Hexose Sugars Research Articles

Process Intensification and Reaction Kinetics for Synthesis of 5‐Hydroxymethyl Furfural Using DICAT‐1 Solid Acid Catalyst in a Batch Reactor

Abstract5‐HMF is a potential platform multifaceted molecule for various industrial applications and can be produced from biomass‐based hexose sugars. In the present study, the indigenously prepared catalyst DICAT‐1 (an acronym derived from DBT‐ICT Center for Energy Biosciences Catalyst #1) has been explored for fructose dehydration, using isopropyl alcohol (IPA) as a green and low boiling point (LBP) organic solvent. The exploration of heterogeneous DICAT‐1 adds specific advantages for 5‐HMF synthesis in terms of high yield, conversion, selectivity, catalyst separation, recycle, reuse, and so forth. Different variants of DICAT‐1, such as DICAT‐1A, DICAT‐1B, DICAT‐1C, and DICAT‐1D, having variable surface acidity were tested for fructose dehydration in the presence of IPA. Of the tested variants, DICAT‐1C provides good yield and selectivity of 5‐HMF. Thus, further process optimization study was conducted to obtain maximum yield, conversion, and selectivity using DICAT‐1C. The intensified process provides maximum 78% yield of 5‐HMF with 83% fructose conversion and 94% selectivity. The catalyst recyclability study showed the consistency in 5‐HMF yield, conversion, and selectivity for five consecutive recycle runs. The study of reaction kinetics showed the first‐order kinetics with an activation energy of 13.16 kJ/mole by using DICAT‐1C catalyst. Thus, the use of easily recyclable and robust catalyst provides an efficient route for production of 5‐HMF in presence of green solvent.

Integrated kinetic modeling of acetone-butanol-ethanol fermentation by Clostridium saccharoperbutilacetonicum N1-4: Co-utilization strategy with cane molasses, biomass-derived sugars, and furaldehydes

Sugar-based byproducts such as molasses and bagasse are abundant resources for large-scale biobutanol commercialization. This study presents a comprehensive kinetic model to analyze ABE (acetone-butanol-ethanol) fermentation by C. saccharoperbutilacetonicum N1-4, emphasizing diverse carbon sources and furaldehyde co-utilization. By leveraging sensitivity analysis against models in the literature and the proposed model as well as parameter optimization using Scilab®, the potential for the simultaneous utilization of sugarcane molasses and bagasse-derived sugars alongside furfural and HMF for butanol synthesis was assessed. These findings revealed that despite the microbial preference for hexose sugars, the incorporation of up to 25% pentose sugars did not significantly hinder butanol production. However, furaldehydes perturbed butyric acid assimilation and acetyl-CoA consumption while enhancing acetone-butanol-ethanol synthesis. The simulation highlighted the peak concentrations of furfural and HMF (0.33 mmol L−1 and 0.03 mmol L−1, respectively), underscoring their conversion to less toxic alcohols, thereby augmenting metabolism. Sensitivity analysis underscores the necessity of enhancing butyric acid assimilation, providing critical insights for optimizing ABE fermentation and advancing biobutanol production from sugar-based byproducts.

Expanded genome and proteome reallocation in a novel, robust Bacillus coagulans strain capable of utilizing pentose and hexose sugars.

Bacillus coagulans, a Gram-positive thermophilic bacterium, is recognized for its probiotic properties and recent development as a microbial cell factory. Despite its importance for biotechnological applications, the current understanding of B. coagulans' robustness is limited, especially for undomesticated strains. To fill this knowledge gap, we characterized the metabolic capability and performed functional genomics and systems analysis of a novel, robust strain, B. coagulans B-768. Genome sequencing revealed that B-768 has the largest B. coagulans genome known to date (3.94 Mbp), about 0.63 Mbp larger than the average genome of sequenced B. coagulans strains, with expanded carbohydrate metabolism and mobilome. Functional genomics identified a well-equipped genetic portfolio for utilizing a wide range of C5 (xylose, arabinose), C6 (glucose, mannose, galactose), and C12 (cellobiose) sugars present in biomass hydrolysates, which was validated experimentally. For growth on individual xylose and glucose, the dominant sugars in biomass hydrolysates, B-768 exhibited distinct phenotypes and proteome profiles. Faster growth and glucose uptake rates resulted in lactate overflow metabolism, which makes B. coagulans a lactate overproducer; however, slower growth and xylose uptake diminished overflow metabolism due to the high energy demand for sugar assimilation. Carbohydrate Transport and Metabolism (COG-G), Translation (COG-J), and Energy Conversion and Production (COG-C) made up 60%-65% of the measured proteomes but were allocated differently when growing on xylose and glucose. The trade-off in proteome reallocation, with high investment in COG-C over COG-G, explains the xylose growth phenotype with significant upregulation of xylose metabolism, pyruvate metabolism, and tricarboxylic acid (TCA) cycle. Strain B-768 tolerates and effectively utilizes inhibitory biomass hydrolysates containing mixed sugars and exhibits hierarchical sugar utilization with glucose as the preferential substrate.IMPORTANCEThe robustness of B. coagulans makes it a valuable microorganism for biotechnology applications; yet, this phenotype is not well understood at the cellular level. Through phenotypic characterization and systems analysis, this study elucidates the functional genomics and robustness of a novel, undomesticated strain, B. coagulans B-768, capable of utilizing inhibitory switchgrass biomass hydrolysates. The genome of B-768, enriched with carbohydrate metabolism genes, demonstrates high regulatory capacity. The coordination of proteome reallocation in Carbohydrate Transport and Metabolism (COG-G), Translation (COG-J), and Energy Conversion and Production (COG-C) is critical for effective cell growth, sugar utilization, and lactate production via overflow metabolism. Overall, B-768 is a novel, robust, and promising B. coagulans strain that can be harnessed as a microbial biomanufacturing platform to produce chemicals and fuels from biomass hydrolysates.

Biohydrogen production from lignocellulosic hydrolysate: Unveiling the synergistic impact of substrate concentration and furfural inhibition.

Lignocellulosic biomass offers substantial potential as an ideal feedstock for dark fermentative hydrogen production due to its sustainability and cost-effectiveness. The current study examined the influence of furfural on fermentative hydrogen production using lignocellulosic hydrolysate in the presence of furfural. Synthetic lignocellulosic hydrolysate, consisting primarily of 76% xylose, 10% glucose, 9% arabinose, and a mixture of other sugars such as galactose and mannose (85% pentose sugars and 15% hexose sugars), was employed as the substrate. Various substrate concentrations ranging from 2 to 32g/L were tested, along with furfural concentrations of 0, 1, and 2g/L. The investigation aimed to assess the effects of initial substrate concentration, initial furfural concentration, furfural-to-biomass ratio (F/B), and furfural-to-substrate ratio (F/S) on biohydrogen production yields. The maximum specific substrate utilization rates at different substrate concentrations were effectively characterized using Haldane's substrate inhibition model. Among the tested concentrations, the 16g/L emerged as the optimal substrate concentration. The initial furfural concentration was identified as the most significant parameter impacting biohydrogen production, with complete inhibition observed at a furfural concentration of 2g/L. Higher F/S ratios at substrate concentrations ranging from 2 to 16g/L resulted in reduced maximum specific hydrogen production rates (MSHPR) and hydrogen yields. Substrate inhibition was observed at 24g/L and 32g/L. Lactate was the predominant metabolite in all batches containing 2-g/L furfural, as well as in batches with 1-g/L furfural and substrate concentrations of 24 and 32g/L. Furfural at a concentration of 1g/L was not inhibitory in any of the batches. Overall, the mixed cultures in this study could efficiently produce hydrogen from lignocellulosic hydrolysates and degrade furfural, providing new insights into fermentative hydrogen-producing bacteria with furfural tolerance.

Characterization of ATP-dependent phosphofructokinase genes during ripening and their modulation by phytohormones during postharvest storage of citrus fruits (Citrus reticulata Blanco.)

The level of sweetness in citrus fruit is crucial for consumer appeal and market competitiveness, determined mainly by soluble sugars and organic acids. ATP-dependent 6-phosphofructokinase is central to regulating sugar metabolism, yet its role in citrus fruit ripening and postharvest storage remains underexplored. We characterized phosphofructokinase genes in citrus, identifying eight genes classified into pyrophosphate-dependent phosphofructokinase (PFP) and ATP-dependent 6-phosphofructokinase (PFK) subgroups using phylogenetic analysis, genomic architectures, and protein motifs. Comparative genomic analysis with other plants highlighted significant protein homology among CitPFKs. The motif analysis indicated conserved phosphofructokinase domains in CitPFK sequences, with upstream promoter regions containing diverse cis-regulatory elements, most notably light-responsive (LREs). The gene expression profiling throughout fruit development and ripening revealed differential patterns, with responses to gibberellic acid and salicylic acid phytohormones during postharvest indicating their roles in regulating CitPFK genes. The analysis of the transcriptome showed high expression of ATP-dependent 6-phosphofructokinase 3 (CitPFK3) during fruit development, indicating a positive role in fruit maturation. Consequently, silencing CitPFK3 through virus-induced gene silencing (VIGS) increased hexose sugar content, suggesting its function in sugar accumulation. These findings improve our understanding of PFKs in citrus, particularly CitPFK3's pivotal role in regulating hexose sugar dynamics and their modulation by exogenous phytohormones after harvest. This study provides a foundation for optimizing soluble sugar regulation to enhance fruit quality and postharvest handling in citrus production.

Mutual Relationships of Nanoconfined Hexoses: Impacts on Hydrodynamic Radius and Anomeric Ratios.

Although all hexose sugars share the same chemical formula, C6H12O6, subtle differences in their stereochemical structures lead to their various biological roles. Due to their prominent role in metabolism, hexose sugars are commonly found in nanoconfined environments. The complexity of authentic nanoconfined biological environments makes it challenging to study how confinement affects their behavior. Here, we present a study using a common model system, AOT reverse micelles, to study hexose sugars in nanoconfinement. We examine how reverse micelles affect the hexoses, how the hexoses affect reverse micelle formation, and the differences between specific hexoses: glucose, mannose, and galactose. We find that addition of glucose, mannose or galactose to reverse micelles that already contain water leaves their size smaller or nearly unchanged. Introducing aqueous hexose solution yields reverse micelles smaller than those prepared with the same volume of water. We use 1H NMR to show how the nanoconfined environment impacts hexose sugars' anomeric ratios. Nanoconfined mannose and galactose display smaller changes in their anomeric ratios compared to glucose. These conclusions may provide insights about the biological roles of each hexose when studied under a more authentic nanoconfined system.

Molecular identification of rhamnolipids produced by Pseudomonas oryzihabitans during biodegradation of crude oil.

The ability to produce biosurfactants plays a meaningful role in the bioavailability of crude oil hydrocarbons and the bioremediation efficiency of crude oil-degrading bacteria. This study aimed to characterize the produced biosurfactants by Pseudomonas oryzihabitans during the biodegradation of crude oil hydrocarbons. The biosurfactants were isolated and then characterized by Fourier transform infrared (FTIR), liquid chromatography-mass-spectrometry (LC-MS), and nuclear magnetic resonance spectroscopy (NMR) analyses. The FTIR results revealed the existence of hydroxyl, carboxyl, and methoxyl groups in the isolated biosurfactants. Also, the LC-MS analysis demonstrated a main di-rhamnolipid (l-rhamnopyranosyll-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoate, Rha-Rha-C10-C10) along with a mono-rhamnolipid (l-rhamnopyranosyl-b-hydroxydecanoylb-hydroxydecanoate, Rha-C10-C10). In agreement with these findings, the NMR analysis confirmed the aromatic, carboxylic, methyl, sulfate moieties, and hexose sugar in the biosurfactants. The emulsion capacity of the biosurfactants decreased the surface tension of the aqueous system from 73.4 mN m-1 to around 33 mN m-1 at 200 mg L-1 as the critical micelle concentration. The emulsification capacity of the biosurfactants in the formation of a stable microemulsion for the diesel-water system at a wide range of pH (2-12), temperature (0-80°C), and salinity (2-20 g L-1 of NaCl) showed their potential use in oil recovery and bioremediation through the use of microbial enhancement. This work showed the ability of Pseudomonas oryzihabitans NC392 cells to produce rhamnolipid molecules during the biodegradation process of crude oil hydrocarbons. These biosurfactants have potential in bioremediation studies as eco-friendly and biodegradable products, and their stability makes them optimal for areas with extreme conditions.

Optimizing dilute acid pretreatment for enhanced recovery and co-fermentation of hexose and pentose sugars for ethanol and butanol production

A systematic study of dilute acid pretreatment of sugarcane bagasse was performed to optimize the recovery of hexose-rich solid and pentose-rich liquid while minimizing fermentation inhibitors. Pretreatment experiments evaluated the effects of pretreatment time (10–90 min) and acid concentration (0.5–3.0 % w/w). The optimized pretreatment (1.4 % w/w sulfuric acid, 56.4 min at 121 °C) led to a solid fraction with 60.4 % cellulose and a liquid stream rich in pentose, in which 92.7 % of sugars was recovered as xylose (17.8 g/L). Besides that, low concentrations of by-products (3 g/L – the sum of acetic and formic acid, furfural, and HMF) were identified in the liquid fraction. Both fractions were integrated into enzymatic processes for further co-fermentation of C5- and C6- sugars. Two fermentative routes were evaluated: simultaneous isomerization of xylose and co-fermentation of glucose and xylulose (SIcF) for 2G ethanol production and acetone-butanol-ethanol (ABE) fermentation of glucose and xylose for biobutanol, utilizing Saccharomyces cerevisiae and Clostridium acetobutylicum, respectively. SIcF yielded 0.48 gethanol/gsugars (24 g/L ethanol) with complete glucose consumption and 58.2 % xylose conversion. ABE fermentation produced 0.36 gABE/gsugars (15.7 g/L ABE and 9.5 g/L butanol), with 96 % glucose consumption and 29.6 % xylose conversion. The proof-of-concept demonstrated that the hydrolysates were satisfactorily fermentable by microorganisms, thus validating the pretreatment optimization for fermentation applications.

Characterization and biological activities of a novel exopolysaccharide produced from Geobacillus thermodenitrificans HBB 111 strain.

In this study, EPS production conditions of Geobacillus thermodenitrificans HBB 111, a thermophilic microorganism, were optimized and the amount of produced EPS (EPS 111) was found to be 44.0mg/L. EPS 111 was purified using ion exchange chromatography and gel filtration chromatography, and a single type of exopolysaccharide was obtained. The structure of the purified EPS 111 was evaluated by TLC, FTIR, NMR, and GC-MS, and it was observed that it contained hexose (glucose, fructose, galactose and mannose) and pentose sugars. From the SEM photographs, it was understood that EPS 111 had an amorphous, rough, and layered structure. It was found that purified EPS 111 had low cytotoxicity (2.3%) and exhibited high antioxidant activity and remarkable antidiabetic, prebiotic and fibrinolytic activities. It is very valuable that the purified EPS 111 in this study offers multiple biological activities compared to the thermophilic EPSs reported in the literature and has a high potential for use in biotechnological and biomedical fields.

Onion (Allium cepa L.) Attracts Scoliid Wasps by Means of Generalist Floral Volatiles.

Onion flowers require pollinator-mediated cross-pollination. However, the cues that pollinators use to locate the flowers are not well understood. The floral scent, along with floral visual cues, might acts as important signal to pollinators in order to locate the floral resources. We used electrophysiological methods combined with behavioural assays to determine which compounds in a floral scent are more attractive and thus biologically important to foraging scollid wasps. The majority of the molecules identified as floral fragrances in onions are common compounds that are already known from other angiosperms, and onion floral scents were predominately composed of aromatic components. The antennae of scoliid wasps responded to a large number of compounds, among them o-cymene, cis-β-ocimene, benzaldehyde and allo-ocimene were behaviourally active. In contrast to other wasp flowers investigated nectar analysis demonstrated the dominance of hexose sugars over sucrose. Our findings provide fresh insights into the floral volatile chemistry of a key vegetable crop grown around the world. We demonstrate here that onion is using generalist floral volatiles to attract floral visitors. This insight could be utilised to make onion blooms more attractive to minor pollinators as well as major pollinators in order to maximise seed set.

The genotype of barley cultivars influences multiple aspects of their associated microbiota via differential root exudate secretion.

Plant-associated microbes play vital roles in promoting plant growth and health, with plants secreting root exudates into the rhizosphere to attract beneficial microbes. Exudate composition defines the nature of microbial recruitment, with different plant species attracting distinct microbiota to enable optimal adaptation to the soil environment. To more closely examine the relationship between plant genotype and microbial recruitment, we analysed the rhizosphere microbiomes of landrace (Chevallier) and modern (NFC Tipple) barley (Hordeum vulgare) cultivars. Distinct differences were observed between the plant-associated microbiomes of the 2 cultivars, with the plant-growth promoting rhizobacterial genus Pseudomonas substantially more abundant in the Tipple rhizosphere. Striking differences were also observed between the phenotypes of recruited Pseudomonas populations, alongside distinct genotypic clustering by cultivar. Cultivar-driven Pseudomonas selection was driven by root exudate composition, with the greater abundance of hexose sugars secreted from Tipple roots attracting microbes better adapted to growth on these metabolites and vice versa. Cultivar-driven selection also operates at the molecular level, with both gene expression and the abundance of ecologically relevant loci differing between Tipple and Chevallier Pseudomonas isolates. Finally, cultivar-driven selection is important for plant health, with both cultivars showing a distinct preference for microbes selected by their genetic siblings in rhizosphere transplantation assays.

Effects of foetal size, sex and developmental stage on adaptive transcriptional responses of skeletal muscle to intrauterine growth restriction in pigs

Intrauterine growth restriction (IUGR) occurs both in humans and domestic species. It has a particularly high incidence in pigs, and is a leading cause of neonatal morbidity and mortality as well as impaired postnatal growth. A key feature of IUGR is impaired muscle development, resulting in decreased meat quality. Understanding the developmental origins of IUGR, particularly at the molecular level, is important for developing effective strategies to mitigate its economic impact on the pig industry and animal welfare. The aim of this study was to characterise transcriptional profiles in the muscle of growth restricted pig foetuses at different gestational days (GD; gestational length ~ 115 days), focusing on selected genes (related to development, tissue injury and metabolism) that were previously identified as dysregulated in muscle of GD90 fetuses. Muscle samples were collected from the lightest foetus (L) and the sex-matched foetus with weight closest to the litter average (AW) from each of 22 Landrace x Large White litters corresponding to GD45 (n = 6), GD60 (n = 8) or GD90 (n = 8), followed by analyses, using RT-PCR and protein immunohistochemistry, of selected gene targets. Expression of the developmental genes, MYOD, RET and ACTN3 were markedly lower, whereas MSTN expression was higher, in the muscle of L relative to AW littermates beginning on GD45. Levels of all tissue injury-associated transcripts analysed (F5, PLG, KNG1, SELL, CCL16) were increased in L muscle on GD60 and, most prominently, on GD90. Among genes involved in metabolic regulation, KLB was expressed at higher levels in L than AW littermates beginning on GD60, whereas both IGFBP1 and AHSG were higher in L littermates on GD90 but only in males. Furthermore, the expression of genes specifically involved in lipid, hexose sugar or iron metabolism increased or, in the case of UCP3, decreased in L littermates on GD60 (UCP3, APOB, ALDOB) or GD90 (PNPLA3, TF), albeit in the case of ALDOB this only involved females. In conclusion, marked dysregulation of genes with critical roles in development in L foetuses can be observed from GD45, whereas for a majority of transcripts associated with tissue injury and metabolism differences between L and AW foetuses were apparent by GD60 or only at GD90, thus identifying different developmental windows for different types of adaptive responses to IUGR in the muscle of porcine foetuses.

Metabolic pathways for glucose and fructose: I synthesis and metabolism of fructose by ovine conceptuses†.

Fructose, the most abundant hexose sugar in fetal fluids and the blood of sheep and other ungulates and cetaceans, is synthesized from glucose via the polyol pathway in trophectoderm and chorion. However, the cell-specific and temporal expression of enzymes for the synthesis and metabolism of fructose in sheep conceptuses (embryo and placental membranes) and placentomes has not been characterized. This study characterized key enzymes involved in fructose synthesis and metabolism by ovine conceptuses throughout pregnancy. Day 17 conceptuses expressed mRNAs for the polyol pathway (SORD and AKR1B1) and glucose and fructose metabolism (HK1, HK2, G6PD, OGT, and FBP), but not those required for gluconeogenesis (G6Pase or PCK). Ovine placentomes also expressed mRNAs for SORD, AKR1B1, HK1, and OGT. Fructose can be metabolized via the ketohexokinase (KHK) pathway, and isoforms, KHK-A and KHK-C, were expressed in ovine conceptuses from Day 16 of pregnancy and placentomes during pregnancy in a cell-specific manner. The KHK-A protein was more abundant in the trophectoderm and cotyledons of placentomes, while KHK-C protein was more abundant in the endoderm of Day 16 conceptuses and the chorionic epithelium in placentomes. Expression of KHK mRNAs in placentomes was greatest at Day 30 of pregnancy (P < 0.05), but not different among days later in gestation. These results provide novel insights into the synthesis and metabolism of fructose via the uninhibited KHK pathway in ovine conceptuses to generate ATP via the tricarboxylic cycle, as well as substrates for the pentose cycle, hexosamine biosynthesis pathway, and one-carbon metabolism required for conceptus development throughout pregnancy.

The Effect of Fermentation Time, pH and Saccharomyces Cerevisiae Concentration for Bioethanol Production from Ulva Reticulata Macroalgae

Research has been carried out on the effect of pH, fermentation time and yeast concentration on bioethanol production through hydrolysis using a CEM (Ceramic Elekctromagnetic Microwave) synthesizer and bioethanol production from Ulva reticulata seaweed. Ulva reticulata seaweed contains carbohydrates in the form of heteropolysaccharides such as glucose, arabinose, ramnose and xylose which are very abundant and suitable for conversion into bioethanol because the people of Timor Island do not use them as food. The carbohydrate content of Ulva reticulata seaweed can be converted into hexose and pentose sugars (glucose, arabinose, ramnose and xylose) through hydrolysis using 3 types of acid catalysts, namely hydrochloric acid (HCl), sulfuric acid (H2SO4) and nitric acid (HNO3). Fermentation was carried out with S. cerevisiae concentration variation of 6; 8; 10; 12 % (v/v) and fermentation time variation of 3; 5; 7; 9 days and a pH variation of 4; 4.5; 5; 5.5 at a temperature of 30 °C. Reducing sugar characterization used the Dinitrosalicylic acid (DNS) reagent, sample surface texture analysis was carried out using Scanning Electron Microscopy (SEM) and ethanol characterization used Gas Chromatography-Flame Ionization Detector (GC-FID). The results of surface texture analysis before and after hydrolysis experienced significant changes. Optimal conditions for hydrolysis of Ulva reticulata seaweed using sulfuric acid (H2SO4) combined with a CEM synthesizer at an acid concentration of 3 % (v/v) with irradiation power of 200 watts for 50 min at a temperature of 150 °C, produced reducing sugar of 97.06 g/L. The results of GC-FID analysis indicated that bioethanol concentration obtained at optimal conditions of pH 4.5 was 42.32 %, S. cerevisiae concentration was 12 % with a bioethanol content of 42.53 % at a fermentation time of 5 days. This research is expected to provide information for researchers and industry to overcome world energy problems and environmental pollution through Ulva reticulata waste. HIGHLIGHTS The results of surface texture analysis before and after hydrolysis experienced significant changes. Optimum conditions for hydrolysis of Ulva reticulata seaweed using sulfuric acid (H2SO4) combined with a CEM synthesizer at an acid concentration of 3 % (v/v) with irradiation power of 200 watts for 50 min at a temperature of 150 °C, producing reducing sugar of 97.06 g /L. GRAPHICAL ABSTRACT

Nutritional Enhancement of Polimaize Lines: Integrating Native Mexican Maize Alleles into High-Yield Varieties

In this study, we evaluated Polimaize lines, named Polimaize, through the integration of alleles from the Native Blue Corn (NBC-JIQ), a local Mexican maize variety indigenous to the northeast region of Michoacán, Mexico, into elite maize lines curated by the International Maize and Wheat Improvement Center (CIMMYT). This crossbreeding aimed to enhance the nutritional profile of maize, particularly in terms of antioxidants and anthocyanins, which are scarce in elite lines. Our results demonstrated a significant increase in these compounds in the Polimaize lines, with variations due to heterosis. Despite these nutritional improvements, some traits showed decreased concentrations compared to parent lines, notably in sucrose and tryptophan, suggesting a potential trade-off. The study also found significant heritability in amino acids and tryptophan, while hexose sugars showed no substantial heritability. The Polimaize variety exhibited high starch content heritability, comparable to elite lines. Field trials confirmed Polimaize’s promising yield and agronomic traits, highlighting its potential for enhancing consumer health and contributing to sustainable agriculture through enriched crop nutritional quality. This project underscores the value of integrating local race alleles into elite lines, offering genetic diversity in maize cultivation.

Ab Initio and Kinetic Modeling of β-d-Xylopyranose under Fast Pyrolysis Conditions.

Lignocellulosic biomass is an abundant renewable resource that can be upgraded to chemical and fuel products through a range of thermal conversion processes. Fast pyrolysis is a promising technology that uses high temperatures and fast heating rates to convert lignocellulose into bio-oils in high yields in the absence of oxygen. Hemicellulose is one of the three major components of lignocellulosic biomass and is a highly branched heteropolymer structure made of pentose, hexose sugars, and sugar acids. In this study, β-d-xylopyranose is proposed as a model structural motif for the essential chemical structure of hemicellulose. The gas-phase pyrolytic reactivity of β-d-xylopyranose is thoroughly investigated using computational strategies rooted in quantum chemistry. In particular, its thermal degradation potential energy surfaces are computed employing Minnesota global hybrid functional M06-2X in conjunction with the 6-311++G(d,p) Pople basis set. Electronic energies are further refined by performing DLPNO-CCSD(T)-F12 single-point calculations on top of M06-2X geometries using the cc-pVTZ-F12 basis set. Conformational analysis for minima and transition states is performed with state-of-the-art semiempirical quantum chemical methods coupled with metadynamics simulations. Key thermodynamic quantities (free energies, barrier heights, enthalpies of formation, and heat capacities) are computed. Rate coefficients for the initial steps of thermal decomposition are computed by means of reaction rate theory. For the first time, a detailed elementary reaction kinetic model for β-d-xylopyranose is developed by utilizing the thermodynamic and kinetic information acquired from the aforementioned calculations. This model specifically targets the initial stages of β-d-xylopyranose pyrolysis in the high-pressure limit, aiming to gain a deeper understanding of its reaction kinetics. This approach establishes a systematic strategy for exploring reactive pathways, evaluating competing parallel reactions, and selectively accepting or discarding pathways based on the analysis. The findings suggest that acyclic d-xylose plays a significant role as an intermediary in the production of key pyrolytic compounds during the pyrolysis of xylose. These compounds include furfural, anhydro-d-xylopyranose, glycolaldehyde, and dihydrofuran-3(2H)-one.

Simultaneous glucose and xylose utilization by an Escherichia coli catabolite repression mutant.

As advances are made toward the industrial feasibility of mass-producing biofuels and commodity chemicals with sugar-fermenting microbes, high feedstock costs continue to inhibit commercial application. Hydrolyzed lignocellulosic biomass represents an ideal feedstock for these purposes as it is cheap and prevalent. However, many microbes, including Escherichia coli, struggle to efficiently utilize this mixture of hexose and pentose sugars due to the regulation of the carbon catabolite repression (CCR) system. CCR causes a sequential utilization of sugars, rather than simultaneous utilization, resulting in reduced carbon yield and complex process implications in fed-batch fermentation. A mutant of the gene encoding the cyclic AMP receptor protein, crp*, has been shown to disable CCR and improve the co-utilization of mixed sugar substrates. Here, we present the strain construction and characterization of a site-specific crp* chromosomal mutant in E. coli BL21 star (DE3). The crp* mutant strain demonstrates simultaneous consumption of glucose and xylose, suggesting a deregulated CCR system. The proteomics further showed that glucose was routed to the C5 carbon utilization pathways to support both de novo nucleotide synthesis and energy production in the crp* mutant strain. Metabolite analyses further show that overflow metabolism contributes to the slower growth in the crp* mutant. This highly characterized strain can be particularly beneficial for chemical production by simultaneously utilizing both C5 and C6 substrates from lignocellulosic biomass.IMPORTANCEAs the need for renewable biofuel and biochemical production processes continues to grow, there is an associated need for microbial technology capable of utilizing cheap, widely available, and renewable carbon substrates. This work details the construction and characterization of the first B-lineage Escherichia coli strain with mutated cyclic AMP receptor protein, Crp*, which deregulates the carbon catabolite repression (CCR) system and enables the co-utilization of multiple sugar sources in the growth medium. In this study, we focus our analysis on glucose and xylose utilization as these two sugars are the primary components in lignocellulosic biomass hydrolysate, a promising renewable carbon feedstock for industrial bioprocesses. This strain is valuable to the field as it enables the use of mixed sugar sources in traditional fed-batch based approaches, whereas the wild-type carbon catabolite repression system leads to biphasic growth and possible buildup of non-preferential sugars, reducing process efficiency at scale.

Isopropanol production via the thermophilic bioconversion of sugars and syngas using metabolically engineered Moorella thermoacetica

BackgroundIsopropanol (IPA) is a commodity chemical used as a solvent or raw material for polymeric products, such as plastics. Currently, IPA production depends largely on high-CO2-emission petrochemical methods that are not sustainable. Therefore, alternative low-CO2 emission methods are required. IPA bioproduction using biomass or waste gas is a promising method.ResultsMoorella thermoacetica, a thermophilic acetogenic microorganism, was genetically engineered to produce IPA. A metabolic pathway related to acetone reduction was selected, and acetone conversion to IPA was achieved via the heterologous expression of secondary alcohol dehydrogenase (sadh) in the thermophilic bacterium. sadh-expressing strains were combined with acetone-producing strains, to obtain an IPA-producing strain. The strain produced IPA as a major product using hexose and pentose sugars as substrates (81% mol-IPA/mol-sugar). Furthermore, IPA was produced from CO, whereas acetate was an abundant byproduct. Fermentation using syngas containing both CO and H2 resulted in higher IPA production at the specific rate of 0.03 h−1. The supply of reducing power for acetone conversion from the gaseous substrates was examined by supplementing acetone to the culture, and the continuous and rapid conversion of acetone to IPA showed a sufficient supply of NADPH for Sadh.ConclusionsThe successful engineering of M. thermoacetica resulted in high IPA production from sugars. M. thermoacetica metabolism showed a high capacity for acetone conversion to IPA in the gaseous substrates, indicating acetone production as the bottleneck in IPA production for further improving the strain. This study provides a platform for IPA production via the metabolic engineering of thermophilic acetogens.

Exploring carbon source related localization and phosphorylation in the Snf1/Mig1 network using population and single cell-based approaches.

The AMPK/SNF1 pathway governs energy balance in eukaryotic cells, notably influencing glucose de-repression. In S. cerevisiae, Snf1 is phosphorylated and hence activated upon glucose depletion. This activation is required but is not sufficient for mediating glucose de-repression, indicating further glucose-dependent regulation mechanisms. Employing fluorescence recovery after photobleaching (FRAP) in conjunction with non-linear mixed effects modelling, we explore the spatial dynamics of Snf1 as well as the relationship between Snf1 phosphorylation and its target Mig1 controlled by hexose sugars. Our results suggest that inactivation of Snf1 modulates Mig1 localization and that the kinetic of Snf1 localization to the nucleus is modulated by the presence of non-fermentable carbon sources. Our data offer insight into the true complexity of regulation of this central signaling pathway in orchestrating cellular responses to fluctuating environmental cues. These insights not only expand our understanding of glucose homeostasis but also pave the way for further studies evaluating the importance of Snf1 localization in relation to its phosphorylation state and regulation of downstream targets.

Comparison of simultaneous saccharification and fermentation with LPMO-supported hybrid hydrolysis and fermentation.

Enzymatic saccharification is used to convert polysaccharides in lignocellulosic biomass to sugars which are then converted to ethanol or other bio-based fermentation products. The efficacy of commercial cellulase preparations can potentially increase if lytic polysaccharide monooxygenase (LPMO) is included. However, as LPMO requires both a reductant and an oxidant, such as molecular oxygen, a reevaluation of process configurations and conditions is warranted. Saccharification and fermentation of pretreated softwood was investigated in demonstration-scale experiments with 10m3 bioreactors using an LPMO-containing cellulase preparation, a xylose-utilizing yeast, and either simultaneous saccharification and fermentation (SSF) or hybrid hydrolysis and fermentation (HHF) with a 24-hour or 48-hour initial phase and with 0.15 vvm aeration before addition of the yeast. The conditions used for HHF, especially with 48h initial phase, resulted in better glucan conversion, but in poorer ethanol productivity and in poorer initial ethanol yield on consumed sugars than the SSF. In the SSF, hexose sugars such as glucose and mannose were consumed faster than xylose, but, in the end of the fermentation >90% of the xylose had been consumed. Chemical analysis of inhibitory pretreatment by-products indicated that the concentrations of heteroaromatic aldehydes (such as furfural), aromatic aldehydes, and an aromatic ketone decreased as a consequence of the aeration. This was attributed mainly to evaporation caused by the gas flow. The results indicate that further research is needed to fully exploit the advantages of LPMO without compromising fermentation conditions.