【摘要】：本研究的目的是尋找精氨酸代謝途徑中與酸脅迫相關的關鍵作用因素。通過構建重組菌株Lactococcus lactis NZ9000(p NZ8148-argG)與Lactococcus lactis NZ9000(pNZ8148-argH)使ArgG和ArgH蛋白在Lactococcus lactis NZ9000中成功表達。與對照菌株相比,重組菌株在多種環(huán)境脅迫下表現出較高的生長性能、存活率以及發(fā)酵性能,其中,乙醇脅迫5 h后,NZ9000(pNZ8148-argG)和NZ9000(p NZ8148-argH)的存活率分別為對照菌株的5.97和4.65倍;酸脅迫5 h后,兩菌株的存活率分別為對照菌株的3.54和7.50倍。生理學性質分析發(fā)現,酸脅迫環(huán)境下,重組菌株細胞中存在較高的胞內pH、胞內NH4+、ATP含量以及H+-ATPase活性。糖酵解途徑關鍵酶活力分析發(fā)現,重組菌株中丙酮酸激酶(PK)、甘油醛3-磷酸脫氫酶(GAPDH)的酶活性高于對照菌株。氨基酸濃度分析發(fā)現,ADI途徑中氨基酸濃度明顯提高,而以天冬氨酸、丙酮酸為前體的天冬氨酸族和丙酮酸族大部分氨基酸濃度均下降。進一步的轉錄分析發(fā)現,天冬氨酸合成、精氨酸代謝相關的基因轉錄水平上調,同時,糖酵解途徑關鍵基因轉錄加強,表明在L.lactis NZ9000中過量表達ArgG和ArgH蛋白可以引發(fā)精氨酸代謝流量的上調,進而提高了細胞的多種脅迫抗性。為進一步提高L.lactis NZ9000的酸脅迫抗性,敲除了精氨酸代謝阻遏蛋白基因argR。酸脅迫(pH 4.0)5 h后,敲除菌株的存活率為原始菌株的11.97倍;argR基因回補實驗則進一步證實了argR基因的敲除可以提高乳酸菌脅迫抗性。結果表明,精氨酸代謝途徑中ArgG和ArgH的過量表達能夠通過促使精氨酸代謝流量的上調提高細胞的多種脅迫抗性。精氨酸合成途徑廣泛存在于多種微生物中,本研究結果為微生物尤其是工業(yè)微生物提高脅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.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：TQ922
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本文編號：2162134