Non-oxidative Pathway Research Articles

A novel effect of sulforaphane on promoting mouse granulosa cells proliferation via the NRF2–TKT pathway

IntroductionGranulosa cells (GCs) is essential for maintaining follicular development. Follicle-stimulating Hormone (FSH) has been demonstrated to effectively promote GCs proliferation, driving the establishment of various superovulation techniques for animal husbandry. However, these techniques face challenges, such as high costs, hormonal imbalances, and an increased risk of early ovarian dysfunction. Therefore, it is important to investigate new methods to improve GCs proliferation. ObjectivesThis study aimed to investigate the effect of sulforaphane (SFN) on ovarian GCs proliferation and the underlying mechanisms. MethodsA comparative transcriptomic analysis of ovaries from the control, SFN, and FSH groups was conducted to identify the primary factors contributing to high proliferative capacity. The role of SFN in the regulation of cell proliferation has been examined in mouse ovarian GCs. Gene interference, overexpression, CUT&TAG technology, and transcriptome analyses were performed to elucidate the underlying mechanisms of the nuclear factor E2-related factor 2 (NRF2)–transketolase (TKT) axis in mediating GCs proliferation. ResultsOur research revealed a previously unknown function of SFN, an isothiocyanate of plant origin that is prevalent in cruciferous vegetables, in facilitating the proliferation of mouse ovarian GCs. The efficacy of SFN in enhancing GCs proliferation is similar to that of FSH. At the mechanistic level, SFN promotes NRF2 to transport to the nucleus, which subsequently activates the key enzyme of the non-oxidative pentose phosphate pathway TKT. This activation is instrumental in generating ribose 5-phosphate, a critical precursor for amino acid and nucleotide biosynthesis that underpins the proliferation of GCs. ConclusionCollectively, our findings delineate a novel pathway by which SFN, through the NRF2-TKT axis, enhances the nucleotide pool and thereby supports the proliferation of mouse GCs, presenting novel avenues for exploration in reproductive biology and agricultural sciences.

Engineering a non-oxidative glycolysis pathway in escherichia coli for high-level citramalate production

BackgroundMethyl methacrylate (MMA) is a key precursor of polymethyl methacrylate, extensively used as a transparent thermoplastic in various industries. Conventional MMA production poses health and environmental risks; hence, citramalate serves as an alternative bacterial compound precursor for MMA production. The highest citramalate titer was previously achieved by Escherichia coli BW25113. However, studies on further improving citramalate production through metabolic engineering are limited, and phage contamination is a persistent problem in E. coli fermentation.ResultsThis study aimed to construct a phage-resistant E. coli BW25113 strain capable of producing high citramalate titers from glucose. First, promoters and heterologous cimA genes were screened, and an effective biosynthetic pathway for citramalate was established by overexpressing MjcimA3.7, a mutated cimA gene from Methanococcus jannaschii, regulated by the BBa_J23100 promoter in E. coli. Subsequently, a phage-resistant E. coli strain was engineered by integrating the Ssp defense system into the genome and mutating key components of the phage infection cycle. Then, the strain was engineered to include the non-oxidative glycolysis pathway while removing the acetate synthesis pathway to enhance the supply of acetyl-CoA. Furthermore, glucose utilization by the strain improved, thereby increasing citramalate production. Ultimately, 110.2 g/L of citramalate was obtained after 80 h fed-batch fermentation. The citramalate yield from glucose and productivity were 0.4 g/g glucose and 1.4 g/(L·h), respectively.ConclusionThis is the highest reported citramalate titer and productivity in E. coli without the addition of expensive yeast extract and additional induction in fed-bath fermentation, emphasizing its potential for practical applications in producing citramalate and its derivatives.

Dysregulation of tetrahydrobiopterin metabolism in myalgic encephalomyelitis/chronic fatigue syndrome by pentose phosphate pathway.

Tetrahydrobiopterin (BH4) and its oxidized derivative dihydrobiopterin (BH2) were found to be strongly elevated in ME/CFS patients with orthostatic intolerance (ME + OI). However, the molecular mechanism of biopterin biogenesis is poorly understood in ME + OI subjects. Here, we report that the activation of the non-oxidative pentose phosphate pathway (PPP) plays a critical role in the biogenesis of biopterins (BH4 and BH2) in ME + OI subjects. Microarray-based gene screening followed by real-time PCR-based validation, ELISA assay, and finally enzyme kinetic studies of glucose-6-phosphate dehydrogenase (G6PDH), transaldolase (TALDO1), and transketolase (TK) enzymes revealed that the augmentation of anaerobic PPP is critical in the regulations of biopterins. To further investigate, we devised a novel cell culture strategy to induce non-oxidative PPP by treating human microglial cells with ribose-5-phosphate (R5P) under a hypoxic condition of 85%N2/10%CO2/5%O2 followed by the analysis of biopterin metabolism via ELISA, immunoblot, and dual immunocytochemical analyses. Moreover, the siRNA knocking down of the taldo1 gene strongly inhibited the bioavailability of phosphoribosyl pyrophosphate (PRPP), reduced the expressions of purine biosynthetic enzymes, attenuated GTP cyclohydrolase 1 (GTPCH1), and suppressed subsequent production of BH4 and its metabolic conversion to BH2 in R5P-treated and hypoxia-induced C20 human microglia cells. These results confirmed that the activation of non-oxidative PPP is indeed required for the upregulation of both BH4 and BH2 via the purine biosynthetic pathway. To test the functional role of ME + OI plasma-derived biopterins, exogenously added plasma samples of ME + OI plasma with high BH4 upregulated inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in human microglial cells indicating that the non-oxidative PPP-induced-biopterins could stimulate inflammatory response in ME + OI patients. Taken together, our current research highlights that the induction of non-oxidative PPP regulates the biogenesis of biopterins contributing to ME/CFS pathogenesis.

Comparative genomic analysis of an emerging Pseudomonadaceae member, Thiopseudomonas alkaliphila.

Thiopseudomonas alkaliphila, an organism recently classified within the Pseudomonadaceae family, has been detected in diverse sources such as human tissues, animal guts, industrial fermenters, and decomposition environments, suggesting a diverse ecological role. However, a large knowledge gap exists in how T. alkaliphila functions. In this comparative genomic analysis, adaptations indicative of habitat specificity among strains and genomic similarity to known opportunistic pathogens are revealed. Genomic investigation reveals a core metabolic utilization of multiple oxidative and non-oxidative catabolic pathways, suggesting adaptability to varied environments and carbon sources. The genomic repertoire of T. alkaliphila includes secondary metabolites, such as antimicrobials and siderophores, indicative of its involvement in microbial competition and resource acquisition. Additionally, the presence of transposases, prophages, plasmids, and Clustered Regularly Interspaced Short Palindromic Repeats-Cas systems in T. alkaliphila genomes suggests mechanisms for horizontal gene transfer and defense against viral predation. This comprehensive genomic analysis expands our understanding on the ecological functions, community interactions, and potential virulence of T. alkaliphila, while emphasizing its adaptability and diverse capabilities across environmental and host-associated ecosystems.IMPORTANCEAs the microbial world continues to be explored, new organisms will emerge with beneficial and/or pathogenetic impact. Thiopseudomonas alkaliphila is a species originally isolated from clinical human tissue and fluid samples but has not been attributed to disease. Since its classification, T. alkaliphila has been found in animal guts, animal waste, decomposing remains, and biogas fermentation reactors. This is the first study to provide an in-depth view of the metabolic potential of publicly available genomes belonging to this species through a comparative genomics and draft pangenome calculation approach. It was found that T. alkaliphila is metabolically versatile and likely adapts to diverse energy sources and environments, which may make it useful for bioremediation and in industrial settings. A range of virulence factors and antibiotic resistances were also detected, suggesting T. alkaliphila may operate as an undescribed opportunistic pathogen.

Retrospective Data Analysis Reveals Unusual Metabolism Pattern of Ethanol in Pediatrics as Compared to Adult and Geriatric Populations.

About 95% of consumed ethanol is metabolized by oxidative pathways. Less than 1% is metabolized via nonoxidative pathways: glucuronidation, sulfation, and the formation of fatty acid esters of ethanol. In neonates, the glucuronidation pathway has been reported to be underdeveloped but matures with age. This work compared the test results of patients' random urine samples submitted to our facility for ethyl glucuronide (EtG) and ethyl sulfate (EtS) measurements across pediatric and adult populations. Test results (n = 63 498) from urine samples tested for EtG and EtS by quantitative liquid chromatography-tandem mass spectrometry at our facility were utilized for this study. EtG and EtS concentrations were compared across the age partitions 0 to 17 years (pediatric), 18 to 80 years (adult), and 81 to 100 years (geriatric). Eight pediatric patients from a tertiary academic hospital contributed clinical context via abstracted clinical information. Across the individual age partitions, 60% to 65% of patients had both EtG and EtS present in urine. Approximately 5% to 10% of patients had only EtG, and 25% to 35% had neither metabolite present. The lowest percentages (<1.5%) had EtS present in the absence of EtG. Markedly, no pediatric patients had only EtS present; compared to the adult population, this was statistically significant (Fisher exact test, P = 0.025). From the data presented in this work, EtG is more prevalent relative to EtS in urine samples of patients assessed for ethanol exposure.

Reversing the directionality of reactions between non-oxidative pentose phosphate pathway and glycolytic pathway boosts mycosporine-like amino acid production in Saccharomyces cerevisiae

BackgroundMycosporine-like amino acids (MAAs) are a class of strongly UV-absorbing compounds produced by cyanobacteria, algae and corals and are promising candidates for natural sunscreen components. Low MAA yields from natural sources, coupled with difficulties in culturing its native producers, have catalyzed synthetic biology-guided approaches to produce MAAs in tractable microbial hosts like Escherichia coli, Saccharomyces cerevisiae and Corynebacterium glutamicum. However, the MAA titres obtained in these hosts are still low, necessitating a thorough understanding of cellular factors regulating MAA production.ResultsTo delineate factors that regulate MAA production, we constructed a shinorine (mycosporine-glycine-serine) producing yeast strain by expressing the four MAA biosynthetic enzymes from Nostoc punctiforme in Saccharomyces cerevisiae. We show that shinorine is produced from the pentose phosphate pathway intermediate sedoheptulose 7-phosphate (S7P), and not from the shikimate pathway intermediate 3-dehydroquinate (3DHQ) as previously suggested. Deletions of transaldolase (TAL1) and phosphofructokinase (PFK1/PFK2) genes boosted S7P/shinorine production via independent mechanisms. Unexpectedly, the enhanced S7P/shinorine production in the PFK mutants was not entirely due to increased flux towards the pentose phosphate pathway. We provide multiple lines of evidence in support of a reversed pathway between glycolysis and the non-oxidative pentose phosphate pathway (NOPPP) that boosts S7P/shinorine production in the phosphofructokinase mutant cells.ConclusionReversing the direction of flux between glycolysis and the NOPPP offers a novel metabolic engineering strategy in Saccharomyces cerevisiae.

Modelling of beer sensory staleness based on flavour instability parameters

Why was the work done: The deterioration of the flavour of fresh beer is challenging for the brewing industry. Despite extensive research on flavour instability, the focus has centred on a limited set of parameters, rather than taking a broader approach.&#x0D; How was the work done: in this study, the intent was to produce a flavour stable beer. Using a low kilning temperature, malt of low staling potential was used in combination with best brewing practice to produce three batches of unpasteurised top fermented pale beers at a pilot scale. Forty-three markers were analysed in the fresh and aged beers (30°C for 15, 30, 60 and 90 days). Staleness was evaluated by a trained sensory panel and multivariate data analysis was used to explore how the markers contribute to staleness.&#x0D; What are the main findings: Repeatability was achieved between replicate brews and, subsequently, staling. Polyphenols, haze, total reactive antioxidant potential (TRAP), iso-α-acids, colour, furfural, 2-methylpropanal and 2-methylbutanal showed a strong correlation with staleness. Staleness doubled after 60 days of storage at 30°C, despite volatile aldehydes remaining below their sensory thresholds, implying a synergistic effect of carbonyls contributing to staleness. A Partial Least Square (PLS) model was established, modelling the sensory staleness from 2-methylpropanal, furfural, TRAP and the trans-/cis-iso-α-acids ratio.&#x0D; Why is the work important: The staling phenomena could be reproduced in beers from parallel brewing trials with only minor variations. The four parameters in PLS modelling indicate that beer staling involves a combination of oxidative and non-oxidative pathways.

Cosmetic application of the stem-bark extract of Bertholletia excelsa H.B.K.

The Amazon has a rich biodiversity where many different plant species can be found. This diversity is an important source of bioactive substances, mainly due to the different structural components of their phytometabolites. Research for natural products is a strategy for the development of new agents in therapeutic applications, especially cosmetic applications, that have better pharmacological potential. Within this perspective, the objective of the study was to investigate the cosmetic application (anti-aging potential) of the stem-bark extract of Bertholletia excelsa H.B.K - (SBEBE), popularly known as the Brazil nut tree, here called SBEBE, a noble plant species of the Amazon that is rich in selenium. Enzymatic, glycation, proliferation, cell-healing, collagen quantification, toxicity and genotoxicity assays were used. Among the enzymes involved in the extracellular matrix of the skin, SBEBE was able to inhibit only elastase (62.67 ± 3.75) when compared to the standard sivelestat (89.04 ± 0.53), and the extract was also able to inhibit both the oxidative and the non-oxidative pathway. When cell toxicity in fibroblasts (MRC-5) and keratinocytes (HACAT) was evaluated, SBEBE did not present toxicity in 24 h of incubation. After this period, the extract showed average cytotoxicity in 48 and 72 h, but not enough to reach the concentration of 50% of MRC-5 fibroblasts. In the trypan blue assay, the extract promoted fibroblast proliferation in 24, 48 and 72 h of incubation, which was evaluated through exponential cell growth, with emphasis mainly on the lowest concentration with results higher than the standard. When the cell healing capacity was evaluated, in 48 h of exposure to fibroblast, SBEBE was able to induce a cell carpet (cell film) in the cell monolayer scratch assay. SBEBE stimulated collagen production at all concentrations tested. In the alkaline comet assay, at the lowest concentration, the extract did not induce DNA damage when compared to the reference drug doxorubicin. This study proved that SBEBE extract can be considered an ally in the treatment of skin anti-ageing as a possible biotechnological, phytocosmetic product.

Multidimensional Optimization of Saccharomyces cerevisiae for Carotenoid Overproduction.

Microbial synthesis of carotenoids is a highly desirable alternative to plant extraction and chemical synthesis. In this study, we investigated multidimensional strategies to improve the carotenoid synthesis in the industrial workhorse of Saccharomyces cerevisiae. First, we rewired the yeast central metabolism by optimizing non-oxidative glycolysis pathway for an improved acetyl-CoA supply. Second, we restricted the consumption of farnesyl pyrophosphate (FPP) by the down-regulation of squalene synthase using the PEST degron. Third, we further explored the human lipid binding/transfer protein saposin B (hSapB)-mediated metabolic sink for an enhanced storage of lipophilic carotenoids. Last, the copper-induced GAL expression system was engineered to function in the yeast-peptone-dextrose medium for an increased biomass accumulation. By combining the abovementioned strategies, the final engineered yeast produced 166.79 ± 10.43 mg/l β-carotene in shake flasks, which was nearly 5-fold improvement of the parental carotenoid-producing strain. Together, we envision that multidimensional strategies reported here might be applicable to other hosts for the future industrial development of carotenoid synthesis from renewable feedstocks.

Bayesian 13C-Metabolic Flux Analysis of Parallel Tracer Experiments in Granulocytes: A Directional Shift within the Non-Oxidative Pentose Phosphate Pathway Supports Phagocytosis.

The pentose phosphate pathway (PPP) plays a key role in the cellular regulation of immune function; however, little is known about the interplay of metabolic adjustments in granulocytes, especially regarding the non-oxidative PPP. For the determination of metabolic mechanisms within glucose metabolism, we propose a novel set of measures for 13C-metabolic flux analysis based on ex vivo parallel tracer experiments ([1,2-13C]glucose, [U-13C]glucose, [4,5,6-13C]glucose) and gas chromatography-mass spectrometry labeling measurements of intracellular metabolites, such as sugar phosphates and their fragments. A detailed constraint analysis showed that the permission range for net and irreversible fluxes was limited to a three-dimensional space. The overall workflow, including its Bayesian flux estimation, resulted in precise flux distributions and pairwise confidence intervals, some of which could be represented as a line due to the strength of their correlation. The principal component analysis that was enabled by these behaviors comprised three components that explained 99.6% of the data variance. It showed that phagocytic stimulation reversed the direction of non-oxidative PPP net fluxes from ribose-5-phosphate biosynthesis toward glycolytic pathways. This process was closely associated with the up-regulation of the oxidative PPP to promote the oxidative burst.

Human papillomavirus type 16 E6 promotes cervical cancer proliferation by upregulating transketolase enzymatic activity through the activation of protein kinase B.

Over 99% of precancerous cervical lesions are associated with human papillomavirus (HPV) infection, with HPV types 16 and 18 (especially type 16) found in over 70% of cervical cancer cases globally. E6, a critical HPV gene, triggers malignant proliferation by degrading p53; however, this mechanism alone cannot fully explain the oncogenic effects of HPV16 E6. Therefore, we aimed to investigate new targets of HPV oncogenic mechanisms. Our results revealed significant changes in nonoxidative pentose phosphate pathway (PPP) metabolites in HPV16-positive cells. However, the role of nonoxidative PPP in HPV-associated cell transformation and tumor development remained unexplored. In this study, we investigated the impact and mechanisms of HPV16 E6 on cervical cancer proliferation using the HPV-negative cervical cancer cell line (C33A). HPV16 E6 was found to promote cervical cancer cell proliferation both in vitro and in vivo, activating the nonoxidative PPP. Transketolase (TKT), a key enzyme in the nonoxidative PPP, is highly expressed in cervical cancer tissues and associated with poor prognosis. HPV16 E6 promotes cervical cancer cell proliferation by upregulating TKT activity through the activation of AKT. In addition, oxythiamine (OT), a TKT inhibitor, hindered tumor growth, with enhanced effects when combined with cisplatin (DDP). In conclusion, HPV16 E6 promotes cervical cancer proliferation by upregulating TKT activity through the activation of AKT. OT demonstrates the potential to inhibit HPV16-positive cervical cancer growth, and when combined with DDP, could further enhance the tumor-suppressive effect of DDP.

Metabolic engineering combined with enzyme engineering for overproduction of ectoine in Escherichia coli

Ectoine, a natural protective agent, is naturally synthesized at low titers by some extreme environment microorganisms that are usually difficult to culture. There is a need for an efficient and eco-friendly ectoine production process. In this study, Escherichia coli BL21(DE3) with the ectABC gene cluster from Halomonas venusta achieved 1.7 g/L ectoine. After optimizing the expression plasmid, 2.1 g/L ectoine was achieved. Besides, the aspartate kinase mutant LysCT311I from Corynebacterium glutamicum and aspartate semialdehyde dehydrogenase from Halomonas elongata were overexpressed to increase precursors supply. Furthermore, the rate-limiting enzyme EctB was semirationally engineered, and the E407D mutation enhanced ectoine production by 13.8 %. To improve acetyl-CoA supply, the non-oxidative glycolysis pathway was introduced. Overall, the optimized strain ECT9-5 produced 67.1 g/L ectoine by fed-batch fermentation with a 0.3 g/g of glucose and the kinetic model resulted in a good fit.

B-316 Retrospective Data Analysis Reveals Unusual Metabolism Pattern of Ethanol in Pediatrics as Compared to Adult and Geriatric Populations

Abstract Background Ethanol is utilized in many pharmaceutical formulations and in the treatment of methanol poisoning. It is also consumed recreationally and is the most abused drug globally. About 95% of consumed ethanol is metabolized by alcohol dehydrogenase into acetaldehyde, followed by conversion of the acetaldehyde into acetic acid by acetaldehyde dehydrogenase. Less than one percent is metabolized via nonoxidative pathways, which may include: glucuronidation, sulfation, and the formation of fatty acid esters of ethanol. In neonates the glucuronidation pathway has been reported to be underdeveloped and matures with age. In this work, the patterns and concentrations of phase II nonoxidative metabolites of ethanol, ethyl glucuronide (EtG) and ethyl sulfate (EtS), were assessed in random urine collections of pediatric, adult, and geriatric patients. Methods Test results (n = 63498) from urine samples tested for EtG and EtS by quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) at our facility were utilized for this study. For both analytes, the lower limit of positivity was 100 ng/mL. The age range under consideration was 0–100 years. Data was partitioned into age groups: 0–17 years, 18–80 years and 81–100 years. The EtG and EtS concentrations across the partitions were compared. Results Across all ages, 60%–65% of patients had both EtG and EtS present in urine. Between 5%–10% had EtG present without EtS, and 25%–35% of patients had neither of the ethanol metabolites present. In all age groups there was a low positivity rate of EtS without the presence of EtG (0%–1%). All age partitions showed a similar pattern with the highest percentages of patients showing the presence of both metabolites, and the lowest percentage of patients showing the presence of EtS only. Markedly, there were no pediatric patient samples that had the sulfated metabolite present in the absence of the glucuronidated metabolite; this was statistically significantly different than this proportion in adults (Fisher’s Exact test, P = 0.025). This finding suggests greater glucuronidation activity in the pediatric population. Conclusion From the data presented in this work, EtG is more prevalent relative to EtS in urine samples of patients assessed for ethanol exposure. This work also shows that glucuronidation may be the predominant pathway for ethanol metabolism in pediatric populations. Although the level and frequency of ethanol exposure, as well as the time between exposure and sampling were unknown for the patients studied, the data offers insights into the relative distribution of EtG and EtS in random urine samples for pediatric and adult populations.

Evaluation of bergenin in amylase enzyme inhibition and lipid uptake in liver cells

Changes in the global economy contribute to a sedentary lifestyle and higher caloric intake, increasing the incidence of metabolic diseases. It is estimated that the number of individuals diagnosed with metabolic diseases will increase by 25% by 2030 and 51% by 2045. Natural products have been tested in metabolic diseases with positive results, as shown by bergenin. Thus, this work aimed to evaluate the possible effects of bergenin in models of metabolic disease through in silico and in vitro experimental procedures. Bergenin showed the ability to inhibit protein glycation by 19.88 ± 4.22% in the oxidative pathway and 8.32 ± 1.85% in the non-oxidative pathway. This study showed for the first time that bergenin has the potential to inhibit the α-amylase enzyme (33.89 ± 0.96%) using in silico and in vitro assays. Bergenin was not able to significantly inhibit lipid uptake by the Oil Red method. After these results, it is understood that bergenin has the potential to be better studied as a therapeutic option in metabolic diseases. However, additional in vivo studies are needed to better elucidate the possible pharmacological effects of bergenin in metabolic diseases.

Improvement of Large-Scale Production of Lignocellulosic Bioethanol through Synthetic Biology Approaches: A Comprehensive Review

This review article explores the use of synthetic biology approaches like gene editing and metabolic engineering to create genetically-modified organisms that can produce commercially-valuable products on large-scale, clean environmental pollutants, and serve as efficient biosensors. The Biobrick system includes a registry of available genetic information and a set of standard protocols for assembling and testing them, which finds various significant uses in synthetic biology, such as in the production of "Fourth generation” biofuel. Genetic engineering techniques may be applied to manipulate crops or microbes for more efficient biofuel production. Such synthetic biology techniques also include modification of lignin structure and content, use of bacterial enzymes abundantly expressed in plants, and manipulation of hemicellulose biosynthesis. However, challenges such as high temperature, efficient conversion of all substrate molecules for maximum product yield, efficient pentose fermentation, and tolerance to acetic acid and bioethanol concentration can all impact the yield of the product. This article explores innovative genetic modification approaches designed to decrease lignin levels and enhance crop digestibility in plants, with a specific focus on the model plant Arabidopsis thaliana and herbaceous plants such as Alfalfa. The aim is to manipulate the Monolignol pathway and modify the CSE gene, known for its ability to decrease lignin content. These modifications have been shown to significantly improve saccharification efficiency and increase the yield of lignocellulosic bioethanol. Besides, the role of xylulose-5-phosphate as an intermediate in the non-oxidative pentose phosphate pathway and the oxidative pentose phosphate pathway, and the use of heterologous xylose transporters and modified sugar transporters as potential solutions to improve the biofuel production efficiency is also discussed in this review article.

Distinct roles of the preparatory and payoff phases of glycolysis in hematopoietic stem cells

In physiological conditions, most adult hematopoietic stem cells (HSCs) maintain a quiescent state. Glycolysis is a metabolic process that can be divided into preparatory and payoff phases. Although the payoff phase maintains HSC function and properties, the role of the preparatory phase remains unknown. In this study, we aimed to investigate whether the preparatory or payoff phases of glycolysis were required for maintenance of quiescent and proliferative HSCs. We used glucose-6-phosphate isomerase (Gpi1) as a representative gene for the preparatory phase and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as a representative gene for the payoff phase of glycolysis. First, we identified that stem cell function and survival were impaired in Gapdh-edited proliferative HSCs. Contrastingly, cell survival was maintained in quiescent Gapdh- and Gpi1-edited HSCs. Gapdh- and Gpi1-defective quiescent HSCs maintained adenosine-triphosphate (ATP) levels by increasing mitochondrial oxidative phosphorylation (OXPHOS), whereas ATP levels were decreased in Gapdh-edited proliferative HSCs. Interestingly, Gpi1-edited proliferative HSCs maintained ATP levels independent of increased OXPHOS. Oxythiamine, a transketolase inhibitor, impaired proliferation of Gpi1-edited HSCs, suggesting that the nonoxidative pentose phosphate pathway (PPP) is an alternative means to maintain glycolytic flux in Gpi1-defective HSCs. Our findings suggest that OXPHOS compensated for glycolytic deficiencies in quiescent HSCs, and that in proliferative HSCs, nonoxidative PPP compensated for defects in the preparatory phase of glycolysis but not for defects in the payoff phase. These findings provide new insights into regulation of HSC metabolism, which could have implications for development of novel therapies for hematologic disorders.

In Vitro Safety Assessment of In-House Synthesized Titanium Dioxide Nanoparticles: Impact of Washing and Temperature Conditions.

Titanium dioxide nanoparticles (TiO2 NPs) have been widely used in food, cosmetics, and biomedical research. However, human safety following exposure to TiO2 NPs remains to be fully understood. The aim of this study was to evaluate the in vitro safety and toxicity of TiO2 NPs synthesized via the Stöber method under different washing and temperature conditions. TiO2 NPs were characterized by their size, shape, surface charge, surface area, crystalline pattern, and band gap. Biological studies were conducted on phagocytic (RAW 264.7) and non-phagocytic (HEK-239) cells. Results showed that washing amorphous as-prepared TiO2 NPs (T1) with ethanol while applying heat at 550 °C (T2) resulted in a reduction in the surface area and charge compared to washing with water (T3) or a higher temperature (800 °C) (T4) and influenced the formation of crystalline structures with the anatase phase in T2 and T3 and rutile/anatase mixture in T4. Biological and toxicological responses varied among TiO2 NPs. T1 was associated with significant cellular internalization and toxicity in both cell types compared to other TiO2 NPs. Furthermore, the formation of the crystalline structure induced toxicity independent of other physicochemical properties. Compared with anatase, the rutile phase (T4) reduced cellular internalization and toxicity. However, comparable levels of reactive oxygen species were generated following exposure to the different types of TiO2, indicating that toxicity is partially driven via non-oxidative pathways. TiO2 NPs were able to trigger an inflammatory response, with varying trends among the two tested cell types. Together, the findings emphasize the importance of standardizing engineered nanomaterial synthesis conditions and evaluating the associated biological and toxicological consequences arising from changes in synthesis conditions.

High-level and -yield production of L-leucine in engineered Escherichia coli by multistep metabolic engineering

L-leucine is an essential amino acid widely used in food and pharmaceutical industries. However, the relatively low production efficiency limits its large-scale application. In this study, we rationally developed an efficient L-leucine-producing Escherichia coli strain. Initially, the L-leucine synthesis pathway was enhanced by overexpressing feedback-resistant 2-isopropylmalate synthase and acetohydroxy acid synthase both derived from Corynebacterium glutamicum, along with two other native enzymes. Next, the pyruvate and acetyl-CoA pools were enriched by deleting competitive pathways, employing the nonoxidative glycolysis pathway, and dynamically modulating the citrate synthase activity, which significantly promoted the L-leucine production and yield to 40.69 g/L and 0.30 g/g glucose, respectively. Then, the redox flux was improved by substituting the native NADPH-dependent acetohydroxy acid isomeroreductase, branched chain amino acid transaminase, and glutamate dehydrogenase with their NADH-dependent equivalents. Finally, L-leucine efflux was accelerated by precise overexpression of the exporter and deletion of the transporter. Under fed-batch conditions, the final strain LXH-21 produced 63.29 g/L of L-leucine, with a yield and productivity of 0.37 g/g glucose and 2.64 g/(L h), respectively. To our knowledge, this study achieved the highest production efficiency of L-leucine to date. The strategies presented here will be useful for engineering E. coli strains for producing L-leucine and related products on an industrial scale.

Enhancing precursor supply and modulating metabolism to achieve high-level production of β-farnesene in Yarrowia lipolytica

β-Farnesene is a sesquiterpene commonly found in essential oils of plants, with applications spanning from agricultural pest control and biofuels to industrial chemicals. The use of renewable substrates in microbial cell factories offers a sustainable approach to β-farnesene biosynthesis. In this study, malic enzyme from Mucor circinelloides was examined for NADPH regeneration, concomitant with the augmentation of cytosolic acetyl-CoA supply by expressing ATP-citrate lyase from Mus musculus and manipulating the citrate pathway via AMP deaminase and isocitrate dehydrogenase. Carbon flux was modulated through the elimination of native 6-phosphofructokinase, while the incorporation of an exogenous non-oxidative glycolysis pathway served to bridge the pentose phosphate pathway with the mevalonate pathway. The resulting orthogonal precursor supply pathway facilitated β-farnesene production, reaching 810 mg/L in shake-flask fermentation. Employing optimal fermentation conditions and feeding strategy, a titer of 28.9 g/L of β-farnesene was attained in a 2 L bioreactor.

The Association between Genetic and Racial Factors in Ethanol Containing Beverages Metabolism and Health Risks in Human

Ethanol containing beverages (alcoholic beverages) are socially acceptable beverages that are expected to provide refreshment, satisfaction and stress relief. Common types of ethanol containing beverages include: wine, beer, and syrup. According to the World Health Organization, alcohol abuse is a risk factor for high mortality and many diseases in humans such as fatty liver, hepatomegaly, cirrhosis, liver cancer, larynx cancer, nasopharynx cancer, stomach and esophageal cancer,… In human, ethanol is eliminated mainly through metabolism in two ways: Oxidative pathway with the participation of enzymes: alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), cytochrome P450 and catalase; non-oxidative pathways involving phospholipase and fatty acid ethyl ester (FAEE) synthase. Alcohol abuse is believed to be caused by genetic factors (ADH, ALDH,...), psychological and social factors. Single nucleotide polymorphisms (SNPs) in genes encoding enzymes involved in ethanol metabolism helped explaining why certain ethnic groups and individuals have higher or lower ethanol-related health risks and development of alcohol abuse syndrome. In Vietnam, using ethanol containing beverages is a habit imbued with traditional culture. At the same time, wine and beer abuse is already a problem and causes many serious consequences for public health, family happiness and social safety. Meanwhile, the concept that drinking alcohol helps relieve nervous tension is common, many people still feel subjectively drinking alcohol to be healthier. Some studies have shown differences about SNPs of gene encoding enzymes involved in ethanol metabolism in Vietnamese. Therefore, it is necessary to study the relationship between SNPs of these genes with alcohol and beer drinking habits leading to abuse and the risks of ethanol-related diseases.