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1.
Bioresour Technol ; 343: 126071, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34606923

RESUMO

One of the potential bioresources for bioethanol production is Napier grass, considering its high cellulose and hemicellulose content. However, the cost of pretreatment hinders the bioethanol produced from being economical. This study examines the effect of hydrothermal process with dilute acid on extruded Napier grass, followed by enzymatic saccharification prior to simultaneous saccharification and co-fermentation (SScF). Extrusion facilitated lignin removal by 30.2 % prior to dilute acid steam explosion. Optimum pretreatment condition was obtained by using 3% sulfuric acid, and 30-min retention time of steam explosion at 190 °C. Ethanol yield of 0.26 g ethanol/g biomass (60.5% fermentation efficiency) was attained by short-term liquefaction and fermentation using a cellulose-hydrolyzing and xylose-assimilating Saccharomyces cerevisiae NBRC1440/B-EC3-X ΔPHO13, despite the presence of inhibitors. This proposed method not only reduced over-degradation of cellulose and hemicellulose, but also eliminated detoxification process and reduced cellulase loading.


Assuntos
Saccharomyces cerevisiae , Xilose , Celulose/metabolismo , Etanol , Fermentação , Hidrólise , Lignina/metabolismo , Saccharomyces cerevisiae/metabolismo , Ácidos Sulfúricos
2.
Bioresour Technol ; 343: 126153, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34673190

RESUMO

Bioethanol is a major biofuel in industry and mainly produced from corn starch with the dry-mill process. However, one of the remaining challenges is how to economically and efficiently exploit the wasted co-products to further improve ethanol production and generate more valuable chemicals. Here, an integrative approach was developed to efficiently utilize the waste cake for ethanol production, accompanied by protein extraction for feed additives. A high-quality protein feed was produced by the ethanol-alkali extraction method (extraction rate up to 46.91%). Notably, by applying two-step chemoenzymatic strategy, the supernatant and solid recycling yield up to 4.1-, 3.8-, and 154-fold improvements of ethanol, glucose, and xylose production, respectively, comparing to non-pretreatment. Moreover, mass balance analysis found this approach significantly contributed 1.74-4.42% (5.96-15.11 kg/ton dry corn) increase of total ethanol production. The gained knowledge about process design holds the potential transferability for other sustainable biowaste management and bioethanol industry.


Assuntos
Biocombustíveis , Etanol , Fermentação , Resíduos/análise , Xilose
3.
Enzyme Microb Technol ; 151: 109921, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34649692

RESUMO

ß-Xylosidases are often inhibited by its reaction product xylose or inactivated by high temperature environment, which limited its application in hemicellulosic biomass conversion to fuel and food processing. Remarkably, some ß-xylosidases from GH39 family are tolerant to xylose. Therefore, it is of great significance to elucidate the effect mechanism of xylose on GH39 ß-xylosidases to improve their application. In this paper, based on the homologous model and prediction of protein active pocket constructed by I-TASSA and PyMOL, two putative xylose tolerance relevant sites (283 and 284) were mutated at the bottom of the protein active pocket, where xylose sensitivity and thermostability of Dictyoglomus thermophilum ß-xylosidase Xln-DT were improved by site-directed mutagenesis. The Xln-DT mutant Xln-DT-284ASP and Xln-DT-284ALA showed high xylose tolerance, with the Ki values of 4602 mM and 3708 mM, respectively, which increased by 9-35% compared with the wildtype Xln-DT. The thermostability of mutant Xln-DT-284ASP was significantly improved at 75 and 85 °C, while the activity of the wild enzyme Xln-DT decreased to 40-20%, the activity of the mutant enzyme still remained 100%. The mutant Xln-DT-284ALA showed excellent stability at pH 4.0-7.0, but Xln-DT-284ASP showed slightly decreased activity. Furthermore, in order to explore the key sites and mechanism of xylose's effect on ß-xylosidase activity, the interaction between xylose and enzyme was simulated by molecular docking. Besides binding to the active sites at the bottom of the substrate channel, xylose can also bind to sites in the middle or entrance of the channel with different affinities, which may determine the xylose inhibition of ß-xylosidase. In conclusion, the improved xylose tolerance of mutant enzyme could be more advantageous in the degradation of hemicellulose and the biotransformation of other natural active substances containing xylose. This study supplies new insights into general mechanism of xylose effect on the activity of GH 39 ß-xylosidases as well as related enzymes that modulate their activity via feedback control mechanism.


Assuntos
Xilose , Xilosidases , Bactérias , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Especificidade por Substrato , Xilosidases/genética , Xilosidases/metabolismo
4.
Nutrients ; 13(10)2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34684319

RESUMO

Demands for novel lactic acid bacteria with potential to be used as probiotics along with healthy fermented plant-based products increase worldwide. In this study, a novel Lactiplantibacillus plantarum P31891 strain with enzymatic capacity to degrade tannins and ferment xylose was used as starter culture for fermentation of a quinoa-based beverage. The probiotic potential of the selected strain was evaluated in healthy volunteers. Twenty participants consumed the beverage for 14 days; microbiota changes in saliva and faecal samples were analyzed by Terminal Restriction Fragment Length Polymorphism (T-RFLP), Next Generation Sequencing (NGS) and qPCR; and gastrointestinal well-being and digestive symptoms were recorded. The results indicated that the consumption of the beverage with Lactiplantibacillus plantarum P31891 in a probiotic dose (1012 CFU/mL) increased the number of Lactobacillus in the feces but not in saliva. Overall, the bacterial community did not seem to be influenced by the bacterium or by the beverage, as expressed by the diversity indexes, but specific genera were affected, as reflected in changes in amplicon sequence variants. Consequently, Lactiplantibacillus plantarum P31891 showed potential to be categorized as a probiotic strain in the fermented quinoa-based beverage.


Assuntos
Chenopodium quinoa/química , Alimentos e Bebidas Fermentados , Lactobacillus plantarum/metabolismo , Microbiota , Xilose/metabolismo , Biodiversidade , Fezes/microbiologia , Humanos , Concentração de Íons de Hidrogênio , Filogenia , Saliva/microbiologia
5.
FASEB J ; 35(11): e21977, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34613640

RESUMO

Xylo-oligosaccharide (XOS), which is considered as a potential prebiotic, exhibits multiple beneficial effects on modulation of gut microbiota, strength of intestinal barrier, and inhibition of intestinal inflammation. The objective of this study is to investigate whether XOS protects against Salmonella infection by modulating gut microbiota, enhancing the intestinal barrier, and resisting colonization. C57BL/6 male mice received water supplementation with 5% XOS for 14 days before Salmonella Typhimurium infection. The results showed that XOS suppressed the Salmonella-induced inflammation, but had limited effects on tight junction molecules and mRNA expression of mucus proteins, except for claudin-1 in the colon. Data of 16S rDNA sequencing indicated that XOS modulated gut microbiota composition by significantly stimulating Bifidobacterium animalis (B. animalis), and reducing Salmonella counts. Therefore, the potential protective effects of B. animalis against Salmonella challenge were investigated as well. Bifidobacterium animalis subsp lactis BB-12 (BB12), which could markedly increase in XOS, was selected to treat mice. Similarly, Salmonella-induced inflammatory reactions were alleviated by BB12 but tight junction molecules and mucin proteins in the colonic tissues were not affected. Administration of BB12 remarkably decreased the copies of Salmonella in cecal digesta post Salmonella infection. Additionally, the decrease concentrations of cecal propionate and total short-chain fatty acids (SCFAs) in Salmonella-infected mice were reversed by BB12 treatment, and propionate performed a strong inhibitory effect on Salmonella growth in vitro. Besides that, BB12 could directly restrict Salmonella proliferation in vitro. Moreover, BB12 reduced the adhesion ability of Salmonella on the Caco-2 cells model. Our results suggest that XOS could be considered as a candidate of functional food to protect against Salmonella infection by stimulating Bifidobacterium, which then resists Salmonella colonization by maintaining the intestinal SCFAs levels and suppressing adhesibility.


Assuntos
Bifidobacterium/efeitos dos fármacos , Inflamação/tratamento farmacológico , Probióticos , Infecções por Salmonella/tratamento farmacológico , Salmonella typhimurium/efeitos dos fármacos , Xilose , Animais , Microbioma Gastrointestinal/efeitos dos fármacos , Intestinos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Probióticos/farmacologia , Probióticos/uso terapêutico , Xilose/análogos & derivados , Xilose/farmacologia , Xilose/uso terapêutico
6.
Appl Microbiol Biotechnol ; 105(19): 7339-7352, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34499201

RESUMO

Herbaspirillum seropedicae is a ß-proteobacterium that establishes as an endophyte in various plants. These bacteria can consume diverse carbon sources, including hexoses and pentoses like D-xylose. D-xylose catabolic pathways have been described in some microorganisms, but databases of genes involved in these routes are limited. This is of special interest in biotechnology, considering that D-xylose is the second most abundant sugar in nature and some microorganisms, including H. seropedicae, are able to accumulate poly-3-hydroxybutyrate when consuming this pentose as a carbon source. In this work, we present a study of D-xylose catabolic pathways in H. seropedicae strain Z69 using RNA-seq analysis and subsequent analysis of phenotypes determined in targeted mutants in corresponding identified genes. G5B88_22805 gene, designated xylB, encodes a NAD+-dependent D-xylose dehydrogenase. Mutant Z69∆xylB was still able to grow on D-xylose, although at a reduced rate. This appears to be due to the expression of an L-arabinose dehydrogenase, encoded by the araB gene (G5B88_05250), that can use D-xylose as a substrate. According to our results, H. seropedicae Z69 uses non-phosphorylative pathways to catabolize D-xylose. The lower portion of metabolism involves co-expression of two routes: the Weimberg pathway that produces α-ketoglutarate and a novel pathway recently described that synthesizes pyruvate and glycolate. This novel pathway appears to contribute to D-xylose metabolism, since a mutant in the last step, Z69∆mhpD, was able to grow on this pentose only after an extended lag phase (40-50 h). KEY POINTS: • xylB gene (G5B88_22805) encodes a NAD+-dependent D-xylose dehydrogenase. • araB gene (G5B88_05250) encodes a L-arabinose dehydrogenase able to recognize D-xylose. • A novel route involving mhpD gene is preferred for D-xylose catabolism.


Assuntos
Biotecnologia , Xilose , Herbaspirillum
7.
Metab Eng ; 68: 119-130, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34592433

RESUMO

Bottlenecks in the efficient conversion of xylose into cost-effective biofuels have limited the widespread use of plant lignocellulose as a renewable feedstock. The yeast Saccharomyces cerevisiae ferments glucose into ethanol with such high metabolic flux that it ferments high concentrations of glucose aerobically, a trait called the Crabtree/Warburg Effect. In contrast to glucose, most engineered S. cerevisiae strains do not ferment xylose at economically viable rates and yields, and they require respiration to achieve sufficient xylose metabolic flux and energy return for growth aerobically. Here, we evolved respiration-deficient S. cerevisiae strains that can grow on and ferment xylose to ethanol aerobically, a trait analogous to the Crabtree/Warburg Effect for glucose. Through genome sequence comparisons and directed engineering, we determined that duplications of genes encoding engineered xylose metabolism enzymes, as well as TKL1, a gene encoding a transketolase in the pentose phosphate pathway, were the causative genetic changes for the evolved phenotype. Reengineered duplications of these enzymes, in combination with deletion mutations in HOG1, ISU1, GRE3, and IRA2, increased the rates of aerobic and anaerobic xylose fermentation. Importantly, we found that these genetic modifications function in another genetic background and increase the rate and yield of xylose-to-ethanol conversion in industrially relevant switchgrass hydrolysate, indicating that these specific genetic modifications may enable the sustainable production of industrial biofuels from yeast. We propose a model for how key regulatory mutations prime yeast for aerobic xylose fermentation by lowering the threshold for overflow metabolism, allowing mutations to increase xylose flux and to redirect it into fermentation products.


Assuntos
Proteínas de Saccharomyces cerevisiae , Xilose , Biocombustíveis , Fermentação , Glucose , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
J Agric Food Chem ; 69(33): 9625-9631, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34382797

RESUMO

Cofactor availability is often a rate-limiting factor in the bioconversion of xylose to xylitol. The overexpression of pentose phosphate pathway genes and the deletion of Embden-Meyerhof-Parnas pathway genes can modulate the glucose metabolic flux and increase the intracellular NADPH supply, enabling Escherichia coli cells to produce xylitol from corncob hydrolysates. The effects of zwf and/or gnd overexpression and pfkA, pfkB, and/or pgi deletion on the intracellular redox environment and xylitol production were examined. The NADPH-enhanced strain 2bpgi produced 162 g/L xylitol from corncob hydrolysates after a 76 h fed-batch fermentation in a 15 L bioreactor, which was 13.3% greater than the 143 g/L xylitol produced by the IS5-d control strain. Additionally, the xylitol productivity and xylitol yield per glucose for 2bpgi were 2.13 g/L/h and 2.50 g/g, respectively. Thus, the genetic modifications in 2bpgi significantly enhanced NADPH regeneration, making 2bpgi a potentially useful strain for the industrial-scale production of xylitol from detoxified corncob hydrolysates.


Assuntos
Via de Pentose Fosfato , Xilitol , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Deleção de Genes , Glucose , Glicólise , NADP/metabolismo , Fosfatos , Xilose
9.
Water Sci Technol ; 84(3): 656-666, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34388125

RESUMO

This study compares the H2 production from glucose, xylose, and acidic hydrolysates of Agave tequilana bagasse as substrates. The fermentation was performed in a granular sludge reactor operated in two phases: (1) model substrates (glucose and xylose) and (2) acidic hydrolysates at 35 °C, pH 4.5 and a hydraulic retention time of 5.5 h with glucose (10 g L-1) and xylose (12 g L-1). A sequencing batch reactor was used to acclimate the biomass between the glucose and xylose continuous fermentation (with a mixture of xylose-glucose) and acidic hydrolysates. During the discontinuous acclimating step, the xylose/glucose ratio increment negatively affected the H2 productivity. Although the continuous H2 production with xylose was negligible, the co-fermentation with glucose (88-12%) allowed H2 productivity of 2,889 ± 502 mL H2 L-1d-1. An acidic hydrolysate concentration of 3.3 gcarbohydrate L-1 showed a three-fold higher H2 productivity than with a concentration of 10 g L-1. The results indicated that xylose, as the only substrate, was challenging to metabolize by the inoculum, and its mixture with glucose improved the H2 productivity. Therefore, the low H2 productivity with hydrolysates could be related to the presence of xylose.


Assuntos
Agave , Xilose , Agave/metabolismo , Celulose/metabolismo , Fermentação , Glucose
10.
J Agric Food Chem ; 69(36): 10648-10656, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34463101

RESUMO

2-Threityl-thiazolidine-4-carboxylic acid (TTCA), a nonvolatile precursor of flavor and color, is considered to be more stable than its isomeric Amadori compound (ARP). The degradation behavior of TTCA favors higher temperatures and pH. In order to adjust and control the thermal degradation of TTCA to improve its food processing adaptability, a TTCA-Xyl thermal reaction model was constructed to explore the effect of extra-added Xyl on the thermal degradation behavior of TTCA. The results confirmed that the extra-added Xyl was involved in the degradation pathway of TTCA and accelerated its depletion, thus promoting the formation of characteristic downstream products of TTCA including some α-dicarbonyl compounds, and consequently accelerating the browning formation. The isotope-labeling technique was further applied to confirm that the added Xyl could trap the Cys released from the decomposition of ARP and formed additional TTCA, which could promote the movement of chemical equilibrium and gradually accelerate the degradation rate of TTCA as well as melanoidins formation. The higher pH value could even promote this phenomenon.


Assuntos
Reação de Maillard , Xilose , Cisteína , Tiazolidinas
11.
Nat Commun ; 12(1): 4975, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404791

RESUMO

Plant cell wall hydrolysates contain not only sugars but also substantial amounts of acetate, a fermentation inhibitor that hinders bioconversion of lignocellulose. Despite the toxic and non-consumable nature of acetate during glucose metabolism, we demonstrate that acetate can be rapidly co-consumed with xylose by engineered Saccharomyces cerevisiae. The co-consumption leads to a metabolic re-configuration that boosts the synthesis of acetyl-CoA derived bioproducts, including triacetic acid lactone (TAL) and vitamin A, in engineered strains. Notably, by co-feeding xylose and acetate, an enginered strain produces 23.91 g/L TAL with a productivity of 0.29 g/L/h in bioreactor fermentation. This strain also completely converts a hemicellulose hydrolysate of switchgrass into 3.55 g/L TAL. These findings establish a versatile strategy that not only transforms an inhibitor into a valuable substrate but also expands the capacity of acetyl-CoA supply in S. cerevisiae for efficient bioconversion of cellulosic biomass.


Assuntos
Parede Celular/metabolismo , Engenharia Metabólica , Polissacarídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilcoenzima A/metabolismo , Biomassa , Reatores Biológicos , Fermentação , Lignina , Pironas/metabolismo , Saccharomyces cerevisiae/genética , Vitamina A/metabolismo , Xilose/metabolismo
12.
J Agric Food Chem ; 69(34): 9924-9933, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34427083

RESUMO

The Maillard reaction performed under a stepwise increase of temperature was applied for researching the inhibition of Maillard browning caused by ellagic acid. Ellagic acid was found effective for the inhibition of melanoidin formation in the xylose-glycine Maillard reaction but depended on its dosage and the point of time it was added in the reaction system. The lightest color of the Maillard reaction products was observed when ellagic acid was added at the 90th min, which was the point of time when the Amadori rearrangement product (ARP) developed the most. LC-ESI-MS/MS analysis results showed a significant tendency of the ellagic acid hydrolysis product to react with the predominant intermediate ARP to yield an adduct. The adduct stabilized the ARP and delayed its decomposition and inhibited the downstream reactions toward browning. After the ARP was depleted, ellagic acid also showed an effect on scavenging some short-chain dicarbonyls which contributed to the inhibition of Maillard browning.


Assuntos
Ácido Elágico , Espectrometria de Massas em Tandem , Produtos Finais de Glicação Avançada , Reação de Maillard , Xilose
13.
Bioresour Technol ; 340: 125677, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34358990

RESUMO

The xylitol production was performed with acidophilic Meyerozyma caribbica. The particle size of 0.02 ± 0.01 to 0.1 ± 0.05 mm was rich in glucose (12.0 ± 0.5 g/L), whereas 0.5 ± 0.25 to 2.0 ± 0.5 mm had a high content of xylose (8.0 ± 0.5 g/L). The xylitol production in the synthetic, non-detoxified and detoxified hydrolysate media was studied (50 ± 0.5 g/L) using 10% v/v non - induced cells of M. caribbica for 120 h. At the end of fermentation with the specific growth rate of 0.056 ± 0.01 (µ), xylitol yields of 45.00 ± 1.00%, 10.00 ± 1.00% and 54.00 ± 1.00% were obtained. The detoxification of the hydrolysate prepared using an identified corncob particle size of 0.5 ± 0.25 to 2.0 ± 0.5 mm could be used as the prospective pretreatment process for ecofriendly and industrial scale production of xylitol with M. caribbica.


Assuntos
Xilose , Zea mays , Fermentação , Hidrólise , Tamanho da Partícula , Polissacarídeos , Estudos Prospectivos , Saccharomycetales , Açúcares , Xilitol
14.
Biosens Bioelectron ; 193: 113573, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34425520

RESUMO

NAD+-dependent dehydrogenase-based biosensors usually suffer from the low accuracy due to the interference of cofactors in the complex environment, such as fermentation samples. Herein, we demonstrate the example of an integrated biosensor device that can be applied for analyzing xylose fermentation samples. The device is composed of one chamber for the elimination of NAD+ and NADH in the fermentation broth and another chamber for the sample analysis. In the first chamber, a cyclic voltammetry method coupled with Ni foam as a working electrode was proven to be effective in removing NAD+ and NADH in the fermentation broth. In the other chamber, xylose dehydrogenase, as the recognition element, and diaphorase, used for the regeneration of bioactive NAD+ mediated by vitamin K3, were co-immobilized on the surface of the magnetic nanoparticles, which was further coated onto a magnetic glassy carbon electrode. The detection range of the constructed biosensor was from 0.5 to 10 g L-1 with a detection limit of 0.01 g L-1 at a signal-to-noise ratio of 3. Moreover, the biosensor achieved high selectivity, recovery, reproducibility, and good long-time stability when analyzing real xylose fermentation samples, suggesting its promising application potential.


Assuntos
Técnicas Biossensoriais , Fermentação , NAD/metabolismo , Oxirredutases , Reprodutibilidade dos Testes , Xilose
15.
Bioresour Technol ; 341: 125817, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34454236

RESUMO

Lipids accumulated in the vegetative tissues of cellulosic feedstocks can be a potential raw material for biodiesel and bioethanol production. In this work, bagasse of genetically engineered sorghum was subjected to liquid hot-water pretreatment at 170, 180, and 190 °C for different reaction time. Under the optimal pretreatment condition (170 °C, 20 min), the residue was enriched in glucan (57.39 ± 2.63 % w/w) and xylan (13.38 ± 0.49 % w/w). The total lipid content of the pretreated residue was 6.81% w/w, similar to that observed in untreated bagasse (6.30% w/w). Pretreatment improved the enzymatic digestibility of bagasse, allowing a recovery of 79% w/w and 86% w/w of glucose and xylose, respectively. The pretreatment and enzymatic saccharification resulted in a 2-fold increase in total lipid in enzymatic residue compared to the original bagasse. Thus, pretreatment and enzymatic hydrolysis enabled high sugar recovery while concentrating triglycerides and free fatty acids in the residue.


Assuntos
Açúcares , Xilose , Hidrólise , Lipídeos , Xilanos
16.
Microb Biotechnol ; 14(5): 1931-1943, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34403199

RESUMO

Pseudomonas putida is a highly solvent-resistant microorganism and useful chassis for the production of value-added compounds from lignocellulosic residues, in particular aromatic compounds that are made from phenylalanine. The use of these agricultural residues requires a two-step treatment to release the components of the polysaccharides of cellulose and hemicellulose as monomeric sugars, the most abundant monomers being glucose and xylose. Pan-genomic studies have shown that Pseudomonas putida metabolizes glucose through three convergent pathways to yield 6-phosphogluconate and subsequently metabolizes it through the Entner-Doudoroff pathway, but the strains do not degrade xylose. The valorization of both sugars is critical from the point of view of economic viability of the process. For this reason, a P. putida strain was endowed with the ability to metabolize xylose via the xylose isomerase pathway, by incorporating heterologous catabolic genes that convert this C5 sugar into intermediates of the pentose phosphate cycle. In addition, the open reading frame T1E_2822, encoding glucose dehydrogenase, was knocked-out to avoid the production of the dead-end product xylonate. We generated a set of DOT-T1E-derived strains that metabolized glucose and xylose simultaneously in culture medium and that reached high cell density with generation times of around 100 min with glucose and around 300 min with xylose. The strains grew in 2G hydrolysates from diluted acid and steam explosion pretreated corn stover and sugarcane straw. During growth, the strains metabolized > 98% of glucose, > 96% xylose and > 85% acetic acid. In 2G hydrolysates P. putida 5PL, a DOT-T1E derivative strain that carries up to five independent mutations to avoid phenylalanine metabolism, accumulated this amino acid in the medium. We constructed P. putida 5PLΔgcd (xylABE) that produced up to 250 mg l-1 of phenylalanine when grown in 2G pretreated corn stover or sugarcane straw. These results support as a proof of concept the potential of P. putida as a chassis for 2G processes.


Assuntos
Aminoácidos Aromáticos , Pseudomonas putida , Glucose , Lignina , Pseudomonas putida/genética , Xilose
17.
Biotechnol J ; 16(11): e2100238, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34418308

RESUMO

Simultaneous co-fermentation of glucose and xylose is a key desired trait of engineered Saccharomyces cerevisiae for efficient and rapid production of biofuels and chemicals. However, glucose strongly inhibits xylose transport by endogenous hexose transporters of S. cerevisiae. We identified structurally distant sugar transporters (Lipomyces starkeyi LST1_205437 and Arabidopsis thaliana AtSWEET7) capable of co-transporting glucose and xylose from previously unexplored oleaginous yeasts and plants. Kinetic analysis showed that LST1_205437 had lenient glucose inhibition on xylose transport and AtSWEET7 transported glucose and xylose simultaneously with no inhibition. Modelling studies of LST1_205437 revealed that Ala335 residue at sugar binding site can accommodates both glucose and xylose. Docking studies with AtSWEET7 revealed that Trp59, Trp183, Asn145, and Asn179 residues stabilized the interactions with sugars, allowing both xylose and glucose to be co-transported. In addition, we altered sugar preference of LST1_205437 by single amino acid mutation at Asn365. Our findings provide a new mechanistic insight on glucose and xylose transport mechanism of sugar transporters and the identified sugar transporters can be employed to develop engineered yeast strains for producing cellulosic biofuels and chemicals.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Glucose , Lipomyces/enzimologia , Proteínas de Transporte de Monossacarídeos/genética , Xilose , Arabidopsis/genética , Fermentação , Cinética , Lipomyces/genética , Saccharomyces cerevisiae/genética
18.
Metab Eng ; 67: 387-395, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34365009

RESUMO

Carbon loss in the form of CO2 is an intrinsic and persistent challenge faced during conventional and advanced biofuel production from biomass feedstocks. Current mechanisms for increasing carbon conservation typically require the provision of reduced co-substrates as additional reducing equivalents. This need can be circumvented, however, by exploiting the natural heterogeneity of lignocellulosic sugars mixtures and strategically using specific fractions to drive complementary CO2 emitting vs. CO2 fixing pathways. As a demonstration of concept, a coculture-coproduction system was developed by pairing two catabolically orthogonal Escherichia coli strains; one converting glucose to ethanol (G2E) and the other xylose to succinate (X2S). 13C-labeling studies reveled that G2E + X2S cocultures were capable of recycling 24% of all evolved CO2 and achieved a carbon conservation efficiency of 77%; significantly higher than the 64% achieved when all sugars are instead converted to just ethanol. In addition to CO2 exchange, the latent exchange of pyruvate between strains was discovered, along with significant carbon rearrangement within X2S.


Assuntos
Dióxido de Carbono , Carbono , Técnicas de Cocultura , Fermentação , Glucose , Xilose
19.
Molecules ; 26(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205848

RESUMO

In this paper, we have performed the Lipozyme 435-catalyzed synthesis of xylose oleate in methyl ethyl ketone (MEK) from xylose and oleic acid. The effects of substrates' molar ratios, reaction temperature, reaction time on esterification rates, and Lipozyme 435 reuse were studied. Results showed that an excess of oleic acid (xylose: oleic acid molar ratio of 1:5) significantly favored the reaction, yielding 98% of xylose conversion and 31% oleic acid conversion after 24 h-reaction (mainly to xylose mono- and dioleate, as confirmed by mass spectrometry). The highest Lipozyme 435 activities occurred between 55 and 70 °C. The predicted Ping Pong Bi Bi kinetic model fitted very well to the experimental data and there was no evidence of inhibitions in the range assessed. The reaction product was purified and presented an emulsion capacity close to that of a commercial sugar ester detergent. Finally, the repeated use of Lipozyme 435 showed a reduction in the reaction yields (by 48 and 19% in the xylose and oleic acid conversions, respectively), after ten 12 h-cycles.


Assuntos
Butanonas/química , Lipase/metabolismo , Xilose/química , Biocatálise , Esterificação , Temperatura Alta , Ácido Oleico/química
20.
Appl Microbiol Biotechnol ; 105(13): 5565-5575, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34215904

RESUMO

Most of the oleaginous microorganisms cannot assimilate xylose in the presence of glucose, which is the major bottleneck in the bioconversion of lignocellulose to biodiesel. Our present study revealed that overexpression of xylose isomerase (XI) gene xylA or xylulokinase (XK) gene xks1 increased the xylose consumption by 25 to 37% and enhanced the lipid content by 8 to 28% during co-fermentation of glucose and xylose. In xylA overexpressing strain Mc-XI, the activity of XI was 1.8-fold higher and the mRNA level of xylA at 24 h and 48 h was 11- and 13-fold higher than that of the control, respectively. In xks1 overexpressing strain Mc-XK, the mRNA level of xks1 was 4- to 11-fold of that of the control strain and the highest XK activity of 950 nmol min-1 mg-1 at 72 h which was 2-fold higher than that of the control. Additionally, expression of a translational fusion of xylA and xks1 further enhanced the xylose utilization rate by 45%. Our results indicated that overexpression of xylA and/or xks1 is a promising strategy to improve the xylose and glucose co-utilization, alleviate the glucose repression, and produce lipid from lignocellulosic biomass in the oleaginous fungus M. circinelloides. KEY POINTS: • Overexpressing xylA or xks1 increased the xylose consumption and the lipid content. • The xylose isomerase activity and the xylA mRNA level were enhanced in strain Mc-XI. • Co-expression of xylA and xks1 further enhanced the xylose utilization rate by 45%.


Assuntos
Glucose , Xilose , Aldose-Cetose Isomerases , Fermentação , Mucor/genética , Fosfotransferases (Aceptor do Grupo Álcool)
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