Fermentation of glucose and xylose in ruminal strains of Butyrivibrio fibrisolvens

Metabolism of glucose and xylose and parameters of growth were investigated in strains of Butyrivibrio fibrisolvens ATCC 19171 and CE 51. In the strain ATCC 19171, the composition of fermentation end-products was the same in cultures supplied with glucose and xylose. The strain CE 51 produced more volatile fatty acids and less lactate from xylose than from glucose. Cells of this strain grown on xylose possessed phosphoketolase activity (EC 4.1.2.9). In both strains the production of cell dry matter and growth rate were higher in cultures supplied with glucose. In xylose-grown cultures butyrivibrios tended to convert more substrate carbon into metabolites and less into cellular material than in cultures grown on glucose.     

Diauxic growth of Clostridium thermosaccharolyticum on glucose and xylose

Abstract Clostridium thermosaccharolyticum growing on medium containing glucose and xylose exhibits classical diauxic growth in which glucose is utilized during the first phase. The lag period between growth phases is associated with induction of synthesis of a xylose transport system together with the enzymes xylose isomerase and xylukokinase. Xylose metabolism by this organism is therefore shown to be inducible and subject to repression by glucose. Xylose utilization by cells adapted to this carbon source is also prevented immediately upon addition of glucose to the culture, suggesting a direct inhibitory effect of glucose on xylose uptake or metabolism.     

Correlation between depression of catabolite control of xylose metabolism and a defect in the phosphoenolpyruvate:mannose phosphotransferase system in Pediococcus halophilus.

Pediococcus halophilus X-160 which lacks catabolite control by glucose was isolated from nature (soy moromi mash). Wild-type strains, in xylose-glucose medium, utilized glucose preferentially over xylose and showed diauxic growth. With wild-type strain I-13, xylose isomerase activity was not induced until glucose was consumed from the medium. Strain X-160, however, utilized xylose concurrently with glucose and did not show diauxic growth. In this strain, xylose isomerase was induced even in the presence of glucose. Glucose transport activity in intact cells of strain X-160 was less than 10% of that assayed in strain I-13. Determinations of glycolytic enzymes did not show any difference responsible for the unique behavior of strain X-160, but the rate of glucose-6-phosphate formation with phosphoenolpyruvate (PEP) as a phosphoryl donor in permeabilized cells was less than 10% of that observed in the wild type. Starved P. halophilus I-13 cells contained the glycolytic intermediates 3-phosphoglycerate, 2-phosphoglycerate, and PEP (PEP pool). These were consumed concomitantly with glucose or 2-deoxyglucose uptake but were not consumed with xylose uptake. The glucose transport system in P. halophilus was identified as a PEP:mannose phosphotransferase system on the basis of the substrate specificity of PEP pool-starved cells. It is concluded that, in P. halophilus, this system is functional as a main glucose transport system and that defects in this system may be responsible for the depression of glucose-mediated catabolite control.     

Evolutionary engineering of Saccharomyces cerevisiae for anaerobic growth on xylose

Xylose utilization is of commercial interest for efficient conversion of abundant plant material to ethanol. Perhaps the most important ethanol-producing organism, Saccharomyces cerevisiae, however, is incapable of xylose utilization. While S. cerevisiae strains have been metabolically engineered to utilize xylose, none of the recombinant strains or any other naturally occurring yeast has been able to grow anaerobically on xylose. Starting with the recombinant S. cerevisiae strain TMB3001 that overexpresses the xylose utilization pathway from Pichia stipitis, in this study we developed a selection procedure for the evolution of strains that are capable of anaerobic growth on xylose alone. Selection was successful only when organisms were first selected for efficient aerobic growth on xylose alone and then slowly adapted to microaerobic conditions and finally anaerobic conditions, which indicated that multiple mutations were necessary. After a total of 460 generations or 266 days of selection, the culture reproduced stably under anaerobic conditions on xylose and consisted primarily of two subpopulations with distinct phenotypes. Clones in the larger subpopulation grew anaerobically on xylose and utilized both xylose and glucose simultaneously in batch culture, but they exhibited impaired growth on glucose. Surprisingly, clones in the smaller subpopulation were incapable of anaerobic growth on xylose. However, as a consequence of their improved xylose catabolism, these clones produced up to 19% more ethanol than the parental TMB3001 strain produced under process-like conditions from a mixture of glucose and xylose.     

Evolutionary Engineering of Saccharomyces cerevisiae for Anaerobic Growth on Xylose

Xylose utilization is of commercial interest for efficient conversion of abundant plant material to ethanol. Perhaps the most important ethanol-producing organism, Saccharomyces cerevisiae, however, is incapable of xylose utilization. While S. cerevisiae strains have been metabolically engineered to utilize xylose, none of the recombinant strains or any other naturally occurring yeast has been able to grow anaerobically on xylose. Starting with the recombinant S. cerevisiae strain TMB3001 that overexpresses the xylose utilization pathway from Pichia stipitis, in this study we developed a selection procedure for the evolution of strains that are capable of anaerobic growth on xylose alone. Selection was successful only when organisms were first selected for efficient aerobic growth on xylose alone and then slowly adapted to microaerobic conditions and finally anaerobic conditions, which indicated that multiple mutations were necessary. After a total of 460 generations or 266 days of selection, the culture reproduced stably under anaerobic conditions on xylose and consisted primarily of two subpopulations with distinct phenotypes. Clones in the larger subpopulation grew anaerobically on xylose and utilized both xylose and glucose simultaneously in batch culture, but they exhibited impaired growth on glucose. Surprisingly, clones in the smaller subpopulation were incapable of anaerobic growth on xylose. However, as a consequence of their improved xylose catabolism, these clones produced up to 19% more ethanol than the parental TMB3001 strain produced under process-like conditions from a mixture of glucose and xylose.     

Investigation of ptsG gene in response to xylose utilization in Corynebacterium glutamicum

Corynebacterium glutamicum strains NC-2 were able to grow on xylose as sole carbon sources in our previous work. Nevertheless, it exhibited the major shortcoming that the xylose consumption was repressed in the presence of glucose. So far, regarding C. glutamicum, there are a number of reports on ptsG gene, the glucose-specific transporter, involved in glucose metabolism. Recently, we found ptsG had influence on xylose utilization and investigated the ptsG gene in response to xylose utilization in C. glutamicum with the aim to improve xylose consumption and simultaneously utilized glucose and xylose. The ptsG-deficient mutant could grow on xylose, while exhibiting noticeably reduced growth on xylose as sole carbon source. A mutant deficient in ptsH, a general PTS gene, exhibited a similar phenomenon. When complementing ptsG gene, the mutant ΔptsG-ptsG restored the ability to grow on xylose similarly to NC-2. These indicate that ptsG gene is not only essential for metabolism on glucose but also important in xylose utilization. A ptsG-overexpressing recombinant strain could not accelerate glucose or xylose metabolism. When strains were aerobically cultured in a sugar mixture of glucose and xylose, glucose and xylose could not be utilized simultaneously. Interestingly, the ΔptsG strain could co-utilize glucose and xylose under oxygen-deprived conditions, though the consumption rate of glucose and xylose dramatically declined. It was the first report of ptsG gene in response to xylose utilization in C. glutamicum.     

Xylose and Glucose Utilization by Bacteroides xylanolyticus X5-1 Cells Grown in Batch and Continuous Culture

During cultivation on a mixture of xylose and glucose, Bacteroides xylanolyticus X5-1 showed neither diauxic growth nor a substrate preference. Xylose-limited continuous-culture cells were able to consume xylose and glucose both as single substrates and as mixed substrates without any lag phase. When glucose was the growth-limiting substrate, the microorganism was unable to consume xylose. However, in the presence of a small amount of glucose or pyruvate, xylose was utilized after a short lag phase. In glucose-limited cells, xylose isomerase was present at low activity but xylulose kinase activity could not be detected. On addition of a mixture of xylose and glucose, xylose isomerase was induced immediately and xylulose kinase was induced after about 30 min. The induction of the two enzymes was sensitive to chloramphenicol, showing de novo synthesis. Xylose uptake in glucose-grown cells was very low, but the uptake rate could be increased when incubated with a xylose-glucose mixture. The increase in the uptake rate was not affected by chloramphenicol, indicating that a constitutive uptake system had to be activated. The inability of B. xylanolyticus X5-1 cells undergoing glucose-limited continuous culture to induce the xylose catabolic pathway after the addition of only xylose probably was caused by energy limitation.     

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.     

Fermentation of xylans by Butyrivibrio fibrisolvens and other ruminal bacteria

The ability of Butyrivibrio fibrisolvens and other ruminal bacteria (6 species, 18 strains) to ferment a crude xylan from wheat straw or to ferment xylans from larchwood or oat spelts was studied. Liquid cultures were monitored for carbohydrate utilization, cell growth (protein), and fermentation acid production. B. fibrisolvens 49, H17c, AcTF2, and D1 grew almost as well on one or more of the xylans as they did on cellobiose-maltose. B. fibrisolvens 12, R28, A38, X10C34, ARD22a, and X6C61 exhibited moderate growth on xylans. Partial fermentation of xylans was observed with Bacteroides ruminicola B14, Bacteroides succinogenes S85, Ruminococcus albus 7, Ruminococcus flavefaciens C94 and FD1, and Succinivibrio dextrinosolvens 22B. All xylans tested appeared to have a small fraction of carbohydrate that supported low levels of growth of nonxylanolytic strains such as Selenomonas ruminantium HD4. Compared to growth on hexoses, the same array of fermentation acids was produced upon growth on xylans for most strains; however, reduced lactate levels were observed for B. fibrisolvens 49 and Selenomonas ruminantium HD4. Measurements of enzyme activities of B. fibrisolvens AcTF2, 49, H17c, and D1 indicated that the xylobiase activities were cell associated and that the xylanase activities were predominantly associated with the culture fluid. The pattern of expression of these enzymes varied both between strains and between the carbon sources on which the strains were grown.     

Fermentation of xylans by Butyrivibrio fibrisolvens and other ruminal bacteria.

The ability of Butyrivibrio fibrisolvens and other ruminal bacteria (6 species, 18 strains) to ferment a crude xylan from wheat straw or to ferment xylans from larchwood or oat spelts was studied. Liquid cultures were monitored for carbohydrate utilization, cell growth (protein), and fermentation acid production. B. fibrisolvens 49, H17c, AcTF2, and D1 grew almost as well on one or more of the xylans as they did on cellobiose-maltose. B. fibrisolvens 12, R28, A38, X10C34, ARD22a, and X6C61 exhibited moderate growth on xylans. Partial fermentation of xylans was observed with Bacteroides ruminicola B14, Bacteroides succinogenes S85, Ruminococcus albus 7, Ruminococcus flavefaciens C94 and FD1, and Succinivibrio dextrinosolvens 22B. All xylans tested appeared to have a small fraction of carbohydrate that supported low levels of growth of nonxylanolytic strains such as Selenomonas ruminantium HD4. Compared to growth on hexoses, the same array of fermentation acids was produced upon growth on xylans for most strains; however, reduced lactate levels were observed for B. fibrisolvens 49 and Selenomonas ruminantium HD4. Measurements of enzyme activities of B. fibrisolvens AcTF2, 49, H17c, and D1 indicated that the xylobiase activities were cell associated and that the xylanase activities were predominantly associated with the culture fluid. The pattern of expression of these enzymes varied both between strains and between the carbon sources on which the strains were grown.     

Growth and fermentation of an anaerobic rumen fungus on various carbon sources and effect of temperature on development.

An anaerobic fungus (strain R1) resembling Neocallimastix spp. was isolated from sheep rumen. When grown on defined medium, the isolate utilized a wide range of polysaccharides and disaccharides, but of the eight monosaccharides tested only fructose, glucose, and xylose supported growth. The organism had doubling times of 5.56 h on glucose and 6.67 h on xylose, and in each case fermentation resulted in production of formate, acetate, lactate, and ethanol. During active growth, formate was a reliable indicator of fungal biomass. Growth on a medium containing glucose and xylose resulted in a doubling time of 8.70 h, but diauxic growth did not occur since both sugars were utilized simultaneously. The optimum temperature for zoospore and immature plant development was 39 degrees C, and no development occurred below 33 degrees C or above 41 degrees C.     

Repression of xylose-specific enzymes by ethanol in Scheffersomyces (Pichia) stipitis and utility of repitching xylose-grown populations to eliminate diauxic lag

During the fermentation of lignocellulosic hydrolyzates to ethanol by native pentose-fermenting yeasts such as Scheffersomyces (Pichia) stipitis NRRL Y-7124 (CBS 5773) and Pachysolen tannophilus NRRL Y-2460, the switch from glucose to xylose uptake results in a diauxic lag unless process strategies to prevent this are applied. When yeast were grown on glucose and resuspended in mixed sugars, the length of this lag was observed to be a function of the glucose concentration consumed (and consequently, the ethanol concentration accumulated) prior to the switch from glucose to xylose fermentation. At glucose concentrations of 95 g/L, the switch to xylose utilization was severely stalled such that efficient xylose fermentation could not occur. Further investigation focused on the impact of ethanol on cellular xylose transport and the induction and maintenance of xylose reductase and xylitol dehydrogenase activities when large cell populations of S. stipitis NRRL Y-7124 were pre-grown on glucose or xylose and then presented mixtures of glucose and xylose for fermentation. Ethanol concentrations around 50 g/L fully repressed enzyme induction although xylose transport into the cells was observed to be occurring. Increasing degrees of repression were documented between 15 and 45 g/L ethanol. Repitched cell populations grown on xylose resulted in faster fermentation rates, particularly on xylose but also on glucose, and eliminated diauxic lag and stalling during mixed sugar conversion by P. tannophilus or S. stipitis, despite ethanol accumulations in the 60 or 70 g/L range, respectively. The process strategy of priming cells on xylose was key to the successful utilization of high mixed sugar concentrations because specific enzymes for xylose utilization could be induced before ethanol concentration accumulated to an inhibitory level.     

Effects of several mycotoxins on specific growth rate of Butyrivibrio fibrisolvens and toxin degradation in vitro

Four strains of Butyrivibrio fibrisolvens did not degrade aflatoxin B1. Acetyl T-2 toxin, T-2 toxin, HT-2 toxin, deoxynivalenol, diacetoxyscirpenol, verrucarin A, zearalenone, and ochratoxin A did not affect the specific growth rate of B. fibrisolvens CE51 significantly, but all were degraded to greater or lesser extents. Breakdown products were produced as a result of deacetylation reactions.     

Effects of several mycotoxins on specific growth rate of Butyrivibrio fibrisolvens and toxin degradation in vitro.

Four strains of Butyrivibrio fibrisolvens did not degrade aflatoxin B1. Acetyl T-2 toxin, T-2 toxin, HT-2 toxin, deoxynivalenol, diacetoxyscirpenol, verrucarin A, zearalenone, and ochratoxin A did not affect the specific growth rate of B. fibrisolvens CE51 significantly, but all were degraded to greater or lesser extents. Breakdown products were produced as a result of deacetylation reactions.     

Expression of a heterologous xylose transporter in a <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain engineered to utilize xylose improves aerobic xylose consumption

The goal of this investigation was to determine the effect of a xylose transport system on glucose and xylose co-consumption as well as total xylose consumption in Saccharomyces cerevisiae. We expressed two heterologous transporters from Arabidopsis thaliana in recombinant xylose-utilizing S. cerevisiae cells. Strains expressing the heterologous transporters were grown on glucose and xylose mixtures. Sugar consumption rates and ethanol concentrations were determined and compared to an isogenic control strain lacking the A. thaliana transporters. Expression of the transporters increased xylose uptake and xylose consumption up to 46% and 40%, respectively. Xylose co-consumption rates (prior to glucose depletion) were also increased by up to 2.5-fold compared to the control strain. Increased xylose consumption correlated with increased ethanol concentration and productivity. During the xylose/glucose co-consumption phase, strains expressing the transporters had up to a 70% increase in ethanol production rate. It was concluded that in these strains, xylose transport was a limiting factor for xylose utilization and that increasing xylose/glucose co-consumption is a viable strategy for improving xylose fermentation.     

Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain

We have recently reported about a Saccharomyces cerevisiae strain that, in addition to the Piromyces XylA xylose isomerase gene, overexpresses the native genes for the conversion of xylulose to glycolytic intermediates. This engineered strain (RWB 217) exhibited unprecedentedly high specific growth rates and ethanol production rates under anaerobic conditions with xylose as the sole carbon source. However, when RWB 217 was grown on glucose-xylose mixtures, a diauxic growth pattern was observed with a relatively slow consumption of xylose in the second growth phase. After prolonged cultivation in an anaerobic, xylose-limited chemostat, a culture with improved xylose uptake kinetics was obtained. This culture also exhibited improved xylose consumption in glucose-xylose mixtures. A further improvement in mixed-sugar utilization was obtained by prolonged anaerobic cultivation in automated sequencing-batch reactors on glucose-xylose mixtures. A final single-strain isolate (RWB 218) rapidly consumed glucose-xylose mixtures anaerobically, in synthetic medium, with a specific rate of xylose consumption exceeding 0.9 gg(-1)h(-1). When the kinetics of zero trans-influx of glucose and xylose of RWB 218 were compared to that of the initial strain, a twofold higher capacity (V(max)) as well as an improved K(m) for xylose was apparent in the selected strain. It is concluded that the kinetics of xylose fermentation are no longer a bottleneck in the industrial production of bioethanol with yeast.     

Cloning of cellobiose phosphoenolpyruvate-dependent phosphotransferase genes: functional expression in recombinant Escherichia coli and identification of a putative binding region for disaccharides.

Genomic libraries from nine cellobiose-metabolizing bacteria were screened for cellobiose utilization. Positive clones were recovered from six libraries, all of which encode phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) proteins. Clones from Bacillus subtilis, Butyrivibrio fibrisolvens, and Klebsiella oxytoca allowed the growth of recombinant Escherichia coli in cellobiose-M9 minimal medium. The K. oxytoca clone, pLOI1906, exhibited an unusually broad substrate range (cellobiose, arbutin, salicin, and methylumbelliferyl derivatives of glucose, cellobiose, mannose, and xylose) and was sequenced. The insert in this plasmid encoded the carboxy-terminal region of a putative regulatory protein, cellobiose permease (single polypeptide), and phospho-beta-glucosidase, which appear to form an operon (casRAB). Subclones allowed both casA and casB to be expressed independently, as evidenced by in vitro complementation. An analysis of the translated sequences from the EIIC domains of cellobiose, aryl-beta-glucoside, and other disaccharide permeases allowed the identification of a 50-amino-acid conserved region. A disaccharide consensus sequence is proposed for the most conserved segment (13 amino acids), which may represent part of the EIIC active site for binding and phosphorylation.     

Absence of diauxie during simultaneous utilization of glucose and Xylose by Sulfolobus acidocaldarius

Sulfolobus acidocaldarius utilizes glucose and xylose as sole carbon sources, but its ability to metabolize these sugars simultaneously is not known. We report the absence of diauxie during growth of S. acidocaldarius on glucose and xylose as co-carbon sources. The presence of glucose did not repress xylose utilization. The organism utilized a mixture of 1 g/liter of each sugar simultaneously with a specific growth rate of 0.079 h(-1) and showed no preference for the order in which it utilized each sugar. The organism grew faster on 2 g/liter xylose (0.074 h(-1)) as the sole carbon source than on an equal amount of glucose (0.022 h(-1)). When grown on a mixture of the two carbon sources, the growth rate of the organism increased from 0.052 h(-1) to 0.085 h(-1) as the ratio of xylose to glucose increased from 0.25 to 4. S. acidocaldarius appeared to utilize a mixture of glucose and xylose at a rate roughly proportional to their concentrations in the medium, resulting in complete utilization of both sugars at about the same time. Gene expression in cells grown on xylose alone was very similar to that in cells grown on a mixture of xylose and glucose and substantially different from that in cells grown on glucose alone. The mechanism by which the organism utilized a mixture of sugars has yet to be elucidated.     

Interactions between rumen bacterial strains during the degradation and utilization of the monosaccharides of barley straw cell-walls

Pure cultures and pair-combinations of strains representative of the rumen cellulolytic species Ruminococcus flavefaciens, Fibrobacter succinogenes and Butyrivibrio fibrisovens were grown on cell-wall materials from barley straw. Of the pure cultures, R. flavefaciens solubilized straw most rapidly. The presence of B. fibrisolvens , which was unable to degrade straw extensively in pure culture, increased the solubilization of dry matter by R. flavefaciens and the solubilization of cell-wall carbohydrates by both R. flavefaciens and F. succinogenes. During fermentation, both R. flavefaciens and F. succinogenes released bound glucose and free and bound arabinose and xylose into solution. The accumulation of these sugars, especially arabinose and xylose, was greatly reduced in co-cultures containing B. fibrisolvens , suggesting that significant interspecies cross feeding of the products of hemicellulose hydrolysis (particularly soluble bound xylose released by F. succinogenes ) occurs during straw degradation by mixed cultures containing this species.     

Contributions of XylR,CcpA and cre to diauxic growth of Bacillus megaterium and to xylose isomerase expression in the presence of glucose and xylose

Bacillus megaterium shows diauxic growth in minimal medium containing glucose and xylose. We have examined the influence of three elements that regulate xyl operon expression on diauxic growth and expression of a xylA-lacZ fusion. xylA is 13-fold repressed during growth on glucose. Induction occurs at the onset of the lag phase after glucose is consumed. Inactivation of xylR yields a two-fold increase in expression of xylA on glucose. Deletion of the catabolite responsive element (cre) has a more pronounced effect, reducing glucose repression from 13-fold in the wild type to about 2.5-fold. When xylR and cre are inactivated together a residual two-fold repression of xylA is found. Inactivation of xylR affects diauxic growth by shortening the lag phase from 70 to 40?min. In-frame deletion of ccpA results in the loss of diauxic growth, an increase in doubling time and simultaneous use of both sugars. In contrast, a strain with an inactivated cre site in xylA exhibits diauxic growth without an apparent lag phase on glucose and xylose, whereas fructose and xylose are consumed simultaneously.