argG、argH和argR基因对Lactococcus lactis NZ9000胁迫抗性的影响
发布时间：2018-08-03 15:09
【摘要】：本研究的目的是寻找精氨酸代谢途径中与酸胁迫相关的关键作用因素。通过构建重组菌株Lactococcus lactis NZ9000(p NZ8148-argG)与Lactococcus lactis NZ9000(pNZ8148-argH)使ArgG和ArgH蛋白在Lactococcus lactis NZ9000中成功表达。与对照菌株相比,重组菌株在多种环境胁迫下表现出较高的生长性能、存活率以及发酵性能,其中,乙醇胁迫5 h后,NZ9000(pNZ8148-argG)和NZ9000(p NZ8148-argH)的存活率分别为对照菌株的5.97和4.65倍;酸胁迫5 h后,两菌株的存活率分别为对照菌株的3.54和7.50倍。生理学性质分析发现,酸胁迫环境下,重组菌株细胞中存在较高的胞内pH、胞内NH4+、ATP含量以及H+-ATPase活性。糖酵解途径关键酶活力分析发现,重组菌株中丙酮酸激酶(PK)、甘油醛3-磷酸脱氢酶(GAPDH)的酶活性高于对照菌株。氨基酸浓度分析发现,ADI途径中氨基酸浓度明显提高,而以天冬氨酸、丙酮酸为前体的天冬氨酸族和丙酮酸族大部分氨基酸浓度均下降。进一步的转录分析发现,天冬氨酸合成、精氨酸代谢相关的基因转录水平上调,同时,糖酵解途径关键基因转录加强,表明在L.lactis NZ9000中过量表达ArgG和ArgH蛋白可以引发精氨酸代谢流量的上调,进而提高了细胞的多种胁迫抗性。为进一步提高L.lactis NZ9000的酸胁迫抗性,敲除了精氨酸代谢阻遏蛋白基因argR。酸胁迫(pH 4.0)5 h后,敲除菌株的存活率为原始菌株的11.97倍;argR基因回补实验则进一步证实了argR基因的敲除可以提高乳酸菌胁迫抗性。结果表明,精氨酸代谢途径中ArgG和ArgH的过量表达能够通过促使精氨酸代谢流量的上调提高细胞的多种胁迫抗性。精氨酸合成途径广泛存在于多种微生物中,本研究结果为微生物尤其是工业微生物提高胁迫抗性提供了新思路。
[Abstract]:The aim of this study was to explore the key factors related to acid stress in arginine metabolic pathway. ArgG and ArgH proteins were successfully expressed in Lactococcus lactis NZ9000 by constructing recombinant strains Lactococcus lactis NZ9000 (p NZ8148-argG) and Lactococcus lactis NZ9000 (pNZ8148-argH). Compared with the control strain, the recombinant strain showed higher growth performance, survival rate and fermentation performance under various environmental stresses. The survival rates of NZ9000 (pNZ8148-argG) and NZ9000 (p NZ8148-argH) were 5.97 and 4.65 times higher than those of the control strain after 5 h ethanol stress, respectively. After 5 h of acid stress, the survival rate of the two strains was 3.54 and 7.50 times higher than that of the control strain, respectively. Physiological properties analysis showed that under acid stress, there were high intracellular pH, intracellular NH4 content and H -ATPase activity in the cells of the recombinant strain. The key enzyme activity analysis of glycolysis pathway showed that the activity of pyruvate kinase (competition) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the recombinant strain was higher than that in the control strain. It was found that the concentration of amino acids in ADI pathway was significantly increased, while the concentrations of aspartic acid and pyruvate as precursors were decreased. Further transcriptional analysis found that aspartic acid synthesis, arginine metabolism related gene transcription level up-regulated, at the same time, glycolysis pathway key genes transcription enhanced, It was suggested that overexpression of ArgG and ArgH proteins in L.lactis NZ9000 could induce the up-regulation of arginine metabolic flow, and thus enhance the resistance of the cells to various stresses. In order to further enhance the acid stress resistance of L.lactis NZ9000, the arginine metabolic repressor gene Arg was knockout. After 5 h of acid stress (pH 4.0), the survival rate of knockout strain was 11.97 times as high as that of original strain. It was further proved that the knockout of argR gene could increase the stress resistance of lactic acid bacteria. The results showed that the overexpression of ArgG and ArgH in arginine metabolic pathway could increase the stress resistance of cells through up-regulation of arginine metabolic flow. Arginine biosynthesis pathway is widely found in many microorganisms. The results of this study provide a new way for microbes, especially industrial microorganisms, to improve stress resistance.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TQ922
，
本文编号：2162134
[Abstract]:The aim of this study was to explore the key factors related to acid stress in arginine metabolic pathway. ArgG and ArgH proteins were successfully expressed in Lactococcus lactis NZ9000 by constructing recombinant strains Lactococcus lactis NZ9000 (p NZ8148-argG) and Lactococcus lactis NZ9000 (pNZ8148-argH). Compared with the control strain, the recombinant strain showed higher growth performance, survival rate and fermentation performance under various environmental stresses. The survival rates of NZ9000 (pNZ8148-argG) and NZ9000 (p NZ8148-argH) were 5.97 and 4.65 times higher than those of the control strain after 5 h ethanol stress, respectively. After 5 h of acid stress, the survival rate of the two strains was 3.54 and 7.50 times higher than that of the control strain, respectively. Physiological properties analysis showed that under acid stress, there were high intracellular pH, intracellular NH4 content and H -ATPase activity in the cells of the recombinant strain. The key enzyme activity analysis of glycolysis pathway showed that the activity of pyruvate kinase (competition) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the recombinant strain was higher than that in the control strain. It was found that the concentration of amino acids in ADI pathway was significantly increased, while the concentrations of aspartic acid and pyruvate as precursors were decreased. Further transcriptional analysis found that aspartic acid synthesis, arginine metabolism related gene transcription level up-regulated, at the same time, glycolysis pathway key genes transcription enhanced, It was suggested that overexpression of ArgG and ArgH proteins in L.lactis NZ9000 could induce the up-regulation of arginine metabolic flow, and thus enhance the resistance of the cells to various stresses. In order to further enhance the acid stress resistance of L.lactis NZ9000, the arginine metabolic repressor gene Arg was knockout. After 5 h of acid stress (pH 4.0), the survival rate of knockout strain was 11.97 times as high as that of original strain. It was further proved that the knockout of argR gene could increase the stress resistance of lactic acid bacteria. The results showed that the overexpression of ArgG and ArgH in arginine metabolic pathway could increase the stress resistance of cells through up-regulation of arginine metabolic flow. Arginine biosynthesis pathway is widely found in many microorganisms. The results of this study provide a new way for microbes, especially industrial microorganisms, to improve stress resistance.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TQ922
，
本文编号：2162134
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