Preliminary Bioengineering Studies Of Escherichia Coli For Production Of 1-butanol

With the deepening concerning in energy and environmental problems,as a potential alternative liquid energy,biobutanol attracts intensive interests in biofuel studies due to its superior physical and chemical property.Since its clearly genetic background and simply genetic operation,Escherichia coli have a huge potential in butanol fermentation.This study chose five key genes,phaA?hbd?crt?ter?adhE1,to construct 4 plasmids pCNA-PT?pRNA-hbd?pENA-crt?pCDA-adhEl mediated by promoter Phya,then transform these 4 plasmids into E.coli Bw25113 for butanol fermentation.But it failed to get butanol.Furthermore,developed the 4 plasmids system by replace the promoter with Pxya,and get 3 new plasmids pCNX-pt?pCDX-adhE1?pENX-crt.Transform these 3 new plasmids and pRNA-hbd into E.coli Bw25113 for butanol fermentation.And the yield of butanol is 0.483g/L.Moreover,with construction of two plasmids pCNA-PHC?pENA-TA mediated by Phya,within gene clusters phaA-hbd-crt and ter-adhE1,the yield of butanol is 0.51g/L.Through compare these two systems,it can be confirmed that butanol producing has a closely related with sequential expression of genes,and has a great influence by the efficiency of promoter.For steadily express butanol synthesis pathway,we construct two plasmids pCNA-PHCF and pENA-TAF.This study developed a novel genetic kit from Red/ET system which facilitates rapid construction of butanol producing E.coli,a commercial available recombination technology.A synthetic butatnol pathway comprising two gene clusters phaA-hbd-crt and ter-adhEl were integrated into the genome of wild-type Escherichia coli,synchronously knock-out two target genes adhE and ldhA.Finally,we obtain the butanol producing recombinant E.coli strain BuS2.Through simple fermentation test of strain BuS2,it was found that TB culture medium is the best substrate so far tested.The concentration of glucose at 2.5%can greatly promote the yield of butanol.And OD600=5.0 is the best condition for butanol fermentation.With the expression of butanol pathway,we also found numerous side products were produced,like butyrate,formic acid,acetone and ethanol.By single gene expression in E.coli,we confirmed that the heterologous phaA is responsible for accumulation of ethanol.The maximum yield of butanol is 1.877 g/L during 32 h,within the best conditions of this study.The novel way of transforming E.coli for butanol production we reported,is important for subsequent butanol fermentation study,and also has application value to other strain engineering works because of its superior features than traditional methods of strain development,including better genetic stability and inducer-free automatic anaerobic expression.Therefore,we believe the methods introduced here can be applied for broader use.