| The acid accumulation from carbon metabolism is a key factor in reducing the physiological activity of microorganisms.However,there is still no targeted control strategies based on the analysis of regulation of anti-acid components.Based on the functional analysis of anti-acid components,the acid-stress tolerance of microbial cells can be effectively improved through targeted strategies of regulation and modification.In this study,Lactococcus lactis NZ9000 was used as the parent strain,and acid-tolerant mutants were obtained through genomic mutation combined with high-throughput screening technology.The transcriptional response processes of L.lactis were explored during acid stress by using transcriptomics approaches.Then,part of the factors associated with acid-stress resistance were initially identified.Finally,the acid tolerance of L.lactis were effectively improved through metabolic engineering strategies,and the specific function of key anti-acid components were analyzed.Major results achieved in this study are highlighted below:(1)L.lactis with acid tolerance was screened by combining mutagenesis breeding and high-throughput techniques.Three acid-tolerant mutants were obtained through chemical and UV mutagenesis combined with high-throughput screening techniques,which with L.lactis NZ9000 as the parent strain.The acid-tolerance analysis showed that the mutant strains(L.lactis WH101,L.lactis WH102 and L.lactis WH103)exhibited 5.5-,3.6-and 4.3-fold higher maximum biomass,respectively,relative to the parent strain at pH 4.5.Moreover,after 5 h of acid shock,the mutant strains showed 22.4-,2.2-,and 1.9-fold higher survival rates,respectively.Finally,further analysis of intracellular physiological response mechanism of the mutant strains showed that L.lactis WH101 maintained higher ATP and NH4~+concentrations and a relatively stable pHi(intracellular pH)during acid stress.(2)The acid-stress response processes were analyzed in L.lactis based on transcriptomics techniques.To reveal the response processes of L.lactis at transcriptional level during acid stress,transcriptome analysis was performed to compare differentally expressed genes between the parent and mutant strains.The significant differentally expressed genes involved in carbon metabolism(kdgA,malQ,and bglX)and transport(msmK,malEFG,rbsAB,fruA,and ptnD)pathways promote central metabolism and supply precursors and energy for cell growth and metabolism in acidic environment;The differentally expressed genes involved in amino acid metabolism(hisCHZ,serB,arcC1C2,ilvABCDN,leuABC,gltBD,glmS,glnA,arcABC1C2,and asnB)and transport(dppA,ctrA,glnPQ,and yjeM)processes,could generate NH3 or alkaline substances through deamination,decarboxylation,etc.,and consume intracellular H+,thereby maintaining the relative balance of intracellular pH;The differentally expressed genes(accABCD and fabDFGHZ)involved in fatty acid synthesis pathways affect the structure and related components of cell membranes,thereby affecting transmembrane transport of related protons during acid stress.(3)The acid-stress tolerance of L.lactis was improved by overexpression of transporters.Based on the transcriptional response of L.lactis during acid stress,overexpression of ABC and amino aicd transporters resulted in enhanced acid-stress tolerance in L.lactis NZ9000.Comparative transcriptomics analysis based on ABC transporters showed that overexpression of ABC transporters significantly increased the transcription level of some common differentally expressed genes,such as cold-shock proteins(csp),fatty acid(fabH)and coA(coaD)biosynthetic pathways,to help microbial cells against acid stress.Also,some specific recombinant strains have their own response processes to acid stress.Moreover,based on the comparative transcriptomic analysis of amino acid transporters,overexpression of CtrA and GlnP proteins enhanced the transport of amino acid(yjeM),arginine metabolism(argGH),and pyrimidine biosynthetic processes.In addition,several genes involved in cold-shock proteins(csp),ribosomal proteins(rpsA and rpmFG),cell division process(rodA and gpsB),ion and amino acid transport,were also up-regulated.(4)Thiamine enhanced the acid-stress tolerance of L.lactis.The mechanisms of thiamine on the acid tolerance in L.lactis were investigated.The acid tolerance of cells was enhanced by addition of thiamine or overexpression of thiT or thiDM genes in the transport and biosynthetic pathways of thiamine.Thiamine could significantly increase the transcription level of key genes in acetoin biosynthesis pathway during acid stress.The enhanced of acetoin biosynthesis consumes more protons,thus maintaining the relative homeostasis of pHi.In addition,thiamine could significantly increase the transcription level of the TPP-dependent enzyme(pyruvate dehydrogenase)in the central metabolic pathway,and strengthen the energy productivity,providing more energy for cells to withstand acid stress.Moreover,thiamine could significantly increase the intracellular amino acid concentration,especially glutamate,and the intracellular ATP concentration was also improved.(5)The tandem expression of single factor anti-acid components was determined based on the interaction analysis of differentially expressed genes.The interaction of differentially expressed genes was analyzed,and the tandem ant-acid components with potential effects were predicted.The effect of predicted tandem ant-acid components on acid stress was determined by constructing engineering strain.Co-expression of GlnP and GlnQ proteins furtherly enhanced the acid tolerance,and the co-expressing strain L.lactis(GlnP/GlnQ)exhibited 16-and 36.6-fold higher survival rates than that of the strain L.lactis(GlnP)and L.lactis(GlnQ),respectively.Then,co-expression of GlnP and GlnQ proteins could furtherly increase the concentration of aspartate,glutamate and ATP. |
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