The Study Of Regulation Strategy Based On The Initiation Codon And Electrical Energy In Escherichia Coli

Diverse valuable targeted product could be produced using microbial cell factories via microbial metabolic engineering and synthetic biology,for example,some bio-based chemicals and biofuels.Since metabolic regulation is one of the research directions in the field of microbial metabolic engineering,it has played an important role in promoting the yield of targeted products,and has become a research hotspot of scientists from all over the world.In this study,the strain Escherichia coli was used to explore the metabolic regulation strategies,which including the research of initial codon library and electrical energy.The main contents are stated in two aspects:First,the effect of combinatorial modulation of initial codons(CMIC)on the synthetic pathway efficiency in Escherichia coli was investigated using reporter protein and zeaxanthin production as the evaluation index.Traditional strategies mostly involve the manipulation of promoters or RBSs,which can encompass long sequences and can be complex to operate.In this work we found that by changing only the three nucleotides of the initiation codons,expression libraries of reporter proteins RFP,GFP and lac Z with a large dynamic range and evenly distributed expression levels could be established in Escherichia coli(E.coli).Thus,a novel strategy that uses combinatorial modulation of initial codons(CMIC)was developed for metabolic pathway optimization and applied to the three genes crt Z,crt Y and crt I of the zeaxanthin synthesis pathway in E.coli.With minimal labor time,a combinatorial library was obtained containing strains with various zeaxanthin production levels,including a strain with a titer of 6.33 mg/L and specific production of 1.24 mg/g DCW–a striking 10-fold improvement over the starting strain.The results demonstrated that CMIC was a feasible technique for conveniently optimizing metabolic pathways.To our best knowledge,this is the less cost time and money metabolic engineering strategy that relies on manipulating the initiation codons for pathway optimization in E.coli.Second,the effects of electricity on the cell metabolism was studied based on the microbial electrosynthsis(MES)platform in Escherichia coli using the succinate production as the evaluation index.Electrochemical energy is a key factor of biosynthesis,previous MES research mainly utilized naturally electroactive microbes to generate non-specific products.In this study,two different electroactive E.coli were constructed,and it was diveided into two parts,in the first part,a successful MES reaction was thus performed using the electroactive E.coli 8739(p Mtr ABC,p Fcc A-Cym A)by expressing mtr ABC,fcc A and cym A from Shewanella oneidensis MR-1,and electricity was utilized in the MES system to produce more reduced fermentation products from glucose,and the lactate yield increased 2.2-fold from 0.10 to 0.22mol/mol glucose,and ethanol increased 1.5-fold from 0.63 to 0.96 mol/mol glucose.Subsequently,an electroactive succinate-producing cell factory was engineered in E.coli T110(p Mtr ABC,p Fcc A-Cym A).The electroactive T110 strain was further improved by incorporating a carbon concentration mechanism(CCM)for increasing the utilization capacity of CO₂.The resulting strain E.coli T110(p Mtr ABC,p Fcc A-Cym A,p BTCA)showed a prominent improvement of succinate yield with electricity,which produced a succinate yield of 1.10 mol/mol glucose,which represented 61.8%improvement over the parent strain E.coli T110.In the second part,the FAD synthesis pathway was manipulated to increase its cellular concentration and thereby improve the electroactivity of E.coli based on the the FAD cofactor.An MES reaction was performed with engineered E.coli 8739(p Rib AB-Ybj I-Yig B,p Rib ECF),and an altered metabolic profile with more reductive fermentation products was obtained,the lactate yield increased 1.8-fold from 0.51 to 0.90 mol/mol glucose,and ethanol increased 1.6-fold from 0.37 to 0.59 mol/mol glucose.An electroactive succinate-producing cell factory was engineered in E.coli T110(p Rib AB-Ybj I-Yig B,p Rib ECF),which can utilize electricity to produce succinate with the yield of 0.97 mol/mol glucose,represented 42.65%increase.The electroactive T110 strain was further improved by incorporating a carbon concentration mechanism(CCM).This engineered strain E.coli T110(p Rib AB-Ybj I-Yig B,p Rib ECF,p BTCA)produced a succinate yield of 1.16mol/mol glucose,which corresponds to 70.59%improvement over the parent strain E.coli T110.Due to the versatility of the E.coli platform,this pioneering research opens the possibility of engineering various other cell factories to utilize electricity for bioproduction of sucicnate,it indicated that the strategy of utilizing electricity to improve targeted compouds yield was feasible and effective.This paper research show that both initial codon optimization and electrical energy utilization can increase the metabolic yield of E.coli,and in the aspects of theory and practice,it provides new strategy and tools for the promotion of metabolic rate and mode.