AbstractPreviously, a native homoethanol pathway was engineered in Escherichia coli B by deletions of competing pathway genes and anaerobic expression of pyruvate dehydrogenase (PDH encoded by aceEF-lpd). The resulting ethanol pathway involves glycolysis, PDH, and alcohol dehydrogenase (AdhE). The E. coli B-derived ethanologenic strain SZ420 was then further improved for ethanol tolerance (up to 40 g l−1 ethanol) through adaptive evolution. However, the resulting ethanol tolerant mutant, SZ470, was still unable to complete fermentation of 75 g l−1 xylose, even though the theoretical maximum ethanol titer would have been less than 40 g l−1 should the fermentation have reached completion. In this study, the cra (encoding for a catabolite repressor activator) and the HSR2 region of rng (encoding for RNase G) were deleted from SZ470 in order to improve xylose fermentation. Deletion of the HSR2 domain resulted in significantly increased mRNA levels (47-fold to 409-fold) of multiple glycolytic genes (pgi, tpiA, gapA, eno), as well as the engineered ethanol pathway genes (aceEF-lpd, adhE) and the transcriptional regulator Fnr (fnr). The higher adhE mRNA level resulted in increased AdhE activity (>twofold). Although not measured, the increase of other mRNAs might also enhance expressions of their encoding proteins. The increased enzymes would then enable the resulting strain, RM10, to achieve increased cell growth and complete fermentation of 75 g l−1 xylose with an 84% improved ethanol titer (35 g l−1), compared to that (19 g l−1) obtained by the parent, SZ470. However, deletion of cra resulted in a negative impact on cell growth and xylose fermentation, suggesting that Cra is important for long-term fermentative cell growth.