Explore the vast range of titles available.

Lignin, the most abundant renewable aromatic compound in nature, is an excellent feedstock for value-added bioproducts manufacturing; while the intrinsic heterogeneity and recalcitrance of which hindered the efficient lignin biorefinery and utilization. Compared with chemical processing, bioprocessing with microbial and enzymatic catalysis is a clean and efficient method for lignin depolymerization and conversion. Generally, lignin bioprocessing involves lignin decomposition to lignin-based aromatics via extracellular microbial enzymes and further converted to value-added bioproducts through microbial metabolism. In the review, the most recent advances in degradation and conversion of lignin to value-added bioproducts catalyzed by microbes and enzymes were summarized. The lignin-degrading microorganisms of white-rot fungi, brown-rot fungi, soft-rot fungi, and bacteria under aerobic and anaerobic conditions were comparatively analyzed. The catalytic metabolism of the microbial lignin-degrading enzymes of laccase, lignin peroxidase, manganese peroxidase, biphenyl bond cleavage enzyme, versatile peroxidase, and β-etherize was discussed. The microbial metabolic process of H-lignin, G-lignin, S-lignin based derivatives, protocatechuic acid, and catechol was reviewed. Lignin was depolymerized to lignin-derived aromatic compounds by the secreted enzymes of fungi and bacteria, and the aromatics were converted to value-added compounds through microbial catalysis and metabolic engineering. The review also proposes new insights for future work to overcome the recalcitrance of lignin and convert it to value-added bioproducts by microbial and enzymatic catalysis.

Background The extremely thermophilic bacterium Caldicellulosiruptor lactoaceticus can degrade and metabolize untreated lignocellulosic biomass containing xylan. The mechanism of the bacterium for degradation of insoluble xylan in untreated biomass has not been revealed. Results In the present study, the only annotated extracellular endo-β-1,4-xylanase (Xyn10B) with multidomain structures in C. lactoaceticus genome was biochemically characterized. Xyn10B contains three N-terminal consecutive family 22 carbohydrate-binding modules (CBMs), one GH10 catalytic domain (CD), two family 9 CBMs and two S-layer homology (SLH) modules in the C-terminal. CBM22a shares 27.1% and 27.2% sequence homology with CBM22b and CBM22c, respectively. The sequence homology between two CBM9 s and two SLHs is 26.8% and 25.6%, respectively. To elucidate the effect of multiple domains on the enzymatic properties of Xyn10B, the truncated variants of which (Xyn10B-TM1: CBM22a-CBM22b-CBM22c-CD10; Xyn10B-TM2: CBM22c-CD10; Xyn10B-TM3: CBM22c-CD10-CBM9a; and Xyn10B-TM4: CD10-CBM9a) were separately reconstructed, recombinantly expressed and biochemically characterized. Enzymatic properties studies showed that the optimal temperature for all four Xyn10B truncations was 65 °C. Compared to Xyn10B-TM3 and Xyn10B-TM4, Xyn10B-TM1 and Xyn10B-TM2 had higher hydrolytic activity, thermostability and affinity on insoluble substrates. It is noteworthy that Xyn10B-TM1 and Xyn10B-TM2 have higher enzymatic activity on insoluble xylan than the soluble counterparts, whereas Xyn10B-TM3 and Xyn10B-TM4 showed opposite characteristics. The kinetic parameters analysis of Xyn10B-TM1 on xylan showed Vmax was 5740, 1300, 1033, and 3925 U/μmol on insoluble oat spelt xylan (OSX), soluble beechwood xylan (BWX), soluble sugar cane xylan (SCX), and soluble corncob xylan (CCX), respectively. The results indicated that CBM22s especially CBM22c promoted the hydrolytic activity, thermostability and affinity on insoluble substrates of the Xyn10B truncations. The functions of CBM22, CBM9, CD and SLH are different, while contribute synergetically to the thermostability, protein structure integrity, substrate binding, and high hydrolytic activity on insoluble xylan of untreated lignocellulosic biomass. The domains of CBM22, CBM9, CD and SLH have different characteristics, which synergistically promote the thermostability, protein structure integrity, affinity on insoluble substrates and enzymatic activity properties of Xyn10B. Conclusions The extracellular endo-β-1,4-xylanase with multidomain structures of CBM, CD and SLH promote the biodegradation of insoluble xylan in untreated lignocellulosic biomass by thermophilic C. lactoaceticus.

Acetoin (3-hydroxy-2-butanone) is an important bio-based platform chemical with wide applications. In vitro enzyme catalysed synthesis exhibits great feasibility in the production of chemicals with high purity. In the present work, a synthetic pathway involving a two-step continuous reaction was constructed in vitro for acetoin production from pyruvate at improved temperature. Thermostable candidates, acetolactate synthase (coAHASL1 and coAHASL2 from Caldicellulosiruptor owensensis OL) and α-acetolactate decarboxylase (bsALDC from Bacillus subtilis IPE5-4) were cloned, heterologously expressed, and characterized. All the enzymes showed maximum activities at 65–70 °C and pH of 6.5. Enzyme kinetics analysis showed that coAHASL1 had a higher activity but lower affinity against pyruvate than that of coAHASL2. In addition, the activities of coAHASL1 and bsALDC were promoted by Mn 2+ and NADPH. The cascade enzymatic reaction was optimized by using coAHASL1 and bsALDC based on their kinetic properties. Under optimal conditions, a maximum concentration of 3.36 ± 0.26 mM acetoin was produced from 10 mM pyruvate after reaction for 24 h at 65 °C. The productivity of acetoin was 0.14 mM h −1 , and the yield was 67.80% compared with the theoretical value. The results confirmed the feasibility of synthesis of acetoin from pyruvate with a cell-free enzyme catalysed system at improved temperature.

Background:Acetoin (3-hydroxy-2-butanone), the precursor of biofuel 2,3-butanediol, is an important bio-based platform chemical with wide applications. Fermenting the low-cost and renewable plant biomass is undoubtedly a promising strategy for acetoin production. Isothermal simultaneous saccharification and fermentation (SSF) is regarded as an efficient method for bioconversion of lignocellulosic biomass, in which the temperature optima fitting for both lignocellulose-degrading enzymes and microbial strains.Results:A thermotolerant (up to 52°C) acetoin producer Bacillus subtilis IPE5-4 which simultaneously consumed glucose and xylose was isolated and identified. By compound mutagenesis, the mutant IPE5-4-UD-4 with higher acetoin productivity was selected. When fermenting at 50°C in a 5-L bioreactor using glucose as the feedstock by strain IPE5-4-UD-4, the acetoin concentration reached 28.83 ± 0.91 g L−1 with the acetoin yield and productivity of 0.34 g g−1 glucose and 0.60 g L−1h−1, respectively. Furthermore, an optimized and thermophilic SSF process operating at 50°C was conducted for acetoin production from alkali-pretreated corncob (APC). An acetoin concentration of 12.55 ± 0.28 g L−1 was achieved by strain IPE5-4-UD-4 in shake flask SSF, with the acetoin yield and productivity of 0.25 g g−1 APC and 0.17 g L−1 h−1. Meanwhile, the utilization of cellulose and hemicellulose in the SSF approach reached 96.34 and 93.29%, respectively. When further fermented at 50°C in a 5-L bioreactor, the concentration of acetoin reached the maximum of 22.76 ± 1.16 g L−1, with the acetoin yield and productivity reaching, respectively, 0.46 g g−1 APC and 0.38 g L−1 h−1. This was by far the highest acetoin yield in SSF from lignocellulosic biomass.Conclusions:This thermophilic SSF process provided an efficient and economical route for acetoin production from lignocellulosic biomass at ideal temperature for both enzymatic hydrolysis and microbial fermentation.

Background Switchgrass (Panicum virgatum L.), a C4 perennial grass, has been recognized as one of the most potentially important lignocellulose biofuel crops. MicroRNA319 (miR319) plays a key role in plant development, abiotic resistance, and cell wall biosynthesis by repressing expression of its target TCP genes. We hypothesized miR319–TCP pathway could play important roles in switchgrass feedstock characteristics for biofuel production, and produced switchgrass transgenic plants overexpressing miR319 (by ectopic expressing Osa-MIR319b gene), blocking miR319 (by overexpressing a target mimicry of miR319/MIM319) and repression of miR319 target gene PvPCF5. Plant phenotype, biomass yield, and feedstock quality of transgenic plants were analyzed. Results Overexpression of miR319 in switchgrass promoted leaf elongation and expansion of transgenic plants, increased plant height, stem diameter, and resulted in a significant increase in plant biomass yield. Transgenic plants overexpressing of miR319 reduced lignin content, showed significantly higher enzymatic hydrolysis efficiency compared to the wild type plant. However, opposite results were observed in the MIM319 plants. Furthermore, suppression of miR319 target gene PvPCF5 activity also reduced lignin content, increased lignin monomer S/G ratio and the proportion of β-O-4 linkages, while significantly improving the sugar production per plant. Quantitative real-time (qRT-PCR) analysis indicated that expression of PvMYB58/63B and PvHCT with predicted TCP binding sites in their promoter regions was negatively regulated by miR319–PvPCF5 module. Conclusions MiR319–PvPCF5 module plays positive roles in regulating biomass yield and quality of switchgrass. It can be utilized as a candidate molecular tool in regulating biomass yield and feedstock quality. The finding could also be transferred to other grasses for forage quality improvement through genetic manipulation.

The xylanolytic extremely thermophilic bacterium Caldicellulosiruptor owensensis provides a promising platform for xylan utilization. In the present study, two novel xylanolytic enzymes, GH10 endo-β-1,4-xylanase (Coxyn A) and GH39 β-1,4-xylosidase (Coxyl A) encoded in one gene cluster of C.owensensis were heterogeneously expressed and biochemically characterized. The optimum temperature of the two xylanlytic enzymes was 75°C, and the respective optimum pH for Coxyn A and Coxyl A was 7.0 and 5.0. The difference of Coxyn A and Coxyl A in solution was existing as monomer and homodimer respectively, it was also observed in predicted secondary structure. Under optimum condition, the catalytic efficiency (kcat/Km) of Coxyn A was 366 mg ml(-1) s(-1) on beechwood xylan, and the catalytic efficiency (kcat/Km) of Coxyl A was 2253 mM(-1) s(-1) on pNP-β-D-xylopyranoside. Coxyn A degraded xylan to oligosaccharides, which were converted to monomer by Coxyl A. The two intracellular enzymes might be responsible for xylooligosaccharides utilization in C.owensensis, also provide a potential way for xylan degradation in vitro.

Caldicellulosiruptor kronotskyensis grows on lignocellulosic biomass by the catalysis of intrinsic glycoside hydrolase and has potential application for consolidated bioprocessing. In current study, two predicted extra- (Xyn10A) and intracellular (Xyn10B) xylanase from C. kronotskyensis were comparatively characterized. Xyn10A and Xyn10B share GH10 catalytic domain with similarity of 41%, while the former contains two tandem N-terminus CBM22s. Xyn10A showed higher hydrolytic capability than Xyn10B on both beechwood xylan (BWX) and oat spelt xylan (OSX). Truncation mutation experiments revealed the importance of CBMs for hydrolytic activity, substrate binding and thermostability of Xyn10A.While the quantity of CBM was not directly related to bind and thermostability. Although CBM was considered to be crucial for substrate binding, Xyn10B and Xyn10A as well as truncations performed similar binding affinity to insoluble substrate OSX. Analysis of point mutation revealed similar key residues, Glu493, Glu601 and Trp658 for Xyn10A and Glu139, Glu247 and Trp305 for Xyn10B. Both Xyn10A and Xyn10B exhibited hydrolytic activity on the mechanical pretreated corncob. After pre-digested by Xyn10A or Xyn10B, the micropores inthe the mechanical pretreated corncob were observed, which enhanced the accessibility for cellulase. Compared with corncob hydrolyzed with cellulase alone, enhanced hydrolytic performance of was observed after pre-digestion by Xyn10A or Xyn10B.

Flavin-dependent monooxygenases (FMOs) are versatile oxidative biocatalysts that catalyze a wide array of oxygenation reactions, such as hydroxylation, epoxidation, Baeyer–Villiger oxidation, and halogenation. These enzymes utilize flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as cofactors to mediate selective incorporation of oxygen into diverse substrates. Owing to their remarkable chemo-, regio-, and stereoselectivity, FMOs have attracted increasing attention as powerful tools for biomanufacturing. Recent advances in enzyme engineering, structural biology, and computational design have expanded the catalytic diversity of FMOs and enabled their integration into biocatalysis frameworks. Moreover, developments in cofactor regeneration, directed evolution, and cell-free biotransformation have improved FMOs’ catalytic efficiency and scalability. Despite these advances, challenges such as limited thermostability, oxygen transfer efficiency, and substrate scope remain obstacles for industrial applications of FMOs. This review summarizes the structural characteristics, catalytic mechanisms, and engineering strategies of FMOs, highlights recent progress in their integration into biocatalysis platforms, and discusses current limitations and possible solutions. Insights into improving FMO catalytic performance and expanding their potential as next-generation biocatalysts for biosynthesis will be provided.

Caldicellulosiruptor lactoaceticus 6A, an anaerobic and extremely thermophilic bacterium, uses natural xylan as carbon source. The encoded genes of C. lactoaceticus 6A for glycoside hydrolase (GH) provide a platform for xylan degradation. The GH family 10 xylanase (Xyn10A) and GH67 α-glucuronidase (Agu67A) from C. lactoaceticus 6A were heterologously expressed, purified and characterized. Both Xyn10A and Agu67A are predicted as intracellular enzymes as no signal peptides identified. Xyn10A and Agu67A had molecular weight of 47.0 kDa and 80.0 kDa respectively as determined by SDS-PAGE, while both appeared as homodimer when analyzed by gel filtration. Xyn10A displayed the highest activity at 80 °C and pH 6.5, as 75 °C and pH 6.5 for Agu67A. Xyn10A had good stability at 75 °C, 80 °C, and pH 4.5-8.5, respectively, and was sensitive to various metal ions and reagents. Xyn10A possessed hydrolytic activity towards xylo-oligosaccharides (XOs) and beechwood xylan. At optimum conditions, the specific activity of Xyn10A was 44.6 IU/mg with beechwood xylan as substrate, and liberated branched XOs, xylobiose, and xylose. Agu67A was active on branched XOs with methyl-glucuronic acids (MeGlcA) sub-chains, and primarily generated XOs equivalents and MeGlcA. The specific activity of Agu67A was 1.3 IU/mg with aldobiouronic acid as substrate. The synergistic action of Xyn10A and Agu67A was observed with MeGlcA branched XOs and xylan as substrates, both backbone and branched chain of substrates were degraded, and liberated xylose, xylobiose, and MeGlcA. The synergism of Xyn10A and Agu67A provided not only a thermophilic method for natural xylan degradation, but also insight into the mechanisms for xylan utilization of C. lactoaceticus.

Carbon dioxide is a major greenhouse gas, and its fixation and transformation are receiving increasing attention. Biofixation of CO2 is an eco–friendly and efficient way to reduce CO2, and six natural CO2 fixation pathways have been identified in microorganisms and plants. In this review, the six pathways along with the most recent identified variant pathway were firstly comparatively characterized. The key metabolic process and enzymes of the CO2 fixation pathways were also summarized. Next, the enzymes of Rubiscos, biotin-dependent carboxylases, CO dehydrogenase/acetyl-CoA synthase, and 2-oxoacid:ferredoxin oxidoreductases, for transforming inorganic carbon (CO2, CO, and bicarbonate) to organic chemicals, were specially analyzed. Then, the factors including enzyme properties, CO2 concentrating, energy, and reducing power requirements that affect the efficiency of CO2 fixation were discussed. Recent progress in improving CO2 fixation through enzyme and metabolic engineering was then summarized. The artificial CO2 fixation pathways with thermodynamical and/or energetical advantages or benefits and their applications in biosynthesis were included as well. The challenges and prospects of CO2 biofixation and conversion are discussed.