Research Of Environment-friendly Escherichia Coli Strain For High-effecicent Isobutanol Producation

In present work,an initial isobutanol-producing strain has been constructed with synthetic biology method.And then the high-effecicent environment-friendly E.coli isobutanol-producing strain has been achieved by figuring out the redox state imbalance and residual glucose transform problem under the rational guidance of genome-scale metabolite network model and metabolome methods,to make the residual glucose decrease to 0 g/L.A starting isobutanol-producing strain LA02 was engineered with only 2.7 g/L isobutanol produced.Then,the genome-scale metabolic modeling was specially carried out for the redox cofactor metabolism by combining flux balance analysis and minimization of metabolic adjustment,and the reaction catalyzed by the glyceraldehyde-3-phosphate dehydrogenase was predicted as the key target for redox status improvement.Then,a gapN-encoding NADP~+-dependent glyceraldehyde-3-phosphate dehydrogenase pathway was constructed and then fine-tuned using constitutive promoters.The best strain LA09 was obtained.The NADPH/NADP+ratios of strain LA09 reached to 0.67 at exponential phase and0.64 at stationary phase.And the isobutanol titer was increased by 221%to 8.68 g/L.However,1.36 g/L the residual glucose was still present in culture medium.In order to overcome the problem of residual glucose,the environment-friendly E.coli isobutanol-producing strain would be constructed with the residual glucose decreased to 0 g/L.Batch fermentation with 20~80 g/L initial glucose concentration was performed,the results showed 40 g/L was the best concentration of glucose for isobutanol production due to the inhibition of high concentration of glucose.The changes of intracellular metabolites in strain LA09 was detected by Gas/Liquid chromatography-Mass spectrometry.Through the Principal component analysis and Cluster analysis,the top 15 marker metabolites were obtained,which were related to the cofactor metabolism,glycolysis metabolism and osmotic pressure.Specially,the comparative analysis shows that in the glycolytic pathway the increase level of upstream metabolites of the reaction catalyzed by phosphofructokinase was1.91~2.68,which is much higher than the increase level(1.16~1.36)of downstream metabolites.Thus,the reaction catalyzed by phosphofructokinase was identified as the rate-limiting step of strain for catalyzing glucose to isobutanol.Then,heterologous artificial ED pathway has been rationally designed and constructed,and the artificial promoter was applied to match the upstream and downstream of the ED pathways for reducing the level of toxic 2-keto-3-deoxy-6-phospho-gluconate.Finally,the best environment-friendly isobutanol-producing strain E.coli ED02 was obtained.With the initial sugar concentration of 40 g/L,the residual sugar was rapid consumed by E.coli ED02 to 0 g/L,the average glucose absorption rate was increased by 19.8%to 1.33 g/L/h compared with that of strain LA09,and the fermentation time was greatly shortened to 30 h.the isobutanol titer of E.coli ED02was increased by 47%and 56%to 13.67 g/L,compared with the control strain E.coli Control1(9.30 g/L)and E.coli LA09(8.74 g/L),respectively.