RESUMO
BACKGROUND: Lignocellulosic ethanol could offer a sustainable source to meet the increasing worldwide demand for fuel. However, efficient and simultaneous metabolism of all types of sugars in lignocellulosic hydrolysates by ethanol-producing strains is still a challenge. RESULTS: An engineered strain Escherichia coli B0013-2021HPA with regulated glucose utilization, which could use all monosaccharides in lignocellulosic hydrolysates except glucose for cell growth and glucose for ethanol production, was constructed. In E. coli B0013-2021HPA, pta-ackA, ldhA and pflB were deleted to block the formation of acetate, lactate and formate and additional three mutations at glk, ptsG and manZ generated to block the glucose uptake and catabolism, followed by the replacement of the wild-type frdA locus with the ptsG expression cassette under the control of the temperature-inducible λ pR and pL promoters, and the final introduction of pEtac-PA carrying Zymomonas mobilis pdc and adhB for the ethanol pathway. B0013-2021HPA was able to utilize almost all xylose, galactose and arabinose but not glucose for cell propagation at 34 °C and converted all sugars to ethanol at 42 °C under oxygen-limited fermentation conditions. CONCLUSIONS: Engineered E. coli strain with regulated glucose utilization showed efficient metabolism of mixed sugars in lignocellulosic hydrolysates and thus higher productivity of ethanol production.
Assuntos
Escherichia coli/metabolismo , Etanol/metabolismo , Glucose/metabolismo , Lignina/metabolismoRESUMO
OBJECTIVES: To develop a xylose-nonutilizing Escherichia coli strain for ethanol production and xylose recovery. RESULTS: Xylose-nonutilizing E. coli CICIM B0013-2012 was successfully constructed from E. coli B0013-1030 (pta-ack, ldhA, pflB, xylH) by deletion of frdA, xylA and xylE. It exhibited robust growth on plates containing glucose, arabinose or galactose, but failed to grow on xylose. The ethanol synthesis pathway was then introduced into B0013-2012 to create an ethanologenic strain B0013-2012PA. In shaking flask fermentation, B0013-2012PA fermented glucose to ethanol with the yield of 48.4 g/100 g sugar while xylose remained in the broth. In a 7-l bioreactor, B0013-2012PA fermented glucose, galactose and arabinose in the simulated corncob hydrolysate to 53.4 g/l ethanol with the yield of 48.9 g/100 g sugars and left 69.6 g/l xylose in the broth, representing 98.6% of the total xylose in the simulated corncob hydrolysate. CONCLUSIONS: By using newly constructed strain B0013-2012PA, we successfully developed an efficient bioprocess for ethanol production and xylose recovery from the simulated corncob hydrolysate.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Etanol/metabolismo , Xilose/química , Zea mays/química , Técnicas de Cultura Celular por Lotes , Reatores Biológicos/microbiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Engenharia Genética , Glucose/metabolismo , HidróliseRESUMO
Efficient production of D-lactate by engineered Escherichia coli entails balancing cell growth and product synthesis. To develop a metabolic switch to implement a desirable transition from cell growth to product fermentation, a thiamine auxotroph B0013-080A was constructed in a highly efficient D-lactate producer E. coli strain B0013-070. This was achieved by inactivation of thiE, a gene encoding a thiamine phosphate synthase for biosynthesis of thiamine monophosphate. The resultant mutant B0013-080A failed to grow on the medium in the absence of thiamine yet growth was restored when exogenous thiamine was provided. A linear relationship between cell mass formation and amount of thiamine supplemented was mathematically determined in a shake flask experiment and confirmed in a 7-L bioreactor system. This calculation revealed that â¼ 95-96 thiamine molecules per cell were required to satisfy cell growth. This relationship was employed to develop a novel fermentation process for D-lactate production by using thiamine as a limiting condition. A D-lactate productivity of 4.11 g · L(-1) · h(-1) from glycerol under microaerobic condition and 3.66 g · L(-1) · h(-1) from glucose under anaerobic condition was achieved which is 19.1% and 10.2% higher respectively than the parental strain. These results revealed a convenient and reliable method to control cell growth and improve D-lactate fermentation. This control strategy could be applied to other biotechnological processes that require optimal allocation of carbon between cell growth and product formation.
Assuntos
Escherichia coli/metabolismo , Ácido Láctico/metabolismo , Tiamina Pirofosfato/metabolismo , Meios de Cultura/química , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Engenharia Metabólica/métodosRESUMO
Whereas osmotic stress response induced by solutes has been well-characterized in fungi, less is known about the other activities of environmentally ubiquitous substances. The latest methodologies to define, identify and quantify chaotropicity, i.e. substance-induced destabilization of macromolecular systems, now enable new insights into microbial stress biology (Cray et al. in Curr Opin Biotechnol 33:228-259, 2015a, doi: 10.1016/j.copbio.2015.02.010 ; Ball and Hallsworth in Phys Chem Chem Phys 17:8297-8305, 2015, doi: 10.1039/C4CP04564E ; Cray et al. in Environ Microbiol 15:287-296, 2013a, doi: 10.1111/1462-2920.12018 ). We used Aspergillus wentii, a paradigm for extreme solute-tolerant fungal xerophiles, alongside yeast cell and enzyme models (Saccharomyces cerevisiae and glucose-6-phosphate dehydrogenase) and an agar-gelation assay, to determine growth-rate inhibition, intracellular compatible solutes, cell turgor, inhibition of enzyme activity, substrate water activity, and stressor chaotropicity for 12 chemically diverse solutes. These stressors were found to be: (i) osmotically active (and typically macromolecule-stabilizing kosmotropes), including NaCl and sorbitol; (ii) weakly to moderately chaotropic and non-osmotic, these were ethanol, urea, ethylene glycol; (iii) highly chaotropic and osmotically active, i.e. NH4NO3, MgCl2, guanidine hydrochloride, and CaCl2; or (iv) inhibitory due primarily to low water activity, i.e. glycerol. At ≤0.974 water activity, Aspergillus cultured on osmotically active stressors accumulated low-M r polyols to ≥100 mg g dry weight(-1). Lower-M r polyols (i.e. glycerol, erythritol and arabitol) were shown to be more effective for osmotic adjustment; for higher-M r polyols such as mannitol, and the disaccharide trehalose, water-activity values for saturated solutions are too high to be effective; i.e. 0.978 and 0.970 (25 ºC). The highly chaotropic, osmotically active substances exhibited a stressful level of chaotropicity at physiologically relevant concentrations (20.0-85.7 kJ kg(-1)). We hypothesized that the kosmotropicity of compatible solutes can neutralize chaotropicity, and tested this via in-vitro agar-gelation assays for the model chaotropes urea, NH4NO3, phenol and MgCl2. Of the kosmotropic compatible solutes, the most-effective protectants were trimethylamine oxide and betaine; but proline, dimethyl sulfoxide, sorbitol, and trehalose were also effective, depending on the chaotrope. Glycerol, by contrast (a chaotropic compatible solute used as a negative control) was relatively ineffective. The kosmotropic activity of compatible solutes is discussed as one mechanism by which these substances can mitigate the activities of chaotropic stressors in vivo. Collectively, these data demonstrate that some substances concomitantly induce chaotropicity-mediated and osmotic stresses, and that compatible solutes ultimately define the biotic window for fungal growth and metabolism. The findings have implications for the validity of ecophysiological classifications such as 'halophile' and 'polyextremophile'; potential contamination of life-support systems used for space exploration; and control of mycotoxigenic fungi in the food-supply chain.
Assuntos
Adaptação Biológica , Aspergillus/fisiologia , Pressão Osmótica , Estresse Fisiológico , Catálise , Glucosefosfato Desidrogenase/metabolismo , Polímeros/metabolismoRESUMO
UNLABELLED: L-Lactic acid, one of the most important chiral molecules and organic acids, is produced via pyruvate from carbohydrates in diverse microorganisms catalyzed by an NAD+-dependent L-lactate dehydrogenase. Naturally, Escherichia coli does not produce L-lactate in noticeable amounts, but can catabolize it via a dehydrogenation reaction mediated by an FMN-dependent L-lactate dehydrogenase. In aims to make the E. coli strain to produce L-lactate, three L-lactate dehydrogenase genes from different bacteria were cloned and expressed. The L-lactate producing strains, 090B1 (B0013-070, ΔldhA::diflldD::Pldh-ldhLca), 090B2 (B0013-070, ΔldhA::diflldD::Pldh-ldhStrb) and 090B3 (B0013-070, ΔldhA::diflldD::Pldh-ldhBcoa) were developed from a previously developed D-lactate over-producing strain, E. coli strain B0013-070 (ack-ptappspflBdldpoxBadhEfrdA) by: (1) deleting ldhA to block D-lactate formation, (2) deleting lldD to block the conversion of L-lactate to pyruvate, and (3) expressing an L-lactate dehydrogenase (L-LDH) to convert pyruvate to L-lactate under the control of the ldhA promoter. Fermentation tests were carried out in a shaking flask and in a 25-l bioreactor. Strains 090B1, 090B2 or 090B3 were shown to metabolize glucose to L-lactate instead of D-lactate. However, L-lactate yield and cell growth rates were significantly different among the metabolically engineered strains which can be attributed to a variation between temperature optimum for cell growth and temperature optimum for enzymatic activity of individual L-LDH. In a temperature-shifting fermentation process (cells grown at 37°C and L-lactate formed at 42°C), E. coli 090B3 was able to produce 142.2 g/l of L-lactate with no more than 1.2 g/l of by-products (mainly acetate, pyruvate and succinate) accumulated. In conclusion, the production of lactate by E. coli is limited by the competition relationship between cell growth and lactate synthesis. Enzymatic properties, especially the thermodynamics of an L-LDH can be effectively used as a factor to regulate a metabolic pathway and its metabolic flux for efficient L-lactate production. HIGHLIGHTS: The enzymatic thermodynamics was used as a tool for metabolic regulation. Minimizing the activity of L-lactate dehydrogenase in growth phase improved biomass accumulation. Maximizing the activity of L-lactate dehydrogenase improved lactate productivity in production phase.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Láctico/biossíntese , Temperatura , Técnicas de Cultura Celular por Lotes , Biomassa , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , L-Lactato Desidrogenase/deficiência , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Engenharia Metabólica , Regiões Promotoras Genéticas , Piruvatos/metabolismo , Estereoisomerismo , TermodinâmicaRESUMO
During alcoholic fermentation, higher alcohols, esters, and acids are formed from amino acids via the Ehrlich pathway by yeast, but many of the genes encoding the enzymes have not yet been identified. When the BAT1/2 genes, encoding transaminases that deaminate amino acids in the first step of the Ehrlich pathway are deleted, higher metabolite formation is significantly decreased. Screening yeast strains with deletions of genes encoding decarboxylases, dehydrogenases, and reductases revealed nine genes whose absence had the most significant impact on higher alcohol production. The seven most promising genes (AAD6, BAT2, HOM2, PAD1, PRO2, SPE1, and THI3) were further investigated by constructing double- and triple-deletion mutants. All double-deletion strains showed a greater decrease in isobutanol, isoamyl alcohol, isobutyric, and isovaleric acid production than the corresponding single deletion strains with the double-deletion strains in combination with ∆bat2 and the ∆hom2-∆aad6 strain revealing the greatest impact. BAT2 is the dominant gene in these deletion strains and this suggests the initial transaminase step of the Ehrlich pathway is rate-limiting. The triple-deletion strains in combination with BAT2 (∆bat2-∆thi3-∆aad6 and ∆bat2-∆thi3-∆hom2) had the greatest impact on the end metabolite production with the exception of isoamyl alcohol and isovaleric acid. The strain deleted for two dehydrogenases and a reductase (∆hom2-∆pro2-∆aad6) had a greater effect on the levels of these two compounds. This study contributes to the elucidation of the Ehrlich pathway and its significance for aroma production by fermenting yeast cells.
Assuntos
Odorantes , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Primers do DNA , Deleção de Genes , Expressão Gênica , Genes Fúngicos , Saccharomyces cerevisiae/genéticaRESUMO
During a fermentation process, the formation of the desired product during the cell growth phase competes with the biomass for substrates or inhibits cell growth directly, which results in a decrease in production efficiency. A genetic switch is required to precisely separate growth from production and to simplify the fermentation process. The ldhA promoter, which encodes the fermentative D-lactate dehydrogenase (LDH) in the lactate producer Escherichia coli CICIM B0013-070 (ack-pta pps pflB dld poxB adhE frdA), was replaced with the λ p(R) and p(L) promoters (as a genetic switch) using genomic recombination and the thermo-controllable strain B0013-070B (B0013-070, ldhAp::kan-cI(ts)857-p(R)-p(L)), which could produce two-fold higher LDH activity at 42°C than the B0013-070 strain, was created. When the genetic switch was turned off at 33°C, strain B0013-070B produced 10% more biomass aerobically than strain B0013-070 and produced only trace levels of lactate which could reduce the growth inhibition caused by oxygen insufficiency in large scale fermentation. However, 42°C is the most efficient temperature for switching on lactate production. The volumetric productivity of B0013-070B improved by 9% compared to that of strain B0013-070 when it was grown aerobically at 33°C with a short thermo-induction at 42°C and then switched to the production phase at 42°C. In a bioreactor experiment using scaled-up conditions that were optimized in a shake flask experiment, strain B0013-070B produced 122.8 g/l D-lactate with an increased oxygen-limited productivity of 0.89 g/g·h. The results revealed the effectiveness of using a genetic switch to regulate cell growth and the production of a metabolic compound.
Assuntos
Escherichia coli , Ácido Láctico/biossíntese , Aerobiose/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Engenharia Genética , L-Lactato Desidrogenase/biossíntese , L-Lactato Desidrogenase/genética , Regiões Promotoras Genéticas/genéticaRESUMO
Saccharomyces cerevisiae maintains a redox balance under fermentative growth conditions by re-oxidizing NADH formed during glycolysis through ethanol formation. Excess NADH stimulates the synthesis of mainly glycerol, but also of other compounds. Here, we investigated the production of primary and secondary metabolites in S. cerevisiae strains where the glycerol production pathway was inactivated through deletion of the two glycerol-3-phosphate dehydrogenases genes (GPD1/GPD2) and replaced with alternative NAD(+)-generating pathways. While these modifications decreased fermentative ability compared to the wild-type strain, all improved growth and/or fermentative ability of the gpd1Δgpd2Δ strain in self-generated anaerobic high sugar medium. The partial NAD(+) regeneration ability of the mutants resulted in significant amounts of alternative products, but at lower yields than glycerol. Compared to the wild-type strain, pyruvate production increased in most genetically manipulated strains, whereas acetate and succinate production decreased in all strains. Malate production was similar in all strains. Isobutanol production increased substantially in all genetically manipulated strains compared to the wild-type strain, whereas only mutant strains expressing the sorbitol producing SOR1 and srlD genes showed increases in isoamyl alcohol and 2-phenyl alcohol. A marked reduction in ethyl acetate concentration was observed in the genetically manipulated strains, while isobutyric acid increased. The synthesis of some primary and secondary metabolites appears more readily influenced by the NAD(+)/NADH availability. The data provide an initial assessment of the impact of redox balance on the production of primary and secondary metabolites which play an essential role in the flavour and aroma character of beverages.
Assuntos
Aromatizantes/metabolismo , Glicerol/metabolismo , NAD/metabolismo , Saccharomyces cerevisiae/metabolismo , Metabolismo dos Carboidratos , Meios de Cultura/química , Etanol/metabolismo , Fermentação , Deleção de Genes , Glicerol-3-Fosfato Desidrogenase (NAD+)/deficiência , Redes e Vias Metabólicas/genética , Oxirredução , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiaeRESUMO
Fine tuning of the key enzymes to moderate rather than high expression levels could overproduce the desired metabolic products without inhibiting cell growth. The aims of this investigation were to regulate rates of lactate production and cell growth in recombinant Escherichia coli through promoter engineering and to evaluate the transcriptional function of the upstream region of ldhA (encoding fermentative lactate dehydrogenase in E. coli). Twelve ldhA genes with sequentially shortened chromosomal upstream regions were cloned in an ldhA deletion, E. coli CICIM B0013-080C (ack-pta pps pflB dld poxB adhE frdA ldhA). The varied ldhA upstream regions were further analyzed using program NNPP2.2 (Neural Network Promoter Prediction 2.2) to predict the possible promoter regions. Two-phase fermentations (aerobic growth and oxygen-limited production) of these strains showed that shortening the ldhA upstream sequence from 291 to 106 bp successively reduced aerobic lactate synthesis and the inhibition effect on cell growth during the first phase. Simultaneously, oxygen-limited lactate productivity was increased during the second phase. The putative promoter downstream of the -96 site of ldhA could function as a transcriptional promoter or regulator. B0013-080C/pTH-rrnB-ldhA8, with the 72-bp upstream segment of ldhA, could be grown at a high rate and achieve a high oxygen-limited lactate productivity of 1.09 g g(-1) h(-1). No transcriptional promoting region was apparent downstream of the -61 site of ldhA. We identified the latent transcription regions in the ldhA upstream sequence, which will help to understand regulation of ldhA expression.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Ácido Láctico/biossíntese , Transcrição Gênica/genética , Sequência de Bases , Clonagem Molecular , Escherichia coli/citologia , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/metabolismo , Fermentação , L-Lactato Desidrogenase/biossíntese , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genéticaRESUMO
In order to rationally manipulate the cellular metabolism of Escherichia coli for D: -lactate production, single-gene and multiple-gene deletions with mutations in acetate kinase (ackA), phosphotransacetylase (pta), phosphoenolpyruvate synthase (pps), pyruvate formate lyase (pflB), FAD-binding D-lactate dehydrogenase (dld), pyruvate oxidase (poxB), alcohol dehydrogenase (adhE), and fumarate reductase (frdA) were tested for their effects in two-phase fermentations (aerobic growth and oxygen-limited production). Lactate yield and productivity could be improved by single-gene deletions of ackA, pta, pflB, dld, poxB, and frdA in the wild type E. coli strain but were unfavorably affected by deletions of pps and adhE. However, fermentation experiments with multiple-gene mutant strains showed that deletion of pps in addition to ackA-pta deletions had no effect on lactate production, whereas the additional deletion of adhE in E. coli B0013-050 (ackA-pta pps pflB dld poxB) increased lactate yield. Deletion of all eight genes in E. coli B0013 to produce B0013-070 (ackA-pta pps pflB dld poxB adhE frdA) increased lactate yield and productivity by twofold and reduced yields of acetate, succinate, formate, and ethanol by 95, 89, 100, and 93%, respectively. When tested in a bioreactor, E. coli B0013-070 produced 125 g/l D-lactate with an increased oxygen-limited lactate productivity of 0.61 g/g h (2.1-fold greater than E. coli B0013). These kinetic properties of D-lactate production are among the highest reported and the results have revealed which genetic manipulations improved D-lactate production by E. coli.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética , Ácido Láctico/metabolismo , Redes e Vias Metabólicas/genética , Aerobiose , Anaerobiose , Proteínas de Escherichia coli/genética , Fermentação , Deleção de Genes , Mutação , Organismos Geneticamente ModificadosRESUMO
Glycerol is a major by-product of ethanol fermentation by Saccharomyces cerevisiae and typically 2-3% of the sugar fermented is converted to glycerol. Replacing the NAD(+)-regenerating glycerol pathway in S. cerevisiae with alternative NADH reoxidation pathways may be useful to produce metabolites of biotechnological relevance. Under fermentative conditions yeast reoxidizes excess NADH through glycerol production which involves NADH-dependent glycerol-3-phosphate dehydrogenases (Gpd1p and Gpd2p). Deletion of these two genes limits fermentative activity under anaerobic conditions due to accumulation of NADH. We investigated the possibility of converting this excess NADH to NAD(+) by transforming a double mutant (gpd1∆gpd2∆) with alternative oxidoreductase genes that might restore the redox balance and produce either sorbitol or propane-1,2-diol. All of the modifications improved fermentative ability and/or growth of the double mutant strain in a self-generated anaerobic high sugar medium. However, these strain properties were not restored to the level of the parental wild-type strain. The results indicate an apparent partial NAD(+) regeneration ability and formation of significant amounts of the commodity chemicals like sorbitol or propane-1,2-diol. The ethanol yields were maintained between 46 and 48% of the sugar mixture. Other factors apart from the maintenance of the redox balance appeared to influence the growth and production of the alternative products by the genetically manipulated strains.
Assuntos
Etanol/metabolismo , Fermentação , Glicerol/metabolismo , Saccharomyces cerevisiae/metabolismo , Metabolismo dos Carboidratos , Glicerolfosfato Desidrogenase/genética , Glicerolfosfato Desidrogenase/metabolismo , NAD/metabolismo , Oxirredução , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The growth and accumulation of intracellular polyols at reduced water activity (a(w)) were studied in 40 basidiomycetous yeast strains. The growth of most strains showed greater tolerance to NaCl than sorbitol at the same a(w). No strain was able to grow below 0.90a(w). (13)C nuclear magnetic resonance spectroscopy revealed that glycerol was the major solute accumulated intracellularly by all the yeasts when grown to 0.96a(w) (NaCl). Arabitol or mannitol was also accumulated in some yeasts, whereas a few only accumulated glycerol. Analysis of six yeasts in detail revealed that the intracellular glycerol concentrations of five yeasts increased significantly when grown at 0.96a(w) (NaCl or sorbitol) compared with growth at 0.998a(w). Arabitol and mannitol concentrations also increased, but not to the same degree. Intracellular potassium concentrations decreased when grown at 0.96a(w) (NaCl or sorbitol) and sodium increased, but only when grown at 0.96a(w) (NaCl). The survival of nine strains was evaluated in soil cultures and it was found that all grew at 100% field capacity, whereas at lower field capacity, only some strains grew or survived. The growth of basidiomycetous yeasts appears to be more sensitive to reduced a(w) than ascomycetous yeasts.
Assuntos
Basidiomycota/fisiologia , Pressão Osmótica , Basidiomycota/química , Citosol/química , Glicerol/análise , Espectroscopia de Ressonância Magnética , Manitol/análise , Potássio/análise , Cloreto de Sódio/metabolismo , Sorbitol/metabolismo , Estresse Fisiológico , Álcoois Açúcares/análiseRESUMO
A rapid screening method for the evaluation of the major fermentation products of Saccharomyces wine yeasts was developed using Fourier transform infrared spectroscopy and principal component factor analysis. Calibration equations for the quantification of volatile acidity, glycerol, ethanol, reducing sugar and glucose concentrations in fermented Chenin blanc and synthetic musts were derived from the Fourier transform infrared spectra of small-scale fermentations. The accuracy of quantification of volatile acidity in both Chenin blanc and synthetic must was excellent, and the standard error of prediction was 0.07 g l(-1) and 0.08 g l(-1), respectively. The respective standard error of prediction in Chenin blanc and synthetic musts for ethanol was 0.32% v/v and 0.31% v/v, for glycerol was 0.38 g l(-1) and 0.32 g l(-1), for reducing sugar in Chenin blanc must was 0.56 g l(-1) and for glucose in synthetic must was 0.39 g l(-1). These values were in agreement with the accuracy obtained by the respective reference methods used for the quantification of the components. The screening method was applied to quantify the fermentation products of glycerol-overproducing hybrid yeasts and commercial wine yeasts. Principal component factor analysis of the fermentation data facilitated an overall comparison of the fermentation profiles (in terms of the components tested) of the strains. The potential of Fourier transform infrared spectroscopy as a tool to rapidly screen the fermentative properties of wine yeasts and to speed up the evaluation processes in the initial stages of yeast strain development programs is shown.
Assuntos
Saccharomyces cerevisiae/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Vinho/microbiologia , Etanol/análise , Etanol/metabolismo , Fermentação , Glucose/análise , Glucose/metabolismo , Glicerol/metabolismo , Microbiologia Industrial/métodos , Análise de Componente Principal , Vinho/análiseRESUMO
The non-cellulolytic Thermomyces lanuginosus is a widespread and frequently isolated thermophilic fungus. Several strains of this fungus have been reported to produce high levels of cellulase-free beta-xylanase both in shake-flask and bioreactor cultivations but intraspecies variability in terms of beta-xylanase production is apparent. Furthermore all strains produce low extracellular levels of other hemicellulases involved in hemicellulose hydrolysis. Crude and purified hemicellulases from this fungus are stable at high temperatures in the range of 50-80 degrees C and over a broad pH range (3-12). Various strains are reported to produce a single xylanase with molecular masses varying between 23 and 29 kDa and pI values between 3.7 and 4.1. The gene encoding the T. lanuginosus xylanase has been cloned and sequenced and is shown to be a member of family 11 glycosyl hydrolases. The crystal structure of the xylanase indicates that the enzyme consists of two beta-sheets and one alpha-helix and forms a rigid complex with the three central sugars of xyloheptaose whereas the peripheral sugars might assume different configurations thereby allowing branched xylan chains to be accepted. The presence of an extra disulfide bridge between the beta-strand and the alpha-helix, as well as to an increase in the density of charged residues throughout the xylanase might contribute to the thermostability. The ability of T. lanuginosus to produce high levels of cellulase-free thermostable xylanase has made the fungus an attractive source of thermostable xylanase with potential as a bleach-boosting agent in the pulp and paper industry and as an additive in the baking industry.
Assuntos
Ascomicetos/enzimologia , Glicosídeo Hidrolases , Polissacarídeos/metabolismo , Xilosidases/metabolismo , Sequência de Aminoácidos , Ascomicetos/classificação , Ascomicetos/genética , Ascomicetos/fisiologia , Proteínas Fúngicas/análise , Proteínas Fúngicas/química , Glicosídeo Hidrolases/biossíntese , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Dados de Sequência Molecular , Alinhamento de Sequência , Temperatura , Xilano Endo-1,3-beta-Xilosidase , Xilosidases/biossíntese , Xilosidases/classificaçãoRESUMO
Principal component analysis (PCA) was used to identify the main sources of variation in the Fourier transform infrared (FT-IR) spectra of 329 wines of various styles. The FT-IR spectra were gathered using a specialized WineScan instrument. The main sources of variation included the reducing sugar and alcohol content of the samples, as well as the stage of fermentation and the maturation period of the wines. The implications of the variation between the different wine styles for the design of calibration models with accurate predictive abilities were investigated using glycerol calibration in wine as a model system. PCA enabled the identification and interpretation of samples that were poorly predicted by the calibration models, as well as the detection of individual samples in the sample set that had atypical spectra (i.e., outlier samples). The Soft Independent Modeling of Class Analogy (SIMCA) approach was used to establish a model for the classification of the outlier samples. A glycerol calibration for wine was developed (reducing sugar content < 30 g/L, alcohol > 8% v/v) with satisfactory predictive ability (SEP = 0.40 g/L). The RPD value (ratio of the standard deviation of the data to the standard error of prediction) was 5.6, indicating that the calibration is suitable for quantification purposes. A calibration for glycerol in special late harvest and noble late harvest wines (RS 31-147 g/L, alcohol > 11.6% v/v) with a prediction error SECV = 0.65 g/L, was also established. This study yielded an analytical strategy that combined the careful design of calibration sets with measures that facilitated the early detection and interpretation of poorly predicted samples and outlier samples in a sample set. The strategy provided a powerful means of quality control, which is necessary for the generation of accurate prediction data and therefore for the successful implementation of FT-IR in the routine analytical laboratory.
Assuntos
Glicerol/análise , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Vinho/análise , Vinho/classificação , Calibragem , Análise de Componente PrincipalRESUMO
Background: Protein glutaminase specifically deamidates glutamine residue in protein and therefore significantly improves protein solubility and colloidal stability of protein solution. In order to improve its preparation efficiency, we exploited the possibility for its secretory expression mediated by twin-arginine translocation (Tat) pathway in Bacillus licheniformis. Results: The B. licheniformis genome-wide twin-arginine signal peptides were analyzed. Of which, eleven candidates were cloned for construction of expression vectors to mediate the expression of Chryseobacterium proteolyticum protein glutaminase (PGA). The signal peptide of GlmU was confirmed that it significantly mediated PGA secretion into media with the maximum activity of 0.16 U/ml in Bacillus subtilis WB600. A mutant GlmU-R, being replaced the third residue aspartic acid of GlmU twin-arginine signal peptide with arginine by site-directed mutagenesis, mediated the improved secretion of PGA with about 40% increased (0.23 U/ml). In B. licheniformis CBBD302, GlmU-R mediated PGA expression in active form with the maximum yield of 6.8 U/ml in a 25-l bioreactor. Conclusions: PGA can be produced and secreted efficiently in active form via Tat pathway of B. licheniformis, an alternative expression system for the industrial-scale production of PGA.
Assuntos
Bacillus licheniformis/enzimologia , Glutaminase/metabolismo , Arginina , Plasmídeos , Prostaglandinas A/química , Bacillus subtilis , Sinais Direcionadores de Proteínas , Sequência de Bases , Mutagênese Sítio-Dirigida , Ácido Aspártico , Escherichia coli , Bacillus licheniformis/genética , Glutaminase/genéticaRESUMO
Background: Burdock (Arctium lappa L.) is a fructan-rich plant with prebiotic potential. The aim of this study was to develop an efficient enzymatic route to prepare fructooligosaccharides (FOS)-rich and highly antioxidative syrup using burdock root as a raw material. Results: Endo-inulinase significantly improved the yield of FOS 2.4-fold while tannase pretreatment further increased the yield of FOS 2.8-fold. Other enzymes, including endo-polygalacturonase, endo-glucanase and endo-xylanase, were able to increase the yield of total soluble sugar by 11.1% (w/w). By this process, a new enzymatic process for burdock syrup was developed and the yield of burdock syrup increased by 25% (w/w), whereas with FOS, total soluble sugars, total soluble protein and total soluble polyphenols were enhanced to 28.8%, 53.3%, 8.9% and 3.3% (w/w), respectively. Additionally, the scavenging abilities of DPPH and hydroxyl radicals, and total antioxidant capacity of the syrup were increased by 23.7%, 51.8% and 35.4%, respectively. Conclusions: Our results could be applied to the development of efficient extraction of valuable products from agricultural materials using enzyme-mediated methods.
Assuntos
Oligossacarídeos/química , Raízes de Plantas/química , Frutose/química , Glicosídeo Hidrolases/metabolismo , Antioxidantes/química , Oligossacarídeos/metabolismo , Poligalacturonase/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Cromatografia Líquida de Alta Pressão , Radical Hidroxila , Arctium , Alimento Funcional , Polifenóis , Frutose/metabolismo , Antioxidantes/metabolismoRESUMO
In order to decrease carbon emissions and negative environmental impacts of various pollutants, more bulk and/or fine chemicals are produced by bioprocesses, replacing the traditional energy and fossil based intensive route. The Gram-negative rod-shaped bacterium, Escherichia coli has been studied extensively on a fundamental and applied level and has become a predominant host microorganism for industrial applications. Furthermore, metabolic engineering of E. coli for the enhanced biochemical production has been significantly promoted by the integrated use of recent developments in systems biology, synthetic biology and evolutionary engineering. In this review, we focus on recent efforts devoted to the use of genetically engineered E. coli as a sustainable platform for the production of industrially important biochemicals such as biofuels, organic acids, amino acids, sugar alcohols and biopolymers. In addition, representative secondary metabolites produced by E. coli will be systematically discussed and the successful strategies for strain improvements will be highlighted. Moreover, this review presents guidelines for future developments in the bio-based chemical production using E. coli as an industrial platform.
Assuntos
Escherichia coli , Microbiologia Industrial , Engenharia Metabólica , Biocombustíveis , Biopolímeros/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Compostos Orgânicos/metabolismoRESUMO
Background: ß-Galactosidases catalyze both hydrolytic and transgalactosylation reactions and therefore have many applications in food, medical, and biotechnological fields. Aspergillus niger has been a main source of ß-galactosidase, but the properties of this enzyme are incompletely studied. Results: Three new ß-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned, expressed, and biochemically characterized. In addition to the known activity of LacA encoded by lacA, three putative ß-galactosidases, designated as LacB, LacC, and LacE encoded by the genes lacB, lacC, and lacE, respectively, were successfully cloned, sequenced, and expressed and secreted by Pichia pastoris. These three proteins and LacA have N-terminal signal sequences and are therefore predicted to be extracellular enzymes. They have the typical structure of fungal ß-galactosidases with defined hydrolytic and transgalactosylation activities on lactose. However, their activity properties differed. In particular, LacB and lacE displayed maximum hydrolytic activity at pH 45 and 50°C, while LacC exhibited maximum activity at pH 3.5 and 60°C. All ß-galactosidases performed transgalactosylation activity optimally in an acidic environment. Conclusions: Three new ß-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned and biochemically characterized. In addition to the known LacA, A. niger has at least three ß-galactosidase family members with remarkably different biochemical properties.
Assuntos
Aspergillus niger/enzimologia , beta-Galactosidase/química , Especificidade por Substrato , Cinética , Sequência de Aminoácidos , Clonagem Molecular , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
In this study, a novel rapid and efficient DNA extraction method based on alkaline lysis, which can deal with a large number of filamentous fungal isolates in the same batch, was established. The filamentous fungal genomic DNA required only 20 min to prepare and can be directly used as a template for PCR amplification. The amplified internal transcribed spacer regions were easy to identify by analysis. The extracted DNA also can be used to amplify other protein-coding genes for fungal identification. This method can be used for rapid systematic identification of filamentous fungal isolates.