Efficient production of hyaluronic acid by Streptococcus zooepidemicus using two-stage semi-continuous fermentation.

Hyaluronic acid is a kind of mucopolysaccharide that has wide applications in cosmetics, health food, and orthopedics. Using Streptococcus zooepidemicus ATCC 39920 as parent, a beneficial mutant SZ07 was obtained by UV mutagenesis, giving 1.42 g/L hyaluronic acid in shake flasks. To enhance the efficiency of hyaluronic acid production, a semi-continuous fermentation process consisted of two-stage 3-L bioreactors was developed, in which 1.01 g/L/h productivity and 14.60 g/L hyaluronic acid were obtained. To further enhance the titer of hyaluronic acid, recombinant hyaluronidase SzHYal was added into 2nd stage bioreactor at 6 h to reduce the viscosity of broth. The highest hyaluronic acid titer of 29.38 g/L was achieved with a productivity of 1.13 g/L/h at 300 U/L SzHYal after 24 h. This newly developed semi-continuous fermentation process provides a promising strategy for the industrial production of hyaluronic acid and related polysaccharides.

Self-Sufficient In Vitro Multi-Enzyme Cascade for Efficient Synthesis of Danshensu from l-DOPA.

Danshensu (DSS), a traditional Chinese medicine, is widely used for the treatment of cardiovascular and cancer diseases. Here, a one-pot multi-enzyme cascade pathway was designed for DSS synthesis from l-DOPA using tyrosine aminotransferase from Escherichia coli (EcTyrB) and d-isomer-specific 2-hydroxyacid dehydrogenase from Lactobacillus frumenti (LfD2-HDH). Glutamate dehydrogenase from Clostridium difficile (CdgluD) was also introduced for a self-sufficient system of α-ketoglutaric acid and NADH. Under optimal conditions (35 °C, pH 7.0, EcTyrB:LfD2-HDH:CdgluD = 3:2:1, glutamate:NAD+ = 1:1), 98.3% yield (at 20 mM l-DOPA) and space-time yield of 6.61 g L-1 h-1 (at 40 mM l-DOPA) were achieved. Decreased yields of DSS at elevated l-DOPA concentrations (100 mM) could be attributed to an inhibited CdgluD activity caused by NH4+ accumulation. This developed multi-enzyme cascade pathway (including EcTyrB, LfD2-HDH, and CdgluD) provides an efficient and sustainable approach for the production of DSS from l-DOPA.

Sustainable one-pot chemo-enzymatic synthesis of chiral furan amino acid from biomass via magnetic solid acid and threonine aldolase.

Sustainable synthesis of valuable noncanonical amino acids from renewable feedstocks is of great importance. Here, a feasible chemo-enzymatic procedure was developed for the synthesis of chiral ß-(2-furyl)serine from biomass catalyzed by a solid acid catalyst and immobilized E. coli whole-cell harboring l-threonine aldolase. A novel magnetic solid acid catalyst Fe3O4@MCM-41/SO42- was successfully synthesized for conversion of corncob into furfural in an aqueous system. Under the optimum conditions, furfural yield of 63.6% was achieved in 40 min at 180 ℃ with 2.0% catalyst (w/w). Furthermore, biomass-derived furfural was converted into an aldol-addition product ß-(2-furyl)serine with 73.6% yield, 99% ee and 20% de by immobilized cells in 6 h. The magnetic solid acid and biocatalyst can be readily recovered and efficiently reused for five consecutive cycles without significant loss on product yields. This chemo-enzymatic route can be attractive for producing noncanonical amino acids from biomass.

High-Throughput Screening Method for Directed Evolution and Characterization of Aldol Activity of D-Threonine Aldolase.

A rapid and reliable method for the determination of aldol condensation activity of threonine aldolases (TAs) toward aldehydes and glycine was developed. This 2,4-dinitrophenylhydrazine (DNPH) method has high sensitivity and low background disturbance and can be spectrophotometrically measured for high-throughput screening and characterization of TAs. For 4-methylsulfonyl benzaldehyde (MSB), the maximum absorbance peak was observed at around 485 nm. Site-directed saturation mutagenesis libraries of D-threonine aldolase from Alcaligenes xylosoxidans CGMCC 1.4257 (AxDTA) was constructed and screened with this DNPH method for increased aldol activity toward MSB. Two beneficial variants AxDTAD321C and AxDTAN101G were identified. Substrate specificity of AxDTA and variants toward nineteen aldehydes with different substituents was facilely characterized employing this DNPH method. Furthermore, AxDTA variants displayed enhanced catalytic performance and selectivity in aldol reaction. Consequently, our study provides a rapid screening and characterization method for TAs with potential applications in preparation of chiral ß-hydroxy-α-amino acids.

Facilely reducing recalcitrance of lignocellulosic biomass by a newly developed ethylamine-based deep eutectic solvent for biobutanol fermentation.

BACKGROUND: Biobutanol is promising and renewable alternative to traditional fossil fuels and could be produced by Clostridium species from lignocellulosic biomass. However, biomass is recalcitrant to be hydrolyzed into fermentable sugars attributed to the densely packed structure by layers of lignin. Development of pretreatment reagents and processes for increasing surface area, removing hemicellulose and lignin, and enhancing the relative content of cellulose is currently an area of great interest. Deep eutectic solvents (DESs), a new class of green solvents, are effective in the pretreatment of lignocellulosic biomass. However, it remains challenging to achieve high titers of total sugars and usually requires combinatorial pretreatment with other reagents. In this study, we aim to develop novel DESs with high application potential in biomass pretreatment and high biocompatibility for biobutanol fermentation. RESULTS: Several DESs with betaine chloride and ethylamine chloride (EaCl) as hydrogen bond acceptors were synthesized. Among them, EaCl:LAC with lactic acid as hydrogen bond donor displayed the best performance in the pretreatment of corncob. Only by single pretreatment with EaCl:LAC, total sugars as high as 53.5 g L-1 could be reached. Consecutive batches for pretreatment of corncob were performed using gradiently decreased cellulase by 5 FPU g-1. At the end of the sixth batch, the concentration and specific yield of total sugars were 58.8 g L-1 and 706 g kg-1 pretreated corncob, saving a total of 50% cellulase. Utilizing hydrolysate as carbon source, butanol titer of 10.4 g L-1 was achieved with butanol yield of 137 g kg-1 pretreated corncob by Clostridium saccharobutylicum DSM13864. CONCLUSIONS: Ethylamine and lactic acid-based deep eutectic solvent is promising in pretreatment of corncob with high total sugar concentrations and compatible for biobutanol fermentation. This study provides an efficient pretreatment reagent for facilely reducing recalcitrance of lignocellulosic materials and a promising process for biobutanol fermentation from renewable biomass.

Engineering of Cyclodextrin Glycosyltransferase Reveals pH-Regulated Mechanism of Enhanced Long-Chain Glycosylated Sophoricoside Specificity.

Sophoricoside glycosylated derivatives, especially long-chain glycosylated sophoricosides (LCGS), have greatly improved water solubility compared with sophoricoside. Here, cyclodextrin glycosyltransferase from Paenibacillus macerans (PmCGTase) was employed for sophoricoside glycosylation. Saturation mutagenesis of alanine 156, alanine 166, glycine 173, and leucine 174 was performed due to their nonconservative properties among α-, ß-, and γ-CGTases with different product specificities. Variants L174P, A156V/L174P, and A156V/L174P/A166Y greatly improved the product specificity for LCGS. pH significantly affected the extent of glycosylation catalyzed by the variants. Further investigations revealed that the pH-regulated mechanism for LCGS synthesis mainly depends on a disproportionation route at a lower pH (pH 4) and a cyclization-coupling route at a higher pH (pH 8) and equivalent effects of cyclization-coupling and disproportionation routes at pH 5. Whereas short-chain glycosylated sophoricosides (SCGS) are primarily produced via disproportionation of maltodextrin at pH 4 and secondary disproportionation of LCGS at pH 8. At pH 5, SCGS synthesis mainly depends on a hydrolysis route by the wild type (WT) and a secondary disproportionation route by variant A156V/L174P/A166Y. Kinetics analysis showed a decreased Km value of variant A156V/L174P/A166Y. Dynamics simulation results demonstrated that the improved LCGS specificity of the variant is possibly attributed to the enhanced affinity to long-chain substrates, which may be caused by the changes of hydrogen bond interactions at the -5, -6, and -7 subsites. Our results reveal a pH-regulated mechanism for product specificity of CGTase and provide guidance for engineering CGTase toward products with different sugar chain lengths.IMPORTANCE The low water solubility of sophoricoside seriously limits its applications in the food and pharmaceutical industries. Long-chain glycosylated sophoricosides show greatly improved water solubility. Here, the product specificity of cyclodextrin glycosyltransferase (CGTase) for long-chain glycosylated sophoricosides was significantly affected by pH. Our results reveal the pH-regulated mechanism of the glycosylated product specificity of CGTase. This work adds to our understanding of the synthesis of long-chain glycosylated sophoricosides and provides guidance for exploring related product specificity of CGTase based on pH regulation.

Enhancing soluble expression of sucrose phosphorylase in Escherichia coli by molecular chaperones.

Sucrose phosphorylase (SPase, EC 2.4.1.7) has a wide range of application in food, cosmetics, and pharmaceutical industries because of its broad substrate specificity. However, low SPase yields produced by wild-type strains cannot meet industrial requirements due to their complex metabolic regulation mechanisms. In this study, spase gene from Thermoanaerobacterium thermosaccharolyticum was cloned and expressed in Escherichia coli BL21 (DE3), leading to 7.05 U/mL (3.71 U/mg) of T. thermosaccharolyticum SPase (TtSPase) under optimum conditions. Co-expression of molecular chaperone teams pGro7 (GroES-GroEL), pG-KJE8 (DnaK-DnaJ-GrpE and GroES-GroEL), and pG-TF2 (GroES-GroEL-Tig) significantly enhanced the TtSPase activities to 18.5 U/mg (59.2 U/mL), 9.52 U/mg (28.6 U/mL), and 25.7 U/mg (64.5 U/mL), respectively. Results suggested that GroES-GroEL chaperone combination could regulate protein folding processes and protect misfolded proteins from aggregation. The enzymatic characterization results showed that TtSPase had an optimal temperature of 60 °C and optimal pH of 6.5. In particular, it had high thermostability of T5030 = 67 °C and half-life (t1/2 at 70 °C) of 19 min. Furthermore, purified TtSPase was used for hydroquinone transglycosylation and 21% of molar production yield of α-arbutin was obtained. This study provides a TtSPase with high thermostability for potential industrial applications, and develops an effective strategy for improving soluble TtSPase production in E. coli.

CRISPR-Cpf1-Assisted Engineering of Corynebacterium glutamicum SNK118 for Enhanced L-Ornithine Production by NADP-Dependent Glyceraldehyde-3-Phosphate Dehydrogenase and NADH-Dependent Glutamate Dehydrogenase.

Here, Corynebacterium glutamicum SNK118 was metabolically engineered for L-ornithine production through CRISPR-Cpf1-based genome manipulation and plasmid-based heterologous overexpression. Genes argF, argR, and ncgl2228 were deleted to block the degradation of L-ornithine, eliminate the global transcriptional repression, and alleviate the competitive branch pathway, respectively. Overexpression of CsgapC (NADP-dependent glyceraldehyde 3-phosphate dehydrogenases gene from Clostridium saccharobutylicum DSM 13864) and BsrocG (NADH-dependent glutamate dehydrogenase gene from Bacillus subtilis HB-1) resulted markedly increased ornithine biosynthesis. Eventually, the engineered strain KBJ11 (SNK118ΔargRΔargFΔncgl2228/pXMJ19-CsgapC-BsrocG) was constructed for L-ornithine overproduction. In fed-batch fermentation, L-ornithine of 88.26 g/L with productivity of 1.23 g/L/h (over 72 h) and yield of 0.414 g/g glucose was achieved by strain KBJ11 in a 10-L bioreactor. Our result represents the highest titer and yield of L-ornithine production by microbial fermentation. This study suggests that heterologous expression of CsgapC and BsrocG could promote L-ornithine production by C. glutamicum strains.

Composite coal fly ash solid acid catalyst in synergy with chloride for biphasic preparation of furfural from corn stover hydrolysate.

A solid acid catalyst SO42-/SnO2-Al2O3-CFA was synthesized based on industrial waste coal fly ash (CFA) as carrier and applied in the conversion of oxalic acid pretreated corn stover hydrolysate to produce furfural. Physical properties of the solid acid catalyst were characterized by SEM, FTIR, XRD, BET, EDAX, and NH3-TPD. Highly wrinkled structure of SO42-/SnO2-Al2O3-CFA could provide more specific surface area for the covalent linkage between SiO2 and SnO2. Factors influencing the efficacy of SO42-/SnO2-Al2O3-CFA were systematically explored. The highest furfural yield of 84.7% was reached in NH4Cl-toluene biphasic system at 180 °C for 30 min. The recyclability of SO42-/SnO2-Al2O3-CFA and toluene could be achieved for five batches with stable performance in transformation of xylose-rich corn stover hydrolysate. This study provided a novel solid acid catalyst with promising potential in the synthesis of furfural from corn stover.

Metabolic engineering of Corynebacterium glutamicum for improved L-arginine synthesis by enhancing NADPH supply.

Corynebacterium glutamicum SNK 118 was metabolically engineered with improved L-arginine titer. Considering the crucial role of NADPH level in L-arginine production, pntAB (membrane-bound transhydrogenase) and ppnK (NAD+ kinase) were co-expressed to increase the intracellular NADPH pool. Expression of pntAB exhibited significant effects on NADPH supply and L-arginine synthesis. Furthermore, argR and farR, encoding arginine repressor ArgR and transcriptional regulator FarR, respectively, were removed from the genome of C. glutamicum. The competitive branch pathway gene ldh was also deleted. Eventually, an engineered C. glutamicum JML07 was obtained for L-arginine production. Fed-batch fermentation in 5-L bioreactor employing strain JML07 allowed production of 67.01 g L-1L-arginine with productivity of 0.89 g L-1 h-1 and yield of 0.35 g g-1 glucose. This study provides a productive L-arginine fermentation strain and an effective cofactor manipulating strategy for promoting the biosynthesis of NADPH-dependent metabolites.

Improving Soluble Expression of Tyrosine Decarboxylase from Lactobacillus brevis for Tyramine Synthesis with High Total Turnover Number.

The soluble expression of tyrosine decarboxylase (TDC) in heterologous host is often challenging. Here, acidic condition was found to be favorable for improving the soluble expression of TDC from Lactobacillus brevis in Escherichia coli, while addition of carbohydrates (such as glucose, arabinose, and fructose) was vital for decreasing the insoluble fraction. By simple pH control and addition of glucose, the specific activity of TDC in crude extract was enhanced to 46.3 U mg-1, 3.67-fold of that produced from LB medium. Optimization of the reaction conditions revealed that Tween-80 was effective in improving the tyramine production catalyzed by TDC, especially at high tyrosine loadings. As much as 400 mM tyrosine could be completely converted into tyramine with a substrate to catalyst ratio of 29.0 g g-1 and total turnover number of 23,300. This study provides efficient strategies for the highly soluble expression of TDC and biocatalytic production of tyramine.

Detoxification of furfural residues hydrolysate for butanol fermentation by Clostridium saccharobutylicum DSM 13864.

The toxicity of furfural residues (FRs) hydrolysate is a major obstacle in its application. This work focused on the detoxification of FRs hydrolysate and its application in butanol fermentation. Combination of activated carbon and resin 717 was appropriate for the detoxification of hydrolysate. Mixed sterilization of FRs hydrolysate and corn steep liquor (CSL) was better than the separate ones, since proteins in CSL could adsorb and remove toxic components during sterilization. The results further confirmed that simultaneous sterilization of activated carbon + resin and fermentation medium was more efficient for detoxification and butanol production, in which 76.4% of phenolic compounds and 99.3% of Maillard reaction products were removed, 8.48 g/L butanol and 12.61 g/L total solvent were obtained. This study provides feasible and economic approaches for the detoxification of FRs hydrolysate and its application in butanol production.

Identification of d-carbamoylase for biocatalytic cascade synthesis of d-tryptophan featuring high enantioselectivity.

In this study, an enantioselective d-carbamoylase (AcHyuC) was identified from Arthrobacter crystallopoietes with optimum pH of 8.5, much more compatible with hydantoinase process than other reported d-N-carbamoylases. AcHyuC has a substrate preference for aromatic carbamoyl-compounds. The dynamic kinetic resolution (DKR) cascade was developed by combining this AcHyuC with hydantoin racemase from Arthrobacter aurescens (AaHyuA) and d-hydantoinase from Agrobacterium tumefaciens (AtHyuH) for enantioselective resolution of l-indolylmethylhydantoin into d-Trp. The optimum pH of DKR cascade reaction was determined to be 8.0, and PEG 400 could facilitate the reaction. As much as 80mM l-indolylmethylhydantoin could be fully converted to d-Trp within 12h at 0.5L scale, with 99.4% yield, >99.9% e.e. and productivity of 36.6gL-1d-1. This study provides a new d-carbamoylase compatible with the DKR cascade for efficient production of optically pure d-Trp from l-indolylmethylhydantoin.

High production of genistein diglucoside derivative using cyclodextrin glycosyltransferase from Paenibacillus macerans.

Genistein has been regarded as one important soy isoflavone with multiple health benefits, whereas its applications are limited by the low hydrophilicity. To improve the water solubility, codon optimized cyclodextrin glycosyltransferase from Paenibacillus macerans was employed for genistein transglycosylation in this study. At least four transglycosylation products were produced and identified by HPLC and LC-MS: genistein monoglucoside, diglucoside, triglucoside, and tetraglucoside derivatives. Obviously, the yields of genistein monoglucoside and genistein diglucoside exhibited great superiority compared with other two products. To maximize the yield of genistein diglucoside, various reaction conditions such as genistein dissolvents, glycosyl donors, substrates concentrations and ratios, enzyme concentrations, reaction pH, temperature, and time were optimized. Finally, the yield of genistein diglucoside was enhanced by 1.5-fold under the optimum reaction system. Our study demonstrates that the production of genistein diglucoside could be specifically enhanced, which is one important genistein derivative with better water solubility and stability.

Engineering coenzyme A-dependent pathway from Clostridium saccharobutylicum in Escherichia coli for butanol production.

Clostridium saccharobutylicum has been proved to be efficient in butanol fermentation from various feedstocks. Whereas, lack of genetic manipulation system has severely hindered the engineering of C. saccharobutylicum for more extensive applications. In this study, recombinant Escherichia coli harboring heterologous coenzyme A-dependent pathway from C. saccharobutylicum DSM 13864 was constructed, which consisted of solventogenic pathway genes: acetoacetyl-CoA thiolase (thlA), aldehyde/alcohol dehydrogenase (adhE2) and bcs-operon (crt-bcd1-etfB2-fixB2-hbd). Then, a butanol titer of 67mg/L was attained. After replacing thlA with acetyl-CoA acetyltransferase (atoB) from E. coli and deleting the competitive branch genes lactate dehydrogenase (ldhA), aldehyde/alcohol dehydrogenase (adhE1) and fumarate reductase (frdBC), the butanol titer was successfully improved for 3.8-fold (254mg/L). Under the optimum fermentation conditions, the final butanol titer reached 584mg/L after 120h. This result demonstrates the feasibility of adapting CoA-dependent solventogenic pathway from C. saccharobutylicum in E. coli for butanol synthesis.

Enhanced curdlan production with nitrogen feeding during polysaccharide synthesis by Rhizobium radiobacter.

Curdlan is a secondary metabolite synthesized by Agrobacterium sp. and some other bacteria. A newly isolated exopolysaccharide-producing strain was identified to be Rhizobium radiobacter CGMCC 12099. The polysaccharide product was confirmed to be curdlan with a molecule weight of 1.4×10(5)Da, and its molecular structure was determined by HPLC and infrared spectrum. Although nitrogen source is necessary for cell reproduction, curdlan production is largely dependent on nitrogen limitation, as well as cell vitality. Here, a nitrogen feeding strategy was investigated to elevate the curdlan production by R. radiobacter. The optimal concentration and addition time of (NH4)2HPO4 were investigated. The results showed that the enhanced cell density was correlated to the amount of (NH4)2HPO4 added. Also, nitrogen addition in earlier fermentation stage was beneficial to the cell growth and curdlan production. Furthermore, continuously feeding strategy was employed by feeding (NH4)2HPO4 at a constant rate of 1.24g/h at 35(th)h of fermentation for 9h, achieving a final curdlan production of 65.27g/L, productivity of 0.544g/L/h and glucose conversion rate of 38.89%. The curdlan production was improved by 2.1 times compared with that without nitrogen addition. This study provides a feasible and cheap nitrogen feeding strategy to enhance curdlan production.

Arginine deiminase: recent advances in discovery, crystal structure, and protein engineering for improved properties as an anti-tumor drug.

Arginine deiminase (ADI) is an important arginine-degrading enzyme with wide applications, in particular as an anti-cancer agent for the therapy of arginine-auxotrophic tumors. In recent years, novel ADIs with excellent properties have been identified from various organisms, and crystal structures of ADI were investigated. To satisfy the requirements of potential therapeutic applications, protein engineering has been performed to improve the activity and properties of ADIs. In this mini-review, we systematically summarized the latest progress on identification and crystal structure of ADIs, and protein engineering strategies for improved enzymatic properties, such as pH optimum, K m and k cat values, and thermostability. We also outlined the PEGylation of ADI for improved circulating half-life and immunogenicity, as well as their performance in clinical trials. Finally, perspectives on extracellular secretion and property improvement of ADI were discussed.

Characterization and Soluble Expression of D-Hydantoinase from Pseudomonas fluorescens for the Synthesis of D-Amino Acids.

An active D-hydantoinase from Pseudomonas fluorescens was heterogeneously overexpressed in Escherichia coli BL21(DE3) and designated as D-PfHYD. Sequence and consensus analysis suggests that D-PfHYD belongs to the dihydropyrimidinase/hydantoinase family and possesses catalytic residues for metal ion and hydantoin binding. D-PfHYD was purified to homogeneity by nickel affinity chromatography for characterization. D-PfHYD is a homotetramer with molecular weight of 215 kDa and specific activity of 20.9 U mg(-1). D-PfHYD showed the highest activity at pH 9.0 and 60 °C. Metal ions such as Mn(2+), Fe(2+), and Fe(3+) could activate D-PfHYD with 20 % improvement. Substrate specificity analysis revealed that purified D-PfHYD preferred aliphatic to aromatic 5'-monosubstituted hydantoins. Among various strategies tested, chaperone GroES-GroEL was efficient in improving the soluble expression of D-PfHYD. Employing 1.0 g L(-1) recombinant E. coli BL21(DE3)-pET28-hyd/pGRO7 dry cells, 100 mM isobutyl hydantoin was converted into D-isoleucine with 98.7 % enantiomeric excess (ee), isolation yield of 78.3 %, and substrate to biocatalyst ratio of 15.6. Our results suggest that recombinant D-PfHYD could be potentially applied in the synthesis of D-amino acids.

Simultaneous saccharification and fermentation of dilute alkaline-pretreated corn stover for enhanced butanol production by Clostridium saccharobutylicum DSM 13864.

Simultaneous saccharification and fermentation (SSF) process was applied for biobutanol production by Clostridium saccharobutylicum DSM 13864 from corn stover (CS). The key influential factors in SSF process, including corn steep liquor concentration, dry biomass and enzyme loading, SSF temperature, inoculation size and pre-hydrolysis time were optimized. In 5-L bioreactor with SSF process, butanol titer and productivity of 12.3 g/L and 0.257 g/L/h were achieved at 48 h, which were 20.6% and 21.2% higher than those in separate hydrolysis and fermentation (SHF), respectively. The butanol yield reached 0.175 g/g pretreated CS in SSF, representing 50.9% increase than that in SHF (0.116 g/g pretreated CS). This study proves the feasibility of efficient and economic production of biobutanol from CS by SSF.

Enhancing cellulose accessibility of corn stover by deep eutectic solvent pretreatment for butanol fermentation.

In this study, an effective corn stover (CS) pretreatment method was developed for biobutanol fermentation. Deep eutectic solvents (DESs), consisted of quaternary ammonium salts and hydrogen donors, display similar properties to room temperature ionic liquid. Seven DESs with different hydrogen donors were facilely synthesized. Choline chloride:formic acid (ChCl:formic acid), an acidic DES, displayed excellent performance in the pretreatment of corn stover by removal of hemicellulose and lignin as confirmed by SEM, FTIR and XRD analysis. After optimization, glucose released from pretreated CS reached 17.0 g L(-1) and yield of 99%. The CS hydrolysate was successfully utilized in butanol fermentation by Clostridium saccharobutylicum DSM 13864, achieving butanol titer of 5.63 g L(-1) with a yield of 0.17 g g(-1) total sugar and productivity of 0.12 g L(-1)h(-1). This study demonstrates DES could be used as a promising and biocompatible pretreatment method for the conversion of lignocellulosic biomass into biofuel.