Silencing of NADPH-Dependent Oxidoreductase Genes (yqhDanddkgA) in Furfural-Resistant EthanologenicEscherichia coli

Abstract

Low concentrations of furfural are formed as a side product during the dilute acid hydrolysis of hemicellulose. Growth is inhibited by exposure to furfural but resumes after the complete reduction of furfural to the less toxic furfuryl alcohol. Growth-based selection was used to isolate a furfural-resistant mutant of ethanologenic Escherichia coli LY180, designated strain EMFR9. Based on mRNA expression levels in the parent and mutant in response to furfural challenge, genes encoding 12 oxidoreductases were found to vary by more than twofold (eight were higher in EMFR9; four were higher in the parent). All 12 genes were cloned. When expressed from plasmids, none of the eight genes in the first group increased furfural tolerance in the parent (LY180). Expression of three of the silenced genes (yqhD, dkgA, and yqfA) in EMFR9 was found to decrease furfural tolerance compared to that in the parent. Purified enzymes encoded by yqhD and dkgA were shown to have NADPH-dependent furfural reductase activity. Both exhibited low K m values for NADPH (8 M and 23 M, respectively), similar to those of biosynthetic reactions. Furfural reductase activity was not associated with yqfA. Deleting yqhD and dkgA in the parent (LY180) increased furfural tolerance, but not to the same extent observed in the mutant EMFR9. Together, these results suggest that the process of reducing furfural by using an enzyme with a low K m for NADPH rather than a direct inhibitory action is the primary cause for growth inhibition by low concentrations of furfural.

Hybrid thermochemical processing: fermentation of pyrolysis-derived bio-oil

“…Furfural toxicity in E. coli was attributed to NADPH depletion by the furfural reductase enzyme YqhD, which converts furfural to furfuryl alcohol (Miller et al 2009a;Miller et al 2009b). Silencing of this enzyme increased not only tolerance to furfural but also to the closely-related 5-hydroxymethyl furfural by increasing NADPH availability for biosynthesis (Miller et al 2010).…”

YqhD: a broad-substrate range aldehyde reductase with various applications in production of biorenewable fuels and chemicals

“…Lignocellulosic biomass is an attractive source of sugars for fermentation, but treatment of this biomass to release the fermentable sugars also releases inhibitory compounds, such as the aldehyde furfural. Reverse engineering of a furfural-tolerant mutant of ethanologenic E. coli W, via transcriptome and phenotype analysis, revealed that yqhD expression was decreased 50-fold and this transcriptional silencing was crucial to the tolerant phenotype (Miller et al 2009b). Deletion of yqhD from the parent strain conferred furfural tolerance and restoration of yqhD expression in the evolved strain restored sensitivity.…”

“…This link between furfural tolerance and YqhD-mediated furfural reduction was attributed to the finite availability of NADPH for biosynthesis (Miller et al 2009b). Conversion of sulfate to sulfide, critical for cysteine biosynthesis, is NADPH-intensive; depletion of NADPH by YqhD with its low NADPH Km (8 µM) leaves insufficient NADPH for cysteine biosynthesis (Miller et al 2009a).…”

“…Three different projects involving whole-cell directed engineering showed perturbed expression of yqhD. As discussed above, evolution of an E. coli W derivative for furfural tolerance resulted in a nearly 50-fold decrease in yqhD transcript abundance (Miller et al 2009b). Contrastingly, when Riehle et al evolved three parallel E. coli lines for thermotolerance, yqhD transcript abundance was increased approximately 4-fold in two of these lines relative to the parental E. coli B-derived strain (Riehle et al 2003;Riehle et al 2005).…”

“…This perturbed expression of yqhD was shown to be critical for furfural and glyoxal tolerance (Lee et al 2010;Miller et al 2009b), but the contribution of increased yqhD expression to thermotolerance has not been determined.…”