Metabolic Engineering Of Escherichia Coli For The Production Of Isobutanol With Acetic Acid As Sole Carbon Source

In order to meet the growing global energy demand and reduce the negative impact on the environment,it is particularly important to find alternatives to traditional fossil fuels.More and more attention has been paid to the biofuels such as higher alcohols as potential substitutes for traditional gasoline and diesel.Isobutanol is a C4 alcohol which is used for gasoline blend and precursor of rubber synthesis.Compared with ethanol and other alcohols,isobutanol has lower vapor pressure,lower hygroscopicity and higher energy density,and its octane number is also higher than that of ordinary fuels.These characteristics make isobutanol a potential biofuel.At present,glucose is the most commonly used carbon source for isobutanol production by recombinant E.coli.Acetic acetic acid,the most common volatile fatty acid on the market,has lower cost compared with glucose,and has a clear metabolic pathway in E.coli,so acetic acid has the potential to replace glucose as a cheap carbon source.In order to use acetic acid as the sole carbon source for isobutanol production,wild-type Escherichia coli BW25113 was carefully engineered in this study.The competitive pathway was first blocke by knocking out ldhA gene encoding lactate dehydrogenase,pfl B encoding pyruvate formate lyase,adhE encoding aldehyde-alcohol dehydrogenase and poxB encoding pyruvate oxidase,and strain BW?PPAL was obtained.In order to improve the expression level of key enzymes in isobutanol synthesis pathway of E.coli,coexistence plasmids pTrc99a and pCL1920 were employed to overexpress kivD?2-keto-acid decarboxylase?,adhA?ethanol dehydrogenase?,ilvC?acetyllactate isoreductase?,ilvD?dihydroxyacid dehydratase?,alsS?acetolactate synthetase?,pfo?pyruvate flavoxyredoxin oxidoreductase?,acs?acetyl-coenzyme A synthetase?,pckA?phosphoenolpyruvate carboxykinase?,maeB?malate dehydrogenase?,yfjB?NAD kinase?,pntAB?transhydrogenase?.Through different gene combinations,four recombinant plasmids were constructed.Then,four recombinant plasmids were transformed into BW?PPAL strain respectively,and WY001?NH001?WY002 and NH002 were obtained.After48 h batch fermentation,four recombinant strains were able to synthesize isobutanol with sodium acetate as sole carbon source,and WY002 strain had the highest isobutanol accumulation of 137 mg/L.Optimization of batch fermentation and sodium acetate oncentration,under the condition of 50 mM sodium acetate concentration,strain WY002 can produce 157 mg/L isobutanol,thus successfully realizing the production of isobutanol by recombinant Escherichia coli using acetic acid as sole carbon source.In addition to isobutanol,succinate,an important biological compound,was also synthesized by recombinant E.coli using acetic acid as sole carbon source.By deletion of sdhAB encoding succinate dehydrogenase,iclR encoding the isocitrate lyase regulator,and maeB encoding the malic enzyme,and overexpression of acs encoding acetyl-CoA synthetase,gltA encoding citrate synthase,and acnB encoding aconitate hydratase in the wild-type Escherichia coli MG1655,recombinant strain WCY-7 was constructed,which could accumulate 11.23 mM succinate with 50 mM sodium acetate.In wild-type Escherichia coli,the transport and phosphorylation of glucose is mainly accomplished by the phosphoenolpyruvate-dependent glucose-specific phosphotransferase system(PTS^Glc).Therefore,PTS^Glc frequently selected as a target for engineering to increase the intracellular level of phosphoenolpyruvate.In this study,the effects of low glucose concentration on the growth,glucose consumption and acetic acid accumulation of PTS^Glclc mutant were studied systematically.It was found that the biomass of most mutants was not affected,but the glucose consumption rate was significantly reduced,and a small amount of acetic acid was secreted.However,the growth,glucose consumption and acetic acid secretion of strain MG1655I with ptsI deficiency were significantly affected.Therefore,the fermentation performance of different PTS^Glc mutants with 5 g/L glucose as substrate is significantly different from that under 20 g/L glucose,which is an effective supplement to the knowledge of E.coli PTS^Glc system.