| Due to its excellent capability to ferment five and six-carbon sugars, Escherichia coli has been considered one of the platform organisms to be engineered for production of bioethanol. Nevertheless, the genetically engineered non-transgenic E. coli KC01 (ldhA pflB ackA frdBC pdhR::pflBp6-aceEF-lpd) lacks production efficiency of homoethanol fermentation from xylose. In this study, we improved the performance of KC01 through adaptive evolution for enhanced ethanol tolerance, and improved the gene expression of alcohol dehydrogenase (adhE) through deletion of the RNase G HSRII domain.;During ∼350 generations of adaptive evolution, a gradually increased concentration of ethanol was used as a selection pressure to enrich ethanol tolerant mutants. The evolved mutant, E. coli SZ470, was able to grow anaerobically at 40 g L-1 of ethanol, a two-fold improvement over that of parent KC01. When compared to KC01 on a small scale (500 ml) xylose (50 g L-1) fermentation , SZ470 achieved 67% higher cell mass, 48% faster volumetric ethanol productivity, and a 50% shorter fermentation time to complete the fermentation with an ethanol titer of 23.5 g L-1 and a yield of 94%.;SZ470 was further improved by the deletion of the RNase G HSRII domain (rng HSRII). This deletion enhanced adhE expression by 409 fold and AdhE activity by ∼2 fold. The resulting strain, RM10, completed 75 g L-1 xylose fermentation in 84 hours with a maximum ethanol titer of 34.23 g L-1 (94% yield), representing an improvement of 82% over those of SZ470 (18.84 g L-1 ). These results demonstrated that an industry-oriented non-transgenic E. coli strain could be developed through incremental improvements of desired traits by a combination of molecular biology and traditional microbiology techniques. |
