Effective Synthesis of High-Integrity mRNA Using In Vitro Transcription.

mRNA vaccines are entering a period of rapid development. However, their synthesis is still plagued by challenges related to mRNA impurities and fragments (incomplete mRNA). Most impurities of mRNA products transcribed in vitro are mRNA fragments. Only full-length mRNA transcripts containing both a 5'-cap and a 3'-poly(A) structure are viable for in vivo expression. Therefore, RNA fragments are the primary product-related impurities that significantly hinder mRNA efficacy and must be effectively controlled; these species are believed to originate from either mRNA hydrolysis or premature transcriptional termination. In the manufacturing of commercial mRNA vaccines, T7 RNA polymerase-catalyzed in vitro transcription (IVT) synthesis is a well-established method for synthesizing long RNA transcripts. This study identified a pivotal domain on the T7 RNA polymerase that is associated with erroneous mRNA release. By leveraging the advantageous properties of a T7 RNA polymerase mutant and precisely optimized IVT process parameters, we successfully achieved an mRNA integrity exceeding 91%, thereby further unlocking the immense potential of mRNA therapeutics.

Antibacterial, antioxidant, Cr(VI) adsorption and dye adsorption effects of biochar-based silver nanoparticles­sodium alginate-tannic acid composite gel beads.

Ultrasonic extraction of Osmanthus fragrans was used for reducing Ag+ to prepare AgNPs, which were further loaded on barley distiller's grains shell biochar. By supplementary of sodium alginate and tannic acid, composite gel beads were prepared. The physical properties of biochar-based AgNPs­sodium alginate-tannic acid composite gel beads (C-Ag/SA/TA) were characterized. SEM, FTIR, and XRD showed that biochar-based AgNPs were compatible with sodium alginate-tannic acid. CAg greatly improved the dissolution, swelling, and expansion of gel beads. Through the analysis by the agar diffusion method, C-Ag/SA/TA gel beads had high antibacterial activity (inhibition zone: 22 mm against Escherichia coli and 20 mm against Staphylococcus aureus). It was observed that C-Ag/SA/TA composite gel beads had high antioxidant capacity and the free radical scavenging rate reached 89.0 %. The dye adsorption performance of gel beads was studied by establishing a kinetic model. The maximum adsorption capacities of C-Ag/SA/TA gel beads for methylene blue and Congo red were 166.57 and 318.06 mg/g, respectively. The removal rate of Cr(VI) reached 96.4 %. These results indicated that the prepared composite gel beads had a high adsorption capacity for dyes and metal ions. Overall, C-Ag/SA/TA composite gel beads were biocompatible and had potential applications in environmental pollution treatment.

Taurine-mediated gene transcription and cell membrane permeability reinforced co-production of bioethanol and Monascus azaphilone pigments for a newly isolated Monascus purpureus.

BACKGROUND: Taurine, a semi-essential micronutrient, could be utilized as a sulfur source for some bacteria; however, little is known about its effect on the accumulation of fermentation products. Here, it investigated the effect of taurine on co-production of bioethanol and Monascus azaphilone pigments (MonAzPs) for a fungus. RESULTS: A newly isolated fungus of 98.92% identity with Monascus purpureus co-produced 23.43 g/L bioethanol and 66.12, 78.01 and 62.37 U/mL red, yellow and orange MonAzPs for 3 d in synthetic medium (SM). Taurine enhanced bioethanol titer, ethanol productivity and ethanol yield at the maximum by 1.56, 1.58 and 1.60 times than those of the control in corn stover hydrolysates (CSH), and red, yellow and orange MonAzPs were raised by 1.24, 1.26 and 1.29 times, respectively. Taurine was consumed extremely small quantities for M. purpureus and its promotional effect was not universal for the other two biorefinery fermenting strains. Taurine intensified the gene transcription of glycolysis (glucokinase, phosphoglycerate mutase, enolase and alcohol dehydrogenase) and MonAzPs biosynthesis (serine hydrolases, C-11-ketoreductase, FAD-dependent monooxygenase, 4-O-acyltransferase, deacetylase, NAD(P)H-dependent oxidoredutase, FAD-dependent oxidoredutase, enoyl reductase and fatty acid synthase) through de novo RNA-Seq assays. Furthermore, taurine improved cell membrane permeability through changing cell membrane structure by microscopic imaging assays. CONCLUSIONS: Taurine reinforced co-production of bioethanol and MonAzPs by increasing gene transcription level and cell membrane permeability for M. purpureus. This work would offer an innovative, efficient and taurine-based co-production system for mass accumulation of the value-added biofuels and biochemicals from lignocellulosic biomass.

Chemoenzymatic Asymmetric Synthesis of Chiral Triazole Fungicide (R)-Tebuconazole in High Optical Purity Mediated by an Epoxide Hydrolase from Rhodotorula paludigensis.

Tebuconazole is a chiral triazole fungicide used globally in agriculture as a racemic mixture, but its enantiomers exhibit significant enantioselective dissimilarities in bioactivity and environmental behaviors. The steric hindrance caused by the tert-butyl group makes it a great challenge to synthesize tebuconazole enantiomers. Here, we designed a simple chemoenzymatic approach for the asymmetric synthesis of (R)-tebuconazole, which includes the biocatalytic resolution of racemic epoxy-precursor (2-tert-butyl-2-[2-(4-chlorophenyl)ethyl] oxirane, rac-1a) by Escherichia coli/Rpeh whole cells expressed epoxide hydrolase from Rhodotorula paludigensis (RpEH), followed by a one-step chemocatalytic synthesis of (R)-tebuconazole. It was observed that (S)-1a was preferentially hydrolyzed by E. coli/Rpeh, whereas (R)-1a was retained with a specific activity of 103.8 U/g wet cells and a moderate enantiomeric ratio (E value) of 13.4, which was remarkably improved to 43.8 after optimizing the reaction conditions. Additionally, a gram-scale resolution of 200 mM rac-1a was performed using 150 mg/mL E. coli/Rpeh wet cells, resulting in the retention of (R)-1a in a 97.0% ees, a 42.5% yields, and a 40.5 g/L/d space-time yield. Subsequently, the synthesis of highly optical purity (R)-tebuconazole (>99% ee) was easily achieved through the chemocatalytic ring-opening of the epoxy-precursor (R)-1a with 1,2,4-triazole. To elucidate insight into the enantioselectivity, molecular docking simulations revealed that the unique L-shaped substrate-binding pocket of RpEH plays a crucial role in the enantioselective recognition of bulky 2,2-disubstituted oxirane 1a.

Effective synthesis of circRNA via a thermostable T7 RNA polymerase variant as the catalyst.

Introduction: Circular RNAs (circRNAs) are endogenous noncoding RNAs (ncRNAs) with transcriptional lengths ranging from hundreds to thousands. circRNAs have attracted attention owing to their stable structure and ability to treat complicated diseases. Our objective was to create a one-step reaction for circRNA synthesis using wild-type T7 RNA polymerase as the catalyst. However, T7 RNA polymerase is thermally unstable, and we streamlined circRNA synthesis via consensus and folding free energy calculations for hotspot selection. Because of the thermal instability, the permuted intron and exon (PIE) method for circRNA synthesis is conducted via tandem catalysis with a transcription reaction at a low temperature and linear RNA precursor cyclization at a high temperature. Methods: To streamline the process, a multisite mutant T7 RNA polymerase (S430P, N433T, S633P, F849I, F880Y, and G788A) with significantly improved thermostability was constructed, and G788A was used. Results: The resulting mutant exhibited stable activity at 45°C for over an hour, enabling the implementation of a one-pot transcription and cyclization reaction. The simplified circRNA production process demonstrated an efficiency comparable to that of the conventional two-step reaction, with a cyclization rate exceeding 95% and reduced production of immunostimulatory dsRNA byproducts.

Dynamic regulation and cofactor engineering of escherichia coli to enhance production of glycolate from corn stover hydrolysate.

Glycolic acid is widely employed in chemical cleaning, the production of polyglycolic acid-lactic acid, and polyglycolic acid. Currently, the bottleneck of glycolate biosynthesis lies on the imbalance of metabolic flux and the deficiency of NADPH. In this study, a dynamic regulation system was developed and optimized to enhance the metabolic flux from glucose to glycolate. Additionally, the knockout of transhydrogenase (sthA), along with the overexpression of pyridine nucleotide transhydrogenase (pntAB) and the implementation of the Entner-Doudoroff pathway, were performed to further increase the production of the NADPH, thereby increasing the titer of glycolate to 5.6 g/L. To produce glycolate from corn stover hydrolysate, carbon catabolite repression was alleviated and glucose utilization was accelerated. The final strain, E. coli Mgly10-245, is inducer-free, achieving a glycolate titer of 46.1 g/L using corn stover hydrolysate (77.1 % of theoretical yield). These findings will contribute to the advancement of industrial glycolate production.

Improving saccharification efficiency of corn stover through ferric chloride-deep eutectic solvent pretreatment.

An effective deep eutectic solvent (DES) and Iron(III) chloride (FeCl3) combination pretreatment system was developed to improve the removal efficiency of lignin and hemicellulose from corn stover (CS) and enhance its saccharification. N-(2-hydroxyethyl)ethylenediamine (NE) was selected as the hydrogen-bond-donor for preparing ChCl-based DES (ChCl:NE), and a mixture of ChCl:NE (60 wt%) and FeCl3 (0.5 wt%) was utilized for combination pretreatment of CS at 110 ℃ for 50 min. FeCl3/ChCl:NE effectively removed lignin (87.0 %) and xylan (55.9 %) and the enzymatic hydrolysis activity of FeCl3/ChCl:NE-treated CS was 5.5 times that of CS. The reducing sugar yield of pretreated CS was 98.6 %. FeCl3/ChCl:NE significantly disrupted the crystal structure of cellulose in CS and improved the removal of lignin and hemicellulose, enhancing the conversion of cellulose and hemicellulose into monomeric sugars. Overall, this combination of FeCl3 and DES pretreatment methods has high application potential for the biological refining of lignocellulose.

Combination of solid acid and solvent pretreatment for co-production of furfural, xylooligosaccharide and reducing sugars from Phyllostachys edulis.

The efficient utilization of biomass resources has gained widespread attention in current research. This study focused on the conversion of hemicellulose into xylo-oligosaccharides and furfural, as well as enhanced cellulose saccharification and lignin removal from residual biomass. The solid acid catalyst AT-Sn-MMT was prepared by sulfonation and tin ion loading of montmorillonite K-10. In a mixture of deep eutectic solvent and γ-valerolactone (3:7, v/v), AT-Sn-MMT was used to catalyze Phyllostachys edulis (PE) at 160 °C for 20 min, obtaining a furfural yield of 85.7 % and 1.5 g/L xylo-oligosaccharides. The delignification of pretreated PE was 59.5 %, reaching an accessibility of 221.3 g dye/g material. While the enzymatic saccharification efficiency was increased to 73.1 %. This work drew on the merits of solid acid catalysts and mixed solvent systems, and this constructed pretreatment method could be efficiently applied for co-production of reducing sugars, xylooligosaccharide and furfural, realizing the efficient valorization of PE.

Antibacterial, antioxidant and fruit packaging ability of biochar-based silver nanoparticles-polyvinyl alcohol-chitosan composite film.

Silver nanoparticles were prepared by loading Ag+ into biochar of waste barley distillers' grains shell by reduction with trisodium citrate, and this silver-loaded biochar was introduced into polyvinyl alcohol-chitosan. Various analysis with Fourier Transform Infrared spectroscopy, X-ray diffraction, Thermogravimetric analysis, and water contact angle revealed that biochar-based silver nanoparticle was incorporated into the polyvinyl alcohol-chitosan film, the biochar-based silver nanoparticles-polyvinyl alcohol-chitosan (C-Ag-loaded PVA/CS) composite film had good thermostability and hydrophobicity. Through the analysis via disk diffusion method, the composite containing 3 % of biochar-based silver nanoparticles-polyvinyl alcohol-chitosan had high antibacterial activity (inhibition zone: 18 mm against E. coli and 15 mm against S. aureus), and the bacterial membrane permeability was measured, indicating that C-Ag-loaded PVA/CS composite film could destroy the cell membrane, release intracellular substances, and have high antioxidant activity. During the storage, the weight loss rate of the biochar-based silver nanoparticles-polyvinyl alcohol-chitosan plastic wrap group was 0.14 %, and the titratable acid content only decreased by 0.061 %, which had a good effect on extending the shelf life of blueberries. The C-Ag-loaded PVA/CS composite film could also delay deterioration of blueberries and prolong storage time. Overall, this composite film had potential in food packaging and extending food shelf-life aspects.

Co-production of 2,5-dihydroxymethylfuran and furfuralcohol from sugarcane bagasse via chemobiocatalytic approach in a sustainable system.

2,5-Dihydroxymethylfuran and furfuryl alcohol serve as versatile building-blocks in pharmaceuticals, polymers, and value-added intermediates. To develop an efficient and sustainable method for their production from biomass, a combined approach using deep eutectic solvent Citric acid:Betaine (CTA:BT) for bagasse catalysis and recombinant E. coli SCFD23 for bioreduction of bagasse-derived 5-hydroxymethylfurfural and furfural was devised. Bagasse was effectively transformed into 5-hydroxymethylfurfural (48 mM) and furfural (14 mM) in CTA:BT (8 wt%)-water at 170 °C for 30 min. Bioreduction of 5-hydroxymethylfurfural and furfural by SCFD23 cell co-expressing formate dehydrogenase and NAD(P)H-dependent aldehyde reductase (SsCR) yielded 2,5-dihydroxymethylfuran (90.0 % yield) and furfuryl alcohol (99.0 % yield) in 6 h, using biomass-derived formic acid, xylose and glucose as co-substrates. Molecular docking confirmed the stable binding and reductase activity of SsCR with the biomass-derived 5-hydroxymethylfurfural and furfural. An efficient and eco-friendly chemobiological approach was applied for co-production of 2,5-dihydroxymethylfuran and furfuryl alcohol from biomass in one-pot two-step reaction.

Efficient co-production of reducing sugars and xylooligosaccharides via clean hydrothermal pretreatment of rape straw.

Hydrothermal treatment was applied to pretreat rape straw for the efficient co-production of reducing sugars and xylooligosaccharides. It was observed that hydrothermal treatment using water as solvent and catalyst destructed the compact structure of rape straw and increased its enzymatic digestion efficiency from 24.6% to 92.0%. Xylooligosaccharide (3.3 g/L) was acquired after the treatment under 200 °C for 60 min (severity factor Log Ro = 4.7). With increasing pretreatment intensity from 3.1 to 5.4, the hemicellulose removal increased from 14.4% to 100%, and the delignification was raised from 12% to 44%. Various characterization proved that the surface morphology of treated material showed a porous shape, while the cellulose accessibility, lignin surface area and lignin hydrophobicity were greatly improved. Consequently, hydrothermal pretreatment played a vital role in the sustainable transformation of biomass to valuable biobased compounds, and had a wide range of application prospects in lignocellulosic biorefining.

Employing deep eutectic solvent synthesized by cetyltrimethylammonium bromide and ethylene glycol to advance enzymatic hydrolysis efficiency of rape straw.

An efficient deep eutectic solvent (DES) was synthesized by cetyltrimethylammonium bromide (CTAB) and ethylene glycol (EG) and employed to treat rape straw (RS) for advancing enzymatic saccharification in this work. By optimizing the pretreatment parameters, the results displayed that the novel DES was strongly selective towards removing lignin and xylan while preserving cellulose. Under optimum conditions with 1:6 of CTAB: EG in DES, 180 °C and 80 min, the enzymatic hydrolysis efficiency of RS was enhanced by 46.0% due to the 62.2% of delignification and 53.2% of xylan removal during CTAB: EG pretreatment. In terms of the recalcitrant structure of RS, DES pretreatment caused the increment of cellulosic accessibility, reduction of hydrophobicity and surface area of lignin, and migration of cellulosic crystalline structure, which was associated with its enzymatic hydrolysis efficiency. Overall, this study presented an emerging method for the effective fractionation and valorization of lignocellulosic biomass within biorefinery technology.

Ultraviolet blocking ability, antioxidant and antibacterial properties of newly prepared polyvinyl alcohol-nanocellulose­silver nanoparticles-ChunJian peel extract composite film.

In this work, nanocellulose (CNC) from waste water chestnut (WCT) shell was firstly used for preparing nanocomposite films, by using ChunJian peel extract (CJPE) as a green reducing agent to synthesize silver nanoparticles (AgNPs), and then loading them into polyvinyl alcohol-nanocellulose (PVA-CNC) matrix, a multifunctional nanocomposite material that could be used in food packaging was developed. The prepared films were tested for mechanical strength, barrier properties, thermal properties, antibacterial, antioxidant and biocompatibility through various characterizations. The PVA-CNC-AgNPs-CJPE film had good thermostability, mechanical strength, barrier properties, and biocompatibility. Compared with pure PVA film and PVA-CNC film, PVA-CNC-AgNPs-CJPE could shield over 95 % of the UVB (320-275 nm) spectrum and UVC (275-200 nm) spectrum and most of the UVA (400-320 nm). By disk diffusion analysis, the inhibition zones of PVA-CNC-AgNPs-CJPE film against E. coli, P. aeruginosa, S. aureus and E. faecalis were 22.3 mm, 25.0 mm, 22.0 mm and 19.3 mm, respectively. The milk antibacterial simulation test confirmed that PVA-CNC-AgNPs-CJPE film could effectively limit bacterial reproduction and prolong the shelf life of milk. PVA-CNC-AgNPs-CJPE film had excellent UV barrier properties, good antioxidant properties and high-efficiency antibacterial activity, which is expected to be widely used in sustainable nanocomposite food packaging.

Efficient co-production of xylooligosaccharides, furfural and reducing sugars from yellow bamboo via the pretreatment with biochar-based catalyst.

The research on the efficient use of biomass to produce chemical products has received extensive attention. In this work, a novel heterogeneous biocarbon-based heterogeneous catalyst AT-Sn-YB was prepared using yellow bamboo (YB) as a carrier, and its physical properties were proved to be good by various characterization and stability experiments. In the γ-valerolactone/water (3:1, v/v) medium containing 100 mM CuCl2, the use of AT-Sn-YB (3.6 wt%) under 170 °C for 20 min was applied to catalyze YB into furfural (80.3% yield), accompanied with 2.8 g/L xylooligosaccharides. The YB solid residue obtained from treatment was efficiently saccharified to reducing sugars (17.2 g/L). Accordingly, comprehensive understanding of efficiently co-producing xylooligosaccharides, furfural and reducing sugars from YB was demonstrated via the pretreatment with biochar-based catalyst. This study innovatively used a new type of solid acid to complete the efficient co-production of chemical products, and realized the value-added utilization of yellow bamboo.

Chemobiocatalytic transfromation of biomass into furfurylamine with mixed amine donor in an eco-friendly medium.

Biobased furfurylamine (FAM) is a versatile platform molecule for producing additives, pharmaceuticals, and pesticides. Recombinant E. coli HNND-AlaDH was created by co-expressing L-alanine dehydrogenase (AlaDH) and mutated Aspergillus terreus ω-transaminase (HNND), aiming to convert furfural (FUR) into FAM using inexpensive L-alanine and isopropylamine as mixed amine donors. In ChCl:FA:OA (10 wt%), pineapple peel, bagasse, barley shell, peanut shell, and corn stalk could be efficiently transformed into FUR under 170 °C for 10 min. Pineapple peel produced a high titer of FUR (183.3 mM). Additionally, the viscosity, surface tension and polarity of ChCl:FA:OA were explored. The biomass-derived FUR was fully transformed to FAM by HNND-AlaDH with amine donor (1:1:1 of L-Ala/isopropylamine/FUR mol/mol/mol) within 300 min. Accordingly, the FAM productivity was 0.58 g/(g xylan in pineapple peel). This chemobiocatalytic strategy established through the combination of chemocatalysis and biocatalysis could be applied to convert renewable biomass into valuable organic amines.

Efficient production of hydroxymethyl-2-furfurylamine by chemoenzymatic cascade catalysis of bread waste in a sustainable approach.

In this study, efficient and sustainable conversion of waste bread (WB) to 5-hydroxymethyl-2-furoamine (HMFA) was achieved in a cascade reaction in betaine:malonic acid (B:MA) - water. 5-HMF (30.3 wt% yield) was synthesized from WB (40.0 g/L) in B:MA - water (B:MA, 18 wt%) in 45 min at 190 °C. By using the newly created recombinant E. coli HNILGD-AlaDH cells expressing L-alanine dehydrogenase (AlaDH) and ω-transaminase mutant HNILGD as biocatalyst, the WB-valorized 5-HMF was biologically aminated into HMFA in a high yield (92.1%) at 35 °C for 12 h through in situ removal of the amino transfer by-products of the amine donor, greatly reducing amine donor dosage (from D-Ala/5-HMF = 16/1 to D-Ala/5-HMF = 2/1, mol/mol) and improving the productivity of HMFA (0.282 g HMFA per g WB). This two-step chemical-enzymatic cascade reaction strategy with B:MA and HNILGD-AlaDH whole-cell provides a new idea for the chemoenzymatic synthesis of valuable furan chemicals from waste biomass.

Biological valorization of lignin-derived vanillin to vanillylamine by recombinant E. coli expressing ω-transaminase and alanine dehydrogenase in a petroleum ether-water system.

Vanillylamine, as an important drug precursor and fine chemical intermediate, has great economic value. By constructing a strategy of double enzyme co-expression, one newly constructed recombinant E. coli HNIQLE-AlaDH expressing ω-transaminase from Aspergillus terreus and alanine dehydrogenase from Bacillus subtilis was firstly used aminate lignin-derived vanillin to vanillylamine by using a relatively low dosage of amine donors (vanillin:L-alanine:isopropylamine = 1:1:1, mol/mol/mol). In addition, in a two-phase system (water:petroleum ether = 80:20 v/v), the bioconversion of vanillin to vanillylamine was catalyzed by HNIQLE-AlaDH cell under the ambient condition, and the vanillylamine yield was 71.5%, respectively. This double-enzyme HNIQLE-AlaDH catalytic strategy was applied to catalyze the bioamination of furfural and 5-hydroxymethylfurfural with high amination efficiency. It showed that the double-enzyme catalytic strategy in this study promoted L-alanine to replace D-alanine to participate in bioamination of vanillin and its derivatives, showing a great prospect in the green biosynthesis of biobased chemicals from biomass.

Comprehensive understanding of enzymatic saccharification of Betaine:Lactic acid-pretreated sugarcane bagasse.

Green solvents, especially deep eutectic solvents (DESs), are widely applied to pretreat biomass for enhancing its enzymatic hydrolysis. In this work, lactic acid was selected as the hydrogen-bond-donor to prepare Betaine-base DES (Betaine:LA), The DES was utilized to pretreat sugarcane bagasse (SCB) at 160 ℃ for 80 min (severity factor LogR0 = 3.67). The influences of Betaine:LA treatment on the chemical composition, crystal and microstructure structure of cellulose, and cellulase digestion were investigated. The results showed that the lignin (47.1%) and xylan (44.6%) were removed, the cellulase digestibility of Betaine:LA-treated SCB was 4.2 times that of the raw material. This improved efficiency was attributed to the enhanced accessibility of cellulose, the weakened surface area of lignin, the declined hydrophobicity, and the decreased crystallinity of cellulose. Several compelling linear correlations were fitted between enzymatic hydrolysis and these alterations of physicochemical features, comprehensively understanding enzymatic saccharification of Betaine:LA-pretreated SCB.

Efficient antibacterial and dye adsorption by novel fish scale silver biochar composite gel.

A silver-loaded carbon-chitosan-polyvinyl alcohol gel (C/CTS/PVA) was designed for suppressing microbial growth and dye adsorption. The antibacterial test results showed that C/CTS/PVA gel had a good antibacterial ability against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The inhibition rate in water was 100 %, and the antibacterial rate remained above 95 % within 35 days after preparation. The tight spatial structure provided by the adhesive effect of PVA and CTS effectively prevented water loss and enhanced the stability of the gel. The adsorption curves of the gel were fitted by establishing the pseudo-first order and pseudo-second order kinetic models. The adsorption curves were more consistent with the pseudo-second-order kinetic model. The best adsorption effect for Malachite green was 128.12 mg/g. C/CTS/PVA gel had a remarkable adsorption effect on Malachite green, Congo red, Methyl orange, and Methylene blue. In general, C/CTS/PVA gels have great potential for the treatment of sewage in the future.

Combined sulfuric acid and choline chloride/glycerol pretreatment for efficiently enhancing enzymatic saccharification of reed stalk.

In this study, an efficient combination of pretreatment solvents involving Choline chloride/Glycerol (ChCl/Gly) and H2SO4 was firstly developed to assess the pretreatment performance and determine optimal pretreatment conditions. The results illustrated that the H2SO4-[ChCl/Gly] combination efficiently removed lignin (52.6%) and xylan (80.5%) from the pretreated reed stalk, and subsequent enzymatic hydrolysis yielded 91.1% of glucose. Furthermore, several characterizations were conducted to examine the structural and morphological changes of the reed stalk, revealing apparently enhanced accessibility (128.4 to 522.6 mg/g), reduced lignin surface area (357.9 to 229.5 m2/g), and substantial changes on biomass surface. Based on the aforementioned study, possible mechanisms for the H2SO4-[ChCl/Gly] pretreatment of reed stalks were proposed. The comprehensive understanding of combined H2SO4-[ChCl/Gly] pretreatment system for enhancing the saccharification of the reed stalk was interpreted in this work. Overall, this novel approach could be efficiently applied to pretreat and saccharify reed stalks, empowering the biomass refining industry.