RESUMO
D-glucaric acid is an important organic acid with numerous applications in therapy, food, and materials, contributing significantly to its substantial market value. The biosynthesis of D-glucaric acid (GA) from renewable sources such as glucose has garnered significant attention due to its potential for sustainable and cost-effective production. This review summarizes the current understanding of the cell factories for GA production in different chassis strains, from static to dynamic control strategies for regulating their metabolic networks. We highlight recent advances in the optimization of D-glucaric acid biosynthesis, including metabolic dynamic control, alternative feedstocks, metabolic compartments, and so on. Additionally, we compare the differences between different chassis strains and discuss the challenges that each chassis strain must overcome to achieve highly efficient GA productions. In this review, the processes of engineering a desirable cell factory for highly efficient GA production are just like an epitome of metabolic engineering of strains for chemical biosynthesis, inferring general trends for industrial chassis strain developments.
Assuntos
Ácido Glucárico , Engenharia Metabólica , Redes e Vias Metabólicas , Engenharia Metabólica/métodos , Ácido Glucárico/metabolismo , Redes e Vias Metabólicas/genética , Glucose/metabolismo , Microbiologia Industrial/métodosRESUMO
Glucaric acid (GA) is a value-added chemical and can be used to manufacture food additives, anticancer drugs, and polymers. The non-genetic cell-to-cell variations in GA biosynthesis are naturally inherent, indicating the presence of both high- and low-performance cells in culture. Low-performance cells can lead to nutrient waste and inefficient production. Furthermore, myo-inositol oxygenase (MIOX) is a key rate-limiting enzyme with the problem of low stability and activity in GA production. Therefore, eliminating cell-to-cell variations and increasing MIOX stability can select high-performance cells and improve GA production. In this study, an in vivo GA bioselector was constructed based on GA biosensor and tetracycline efflux pump protein TetA to continuously select GA-efficient production strains. Additionally, the upper limit of the GA biosensor was improved to 40 g/L based on ribosome-binding site optimization, achieving efficient enrichment of GA high-performance cells. A small ubiquitin-like modifier (SUMO) enhanced MIOX stability and activity. Overall, we used the GA bioselector and SUMO-MIOX fusion in fed-batch GA production and achieved a 5.52-g/L titer in Escherichia coli, which was 17-fold higher than that of the original strain.IMPORTANCEGlucaric acid is a non-toxic valuable product that was mainly synthesized by chemical methods. Due to the problems of non-selectivity, inefficiency, and environmental pollution, GA biosynthesis has attracted significant attention. The non-genetic cell-to-cell variations and MIOX stability were both critical factors for GA production. In addition, the high detection limit of the GA biosensor was a key condition for performing high-throughput screening of GA-efficient production strains. To increase GA titer, this work eliminated the cell-to-cell variations by GA bioselector constructed based on GA biosensor and TetA, and improved the stability and activity of MIOX in the GA biosynthetic pathway through fusing the SUMO to MIOX. Finally, these approaches improved the GA production by 17-fold to 5.52 g/L at 65 h. This study represents a significant step toward the industrial application of GA biosynthetic pathways in E. coli.
Assuntos
Escherichia coli , Ácido Glucárico , Inositol Oxigenase , Inositol , Escherichia coli/genética , Escherichia coli/metabolismo , Inositol/metabolismo , Inositol Oxigenase/metabolismo , Inositol Oxigenase/genética , Ácido Glucárico/metabolismo , Engenharia Metabólica , Técnicas BiossensoriaisRESUMO
D-Glucaric acid is a potential biobased platform chemical. Previously mainly Escherichia coli, but also the yeast Saccharomyces cerevisiae, and Pichia pastoris, have been engineered for conversion of D-glucose to D-glucaric acid via myo-inositol. One reason for low yields from the yeast strains is the strong flux towards glycolysis. Thus, to decrease the flux of D-glucose to biomass, and to increase D-glucaric acid yield, the four step D-glucaric acid pathway was introduced into a phosphoglucose isomerase deficient (Pgi1p-deficient) Saccharomyces cerevisiae strain. High D-glucose concentrations are toxic to the Pgi1p-deficient strains, so various feeding strategies and use of polymeric substrates were studied. Uniformly labelled 13C-glucose confirmed conversion of D-glucose to D-glucaric acid. In batch bioreactor cultures with pulsed D-fructose and ethanol provision 1.3 g D-glucaric acid L-1 was produced. The D-glucaric acid titer (0.71 g D-glucaric acid L-1) was lower in nitrogen limited conditions, but the yield, 0.23 g D-glucaric acid [g D-glucose consumed]-1, was among the highest that has so far been reported from yeast. Accumulation of myo-inositol indicated that myo-inositol oxygenase activity was limiting, and that there would be potential to even higher yield. The Pgi1p-deficiency in S. cerevisiae provides an approach that in combination with other reported modifications and bioprocess strategies would promote the development of high yield D-glucaric acid yeast strains.
Assuntos
Glucose-6-Fosfato Isomerase , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/metabolismo , Ácido Glucárico/metabolismo , Escherichia coli/metabolismo , Inositol/metabolismo , Glucose/metabolismoRESUMO
Glucaric acid is a valuable chemical with applications in the detergent, polymer, pharmaceutical and food industries. In this study, two key enzymes for glucaric acid biosynthesis, MIOX4 (myo-inositol oxygenase) and Udh (uronate dehydrogenase), were fused and expressed with different peptide linkers. It was found that a strain harboring the fusion protein MIOX4-Udh linked by the peptide (EA3K)3 produced the highest glucaric acid titer and thereby resulted in glucaric acid production that was 5.7-fold higher than that of the free enzymes. Next, the fusion protein MIOX4-Udh linked by (EA3K)3 was integrated into delta sequence sites of the Saccharomyces cerevisiae opi1 mutant, and a strain, GA16, that produced a glucaric acid titer of 4.9 g/L in a shake flask fermentation was identified by a high-throughput screening method using an Escherichia coli glucaric acid biosensor. Strain improvement by further engineering was performed to regulate the metabolic flux of myo-inositol to increase the supply of glucaric acid precursors. The downregulation of ZWF1 and the overexpression of INM1 and ITR1 increased glucaric acid production significantly, and glucaric acid production was increased to 8.49 g/L in the final strain GA-ZII in a shake flask fermentation. Finally, in a 5-L bioreactor, GA-ZII produced a glucaric acid titer of 15.6 g/L through fed-batch fermentation. IMPORTANCE Glucaric acid is a value-added dicarboxylic acid that was synthesized mainly through the oxidation of glucose chemically. Due to the problems of the low selectivity, by-products, and highly polluting waste of this process, producing glucaric acid biologically has attracted great attention. The activity of key enzymes and the intracellular myo-inositol level were both rate-limiting factors for glucaric acid biosynthesis. To increase glucaric acid production, this work improved the activity of the key enzymes in the glucaric acid biosynthetic pathway through the expression of a fusion of Arabidopsis thaliana MIOX4 and Pseudomonas syringae Udh as well as a delta sequence-based integration. Furthermore, intracellular myo-inositol flux was optimized by a series of metabolic strategies to increase the myo-inositol supply, which improved glucaric acid production to a higher level. This study provided a way for constructing a glucaric acid-producing strain with good synthetic performance, making glucaric acid production biologically in yeast cells much more competitive.
Assuntos
Ácido Glucárico , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ácido Glucárico/metabolismo , Escherichia coli/genética , Vias Biossintéticas , Fermentação , Inositol/metabolismo , Engenharia Metabólica/métodosRESUMO
Fusion protein combined the oligopeptide (HQAFFHA) with the C terminus of α-glucuronidase from Thermotoga maritima was produced in E. coli and purified for characterization and applications of glucuronic and glucaric acid production. The fusion protein with oligopeptide exhibited a 2.97-fold higher specific activity than individual protein. Their catalytic efficiency kcat/Km and kcat increased from 469.3 ± 2.6 s-1 (g mL-1)-1 and 62.4 ± 0.9 s-1 to 2209.5 ± 26.3 s-1 (g mL-1)-1 and 293.9 ± 4.9 s-1, respectively. Fusion protein had similar temperature and pH profiles to those without oligopeptide, but the thermal stability decreases and the pH stability shifts to alkaline. Using beech xylan hydrolysate as a substrate, the glucuronic acid yield of fusion enzyme increased by 9.94% compared with its parent at 65 °C pH 8.5 for 10 h, and can hydrolyze corn cob xylan with xylanase to obtain glucuronic acid, and can be combined with uronate dehydrogenase to obtain high-added value glucaric acid. Homologous modeling analysis revealed the factors contributing to the high catalytic efficiency of fusion enzyme. These results show that the peptide fusion strategy described here may be useful for improving the catalytic efficiency and stability of other industrial enzymes, and has great potential for producing high value-added products from agricultural waste.
Assuntos
Thermotoga maritima , Xilanos , Xilanos/metabolismo , Escherichia coli/metabolismo , Oligopeptídeos/metabolismo , Ácido Glucárico/metabolismoRESUMO
As an important dicarboxylic acids existing in nature, glucaric acid has been widely used in medical, health, and polymer materials industry, therefore it is considered as one of the "top value-added chemicals from biomass". In this study, using Saccharomyces cerevisiae as a chassis microorganism, the effects of overexpression of myo-inositol transporter Itr1, fusional expression of inositol oxygenase MIOX4 and uronate dehydrogenase Udh, and down-expression of glucose-6-phosphate dehydrogenase gene ZWF1 on the glucaric acid production were investigated. The results showed that the yield of glucaric acid was increased by 26% compared with the original strain Bga-3 under shake flask fermentation after overexpressing myo-inositol transporter Itr1. The yield of glucaric acid was increased by 40% compared with Bga-3 strain by expressing the MIOX4-Udh fusion protein. On these basis, the production of glucaric acid reached 5.5 g/L, which was 60% higher than that of Bga-3 strain. In a 5 L fermenter, the highest yield of glucaric acid was 10.85 g/L, which was increased 80% compared with that of Bga-3 strain. The application of the above metabolic engineering strategy improved the pathway efficiency and the yield of glucaric acid, which may serve as a reference for engineering S. cerevisiae to produce other chemicals.
Assuntos
Ácido Glucárico , Saccharomyces cerevisiae , Fermentação , Ácido Glucárico/metabolismo , Inositol Oxigenase/genética , Engenharia Metabólica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Metabolic pathways are commonly organized by sequestration into discrete cellular compartments. Compartments prevent unfavorable interactions with other pathways and provide local environments conducive to the activity of encapsulated enzymes. Such compartments are also useful synthetic biology tools for examining enzyme/pathway behavior and for metabolic engineering. Here, we expand the intracellular compartmentalization toolbox for budding yeast (Saccharomyces cerevisiae) with Murine polyomavirus virus-like particles (MPyV VLPs). The MPyV system has two components: VP1 which self-assembles into the compartment shell and a short anchor, VP2C, which mediates cargo protein encapsulation via binding to the inner surface of the VP1 shell. Destabilized green fluorescent protein (GFP) fused to VP2C was specifically sorted into VLPs and thereby protected from host-mediated degradation. An engineered VP1 variant displayed improved cargo capture properties and differential subcellular localization compared to wild-type VP1. To demonstrate their ability to function as a metabolic compartment, MPyV VLPs were used to encapsulate myo-inositol oxygenase (MIOX), an unstable and rate-limiting enzyme in d-glucaric acid biosynthesis. Strains with encapsulated MIOX produced â¼20% more d-glucaric acid compared to controls expressing "free" MIOXâdespite accumulating dramatically less expressed proteinâand also grew to higher cell densities. This is the first demonstration in yeast of an artificial biocatalytic compartment that can participate in a metabolic pathway and establishes the MPyV platform as a promising synthetic biology tool for yeast engineering.
Assuntos
Polyomavirus , Saccharomyces cerevisiae , Animais , Proteínas do Capsídeo/metabolismo , Ácido Glucárico/metabolismo , Inositol Oxigenase/metabolismo , Redes e Vias Metabólicas , Camundongos , Polyomavirus/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
myo-Inositol oxygenase (Miox) is a rate-limiting enzyme for glucaric acid production via microbial fermentation. The enzyme converts myo-inositol to glucuronate, which is further converted to glucaric acid, a natural compound with industrial uses that range from detergents to pharmaceutical synthesis to polymeric materials. More than 2,000 Miox sequences are available in the Uniprot database but only thirteen are classified as reviewed in Swiss-Prot (August 2019). In this study, sequence similarity networks were used to identify new homologues to be expressed in Saccharomyces cerevisiae for glucaric acid production. The expression of four homologues did not lead to product formation. Some of these enzymes may have a defective "dynamic lid" - a structural feature important to close the reaction site - which might explain the lack of activity. Thirty-one selected Miox sequences did allow for product formation, of which twenty-five were characterized for the first time. Expression of Talaromyces marneffei Miox led to the accumulation of 1.76⯱â¯0.33â¯g glucaric acid/L from 20â¯g glucose/L and 10â¯g/L myo-inositol. Specific glucaric acid titer with TmMiox increased 44 % compared to the often-used Arabidopsis thaliana variant AtMiox4 (0.258 vs. 0.179â¯g glucaric acid/g biomass). AtMiox4 activity decreased from 12.47 to 0.40â¯nmol/min/mg protein when cells exited exponential phase during growth on glucose, highlighting the importance of future research on Miox stability in order to further improve microbial production of glucaric acid.
Assuntos
Bioprospecção/métodos , Ácido Glucárico/metabolismo , Inositol Oxigenase/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Biomassa , Bases de Dados de Proteínas , Estabilidade Enzimática , Fermentação , Fungos/classificação , Fungos/enzimologia , Fungos/genética , Glucose/metabolismo , Inositol/metabolismo , Inositol Oxigenase/química , Inositol Oxigenase/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Talaromyces/enzimologia , Talaromyces/genéticaRESUMO
Dynamic regulation is a promising strategy for fine-tuning metabolic fluxes in microbial cell factories. However, few of these synthetic regulatory systems have been developed for central carbon metabolites. Here we created a set of programmable and bifunctional pyruvate-responsive genetic circuits for dynamic dual control (activation and inhibition) of central metabolism in Bacillus subtilis. We used these genetic circuits to design a feedback loop control system that relies on the intracellular concentration of pyruvate to fine-tune the target metabolic modules, leading to the glucaric acid titer increasing from 207 to 527 mg l-1. The designed logic gate-based circuits were enabled by the characterization of a new antisense transcription mechanism in B. subtilis. In addition, a further increase to 802 mg l-1 was achieved by blocking the formation of by-products. Here, the constructed pyruvate-responsive genetic circuits are presented as effective tools for the dynamic control of central metabolism of microbial cell factories.
Assuntos
Proteínas de Bactérias/genética , Redes Reguladoras de Genes/efeitos dos fármacos , Ácido Pirúvico/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Biblioteca Genômica , Ácido Glucárico/metabolismo , Glucose/metabolismo , Histidina/química , Inositol/metabolismo , Lógica , Engenharia Metabólica/métodos , Metaboloma/genética , Modelos Genéticos , Oligopeptídeos/química , Fatores de Transcrição , Transcrição GênicaRESUMO
OBJECTIVE: To enhance the glucaric acid (GA) production in Saccharomyces cerevisiae, the Vitreoscilla hemoglobin was employed to reinforce cellular oxygen supplement. Additionally, the pH-free fermentation strategy was engaged to lower the cost brought by base feeding during the acid-accumulated and long-period glucaric acid production. RESULTS: Recombinant yeast Bga-4 was constructed harboring Vitreoscilla hemoglobin on the basis of previous Bga-3. Higher glucose uptake rate, growth rate, and ethanol reuse rate were achieved in Bga-4 in shake-flask fermentation than those in Bga-3. Furthermore, the fed-batch fermentation in a 5-L bioreactor was performed without pH control, resulting in a final glucaric acid titer of 6.38 g/L. CONCLUSIONS: Both the GA titer and biomass were enhanced along with the efficiency of ethanol re-utilization in the presence of VHb. Moreover, the absence of base feeding for long-period fermentation reduced production cost, which is meaningful for industrial applications.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácido Glucárico/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Hemoglobinas Truncadas/genética , Hemoglobinas Truncadas/metabolismo , Técnicas de Cultura Celular por Lotes , Biomassa , Reatores Biológicos/microbiologia , Clonagem Molecular , Fermentação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
D-glucaric acid (GA) has been identified as among promising biotechnological alternatives to oil-based chemicals. GA and its derivatives are widely used in food additives, dietary supplements, drugs, detergents, corrosion inhibitors and biodegradable materials. The increasing availability of a GA market is improving the cost-effectiveness and efficiency of various biosynthetic pathways. In this study, an engineered Escherichia coli strain GA10 was constructed by systematic metabolic engineering. This involved redirecting metabolic flux into the GA biosynthetic pathways, blocking the conversion pathways of d-glucuronic acid (GlcA) and GA into by-products, introducing an in situ NAD+ regeneration system and fine-tuning the activity of the key enzyme, myo-inositol oxygenase (Miox). Subsequently, the culture medium was optimized to achieve the best performance of the GA10 strain. GA was produced at 5.35â¯g/L (extracellular and intracellular), with a maximized yield of â¼0.46â¯mol/mol on d-glucose and glycerol, by batch fermentation. This work demonstrates efficient biosynthetic pathways of GA in E. coli by metabolic engineering and should accelerate the application of GA biosynthetic pathways in industrial processes.
Assuntos
Escherichia coli/metabolismo , Ácido Glucárico/metabolismo , Engenharia Metabólica , Vias Biossintéticas , Biotecnologia , Escherichia coli/enzimologia , Inositol Oxigenase/metabolismoRESUMO
The unbalanced distribution of carbon flux in microbial cell factories can lead to inefficient production and poor cell growth. Uncoupling cell growth and chemical synthesis can therefore improve microbial cell factory efficiency. Such uncoupling, which requires precise manipulation of carbon fluxes, can be achieved by up-regulating or down-regulating the expression of enzymes of various pathways. In this study, a dynamic turn-off switch (dTFS) and a dynamic turn-on switch (dTNS) were constructed using growth phase-dependent promoters and degrons. By combining the dTFS and dTNS, a bifunctional molecular switch that could orthogonally regulate two target proteins was introduced. This bifunctional molecular switch was used to uncouple cell growth from shikimic acid and D-glucaric acid synthesis, resulting in the production of 14.33 g/L shikimic acid and the highest reported productivity of D-glucaric acid (0.0325 g/L/h) in Escherichia coli MG1655. This proved that the bifunctional molecular switch could rewire carbon fluxes by controlling target protein abundance.
Assuntos
Carbono/metabolismo , Escherichia coli , Ácido Glucárico/metabolismo , Engenharia Metabólica , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
BACKGROUND: The aim of this article was to evaluate the usefulness of sequential dual-time-point 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography/computed tomography (DTP [18F]FDG PET/CT) in distinguishing physiologic, inflammatory and malignant palatine tonsils as difficult to differentiate in the oncological practice. METHODS: A total of 90 patients before the treatment underwent sequential DTP [18F]FDG PET/CT examinations. We analyzed 104 structures in 90 patients: 31 physiologic tonsils, 28 histopathologically confirmed inflammatory tonsils of non-specified origin, 31 histopathologically confirmed palatine tonsils cancer and 14 non-malignant contralateral tonsils in patients with histopathologically confirmed unilateral palatine tonsil malignancy. Patients underwent sequential [18F]FDG PET/CT examinations at 60 and 90 minutes post-injection of the [18F]FDG. We analyzed the SUVmax and SUVmean values at 60 and 90 minutes post-injection changes over time and the Retention Index (RI-SUVmax). To find the predictive SUV value and the RI cut-off between physiology, inflammatory and malignancy, we used the ROC analysis. RESULTS: The average SUVmax values at 60 and 90minutes post-injection within physiologic palatine tonsils were 1.36±0.26 and 1.31±0.26, respectively, P>0.05. The average SUVmax values at 60 and 90 minutes post-injection within inflammatory and malignant tonsils were 3.74±1.45, 3.80±1.47 (P>0.05) and 5.19±2.19, 5.81±2.50 (P<0.05), respectively. The RI-SUVmax fluctuation over time were 5±28% within physiologic, -4±11% within contralateral non-malignant tonsils in patients with one tonsil involved, 2±11% within inflammatory and 13±13% within malignant tonsils. CONCLUSIONS: The sequential dual-time-point [18F]FDG PET/CT examinations may increase the sensitivity and the specificity of the PET/CT method in differential palatine tonsils diagnosis.
Assuntos
Fluordesoxiglucose F18 , Ácido Glucárico/metabolismo , Tonsila Palatina/diagnóstico por imagem , Tonsila Palatina/metabolismo , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Adulto , Idoso , Idoso de 80 Anos ou mais , Transporte Biológico , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Pessoa de Meia-Idade , Curva ROC , Adulto JovemRESUMO
Glucaric acid (GlucA) is a valuable glucose-derived chemical with promising applications as a biodegradable and biocompatible chemical in the manufacturing of plastics, detergents and drugs. Recently, there has been a significant focus on producing GlucA microbially (in vivo) from renewable materials such as glucose, sucrose and myo-inositol. However, these in vivo GlucA production processes generally lack efficiency due to toxicity problems, metabolite competition and suboptimal enzyme ratios. Synthetic biology and accompanying cell-free biocatalysis have been proposed as a viable approach to overcome many of these limitations. However, cell-free biocatalysis faces its own limitations for industrial applications due to high enzyme costs and cofactor consumption. We have constructed a cell-free GlucA pathway and demonstrated a novel framework to overcome limitations of cell-free biocatalysis by i) the combination of both thermostable and mesophilic enzymes, ii) incorporation of a cofactor regeneration system and iii) immobilisation and recycling of the pathway enzymes. The cell-free production of GlucA was achieved from glucose-1-phosphate with a titre of 14.1⯱â¯0.9â¯mM (3.0⯱â¯0.2â¯gâ¯l-1) and a molar yield of 35.2⯱â¯2.3% using non-immobilised enzymes, and a titre of 8.1⯱â¯0.2â¯mM (1.70⯱â¯0.04â¯gâ¯l-1) and a molar yield of 20.2⯱â¯0.5% using immobilised enzymes with a total reaction time of 10â¯h. The resulting productivities (0.30⯱â¯0.02â¯g/h/l for free enzymes and 0.170⯱â¯0.004â¯g/h/l for immobilised enzymes) are the highest productivities so far reported for glucaric acid production using a synthetic enzyme pathway.
Assuntos
Biocatálise , Ácido Glucárico/metabolismo , Engenharia Metabólica , Biologia Sintética , Sistema Livre de Células/enzimologiaRESUMO
Short-chain fatty acids (SCFAs) are produced by the fermentation of dietary fiber by the gut microbiota and are beneficial to the health of the body. Insufficient SCFAs productions are associated with type 2 diabetes (T2D). We used a long-term high-fat diet to simulate the pathogenesis of T2D and studied the effects of baicalin on gut microbiota and metabolites in mice as well as its mechanism, providing a theoretical basis for the treatment of T2D. Baicalin groups were given 200â¯mg/kg/day, and control groups were given an equal volume of 0.5% sodium carboxymethyl cellulose solution for 15 weeks. 16S rRNA amplicon pyrosequences was performed to evaluate the gut microbiota composition, and gas chromatography was used to detect SCFAs in stool samples in the different experimental groups. The abundance of gut microbiota in the high-fat model group was altered, and was associated with a decreased production of SCFAs. The microbiota abundance of the baicalin group was closer to that of the control group, increasing the population of SCFA-producing bacteria spp and improving metabolic syndrome, including abnormal glucose and lipid metabolism caused by a high-fat diet. Baicalin may improve abnormalities in glycolipid metabolism by affecting the production of SCFAs.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Flavonoides/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Ácidos Graxos Voláteis/metabolismo , Fezes/química , Flavonoides/uso terapêutico , Microbioma Gastrointestinal/efeitos dos fármacos , Ácido Glucárico/metabolismo , Glucose/metabolismo , Hiperlipidemias/induzido quimicamente , Hiperlipidemias/tratamento farmacológico , Hiperlipidemias/metabolismo , Hiperlipidemias/microbiologia , Mucosa Intestinal/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/induzido quimicamente , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Obesidade/microbiologiaRESUMO
Thraustochytrids, a group of marine protists, are continuously gaining attention due to their capability in producing lipids for various biotechnological applications towards foods, medicines, chemicals, and biofuels. Although various substrates, predominantly glucose, have been used as carbon source for this microalga, it is desirable to adopt cheaper and more diversified substrate to expand their application range. In this study, we aimed to examine the ability of acetate, which can be easily generated from various resources by acetogenic microorganisms, as a substrate of Aurantiochytrium limacinum SR21. As a result of flask-scale analysis, specific growth rates (µ) of the strain SR21 grown in 3% acetate- or glucose-based medium were 0.55 and 0.98 h-1, respectively. The maximum yield of total fatty acid in acetate medium was 4.8 g/L at 48 h while that in glucose medium was 6.8 g/L at 30 h, indicating that acetate has potential as substrate. Metabolome analysis was performed to comprehensively elucidate characteristic metabolic fluctuations caused by acetate assimilation and identify targets to improve the fatty acid productivity from acetate. It was found that the use of glyoxylate cycle, which bypasses release of energy molecules such as NADH and GTP, and the inhibition of utilization of compounds from TCA cycle for anabolic reactions, may cause the slow growth in acetate which has an effect also in lipid productivity. The activity of the pentose phosphate pathway was found to be weak in acetate cultivation, thus NADPH was mainly produced in malate-pyruvate cycle. Lastly, mevalonate pathway was found to be activated in acetate cultivation which additionally competes with acetyl-CoA as starting material of fatty acid synthesis.
Assuntos
Acetatos/metabolismo , Meios de Cultura , Ácidos Graxos/biossíntese , Fermentação/fisiologia , Metabolismo dos Lipídeos/fisiologia , Estramenópilas/metabolismo , Acetilcoenzima A/metabolismo , Meios de Cultura/química , Ácido Glucárico/metabolismo , Ácido Mevalônico/metabolismo , NADP/biossíntese , Via de Pentose Fosfato , Estramenópilas/crescimento & desenvolvimentoRESUMO
Glucaric acid (GlucA) has been identified as one of the top 10 potential bio-based chemicals for replacement of oil-based chemicals. Several synthetic enzyme pathways have been engineered in bacteria and yeast to produce GlucA from glucose and myo-inositol. However, the yields and titres achieved with these systems remain too low for the requirements of a bio-based GlucA industry. A major limitation for the optimisation of GlucA production via synthetic enzymatic pathways are the laborious analytical procedures required to detect the final product (GlucA) and pathway intermediates. We have developed a novel method for the simple and simultaneous analysis of GlucA and pathway intermediates to address this limitation using mixed mode (MM) HILIC and weak anion exchange chromatography (WAX), referred to as MM HILIC/WAX, coupled with RID. Isocratic mobile phase conditions and the sample solvent were optimised for the separation of GlucA, glucose-1-phosphate (G1P), glucose-6-phosphate (G6P), inositol-1-phosphate (I1P), myo-inositol and glucuronic acid (GA). The method showed good repeatability, precision and excellent accuracy with detection and quantitation limits (LOD and LOQ) of 1.5-2 and 577â¯mM, respectively. The method developed was used for monitoring the enzymatic synthesis of the final step in the GlucA pathway, and showed that GlucA was produced from GA with near 100% conversion and a titre of 9.2â¯gâ¯L-1.
Assuntos
Aldeído Oxirredutases/metabolismo , Biocatálise , Cromatografia Líquida/métodos , Ácido Glucárico/metabolismo , Configuração de Carboidratos , Escherichia coli/enzimologia , Ácido Glucárico/química , Rhizobiaceae/enzimologiaRESUMO
Endothelial functionality profoundly contributes to cardiovascular health. The effects of flavonoids shown to improve endothelial performance include regulating blood pressure by modulating endothelial nitric oxide synthase and NADPH oxidases, but their impact on glucose uptake and metabolism has not been explored. We treated human umbilical vein endothelial cells (HUVEC) with the flavonoid quercetin and its circulating metabolites acutely and chronically, then assessed glucose uptake, glucose metabolism, gene transcription and protein expression. Acute treatment had no effect on glucose uptake, ruling out any direct interaction with sugar transporters. Long term treatment with quercetin, but not quercetin 3-O-glucuronide or 3'-O-sulfate, significantly increased glucose uptake. Heme oxygenase-1 (HO-1) was induced by quercetin but not its conjugates, but was not implicated in the glucose uptake stimulation since hemin, a classical inducer of HO-1, did not affect glucose metabolism. Quercetin increased stability of the transcription factor hypoxia induced factor 1α (HIF1α), a powerful stimulant of glucose metabolism, which was also paralleled by treatment with a prolyl-4-hydroxylase inhibitor dimethyloxalylglycine (DMOG). 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), which regulates the rate of glycolysis, was upregulated by both quercetin and DMOG. Pyruvate dehydrogenase kinase (PDK) isoforms regulate pyruvate dehydrogenase; PDK2 and PDK4 were down-regulated by both effectors, but only DMOG also upregulated PDK1 and PDK3. Quercetin, but not DMOG, increased glucose-6-phosphate dehydrogenase. Chronic quercetin treatment also stimulated glucose transport across the HUVEC monolyer in a 3D culture model. Gene expression of several flavonoid transporters was repressed by quercetin, but this was either abolished (Organic anion transporter polypeptide 4C1) or reversed (Multidrug resistance gene 1) by both conjugates. We conclude that quercetin and its circulating metabolites differentially modulate glucose uptake/metabolism in endothelial cells, through effects on HIF1α and transcriptional regulation of energy metabolism.
Assuntos
Células Endoteliais/metabolismo , Ácido Glucárico/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Quercetina/análogos & derivados , Quercetina/farmacologia , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica , Glucosefosfato Desidrogenase/metabolismo , Heme Oxigenase-1/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Estabilidade Proteica , Quercetina/química , Transdução de SinaisRESUMO
Glucaric acid (GA) is a major value-added chemicals feedstock and additive, especially in the food, cosmetics, and pharmaceutical industries. The increasing demand for GA is driving the search for a more efficient and less costly production pathway. In this study, a new inâ vitro multi-enzyme cascade system was developed, which converts sucrose efficiently to GA in a single vessel. The inâ vitro system, which does not require adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide (NAD+ ) supplementation, contains seven enzymes. All enzymes were chosen from the BRENDA and NCBI databases and were expressed efficiently in Escherichia coli BL21(DE3). All seven enzymes were combined in an inâ vitro cascade system, and the reaction conditions were optimized. Under the optimized conditions, the inâ vitro seven-enzyme cascade system converted 50â mm sucrose to 34.8â mm GA with high efficiency (75 % of the theoretical yield). This system represents an alternative pathway for more efficient and less costly production of GA, which could be adapted for the synthesis of other value-added chemicals.
Assuntos
Ácido Glucárico/metabolismo , Engenharia Metabólica/métodos , Sacarose/metabolismo , Biotransformação , Escherichia coli/enzimologia , Escherichia coli/metabolismoRESUMO
A thermostable uronate dehydrogenase Tb-UDH from Thermobispora bispora was over-expressed in Escherichia coli using the T7 polymerase expression system. The Tb-UDH was purified by metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on glucuronic acid was found at 60 °C and pH 7.0. The purified enzyme retained over 58% of its activity after holding a pH ranging from 7.0 to 7.5 for 1 h at 60 °C. The Km and Vmax values of the purified Tb-UDH for Glucuronic acid (GluUA) were 0.165 mM and 117.7 U mg-1, respectively, those for galacturonic acid (GalUA) were 0.115 mM and 104.2 U mg-1, respectively, and those for NAD+ were 0.120 mM and 133.3 U mg-1, respectively; the turnover number (kcat) with GluUA as a substrate was higher than that with GalUA; however, the Michaelis constant (Km) for GalUA was lower than that for GluUA. After 60 min of incubation at 50 °C, Tb-UDH exhibited a conversion ratio for glucuronic acid to the glucaric acid of 84% on chemical reagent and 81.3% on hydrolysates from breech xylans formed by xylanase and α-glucuronidase. This work shows that biocatalytic routes have great potential for the conversion of hemicellulose substrate into value-added products derived from renewable biomass. TOC GRAPHIC: (A) The structure of the xylan is described and the site of action of the xylan degrading enzyme is indicated. (B) The effect of substrate concentration on recombinant Tb-UDH activity when galacturonic acid was used as substrate. (C) SDS-PAGE analysis of E. coli BL21 (DE3) harboring pET-20b(+) and pET-20b-Tb-UDH. (D) Oxidative conversion of glucuronic acid from a beechwood xylan to glucaric acid.