Bioprospecting of Native Efflux Pumps To Enhance Furfural Tolerance in Ethanologenic Escherichia coli.
ABSTRACT: Efficient microbial conversion of lignocellulose into valuable products is often hindered by the presence of furfural, a dehydration product of pentoses in hemicellulose sugar syrups derived from woody biomass. For a cost-effective lignocellulose microbial conversion, robust biocatalysts are needed that can tolerate toxic inhibitors while maintaining optimal metabolic activities. A comprehensive plasmid-based library encoding native multidrug resistance (MDR) efflux pumps, porins, and select exporters from Escherichia coli was screened for furfural tolerance in an ethanologenic E. coli strain. Small multidrug resistance (SMR) pumps, such as SugE and MdtJI, as well as a lactate/glycolate:H+ symporter, LldP, conferred furfural tolerance in liquid culture tests. Expression of the SMR pump potentially increased furfural efflux and cellular viability upon furfural assault, suggesting novel activities for SMR pumps as furfural efflux proteins. Furthermore, induced expression of mdtJI enhanced ethanol fermentative production of LY180 in the presence of furfural or 5-hydroxymethylfurfural, further demonstrating the applications of SMR pumps. This work describes an effective approach to identify useful efflux systems with desired activities for nonnative toxic chemicals and provides a platform to further enhance furfural efflux by protein engineering and mutagenesis.IMPORTANCE Lignocellulosic biomass, especially agricultural residues, represents an important potential feedstock for microbial production of renewable fuels and chemicals. During the deconstruction of hemicellulose by thermochemical processes, side products that inhibit cell growth and production, such as furan aldehydes, are generated, limiting cost-effective lignocellulose conversion. Here, we developed a new approach to increase cellular tolerance by expressing multidrug resistance (MDR) pumps with putative efflux activities for furan aldehydes. The developed plasmid library and screening methods may facilitate new discoveries of MDR pumps for diverse toxic chemicals important for microbial conversion.
Project description:The sugar dehydration products, furfural and 5-(hydroxymethyl)furfural (HMF), are commonly formed during high-temperature processing of lignocellulose, most often in thermochemical pretreatment, liquefaction, or pyrolysis. Typically, these two aldehydes are considered major inhibitors in microbial conversion processes. Many microbes can convert these compounds to their less toxic, dead-end alcohol counterparts, furfuryl alcohol and 5-(hydroxymethyl)furfuryl alcohol. Recently, the genes responsible for aerobic catabolism of furfural and HMF were discovered in Cupriavidus basilensis HMF14 to enable complete conversion of these compounds to the TCA cycle intermediate, 2-oxo-glutarate. In this work, we engineer the robust soil microbe, Pseudomonas putida KT2440, to utilize furfural and HMF as sole carbon and energy sources via complete genomic integration of the 12 kB hmf gene cluster previously reported from Burkholderia phytofirmans. The common intermediate, 2-furoic acid, is shown to be a bottleneck for both furfural and HMF metabolism. When cultured on biomass hydrolysate containing representative amounts of furfural and HMF from dilute-acid pretreatment, the engineered strain outperforms the wild type microbe in terms of reduced lag time and enhanced growth rates due to catabolism of furfural and HMF. Overall, this study demonstrates that an approach for biological conversion of furfural and HMF, relative to the typical production of dead-end alcohols, enables both enhanced carbon conversion and substantially improves tolerance to hydrolysate inhibitors. This approach should find general utility both in emerging aerobic processes for the production of fuels and chemicals from biomass-derived sugars and in the biological conversion of high-temperature biomass streams from liquefaction or pyrolysis where furfural and HMF are much more abundant than in biomass hydrolysates from pretreatment.
Project description:Lignocellulosic biomass as a potential alternative to fossil resource for the production of valuable chemicals and fuels has attracted substantial attention, while reducing the recalcitrance of lignocellulosic biomass is still challenging due to the complex and cross-linking structure of biomass. Solvent system plays important roles in the pretreatment of lignocellulose, enabling the transformation of solid biomass to liquid fluid with better mass and heat transfer, as well as in the selective formation of target products. In particular, H2O/tetrahydrofuran (H2O/THF) system has recently been widely applied in lignocellulose valorization, which has been proved to exhibit outstanding efficiency for the conversion of lignocellulose, solubilization of the intermediates and products, and shifting reaction equilibrium, thereby significantly improving the yield and selectivity of target products, as well as the full utilization of lignocellulose. In addition, THF shows low toxicity, and is known as a renewable solvent which can be produced from bio-derived chemicals. Herein, this review concentrates on the advances of H2O/THF system in lignocellulose valorization in recent years. Several aspects relative to the roles of H2O/THF system are discussed as follows: the pretreatment of lignin, conversion of hemicellulose and cellulose components in lignocelluloses, and the promoting formation of valuable chemicals like furfural, 5-hydroxymethyl furfural (HMF), levulinic acid, and so on, as well as the inhibiting role in humins formation. This review might provide useful information for the design of effective solvent system in full utilization of lignocellulosic biomass.
Project description:BACKGROUND:Furfural and 5-hydroxymethylfurfural (HMF) are the two major furan aldehyde inhibitors generated from lignocellulose dilute acid pretreatment which significantly inhibit subsequent microbial cell growth and ethanol fermentation. Zymomonas mobilis is an important strain for cellulosic ethanol fermentation but can be severely inhibited by furfural and (or) HMF. Previous study showed that Z. mobilis contains its native oxidoreductases to catalyze the conversion of furfural and HMF, but the corresponding genes have not been identified. RESULTS:This study identified a NADPH-dependent alcohol dehydrogenase gene ZMO1771 from Z. mobilis ZM4, which is responsible for the efficient reduction of furfural and HMF. Over-expression of ZMO1771 in Z. mobilis significantly increased the conversion rate to both furfural and HMF and resulted in an accelerated cell growth and improved ethanol productivity in corn stover hydrolysate. Further, the ethanol fermentation performance was enhanced again by co-expression of the transhydrogenase gene udhA with ZMO1771 by elevating the NADPH availability. CONCLUSIONS:A genetically modified Z. mobilis by co-expressing alcohol dehydrogenase gene ZMO1771 with transhydrogenase gene udhA showed enhanced conversion rate of furfural and HMF and accelerated ethanol fermentability from lignocellulosic hydrolysate. The results presented in this study provide an important method on constructing robust strains for efficient ethanol fermentation from lignocellulose feedstock. GRAPHICAL ABSTRACT:
Project description:The conversion of lignocellulose into its building blocks and their further transformation into valuable platform chemicals (e.?g., furfural) are key technologies to move towards the use of renewable resources. This paper explored the disentanglement of lignocellulose into hemicellulose-derived sugars, cellulose, and lignin in a biphasic solvent system (water/2-methyltetrahydrofuran) using phosphoric acid as recyclable catalyst. Integrated with the biomass fractionation, in a second step hemicellulose-derived sugars (mainly xylose) were converted to furfural, which was in?situ extracted into 2-methyltetrahydrofuran with high selectivity (70?%) and yield (56?wt?%). To further increase the economic feasibility of the process, a downstream and recycling strategy enabled recovery of phosphoric acid without loss of process efficiency over four consecutive cycles. This outlines a more efficient and sustainable use of phosphoric acid as catalyst, as its inherent costs can be significantly lowered.
Project description:BACKGROUND:Furfural is the prevalent microbial inhibitor generated during pretreatment and hydrolysis of lignocellulose biomass to monomeric sugars, but the response of acetone butanol ethanol (ABE) producing Clostridium beijerinckii NCIMB 8052 to this compound at the molecular level is unknown. To discern the effect of furfural on C. beijerinckii and to gain insight into molecular mechanisms of action and detoxification, physiological changes of furfural-stressed cultures during acetone butanol ethanol (ABE) fermentation were studied, and differentially expressed genes were profiled by genome-wide transcriptional analysis. RESULTS:A total of 5,003 C. beijerinckii NCIMB 8052 genes capturing about 99.7% of the genome were examined. About 111 genes were differentially expressed (up- or down-regulated) by C. beijerinckii when it was challenged with furfural at acidogenic growth phase compared with 721 genes that were differentially expressed (up- or down-regulated) when C. beijerinckii was challenged with furfural at solventogenic growth phase. The differentially expressed genes include genes related to redox and cofactors, membrane transporters, carbohydrate, amino sugar and nucleotide sugar metabolisms, heat shock proteins, DNA repair, and two-component signal transduction system. While C. beijerinckii exposed to furfural stress during the acidogenic growth phase produced 13% more ABE than the unstressed control, ABE production by C. beijerinckii ceased following exposure to furfural stress during the solventogenic growth phase. CONCLUSION:Genome-wide transcriptional response of C. beijerinckii to furfural stress was investigated for the first time using microarray analysis. Stresses emanating from ABE accumulation in the fermentation medium; redox balance perturbations; and repression of genes that code for the phosphotransferase system, cell motility and flagellar proteins (and combinations thereof) may have caused the premature termination of C. beijerinckii 8052 growth and ABE production following furfural challenge at the solventogenic phase.This study provides insights into basis for metabolic engineering of C. beijerinckii NCIMB 8052 for enhanced tolerance of lignocellulose-derived microbial inhibitory compounds, thereby improving bioconversion of lignocellulose biomass hydrolysates to biofuels and chemicals. Indeed, two enzymes encoded by Cbei_3974 and Cbei_3904 belonging to aldo/keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) families have been identified to be involved in furfural detoxification and tolerance.
Project description:In situ detoxification of lignocellulose-derived microbial inhibitory compounds is an economical strategy for the fermentation of lignocellulose-derived sugars to fuels and chemicals. In this study, we investigated homologous integration and constitutive expression of Cbei_3974 and Cbei_3904, which encode aldo-keto reductase and previously annotated short chain dehydrogenase/reductase, respectively, in Clostridium beijerinckii NCIMB 8052 (Cb), resulting in two strains: Cb_3974 and Cb_3904. Expression of Cbei_3974 led to 2-fold increase in furfural detoxification relative to Cb_3904 and Cb_wild type. Correspondingly, butanol production was up to 1.2-fold greater in furfural-challenged cultures of Cb_3974 relative to Cb_3904 and Cb_wild type. With 4-hydroxybezaldehyde and syringaldehyde supplementation, Cb_3974 showed up to 2.4-fold increase in butanol concentration when compared to Cb_3904 and Cb_wild type. Syringic and vanillic acids were considerably less deleterious to all three strains of Cb tested. Overall, Cb_3974 showed greater tolerance to furfural, 4-hydroxybezaldehyde, and syringaldehyde with improved capacity for butanol production. Hence, development of Cb_3974 represents a significant progress towards engineering solventogenic Clostridium species that are tolerant to lignocellulosic biomass hydrolysates as substrates for ABE fermentation.
Project description:Active efflux of drugs mediated by efflux pumps that confer drug resistance is one of the mechanisms developed by bacteria to counter the adverse effects of antibiotics and chemicals. To understand these efflux mechanisms in Mycobacterium tuberculosis, we generated knockout (KO) mutants of four efflux pumps of the pathogen belonging to different classes. We measured the MICs and kill values of two different compound classes on the wild type (WT) and the efflux pump (EP) KO mutants in the presence and absence of the efflux inhibitors verapamil and l-phenylalanyl-l-arginyl-?-naphthylamide (PA?N). Among the pumps studied, the efflux pumps belonging to the ABC (ATP-binding cassette) class, encoded by Rv1218c, and the SMR (small multidrug resistance) class, encoded by Rv3065, appear to play important roles in mediating the efflux of different chemical classes and antibiotics. Efflux pumps encoded by Rv0849 and Rv1258c also mediate the efflux of these compounds, but to a lesser extent. Increased killing is observed in WT M. tuberculosis cells by these compounds in the presence of either verapamil or PA?N. The efflux pump KO mutants were more susceptible to these compounds in the presence of efflux inhibitors. We have shown that these four efflux pumps of M. tuberculosis play a vital role in mediating efflux of different chemical scaffolds. Inhibitors of one or several of these efflux pumps could have a significant impact in the treatment of tuberculosis. The identification and characterization of Rv0849, a new efflux pump belonging to the MFS (major facilitator superfamily) class, are reported.
Project description:Intestinal epithelial cell (IEC) shedding is a fundamental response to intestinal damage, yet underlying mechanisms and functions have been difficult to define. Here we model chronic intestinal damage in zebrafish larvae using the nonsteroidal antiinflammatory drug (NSAID) Glafenine. Glafenine induced the unfolded protein response (UPR) and inflammatory pathways in IECs, leading to delamination. Glafenine-induced inflammation was augmented by microbial colonization and associated with changes in intestinal and environmental microbiotas. IEC shedding was a UPR-dependent protective response to Glafenine that restricts inflammation and promotes animal survival. Other NSAIDs did not induce IEC delamination; however, Glafenine also displays off-target inhibition of multidrug resistance (MDR) efflux pumps. We found a subset of MDR inhibitors also induced IEC delamination, implicating MDR efflux pumps as cellular targets underlying Glafenine-induced enteropathy. These results implicate IEC delamination as a protective UPR-mediated response to chemical injury, and uncover an essential role for MDR efflux pumps in intestinal homeostasis.
Project description:Over the last decade, MDR (multidrug resistance) has increased worldwide in microbial pathogens by efflux mechanisms, leading to treatment failures in human infections. Several Gram-negative bacteria efflux pumps have been described. These proteinaceous channels are capable of expelling structurally different drugs across the envelope and conferring antibiotic resistance in various bacterial pathogens. Combating antibiotic resistance is an urgency and the blocking of efflux pumps is an attractive response to the emergence of MDR phenotypes in infectious bacteria. In the present study, various alkylaminoquinolines were tested as potential inhibitors of drug transporters. We showed that alkylaminoquinolines are capable of restoring susceptibilities to structurally unrelated antibiotics in clinical isolates of MDR Gram-negative bacteria. Antibiotic efflux studies indicated that 7-nitro-8-methyl-4-[2'-(piperidino)ethyl]aminoquinoline acts as an inhibitor of the AcrAB-TolC efflux pump and restores a high level of intracellular drug concentration. Inhibitory activity of this alkylaminoquinoline is observed on clinical isolates showing different resistance phenotypes.
Project description:TolC is the outer-membrane component of several multidrug resistance (MDR) efflux pumps and plays an important role in the survival and virulence of many gram-negative bacterial animal pathogens. We have identified and characterized the outer-membrane protein-encoding gene tolC in the bacterial plant pathogen Erwinia chrysanthemi EC16. The gene was found to encode a 51-kDa protein with 70% identity to its Escherichia coli homologue. The E. chrysanthemi gene was able to functionally complement the E. coli tolC gene with respect to its role in MDR efflux pumps. A tolC mutant of E. chrysanthemi was found to be extremely sensitive to antimicrobial agents, including several plant-derived chemicals. This mutant was unable to grow in planta and its ability to cause plant tissue maceration was severely compromised. The tolC mutant was shown to be defective in the efflux of berberine, a model antimicrobial plant chemical. These results suggest that by conferring resistance to the antimicrobial compounds produced by plants, the E. chrysanthemi tolC plays an important role in the survival and colonization of the pathogen in plant tissue.