Multiple Sugars Research Articles

Bioconversion of hemicellulose hydrolysate of sweet sorghum bagasse to ethanol by using Pichia stipitis NCIM 3497 and Debaryomyces hansenii sp.

Production of ethanol from concentrated D-xylose solutions and hemicellulose hydrolysate of sweet sorghum bagasse was achieved by using Pichia stipitis NCIM 3497 and an isolated yeast Debaryomyces hansenii sp. These yeasts werecapable of producing ethanol from solutions containing 800 g/L D-xylose, and the optimum sugar concentration was found to be 150 g/L at pH 4, 30oC, with a production time of 72 hours. These yeasts were capable of utilizing multiple sugars. Hemicellulose hydrolysates of sweet sorghum bagasse were obtained by dilute acid hydrolysis and autohydrolysis including steam explosion treatment. The hydrolysate was treated by an over-liming process for detoxification and pH adjustment. Ethanol yield from hemicellulose hydrolysate was found to be higher than that of synthetic medium containing D-xylose. These yeasts can be used in production of ethanol from concentrated hemicellulose hydrolysates containing high pentose sugars obtained while treating lignocellulosic biomass at high substrate concentrations.

Seryl‐phosphorylated HPr regulates CcpA‐independent carbon catabolite repression in conjunction with PTS permeases in Streptococcus mutans

Carbohydrate catabolite repression (CCR) in Streptococcus mutans can be independent of catabolite control protein A (CcpA) and requires specific components of phosphoenolpyruvate-dependent sugar:phosphotransferase system (PTS) permeases. Here, the effects of various ptsH (HPr) and hprK (HPr kinase/phosphatase) mutations on growth and CCR were evaluated. An hprKV265F mutation, which enhanced Ser46 phosphorylation of HPr, inhibited growth on multiple PTS sugars. A ptsHS46A mutation reversed the effects of hprKV265F in most cases. A strain carrying a ptsHS46D mutation, which mimics HPr(Ser-P), presented with more severe growth defects than the hprKV265F mutant. The hprKV265F mutant enhanced CCR of the fruA and levD operons, a phenotype reversible by the ptsHS46A mutation. The effects of the hprKV265F mutation on fruA and levD expression were independent of CcpA, but dependent on ManL (IIAB(Man)) and, to a lesser extent, on FruI (IIABC(Fru)), in a carbohydrate-specific fashion. Expression of the Bacillus subtilis ptsG gene in the manL mutant did not restore CCR of the lev or fru operons. The hprKV265F mutation inhibited growth on cellobiose and lactose, but only the transcription of the cel operon was decreased. Thus, in S. mutans, serine-phosphorylated HPr functions in concert with particular PTS permeases to prioritize carbohydrate utilization by modulating sugar transport and transcription of catabolic operons.

Regulation of arabinose and xylose metabolism in Escherichia coli.

Bacteria such as Escherichia coli will often consume one sugar at a time when fed multiple sugars, in a process known as carbon catabolite repression. The classic example involves glucose and lactose, where E. coli will first consume glucose, and only when it has consumed all of the glucose will it begin to consume lactose. In addition to that of lactose, glucose also represses the consumption of many other sugars, including arabinose and xylose. In this work, we characterized a second hierarchy in E. coli, that between arabinose and xylose. We show that, when grown in a mixture of the two pentoses, E. coli will consume arabinose before it consumes xylose. Consistent with a mechanism involving catabolite repression, the expression of the xylose metabolic genes is repressed in the presence of arabinose. We found that this repression is AraC dependent and involves a mechanism where arabinose-bound AraC binds to the xylose promoters and represses gene expression. Collectively, these results demonstrate that sugar utilization in E. coli involves multiple layers of regulation, where cells will consume first glucose, then arabinose, and finally xylose. These results may be pertinent in the metabolic engineering of E. coli strains capable of producing chemical and biofuels from mixtures of hexose and pentose sugars derived from plant biomass.

Sessions 2 and 7: Advances in Microbial Science and Technology

The Microbial Science and Technology Sessions emphasized a variety of approaches used for development of superior recombinant microorganisms for fermentation of multiple sugars typically present in any lignocellulosic hydrolyzate to fuels and chemicals. Efficient conversion of lignocellulosic biomass to liquid transportation fuels is a major goal for many in these sessions. The Session 2 contained six oral presentations and 62 poster presentations and Session 7 contained an additional six oral presentations and 49 posters. A brief overview of each oral Session and a summary of topics presented during the poster sessions are provided below. Session 2 opened with a comparison of different pretreatments for corn stover, followed by fermentation using different recombinant organisms. Next, the metabolic engineering of a novel thermophilic Geobacillus sp. capable of high-yield ethanol production was discussed. The pathway for D-galacturonic acid catabolism in fungi was elucidated in the third presentation and determined to be different from any previously described pathway. Butanol production from engineered Escherichia coli and improvements in solventogenic Clostridium butanol production were the topics for another presentation. Metabolic pathway engineering of Thermoanaerobacterium saccharolyticum for efficient ethanol production and reduction of cellulase loading emphasized a consolidated bioprocessing approach. New methods for isolating novel biocatalysts and enzymes from lignocellulosedegrading insects rounded out this first session. Appl Biochem Biotechnol (2009) 153:11–12 DOI 10.1007/s12010-009-8609-9

Functional Redundancy between Two Caenorhabditis elegans Nucleotide Sugar Transporters with a Novel Transport Mechanism

Transporters of nucleotide sugars regulate the availability of these substrates required for glycosylation reactions in the lumen of the Golgi apparatus and play an important role in the development of multicellular organisms. Caenorhabditis elegans has seven different sugars in its glycoconjugates, although 18 putative nucleotide sugar transporters are encoded in the genome. Among these, SQV-7, SRF-3, and CO3H5.2 exhibit partially overlapping substrate specificity and expression patterns. We now report evidence of functional redundancy between transporters CO3H5.2 and SRF-3. Reducing the activity of the CO3H5.2 gene product by RNA interference (RNAi) in SRF-3 mutants results in oocyte accumulation and abnormal gonad morphology, whereas comparable RNAi treatment of wild type or RNAi hypersensitive C. elegans strains does not cause detectable defects. We hypothesize this genetic enhancement to be a mechanism to ensure adequate glycoconjugate biosynthesis required for normal tissue development in multicellular organisms. Furthermore, we show that transporters SRF-3 and CO3H5.2, which are closely related in the phylogenetic tree, share a simultaneous and independent substrate transport mechanism that is different from the competitive one previously demonstrated for transporter SQV-7, which shares a lower amino acid sequence identity with CO3H5.2 and SRF-3. Therefore, different mechanisms for transporting multiple nucleotide sugars may have evolved parallel to transporter amino acid divergence.

A Drosophila gustatory receptor required for the responses to sucrose, glucose, and maltose identified by mRNA tagging

In Drosophila , detection of tastants is thought to be mediated by members of a family of 68 gustatory receptors (Grs). However, only one receptor, Gr5a, has been associated with a sugar, and it appears to be activated specifically by trehalose. It is unclear whether other sugar receptors are activated by single or multiple sugars. Currently, no Grs are known to colocalize with Gr5a. Such Grs would be candidate sugar receptors because Gr5a-expressing cells function in the responses to attractive tastants. Here we use an “mRNA tagging” approach to identify Gr RNAs that are coexpressed with Gr5a . We found that all seven Gr s most related to Gr5a ( Gr64a-f and Gr61a ) were expressed in Gr5a -expressing cells, whereas none of the other Gr s examined were enriched in these Gr neurons (GRNs). We characterized the role of one Gr5a-related receptor, Gr64a, and found that it was required for the behavioral responses to glucose, sucrose, and maltose. Gr64a was required for GRN function because action potentials induced by these sugars were dependent on expression of Gr64a in GRNs. These data demonstrate that multiple Grs are coexpressed with Gr5a and that Drosophila Gr64a is required for the responses to multiple sugars.

Sugar microanalysis by HPLC with benzoylation: Improvement via introduction of a C-8 cartridge and a high efficiency ODS column

An HPLC protocol for sugar microanalysis based on the formation of ultraviolet-absorbing benzoyl chloride derivatives was improved. Here, samples were prepared with a C-8 cartridge and analyzed with a high efficiency ODS column, in which porous spherical silica particles 3 μm in diameter were packed. These devices allowed us to simultaneously quantify multiple sugars and sugar alcohols up to 10 ng/ml and to provide satisfactory separations of some sugars, such as fructose and myo-inositol and sorbitol and mannitol. This protocol, which does not require special apparatuses, should become a powerful tool in sugar research.

Involvement of Streptococcus gordonii beta-glucoside metabolism systems in adhesion, biofilm formation, and in vivo gene expression.

Streptococcus gordonii genes involved in beta-glucoside metabolism are induced in vivo on infected heart valves during experimental endocarditis and in vitro during biofilm formation on saliva-coated hydroxyapatite (sHA). To determine the roles of beta-glucoside metabolism systems in biofilm formation, the loci of these induced genes were analyzed. To confirm the function of genes in each locus, strains were constructed with gene inactivation, deletion, and/or reporter gene fusions. Four novel systems responsible for beta-glucoside metabolism were identified, including three phosphoenolpyruvate-dependent phosphotransferase systems (PTS) and a binding protein-dependent sugar uptake system for metabolizing multiple sugars, including beta-glucosides. Utilization of arbutin and esculin, aryl-beta-glucosides, was defective in some mutants. Esculin and oligochitosaccharides induced genes in one of the three beta-glucoside metabolism PTS and in four other genetic loci. Mutation of genes in any of the four systems affected in vitro adhesion to sHA, biofilm formation on plastic surfaces, and/or growth rate in liquid medium. Therefore, genes associated with beta-glucoside metabolism may regulate S. gordonii in vitro adhesion, biofilm formation, growth, and in vivo colonization.

Metabolic Switching in the Sugar Phosphotransferase System of Escherichia coli

Bacteria grown in a mixture of multiple sugars will first metabolize a preferred sugar until it is nearly depleted, only then turning to other carbon sources in the medium. This sharp switching of metabolic preference is characteristic of systems that optimize fitness. Here we consider the mechanism by which switching can occur in the Escherichia coli phosphotransferase system (PTS), which regulates the uptake and metabolism of several sugars. Using a model combining the description of fast biochemical processes and slower genetic regulation, we derive metabolic phase diagrams for the uptake of two PTS sugars, indicating regions of distinct sugar preference as a function of external sugar concentrations. We then propose a classification of bacterial phenotypes based on the topology of the metabolic phase diagram, and enumerate the possible topologically distinct phenotypes that can be achieved through mutations of the PTS. This procedure reveals that there is only one nontrivial switching phenotype that is insensitive to large changes in biochemical parameters. This phenotype exhibits diauxic growth, a manifestation of the winner-take-all dynamics enforced by PTS architecture. Winner-take-all behavior is implemented by the induction of sugar-specific operons, combined with competition between sugars for limited phosphoryl flux. We propose that flux-limited competition could be a common mechanism for introducing repressive interactions in cellular networks, and we argue that switching behavior similar to that described here should occur generically in systems that implement such a mechanism.

Characterization of Multicomponent Monosaccharide Solutions Using an Enzyme-Based Sensor Array

We report the development of a sensor for rapidly and simultaneously measuring multiple sugars in aqueous samples. In this strategy, enzyme-based assays are localized within an array of individually addressable sites on a micromachined silicon chip. Microspheres derivatized with monosaccharide-specific dehydrogenases are distributed to pyramidal cavities anisotropically etched in a wafer of silicon (100) and are exposed to sample solution that is forced through the cavities by a liquid chromatography pumping system. Production of fluorescent reporter molecules is monitored under stopped-flow conditions when localized dehydrogenase enzyme systems are exposed to their target sugars. We demonstrate the capability of this analysis strategy to quantify β-d-glucose and β-d-galactose at low micromolar to millimolar levels, with no detectable cross-talk between assay sites. Analysis is achieved either through fluorescence detection of an initial dehydrogenase product (NADH, NADPH) or by production of a secondary fluorescent product created by hydride transfer from the reduced nicotinamide cofactor to a fluorogenic reagent. The array format of this sensor provides capabilities for redundant analysis of sugars and for monitoring levels of other solution components known to affect the activity of enzymes. The use of this strategy to normalize raw fluorescence signals is demonstrated by the determination of glucose and pH on a single chip. Alternatively, uncertainties in the activity of an immobilized enzyme can be accounted for using standard additions, an approach used here in the determination of serum glucose.

Determining sugar composition of food gum polysaccharides by HPTLC

Most commercially important food gums, including gum arabic, guar gum, and carrageenan are polysaccharides which consist of multiple sugars, including uronic acids. For the first time, the sugars in these and other gums were determined by comparison ofR F values with standards by HPTLC on Si 50000 plates after acid hydrolysis of the polysaccharides. The solvent system consisted of npropanol∶water∶triethylamine∶30% NH3 (80∶20∶0.2∶4). Analysis was rapid, as the separations were accomplished using a single plate development and the sugars were located by simple charring. Sugar separations on Si 50000 with this solvent system are more efficient than others and provide the additional advantage over bonded-phase silicas in that sugars can be detected using aggressive methods.

SQV-7, a protein involved in Caenorhabditis elegans epithelial invagination and early embryogenesis, transports UDP-glucuronic acid, UDP-N- acetylgalactosamine, and UDP-galactose.

Caenorhabditis elegans sqv mutants are defective in vulval epithelial invagination and have a severe reduction in hermaphrodite fertility. The gene sqv-7 encodes a multitransmembrane hydrophobic protein resembling nucleotide sugar transporters of the Golgi membrane. A Golgi vesicle enriched fraction of Saccharomyces cerevisiae expressing SQV-7 transported UDP-glucuronic acid, UDP-N-acetylgalactosamine, and UDP-galactose (Gal) in a temperature-dependent and saturable manner. These nucleotide sugars are competitive, alternate, noncooperative substrates. The two mutant sqv-7 missense alleles resulted in a severe reduction of these three transport activities. SQV-7 did not transport CMP-sialic acid, GDP-fucose, UDP-N-acetylglucosamine, UDP-glucose, or GDP-mannose. SQV-7 is able to transport UDP-Gal in vivo, as shown by its ability to complement the phenotype of Madin-Darby canine kidney ricin resistant cells, a mammalian cell line deficient in UDP-Gal transport into the Golgi. These results demonstrate that unlike most nucleotide sugar transporters, SQV-7 can transport multiple distinct nucleotide sugars. We propose that SQV-7 translocates multiple nucleotide sugars into the Golgi lumen for the biosynthesis of glycoconjugates that play a pivotal role in development.

The diversity of lectin-detectable sugar residues on root hair tips of selected legumes correlates with the diversity of their host ranges for rhizobia

Summary. Four legumes and two nonlegumes were investigated for the presence of sugars at the tips of their root hairs, using commercially available lectins which have specific affinities for certain sugars. It was found that while only one lectin (RCA-I, which binds to [3D-galactose) bound to narrow-host-range legumes and one nonlegume, five out of ten lectins tested bound to the root hair tips of the broad-host-range legume siratro (Macroptilium atropurpureum). None of the lectins tested bound to any part of Arabidopsis roots. Binding of lectins (and therefore the presence of sugars) only at the tips of growing root hairs has led us to deduce that the sugars are the glyco moiety of membrane-bound glycoproteins that are recycled at the base of the tip apical dome, along with excess plasma membrane that is known to be recycled there. As many kinds of signal transduction molecules are membrane-bound glycoproteins, we suggest that these sugars may be involved in early interactions with rhizobia, and that the broad-host-range legume siratro has more kinds of sugars to cope with the wide range of rhizobia it is able to accept for symbiotic interactions. As far as we know, this is the first report of multiple sugars at the same surface area of a tip-growing plant cell.

Fuel ethanol production from corn fiber current status and technical prospects

Corn fiber, which consists of about 20% starch, 14% cellulose, and 35% hemicellulose, has the potential to serve as a low cost feedstock for production of fuel ethanol. Currently, the use of corn fiber to produce fuel ethanol faces significant technical and economic challenges. Its success depends largely on the development of environmentally friendly pretreatment procedures, highly effective enzyme systems for conversion of pretreated corn fiber to fermentable sugars, and efficient microorganisms to convert multiple sugars to ethanol. Several promising pretreatment and enzymatic processes for conversion of corn fiber cellulose, hemicellulose, and remaining starch to fermentable sugars were evaluated. These hydrolyzates were then examined for ethanol production in bioreactors, using genetically modified bacteria and yeast. Several novel enzymes were also developed for use in pretreated corn fiber saccharification.

A mathematical model of ethanol fermentation from cheese whey

A cybernetic model for microbial growth on mixed substrates, was used to simulate the anaerobic fermentation of cheese whey and multiple sugars in semisynthetic media by Kluyveromyces marxianus CBS 397. The model simulations quite successfully predicted the observed behavior in batch and during transients in continuous operation, in single-substrate systems as well as in media involving multiple substrates, and in semisynthetic and reconstituted cheese whey solutions. The results of simulations and their comparison with the experimental data are presented.

Stabilization of pet operon plasmids and ethanol production in Escherichia coli strains lacking lactate dehydrogenase and pyruvate formate-lyase activities.

In the last decade, a major goal of research in biofuels has been to metabolically engineer microorganisms to ferment multiple sugars from biomass or agricultural wastes to fuel ethanol. Escherichia coli strains genetically engineered to contain the pet operon (Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase B genes) produce high levels of ethanol. Strains carrying the pet operon in plasmid (e.g., E. coli B/pLOI297) or in chromosomal (e.g., E. coli KO11) sites require antibiotics in the media to maintain genetic stability and high ethanol productivity. To overcome this requirement, we used the conditionally lethal E. coli strain FMJ39, which carries mutations for lactate dehydrogenase and pyruvate formate lyase and grows aerobically but is incapable of anaerobic growth unless these mutations are complemented. E. coli FBR1 and FBR2 were created by transforming E. coli FMJ39 with the pet operon plasmids pLOI295 and pLOI297, respectively. Both strains were capable of anaerobic growth and displayed no apparent pet plasmid losses after 60 generations in serially transferred (nine times) anaerobic batch cultures. In contrast, similar aerobic cultures rapidly lost plasmids. In high-cell-density batch fermentations, 3.8% (wt/vol) ethanol (strain FBR1) and 4.4% (wt/vol) ethanol (strain FBR2) were made from 10% glucose. Anaerobic, glucose-limited continuous cultures of strain FBR2 grown for 20 days (51 generations; 23 with tetracycline and then 28 after tetracycline removal) showed no loss of antibiotic resistance. Anaerobic, serially transferred batch cultures and high-density fermentations were inoculated with cells taken at 57 generations from the previous continuous culture. Both cultures continued to produce high levels of ethanol in the absence of tetracycline. The genetic stability conferred by selective pressure for pet-containing cells without requirement for antibiotics suggests potential commercial suitability for E. coli FBR1 and FBR2.

A binding protein-dependent transport system in Streptococcus mutans responsible for multiple sugar metabolism.

An 11-kilobase gene region of Streptococcus mutans has been identified which contains eight contiguous genes involved with the uptake and metabolism of multiple sugars (the msm system). Sequence analysis of this region indicates that several of these genes specify proteins with strong homology to components of periplasmic binding protein-dependent transport systems of Gram-negative bacteria. Additionally, this operon is controlled by a regulatory gene (msmR) that acts as a positive effector. The proteins specified by the structural genes of the msm operon include alpha-galactosidase (aga), a "periplasmic-like" sugar-binding protein (msmE), two membrane proteins (msmF, msmG), sucrose phosphorylase (gtfA), an ATP-binding protein (msmK), and dextran glucosidase (dexB). Insertional inactivation of each of these genes along with uptake data indicate that this system is responsible for the uptake of melibiose, raffinose, and isomaltotriose and the metabolism of melibiose, sucrose, and isomaltosaccharides.

A new model of uptake of multiple sugars by S. cerevisiae

A new model of uptake of multiple sugars by S. cerevisiae

A new model of uptake of multiple sugars by S. cerevisiae

Recent studies into the mechanisms of uptake of glucose and fructose in S. cerevisiae suggest the existence of at least two transport systems. Based on novel results from our work with glucose, fructose and sucrose, a model of multiple sugars uptake has been developed. This model utilizes a switching concept during the course of batch experiment, the consequences of which are discussed.

The mechanism of uptake of multiple sugars by Saccharomyces cerevisiae in batch culture under fully aerobic conditions

Kinetic studies of the aerobic batch growth of S. cerevisiae on a glucose substrate, a fructose substrate and a substrate consisting of an equimolar mixture of the two sugars are discussed with reference to possible transport mechanisms. Two systems of uptake were found to be present; one resulting in equal uptake of both glucose and fructose, the other in preferential uptake of glucose to fructose.