Monday, August 13, 2012
Columbia Hall, Terrace Level (Washington Hilton)
Previously, a native homoethanol pathway was engineered in Escherichia coli B by deletions of competing pathway genes and anaerobically expression of pyruvate dehydrogenase (PDH encoded by aceEF-lpd). This E. coli B derived ethanologenic strain SZ420 was further improved for ethanol tolerance (up to 40 g L-1 ethanol) through adaptive evolution. However, the 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, deletion of the HSR2 region of rng (encoding for RNase G) from SZ470 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 (> two fold). Although not measured, the increase of other mRNAs might also enhance expressions of their encoding proteins. These increased enzymes enabled the resulting strain, RM10, to 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.