Construction And Characteration Of An Ethanologenic Eescherichia Coli Strain Deficient In Lactate Dehydrogenase And Pyruvate-formate Lyase Genes

Lignocellulosic biomass is an attractive alternate to petroleum for production of biofuel. This conversion of biomass would require a new generation of microbial biocatalysts that can convert all the sugars present in the biomass to the desired compound. Eescherichia coli which has a extensive background of knowledge, is easy for genetic manipulation and able to utilize a wide range of carbon sources (cellulose and hemicellulose hydrolyzed), is selected for further research in this study. An ethanol-torerant E. coli MG1655 is isolated as a starting strain to knock out the pyruvate formate lyase gene (pflB) and alcohol dehydrogenase gene (ldhA), and to develop an ethanologenic strain using molecular technologies.To enhance the ethanol tolerance of MG1655 strain, directed evolution was adopted by inoculating and transferring the strain serially into LB medium with increasing concentrations of ethanol. A final mutant that can tolerate up to 6.5% (w/v) of ethanol was obtained, which was selected to express the ethanol production genes from Zommonas mobilis.Z. mobilis pdc and adhB genes were cloned in pZY507 to yield the tandem plasmid, P4, with both genes expressed under the lacIq-tac regulation system. P4 was introduced by transformation into both the selected ethanol-tolerant mutant, resulting recombinant was named MG1655E-P4, which can grow under 6.5% of ethanol, produced ethanol as main product, productivity of which researched 56.8% of the theory value.To reduce the organic acids produced and improve ethanol productivity during fermentation, Gene pflB, which is highly expressed in E. coli MG1655E, was eliminated by using recombinase ofλphage system, the developed MP mutant showed a drastic decrease in acetate production (10.4% of parental strain), but an increase of 20.6% in lactate. To further reduce the lactate formation, the pflB and ldhA deficient double mutant (MPL) was constructed. In anaerobic condition, the growth of pflB- mutant was lower than the strain MG1655E, and the double mutant could not grow under the anaerobic condition, while both strains showed no siganificant difference with the strain MG1655E in aerobic growth. When the plasmid P4 was introduced into MP and MPL, respectively. The resulting strains MP-P4 and MPL-P4 could grows well in both aerobic and anaerobic circumstances.Ethanol production by strain MPL-P4 was evaluated in M9 medium added with 10% of glucose, the amount of acetate was greatly reduced and no lactate was detected in the culture at 66 hours, the final concentration of ethanol reached 6.0%, 93% of the theory value, a 23% improvement over MP-P4.