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.

During metabolic evolution to improve succinate production in Escherichia coli strains, significant changes in cellular metabolism were acquired that increased energy efficiency in two respects. The energyconserving phosphoenolpyruvate (PEP) carboxykinase (pck), which normally functions in the reverse direction (gluconeogenesis; glucose repressed) during the oxidative metabolism of organic acids, evolved to become the major carboxylation pathway for succinate production. Both PCK enzyme activity and gene expression levels increased significantly in two stages because of several mutations during the metabolic evolution process. High-level expression of this enzymedominated CO 2 fixation and increased ATP yield (1 ATP per oxaloacetate). In addition, the native PEP-dependent phosphotransferase system for glucose uptake was inactivated by a mutation in ptsI. This glucose transport function was replaced by increased expression of the GalP permease (galP) and glucokinase (glk). Results of deleting individual transport genes confirmed that GalP served as the dominant glucose transporter in evolved strains. Using this alternative transport system would increase the pool of PEP available for redox balance. This change would also increase energy efficiency by eliminating the need to produce additional PEP from pyruvate, a reaction that requires two ATP equivalents. Together, these changes converted the wild-type E. coli fermentation pathway for succinate into a functional equivalent of the native pathway that nature evolved in succinate-producing rumen bacteria.biocatalyst ͉ metabolic engineering ͉ succinic acid S uccinate, a four-carbon dicarboxylic acid, is currently used as a specialty chemical in the food, agricultural, and pharmaceutical industries (1). Succinic acid can also serve as a starting point for the synthesis of many commodity chemicals used in plastics and solvents with a potential global market of $15 billion (2). Although succinate is primarily produced from petroleum, recent increases in costs have generated considerable interest in the fermentative production of succinate from sugars using Escherichia coli and other biocatalysts (2, 3).The yield of succinate from glucose fermentation is primarily determined by carbon partitioning at the phosphoenolpyruvate (PEP) node (Fig. 1A). In rumen bacteria such as Anaerobiospirillum succiniciproducens (4), Actinobacillus succinogenes (5, 6) and Mannheimia succiniciproducens (7,8), more than half of the phosphoenolpyruvate formed from glucose is carboxylated to oxaloacetate and converted to succinate, the primary fermentation product. However, requirements for complex nutrients by these bacteria increase both the cost and process complexity. Native strains of E. coli ferment glucose effectively in simple mineral salts medium but produce succinate only as a minor product (9). In E. coli, half of the PEP from glucose is metabolized directly to pyruvate by the PEP-dependent phosphotransferase system for glucose uptake. Most of the remaining PEP is used for ATP produc...

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...

Carboxylic acids are an attractive biorenewable chemical. However, like many other fermentatively produced compounds, they are inhibitory to the biocatalyst. An understanding of the mechanism of toxicity can aid in mitigating this problem. Here, we show that hexanoic and octanoic acids are completely inhibitory to Escherichia coli MG1655 in minimal medium at a concentration of 40 mM, while decanoic acid was inhibitory at 20 mM. This growth inhibition is pHdependent and is accompanied by a significant change in the fluorescence polarization (fluidity) and integrity. This inhibition and sensitivity to membrane fluidization, but not to damage of membrane integrity, can be at least partially mitigated during short-term adaptation to octanoic acid. This short-term adaptation was accompanied by a change in membrane lipid composition and a decrease in cell surface hydrophobicity. Specifically, the saturated/unsaturated lipid ratio decreased and the average lipid length increased. A fatty acid-producing strain exhibited an increase in membrane leakage as the product titer increased, but no change in membrane fluidity. These results highlight the importance of the cell membrane as a target for future metabolic engineering efforts for enabling resistance and tolerance of desirable biorenewable compounds, such as carboxylic acids. Knowledge of these effects can help in the engineering of robust biocatalysts for biorenewable chemicals production.

Nitric oxide (NO) is used by mammalian immune systems to counter microbial invasions and is produced by bacteria during denitrification. As a defense, microorganisms possess a complex network to cope with NO. Here we report a combined transcriptomic, chemical, and phenotypic approach to identify direct NO targets and construct the biochemical response network. In particular, network component analysis was used to identify transcription factors that are perturbed by NO. Such information was screened with potential NO reaction mechanisms and phenotypic data from genetic knockouts to identify active chemistry and direct NO targets in Escherichia coli. This approach identified the comprehensive E. coli NO response network and evinced that NO halts bacterial growth via inhibition of the branched-chain amino acid biosynthesis enzyme dihydroxyacid dehydratase. Because mammals do not synthesize branched-chain amino acids, inhibition of dihydroxyacid dehydratase may have served to foster the role of NO in the immune arsenal.systems biology ͉ chemoinformatics M ammals possess complex immune systems that have evolved to prevent microbial invasion. Nitric oxide (NO) is one of the key chemicals used by mammalian cells to combat infections (1). Although NO is known to act in a bacteriostatic fashion (2), the mechanism underlying this action is not completely understood. NO interferes with biological processes either directly, by reacting with metal centers and free radicals, or indirectly, by promoting the formation of reactive nitrogen oxide species (RNOS), such as peroxynitrite and N 2 O 3 (3). NO has been reported to react with various protein Fe-S clusters (4-7), and this reactivity has been implicated in the inhibition of tumor proliferation (8)(9)(10). NO also binds to the metal centers of respiratory enzymes, inhibiting bacterial respiration (11-13). Because NO is used to combat Escherichia coli in low oxygen environments, it is likely that respiration is not the only system targeted by NO.E. coli contains NO-consuming proteins NO reductase NorV (14), NO oxidase HmpA (15, 16), and cytochrome c nitrite reductase NrfA (17). Expression of norV and hmpA is increased in response to NO by NO-specific transcription factors (TFs) NorR (18) and NsrR, respectively (19,20). Our goal here was to identify the comprehensive NO response network, consisting of the direct NO targets leading to bacteriostasis, the response network resulting from bacteriostasis, bacterial NO sensors for self-defense, and the response network for self-defense.Previous genome-scale studies have used DNA microarrays to identify the global genetic response to [21][22][23][24]. Although transcriptome analysis provides substantial information regarding changes in gene expression, examination of individual genes cannot distinguish primary from secondary effects and does not directly identify the TFs and pathways leading to the transcriptional changes. Because many promoters are controlled by multiple TFs, the identity of the TF responsible for mediating transcr...