Regulation Of Aspartate Metabolic Pathways For Improving Acid Stress Resistance In Lactococcus Lactis NZ9000

Lactic acid bacteria?LAB?are excellent probiotics,and widely used in food,medicine,and other fields.However,LAB are confronted with severe acid stress during fermentation,storage and digestion,which critically affects its growth and probiotic function,so it is particularly urgent to improve acid stress resistance in LAB.In the previous study,We first found that the accumulation of aspartate can effectively improve acid stress resistance in LAB.However,the mechanism of aspartate is still not clear.This study confirmed that aspartate can effectively improve acid stress resistance in Lactococcus lactis NZ9000 and analyzed the mechanism of aspartate metabolic pathways by exogenous addition of aspartate;and by individually overexpressing the L-asparaginase and transporter AcaP to increase intracellular aspartate accumulation,and then improve the acid stress resistance in L.lactis NZ9000.First of all,we found that it can improve acid stress resistance in L.lactis NZ9000effectively by exogenous addition of aspartate.Under pH 6.0,5.5 and 5.0,the biomass of treatment group increase by 23.9%,63.3%and 463.0%respectively;Survival rate experiment showed that treatment group was 75.0 times higher compared with control group with pH 4.0for 3.5 h.We studied the effects of aspartate on the transcriptional level of key genes in energy production and amino acid metabolism pathways by fluorescence quantitative PCR.Results showed that the genes transcriptional level in the glycolytic pathway were up-regulated;glutamic acid decarboxylase gene transcription was up-regulated;aspartate mainly generated oxaloacetate and glutamate with transaminase and its transcriptional level was 2.2 times higher compared with control group.The addition of aspartate increased the intracellular glutamate and ATP which were 2.3-fold and 42.0-fold that of the control group under pH 4.0 for 3.5 h.Therefore,it was confirmed that aspartate can accelerate the metabolism of glycolysis,and mainly generates oxaloacetate and glutamate with transaminase.Oxaloacetate and glutamate can further participate in the tricarboxylic acid cycle and glutamate decarboxylase metabolic pathway,and increase the acid stress resistance in L.lactis NZ9000.Asparagine removes amino group to generate aspartate by L-asparaginase.Next,we constructed recombination strain,L.lactis NZ9000-pNZ8148-ansB by overexpressing of the L-asparaginase.Compared with control strain,the activity of L-asparaginase were 5.6 times higher under normal condition and the content of aspartate were 1.5 times higher with control strain.Under pH 3.6,compared with control strain,the survival rate of recombination strain was 11.1 times higher;we found that the gene transcriptional level of glyceraldehyde-3-phosphate dehydrogenase?GAPDH?,glutamate decarboxylase and aspartate aminotransferase?Asp B?of recombination strain were 9.3-fold,8.3-fold and3.0-fold respectively that of the control strain by fluorescence quantitative PCR;we found that the content of intracellular ATP and NH₄⁺were 2.4 and 3.8 times higher than the control strain by intracellular microenvironment analysis.Thus,it was confirmed that L-asparaginase can improve acid stress resistance in L.lactis NZ9000 by accelerating the metabolism of glycolysis,glutamate and aspartate.Finally,we constructed recombination strain,L.lactis NZ9000-pNZ8148-ylcA by overexpressing of acidic amino acids transporter AcaP.Compared with control strain,the content of aspartate was 1.8 times higher under normal condition.Under pH 3.6,compared with control strain,the survival rate of recombination strain was 12.0 times higher;we found that the gene transcriptional level of glyceraldehyde-3-phosphate dehydrogenase?GAPDH?,citrate synthase,aspartate aminotransferase?Asp B?and asparginase?AnsB?were 5.7-fold,4.7-fold,10.8-fold and 57.2-fold respectively that of the control strain by fluorescence quantitative PCR;we found that the content of intracellular ATP was 4.2 times higher than the control strain;the pH_i and transmembrane potential????of recombination strain were more balanced by intracellular microenvironment analysis.Therefore,it was confirmed that acidic amino acids transporter AcaP can efficiently improve acid stress resistance in L.lactis NZ9000 by accelerating the metabolism of glycolysis and aspartate.In this study,we explored the mechanism of aspartate metabolic pathways and confirmed that increasing intracellular aspartate was an effective method for improving acid stress resistance in L.lactis NZ9000.This study can further perfecting the theoretical basis of amino acids metabolism in improving acid stress resistance in lactic acid bacteria and provide a new method for construction of acid stress resistance industrial microorganisms.