Engineering Global Regulators To Improve Sodium Acetate And Isobutanol Tolerances And Their Mechanisms In Escherichia Coli

In the industrial fermentation,the presence and accumulation of multiple stresses inhibit cell growth,yield and productivity.Hence,it is very meaningful to enchance microbial tolerance in industrial applications.In this study,we obtained sodium acetate or isobutanol-tolerance mutant strains from Escherichia coli by screening,isolating and reconstructing from the global regulators mutation library.Moreover,the key tolerance global regulator mutations were evaluated for various stress and identified strains with multiple tolerances.Finally,the mechanism of tolerance to sodium acetate and isobutanol in E.coli was studied by transcriptome sequencing.Based on the global regulators mutation library about E.coli populations,we obtained eight potential tolerance mutations resistant to sodium acetate and five resistant to isobutanol,respectively.Combined with mutation reconstruction and tolerance analysis,one acetate and three isobutanol tolerant mutations were identified.By evaluation of multiple tolerance(acetate,furfural,isobutanol,sodium chloride)for 20 global regulator mutations,we found that two mutations conferred multiple tolerances significantly,including N-CRP-E182D and N-CRP-G142I.The maximum biomass biomass(OD₆₀₀)of N-CRP-E182D was increased by 2266.67%,111.57%and77.91%under 30 g/L sodium acetate,0.6 mol/L sodium chloride and 8 g/L isobutanol,respectively.And N-CRP-G142I showed 1833.33%,35.53%and 74.42%increase in maximum biomass(OD₆₀₀)under 30 g/L sodium acetate,0.6 mol/L sodium chloride and 8 g/L isobutanol,respectively.The global regulator mutation CRP(E182D)conferred acetic acid tolerance in E.coli by multiple regulating metabolisms using RNA-seq and genetic engineering.The multiple regulating metabolisms included amino acid metabolism,supplementation with isoleucine,valine or overexpression of serine and threonine deaminase IlvA increased acetic acid tolerance;Up-regulation of nitrate reductase(NarGHI)and nitrite reductase(NirBD)in nitrogen metabolism conferred E.coli acetic acid tolerance;Different expression of ABC transpoter,low-expression of sRNAs and other related mechanisms such as overexpression nad A,pnuc and dps resulted in the acetic acid tolerance in E.coli.Based on transcriptome analysis,the global regulator mutation CRP(G142I)may conferred tolerance to isobutanol in E.coli by multiple regulating metabolisms,including change of the aerobic state by regulating carbon metabolism,improvement of cell membrane structure by fatty acid metabolism,down-regulation of the ABC transport,bacterial chemotaxis and flagella metabolism by restricting wastage during nonessential metabolic process.