Abstract

Efficient ethanol production will require a recombinant to able to ferment a variety of sugars (pentoses, and hexoses), less formation of by-products, as well as to tolerate high ethanol stress. In this study, a mutant (MGE) that can grow in 60 g ethanol/l was selected from Escherichia coli MG1655 by enrichment method with increasing concentrations of ethanol. The ethanol-tolerant mutant was used as the host to develop the ethanologenic recombinant by knockout of pyruvate formate lyase (pflB) and lactate dehydrogenase (ldhA) genes, and expression ofZymomonas mobilis alcohol dehydrogenase and pyruvate decarboxylase genes in plasmid pZY507bc. The resultant recombinant (GMEPLbc) showed the genetic stability of Z. mobilis genes in glucose medium without antibiotics under anaerobic conditions, and generated little acetic acid (3.6 mM), no formic acid and lactic acid. The ethanol production by GMEPLbc were 41.6 and 35.8 g ethanol/l from 100 g/L glucose and 100 g/L xylose during fermentation in M9 mineral medium, 37.0 and 36.5% more than that of the ethanol-sensitive strain carrying pZY507bc alone, respectively. Our results indicated that enhancement of ethanol tolerance and inactivation of pflB and ldhA are advantageous in the production of ethanol.

Key words: Escherichia coli, ethanol production, ethanol-tolerance, Gene knockout, metabolic engineering.