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1.
Anal Chem ; 94(37): 12757-12761, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36069721

RESUMO

Most interesting problems in chemistry, biology, and pharmacy involve mixtures. However, analysis of such mixtures by NMR remains a challenge, often requiring the mixture components to be physically separated before analysis. A variety of methods have been proposed that exploit species-specific properties such as diffusion and relaxation to distinguish between the signals of different components in a mixture without the need for laborious separation. However, these methods can struggle to distinguish between components when signals overlap. Here, we exploit the relaxation properties of selected nuclei to distinguish between different components of a mixture while using pure shift methods to increase spectral resolution by up to an order of magnitude, greatly reducing signal overlap. The advantages of the new method are demonstrated in a mixture of d-xylose and l-arabinose, distinguishing unambiguously between the five major species present.


Assuntos
Arabinose , Xilose , Difusão , Espectroscopia de Ressonância Magnética/métodos
2.
Bioprocess Biosyst Eng ; 45(10): 1705-1717, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36063213

RESUMO

As a promising feedstock, alkali-extracted xylan from lignocellulosic biomass is desired for producing xylose, which can be used for renewable biofuels production. In this study, an efficient pathway has been established for low-cost and high-yield production of xylose by hydrolysis of alkali-extracted xylan from agricultural wastes using an endo-1,4-xylanase (XYLA) from Bacillus safensis TCCC 111022 and a ß-xylosidase (XYLO) from B. pumilus TCCC 11573. The optimum activities of recombinant XYLA (rXYLA) and XYLO (rXYLO) were 60 â„ƒ and pH 8.0, and 30 â„ƒ and pH 7.0, respectively. They were stable over a broad pH range (pH 6.0-11.0 and 7.0-10.0). rXYLO showed a relatively high xylose tolerance up to 100 mM. Furthermore, the yield of xylose from wheat straw, rice straw, corn stover, corncob and sugarcane bagasse by rXYLA and rXYLO was 63.77%, 71.76%, 68.55%, 53.81%, and 58.58%, respectively. This study demonstrated a strategy to produce xylose from agricultural wastes by integrating alkali-extracted xylan and enzymatic hydrolysis.


Assuntos
Bacillus , Saccharum , Xilosidases , Álcalis , Bacillus/metabolismo , Biocombustíveis , Celulose , Endo-1,4-beta-Xilanases/metabolismo , Hidrólise , Saccharum/metabolismo , Xilanos , Xilose/metabolismo , Xilosidases/metabolismo
3.
Int J Mol Sci ; 23(18)2022 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-36142202

RESUMO

To explore the protective effect of dietary ß-glucan (BGL) supplementation on intestinal epithelium exposure to enterotoxigenic Escherichia coli (ETEC), thirty-two weaned pigs were assigned to four groups. Pigs were fed with a basal diet or basal diet containing 500 mg/kg BGL, and were orally infused with ETEC or culture medium. Results showed BGL supplementation had no influence on growth performance in weaned pigs. However, BGL supplementation increased the absorption of D-xylose, and significantly decreased the serum concentrations of D-lactate and diamine oxidase (DAO) in the ETEC-challenged pigs (p < 0.05). Interestingly, BGL significantly increased the abundance of the zonula occludens-1-(ZO-1) in the jejunal epithelium upon ETEC challenge (p < 0.05). BGL supplementation also increased the number of S-phase cells and the number of sIgA-positive cells, but significantly decreased the number of total apoptotic cells in the jejunal epithelium upon ETEC challenge (p < 0.05). Moreover, BGL significantly increased the duodenal catalase (CAT) activity and the ileal total superoxide dismutase (T-SOD) activity in the ETEC-challenged pigs (p < 0.05). Importantly, BGL significantly decreased the expression levels of critical inflammation related proteins such as the tumor necrosis factor-α (TNF-α), interlukin-6 (IL-6), myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB) in the jejunal and ileal mucosa upon ETEC challenge (p < 0.05). BGL also elevated the propanoic acid content and the abundance of Lactobacillus and Bacillus in the colon upon ETEC challenge (p < 0.05). These results suggested BGL could alleviate the ETEC-induced intestinal epithelium injury, which may be associated with suppressed inflammation and improved intestinal immunity and antioxidant capacity, as well as the improved intestinal macrobiotic.


Assuntos
Amina Oxidase (contendo Cobre) , Escherichia coli Enterotoxigênica , Infecções por Escherichia coli , Doenças dos Suínos , beta-Glucanas , Agrobacterium/metabolismo , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Antioxidantes/farmacologia , Catalase/metabolismo , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/prevenção & controle , Infecções por Escherichia coli/veterinária , Imunoglobulina A Secretora/metabolismo , Inflamação/patologia , Interleucina-6/metabolismo , Mucosa Intestinal/metabolismo , Lactatos/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Propionatos/farmacologia , Superóxido Dismutase/metabolismo , Suínos , Doenças dos Suínos/tratamento farmacológico , Doenças dos Suínos/prevenção & controle , Fator de Necrose Tumoral alfa/metabolismo , Xilose/metabolismo , beta-Glucanas/metabolismo
4.
Int J Mol Sci ; 23(18)2022 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-36142298

RESUMO

The edible fungus Tremella fuciformis was shown to have a high molecular weight (1.87 × 103 kDa) bioactive polysaccharide, denoted as TFP-F1. Monosaccharide composition and NMR analysis of the polysaccharide and its derivatives indicated it contained fucose (Fucp), xylose (Xylp), mannose (Manp), and glucuronic acid (GlcAp) in a ratio of 0.9:1.0:3.2:1.2. Using IR, NMR, and GC-MS spectroscopic data, the structure of TFP-F1 was elucidated as {→3)-[ß-D-GlcAp-(1→2)]-α-D-Manp-(1→3)-α-D-Manp-(1→3)-[α-L-Fucp-(1→2)-ß-D-Xylp-(1→2)]-α-D-Manp-(1→}n, with partial acetylation of C6-OH in mannoses. Furthermore, at a concentration of 1 µg/mL, TFP-F1 was found to stimulate the secretion of TNF-α and IL-6 in J774A.1 macrophage cells in vitro via interaction with toll-like receptor 4 (TLR4). The removal of O-acetyl groups led to the loss of immunomodulatory activities, demonstrating that O-acetyl groups play an essential role in enhancing the production of pro-inflammatory cytokines.


Assuntos
Receptor 4 Toll-Like , Fator de Necrose Tumoral alfa , Acetilação , Basidiomycota , Citocinas , Carboidratos da Dieta , Fucose , Ácido Glucurônico , Imunomodulação , Interleucina-6 , Manose , Monossacarídeos , Polissacarídeos/química , Polissacarídeos/farmacologia , Xilose
5.
Int J Mol Sci ; 23(18)2022 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-36142342

RESUMO

It has been recently proven that xylooligosaccharides (XOS) with prebiotic properties have diverse beneficial biological effects including immunomodulatory and antitumor activities. The present article focused on the chemical and biological evaluation of corn-derived commercially available XOS and aimed to elucidate their cytotoxicity and inhibitory potential against tumor cells. Spectrophotometric chemical analyses, Fourier transform infrared spectroscopy, and high-performance liquid chromatography analyses were performed. Antioxidant activity was determined by measuring the oxygen radical absorbance capacity and hydroxyl radical averting capacity. In vitro cytotoxicity assays with human cell lines derived from normal and tumor tissues, assessments of ATP production, mitochondrial membrane potential specific staining, cytokine assays, and molecular docking were used to evaluate the biological activity of XOS. The sample showed significant antioxidant activity, and it was determined that most xylose oligomers in it are composed of six units. XOS exhibited antitumor activity with pronounced inhibitory effect on lysosomes, but mitochondrial functionality was also affected. The production of proinflammatory cytokines by lipopolysaccharide-stimulated U-937 cells was reduced by XOS treatment, which suggested the involvement of Toll-like receptor 4 (TLR4)-mediated signaling in the mechanism of XOS action. Molecular docking analyses confirmed the potential inhibitory interaction between the sample and TLR4. In addition, XOS treatment had significant tumor-cell-specific influence on the glutathione antioxidant system, affecting its balance and thus contributing to the inhibition of cellular viability. The present study elucidated the tumor-inhibitory potential of commercially available XOS that could be utilized in pharmaceutical and food industry providing disease-preventive and therapeutic benefits.


Assuntos
Antioxidantes , Receptor 4 Toll-Like , Trifosfato de Adenosina , Antioxidantes/metabolismo , Citocinas , Glucuronatos/metabolismo , Glutationa , Humanos , Radical Hidroxila , Lipopolissacarídeos , Simulação de Acoplamento Molecular , Oligossacarídeos/química , Preparações Farmacêuticas , Xilose
6.
Molecules ; 27(18)2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36144482

RESUMO

The Maillard reaction kinetics in the confined volume of the thin film produced by ESI microdroplet deposition was studied by mass spectrometry. The almost exclusive formation of the Amadori product from the reaction of D-xylose and D-glucose toward L-glycine and L-lysine was demonstrated. The thin film Maillard reaction occurred at a mild synthetic condition under which the same process in solution was not observed. The comparison of the thin film kinetics with that of the reaction performed in solution showed strong thin film rate acceleration factors.


Assuntos
Reação de Maillard , Xilose , Glucose , Glicina/química , Lisina/análise , Xilose/química
7.
Food Res Int ; 160: 111704, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36076455

RESUMO

A similar color change to meat would promote the appetite of consumers for meat analogs. In this study, three-dimensional (3D) printable colorant-containing meat analogs were developed to mimic the red and brown color of meat before and after cooking. These color changes were realized based on the thermal lability of beet red and the Maillard reaction. Moreover, we studied the influence of the addition of xylose on the shear modulus, printing performance, color, texture, chemical structure, and microstructure of colorant-containing meat analogs. Upon increasing the amount of xylose added, the raw colorant-containing meat analogs showed an increase in shear modulus, dimensional stability, and hardness; whereas, cooked colorant-containing meat analogs showed an increase in hardness, and a decrease in lightness, yellowness, hue angle, and chroma. Moreover, adding xylose changed the interactions and microstructure of colorant-containing meat analogs, leading to texture changes. This study would provide guidelines for the color and texture change by adding reducing saccharides to protein-rich gels.


Assuntos
Vigna , Xilose , Cor , Carne , Impressão Tridimensional
8.
Microb Cell Fact ; 21(1): 198, 2022 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-36153615

RESUMO

BACKGROUND: 5-hydroxytryptophan (5-HTP), the direct biosynthetic precursor of the neurotransmitter 5-hydroxytryptamine, has been shown to have unique efficacy in the treatment of a variety of disorders, including depression, insomnia, and chronic headaches, and is one of the most commercially valuable amino acid derivatives. However, microbial fermentation for 5-HTP production continues to face many challenges, including low titer/yield and the presence of the intermediate L-tryptophan (L-Trp), owing to the complexity and low activity of heterologous expression in prokaryotes. Therefore, there is a need to construct an efficient microbial cell factory for 5-HTP production. RESULTS: We describe the systematic modular engineering of wild-type Escherichia coli for the efficient fermentation of 5-HTP from glucose. First, a xylose-induced T7 RNA polymerase-PT7 promoter system was constructed to ensure the efficient expression of each key heterologous pathway in E. coli. Next, a new tryptophan hydroxylase mutant was used to construct an efficient tryptophan hydroxylation module, and the cofactor tetrahydrobiopterin synthesis and regeneration pathway was expressed in combination. The L-Trp synthesis module was constructed by modifying the key metabolic nodes of tryptophan biosynthesis, and the heterologous synthesis of 5-HTP was achieved. Finally, the NAD(P)H regeneration module was constructed by the moderate expression of the heterologous GDHesi pathway, which successfully reduced the surplus of the intermediate L-Trp. The final engineered strain HTP11 was able to produce 8.58 g/L 5-HTP in a 5-L bioreactor with a yield of 0.095 g/g glucose and a maximum real-time productivity of 0.48 g/L/h, the highest values reported by microbial fermentation. CONCLUSION: In this study, we demonstrate the successful design of a cell factory for high-level 5-HTP production, combined with simple processes that have potential for use in industrial applications in the future. Thus, this study provides a reference for the production of high-value amino acid derivatives using a systematic modular engineering strategy and a basis for an efficient engineered strain development of 5-HTP high-value derivatives.


Assuntos
5-Hidroxitriptofano , Engenharia Metabólica , 5-Hidroxitriptofano/genética , 5-Hidroxitriptofano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Glucose/metabolismo , NAD/metabolismo , Neurotransmissores/metabolismo , Serotonina/metabolismo , Triptofano/metabolismo , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismo , Xilose/metabolismo
9.
World J Microbiol Biotechnol ; 38(12): 229, 2022 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-36149541

RESUMO

This research investigated the characteristics of Zalaria obscura LS31012019 in terms of growth ability in different media (SDB, YPD and TSB) and temperatures (22, 25 and 37 °C), utilization of several carbon sources (Glucose, Fructose, Lactose, Sucrose, Xylose, Glycerol and Mannitol at 5, 2 and 1%) and several biochemical features (total protein content, Glutathione, pigments), in comparison with those of the phylogenetically related Aureobasidium pullulans ATCC 15233. The best growth of Z. obscura LS31012019 was obtained in YPD at 25 °C with the highest OD value (0.45) after 144 h of incubation, similar to that of A. pullulans ATCC 15233 (0.48). Glucose resulted the preferred carbon source for both the considered yeasts but also sucrose resulted in efficacy supporting the growth of Z. obscura LS31012019 and A. pullulans ATCC 15233, for their ability in converting sucrose to glucose and fructose and the latter into glucose. Interestingly, Z. obscura LS31012019 utilized also glycerol and mannitol. The biochemical analysis showed the similarity of protein profile in Z. obscura LS31012019 and A. pullulans ATCC 15233 (from 90 to 20 kDa) and a reduced GSH content (0.321 and 0.233 µmol/mg). The pigments extraction with hexane generated a yellow oleaginous pellet in both the strains, while a yellow solid matrix more intensely coloured in A. pullulans ATTC 15233 was visible with the following solvent extractions. Overall, our data showed that Z. obscura LS31012019 can grow in different media and temperatures and utilize carbon sources apart from glucose and sucrose, shifting to a non-fermentative metabolism. These results improve the information regarding the characteristics of Z. obscura, opening a new field of investigation for the possible application of new species of black yeasts in human application.


Assuntos
Glicerol , Xilose , Ascomicetos , Carbono/metabolismo , Frutose , Glucose/metabolismo , Glutationa , Glicerol/metabolismo , Hexanos , Humanos , Lactose , Manitol , Solventes , Sacarose/metabolismo
10.
PLoS One ; 17(9): e0274420, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36107941

RESUMO

UDP-glucose dehydrogenase (UGDH) generates essential precursors of hyaluronic acid (HA) synthesis, however mechanisms regulating its activity are unclear. We used enzyme histostaining and quantitative image analysis to test whether cytokines that stimulate HA synthesis upregulate UGDH activity. Fibroblast-like synoviocytes (FLS, from N = 6 human donors with knee pain) were cultured, freeze-thawed, and incubated for 1 hour with UDP-glucose, NAD+ and nitroblue tetrazolium (NBT) which allows UGDH to generate NADH, and NADH to reduce NBT to a blue stain. Compared to serum-free medium, FLS treated with PDGF showed 3-fold higher UGDH activity and 6-fold higher HA release, but IL-1beta/TGF-beta1 induced 27-fold higher HA release without enhancing UGDH activity. In selected proliferating cells, UGDH activity was lost in the cytosol, but preserved in the nucleus. Cell-free assays led us to discover that diaphorase, a cytosolic enzyme, or glutathione reductase, a nuclear enzyme, was necessary and sufficient for NADH to reduce NBT to a blue formazan dye in a 1-hour timeframe. Primary synovial fibroblasts and transformed A549 fibroblasts showed constitutive diaphorase/GR staining activity that varied according to supplied NADH levels, with relatively stronger UGDH and diaphorase activity in A549 cells. Unilateral knee injury in New Zealand White rabbits (N = 3) stimulated a coordinated increase in synovial membrane UGDH and diaphorase activity, but higher synovial fluid HA in only 2 out of 3 injured joints. UGDH activity (but not diaphorase) was abolished by N-ethyl maleimide, and inhibited by peroxide or UDP-xylose. Our results do not support the hypothesis that UGDH is a rate-liming enzyme for HA synthesis under catabolic inflammatory conditions that can oxidize and inactivate the UGDH active site cysteine. Our novel data suggest a model where UGDH activity is controlled by a redox switch, where intracellular peroxide inactivates, and high glutathione and diaphorase promote UGDH activity by maintaining the active site cysteine in a reduced state, and by recycling NAD+ from NADH.


Assuntos
Sinoviócitos , Animais , Cisteína/metabolismo , Fibroblastos/metabolismo , Formazans , Glucose/farmacologia , Glucose Desidrogenase/metabolismo , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Ácido Hialurônico/farmacologia , Maleimidas , NAD/metabolismo , Nitroazul de Tetrazólio , Oxirredução , Peróxidos , Coelhos , Sinoviócitos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Difosfato de Uridina/metabolismo , Uridina Difosfato Glucose Desidrogenase/química , Uridina Difosfato Glucose Desidrogenase/metabolismo , Xilose
11.
J Agric Food Chem ; 70(38): 12085-12094, 2022 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-36103687

RESUMO

Issatchenkia orientalis, exhibiting high tolerance against harsh environmental conditions, is a promising metabolic engineering host for producing fuels and chemicals from cellulosic hydrolysates containing fermentation inhibitors under acidic conditions. Although genetic tools for I. orientalis exist, they require auxotrophic mutants so that the selection of a host strain is limited. We developed a drug resistance gene (cloNAT)-based genome-editing method for engineering any I. orientalis strains and engineered I. orientalis strains isolated from various sources for xylose fermentation. Specifically, xylose reductase, xylitol dehydrogenase, and xylulokinase from Scheffersomyces stipitis were integrated into an intended chromosomal locus in four I. orientalis strains (SD108, IO21, IO45, and IO46) through Cas9-based genome editing. The resulting strains (SD108X, IO21X, IO45X, and IO46X) efficiently produced ethanol from cellulosic and hemicellulosic hydrolysates even though the pH adjustment and nitrogen source were not provided. As they presented different fermenting capacities, selection of a host I. orientalis strain was crucial for producing fuels and chemicals using cellulosic hydrolysates.


Assuntos
Engenharia Metabólica , Xilose , Aldeído Redutase/genética , Sistemas CRISPR-Cas , D-Xilulose Redutase/genética , Etanol/metabolismo , Fermentação , Engenharia Metabólica/métodos , Nitrogênio/metabolismo , Pichia , Saccharomyces cerevisiae/metabolismo , Xilose/metabolismo
12.
J Agric Food Chem ; 70(38): 12164-12171, 2022 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-36124743

RESUMO

The browning inhibition of cysteine on the Maillard reaction of glycine-xylose performed under stepwise increased temperature was investigated. The browning degrees of the final products prepared with cysteine addition at different time points were found dissimilar, and the addition time point of cysteine yielding the lightest browning products was consistent with the time when the glycine-xylose Amadori rearrangement product (GX-ARP) reached its maximum yield. To clarify the reason for browning inhibition caused by cysteine, the evolution of key browning precursors formed in the GX-ARP model with cysteine involved was investigated by HPLC with a diode array detector. The results on the browning degree of the thermal reaction products of GX-ARP with cysteine addition showed great inhibition of α-dicarbonyl generation, which resulted in a significant increase in the activation energy of GX-ARP conversion to browning formation during heat treatment. Strong evidence suggested that the additional cysteine got involved in GX-ARP degradation and reacted with the deoxyosones derived from GX-ARP to yield cyclic 2-threityl-thiazolidine-4-carboxylic acid (TTCA). TTCA formation shunted the degradation of deoxyosones into short-chain α-dicarbonyls, which were important browning precursors, and consequently inhibited the Maillard browning.


Assuntos
Cisteína , Xilose , Glicina , Reação de Maillard , Tiazolidinas
13.
Nat Commun ; 13(1): 4925, 2022 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-35995792

RESUMO

Muconic acid is a bioprivileged molecule that can be converted into direct replacement chemicals for incumbent petrochemicals and performance-advantaged bioproducts. In this study, Pseudomonas putida KT2440 is engineered to convert glucose and xylose, the primary carbohydrates in lignocellulosic hydrolysates, to muconic acid using a model-guided strategy to maximize the theoretical yield. Using adaptive laboratory evolution (ALE) and metabolic engineering in a strain engineered to express the D-xylose isomerase pathway, we demonstrate that mutations in the heterologous D-xylose:H+ symporter (XylE), increased expression of a major facilitator superfamily transporter (PP_2569), and overexpression of aroB encoding the native 3-dehydroquinate synthase, enable efficient muconic acid production from glucose and xylose simultaneously. Using the rationally engineered strain, we produce 33.7 g L-1 muconate at 0.18 g L-1 h-1 and a 46% molar yield (92% of the maximum theoretical yield). This engineering strategy is promising for the production of other shikimate pathway-derived compounds from lignocellulosic sugars.


Assuntos
Pseudomonas putida , Xilose , Fermentação , Glucose/metabolismo , Engenharia Metabólica , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Ácido Sórbico/análogos & derivados , Xilose/metabolismo
14.
Microb Cell Fact ; 21(1): 162, 2022 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-35964033

RESUMO

BACKGROUND: Fuel ethanol from lignocellulose could be important source of renewable energy. However, to make the process feasible, more efficient microbial fermentation of pentose sugars, mainly xylose, should be achieved. The native xylose-fermenting thermotolerant yeast Ogataea polymorpha is a promising organism for further development. Efficacy of xylose alcoholic fermentation by O. polymorpha was significantly improved by metabolic engineering. Still, genes involved in regulation of xylose fermentation are insufficiently studied. RESULTS: We isolated an insertional mutant of O. polymorpha with impaired ethanol production from xylose. The insertion occurred in the gene HXS1 that encodes hexose transporter-like sensor, a close homolog of Saccharomyces cerevisiae sensors Snf3 and Rgt2. The role of this gene in xylose utilization and fermentation was not previously elucidated. We additionally analyzed O. polymorpha strains with the deletion and overexpression of the corresponding gene. Strains with deletion of the HXS1 gene had slower rate of glucose and xylose consumption and produced 4 times less ethanol than the wild-type strain, whereas overexpression of HXS1 led to 10% increase of ethanol production from glucose and more than 2 times increase of ethanol production from xylose. We also constructed strains of O. polymorpha with overexpression of the gene AZF1 homologous to S. cerevisiae AZF1 gene which encodes transcription activator involved in carbohydrate sensing. Such transformants produced 10% more ethanol in glucose medium and 2.4 times more ethanol in xylose medium. Besides, we deleted the AZF1 gene in O. polymorpha. Ethanol accumulation in xylose and glucose media in such deletion strains dropped 1.5 and 1.8 times respectively. Overexpression of the HXS1 and AZF1 genes was also obtained in the advanced ethanol producer from xylose. The corresponding strains were characterized by 20-40% elevated ethanol accumulation in xylose medium. To understand underlying mechanisms of the observed phenotypes, specific enzymatic activities were evaluated in the isolated recombinant strains. CONCLUSIONS: This paper shows the important role of hexose sensor Hxs1 and transcription factor Azf1 in xylose and glucose alcoholic fermentation in the native xylose-fermenting yeast O. polymorpha and suggests potential importance of the corresponding genes for construction of the advanced ethanol producers from the major sugars of lignocellulose.


Assuntos
Proteínas Fúngicas/metabolismo , Xilose , Etanol/metabolismo , Glucose/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Pichia/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Xilose/metabolismo
15.
Bioorg Chem ; 128: 106073, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35939856

RESUMO

Development of a synthetic route applicable to d-ribose and d-xylose enabled the synthesis of cleistanolate putative structure, its five stereoisomers, and led to revision and confirmation of absolute stereochemistry of the natural product. Key steps of the synthesis included zinc-mediated THF ring-opening and stereoselective dihydroxylation under the Upjohn conditions. The first total synthesis of cleistanolate was completed in eight steps starting from d-xylose. The C-5 stereocenter of the natural product was assigned the correct (5S)-stereochemistry. Cytotoxicity of natural product was briefly investigated.


Assuntos
Antineoplásicos , Produtos Biológicos , Produtos Biológicos/química , Lactonas/química , Estrutura Molecular , Estereoisomerismo , Xilose
16.
ACS Synth Biol ; 11(9): 3004-3014, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36037444

RESUMO

As a new method of diagnosis and treatment for intestinal diseases, intelligent engineered bacteria based on synthetic biology have been developed vigorously in recent years. However, how to deal with the engineered bacteria in vivo after completing the tasks is an urgent problem to be resolved. In this study, we constructed a thiosulfate (a biomarker of inflammatory bowel disease)-responsive engineered bacteria to generate two signals, sfGFP (monitoring) and gain-of-function (translation activation) mutation (ACG to ATG), in the initiation codon of lysisE (recording) via the CRISPR/Cas9-mediated base editing system. Once these two signals were detected, xylose could be added to induce lysis E expression, resulting in the destruction of the edited bacteria and the release of AvCystain simultaneously. Overall, our innovative engineered bacteria can record instant and historical information of the disease, and especially, the edited bacteria can be artificially attenuated and release drug in situ when needed, ultimately serving as a disposable and recyclable candidate for more types of diseases.


Assuntos
Edição de Genes , Enteropatias , Bactérias/genética , Sistemas CRISPR-Cas/genética , Códon de Iniciação , Edição de Genes/métodos , Humanos , Enteropatias/genética , Prebióticos , Tiossulfatos , Xilose
17.
J Agric Food Chem ; 70(34): 10604-10610, 2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-35977412

RESUMO

To enable its structural characterization by nuclear magnetic resonance (NMR) spectroscopy, the native structure of cereal water-unextractable arabinoxylan (WU-AX) is typically disrupted by alkali or enzymatic treatments. Here, WU-AX in the wheat flour unextractable cell wall material (UCWM) containing 40.9% ± 1.5 arabinoxylan with an arabinose-to-xylose ratio of 0.62 ± 0.04 was characterized by high-resolution solid-state NMR without disrupting its native structure. Hydration of the UCWM (1.7 mg H2O/mg UCWM) in combination with specific optimizations in the NMR methodology enabled analysis by solid-state 13C NMR with magic angle spinning and 1H high-power decoupling (13C HPDEC MAS NMR) which provided sufficiently high resolution to allow for carbon atom assignments. Spectral resonances of C-1 from arabinose and xylose residues of WU-AX were here assigned to the solid state. The proportions of un-, mono-, and di-substituted xyloses were 59.2, 19.5, and 21.2%, respectively. 13C HPDEC MAS NMR showed the presence of solid-state fractions with different mobilities in the UCWM. This study presents the first solid-state NMR spectrum of wheat WU-AX with sufficient resolution to enable assignment without prior WU-AX solubilization.


Assuntos
Farinha , Triticum , Arabinose/análise , Parede Celular/química , Farinha/análise , Espectroscopia de Ressonância Magnética , Triticum/química , Água/química , Xilanos/química , Xilose
18.
Microb Cell Fact ; 21(1): 154, 2022 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-35933385

RESUMO

BACKGROUND: Sugar hydrolysates from lignocellulosic biomass are majorly composed of glucose and xylose that can be fermented to biofuels. Bacteria, despite having the natural ability to consume xylose are unable to consume it in presence of glucose due to a carbon catabolite repression (CCR) mechanism. This leads to overall reduced productivity as well as incomplete xylose utilization due to ethanol build-up from glucose utilization. In our effort to develop a strain for simultaneous fermentation of glucose and xylose into ethanol, we deleted ptsG in ethanologenic E. coli SSK42 to make it deficient in CCR and performed adaptive laboratory evolution to achieve accelerated growth rate, sugar consumption and ethanol production. Finally, we performed proteomics study to identify changes that might have been responsible for the observed improved phenotype of the evolved strain. RESULTS: The parental strain of SSK42, i.e., wild-type E. coli B, did not co-utilize glucose and xylose as expected. After deleting the ptsG gene encoding the EIIBCGlc subunit of PTS system, glucose consumption is severely affected in wild-type E. coli B. However, the ethanologenic, SSK42 strain, which was evolved in our earlier study on both glucose and xylose, didn't show such a drastic effect of EIIBCGlc deletion, instead consumed glucose first, followed by xylose without delay for switching from one sugar to another. To improve growth on xylose and co-utilization capabilities, the ptsG deleted SSK42 was evolved on xylose. The strain evolved for 78 generations, strain SCD78, displayed significant co-utilization of glucose and xylose sugars. At the bioreactor level, the strain SCD78 produced 3-times the ethanol titer of the parent strain with significant glucose-xylose co-utilization. The rate of glucose and xylose consumption also increased 3.4-fold and 3-fold, respectively. Proteome data indicates significant upregulation of TCA cycle proteins, respiration-related proteins, and some transporters, which may have a role in increasing the total sugar consumption and co-utilization of sugars. CONCLUSION: Through adaptive evolution, we have obtained a strain that has a significant glucose-xylose co-utilization phenotype with 3-fold higher total sugar consumption rate and ethanol production rate compared to the unevolved strain. This study also points out that adaptation on xylose is enough to impart glucose-xylose co-utilization property in CCR compromised ethanologenic strain SSK42.


Assuntos
Repressão Catabólica , Xilose , Açúcares da Dieta/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Etanol/metabolismo , Fermentação , Glucose/metabolismo , Açúcares/metabolismo , Xilose/metabolismo
19.
Bioprocess Biosyst Eng ; 45(10): 1635-1644, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35974197

RESUMO

L-Asparaginase (L-ASNase) is an enzyme applied in the treatment of lymphoid malignancies. However, an innovative L-ASNase with high yield and lower side effects than the commercially available preparations are still a market requirement. Here, a new-engineered Bacillus subtilis strain was evaluated for Aliivibrio fischeri L-ASNase II production, being the bioprocess development and the enzyme characterization studied. The pBS0E plasmid replicative in Bacillus sp and containing PxylA promoter inducible by xylose and its repressive molecule sequence (XylR) was used for the genetic modification. Initially, cultivations were carried out in orbital shaker, and then the process was scaled up to stirred tank bioreactor (STB). After the bioprocess, the cells were recovered and submitted to ultrasound sonication for cells disruption and intracellular enzyme recovery. The enzymatic extract was characterized to assess its biochemical, kinetic and thermal properties using L-Asparagine and L-Glutamine as substrates. The results indicated the potential enzyme production in STB achieving L-ASNase activity up to 1.539 U mL-1. The enzymatic extract showed an optimum pH of 7.5, high L-Asparagine affinity (Km = 1.2275 mmol L-1) and low L-Glutaminase activity (0.568-0.738 U mL-1). In addition, thermal inactivation was analyzed by two different Kinect models to elucidate inactivation mechanisms, low kinetic thermal inactivation constants for 25 ºC and 37 ºC (0.128 and 0.148 h-1, respectively) indicate an elevated stability. The findings herein show that the produced recombinant L-ASNase has potential to be applied for pharmaceutical purposes.


Assuntos
Antineoplásicos , Produtos Biológicos , Aliivibrio fischeri , Antineoplásicos/química , Asparaginase/química , Asparaginase/genética , Asparaginase/uso terapêutico , Asparagina , Bacillus subtilis/genética , Glutaminase , Glutamina , Preparações Farmacêuticas , Xilose
20.
FEMS Yeast Res ; 21(1)2022 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-35918180

RESUMO

Sugar transporter research focuses on the sugar uptake into cells. Under certain physiological conditions, however, the intracellular accumulation and secretion of carbohydrates (efflux) are relevant processes in many cell types. Currently, no cell-based system is available for specifically investigating glucose efflux. Therefore, we designed a system based on a hexose transporter-deficient Saccharomyces cerevisiae strain, in which the disaccharide maltose is provided as a donor of intracellular glucose. By deleting the hexokinase genes, we prevented the metabolization of glucose, and thereby achieved the accumulation of growth-inhibitory glucose levels inside the cells. When a permease mediating glucose efflux is expressed in this system, the inhibitory effect is relieved proportionally to the capacity of the introduced transporter. The assay is thereby suitable for screening of transporters and quantitative analyses of their glucose efflux capacities. Moreover, by simultaneous provision of intracellular glucose and extracellular xylose, we investigated how each sugar influences the transport of the other one from the opposite side of the membrane. Thereby, we could show that the xylose transporter variant Gal2N376F is insensitive not only to extracellular but also to intracellular glucose. Considering the importance of sugar transporters in biotechnology, the assay could facilitate new developments in a variety of applications.


Assuntos
Saccharomyces cerevisiae , Xilose , Carboidratos , Glucose/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Maltose/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Açúcares/metabolismo , Xilose/metabolismo
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