Influence Of ArgG, ArgH And ArgR Genes On Stress Tolerance In Lactococcus Lactis NZ9000

The aim of the present study was to find key factors of arginine metabolism on the stress tolerance. Protein ArgG and ArgH were overexpressed successfully by constructing recombinant strains Lactococcus lactis NZ9000(pNZ8148-argG) and Lactococcus lactis NZ9000(pNZ8148-argH). The recombinant strains exhibited higher growth performance, viability and fermentation performance compared with the control strain under environmental stresses. After ethanol stress for 5 h, survival rate of NZ9000(p NZ8148-argG) and NZ9000(pNZ8148-argH) were respectively 4.97 and 3.65 times higher than the control; and under acid stress for 5 h, the survival rates of the two recombinant strains were 2.54- and 6.50-fold higher than the control. Analysis of the physiological data showed that the recombinant cells exhibited higher intracellular pH, intracellular NH4+ and ATP content, and H+-ATPase activity under acid stress. Key enzyme analysis of glycolytic pathway found that the enzyme activity of pyruvate kinase(PK) and glyceraldehyde 3-phosphate dehydrogenase(GAPDH) in the recombinant strains were higher than the control strain. Concentration analysis of amino acid reflected that the content of amino acid in arginine deiminase(ADI) pathway was significantly increased, and content of amino acids, which synthesized from aspartate or pyruvate, in aspartate family and pyruvate family decreased. Further transcriptional analysis showed that the expression of the genes related to aspartate synthesis, arginine catabolism and key genes in glycolysis pathway were up-regulated. The results reflected that ArgG or ArgH overexpression in L. lactis NZ9000 can enhance arginine metabolism flux and up-regulated arginine metabolism flux can improve the multiple-stress tolerance of cells. To further enhance the tolerance of L. lactis NZ9000 under acid stress, arginine metabolism repressor protein gene argR was knocked out. For argR gene knockout strain, the survival rate was 10.97 times higher than the original strain under acid stress(pH 4.0). The anaplerotic flux of argR in knockout strain also reflected the knockout of arg R gene can enhance stress tolerance of lactic acid bacteria. These results suggest that the overexpression of ArgG or ArgH protein can accelerate arginine metabolism flux so as to improve the multiple-stress tolerance of cells. As arginine synthesis pathway widely exists in various kinds of microorganisms, results presented in this study provide new idea to enhance stress tolerance of tons of microorganisms especially industrial strains.