Glucose Repression Effect Research Articles

Improvement of Enantiomeric l-Lactic Acid Production from Mixed Hexose-Pentose Sugars by Coculture of Enterococcus mundtii WX1 and Lactobacillus rhamnosus SCJ9

Among 39 pentose-utilizing lactic acid bacteria (LAB) selected from acid-forming bacteria from the midgut of Eri silkworm, the isolate WX1 was selected with the highest capability to produce optically pure l-lactic acid (l-LA) from glucose, xylose and arabinose with furfural-tolerant properties. The isolate WX1 was identified as Enterococcus mundtii based on 16S rDNA sequence analysis. The conversion yields of l-LA from glucose and xylose by E. mundtii WX1 were 0.97 and 0.68 g/g substrate, respectively. Furthermore, l-LA production by E. mundtii WX1 in various glucose-xylose mixtures indicated glucose repression effect on xylose consumption. The coculture of E. mundtii WX1 and Lactobacillus rhamnosus SCJ9, a homofermentative LAB capable of producing l-LA from glucose clearly showed an improvement of l-LA production from 30 g/L total glucose-xylose (6:4). The results from Plackett–Burman design (PBD) indicated that Tween 80, MnSO4 and yeast extract (YE) were three medium components that significantly influenced (p < 0.05) l-LA production using the coculture strategy in the presence of 2 g/L furfural. Optimal concentrations of these variables revealed by central composite design (CCD) and response surface methodology (RSM) were 20.61 g/L YE, 1.44 g/L Tween 80 and 1.27 g/L MnSO4. Based on the optimized medium with 30 g/L total glucose-xylose (6:4), the maximum experimental l-LA value of 23.59 g/L reflecting 0.76 g/g substrate were achieved from 48 h fermentation at 37 °C. l-LA produced by coculture cultivated under standard MRS medium and new optimized conditions were 1.28 and 1.53 times higher than that obtained from single culture by E. mundtii WX1, respectively. This study provides the foundations for practical applications of coculture in bioconversion of lignocellulose particularly glucose-xylose-rich corn stover to l-LA.

High temperature xylitol production through simultaneous co-utilization of glucose and xylose by engineered Kluyveromyces marxianus

Abstract Xylitol production from lignocellulose-hydrolyzed glucose and xylose is a sustainable and environment-friendly process. In this study, Kluyveromyces marxianus YZB194, constructed by overexpressing ScGAL2N376F from Saccharomyces cerevisiae and NcXYL1 from Neurospora crassa, and deleting GPD1, KU70, PGI1, and XYL2, was used for xylitol production by co-utilizing glucose and xylose. By deleting PGI1, the glucose repression effect on xylose in K. marxianus was released, besides, glucose metabolism in YZB194 was refactored through the pentose phosphate pathway, a pathway yielding the largest supply of NADPH in yeast. The metabolic pathway of xylitol was blocked by deleting of XYL2. By simultaneously co-utilizing 70 g/L glucose and 140 g/L xylose, 139.96 g/L xylitol with a productivity of 0.83 g/L/h was obtained by YZB194 at 37 °C under aerobic conditions. Through fed-batch fermentation in a fermenter at 42 °C, 203.57 g/L xylitol with a productivity of 2.99 g/L/h and yield on xylose of 0.99 g/g were obtained using K. marxianus YZB194. A platform for xylitol production by co-utilizing glucose and xylose via rewriting the glucose metabolism pathway was implemented.

Glucose repression can be alleviated by reducing glucose phosphorylation rate in Saccharomyces cerevisiae

Microorganisms commonly exhibit preferential glucose consumption and diauxic growth when cultured in mixtures of glucose and other sugars. Although various genetic perturbations have alleviated the effects of glucose repression on consumption of specific sugars, a broadly applicable mechanism remains unknown. Here, we report that a reduction in the rate of glucose phosphorylation alleviates the effects of glucose repression in Saccharomyces cerevisiae. Through adaptive evolution under a mixture of xylose and the glucose analog 2-deoxyglucose, we isolated a mutant strain capable of simultaneously consuming glucose and xylose. Genome sequencing of the evolved mutant followed by CRISPR/Cas9-based reverse engineering revealed that mutations in the glucose phosphorylating enzymes (Hxk1, Hxk2, Glk1) were sufficient to confer simultaneous glucose and xylose utilization. We then found that varying hexokinase expression with an inducible promoter led to the simultaneous utilization of glucose and xylose. Interestingly, no mutations in sugar transporters occurred during the evolution, and no specific transporter played an indispensable role in simultaneous sugar utilization. Additionally, we demonstrated that slowing glucose consumption also enabled simultaneous utilization of glucose and galactose. These results suggest that the rate of intracellular glucose phosphorylation is a decisive factor for metabolic regulations of mixed sugars.

Glucose repression in Saccharomyces cerevisiae.

Glucose is the primary source of energy for the budding yeast Saccharomyces cerevisiae. Although yeast cells can utilize a wide range of carbon sources, presence of glucose suppresses molecular activities involved in the use of alternate carbon sources as well as it represses respiration and gluconeogenesis. This dominant effect of glucose on yeast carbon metabolism is coordinated by several signaling and metabolic interactions that mainly regulate transcriptional activity but are also effective at post-transcriptional and post-translational levels. This review describes effects of glucose repression on yeast carbon metabolism with a focus on roles of the Snf3/Rgt2 glucose-sensing pathway and Snf1 signal transduction in establishment and relief of glucose repression.

Meta-analysis and functional validation of nutritional requirements of solventogenic Clostridia growing under butanol stress conditions and coutilization of D-glucose and D-xylose.

Recent advances in systems biology, omics, and computational studies allow us to carry out data mining for improving biofuel production bioprocesses. Of particular interest are bioprocesses that center on microbial capabilities to biotransform both the hexose and pentose fractions present in crop residues. This called for a systematic exploration of the components of the media to obtain higher-density cultures and more-productive fermentation operations than are currently found. By using a meta-analysis approach of the transcriptional responses to butanol stress, we identified the nutritional requirements of solvent-tolerant strain Clostridium beijerinckii SA-1 (ATCC 35702). The nutritional requirements identified were later validated using the chemostat pulse-and-shift technique. C. beijerinckii SA-1 was cultivated in a two-stage single-feed-stream continuous production system to test the proposed validated medium formulation, and the coutilization of D-glucose and D-xylose was evaluated by taking advantage of the well-known ability of solventogenic clostridia to utilize a large variety of carbon sources such as mono-, oligo-, and polysaccharides containing pentose and hexose sugars. Our results indicated that C. beijerinckii SA-1 was able to coferment hexose/pentose sugar mixtures in the absence of a glucose repression effect. In addition, our analysis suggests that the solvent and acid resistance mechanisms found in this strain are differentially regulated compared to strain NRRL B-527 and are outlined as the basis of the analysis toward optimizing butanol production.

Cloning of novel laccase isozyme genes from Trametes sp. AH28-2 and analyses of their differential expression

Three novel laccase isozyme genes, lacA, lacB, and lacC, have been identified from basidiomycete Trametes sp. AH28-2. These genes display a high similarity with other basidiomycete laccases at the amino acid level. An inferred TATA box and several putative CAAT, MRE, XRE, and CreA consensus sequences were identified in the lacA, lacB, and lacC promoter regions. Different from the TATA boxes of lacA and lacB at about -100, the TATA box of lacC is located at -172. For all the isozymes, copper ion is essential for laccase synthesis in Trametes sp. AH28-2. More interestingly, different aromatic compounds can selectively induce the production of distinct laccase isozymes, with o-toluidine inducing the expression of laccase A (LacA) while 3,5-dihydroxytoluene mainly stimulating the production of laccase B (LacB). Quantitative reverse transcriptase-polymerase chain reaction showed that the accumulation of laccase messenger RNA transcripts is accompanied by the increase of corresponding enzyme activity in cultures. The glucose-repression effect on laccase expression in Trametes sp. AH28-2 was also observed. Furthermore, lower Cu2+ concentration (lower than 0.5 mM) can induce LacA and a novel laccase (LacC), and the latter will disappear when Cu2+ concentration is increased up to 1-2 mM. Upon induction by 3,5-dihydroxytoluene, the ratio of LacA to LacB decreased in the later phase of induction.

Glucose repression of PRX1 expression is mediated by Tor1p and Ras2p through inhibition of Msn2/4p in Saccharomyces cerevisiae

Expression of mitochondrial thioredoxin peroxidase (Prx1p) from Saccharomyces cerevisiae is subjected to complex transcriptional regulation and is responsive to the levels of several compounds such as glucose and peroxides. We have previously shown that glucose represses the expression of mitochondrial thioredoxin peroxidase gene ( PRX1) in a process mediated by cAMP/protein kinase A (PKA) and Msn2/4p. Here, we show by northern blot and reporter gene (β-galactosidase) assays that deletion of genes encoding Tor1p and Ras2p resulted in increased PRX1 expression, indicating that these proteins are also mediators of the glucose repression effect. We also identified the position of the stress transcription responsive element (STRE) in the PRX1 promoter, which is recognized by Msn2p and Msn4p activators. Mutation of AGGGG sequence at position −116 to −112 caused a high drop in PRX1 expression under respiratory conditions and in strains containing deletions of TOR1 or RAS2, confirming the finding that this sequence is a STRE.

The effect of CreA in glucose and xylose catabolism in Aspergillus nidulans.

The catabolism of glucose and xylose was studied in a wild type and creA deleted (carbon catabolite de-repressed) strain of Aspergillus nidulans. Both strains were cultivated in bioreactors with either glucose or xylose as the sole carbon source, or in the presence of both sugars. In the cultivations on single carbon sources, it was demonstrated that xylose acted as a carbon catabolite repressor (xylose cultivations), while the enzymes in the xylose utilisation pathway were also subject to repression in the presence of glucose (glucose cultivations). In the wild type strain growing on the sugar mixture, glucose repression of xylose utilisation was observed; with xylose utilisation occurring only after glucose was depleted. This phenomenon was not seen in the creA deleted strain, where glucose and xylose were catabolised simultaneously. Measurement of key metabolites and the activities of key enzymes in the xylose utilisation pathway revealed that xylose metabolism was occurring in the creA deleted strain, even at high glucose concentrations. Conversely, in the wild type strain, activities of the key enzymes for xylose metabolism increased only when the effects of glucose repression had been relieved. Xylose was both a repressor and an inducer of xylanases at the same time. The creA mutation seemed to have pleiotropic effects on carbohydratases and carbon catabolism.

The onset of fermentative metabolism in continuous cultures depends on the catabolite repression properties of saccharomyces cerevisiae

In glucose-limited continuous cultures, a Crabtree positive yeast such as Saccharomyces cerevisiae displays respiratory metabolism at low dilution rates ( D) and respirofermentative metabolism at high D. We hypothesized that the onset of fermentative metabolism is related with the catabolite repression or glucose repression effect. To test this hypothesis, we have investigated the physiological behavior in glucose-limited continuous cultures of S. cerevisiae strain CEN.PK122 and isogenic mutants, snf1 ( cat1) and snf4 ( cat3), defective in proteins involved in the release from glucose repression and the mutant in glucose repression mig1. We analyzed the behavior of the wild type and mutant strains at steady state in chemostat cultures as a function of D. Wild-type cells displayed respiratory metabolism up to a D of 0.2 h −1. snf1 and snf4 mutants started fermenting after a D of 0.1 and 0.15 h −1, respectively. The latter behavior was not due to an impairment of respiration since their specific rate of oxygen consumption was similar or even higher than that shown by the wild type. The snf1 strain displayed much lower yields than the wild type and the other mutants in the whole range of D studied. We conclude that the onset of fermentative metabolism in yeast growing in chemostat cultures is related with glucose repression.

Modelling of ethanol production by Saccharomyces cerevisiae from a glucose and maltose mixture

A model for ethanol production from a glucose-maltose mixture has been proposed, which includes a term representing the glucose repression effect on maltose consumption. The model parameters were estimated from batch experimental data. Results of sensitivity analysis on the Monod constants for glucose and maltose, and the repression constant, showed that ±10% changes in these three parameters caused no significant effect on data fitting.

THE INTERACTION OF GENETIC AND ENVIRONMENTAL FACTORS IN THE CONTROL OF AMYLASE GENE EXPRESSION IN DROSOPHILA MELANOGASTER

A number of previous studies have established that amylase activity can vary between Drosophila strains which are maintained under identical laboratory conditions. In addition, we have recently shown that all strains examined so far are subject to glucose repression of amylase activity. In this study, we show that the degree of glucose repression can vary between strains. Moreover, the glucose repression effect is much more pronounced in larvae than in adult flies. Our results lead to the conclusion that the strain-specific differences in activity and the dietary effects are not independent phenomena. These results have implications for the interpretation of many studies on amylase activity variation, including those experiments which have been designed to link amylase activity variations with fitness differences in nature. A question that naturally arises concerns the molecular basis for these strain-specific variations in the degree of glucose repression of this eukaryotic gene.

Purification and properties of a novel polyol dehydrogenase of bacterial origin

Abstract A bacterium, as yet unidentified, has been isolated from floor dust by direct selection on minimal agar using l -glucitol ( d -gulitol) as the sole carbon energy source. The bacterium possesses a constitutive enzyme which catalyzes the reaction: l -glucitol + NAD + → d -sorbose + NADH + H + . A new species of enzyme has been induced by l -arabinitol or ribitol, but not l - or d -glucitol, and the induction is only partially counteracted by the glucose-repression effect. The constitutive enzyme was purified by fractionation on Sephadex G-200 gel and chromatography on DEAE Biogel A. The enzyme required NAD + , but not NADP + , as a cofactor. It oxidizes also ribitol, xylitol and l -arabinitol, but not d -arabinitol, lactitol or a variety of other commercially available alditols. The enzyme is not inhibited by 10 mM sodium azide but is totally inhibited by 0.1 mM potassium ferricyanide.

Effects of Glucose Repression and Anaerobiosis on the Activities and Subcellular Distribution of Tricarboxylic Acid Cycle and Associated Enzymes in Saccharomyces carlsbergensis

Cell-free extracts were prepared from sphaeroplasts of aerobically and anaerobically grown, glucose-derepressed and glucose-repressed Saccharomyces carlsbergensis. The activities of the enzymes of the tricarboxylic acid cycle and of related enzymes were measured, together with their distributions after differential centrifugation. Glucose repression lowered activities of enzymes of the tricarboxylic acid cycle by 60 to 89 % under aerobic conditions. Activities were still further decreased under anaerobic glucose-derepressed conditions. 2-Oxoglutarate decarboxylase activity was not detected after anaerobic growth; malate synthase and isocitrate lyase activities were not detected in organisms grown either aerobically or anaerobically. Glucose had little effect on the activities of tricarboxylic acid cycle enzymes under anaerobic conditions, with the exception of citrate synthase whose activity increased under anaerobic glucose-repressed conditions. Differential centrifugation of cell-free extracts showed different distribution patterns of the enzymes under the four growth conditions. The distribution patterns and enzyme activities reflected the differential effects of glucose repression and/or anaerobiosis.

Regulation of beta-1,3-glucanase synthesis in Penicillium italicum

The filamentous fungus Penicillium italicum produced a certain level of beta-1,3-glucanase during active growth in a glucose-supplemented medium; however, at a low glucose concentration (2 to 10 mM), derepression took place and the specific activity of the enzyme increased significantly. Derepressed cells (incubated in a glucose-limited medium) accumulated a capacity for the synthesis of beta-1,3-glucanase, which led to a subsequent increase in the specific activity even when the cells were transferred to a medium with an excess of glucose (180 mM). Two protein synthesis inhibitors, cycloheximide and trichodermin, immediately stopped the increase in specific activity when added to derepressed cells. On the other hand, 8-hydroxyquinoline, an RNA a synthesis inhibitor, acted differently, since it permitted the specific activity to increase for some time after being added to depressed cells. Moreover, the concentration of glucose did not affect the 8-hydroxyquinoline-insensitive synthesis of beta-1,3-glucanase. It is concluded that the glucose repression effect on beta-1,3-glucanase production must be exerted at a pretranslational level that could be either mRNA synthesis or some stage of the process involved in its maturation or stabilization.

Effects of glucose repression of the transmission and recombination of mitochondrial genes in yeast (Saccharomyces cerevisiae).

Matings of a number of Saccharomyces cerevisiae stocks give different output ratios of mitochondrial genotypes depending on whether the cells are glucose-repressed or derepressed. The effects of glucose repression are independent of cellular mating type and mitochondrial genotype, and take place at least in part after zygotes are formed. An explanation is proposed in terms of changes in the relative numbers of mitochondrial DNA molecules contributed by the a and alpha parents, modified by selective replication or destruction of molecules inside the zygote.

Formation of the yeast mitochondrial membrane. 2. Effects of glucose repression on mitochondrial protein synthesis.

Amino acid incorporation in vitro by isolated yeast mitochondria could be supported by either an external ATP‐regenerating system or by the respiratory‐chain‐linked synthesis of ATP. However, the incorporation rates obtained with ATP and succinate were almost double those obtained with the ATP‐regenerating system and were inhibited by either antimycin A or oligomycin. In either system, addition of chloramphenicol, erythromycin or ethidium bromide caused an 80–90% inhibition of incorporation, actinomycin D and cordycepin caused a 20% inhibition, while rifamycin or cycloheximide had no effect.The rate of amino acid incorporation in vitro by isolated mitochondria was not subject to glucose derepression. In contrast, the rate of cycloheximide‐insensitive protein synthesis into mitochondrial membranes in vivo was lowered over 90% in repressed cells. Analysis of the labeled mitochondrial membranes by sodium dodecylsulfate gel electrophoresis indicated that the products of mitochondrial protein synthesis were qualitatively identical, but quantitatively different, in glucose‐repressed and derepressed cells. For example, a two‐fold increase in labeling into both the two peaks of highest molecular weight as well as the peak of lowest molecular weight was observed in mitochondrial membranes obtained from repressed cells as compared to mitochondrial membranes obtained from partially and fully derepressed cells.The rate of amino acid incorporation in vitro as well as the labeling of mitochondrial membranes in vivo was stimulated 50—100% in partially derepressed yeast cells which had been grown in medium containing chloramphenicol for three hours, washed, and allowed to grow for one hour in fresh medium without chloramphenicol. The stimulation of labeling in vivo resulted in a threefold increase of labeling into the peak of lowest molecular weight with a concomitant decrease of labeling into the peaks of high molecular weight suggesting that the accumulation of mitochondrial proteins synthesized in the cytoplasm, when chloramphenicol is present in the medium, may stimulate the synthesis of certain specific mitochondrial proteins. Furthermore, growth of the cells in the presence of cycloheximide, which would result in a depletion of the pool of mitochondrial proteins synthesized in the cytoplasm, followed by growth in fresh medium without cycloheximide, resulted in a 50% decrease in the incorporation rate in vitro.

Carboxylase levels and carbon dioxide fixation in baker's yeast.

Levels of pyruvate carboxylase (PC), phosphopyruvate carboxylase (PEPC), and malate dehydrogenase (decarboxylating) were compared in wild-type bakers' yeast (I), a cytoplasmic-respiratory mutant (II), a biotin-deficient wild-type yeast (III), and a biotin-deficient respiratory mutant (IV). PC activities were greatly reduced in III and IV, whereas PEPC was reduced in II and IV. Malate dehydrogenase (decarboxylating) could not be detected in any of the yeasts. With yeast I growing on glucose as the sole carbon source, PEPC decreased to negligible levels during the logarithmic phase of growth (glucose repression effect), whereas PC increased. Both enzymes reverted to their original levels during the stationary phase, when glucose in the medium was exhausted. In agreement with the leading role of PC for CO(2) assimilation, the rates of (14)CO(2) fixation in yeasts I and II were approximately equal and were much higher than that in yeast IV. With I and II, most of the (14)C was distributed similarly in oxalacetate derivatives; with yeast IV, most of (14)C appeared in a compound apparently unrelated to CO(2) fixation via C(4)-dicarboxylic acids.