Partial deletion ofrng(RNase G)-enhanced homoethanol fermentation of xylose by the non-transgenicEscherichia coliRM10

Abstract

Previously, 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.

Thymol tolerance in Escherichia coli induces morphological, metabolic and genetic changes

“…Different mutations reported in RNase G in the S1-like RNA-binding domain resulted in slowed growth of E. coli cultures [47]. Moreover, partial deletion of rng RNA-binding domain has been shown to enhance homoethanol fermentation [48]. It is possible that our reported missense mutation in RNase G that lies in the same domain would similarly support the metabolic shift to fermentation by alcohol dehydrogenase sustained expression and the noted slowed growth.…”

Identification of a transporter Slr0982 involved in ethanol tolerance in cyanobacterium Synechocystis sp. PCC 6803

“…Bioethanol production through microbiological processes has drawn greater attention in recent years due to increasing cost of non-renewable fossil fuels and environmental concerns related to over-utilization of fossil fuels (Hahn-Hagerdal et al, 2006 ). Various native or engineered microorganisms, such as Saccharomyces cerevisiae (Eiadpum et al, 2012 ; Yu et al, 2012 ), Zymomonas mobiles (Hayashi et al, 2012 ; Letti et al, 2012 ) and even Escherichia coli (Zhou et al, 2008 ; Manow et al, 2012 ) have been employed for ethanol production. As an alternative, photosynthetic cyanobacteria have been recently engineered by various synthetic biology tools into an “autotrophic microbial cell factory” to produce biofuels and fine chemicals directly from CO 2 using solar energy (Angermayr et al, 2009 ; Ducat et al, 2011 ; Ruffing, 2011 ; Machado and Atsumi, 2012 ; Oliver and Atsumi, 2014 ), which provides a complementary approach to the above heterotrophic microorganisms.…”

A Recurrent Silent Mutation Implicates fecA in Ethanol Tolerance by Escherichia coli

“…Beyond membrane lipid metabolism, published literature regarding ethanol tolerant E. coli and other ethanologenic microbes suggests that, in principle, alterations in sugar transport, increased TCA activity, transcriptional regulation of electron transport components or fermentation enzymes, altered expression of transcriptional regulators such as FNR, increased peptidoglycan synthesis, elevated biosynthesis or transport of various amino acids, increased betaine production, and uptake or retention of metals such as iron or zinc can contribute to ethanol tolerance (cf. [ 28 , 29 , 57 – 59 ]) reviewed in [ 60 ]. Indeed, changes to genes in a number of those categories are represented in our collection of ethanol tolerant strains.…”