OBJECTIVE: New characterized carbohydrate-active enzymes are needed for use as tools to discriminate complex carbohydrate structural features. Fungal glycoside hydrolase family 3 (GH3) β-xylosidases have been shown to be useful for the structural elucidation of glucuronic acid (GlcA) and arabinofuranose (Araf) substituted oligoxylosides. A homolog of these GH3 fungal enzymes from the bacterium Segatella baroniae (basonym Prevotella bryantii), Xyl3C, has been previously characterized, but those studies did not address important functional specificity features. In an interest to utilize this enzyme for laboratory methods intended to discriminate the structure of the non-reducing terminus of substituted xylooligosaccharides, we have further characterized this GH3 xylosidase.
RESULTS: In addition to verification of basic functional characteristics of this xylosidase we have determined its mode of action as it relates to non-reducing end xylose release from GlcA and Araf substituted oligoxylosides. Xyl3C cleaves xylose from the non-reducing terminus of β-1,4-xylan until occurrence of a penultimate substituted xylose. If this substitution is O2 linked, then Xyl3C removes the non-reducing xylose to leave the substituted xylose as the new non-reducing terminus. However, if the substitution is O3 linked, Xyl3C does not hydrolyze, thus leaving the substitution one-xylose (penultimate) from the non-reducing terminus. Hence, Xyl3C enables discrimination between O2 and O3 linked substitutions on the xylose penultimate to the non-reducing end. These findings are contrasted using a homologous enzyme also from S. baroniae, Xyl3B, which is found to yield a penultimate substituted nonreducing terminus regardless of which GlcA or Araf substitution exists.

This study proposed a two-stage pressurized microwave hydrothermal treatment with a catalyst, followed by enzymatic saccharification, as a pretreatment method for efficiently converting cellulose and hemicellulose from rice straw into glucose and xylose. The use of various inorganic salts and dilute sulfuric acid as catalysts enhances sugar production. Using 1 wt% sulfuric acid as a catalyst at 150 °C for 5 min for the first-stage and then 180 °C for 5 min for the second-stage yielded the highest sugar production from rice straw compared with other inorganic salts tested. The filtrate and enzymatic saccharification solution contained a total sugar of 0.434 g/g-untreated rice straw (i.e. 0.302 g-glucose/g-untreated rice straw and 0.132 g-xylose/g-untreated rice straw). When inorganic salts such as NaCl, MgCl2, CaCl2, and FeCl3 were used as catalysts, the highest sugar yield of 0.414 g/g-untreated rice straw (i.e. 0.310 g-glucose/g-untreated rice straw and 0.104 g-xylose/g-untreated rice straw) was obtained when using 1 wt% FeCl3 at 170 °C for 5 min in the first-stage and 190 °C for 5 min in the second-stage, with a value close to that of 1 wt% sulfuric acid. These findings suggest that two-stage treatment with a catalyst is a suitable pretreatment method for the production of glucose and xylose from rice straw owing to the different hydrolysis temperatures of cellulose and hemicellulose.

d-Allulose, a C-3 epimer of d-fructose, has great market potential in food, healthcare, and medicine due to its excellent biochemical and physiological properties. Microbial fermentation for d-allulose production is being developed, which contributes to cost savings and environmental protection. A novel metabolic pathway for the biosynthesis of d-allulose from a d-xylose-methanol mixture has shown potential for industrial application. In this study, an artificial antisense RNA (asRNA) was introduced into engineered Escherichia coli to diminish the flow of pentose phosphate (PP) pathway, while the UDP-glucose-4-epimerase (GalE) was knocked out to prevent the synthesis of byproducts. As a result, the d-allulose yield on d-xylose was increased by 35.1%. Then, we designed a d-xylose-sensitive translation control system to regulate the expression of the formaldehyde detoxification operon (FrmRAB), achieving self-inductive detoxification by cells. Finally, fed-batch fermentation was carried out to improve the productivity of the cell factory. The d-allulose titer reached 98.6 mM, with a yield of 0.615 mM/mM on d-xylose and a productivity of 0.969 mM/h.

Lignocellulosic biomass is a valuable, renewable substrate for the synthesis of polyhydroxybutyrate (PHB), an ecofriendly biopolymer. In this study, bacterial strain E5-3 was isolated from soil in Japan; it was identified as Burkholderia ambifaria strain E5-3 by 16 S rRNA gene sequencing. The strain showed optimal growth at 37 °C with an initial pH of 9. It demonstrated diverse metabolic ability, processing a broad range of carbon substrates, including xylose, glucose, sucrose, glycerol, cellobiose, and, notably, palm oil. Palm oil induced the highest cellular growth, with a PHB content of 65% wt. The strain exhibited inherent tolerance to potential fermentation inhibitors derived from lignocellulosic hydrolysate, withstanding 3 g/L 5-hydroxymethylfurfural and 1.25 g/L acetic acid. Employing a fed-batch fermentation strategy with a combination of glucose, xylose, and cellobiose resulted in PHB production 2.7-times that in traditional batch fermentation. The use of oil palm trunk hydrolysate, without inhibitor pretreatment, in a fed-batch fermentation setup led to significant cell growth with a PHB content of 45% wt, equivalent to 10 g/L. The physicochemical attributes of xylose-derived PHB produced by strain E5-3 included a molecular weight of 722 kDa, a number-average molecular weight of 191 kDa, and a polydispersity index of 3.78. The amorphous structure of this PHB displayed a glass transition temperature of 4.59 °C, while its crystalline counterpart had a melting point of 171.03 °C. This research highlights the potential of lignocellulosic feedstocks, especially oil palm trunk hydrolysate, for PHB production through fed-batch fermentation by B. ambifaria strain E5-3, which has high inhibitor tolerance.

Four yeast isolates were obtained from rotting wood and galleries of passalid beetles collected in different sites of the Brazilian Amazonian Rainforest in Brazil. This yeast produces unconjugated allantoid asci each with a single elongated ascospore with curved ends. Sequence analysis of the internal transcribed spacer-5.8 S region and the D1/D2 domains of the large subunit ribosomal RNA (rRNA) gene showed that the isolates represent a novel species of the genus Spathaspora. The novel species is phylogenetically related to a subclade containing Spathaspora arborariae and Spathaspora suhii. Phylogenomic analysis based on 1884 single-copy orthologs for a set of Spathaspora species whose whole genome sequences are available confirmed that the novel species represented by strain UFMG-CM-Y285 is phylogenetically close to Sp. arborariae. The name Spathaspora marinasilvae sp. nov. is proposed to accommodate the novel species. The holotype of Sp. marinasilvae is CBS 13467 T (MycoBank 852799). The novel species was able to accumulate xylitol and produce ethanol from  d-xylose, a trait of biotechnological interest common to several species of the genus Spathaspora.

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The first comparative pre-treatment study of Miscanthus (Mxg) and sugarcane bagasse (SCB) using steam explosion (SE) and pressurised disc refining (PDR) pretreatment to optimise xylose and xylo-oligosaccharide release is described. The current investigation aimed to 1) Develop optimised batch-wise steam explosion parameters for Mxg and SCB, 2) Scale from static batch steam explosion to dynamic continuous pressurised disc refining, 3) Identify, understand, and circumvent scale-up production hurdles. Optimised SE parameters released 82% (Mxg) and 100% (SCB) of the available xylan. Scaling to PDR, Miscanthus yielded 85% xylan, highlighting how robust scouting assessments for boundary process parameters can result in successful technical transfer. In contrast, SCB technical transfer was not straightforward, with significant differences observed between the two processes, 100% (SE) and 58% (PDR). This report underlines the importance of feedstock-specific pretreatment strategies to underpin process development, scale-up, and optimisation of carbohydrate release from biomass.

Glucose and xylose are two major components of lignocellulose. Simultaneous consumption of glucose and xylose is critical for engineered microorganisms to produce fuels and chemicals from lignocellulosic biomass. Although many production limitations have been resolved, glucose-induced inhibition of xylose transport remains a challenge. In this study, a cell growth-based screening strategy was designed to identify xylose transporters uninhibited by glucose. The glucose pathway was genetically blocked in Escherichia coli so that glucose functions only as an inhibitor and cells need xylose as the carbon source for survival. Through adaptive evolution, omics analysis and reverse metabolic engineering, a new phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) galactitol transporter (GalABC, encoded by EcolC_1640, EcolC_1641, and EcolC_1642 genes) that is not inhibited by glucose was identified. Inactivation of adenylate cyclase led to increased expression of the EcolC_1642 gene, and a point mutation in gene EcolC_1642 (N13S) further enhanced xylose transport. During the second round of gene mining, AraE and a new ABC transporter (AraFGH) of xylose were identified. A point mutation in the transcription regulator araC (L156I) caused increased expression of araE and araFGH genes without arabinose induction, and a point mutation in araE (D223Y) further enhanced xylose transport. These newly identified xylose transporters can support the simultaneous consumption of glucose and xylose and have potential use in producing chemicals from lignocellulose.

UNASSIGNED: Pectobacterium betavasculorum is a member of the Pectobacerium genus that inhabits a variety of niches and is found in all climates. Bacteria from the Pectobacterium genus can cause soft rot disease on various plants due to the secretion of plant cell wall degrading enzymes (PCWDEs). The species P. betavasculorum is responsible for the vascular necrosis of sugar beet and soft rot of many vegetables. It also infects sunflowers and artichokes. The main sugar present in sugar beet is sucrose while xylose is one of the main sugars in artichoke and sunflower.
UNASSIGNED: In our work, we applied metabolomic studies coupled with genomics to investigate the metabolism of P. betavasculorum in the presence of xylose and sucrose as the only carbon source. The ability of the strains to use various sugars as the only carbon source were confirmed by the polypyridyl complex of Ru(II) method in 96-well plates.
UNASSIGNED: Our studies provided information on the metabolic pathways active during the degradation of those substrates. It was observed that different metabolic pathways are upregulated in the presence of xylose in comparison to sucrose.
UNASSIGNED: The presence of xylose enhances extracellular metabolism of sugars and glycerol as well as stimulates EPS and IPS synthesis. In contrast, in the presence of sucrose the intensive extracellular metabolism of amines and amino acids is promoted.

Lignocellulose is mainly composed of hydrophobic lignin and hydrophilic polysaccharide polymers, contributing to an indispensable carbon resource for green biorefineries1,2. When chemically treated, lignin is compromised owing to detrimental intra- and intermolecular crosslinking that hampers downstream process3,4. The current valorization paradigms aim to avoid the formation of new C-C bonds, referred to as condensation, by blocking or stabilizing the vulnerable moieties of lignin5-7. Although there have been efforts to enhance biomass utilization through the incorporation of phenolic additives8,9, exploiting lignin\'s proclivity towards condensation remains unproven for valorizing both lignin and carbohydrates to high-value products. Here we leverage the proclivity by directing the C-C bond formation in a catalytic arylation pathway using lignin-derived phenols with high nucleophilicity. The selectively condensed lignin, isolated in near-quantitative yields while preserving its prominent cleavable β-ether units, can be unlocked in a tandem catalytic process involving aryl migration and transfer hydrogenation. Lignin in wood is thereby converted to benign bisphenols (34-48 wt%) that represent performance-advantaged replacements for their fossil-based counterparts. Delignified pulp from cellulose and xylose from xylan are co-produced for textile fibres and renewable chemicals. This condensation-driven strategy represents a key advancement complementary to other promising monophenol-oriented approaches targeting valuable platform chemicals and materials, thereby contributing to holistic biomass valorization.