Mixture Of Glucose Research Articles

Metabolic engineering of Corynebacterium glutamicum for the production of anthranilate from glucose and xylose.

Anthranilate and its derivatives are important basic chemicals for the synthesis of polyurethanes as well as various dyes and food additives. Today, anthranilate is mainly chemically produced from petroleum-derived xylene, but this shikimate pathway intermediate could be also obtained biotechnologically. In this study, Corynebacterium glutamicum was engineered for the microbial production of anthranilate from a carbon source mixture of glucose and xylose. First, a feedback-resistant 3-deoxy-arabinoheptulosonate-7-phosphate synthase from Escherichia coli, catalysing the first step of the shikimate pathway, was functionally introduced into C. glutamicum to enable anthranilate production. Modulation of the translation efficiency of the genes for the shikimate kinase (aroK) and the anthranilate phosphoribosyltransferase (trpD) improved product formation. Deletion of two genes, one for a putative phosphatase (nagD) and one for a quinate/shikimate dehydrogenase (qsuD), abolished by-product formation of glycerol and quinate. However, the introduction of an engineered anthranilate synthase (TrpEG) unresponsive to feedback inhibition by tryptophan had the most pronounced effect on anthranilate production. Component I of this enzyme (TrpE) was engineered using a biosensor-based invivo screening strategy for identifying variants with increased feedback resistance in a semi-rational library of TrpE muteins. The final strain accumulated up to 5.9 g/L (43 mM) anthranilate in a defined CGXII medium from a mixture of glucose and xylose in bioreactor cultivations. We believe that the constructed C. glutamicum variants are not only limited to anthranilate production but could also be suitable for the synthesis of other biotechnologically interesting shikimate pathway intermediates or any other aromatic compound derived thereof.

Proteomic Analysis of Pleurotus ostreatus Grown on Glucose and Xylose Mixtures in Submerged Fermentation Provides Insights into Differentiated Mycelial Composition

Proteomic Analysis of Pleurotus ostreatus Grown on Glucose and Xylose Mixtures in Submerged Fermentation Provides Insights into Differentiated Mycelial Composition

Formulation of 10% Glucose as a Mixture of Practicum Materials for Pregnancy Diabetes Mellitus Examination Skills in the Laboratory

In laboratory-based learning, the use of formulated materials is crucial. A formulated glucose solution, with its predetermined composition, ready for use in observing Diabetes Mellitus (DM) scenarios in the laboratory, enables students to focus on direct experiences in simulating real-world situations. This enhances learning efficiency and provides greater opportunities for practical experiences. The objective of this research is to develop a formulation of a glucose 10% and aquadest mixture as synthetic urine to detect DM in laboratory skills activities. The experiment involved testing and observing a mixture of glucose 10% and aquadest. The resulting formula was tested using test strips and a heating procedure with Benedict's reagent. Five formulations, each prepared with a volume of 100 ml, yielded observations ranging from positive 1 to positive 4. Formula I, a mixture of 5 ml glucose 10% and 95 ml aquadest, resulted in a positive 4 observation. Formula II, with 4 ml glucose 10% and 96 ml aquadest, yielded a positive 3 observation. Formula III, with 3 ml glucose 10% and 97 ml aquadest, resulted in a positive 2 observation. Formula IV, with 2 ml glucose 10% and 98 ml aquadest, also produced a positive 2 observation. Formula V, a mixture of 1 ml glucose 10% and 99 ml aquadest, resulted in a positive 1 observation. It is recommended to prepare the mixture just before the activity, ensuring the cleanliness of containers to maintain the stability of the materials used. Further studies are needed to refine the formula with additional parameters

1,4-Dioxane intervention enables simultaneous valorization of biomass-based C5 and C6 sugars to furfural over Hβ zeolite

The simultaneous valorization of biomass-based C5 and C6 sugars to furfural, a versatile intermediate, is of great significance to fully utilize biomass components. Inability to effectively transform C6 sugars remains a major barrier toward this challenging goal. We herein report the 1,4-dioxane-mediated solvent system with Hβ zeolite catalyst for the transformation of glucose giving high yield to furfural (57%). The maintenance of intrinsic balanced Lewis and Brønsted acid sites from Hβ zeolite that is credited to the positive solvent effect of 1,4-dioxane is highly conductive to the selective C-C bond cleavage of glucose. Also importantly, the introduction of proper amount of H2O into the reaction system results in a high formation rate and an improved selectivity toward furfural due to the induction of highly reactive acyclic glucose. Glucose-to-furfural through 3-deoxyglucose should be more favorable than that via fructose. Impressively, the developed protocol can also be adapted to C5 sugars dehydration, and the one-pot conversion of glucose and xylose mixtures gains a furfural yield as high as 63%. The activation energy barrier for furfural production is higher from glucose compared to xylose. This work not only develops a promising protocol for furfural production simultaneously from C5 and C6 sugars but also provides insights into the mechanistic role of the solvent 1,4-dioxane to availably manipulate the acid-catalyzed conversion of C6 sugars.

Engineering a Synthetic Escherichia coli Coculture for Compartmentalized de novo Biosynthesis of Isobutyl Butyrate from Mixed Sugars.

Short-chain esters are versatile chemicals that can be used as flavors, fragrances, solvents, and fuels. The de novo ester biosynthesis consists of diverging and converging pathway submodules, which is challenging to engineer to achieve optimal metabolic fluxes and selective product synthesis. Compartmentalizing the pathway submodules into specialist cells that facilitate pathway modularization and labor division is a promising solution. Here, we engineered a synthetic Escherichia coli coculture with the compartmentalized sugar utilization and ester biosynthesis pathways to produce isobutyl butyrate from a mixture of glucose and xylose. To compartmentalize the sugar-utilizing pathway submodules, we engineered a xylose-utilizing E. coli specialist that selectively consumes xylose over glucose and bypasses carbon catabolite repression (CCR) while leveraging the native CCR machinery to activate a glucose-utilizing E. coli specialist. We found that the compartmentalization of sugar catabolism enabled simultaneous co-utilization of glucose and xylose by a coculture of the two E. coli specialists, improving the stability of the coculture population. Next, we modularized the isobutyl butyrate pathway into the isobutanol, butyl-CoA, and ester condensation submodules, where we distributed the isobutanol submodule to the glucose-utilizing specialist and the other submodules to the xylose-utilizing specialist. Upon compartmentalization of the isobutyl butyrate pathway submodules into these sugar-utilizing specialist cells, a robust synthetic coculture was engineered to selectively produce isobutyl butyrate, reduce the biosynthesis of unwanted ester byproducts, and improve the production titer as compared to the monoculture.

VALIDATION OF AN ANALYTICAL METHOD FOR THE QUANTIFICATION OF 1-KESTOSE, GLUCOSE AND SUCROSE IN THE REACTION PRODUCTS OF A FRUCTOSYLTRANSFERASE

The enzymatic synthesis of fructooligosaccharides (FOS) from sucrose by the action of fructosyltransferases produces a mixture of different carbohydrates. The concentration of each carbohydrate involved must be quantified to follow its variation as the kinetics of the reaction progresses. This requires a method such as high-performance liquid chromatography (HPLC) with refractive index detector (IR). The aim of this work was to validate the HPLC-IR analytical method for the quantification of total and individual carbohydrates in mixtures of 1-kestose, sucrose and glucose. For this study, the validation parameters recommended by the FDA (Food and Drug Administration), the ICH (International Council for Harmonisation) and the EMEA (European Medicines Agency) were followed. The method was able to quantify carbohydrates with adequate levels of precision, accuracy and linearity in ranges of 1.6-8.7, 4.3-22.0 and 7.0-41.0 mg/mL for glucose, 1-kestose and sucrose, respectively. The lowest limit of detection was for glucose (0.2 mg/mL) and the highest for sucrose (0.8 mg/mL), while the lowest and highest limit of quantification were also for glucose (0.6 mg/mL) and sucrose (1.8 mg/mL). The detection (0.7 mg/mL) and quantification (1.4 mg/mL) limits for 1-kestose were intermediate. The HPLC-IR method was used to calculate the concentration of the carbohydrates involved in the reaction kinetics of a recombinant fructosyltransferase (Sa1-SSTrec). This analytical technique can be implemented for the analysis of samples from the FOS production process at both laboratory and industrial scale, but also the technique is useful to detect 1-kestose in functional foods based in fruit juices.

Engineering polysaccharide hydrolases in the product-releasing cleft to alter their product profiles

Polysaccharide hydrolases are enzymes capable of hydrolyzing polysaccharides to generate oligosaccharides that have diverse applications in the food, feed and pharmaceutical industries. However, the detailed mechanisms governing the compositions of their hydrolysates remain poorly understood. Previously, we identified a novel neopullulase Amy117, which exclusively converts pullulan to panose by specifically cleaving α-1,4-glycosidic bonds. Yet, several enzymes with high homology to Amy117 produce a mixture of glucose, maltose and panose during pullulan hydrolysis. To explore this particular phenomenon, we compared the sequences and structures between Amy117 and the maltose amylase ThMA, and identified a specific residue Thr299 in Amy117 (equivalent to His294 in ThMA) within the product-releasing cleft of Amy117, which might be responsible for this characteristic feature. Using structure-based rational design, we have successfully converted the product profiles of pullulan hydrolysates between Amy117 and ThMA by simply altering this key residue. Molecular docking analysis indicated that the key residue at the product-releasing outlet altered the product profile by affecting the panose release rate. Moreover, we modeled the long-chain pullulan substrate G8 to examine its potential conformations and found that G8 might undergo a conformational change in the narrow cleft that allows the Amy117 variant to specifically recognize α-1,6-glycosidic bonds.

Authigenic Mineral Formation in Aquifers near the Uranium Sludge Storage Facility of Chepetsky Mechanical Plant during the Formation of a Biogeochemical Barrier in a Laboratory and Field Experiment

In this work, authigenic microbial mineral formation in groundwater near the uranium sludge storage at SC Chepetsky Mechanical Plant (ChMP) (Glazov, Russia) was analysed in field and laboratory experiments using thermodynamic modelling when the microbial community was activated by a mixture of acetate, glucose and whey. It was found that the mineral basis of the barrier consisted of aggregated soil particles with freshly deposited phases of carbonate and sulphide minerals of different degrees of crystallinity. An important factor in the formation of calcium phases is microbial denitrification, which is accompanied by an increase in pH values of the medium. The main factors of uranium immobilisation in the biogeochemical barrier were revealed, including its reduction to insoluble forms of uranium dioxide, adsorption on ferrous and sulphide-ferrous minerals, as well as the formation of phosphate phases through the addition of phosphorus-containing whey and co-precipitation or co-crystallisation in calcite phases.

Tocopherol Content of Euglena sp. Isolated from Yogyakarta under Glucose and Ethanol Mixture Treatment

Euglena sp. is a microalgae with significant potential for utilization as a high-value product because of the presence of protein, lipid, paramylon, and other compounds. Even though these microalgae may be found in freshwater, research on enhancing Euglena sp. cultivation is still limited in Indonesia. Tocopherols are antioxidants that can effectively protect against diseases caused by oxidative stress. The isomer of tocopherol with the highest biological activity is α-tocopherol. Euglena sp. cells had the highest levels of α-tocopherol compared to other microorganisms. Scientists are continuously trying to determine how to obtain a high α-tocopherol concentration and a significant Euglena cell biomass. Photosynthetic organisms culture has been found to boost α-tocopherol content in Euglena sp., although heterotrophic culture can potentially increase biomass. This study used photoheterotrophic culture with a mixture of glucose and ethanol to increase the α-tocopherol and biomass concentration inside the culture of the local strain of Euglena sp. The addition of treatments in a glucose and ethanol combination with levels of 3:2; 2.5: 2.5; 2: 2; and 0:0 (control) g L-1 was used in this study to assess the impact of Euglena sp. culture on growth, biomass, and α-tocopherol concentration. According to the findings of this study, the 3:2 treatment produced the most significant specific growth rate and biomass, including 0.992 (OD680/OD680/day) and 8.480 (g L-1). In contrast, the 2.5:2.5 treatment produced the highest α-tocopherol content, specifically 7.09±0.096 mg L-1.

A field bioassay for assessing ivermectin bio-efficacy in wild malaria vectors

BackgroundIvermectin (IVM) mass drug administration is a candidate complementary malaria vector control tool. Ingestion of blood from IVM treated hosts results in reduced survival in mosquitoes. Estimating bio-efficacy of IVM on wild-caught mosquitoes requires they ingest the drug in a blood meal either through a membrane or direct feeding on a treated host. The latter, has ethical implications, and the former results in low feeding rates. Therefore, there is a need to develop a safe and effective method for IVM bio-efficacy monitoring in wild mosquitoes.MethodsInsectary-reared Anopheles gambiae s.s. were exposed to four IVM doses: 85, 64, 43, 21 ng/ml, and control group (0 ng/ml) in three different solutions: (i) blood, (ii) 10% glucose, (iii) four ratios (1:1, 1:2, 1:4, 1:8) of blood in 10% glucose, and fed through filter paper. Wild-caught An. gambiae s.l. were exposed to 85, 43 and 21 ng/ml IVM in blood and 1:4 ratio of blood-10% glucose mixture. Survival was monitored for 28 days and a pool of mosquitoes from each cohort sacrificed immediately after feeding and weighed to determine mean weight of each meal type.ResultsWhen administered in glucose solution, mosquitocidal effect of IVM was not comparable to the observed effects when similar concentrations were administered in blood. Equal concentrations of IVM administered in blood resulted in pronounced reductions in mosquito survival compared to glucose solution only. However, by adding small amounts of blood to glucose solution, mosquito mortality rates increased resulting in similar effects to what was observed during blood feeding.ConclusionBio-efficacy of ivermectin is strongly dependent on mode of drug delivery to the mosquito and likely influenced by digestive processes. The assay developed in this study is a good candidate for field-based bio-efficacy monitoring: wild mosquitoes readily feed on the solution, the assay can be standardized using pre-selected concentrations and by not involving treated blood hosts (human or animal) variation in individual pharmacokinetic profiles as well as ethical issues are bypassed. Meal volumes did not explain the difference in the lethality of IVM across the different meal types necessitating further research on the underlying mechanisms.

Solvent-free Synthesis of Transition Metals Nanoparticles Decorated Foamy Flakes-like Nitrogen-doped Carbon as Bifunctional-catalyst for High-performance Rechargeable Zn-air Batteries

Transition metal-nitrogen-carbon (M/NC) demonstrates a promising effective electrocatalyst for enhancing oxygen evolution/reduction reactions (OER/ORR). However, synthesizing these catalysts is often complex, time-consuming, and involves hazardous solvents while producing a low yield. This work introduces a versatile, eco-friendly, and straightforward solvent-free method to produce M/NC (M = Co, Ni, and Fe) catalysts in ~3 h using a glucose, urea, and metal nitrate hydrate mixture. The high-yield M/NC catalysts exhibit a porous architecture and uniform distribution of metal nanoparticles within a foaming flakes-like nitrogen-doped carbon matrix. The metal nanoparticles are wrapped with protective nitrogen-doped carbon layers, producing stable active sites. Possessing these unique properties, the obtained M/NC catalysts show high-performance and stable bifunctional OER/ORR. As the best result, Co/NC presents an OER/ORR potential difference (ΔE) of 0.78 V and retains 96% and 89% of its OER and ORR performance after a 10 h stability test. In practical application, the Co/NC-based Zn-air battery depicts a high power density of 184 mW cm−2 and good rechargeability of up to 120 h, outperforming the battery with noble metal-based catalysts. This work sheds light on the versatile, eco-friendly, and scalable synthesis of M/NC catalysts while presenting a strategy to accelerate the generation of inexpensive and highly effective bifunctional OER/ORR catalysts.

Simultaneous carbon catabolite repression governs sugar and aromatic co-utilization in Pseudomonas putida M2.

Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatic compounds, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatic compounds simultaneously; however, during cultivation with glucose and aromatic compounds (p-coumarate and ferulate) mixture, intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during cultivation with aromatic compounds implying simultaneous catabolite repression by sugars and aromatic compounds. Reduction of crc expression via CRISPRi led to faster growth and glucose and p-coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+p-coumarate. The increased abundances of Eda and amino acid biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose+p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only). IMPORTANCE A newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatic compounds making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatic compounds present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures, while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.

CRISPR/Cas9 mediated gene editing of transcription factor ACE1 for enhanced cellulase production in thermophilic fungus Rasamsonia emersonii

BackgroundThe filamentous fungus Rasamsonia emersonii has immense potential to produce biorefinery relevant thermostable cellulase and hemicellulase enzymes using lignocellulosic biomass. Previously in our lab, a hyper-cellulase producing strain of R. emersonii was developed through classical breeding and system biology approaches. ACE1, a pivotal transcription factor in fungi, plays a crucial role in negatively regulating the expression of cellulase genes. In order to identify the role of ACE1 in cellulase production and to further improve the lignocellulolytic enzyme production in R. emersonii, CRISPR/Cas9 mediated disruption of ACE1 gene was employed.ResultsA gene-edited ∆ACE1 strain (GN11) was created, that showed 21.97, 20.70 and 24.63, 9.42, 18.12%, improved endoglucanase, cellobiohydrolase (CBHI), β-glucosidase, FPase, and xylanase, activities, respectively, as compared to parental strain M36. The transcriptional profiling showed that the expression of global regulator (XlnR) and different CAZymes genes including endoglucanases, cellobiohydrolase, β-xylosidase, xylanase, β-glucosidase and lytic polysaccharide mono-oxygenases (LPMOs) were significantly enhanced, suggesting critical roles of ACE1 in negatively regulating the expression of various key genes associated with cellulase production in R. emersonii. Whereas, the disruption of ACE1 significantly down-regulated the expression of CreA repressor gene as also evidenced by 2-deoxyglucose (2-DG) resistance phenotype exhibited by edited strain GN11 as well as appreciably higher constitutive production of cellulases in the presence of glucose and mixture of glucose and disaccharide (MGDs) both in batch and flask fed batch mode of culturing. Furthermore, ∆ACE1 strains were evaluated for the hydrolysis of biorefinery relevant steam/acid pretreated unwashed rice straw slurry (Praj Industries Ltd; 15% substrate loading rate) and were found to be significantly superior when compared to the benchmark enzymes produced by parent strain M36 and Cellic Ctec3.ConclusionsCurrent work uncovers the crucial role of ACE1 in regulating the expression of the various cellulase genes and carbon catabolite repression mechanism in R. emersonii. This study represents the first successful report of utilizing CRISPR/Cas9 genome editing technology to disrupt the ACE1 gene in the thermophlic fungus R. emersonii. The improved methodologies presented in this work might be applied to other commercially important fungal strains for which genetic manipulation tools are limited.

Metabolic engineering of Halomonas bluephagenesis for high-level mevalonate production from glucose and acetate mixture

Mevalonate (MVA) plays a crucial role as a building block for the biosynthesis of isoprenoids. In this study, we engineered Halomonas bluephagenesis to efficiently produce MVA. Firstly, by screening MVA synthetases from eight different species, the two efficient candidate modules, specifically NADPH-dependent mvaESEfa from Enterococcus faecalis and NADH-dependent mvaESLca from Lactobacillus casei, were integrated into the chromosome, leading to the construction of the H. bluephagenesis MVA11. Through the synergetic utilization of glucose and acetate as mixed carbon sources, MVA11 produced 11.2 g/L MVA with a yield of 0.45 g/g (glucose + acetic acid) in the shake flask. Subsequently, 10 beneficial genes out of 50 targets that could promote MVA production were identified using CRISPR interference. The simultaneous repression of rpoN (encoding RNA polymerase sigma-54 factor) and IldD (encoding L-lactate dehydrogenase) increased MVA titer (13.3 g/L) by 19.23% and yield (0.53 g/g (glucose + acetic acid)) by 17.78%, respectively. Furthermore, introducing the non-oxidative glycolysis (NOG) pathway into MVA11 enhanced MVA yield by 12.20%. Ultimately, by combining these strategies, the resultant H. bluephagenesis MVA13/pli-63 produced 13.9 g/L MVA in the shake flask, and the yield increased to 0.56 g/g (glucose + acetic acid), which was the highest reported so far. Under open fed-batch fermentation conditions, H. bluephagenesis MVA13/pli-63 produced 121 g/L of MVA with a yield of 0.42 g/g (glucose + acetic acid), representing the highest reported titer and yield in the bioreactor to date. This study demonstrates that H. bluephagenesis is one of the most favorable chassis for MVA production.

Quantitative Analysis of The High-Yield Hydrolysis of Kelp by Laminarinase and Alginate Lyase.

Kelp is an abundant, farmable biomass-containing laminarin and alginate as major polysaccharides, providing an excellent model substrate to study their deconstruction by simple enzyme mixtures. Our previous study showed strong reactivity of the glycoside hydrolase family 55 during hydrolysis of purified laminarin, raising the question of its reactivity with intact kelp. In this study, we determined that a combination of a single glycoside hydrolase family 55 β-1,3-exoglucanase with a broad-specificity alginate lyase from the polysaccharide lyase family 18 gives efficient hydrolysis of untreated kelp to a mixture of simple sugars, that is, glucose, gentiobiose, mannitol-end glucose, and mannuronic and guluronic acids and their soluble oligomers. Quantitative assignments from nanostructure initiator mass spectrometry (NIMS) and 2D HSQC NMR spectroscopy and analysis of the reaction time-course are provided. The data suggest that binary combinations of enzymes targeted to the unique polysaccharide composition of marine biomass are sufficient to deconstruct kelp into soluble sugars for microbial fermentation.

Co-Fermentation of Glucose–Xylose–Cellobiose–XOS Mixtures Using a Synthetic Consortium of Recombinant Saccharomyces cerevisiae Strains

The efficient conversion of cellulosic sugars is vital for the economically viable production of biofuels/biochemicals from lignocellulosic biomass hydrolysates. Based on comprehensive screening, Saccharomyces cerevisiae RC212 was chosen as the chassis strain for multiple integrations of heterologous β-glucosidase and β-xylosidase genes in the present study. The resulting recombinant BLN26 and LF1 form a binary synthetic consortium, and this co-culture system achieved partial fermentation of four sugars (glucose, xylose, cellobiose, and xylo-oligosaccharides). Then, we developed a ternary S. cerevisiae consortium consisting of LF1, BSGIBX, and 102SB. Almost all four sugars were efficiently fermented to ethanol within 24 h, and the ethanol yield is 0.482 g g−1 based on the consumed sugar. To our knowledge, this study represents the first exploration of the conversion of mixtures of glucose, xylose, cellobiose, and xylo-oligosaccharides by a synthetic consortium of recombinant S. cerevisiae strains. This synthetic consortium and subsequent improved ones have the potential to be used as microbial platforms to produce a wide array of biochemicals from lignocellulosic hydrolysates.

The Influence of Trctf1 Gene Knockout by CRISPR–Cas9 on Cellulase Synthesis by Trichoderma reesei with Various Soluble Inducers

Knockout of the transcriptional repressor Trctf1 is known to enhance the yield of cellulose-induced cellulase synthesis in Trichoderma reesei. However, different inducers possess distinct induction mechanisms, and the effect of Trctf1 on cellulase synthesis with soluble inducers remains unknown. To evaluate the effect of the Trctf1 gene on cellulase synthesis and develop a high-yielding cellulase strain, we established a CRISPR–Cas9 genome editing system in T. reesei Rut C30 using codon-optimized Cas9 protein and in vitro transcribed RNA. This study demonstrated that T. reesei ΔTrctf1 with the Trctf1 gene knocked out showed no statistically significant differences in cellulase, cellobiohydrolase, endoglucanase, and β−glucosidase production when induced with MGD (the mixture of glucose and sophorose). However, when induced with lactose, the activities of these enzymes increased by 20.2%, 12.4%, and 12.9%, respectively, with no statistically significant differences in β−glucosidase activity. The hydrolysis efficiency on corn stover of cellulases produced by T. reesei ΔTrctf1 under different inducers was not significantly different from that of wild-type cellulases, indicating that Trctf1 gene deletion has little effect on the cellulase cocktail. These findings contribute to a better understanding of the molecular mechanisms underlying the regulation of T. reesei cellulase synthesis by different soluble inducers, as well as the construction of high-yield cellulase gene−engineered strains.

Determining the metabolic effects of dietary fat, sugars and fat-sugar interaction using nutritional geometry in a dietary challenge study with male mice

The metabolic effects of sugars and fat lie at the heart of the “carbohydrate vs fat” debate on the global obesity epidemic. Here, we use nutritional geometry to systematically investigate the interaction between dietary fat and the major monosaccharides, fructose and glucose, and their impact on body composition and metabolic health. Male mice (n = 245) are maintained on one of 18 isocaloric diets for 18–19 weeks and their metabolic status is assessed through in vivo procedures and by in vitro assays involving harvested tissue samples. We find that in the setting of low and medium dietary fat content, a 50:50 mixture of fructose and glucose (similar to high-fructose corn syrup) is more obesogenic and metabolically adverse than when either monosaccharide is consumed alone. With increasing dietary fat content, the effects of dietary sugar composition on metabolic status become less pronounced. Moreover, higher fat intake is more harmful for glucose tolerance and insulin sensitivity irrespective of the sugar mix consumed. The type of fat consumed (soy oil vs lard) does not modify these outcomes. Our work shows that both dietary fat and sugars can lead to adverse metabolic outcomes, depending on the dietary context. This study shows how the principles of the two seemingly conflicting models of obesity (the “energy balance model” and the “carbohydrate insulin model”) can be valid, and it will help in progressing towards a unified model of obesity. The main limitations of this study include the use of male mice of a single strain, and not testing the metabolic effects of fructose intake via sugary drinks, which are strongly linked to human obesity.

Validation of a pulp-type candy (Pulpijamay) based on the by-product of the hibiscus flower (Hibiscus sabdariffa)

There is a diversity of sweets in confectionery according to times and countries, according to regions and religions where there is no limitation in the preparation of the sweet depending on the imagination of each confectioner. With the jamaica flower (Hibiscus sabdariffa) various food products are made and currently with pharmaceutical interest, certain products are attributed beneficial qualities in weight loss, favoring the digestive process in a good way, it is slightly laxative and is also diuretic, reason for which it has cleansing and detoxifying effects, despite these benefits, in general only the supernatant is used and the flower is discarded as organic waste. Therefore, in the present research work, it seeks the use of the pulp of the jamaican flower (Hibiscus sabdariffa) developing a sweet, carrying out tests with different binding substances and concentrations, obtaining samples of paste to later be mixed with sucrose, salt and chili pequin. The same procedure was carried out for gum arabic, glucose and the glucose-sucrose mixture. Physicochemical results were obtained from the product, such as pH, ºBrix and the desired viscosity for the Jamaica flower candy.

Increased Flux of Lipid Metabolism Enhances Bioethanol Fermentability and Inhibitor Tolerance of Xylose-Utilizing Zymomonas mobilis

The microbial production of fuel ethanol is an attractive and sustainable biotechnological approach. This study presents a metabolic engineering strategy of Zymomonas mobilis aimed at coproducing bioethanol and fatty acids. The increased flux of fatty acids stabilizes the cell membrane and thus counteracts the progressively higher ethanol toxicity. In a glucose medium, the highest ethanol titer achieved was 146.7 g/kg of broth, surpassing the wild-type Z. mobilis CP4 and angel yeast by 30% and 45%, respectively. The recombinant strain exhibited a total fatty acid titer of 0.4 g/L from 230 g/L total sugar solution (5 L bioreactor), representing a 12-fold increase compared to the wild-type Z. mobilis CP4. Furthermore, when using a 4:2:1 mixture of glucose: xylose: mannose (w/v), an ethanol concentration of 142.8 g/kg of broth was attained, only 2.66% lower than that of the glucose-only medium. These findings highlight the enormous potential of this genetically engineered strain for the sustainable production of ethanol and fatty acids from lignocellulosic renewable carbon sources.