马克斯克鲁维酵母GX-UN120糖转运蛋白基因的克隆及表征

【背景】马克斯克鲁维酵母(Kluyveromyces marxianus)具有完整的木糖代谢途径,可以高效利用木质纤维素中的木糖,因此对其糖转运蛋白基因的研究或可有效解决酵母木糖转运的相关问题。【目的】根据马克斯克鲁维酵母DMKU3-1042中KLMA＿70145和KLMA＿80101基因位点的功能预测,获得马克斯克鲁维酵母GX-UN120相应的糖转运蛋白基因序列并探究其功能。【方法】将转运蛋白基因分别克隆表达至酿酒酵母EBY.VW4000中考察重组菌株生长特性,以此间接评价对应转运蛋白的转运能力。【结果】Km＿SUT2基因编码的糖转运蛋白可有效提高宿主细胞转运木糖、阿拉伯糖、山梨糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖、果糖、蔗糖和半乳糖。类似地,Km＿SUT3基因编码的糖转运蛋白可提高细胞转运木糖、阿拉伯糖、山梨糖、半乳糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖和果糖。然而在葡萄糖存在的条件下,重组菌株对各种碳源的利用均受抑制,但Km＿SUT3转运木糖和核糖过程中受葡萄糖的抑制作用较小。【结论】马克斯克鲁维酵母GX-UN120中转运蛋白Km＿SUT2和Km＿SUT3可...     

Comparative proteome analysis of alkaliphilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. N16-5 grown on different carbon sources

To determine the impact of carbohydrates on the metabolic pathway in alkaliphiles, proteomes were obtained from cultures containing different carbohydrates and were resolved on two-dimensional gel electrophoresis (2-DE). The proteomes were compared to determine differentially expressed proteins. A novel alkaliphilic bacterium (alkaliphilic Bacillus sp. N16-5 isolated from Wudunur Soda Lake, China) was isolated in media with five different carbon sources (glucose, mannose, galactose, arabinose, and xylose). Comparative proteome analysis identified 61 differentially expressed proteins, which were mainly involved in carbohydrate metabolism, amino acid transport, and metabolism, as well as energy production and conversion. The comparison was based on the draft genome sequence of strain N16-5. The abundance of enzymes involved in central metabolism was significantly changed when exposed to various carbohydrates. Notably, catabolite control protein A (CcpA) was up-regulated under all carbon sources compared with glucose. In addition, pentose exhibited a stronger effect than hexose in CcpA-mediated carbon catabolite repression. These results provided a fundamental understanding of carbohydrate metabolism in alkaliphiles.     

Carbohydrate utilization by Trichomonas gallinae: effects on growth and metabolic activity under nongrowth conditions

Trichomonas gallinae used 13 of 29 carbohydrates for growth. Quantitative relationships between final populations, acid production, and cellular glycogen contents varied depending on the substrate. The effect of growth on different carbohydrates on the subsequent utilization of carbohydrates by cells under nongrowth conditions was studied by measuring carbohydrate uptake, changes in cellular glycogen content, and gas production. Two major utilization patterns were found. Cells grown on maltose or starch used these substrates well, but cells grown on other sugars did not. All cells used glucose, fructose, galactose, and mannose, but cells grown on maltose or starch did not use them as well as cells grown on other sugars. All cells used ribose slightly but not xylose or arabinose. Turanose, a disaccharide yielding high populations in growth medium, was not used under nongrowth conditions.     

A rapid method for gas chromatographic analysis of mono- and disaccharide mixtures

A technique is described for the rapid gas-liquid chromatographic analysis of mixtures of carbohydrate trimethylsilyl ethers prepared at mutarotation equilibrium. A mixture containing arabinose, ribose, xylose, fructose, glucose, galactose, mannose, sucrose, maltose, and cellobiose can be determined in less than 16 min using high-rate temperature programming.     

Carbohydrate utilization patterns and substrate preferences in Bacteroides thetaiotaomicron

Specific growth rates of Bacteroides thetaiotaomicron NCTC 10582 with either glucose, arabinose, mannose, galactose or xylose as sole carbon sources were 0.42/h, 0.10/h, 0.38/h, 0.38/h and 0.16/h respectively, suggesting that hexose metabolism was energetically more efficient than pentose fermentation in this bacterium. Batch culture experiments to determine whether carbohydrate utilization was controlled by substrate-induced regulatory mechanisms demonstrated that mannose inhibited uptake of glucose, galactose and arabinose, but had less effect on xylose. Arabinose and xylose were preferentially utilized at high dilution rates (D > 0.26/h) in carbon-limited continuous cultures grown on mixtures of arabinose, xylose, galactose and glucose. When mannose was also present, xylose was co-assimilated at all dilution rates. Under nitrogen-limited conditions, however, mannose repressed uptake of all sugars, showing that its effect on xylose utilization was strongly concentration dependent. Studies with individual D-ZU-14C]-labelled substrates showed that transport systems for glucose, galactose, xylose and mannose were inducible. Measurements to determine incorporation of these sugars into trichloroacetic acid-precipitable material indicated that glucose and mannose were the principal precursor monosaccharides. Xylose was only incorporated into intracellular macromolecules when it served as growth substrate. Phosphoenolpyruvate:phosphotransferase systems were not detected in preliminary experiments to elucidate the mechanisms of sugar uptake, and studies with inhibitors of carbohydrate transport showed no consistent pattern of inhibition with glucose, galactose, xylose and mannose. These results indicate the existence of a variety of different systems involved in sugar transport in B. thetaiotaomicron.     

Assessment of different carbohydrates as exogenous carbon source in cultivation of cyanobacteria

Glucose is the substrate most widely used as exogenous carbon source for heterotrophic cultivation of cyanobacteria. Due to limited information about the use of different carbohydrates as carbon sources to support cyanobacterial heterotrophic metabolism, the objective of this work was to evaluate different monosaccharides (arabinose, fructose, galactose, glucose, mannose and xylose), disaccharides (lactose, maltose, sucrose and trehalose) and polysaccharides (carboxymethylcellulose, cassava starch, Hi-maize^®, maltodextrin Corn Globe 1805^® and xylan) as exogenous carbon source for heterotrophic culture of cyanobacterium Phormidium sp. The batch cultivation using fructose as organic carbon source resulted in the highest (p < 0.05) cell biomass (5,540 mg/L) in parallel with the highest (p < 0.05) substrate yield coefficient (0.67 mg_biomass/mg_fructose). Mannose was the carbon source with the highest (p < 0.05) substrate consumption rate (3,185.7 mg/L/day) and maltodextrin was the carbohydrate with major potential to produce biomass (1,072.8 mg_biomass/L/day) and lipids (160.8 mg_lipids/L/day). Qualitatively, the fatty acid profiles of the lipid extract from Phormidium sp. showed predominance of saturated chains for the cultures grown with most of the carbon sources, with the exception of the ones grown with xylose and maltodextrin.     

Digestibility by sheep of direct cut alfalfa silage made with ozonated cotton stalks

Alfalfa was ensiled either by conventional wilting (WA) or as a mixture with 20% (DM) ozonated cotton stalks (A + OCS), and the digestibility of the silage carbohydrate constituents was examined in sheep. The WA silage had a lower content of glucose, xylose, and total carbohydrates and a slightly higher content of arabinose, galactose and mannose than the A + OCS silage. Stomachs and whole tract carbohydrate digestibility was 52% and 63%, respectively, in both silages. Digestibility of neutral detergent fiber (NDF) glucose and xylose was slightly higher and that of NDF-arabinose, galactose and mannose slightly lower, in the sheep fed A + OCS vs. those fed WA.Digestibility of total NDF monosaccharide components in WA and A + OCS silages were 55% and 57%, respectively, of which 90% were digested in the fore-stomachs. In both silages, NDF-glucose was more digestible than NDF-xylose and NDF-uronic acid, and less digestible than NDF-arabinose and NDF-mannose.Results of this study indicate that a direct-cut and ensiled alfalfa silage made with 20% (DM) OCS is comparable with WA in its potential to supply ruminants with digestible carbohydrates.     

Dietary carbohydrates: role in feeding behavior and growth of the southwestern corn borer, Diatraea grandiosella

The effect of carbohydrates on feeding behaviour and growth and development of the southwestern corn borer, Diatraea grandiosella, was examined. A feeding behavioural biossay based on establishment of first stage larvae on test or control stations within 24 hr was employed. A nutritional bioassay evaluated larval growth rates and metamorphic success of larvae reared on test or control diets. The principal results showed that glucose, fructose, sucrose, and dextrins were feeding stimulants and permitted optimum growth and development. A dietary supplement of amylopectin allowed reasonably good growth while amylose was not a utilizable carbohydrate. The pentoses, arabinose, ribose, and xylose, as well as galactose, mannose, and sorbose, were found to inhibit larval growth. Since of all these sugars only xylose acted as a feeding deterrent it was proposed that at high concentrations they or metabolic derivatives were toxic. The results are discussed in relation to those obtained with several other insect species.     

Studies of the polysaccharides from sunflower heads

Extraction of sunflower heads with ammonium oxalate afforded water-soluble pectin material and water-insoluble glycoprotein material, the carbohydrate portion of which consisted of galacturonic acid and xylose residues; the pectin material defied fractionation with cetylpyridinium chloride. Extraction with hydrochloric acid (pH 1.5) afforded water-soluble and water-insoluble polysaccharide materials. The former, when fractionated with cetylpyridinium chloride, gave a glycoprotein, the carbohydrate moiety of which was composed of galacturonic acid, galactose (major), glucose, arabinose, and xylose, and also a rhamnan. The latter was a glycoprotein, the carbohydrate portion of which consisted of galactose (major), glucose, xylose, and rhamnose residues. Extraction of the sunflower heads with water also gave glycoprotein material, which was fractionated by paper electrophoresis into a glyco-protein, the carbohydrate moiety ofwhich was composed of galacturonic acid (minor), galactose, glucose, xylose, arabinose, and rhamnose (major) residues, and a heteropolysaccharide composed of galactose (major), glucose, xylose, and arabinose residues.     

马齿苋多糖的研究

用苯酚硫酸法对马齿苋多糖含量进行测定,设计正交实验确定马齿苋多糖提取的最佳工艺,马齿苋多糖的提取率高达9.23%。将其多糖分离纯化,进行理化性质试验测试,利用纸层析和气相色谱对马齿苋多糖中的单糖组成做了分析,马齿苋多糖中的单糖组成有葡萄糖、半乳糖、甘露糖、果糖、木糖、阿拉伯糖。     

Proteomics analysis of Bifidobacterium longum NCC2705 growing on glucose, fructose, mannose, xylose, ribose, and galactose

To investigate the molecular mechanisms underlying carbohydrate uptake and connected metabolic pathways of Bifidobacterium longum NCC2705, the proteomic profiles of bacteria grown on different carbon sources including glucose, fructose, mannose, xylose, ribose, and galactose were analyzed. Our results show that all sugars tested were catabolized via the bifid shunt. Sixty-eight proteins that exhibited changes in abundance of threefold or greater were identified by MS. A striking observation was the differential expression of proteins related to the pyruvate metabolism. Further analysis of acetic acid and lactic acid in the culture supernatants by HPLC at the end of fermentation showed that more lactic acid was produced during growth on fructose, ribose, xylose, galactose and more acetic acid was produced during the fermentation of glucose and mannose. Growth experiments revealed that B. longum NCC2705 preferentially used fructose, ribose, xylose, and galactose with higher growth rates over glucose and mannose. Furthermore, five proteins (GroEL, Eno, Tal, Pgm, and BL0033) exhibited clear phosphorylation modifications at serine and/or tyrosine residues. BL0033, a component of an ATP-binding cassette (ABC) transporter, was significantly more abundant in bacteria grown on fructose and, to a lesser extent, ribose and xylose. RT-PCR analysis revealed that all genes of the ABC transporter are induced in the presence of these sugars suggesting that BL0033, BL0034, BL0035, and BL0036 constitute an ABC transporter with fructose as preferred substrate.     

Functional survey for heterologous sugar transport proteins, using Saccharomyces cerevisiae as a host

Molecular transport is a key process in cellular metabolism. This step is often limiting when using a nonnative carbon source, as exemplified by xylose catabolism in Saccharomyces cerevisiae. As a step toward addressing this limitation, this study seeks to characterize monosaccharide transport preference and efficiency. A group of 26 known and putative monosaccharide transport proteins was expressed in a recombinant Saccharomyces cerevisiae host unable to transport several monosaccharides. A growth-based assay was used to detect transport capacity across six different carbon sources (glucose, xylose, galactose, fructose, mannose, and ribose). A mixed glucose-and-xylose cofermentation was performed to determine substrate preference. These experiments identified 10 transporter proteins that function as transporters of one or more of these sugars. Most of these proteins exhibited broad substrate ranges, and glucose was preferred in all cases. The broadest transporters confer the highest growth rates and strongly prefer glucose. This study reports the first molecular characterization of the annotated XUT genes of Scheffersomyces stipitis and open reading frames from the yeasts Yarrowia lipolytica and Debaryomyces hansenii. Finally, a phylogenetic analysis demonstrates that transporter function clusters into three distinct groups. One particular group comprised of D. hansenii XylHP and S. stipitis XUT1 and XUT3 demonstrated moderate transport efficiency and higher xylose preferences.     

The Anaerobic Fungus Neocallimastix sp. Strain L2: Growth and Production of (Hemi)Cellulolytic Enzymes on a Range of Carbohydrate Substrates

The anaerobic fungus Neocallimastix sp. strain L2, isolated from the feces of a llama, was tested for growth on a range of soluble and insoluble carbohydrate substrates. The fungus was able to ferment glucose, cellobiose, fructose, lactose, maltose, sucrose, soluble starch, inulin, filter paper cellulose, and Avicel. No growth was observed on arabinose, galactose, mannose, ribose, xylose, sorbitol, pectin, xylan, glycerol, citrate, soya, and wheat bran. The fermentation products after growth were hydrogen, formate, acetate, ethanol, and lactate. The fermentation pattern was dependent on the carbon source. In general, higher hydrogen production resulted in decreased formation of lactate and ethanol. Recovery of the fermented carbon in products at the end of growth ranged from 50% to 80%. (Hemi)cellulolytic enzyme activities were affected by the carbon source. Highest activities were found in filtrates from cultures grown on cellulose. Growing the fungus on inulin and lactose yielded the lowest cellulolytic activities. Highest specific activities for avicelase, endoglucanase, β-glucosidase, and xylanase were obtained with Avicel as the substrate for growth (0.29, 5.9, 0.57, and 13 IU · mg⁻¹ protein, respectively). Endoglucanase activity banding patterns after SDS-PAGE were very similar for all substrates. Minor differences indicated that enzyme activities may in part be the result of secretion of different sets of isoenzymes. Received: 10 July 1996 / Accepted: 22 July 1996     

A simple procedure for the synthesis of alpha-32P-nucleoside triphosphates.

Chemical analysis of grapefruit (Citrus paradisi) pectic polysaccharides demonstrated that galacturonic acid constitutes 78% by weight of the total carbohydrates found. The remaining 22% was accounted for by a number of sugars which include galactose, glucose, arabinose, xylose, and mannose and, by weight, galactose accounted for almost 50% of the total neutral sugar components found in these pectic polysaccharides. Treatment of pectic polysaccharides with galactose oxidase followed by reduction of oxidized galactose residues with tritiated potassium borohydride resulted in the labeling of pectic polysaccharides. Analysis of the labeled polysaccharides demonstrated that of the total radioactivity incorporated more than 90% was recovered in the galactose residues. These results clearly demonstrate the successful utilization of the galactose oxidase/tritiated potassium borohydride method in labeling plant pectic polysaccharide.     

Carbohydrate composition and physical properties of tobacco phosphodiesterase

Some physical and chemical properties of phosphodiesterase from cultured tobacco cells were studied. The enzyme contained ca 50% carbohydrate consisting of residues of arabinose, glucose, glucosamine, galactose, mannose and xylose. Analyses showed that the enzyme had a sedimentation coefficient, s_20,w of 16 S, a Stokes'radius of 5.7 nm and a calculated specific volume of 0.66 ml/g. The MW of the enzyme was calculated to be 300 000 from these values.     

Utilization of some monosaccharides by two species of Colletotrichum

Summary Utilization of 8 monosaccharides, viz., glucose, fructose, galactose, mannose, sorbose, arabinose, xylose and rhamnose, by some plant pathogenic isolates ofColletotrichum gloeosphorioides andC. dematium has been studied with the help of paper chromatography. Among hexoses, the rate of utilization of glucose, fructose and mannose was fast, whereas, that of galactose was comparatively slow. The rate of assimilation of sorbose was very slow at early stages of incubation, although at later stages this rate showed marked enhancement. The pentoses were utilized readily. The dry weight of mycelial mats showed an increase up to the end of final incubation period (15 days), on sugars which were slowly assimilated. In cases where the sugars were consumed up rapidly, the dry weight at later stages of incubation either became nearly stationary or recorded slight fall.     

Growth of five pathogenic fungi on cell wall-related monosaccharides

Spores of Monilinia fructicola, Colletotrichum coccodes, Botrytis cinerea, Alternaria alternata and Mucor mucedo were transferred to agar plates containing 0.5, 5 or 50 mM concentrations of various plant cell wall-related monosaccharides or glucose. The wall-related monosaccharides tested were arabinose, galactose, galacturonic acid, mannose, rhamnose and xylose. Germ tube length was measured after 24 h. Growth of germ tubes on media containing the different monosaccharides varied substantially among fungi. The most striking response was with M. mucedo; spores were only able to grow on media containing glucose or galactose.Increase in colony size was determined during a 7-day period after subcultures were transferred to media containing 5 mM arabinose, galactose, galacturonic acid, mannose, rhamnose and xylose, each with and without 5 mM glucose. Glucose, alone or in conjunction with any other monosaccharide, was the optimal carbon source for growth. In general, fungi were unable to grow as well on any of the wall-related monosaccharides without glucose. Galacturonic acid and rhamnose, major components of pectic polysaccharides of plant cell walls, were unable to support any growth of M. fructicola cultures. No wall-related monosaccharides supported more than 50% of the growth observed when M. mucedo cultures were grown on glucose.     

Composition of ethanol-insoluble polysaccharides in water extracts of ripening tomatoes

The carbohydrate composition of the 80% ethanol-insoluble polysaccharides (EIP) from water extracts of ‘Rutgers,’ rin (ripening inhibitor) and nor (non-ripening) tomatoes has been determined. The amount of EIP extracted from ‘Rutgers’ fruit increased from 0.34 to 0.61 mg/g fr. wt during ripening little change occurred in rin or nor fruit. The carbohydrate composition (μg/g fr. wt) of EIP from mature green fruit was: galacturonic acid (48); rhamnose (3); arabinose (20); xylose (48); mannose (31); glucose (139); galactose (51). The most obvious changes that accompanied ripening were a 7.4-fold and 4-fold increase in galacturonic acid and rhamnose content, respectively. These changes were attenuated in the ripening mutants. EIP was fractionated into three major peaks by using DEAE-cellulose ion exchange chromatography. The first peak, which was not retained by the column, contained predominantly glucose and mannose, with lower amounts of galacturonic acid and galactose. The two retained peaks which eluted at 0.1 and 0.2 M sodium chloride contained primarily galacturonic acid, xylose, galactose and arabinose. The galacturonic acid content of these two fractions increased substantially during ripening, whereas the other components decreased. No changes were evident in the ripening mutants. No increase in water-soluble polysaccharides high in galactose content was observed during ripening.     

Characterization and regulation of galactose transport in Neurospora crassa

Two galactose uptake systems were found in the mycelia of Neurospora crassa. In glucose-grown mycelia, galactose was transported by a low-affinity (Km = 400 mM) constitutive system which was distinct from the previously described glucose transport system I (R. P. Schneider and W. R. Wiley, J. Bacteriol. 106:479--486, 1971). In carbon-starved mycelia or mycelia incubated with galactose, a second galactose transport activity appeared which required energy, had a high affinity for galactose (Km = 0.7 mM), and was shown to be the same as glucose transport system II. System II also transported mannose, 2-deoxyglucose, xylose, and talose and is therefore a general monosaccharide transport system. System II was derepressed by carbon starvation, completely repressed by glucose, mannose, and 2-deoxyglucose, and partially repressed by fructose and xylose. Incubation with galactose yielded twice as much activity as starvation. This extra induction by galactose required protein synthesis, and represented an increase in activity of system II rather than the induction of another transport system. Glucose, mannose, and 2-deoxyglucose caused rapid degradation of preexisting system II; fructose and xylose caused a slower degradation of activity.