Based On Research Of Transmembrane Protein To Improve Lactococcus Lactis NZ9000 Acid Stress Resistance

Lactic acid bacteria(LAB)are widely used in many fields due to excellent performance.However,during the fermentation process,strains are subjected to acid stress,resulting in a significant decrease in cell growth and metabolic activity,inhibiting strains to play a probiotic function.Therefore,it is particularly urgent to improve the acid tolerance of lactic acid bacteria.Cell membranes,which are the primary target of acid stress,help cells protect against acid stress in multiple ways.However,at present,it is still lack of applying membrane proteins to improve the resistance of lactic acid bacteria.In this paper,multiple membrane protein genes were overexpressed in Lactococcus lactis NZ9000.Then some acid-resistant components related to acid stress resistance were identified through the combination of transcriptomics and molecular biology techniques.The genes which were dramatically increased under acid stress conditions were overexpressed in L.lactis NZ9000.Consequently,the acid-resistant recombinant strains L.lactis(ZitP)and L.lactis(ZitQ)were screened,which showed 14.5 and 9.5-fold higher survival rates,respectively,than that of the control strain L.lactis(pNZ8148)(after 4 h of lactic acid shock at pH 4.0).During acid stress,the recombinant strains maintained a relatively stable intracellular pH and higher ATP concentration,which were 7.7-fold and 11.7-fold than that of the control strain under pH 4.0 for 3 h.To reveal the response conditions of membrane proteins ZitP(metal ABC transporter permease)and ZitQ(metal ABC transporter ATP-binding protein)at transcriptional level under acid-stress condition,transcriptome sequencing was performed to compare different gene-expression between the recombinant and control strains under normal and acid stress conditions in this study.The results showed that significantly up-regulated genes were involved in the ABC transport family proteins(ecfA2 and azlC)and metal ion transport proteins(mtsC and zitS);Some transcriptional regulator(MarR family transcriptional regulator and gene spxA)as well as stress proteins(cspABD2)were dramatically increased,suggesting that differential expression of these genes could help protect cells from lethal environment damage.Based on transcriptomic analysis,there were 7 and 6 membrane protein genes that were significantly up-regulated in L.lactis(ZitP)and L.lactis(ZitQ),respectively,and there were 10 up-regulated genes in common.Combined with molecular biological manipulation,the acid-resistant components secG(preprotein translocase subunit),ecfA2(energy-coupling factor transporter ATPase)and purC(phosphoribosylaminoimidazole-succinocarboxamide synthase)were verified and obtained from the above significantly up-regulated genes.Additionally,the overexpressed recombinant strains showed 20.9,598.7 and 83.2-fold higher survival rates,than that of the control strain L.lactis(pNZ8148),respectively(lactic acid stress at pH 4.0 for 4 h).It was further found that the intracellular ATP contents of L.lactis(SecG)and L.lactis(EcfA2)were also obviously higher,which were 6.5-fold and 4.8-fold than that of the control strain under acid stress,respectively,providing energy for the cells to resist acid stress.It was also found that L.lactis(PurC)could produce more intracellular aspartate,glutamate,threonine and ?-aminobutyric acid under acid stress conditions,promoting cells against acid stress.Next,membrane proteins ZitP and ZitQ were co-overexpressed with acid-resistant components,and the acid tolerance results indicated that the recombinant strains L.lactis(ZitP/SecG),L.lactis(ZitQ/EcfA2),L.lactis(ZitP/PurC)and L.lactis(ZitQ/PurC)exhibited 3.7?54.9?174.3 and 102.8-fold higher survival rates,than that of the control strain L.lactis(pNZ8148Dual),respectively(lactic acid stress at pH 4.0 for 4 h).It was confirmed that the combined expression of the above acid-resistant components could increase the acid tolerance.In this paper,we explored the conditions of membrane protein ZitP and ZitQ to improve the tolerance of lactic acid,and through the combination of transcriptomics and molecular biology techniques,we have discovered and confirmed the anti-acid effect of secG?ecfA2 as well as purC components.It will provide new thoughts for the construction of acid-resistant industrial strains.