Evolution Of DsrA-Hfq Acid-resistant Module And Its Improvement Of Acid Tolerance Of Escherichia Coli

The biological fermentation industry exceeds 300 billion output value,which is one of the main compositions of strategic industry in China.During fermentation,strains often encounter acid stress.Enhancing the acid tolerance of the strain can reduce the amount of neutralizer and wastewater discharge in the fermentation process and downstream processes,as well as promote the green manufacturing.In the past few years,most of the studies focus on the acid resistance mechanisms of the microorganism.The development of synthetic biology provides powerful strategies and tools to improve the acid tolerance of industrial strains by artificial efficient acid resistance modules.In our previous work,we found that non-coding small molecule RNA?sRNA?DsrA and chaperone Hfq are potential acid resistance module.Herein,we conducted in-depth research on the DsrA-Hfq acid-resistant module.By established the directed evolution of the acid resistance module on the genomic context and obtained stable genetically acid tolerance Escherichia coli with DsrA-Hfq module.And then,preliminary analysis of the acid resistance mechanism on the DsrA-Hfq mutant.The mainly results were showed as follows:In this work,we constructed a method successfully for directed evolusion of DsrA-Hfq module in the E.coli genome context by emplied the CRISPR/Cas9 approach and error-prone PCR.Six mutants were obtained after screening and conferred a significant growth adavantage compared with the initial strain MG1655.The final OD₆₀₀ of the mutants increased by 41%?51%?in LBG with 2%glucose?and 51%?72%?in LBG with 7.5%glucose?at p H 4.5.Among the mutants,the final OD of H4 or E11 improved by 12.6%or 11.6%compared with DsrA-Hfq?WT?and can be stably inherited.The survival rate of the mutants increased by 1?100 fold at p H 2.5 comapred to the initial strain,among which H4 mutant strain had no significant increase,and the E11 mutant strain increases of 10 folds.Therefore,the growth assay under moderate acid stress is recommented for the evaluation of the acid tolerance of the strains.Furthermore,we investigated the mechanism under the acid tolerance of the DsrA-Hfq strains.Sequencing results showed that the mutations in both the promoter region and the gene region.The mutantions in the promoter region might affect the transcription level of Hfq.The mutations in the Hfq gene region may change its affinity to the corresponding sRNA.The mutations in the DsrA is presumed to increase the stability of DsrA or the affinity to the Rpo S or H-NS m RNAs,thereby affect the expression of downstream related genes.Quantitative RT-PCR results showed that the transcription levels of the acid resistance relative genes,i.e.gad E,gad B,yba S,hde B and kat E were upregulated.The assay of?-aminobutyric acid release and NH₃ release have also shown that the acid resistance system 2 is active in the strains hoboring DsrA-Hfq.In summary,it is speculated that the increase in acid resistance system 2,chaperone expression and reactive oxygen species removal system might be the mianly constribution for acid tolerance phenotype of the DsrA-Hfq mutant srtains.In this study,we demonstrated successfully directed evolution of DsrA-Hfq module in the E.coli genome context that effectively improve the acid tolerance of the strains.Our study provides a new avenue for engineering of strains with acid tolerance phenotype,as well as a reference for directed evolution of genetic parts or strains.