria isolated from a variety of Italian cheeses[J]. Appl Environ Microbiol,2007,73(22):7 283-7 290.
[14] FRANCIOSI E,CARAFA I,NARDIN T,et al. Biodiversity and gamma-aminobutyric acid production by lactic acid bacteria isolated from traditional alpine raw cow's milk cheeses[J]. Biomed Res Int,2015:625 740.
[15] RADHIKA D,BAJPAI V K,KWANG-HYUN B. Production of gaba(γ-aminobutyric acid)by microorganisms:A review[J]. Brazilian Journal of Microbiology,2012,43(4):1 230-1 241.
[16] KRULWICH T A,SACHS G,PADAN E. Molecular aspects of bacterial p H sensing and homeostasis[J]. Nat Rev Microbiol,2011,9(5):330-343.
[17] LYU Changjiang,ZHAO Weirui,HU Sheng,et al. Physiology-oriented engineering strategy to improve gammaaminobutyrate production in Lactobacillus brevis[J]. J Agric Food Chem,2017,65(4):858-866.
[18]彭春龙,黄俊,赵伟睿,等.酸胁迫下短乳杆菌谷氨酸脱羧酶系统关键基因的表达及酶活性响应[J].高校化学工程学报,2015(2):359-365.
[19] WU Qinglong,TUN Heinming,LAW Yeesong,et al.Common distribution of gad operon in Lactobacillus brevis and its Gad A contributes to efficient GABA synthesis toward cytosolic near-neutral p H[J]. Front Microbiol,2017,8:206.
[20] SU M S,SCHLICHT S,GANZLE M G. Contribution of glutamate decarboxylase in Lactobacillus reuteri to acid resistance and persistence in sourdough fermentation[J].Microb Cell Fact,2011,10 Suppl 1:S8.
[21] YUNES R A,POLUEKTOVA E U,DYACHKOVA M S,et al. GABA production and structure of gad B/gad C genes in Lactobacillus and Bifidobacterium strains from human microbiota[J]. Anaerobe,2016,42:197-204.
[22] KOMATSUZAKI N,NAKAMURA T,KIMURA T,et al.Characterization of glutamate decarboxylase from a high gamma-aminobutyric acid(GABA)-producer,Lactobacillus paracasei[J]. Biosci Biotechnol Biochem,2008,72(2):278-285.
[23] SA H D,PARK J Y,JEONG S J,et al. Characterization of glutamate decarboxylase(GAD)from Lactobacillus sakei A156 isolated from jeot-gal[J]. J Microbiol Biotechnol,2015,25(5):696-703.
[24] DE BIASE D and PENNACCHIETTI E. Glutamate decarboxylase-dependent acid resistance in orally acquired bacteria:function,distribution and biomedical implications of the gad BC operon[J]. Mol Microbiol,2012,86(4):770-786.
[25]李理.植物乳杆菌γ物氨基丁酸产量影响因素及代谢途径的研究[D].哈尔滨:东北农业大学,2015.