Engineering furfural tolerance in
            <i>Escherichia coli</i>
            improves the fermentation of lignocellulosic sugars into renewable chemicals

Self Cite

Expression of genes encoding polyamine transporters from plasmids and polyamine supplements increased furfural tolerance (growth and ethanol production) in ethanologenic Escherichia coli LY180 (in AM1 mineral salts medium containing xylose). This represents a new approach to increase furfural tolerance and may be useful for other organisms. Microarray comparisons of two furfural-resistant mutants (EMFR9 and EMFR35) provided initial evidence for the importance of polyamine transporters. Each mutant contained a single polyamine transporter gene that was upregulated over 100-fold (microarrays) compared to that in the parent LY180, as well as a mutation that silenced the expression of yqhD. Based on these genetic changes, furfural tolerance was substantially reconstructed in the parent, LY180. Deletion of potE in EMFR9 lowered furfural tolerance to that of the parent. Deletion of potE and puuP in LY180 also decreased furfural tolerance, indicating functional importance of the native genes. Of the 8 polyamine transporters (18 genes) cloned and tested, half were beneficial for furfural tolerance (PotE, PuuP, PlaP, and PotABCD). Supplementing AM1 mineral salts medium with individual polyamines (agmatine, putrescine, and cadaverine) also increased furfural tolerance but to a smaller extent. In pH-controlled fermentations, polyamine transporter plasmids were shown to promote the metabolism of furfural and substantially reduce the time required to complete xylose fermentation. This increase in furfural tolerance is proposed to result from polyamine binding to negatively charged cellular constituents such as nucleic acids and phospholipids, providing protection from damage by furfural.

Section: Discussionmentioning

confidence: 99%

“…Genes have been described that increase the resistance of bacterial and yeast biocatalysts to furfural and hydroxymethylfurfural (5,7,(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). However, to date, none have completely solved the toxicity problem.…”

mentioning

confidence: 99%

Polyamine Transporters and Polyamines Increase Furfural Tolerance during Xylose Fermentation with Ethanologenic Escherichia coli Strain LY180

Geddes

Wang

Yomano

et al. 2014

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“…In contrast, ThyX produces THF as a direct product of dTMP synthesis but oxidizes NADPH during this reaction, obviating the need for DHF reduction but not NADPH oxidation (60,71,72). Because both thymidylate synthases result in oxidized NADPH and yet both confer furfural tolerance when overexpressed, these enzymes likely ameliorate furfural toxicity independently of the compound's effect on NADPH levels (59,70,73,74).…”

Section: Discussionmentioning

confidence: 99%

Identification of Genes Conferring Tolerance to Lignocellulose-Derived Inhibitors by Functional Selections in Soil Metagenomes

Forsberg

Patel

Witt

et al. 2016

The production of fuels or chemicals from lignocellulose currently requires thermochemical pretreatment to release fermentable sugars. These harsh conditions also generate numerous small-molecule inhibitors of microbial growth and fermentation, limiting production. We applied small-insert functional metagenomic selections to discover genes that confer microbial tolerance to these inhibitors, identifying both individual genes and general biological processes associated with tolerance to multiple inhibitory compounds. Having screened over 248 Gb of DNA cloned from 16 diverse soil metagenomes, we describe gain-of-function tolerance against acid, alcohol, and aldehyde inhibitors derived from hemicellulose and lignin, demonstrating that uncultured soil microbial communities hold tremendous genetic potential to address the toxicity of pretreated lignocellulose. We recovered genes previously known to confer tolerance to lignocellulosic inhibitors as well as novel genes that confer tolerance via unknown functions. For instance, we implicated galactose metabolism in overcoming the toxicity of lignin monomers and identified a decarboxylase that confers tolerance to ferulic acid; this enzyme has been shown to catalyze the production of 4-vinyl guaiacol, a valuable precursor to vanillin production. These metagenomic tolerance genes can enable the flexible design of hardy microbial catalysts, customized to withstand inhibitors abundant in specific bioprocessing applications. Many lignocellulosic feedstocks (e.g., switchgrass) are preferred to maize, sugarcane, and other traditional food crops for the production of fuels and chemicals because they are able to grow on marginal land, often require little attention or energy input, and do not compete directly with the food supply (1-6). However, lignocellulose requires harsh thermochemical pretreatment methods to liberate fermentable monosaccharides (7-9), producing an additional compendium of compounds inhibitory to microbial growth that ultimately reduce production efficiencies (10-12). These small-molecule inhibitors derived from lignocellulose pretreatment (here called lignocellulosic inhibitors) are typically aldehydes, organic acids, furans, or phenolics and can originate from the cellulosic, hemicellulosic, and lignified fractions of the feedstock (11-15).Engineering hardier microbial production hosts with elevated tolerance to these inhibitors offers potential to ameliorate the toxic effects of these compounds without incurring the high process costs associated with detoxifying the lignocellulosic hydrolysate (16, 17). Unfortunately, the modes of toxicity of many of these toxins are poorly described, and genes conferring tolerance to many of these compounds have not been identified (18,19). An expanded catalog of tolerance-conferring genotypes may shed light on mechanisms of toxicity and enable synthetic biology approaches for the design of diverse production hosts with broadspectrum tolerance.Soil microorganisms, including white-rot fungi (20) and many bacteria (21), ar...

“…The strains, plasmids, and primers used in this study are listed in Table 1 (6,15,16). LB medium containing xylose was used for the construction of ethanol strains.…”

Section: Methodsmentioning

confidence: 99%

Improving Escherichia coli FucO for Furfural Tolerance by Saturation Mutagenesis of Individual Amino Acid Positions

Zheng

Wang

Yomano

et al. 2013

Self Cite

Furfural is an inhibitory side product formed during the depolymerization of hemicellulose with mineral acids. In Escherichia coli, furfural tolerance can be increased by expressing the native fucO gene (encoding lactaldehyde oxidoreductase). This enzyme also catalyzes the NADH-dependent reduction of furfural to the less toxic alcohol. Saturation mutagenesis was combined with growth-based selection to isolate a mutated form of fucO that confers increased furfural tolerance. The mutation responsible, L7F, is located within the interfacial region of FucO homodimers, replacing the most abundant codon for leucine with the most abundant codon for phenylalanine. Plasmid expression of the mutant gene increased FucO activity by more than 10-fold compared to the wild-type fucO gene and doubled the rate of furfural metabolism during fermentation. No inclusion bodies were evident with either the native or the mutated gene. mRNA abundance for the wild-type and mutant fucO genes differed by less than 2-fold. The K m (furfural) for the mutant enzyme was 3-fold lower than that for the native enzyme, increasing efficiency at low substrate concentrations. The L7F mutation is located near the FucO N terminus, within the ribosomal binding region associated with translational initiation. Free-energy calculations for mRNA folding in this region (nucleotides ؊7 to ؉37) were weak for the native gene (؊4.1 kcal mol ؊1 ) but weaker still for the fucO mutant (؊1.0 to ؊0.1 kcal mol ؊1 ). The beneficial L7F mutation in FucO is proposed to increase furfural tolerance by improving gene expression and increasing enzyme effectiveness at low substrate levels.T he enzyme L-1,2-propanediol oxidoreductase (encoded by fucO) is an NADH-linked, iron-dependent group III dehydrogenase. Typically, this enzyme functions only during the catabolism of deoxy sugars, where it catalyzes the reduction of lactaldehyde (1-3). FucO is a homodimer in which each subunit contains an active site (4). This enzyme has a broad substrate range (5) that includes furfural (6), a toxic side product in dilute acid hydrolysates of hemicellulose and an important inhibitor of microbial fermentations (7-9). Expression of fucO from plasmids has been used to improve furfural tolerance in Escherichia coli-based fermentations for ethanol and lactic acid (6). The reduction of furfural to the less toxic alcohol seems essential for growth and fermentation of dilute acid hydrolysates of hemicellulose (10-13).Deoxy sugars such as fucose seldom dominate in the natural environment of E. coli (14). The fuc genes encoding deoxy sugar metabolism remain silent unless induced by fucose or other related sugars in the absence of competing substrates (1, 2). Although a natural product, furfural is unlikely to be an important natural substrate for this enzyme.In this study, we have used site-specific mutagenesis and growth-based selection to identify a fucO mutation that confers a further increase in furfural tolerance. MATERIALS AND METHODSStrains, media, and genetic manipulations. The strains, pl...