Identification of furfural as a key toxin in lignocellulosic hydrolysates and evolution of a tolerant yeast strain

136

A rise in resistance to current antifungals necessitates strategies to identify alternative sources of effective fungicides. We report the discovery of poacic acid, a potent antifungal compound found in lignocellulosic hydrolysates of grasses. Chemical genomics using Saccharomyces cerevisiae showed that loss of cell wall synthesis and maintenance genes conferred increased sensitivity to poacic acid. Morphological analysis revealed that cells treated with poacic acid behaved similarly to cells treated with other cell wall-targeting drugs and mutants with deletions in genes involved in processes related to cell wall biogenesis. Poacic acid causes rapid cell lysis and is synergistic with caspofungin and fluconazole. The cellular target was identified; poacic acid localized to the cell wall and inhibited β-1,3-glucan synthesis in vivo and in vitro, apparently by directly binding β-1,3-glucan. Through its activity on the glucan layer, poacic acid inhibits growth of the fungi Sclerotinia sclerotiorum and Alternaria solani as well as the oomycete Phytophthora sojae. A single application of poacic acid to leaves infected with the broad-range fungal pathogen S. sclerotiorum substantially reduced lesion development. The discovery of poacic acid as a natural antifungal agent targeting β-1,3-glucan highlights the potential side use of products generated in the processing of renewable biomass toward biofuels as a source of valuable bioactive compounds and further clarifies the nature and mechanism of fermentation inhibitors found in lignocellulosic hydrolysates.

mentioning

confidence: 99%

Plant-derived antifungal agent poacic acid targets β-1,3-glucan

Piotrowski

Okada

et al. 2015

136

“…Furfural (2-furaldehyde, CAS number 98-01-1) and 5-(hydroxymethyl)furfural (HMF, CAS number 67-47-0) are key toxic furan derivatives in acid-pretreated lignocellulose hydrolysates (1,2). These furanic aldehydes cause detrimental effects that are mostly ill-understood at the molecular level, but result in a decrease of specific growth rates, ethanol yields, and productivities in both yeasts and bacteria (for review, see refs.…”

mentioning

confidence: 99%

Identification and characterization of the furfural and 5-(hydroxymethyl)furfural degradation pathways of Cupriavidus basilensis HMF14

Koopman

Wierckx

Winde

et al. 2010

222

240

The toxic fermentation inhibitors in lignocellulosic hydrolysates pose significant problems for the production of second-generation biofuels and biochemicals. Among these inhibitors, 5-(hydroxymethyl)furfural (HMF) and furfural are specifically notorious. In this study, we describe the complete molecular identification and characterization of the pathway by which Cupriavidus basilensis HMF14 metabolizes HMF and furfural. The identification of this pathway enabled the construction of an HMF and furfural-metabolizing Pseudomonas putida. The genetic information obtained furthermore enabled us to predict the HMF and furfural degrading capabilities of sequenced bacterial species that had not previously been connected to furanic aldehyde metabolism. These results pave the way for in situ detoxification of lignocellulosic hydrolysates, which is a major step toward improved efficiency of utilization of lignocellulosic feedstock.

“…Aliquots of 1 mL were harvested at an OD 600 of 1 by centrifugation (14,000 × g, 4°C, 2 min), and sample processing and analysis of cell pellets was performed as described above, yielding the flux ratio of glycolysis to pentose phosphate pathway. Onemilliliter supernatant aliquots were harvested by centrifugation (14,000 × g, 2 min) during exponential growth and analyzed with a HPX-87H Aminex, ion-exclusion column (Bio-Rad) on a HPLC HP1100 system (Agilent Technologies) as described previously (48,49), and used to calculate carbon uptake rates converting OD 600 into cellular volume as described above. Absolute glycolytic fluxes were calculated by multiplying the carbon uptake rate with the flux ratio of glycolysis to pentose phosphate pathway.…”

mentioning

confidence: 99%

Functioning of a metabolic flux sensor in Escherichia coli

Kochanowski

Volkmer

Gerosa

et al. 2012

187

183

Regulation of metabolic operation in response to extracellular cues is crucial for cells' survival. Next to the canonical nutrient sensors, which measure the concentration of nutrients, recently intracellular "metabolic flux" was proposed as a novel impetus for metabolic regulation. According to this concept, cells would have molecular systems ("flux sensors") in place that regulate metabolism as a function of the actually occurring metabolic fluxes. Although this resembles an appealing concept, we have not had any experimental evidence for the existence of flux sensors and also we have not known how these flux sensors would work in detail. Here, we show experimental evidence that supports the hypothesis that Escherichia coli is indeed able to measure its glycolytic flux and uses this signal for metabolic regulation. Combining experiment and theory, we show how this flux-sensing function could emerge from an aggregate of several molecular mechanisms: First, the system of reactions of lower glycolysis and the feedforward activation of fructose-1,6-bisphosphate on pyruvate kinase translate flux information into the concentration of the metabolite fructose-1,6-bisphosphate. The interaction of this "flux-signaling metabolite" with the transcription factor Cra then leads to flux-dependent regulation. By responding to glycolytic flux, rather than to the concentration of individual carbon sources, the cell may minimize sensing and regulatory expenses. R egulation of metabolic operation is crucial for cells' survival. The canonical view is that this regulation occurs in response to extracellular cues, where, for instance, nutrient-specific transmembrane or intracellular receptors sense the presence of a nutrient and transfer respective commands to the regulatory machinery (1-5).Recently, however, a novel impetus for metabolic regulation was proposed: cells could regulate their metabolism as a function of the actually occurring intracellular metabolic fluxes (6, 7). According to this concept, changes in extracellular nutrient abundances would first-in a rather passive manner-result in changes in intracellular metabolic fluxes. In a second instance, the metabolic fluxes would be sensed by molecular systems ("flux sensors"), which in turn would transmit the sensed "flux signal" to the regulatory machinery that consequently would adjust metabolic operation (6). On the basis of a detailed mathematical model of Escherichia coli's central metabolism and its regulation, Kotte et al. suggested that this organism would have such flux sensors in place that establish a correlation between a metabolic flux and the concentration of certain so-called flux-signaling metabolites, which in turn affect the activity of transcription factors and thus would allow for transcriptional regulation in a flux-dependent manner.Using intracellular flux to regulate metabolism is an appealing concept as it would omit the need for nutrient-specific sensors for many different nutrients, and would allow the integration of multiple nutrient inputs directl...