Furfural Inhibits Growth by Limiting Sulfur Assimilation in EthanologenicEscherichia coliStrain LY180

Abstract

A wide variety of commercial products can be potentially made from monomeric sugars produced by the dilute acid hydrolysis of lignocellulosic biomass. However, this process is accompanied by side products such as furfural that hinder microbial growth and fermentation. To investigate the mechanism of furfural inhibition, mRNA microarrays of an ethanologenic strain of Escherichia coli (LY180) were compared immediately prior to and 15 min after a moderate furfural challenge. Expression of genes and regulators associated with the biosynthesis of cysteine and methionine was increased by furfural, consistent with a limitation of these critical metabolites. This was in contrast to a general stringent response and decreased expression of many other biosynthetic genes. Of the 20 amino acids individually tested as supplements (100 M each), cysteine and methionine were the most effective in increasing furfural tolerance with serine (precursor of cysteine), histidine, and arginine of lesser benefit. Supplementation with other reduced sulfur sources such as D-cysteine and thiosulfate also increased furfural tolerance. In contrast, supplementation with taurine, a sulfur source that requires 3 molecules of NADPH for sulfur assimilation, was of no benefit. Furfural tolerance was also increased by inserting a plasmid encoding pntAB, a cytoplasmic NADH/NADPH transhydrogenase. Based on these results, a model is proposed for the inhibition of growth in which the reduction of furfural by YqhD, an enzyme with a low K m for NADPH, depletes NADPH sufficiently to limit the assimilation of sulfur into amino acids (cysteine and methionine) by CysIJ (sulfite reductase).Lignocellulose contains up to 70% carbohydrate by weight (35 to 45% cellulose and 20 to 35% hemicellulose) and represents an excellent potential source of sugars for microbial conversion into renewable fuels, plastics, and other chemicals (9,13,15,38). Prior to fermentation, these carbohydrate polymers must be converted to soluble sugars. Hemicellulose can be conveniently hydrolyzed to sugar monomers using dilute mineral acids. However, this process is accompanied by side products that inhibit microbial growth (20,21,29,30,(44)(45)(46). Furfural, the dehydration product of pentose sugars, is one of the most important such inhibitors (1). Previous studies have shown that furfural levels directly correlate with toxicity (20, 21). Overliming treatments that render hemicellulose hydrolysates fermentable also reduce the levels of furfural. Full toxicity in overlimed hydrolysates is restored by the addition of furfural. Of the many components of hydrolysates that have been tested for toxicity, only furfural was found to potentiate the toxicity of other agents in binary combinations (45).A number of approaches have been used to investigate the mechanism of furfural action. Furfural and 5-hydroxymethyl furfural (dehydration product from hexose sugars) have been previously proposed to inhibit growth by damaging DNA (3,16,36), inhibiting glycolysis and glycolytic enzymes (5,11,25), an...

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

“…Cd 2+ has a high affinity for sulfide, resulting in limitation of cysteine biosynthesis (Helbig et al 2008), similar to the effect seen following furfural challenge (Miller et al 2009a). …”

“…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). YqhD is similarly important for tolerance to 5-hydroxymethyl furfural .…”

Microbial Engineering for Aldehyde Synthesis

“…The E. coli strains investigated by Zaldivar et al were not engineered to have minimal aldehyde reductase activity, and later studies from the same group suggested that growth inhibition may be caused by NADPH consumption resulting from aldehyde reduction (78,79). Two genes (dkgA and yqhD) were found to be silenced in an evolved furfural-resistant strain.…”

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).…”