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1.
Appl Environ Microbiol ; 90(6): e0014924, 2024 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-38808978

RESUMO

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 Biossensoriais
2.
Biotechnol Lett ; 46(1): 69-83, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38064042

RESUMO

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/metabolismo
3.
Bioprocess Biosyst Eng ; 47(12): 1999-2010, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-39158597

RESUMO

D-glucaric acid is a platform chemical of great importance and the consolidated bioprocessing (CBP) of lignocellulose by the microbial consortium of Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 features prospects in biomanufacturing it. Here we compared some representative lignocelluloses in Northwest China including corn stover, wheat straw and switchgrass, and the leading pretreatments including steam explosion, subcritical water pretreatment, sodium hydroxide pretreatment, aqueous ammonia pretreatment, lime pretreatment, and diluted sulfuric acid pretreatment. It was found that sodium hydroxide pretreated switchgrass (SHPSG) was the best substrate for D-glucaric acid production, resulting in the highest D-glucaric acid titers, 11.69 ± 0.73 g/L in shake flask and 15.71 ± 0.80 g/L in 10L airlift fermenter, respectively. To the best of our knowledge, this is the highest D-glucaric acid production titer from lignocellulosic biomass. This work offers a paradigm of producing low-cost D-glucaric acid for low-carbon polyethylene 2,5-furandicarboxylate (PEF) and a reference on developing biorefinery in Northwest China.


Assuntos
Ácido Glucárico , Lignina , Saccharomyces cerevisiae , Lignina/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , China , Ácido Glucárico/metabolismo , Consórcios Microbianos , Zea mays/química , Hypocreales/metabolismo , Fermentação , Triticum , Panicum/metabolismo , Hidróxido de Sódio/química
4.
World J Microbiol Biotechnol ; 40(10): 292, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-39112688

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/métodos
5.
Appl Environ Microbiol ; 89(6): e0053523, 2023 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-37212714

RESUMO

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/métodos
6.
Biosci Biotechnol Biochem ; 86(1): 56-67, 2021 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-34669931

RESUMO

In 2004, the US Department of Energy listed d-glucaric acid as one of the top 12 bio-based chemicals and a potential biopolymer building block. In this study, we show that Pseudogluconobacter saccharoketogenes strains can produce d-glucaric acid from d-glucose, although in low yield because of the generation of the byproduct 2-keto-d-gluconic acid in large quantities. To improve d-glucaric acid yield, we generated Rh47-3, a P. saccharoketogenes IFO14464 mutant, which produced d-glucaric acid from d-gluconic acid and d-glucose with 81 and 53 mol% yields, respectively. Furthermore, the key enzymes involved in d-glucaric acid production, alcohol dehydrogenase (Ps-ADH), aldehyde dehydrogenase (Ps-ALDH), and gluconate 2-dehydrogenase (Ps-GADH), were purified and their roles in d-glucaric acid synthesis were evaluated. Ps-ADH and Ps-ALDH catalyzed d-glucaric acid production, which was mediated by d-gluconic acid and d-glucuronic acid pathways. In contrast, Ps-GADH inhibited d-glucaric acid production by promoting the formation of 2-keto-d-gluconic acid from d-glucose.


Assuntos
Bactérias
7.
Metab Eng ; 57: 162-173, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31726216

RESUMO

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€¯0.9 mM (3.0 ±â€¯0.2 g l-1) and a molar yield of 35.2 ±â€¯2.3% using non-immobilised enzymes, and a titre of 8.1 ±â€¯0.2 mM (1.70 ±â€¯0.04 g l-1) and a molar yield of 20.2 ±â€¯0.5% using immobilised enzymes with a total reaction time of 10 h. The resulting productivities (0.30 ±â€¯0.02 g/h/l for free enzymes and 0.170 ±â€¯0.004 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/enzimologia
8.
Biotechnol Lett ; 42(11): 2169-2178, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32691185

RESUMO

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/genética
9.
Metab Eng ; 49: 212-219, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30125674

RESUMO

The development of D-glucaric acid (GA) production in recombinant cells has leapt forward in recent years, and higher throughput screening and selection of better-performing recombinant cells or biocatalysts is in current demand. A biosensor system which converts GA concentration into fluorescence signal in Escherichia coli was developed in 2016, but its application has rarely been reported. Herein, an effective high-throughput screening approach independent of special-purpose devices such as microfluidic platforms was established and tentatively applied. In this one-pot two-strain system, GA producers-bacterial or yeast cells containing the GA biosynthetic pathway-were sorted with the help of another E. coli strain acting as a GA biosensor. The identification of highly active mutants of myo-inositol oxygenase through this system validates its effectiveness in sorting E. coli cells. Subsequently, accurate ranking of the GA synthesis capacity of a small library of Saccharomyces cerevisiae strains containing distinct GA synthesis pathways demonstrated that this optimized one-pot two-strain system may also be used for eukaryotic producer strains. These results will assist in research into metabolic engineering for GA production and development of biosensor applications.


Assuntos
Técnicas Biossensoriais , Escherichia coli , Glutaratos , Inositol Oxigenase , Mutação , Saccharomyces cerevisiae , Escherichia coli/genética , Escherichia coli/metabolismo , Glutaratos/análise , Glutaratos/metabolismo , Inositol Oxigenase/genética , Inositol Oxigenase/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
10.
Microb Cell Fact ; 17(1): 67, 2018 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-29729665

RESUMO

BACKGROUND: Glucaric acid is a high-value-added chemical that can be used in various fields. Because chemical oxidation of glucose to produce glucaric acid is not environmentally friendly, microbial production has attracted increasing interest recently. Biological pathways to synthesize glucaric acid from glucose in both Escherichia coli and Saccharomyces cerevisiae by co-expression of genes encoding myo-inositol-1-phosphate synthase (Ino1), myo-inositol oxygenase (MIOX), and uronate dehydrogenase (Udh) have been constructed. However, low activity and instability of MIOX from Mus musculus was proved to be the bottleneck in this pathway. RESULTS: A more stable miox4 from Arabidopsis thaliana was chosen in the present study. In addition, high copy delta-sequence integration of miox4 into the S. cerevisiae genome was performed to increase its expression level further. Enzymatic assay and quantitative real-time PCR analysis revealed that delta-sequence-based integrative expression increased MIOX4 activity and stability, thus increasing glucaric acid titer about eight times over that of episomal expression. By fed-batch fermentation supplemented with 60 mM (10.8 g/L) inositol, the multi-copy integrative expression S. cerevisiae strain produced 6 g/L (28.6 mM) glucaric acid from myo-inositol, the highest titer that had been ever reported in S. cerevisiae. CONCLUSIONS: In this study, glucaric acid titer was increased to 6 g/L in S. cerevisiae by integrating the miox4 gene from A. thaliana and the udh gene from Pseudomonas syringae into the delta sequence of genomes. Delta-sequence-based integrative expression increased both the number of target gene copies and their stabilities. This approach could be used for a wide range of metabolic pathway engineering applications with S. cerevisiae.


Assuntos
Ácido Glucárico/metabolismo , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/metabolismo
11.
World J Microbiol Biotechnol ; 34(7): 102, 2018 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-29936649

RESUMO

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.


Assuntos
Ascomicetos/metabolismo , Ácido Glucárico/metabolismo , Polissacarídeos/metabolismo , Aldeído Oxirredutases/metabolismo , Cromatografia de Afinidade , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio
12.
European J Org Chem ; 2017(45): 6811-6814, 2017 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-29576740

RESUMO

Glucaric acid has potential applications in food, pharmaceutical and polymer industries yet no methodology exists within the public domain for isolation of this key bio-derived platform molecule as a pure, crystalline solid. Here we demonstrate the difficulties, which arise in doing so and report development of a process for derivation of free-glucaric acid from its Ca2+/K+ glucarate salts, which are both commercially available. Employing Amberlyst-15 (H+) exchange resin and azeotrope drying, powdered glucaric acid is prepared at > 99.96 % purity in 98.7 % dry yield.

13.
Magn Reson Chem ; 54(7): 561-7, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26749401

RESUMO

d-Glucaric acid (GA) is an aldaric acid and consists of an asymmetric acyclic sugar backbone with a carboxyl group positioned at either end of its structure (i.e., the C1 and C6 positions). The purpose of this study was to conduct a conformation analysis of flexible GA as a solution in deuterium oxide by NMR spectroscopy, based on J-resolved conformation analysis using proton-proton ((3) JHH ) and proton-carbon ((2) JCH and (3) JCH ) coupling constants, as well as nuclear overhauser effect spectroscopy (NOESY). The (2) JCH and (3) JCH coupling constants were measured using the J-resolved heteronuclear multiple bond correlation (HMBC) NMR technique. NOESY correlation experiments indicated that H2 and H5 were in close proximity, despite the fact that these protons were separated by too large distance in the fully extended form of the chain structure to provide a NOESY correlation. The validities of the three possible conformers along the three different bonds (i.e., C2C3, C3C4, and C4C5) were evaluated sequentially based on the J-coupling values and the NOESY correlations. The results of these analyses suggested that there were three dominant conformers of GA, including conformer 1, which was H2H3:gauche, H3H4:anti, and H4H5:gauche; conformer 2, which was H2H3:gauche, H3H4:anti, and H4H5:anti; and conformer 3, which was H2H3:gauche, H3H4: gauche, and H4H5:anti. These results also suggested that all three of these conformers exist in equilibrium with each other. Lastly, the results of the current study suggested that the conformational structures of GA in solution were 'bent' rather than being fully extended. Copyright © 2016 John Wiley & Sons, Ltd.


Assuntos
Óxido de Deutério/química , Ácido Glucárico/química , Espectroscopia de Ressonância Magnética , Conformação Molecular , Termodinâmica
14.
Biotechnol Bioeng ; 112(3): 579-87, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25258165

RESUMO

The use of lignocellulosic biomass as a feedstock for microbial fermentation processes presents an opportunity for increasing the yield of bioproducts derived directly from glucose. Lignocellulosic biomass consists of several fermentable sugars, including glucose, xylose, and arabinose. In this study, we investigate the ability of an E. coli Δpgi Δzwf mutant to consume alternative carbon sources (xylose, arabinose, and glycerol) for growth while reserving glucose for product formation. Deletion of pgi and zwf was found to eliminate catabolite repression as well as the ability of E. coli to consume glucose for biomass formation. In addition, the yield from glucose of the bioproduct D-glucaric acid was significantly increased in a Δpgi Δzwf strain.


Assuntos
Técnicas de Cultura de Células/métodos , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Glucose-6-Fosfato Isomerase/genética , Glucose/metabolismo , Engenharia Metabólica/métodos , Biomassa , Reatores Biológicos/microbiologia , Fermentação , Técnicas de Inativação de Genes , Ácido Glucárico/metabolismo
15.
Metab Eng ; 22: 22-31, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24333274

RESUMO

D-glucaric acid has been explored for a myriad of potential uses, including biopolymer production and cancer treatment. A biosynthetic route to produce D-glucaric acid from glucose has been constructed in Escherichia coli (Moon et al., 2009b), and analysis of the pathway revealed myo-inositol oxygenase (MIOX) to be the least active enzyme. To increase pathway productivity, we explored protein fusion tags for increased MIOX solubility and directed evolution for increased MIOX activity. An N-terminal SUMO fusion to MIOX resulted in a 75% increase in D-glucaric acid production from myo-inositol. While our directed evolution efforts did not yield an improved MIOX variant, our screen isolated a 941 bp DNA fragment whose expression led to increased myo-inositol transport and a 65% increase in D-glucaric acid production from myo-inositol. Overall, we report the production of up to 4.85 g/L of D-glucaric acid from 10.8 g/L myo-inositol in recombinant E. coli.


Assuntos
Estabilidade Enzimática , Escherichia coli , Ácido Glucárico/metabolismo , Inositol/metabolismo , Transporte Biológico Ativo/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Inositol Oxigenase/genética , Inositol Oxigenase/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
16.
Front Chem ; 12: 1393860, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38752198

RESUMO

Long-term electrolyses of glucose in a potassium carbonate (K2CO3) aqueous electrolyte have been performed on graphite felt electrodes with TEMPO as a homogeneous catalyst. The influences of the operating conditions (initial concentrations of glucose, TEMPO, and K2CO3 along with applied anode potential) on the conversion, selectivity toward gluconate/glucarate, and faradaic efficiency were assessed first. Then, optimizations of the conversion, selectivity, and faradaic efficiency were performed using design of experiments based on the L9 (34) Taguchi table, which resulted in 84% selectivity toward gluconate with 71% faradaic efficiency for up to 79% glucose conversion. Side products such as glucaric acid were also obtained when the applied potential exceeded 1.5 V vs. reversible hydrogen electrode.

17.
ACS Appl Mater Interfaces ; 16(20): 26107-26120, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38725264

RESUMO

The slow anodic oxygen evolution reaction (OER) significantly limits electrocatalytic water splitting for hydrogen production. We proposed the electrocatalyst for glucose oxidation by Ta-doping NiFe LDH nanosheets to simultaneously obtain glucaric acid (GRA) and hydrogen gas as a useful byproduct. Superior glucose oxidation reaction (GOR) activity is demonstrated by the optimized Ta-NiFe LDH, which has a low overpotential of 192 mV, allowing for a small Tafel slope of 70 mV dec-1 and a current density of 50 mA cm-2. The Ta NiFe LDH-oxidized glucose to GRA with a 72.94% yield and 64.3% Faradaic efficiency at 1.45 VRHE. Herein, we report the Ta NiFe LDH/NF electrode for the GOR&hydrogen evolution reaction (HER), which exhibits a cell voltage of 1.62 V to reach a current density of 10 mA cm-2, which is 250 mV lower compared to OER&HER (1.87 V). This study reveals that GOR is an energy-efficient and cost-effective method for producing H2 and valorizing biomass.

18.
Food Chem ; 460(Pt 3): 140626, 2024 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-39128363

RESUMO

Glucose-derived acids for the further production of value-added medicine, food additives, and polymers, will promote lignocellulosic biomass biorefinery industry. In response to the diversity and complexity, a new method was established by employing high performance anion exchange chromatography (HPAEC) coupled with a CarboPac™ PA200 column, for the precise and fast determination of glucose, gluconic acid, glucuronic acid, 2-ketogluconic acid, 5-ketogluconic acid and glucaric acid. Based on the analysis of tiny varieties in retention behavior, a gradient elution mode was designed and optimized for the quantitative and qualitative analysis. The protocol displayed acceptable linearity (R2 ≥ 0.995), commendable average recovery rate (95.28% âˆ¼ 99.89%), satisfactory precision (RSD% ≤ 1.5%), and sufficient resolution (R > 6). Additionally, this method was successfully applied to the high-value biorefining process, which confirmed the practicability and accuracy. The results demonstrated that HPAEC has good detection performance for glucose and its derivative acids, and provide key identification technical support for the high-value utilization of lignocellulose.


Assuntos
Biomassa , Glucose , Cromatografia por Troca Iônica/métodos , Glucose/análise , Glucose/química , Cromatografia Líquida de Alta Pressão/métodos , Lignina/química , Ácidos/análise , Ácidos/química
20.
Front Chem ; 11: 1114454, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37324557

RESUMO

Photocatalysis has exhibited huge potential in selective conversion of glucose into value-added chemicals. Therefore, modulation of photocatalytic material for selective upgrading of glucose is significant. Here, we have investigated the insertion of different central metal ions, Fe, Co, Mn, and Zn, into porphyrazine loading with SnO2 for access to more efficient transformation of glucose into value-added organic acids in aqueous solution at mild reaction conditions. The best selectivity for organic acids containing glucaric acid, gluconic acid, and formic acid of 85.9% at 41.2% glucose conversion was attained by using the SnO2/CoPz composite after reacting for 3 h. The effects of central metal ions on surficial potential and related possible factors have been studied. Experimental results showed that the introduction of metalloporphyrazine with different central metal ions on the surface of SnO2 has a significant effect on the separation of photogenerated charges, changing the adsorption and desorption of glucose and products on the catalyst surface. The central metal ions of cobalt and iron contributed more to the positive effects toward enhancing conversion of glucose and yields of products, and manganese and zinc contributed more to the negative effects, resulting in the poor yield of products. The differences from the central metals may attribute to the surficial potential change of the composite and the coordination effects between the metal and oxygen atom. An appropriate surficial potential environment of the photocatalyst may achieve a better interactive relationship between the catalyst and reactant, while appropriate ability of producing active species matched with adsorption and desorption abilities would gain a better yield of products. These results have provided valued ideas for designing more efficient photocatalysts in selective oxidation of glucose utilizing clean solar energy in the future.

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