Construction And Application Of Synthetic Acid-tolerance Modules In Escherichia Coli

Industrial microorganisms play an important role in environmentally friendly biofuel,chemical and biomaterial production,which support the annual production value of 300 billion yuan in China.During fermentation,industrial microorganisms produced acidic metabolites which would accumulate in the fermentation medium,leading to the p H of fermentation medium decrease.Microorganisms encounter multiple stresses during fermentation,such as acidic stress,oxidative stress and so on,which inhibit cell growth and decrease fermentation yields due to the damge of DNA,RNA and key metabolic enzymes.Although the issue can be addressed by adding base at the fermentation broth,however,it would enhance the downstream separation costs,the use of neutralizing reagents,and the waste streams.Therefore,engineering acid tolerant strain is a cost-effective strategy to address this issue.On the basis of the principles of modular design of synthetic biology,constructing a synthetic acid-tolerance module achieving a just-enough and just-in-time gene expression could efficiently achieve acid tolerant strain.To fulfil the just-enough and just-in-time regulation,we first construct a synthetic acid responsive promoter library by directed evolution and achieved49 asr promoter variants with dirrerent strengths to regulate the overexpressing acid resistance related genes.The relative strength of asr promoter variants was ranging from 22%to 133%.Considering the mechanism of acid resistance of E.coli,in the end,we selected 4 genes including gad E encoding acid resitance system 2 regulater,hde B encoding periplasmic chaperone,and sod B/kat E encoding intracullar reactive oxygen（ROS）scavengers to construct synthetic acid-tolerance modules.832 strains with the synthetic acid-tolerance modules were screened at p H 5.0 LBG based on cell growth assay,and the final OD₆₀₀ of best strain MG 1124was 151%of the wild type strain.A multi-factors ANOVA analysis was adopted to analyze the key genes in gad E-hde B-sod B-kat E module,demonstrated the key genes for enhancing OD₆₀₀were hde B,sod B and kat E.The transcriptional and biochemical analysis demonstrated that the synthetic acid-tolerance modules could efficiently decrease the intracellular ROS level and increase the ability of proton consumption.We developed a stepwise screening protocol to investigate the effect of acid-tolerance modules on larboratory E.coli in larboratory and lysine producing E.coli in industrial medium.At the beginning,we screened 908 strains harboring synthetic acid-tolerance modules in larboratory E.coli with larboratory medium at automatic growth curve analyzer.And then we selected 33 acid-tolerance modules with advanced growth enhancement performance,and screened in lysine producing E.coli with industrial fermentation medium at micro bioreactor based on the titer of lysine.Finally,the best 4 strains with lysine productivity were performed lysine fermentation at 1.3-L bioreactors.we transformed several advanced acid-tolerance modules into a lysine producing E.coli,and performed a stepwise screening protocol to screen modules which could confer acid tolerance of industial strain.The lysine titer and lysine yield of the best stain SC 3124 were 115%and 1150%of that of the parent strain,and were comparable to that of the parent strain at the neutral condition（p H 6.8）,indicating that the synthetic acid-tolerance module had the potential to apply in industrial production.In this work,we successfully constructed the synthetic acid-tolerance modules with limited genes and developed a stepwise screening protocol to screen modules which were efficient to improve the growth robutnrss and production performance at low p H（decresing 1unit of p H）.In addition,this work provided a successful example for engineering stress tolerant microgram,and the acid responsive promoter variants enriched the element libaray of synthetic biology.