Role of catabolite regulatory mechanisms in control of carbohydrate utilization by the rumen anaerobic fungus Neocallimastix frontalis

Neocallimastix frontalis PN-1 utilized the soluble sugars D-glucose, D-cellobiose, D-fructose, maltose, sucrose, and D-xylose for growth. L-Arabinose, D-galactose, D-mannose, and D-xylitol did not support growth of the fungus. Paired substrate test systems were used to determine whether any two sugars were utilized simultaneously or sequentially. Of the paired monosaccharides tested, glucose was found to be preferentially utilized compared with fructose and xylose. The disaccharides cellobiose and sucrose were preferentially utilized compared with fructose and glucose, respectively, an cellobiose was also the preferred substrate compared with xylose. Xylose was the preferred substrate compared with maltose. In further incubations, the fungus was grown on the substrate utilized last in the two-substrate tests. After moderate growth was attained, the preferred substrate was added to the culture medium. Inhibition of nonpreferred substrate utilization by the addition of the preferred substrate was taken as evidence of catabolite regulation. For the various combinations of substrates tested, fructose and xylose utilization was found to be inhibited in the presence of glucose, indicating that catabolite regulation was involved. No clear-cut inhibition was observed with any of the other substrate combinations tested. The significance of these findings in relation to rumen microbial interactions and competitions is discussed.     

Evidence for catabolite inhibition in regulation of pentose utilization and transport in the ruminal bacterium Selenomonas ruminantium.

Pentose sugars can be an important energy source for ruminal bacteria, but there has been relatively little study regarding the regulation of pentose utilization and transport by these organisms. Selenomonas ruminantium, a prevalent ruminal bacterium, actively metabolizes xylose and arabinose. When strain D was incubated with a combination of glucose and xylose or arabinose, the hexose was preferentially utilized over pentoses, and similar preferences were observed for sucrose and maltose. However, there was simultaneous utilization of cellobiose and pentoses. Continuous-culture studies indicated that at a low dilution rate (0.10 h-1) the organism was able to co-utilize glucose and xylose. This co-utilization was associated with growth rate-dependent decreases in glucose phosphotransferase activity, and it appeared that inhibition of pentose utilization was due to catabolite inhibition by the glucose phosphotransferase transport system. Xylose transport activity in strain D required induction, while arabinose permease synthesis did not require inducer but was subject to repression by glucose. Since an electrical potential or a chemical gradient of protons drove xylose and arabinose uptake, pentose-proton symport systems apparently contributed to transport.     

Kinetic models for growth and product formation on multiple substrates

Hydrolyzates from lignocellulosic biomass contain a mixture of simple sugars; the predominant ones being glucose, cellobiose and xylose. The fermentation of such mixtures to ethanol or other chemicals requires an understanding of how each of these substrates is utilized.Candida lusitaniae can efficiently produce ethanol from both glucose and cellobiose and is an attractive organism for ethanol production. Experiments were performed to obtain kinetic data for ethanol production from glucose, cellobiose and xylose. Various combinations were tested in order to determine kinetic behavior with multiple carbon sources. Glucose was shown to repress the utilization of cellobiose and xylose. However, cellobiose and xylose were simultaneously utilized after glucose depletion. Maximum volumetric ethanol production rates were 0.56, 0.33, and 0.003 g/L-h from glucose, cellobiose and xylose, respectively. A kinetic model based on cAMP mediated catabolite repression was developed. This model adequately described the growth and ethanol production from a mixture of sugars in a batch culture.     

Studies on helicostylum piriforme. II. Utilization of mono-, oligo- and polysaccharides

Summary The utilization of mono-, oligo- and polysaccharides has been studied in detail. Out of the six monosaccharides tested, xylose, glucose, fructose and galactose were completely utilized within the incubation period, while rhamnose and sorbose were detected in the medium till the end of the incubation period.Amongst the four oligosaccharides, maltose and raffinose were utilized satisfactorily. The hydrolytic products of these two sugars were detected in the medium. In spite of all the efforts the hydrolytic products of sucrose and lactose could not be traced in the medium. It was noticed that the fungus, during the period of utilizing maltose, synthesized some additional unknown oligosaccharides. Both dextrin and starch were not completely utilized by the fungus.The addition of sorbose affected the utilization of mono-, oligo-and polysaccharides by reducing the amount of mycelium produced by the fungus.     

Xylose and arabinose utilization by the rumen bacterium Butyrivibrio fibrisolvens

Abstract The rumen bacterium Butyrivibrio fibrisolvens strain D1 co-utilized xylose and glucose in batch culture, but there was a marked preference for glucose over arabinose. When both pentoses were provided, xylose was preferred over arabinose. Strain D1 co-utilized a combination of either pentose and cellobiose, but preferred pentoses over maltose. Pentose sugars were depleted less rapidly in the presence of sucrose than controls containing only pentose. In contrast, B. fibrisolvens strain A38 exhibited a strong preference for disaccharides, including maltose, over either xylose or arabinose. Theoretical maximum growth yields for strain D1v in single-substrate continuous culture were highest for sucrose and cellobiose and the maintenance energy coefficient for arabinose was at least 3.8-fold greater than for other substrates. We suggest that B. fibrisolvens may have evolved a mechanism to utilize certain sugars before arabinose in order to avoid this high maintenance energy expenditure.     

Influence of the presence of Zymomonas anaerobia on the conversion of cellobiose, glucose, and xylose to ethanol by Clostridium saccharolyticum

To convert sugar mixtures containing cellobiose, glucose, and xylose to ethanol in a single step, the possibility of using a coculture consisting of Clostridium saccharolyticum and Zymomonas anaerobia was studied. In monoculture, C. saccharolyticum utilized all three sugars; however, it preferentially utilized glucose and produced acetic acid in addition to ethanol. The formation of acetic acid from the metabolism of glucose inhibited the growth of C. saccharolyticum and, consequently, the utilization of cellobiose and xylose. In monoculture, Z. anaerobia utilized glucose at a rate of 50 g/L day, but it did not ferment cellobiose or xylose. In coculture, Z. anaerobia converted most of the glucose to ethanol during the lag phase of growth of C. saccharolyticum, which then converted cellobiose and xylose to ethanol. The use of this coculture increased both the rate and the efficiency of the conversion of these three sugars to ethanol, and produced relatively small amounts of acetic acid.     

Studies on choanephoraceae. VI. The utilization of mono- and oligosaccharides

Summary The utilization of some mono- and oligosaccharides by the members of Choanephoraceae has been studied in detail. The filtrate was analysed by using circular paper chromatography. Amongst the seven monosaccharides tested, viz., glucose, galactose, fructose, mannose, xylose, sorbose and rhamnose, the first five were completely utilized within the specified period, while sorbose and rhamnose remained in the medium throughout the incubation period. A mixture of glucose, galactose and fructose was found to support better growth of all the present species, than that when these sugars were supplied singly. Out of the four oligosaccharides tested, only maltose could be hydrolysed, and it was completely consumed within the specified period. The other three oligosaccharides, viz., sucrose, lactose and raffinose were not hydrolysed and they remained in the medium throughout the incubation period.     

Sugar utilization by yeast during fermentation

Summary When glucose and fructose are fermented separately, the uptake profiles indicate that both sugars are utilized at similar rates. However, when fermentations are conducted in media containing an equal concentration of glucose and fructose, glucose is utilized at approximately twice the rate of fructose. The preferential uptake of glucose also occurred when sucrose, which was first rapidly hydrolyzed into glucose and fructose by the action of the enzyme invertase, was employed as a substrate. Similar results were observed in the fermentation of brewer's wort and wort containing 30% sucrose and 30% glucose as adjuncts. In addition, the high levels of glucose in the wort exerted severe catabolite repression on maltose utilization in theSaccharmyces uvarum (carlsbergensis) brewing strain. Kinetic analysis of glucose and fructose uptake inSaccharomyces cerevisiae revealed aK _m of 1.6 mM for glucose and 20 mM for fructose. Thus, the yeast strain has a higher affinity for glucose than fructose. Growth on glucose or fructose had no repressible effect on the uptake of either sugar. In addition, glucose inhibited fructose uptake by 60% and likewise fructose inhibited, glucose uptake by 40%. These results indicate that glucose and fructose share the same membrane transport components.     

Comparison of carbohydrate substrate preferences in eight species of bifidobacteria

Eight species of bifidobacteria were tested for their abilities to grow on a range of monosaccharides (glucose, arabinose, xylose, galactose and mannose). In contrast to the other sugars, glucose and galactose were utilized by all species and, in general, specific growth rates were highest on these sugars. Different substrate preferences were observed between species when the bacteria were grown in the presence of all five monosaccharides. For example, glucose and xylose were coutilized by Bifidobacterium longum, whereas glucose repressed uptake of all other sugars in B. bifidum and B. catenulatum. Galactose was the preferred substrate with B. pseudolongum. In B. angulatum, glucose and galactose were utilized simultaneously. B. breve did not grow on arabinose when this sugar provided the sole source of energy. However, glucose and arabinose were preferentially taken up during growth on sugar mixtures.     

Regulation of the synthesis of M protein by sugars, Todd Hewitt broth, and horse serum, in growing cells of Streptococcus pyogenes.

Various sugars were tested for their effect on the differential rate of synthesis of M protein during the growth of Streptococcus pyogenes strain 0055 M12T12. In a semisynthetic medium alone, a high rate of M protein synthesis occurred with glucose as a substrate; decreasing rates of synthesis occurred with sucrose and trehalose, in that order, although the rates of growth were approximately equal with all sugars. A period of derepressed synthesis of M protein occurred in the lag phase of growth and in the stationary period as the substrates were being depleted. Although glucose inhibited the utilization of other sugars, diauxie was not apparent from the growth curves. However, synthesis of M protein followed strong diauxie curves with a reduction in rate of synthesis during the utilization of the second sugar. With glucose as a substrate, 2-deoxyglucose showed a strong permanent repression of M protein synthesis, whereas both glucose and 2-deoxyglucose caused temporary repression when sucrose was the substrate. Horse serum increased the rate of synthesis of M protein in a manner very similar to that caused by adding cyclic AMP, although quantitative analyses suggested that cyclic AMP, per se, was not the effector in horse serum. Addition of Todd Hewitt broth permitted the organisms to grow on phosphorylated sugars. Although the rates of growth on phosphorylated sugars were similar to that obtained with glucose, M protein was not synthesized when a phosphorylated sugar was the sole substrate. The addition of phosphorylated sugars with glucose or sucrose as substrates strongly repressed the synthesis of M protein with glucose-1-phosphate and with fructose 1,6-diphosphate repressing M protein synthesis the most. Clearly, M protein synthesis, which was not required for growth, was preferentially induced by glucose as compared to the other sugars and was dependent upon the metabolic route by which glucose was utilized.     

Catabolite inhibition: a general phenomenon in the control of carbohydrate utilization

When Escherichia coli is grown in synthetic medium with radioactive galactose or lactose as the carbon source, the addition of glucose rapidly inhibited utilization of the radioactive substrate, whether the formation of (14)CO(2) or acid-insoluble products was measured. The inhibition was reversed after the removal of glucose. Experiments with mutants blocked in subsequent steps of galactose and lactose metabolism demonstrated that the inhibition occurs prior to the formation of the first metabolic product. The utilization of a variety of sugars, including maltose, lactose, mannose, galactose, l-arabinose, xylose, and glycerol was inhibited by glucose. Of a number of carbohydrates tested as potential inhibitors, only glucose and, to a lesser extent, glucose-6-phosphate (G-6-P) were capable of inhibiting the utilization of all of the substrates. Glucose did not inhibit G-6-P utilization but G-6-P inhibited glucose utilization. With all substrates, except glycerol, there was a delay before the onset of inhibition by G-6-P. We conclude that E. coli has a general regulatory mechanism, termed catabolite inhibition, which controls the activity of early reactions in carbohydrate metabolism, allowing certain substrates to be utilized preferentially.     

Anaerobic Growth and Fermentation Characteristics of Paecilomyces lilacinus Isolated from Mullet Gut

The anaerobic growth and fermentation of a marine isolate of Paecilomyces lilacinus is described. The fungus was isolated from mullet gut and grew optimally at 30°C and at a salinity of ≥10%. The best growth was obtained with glucose or laminarin as substrate, and the growth yield was 5.0 g (dry weight of fungus) per mol of hexose fermented. Moles of products as a percentage of moles of hexose fermented were acetate, 29.0%; ethanol, 156.6%; CO₂, 108.0%; and lactate, 4.3%. Together these products accounted for >80% of hexose carbon. Hydrogen and formate were not detectable as fermentation end products (<0.5%). Other substrates utilized for growth, although less effectively than laminarin or glucose, included the monosaccharides galactose, fructose, arabinose, and xylose and the disaccharides maltose and cellobiose. No growth of the fungus occurred on cellulose, and of a variety of other polysaccharides tested only xylan supported growth.     

Various Hexoses and di-hexoses Differently Influence Growth,Morphology and Pigment Synthesis in Transformed Root Cultures of Red Beet (Beta vulgaris)

Red beet hairy root cultures, obtained after genetic transformation with Agrobacterium rhizogenes, are completely heterotrophic and synthesize betalaines (BNs). Upon subjecting the hairy roots to treatments containing different sugars (3% w/v) it was found that sucrose was rapidly utilized, followed by maltose, and a very limited use of glucose, but the other hexoses – fructose, lactose, xylose and galactose or glycerol totally suppressed both growth and BN synthesis. No habituation or adaptability to maltose or glucose occurred, evidenced by the lack of growth upon re-culture in respective medium. Glycerol, was not taken up alone, but was utilized to a considerable extent in the presence of low levels of sucrose for growth only but not BN synthesis. Red beet hairy root culture did not exogenously hydrolyse sucrose to hexoses, as there were only traces of reducing sugar present in the medium soon after inoculation, without an increase later, confirmed by HPLC. There was an increase in medium osmolarity in the presence of fructose indicating the exudation of certain compounds from the roots. Red beet hairy roots appear useful as a model system to study sugar metabolism/signalling due to their sensitivity to different sugars that may directly link to morphological changes and BN synthesis.     

Carbon catabolite repression of invertase during batch cultivations of Saccharomyces cerevisiae: the role of glucose, fructose, and mannose

When Saccharomyces cerevisiae are grown on a mixture of glucose and another fermentable sugar such as sucrose, maltose or galactose, the metabolism is diauxic, i.e. glucose is metabolized first, whereas the other sugars are metabolized when glucose is exhausted. This phenomenon is a consequence of glucose repression, or more generally, catabolite repression. Besides glucose, the hexoses fructose and mannose are generally also believed to trigger catabolite repression. In this study, batch fermentations of S. cerevisiae in mixtures of sucrose and either glucose, fructose or mannose were performed. It was found that the utilization of sucrose is inhibited by concentrations of either glucose or fructose higher than 5 g/l, and thus that glucose and fructose are equally capable of exerting catabolite repression. However, sucrose was found to be hydrolyzed to glucose and fructose, even when the mannose concentration was as high as 17 g/l, indicating, that mannose is not a repressing sugar. It is suggested that the capability to trigger catabolite repression is connected to hexokinase PII, which is involved in the in vivo phosphorylation of glucose and fructose. Received: 5 May 1998 / Received revision: 3 August 1998 / Accepted: 8 August 1998     

Energy utilization for polysaccharide synthesis by mixed rumen organisms fermenting soluble carbohydrates

Synthesis of reserve polysaccharide by mixed rumen organisms fermenting glucose, maltose, cellobiose, and xylose has been studied in relation to the adenosine triphosphate energy calculated to be available from substrate fermentation. About 80% of the energy available from glucose and xylose was used for polysaccharide synthesis, whereas, assuming hydrolytic cleavage of the disaccharides, more than 100% was used when cellobiose and maltose were the substrates. If, however, phosphorolytic cleavage of the disaccharides, for which there is evidence, was involved, the energy from both maltose and cellobiose fermentation was used with about the same efficiency as that from glucose and xylose fermentation. The rumen fluid used was collected 24 hr after feeding, and growth of microorganisms in such samples was sufficient to account for utilization of less than 10% of the total energy becoming available during the 40-min incubation period.     

Overlapping substrate specificity for sucrose and maltose of two binding protein-dependent sugar uptake systems in Streptococcus mutans

Sugar metabolism by Streptococcus mutans is associated with tooth decay. The most abundant sugars in the human diet are sucrose and maltose, a derivative of starch. Previously, we reported a binding protein-dependent transport system (msm) in S. mutans that transports sucrose and maltose, but its associated enzymes do not metabolize maltose. By searching the S. mutans genomic sequence for a maltose system (mal), we found a gene cluster encoding proteins with homology to those of msm and the Escherichia coli maltose system. Mutants were constructed by deleting msm or mal, or both, and tested for sugar utilization. Deletion of the mal system diminished the ability of S. mutans to ferment maltose, but deletion of only the mal transporter genes or msm showed reduced utilization of chromogenic maltosides. Maltose, sucrose, glucose, fructose, mannose, and N-acetyl glucosamine inhibited utilization of chromogenic maltosides by the wild-type strain and mutants. In conclusion, the two binding protein-dependent systems in S. mutans appear to transport collaboratively their common substrate sugars, notably sucrose and maltose.     

The transport and mediation mechanisms of the common sugars in Escherichia coli

Escherichia coli can uptake and utilize many common natural sugars to form biomass or valuable target bio-products. Carbon catabolite repression (CCR) will occur and hamper the efficient production of bio-products if E. coli strains are cultivated in a mixture of sugars containing some preferred sugar, such as glucose. Understanding the transport and metabolism mechanisms of the common and inexpensive sugars in E. coli is important for further improving the efficiency of sugar bioconversion and for reducing industrial fermentation costs using the methods of metabolic engineering, synthetic biology and systems biology. In this review, the transport and mediation mechanisms of glucose, fructose, sucrose, xylose and arabinose are discussed and summarized, and the hierarchical utilization principles of these sugars are elucidated.     

Utilization of cellobiose and D-glucose by Clostridium thermocellum ATCC-27405

Cultures of Clostridium thermocellum ATCC-27405, maintained on cellulose and not adapted to grow on glucose utilize cellobiose preferentially over D-glucose, and are only able to initiate growth on D-glucose when the cellobiose has been exhausted from the growth medium. However, D-glucose is the carbon source preferentially utilized when cultures of this microorganism, previously adapted for growth on glucose, are transferred to a medium with equivalent concentrations of both sugars. One reason for the preferential utilization of glucose over that of cellobiose might be the competitive inhibition of cellobiose phosphorylase by intracellular glucose accumulation. When in the glucose-adapted cultures the pressure to grow on glucose as the sole carbon source is again released, both sugars can be simultaneously utilized.     

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.