Utilization Of Mixed Sugars Research Articles

Reciprocal Regulation of l-Arabinose and d-Xylose Metabolism in Escherichia coli.

Glucose is known to inhibit the transport and metabolism of many sugars in Escherichia coli. This mechanism leads to its preferential consumption. Far less is known about the preferential utilization of nonglucose sugars in E. coli. Two exceptions are l-arabinose and d-xylose. Previous studies have shown that l-arabinose inhibits d-xylose metabolism in Escherichia coli. This repression results from l-arabinose-bound AraC binding to the promoter of the d-xylose metabolic genes and inhibiting their expression. This mechanism, however, has not been explored in single cells. Both the l-arabinose and d-xylose utilization systems are known to exhibit a bimodal induction response to their cognate sugar, where mixed populations of cells either expressing the metabolic genes or not are observed at intermediate sugar concentrations. This suggests that l-arabinose can only inhibit d-xylose metabolism in l-arabinose-induced cells. To understand how cross talk between these systems affects their response, we investigated E. coli during growth on mixtures of l-arabinose and d-xylose at single-cell resolution. Our results showed that mixed, multimodal populations of l-arabinose- and d-xylose-induced cells occurred at intermediate sugar concentrations. We also found that d-xylose inhibited the expression of the l-arabinose metabolic genes and that this repression was due to XylR. These results demonstrate that a strict hierarchy does not exist between l-arabinose and d-xylose as previously thought. The results may also aid in the design of E. coli strains capable of simultaneous sugar consumption. Glucose, d-xylose, and l-arabinose are the most abundant sugars in plant biomass. Developing efficient fermentation processes that convert these sugars into chemicals and fuels will require strains capable of coutilizing these sugars. Glucose has long been known to repress the expression of the l-arabinose and d-xylose metabolic genes in Escherichia coli. Recent studies found that l-arabinose also represses the expression of the d-xylose metabolic genes. In the present study, we found that d-xylose also represses the expression of the l-arabinose metabolic genes, leading to mixed populations of cells capable of utilizing l-arabinose and d-xylose. These results further our understanding of mixed-sugar utilization and may aid in strain design.

Alleviation of carbon catabolite repression in Enterobacter aerogenes for efficient utilization of sugarcane molasses for 2,3-butanediol production.

BackgroundDue to its cost-effectiveness and rich sugar composition, sugarcane molasses is considered to be a promising carbon source for biorefinery. However, the sugar mixture in sugarcane molasses is not consumed as efficiently as glucose in microbial fermentation due to complex interactions among their utilizing pathways, such as carbon catabolite repression (CCR). In this study, 2,3-butanediol-producing Enterobacter aerogenes was engineered to alleviate CCR and improve sugar utilization by modulating its carbon preference.ResultsThe gene encoding catabolite repressor/activator (Cra) was deleted in the genome of E. aerogenes to increase the fructose consumption rate. However, the deletion mutation repressed sucrose utilization, resulting in the accumulation of sucrose in the fermentation medium. Cra regulation on expression of the scrAB operon involved in sucrose catabolism was verified by reverse transcription and real-time PCR, and the efficiency of sucrose utilization was restored by disrupting the scrR gene and overexpressing the scrAB operon. In addition, overexpression of the ptsG gene involved in glucose utilization enhanced the glucose preference among mixed sugars, which relieved glucose accumulation in fed-batch fermentation. In fed-batch fermentation using sugarcane molasses, the maximum titer of 2,3-butanediol production by the mutant reached 140.0 g/L at 54 h, which was by far the highest titer of 2,3-butanediol with E. aerogenes achieved through genetic engineering.ConclusionsWe have developed genetically engineered E. aerogenes as a 2,3-butanediol producer that efficiently utilizes sugarcane molasses. The fermentation efficiency was dramatically improved by the alleviation of CCR and modulation of carbon preference. These results offer a metabolic engineering approach for achieving highly efficient utilization of mixed sugars for the biorefinery industry.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0290-3) contains supplementary material, which is available to authorized users.

Co-utilization of glucose, xylose and cellobiose by the oleaginous yeast Cryptococcus curvatus

Simultaneous utilization of mixed sugars is one of the major challenges for biofuel production utilizing lignocellulosic biomass as feedstock. Our previous work proved that the oleaginous yeast Cryptococcus curvatus could efficiently produce lipids, the precursors of hydrocarbons with high energy density, from lignocellulosic hydrolysates. However, the strain's capability of simultaneously utilizing primary sugars was still unknown. Thus, this work comprehensively explored the co-utilization of glucose, xylose and cellobiose by C. curvatus. The results indicated that the consumption of both xylose and cellobiose was repressed by glucose, while xylose and cellobiose could be simultaneously consumed at similar rates. The total sugar consumption rates remained constant at about 0.6 g L−1 h−1 regardless of the sugar composition in the mixtures, and the cell biomass and lipid production by C. curvatus cultured on the different sugar mixtures were similar. Moreover, compared with glucose and xylose, cellobiose could lead to higher dry cell weights and lipid yields, suggesting an excellent carbon source for lipid production. Based on these findings, this study demonstrated an effective approach for alleviating glucose repression for microbial lipid production by C. curvatus through xylose/cellobiose co-utilization which would greatly contribute to a more efficient and economical cellulosic biofuel production.

Insights into acetate toxicity in Zymomonas mobilis 8b using different substrates.

BackgroundLignocellulosic biomass is a promising renewable feedstock for biofuel production. Acetate is one of the major inhibitors liberated from hemicelluloses during hydrolysis. An understanding of the toxic effects of acetate on the fermentation microorganism and the efficient utilization of mixed sugars of glucose and xylose in the presence of hydrolysate inhibitors is crucial for economic biofuel production.ResultsA new microarray was designed including both coding sequences and intergenic regions to investigate the acetate stress responses of Zymomonas mobilis 8b when using single carbon sources of glucose or xylose, or mixed sugars of both glucose and xylose. With the supplementation of exogenous acetate, 8b can utilize all the glucose with a similar ethanol yield, although the growth, final biomass, and ethanol production rate were reduced. However, xylose utilization was inhibited in both media containing xylose or a mixed sugar of glucose and xylose, although the performance of 8b was better in mixed sugar than xylose-only media. The presence of acetate caused genes related to biosynthesis, the flagellar system, and glycolysis to be downregulated, and genes related to stress responses and energy metabolism to be upregulated. Unexpectedly, xylose seems to pose more stress on 8b, recruiting more genes for xylose utilization, than does acetate. Several gene candidates based on transcriptome results were selected for genetic manipulation, and a TonB-dependent receptor knockout mutant was confirmed to have a slight advantage regarding acetate tolerance.ConclusionsOur results indicate Z. mobilis utilized a different mechanism for xylose utilization, with an even more severe impact on Z. mobilis than that caused by acetate treatment. Our study also suggests redox imbalance caused by stressful conditions may trigger a metabolic reaction leading to the accumulation of toxic intermediates such as xylitol, but Z. mobilis manages its carbon and energy metabolism through the control of individual reactions to mitigate the stressful conditions. We have thus provided extensive transcriptomic datasets and gained insights into the molecular responses of Z. mobilis to the inhibitor acetate when grown in different sugar sources, which will facilitate future metabolic modeling studies and strain improvement efforts for better xylose utilization and acetate tolerance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0140-8) contains supplementary material, which is available to authorized users.

Improved xylose and arabinose utilization by an industrial recombinant Saccharomyces cerevisiae strain using evolutionary engineering

BackgroundCost-effective fermentation of lignocellulosic hydrolysate to ethanol by Saccharomyces cerevisiae requires efficient mixed sugar utilization. Notably, the rate and yield of xylose and arabinose co-fermentation to ethanol must be enhanced.ResultsEvolutionary engineering was used to improve the simultaneous conversion of xylose and arabinose to ethanol in a recombinant industrial Saccharomyces cerevisiae strain carrying the heterologous genes for xylose and arabinose utilization pathways integrated in the genome. The evolved strain TMB3130 displayed an increased consumption rate of xylose and arabinose under aerobic and anaerobic conditions. Improved anaerobic ethanol production was achieved at the expense of xylitol and glycerol but arabinose was almost stoichiometrically converted to arabitol. Further characterization of the strain indicated that the selection pressure during prolonged continuous culture in xylose and arabinose medium resulted in the improved transport of xylose and arabinose as well as increased levels of the enzymes from the introduced fungal xylose pathway. No mutation was found in any of the genes from the pentose converting pathways.ConclusionTo the best of our knowledge, this is the first report that characterizes the molecular mechanisms for improved mixed-pentose utilization obtained by evolutionary engineering of a recombinant S. cerevisiae strain. Increased transport of pentoses and increased activities of xylose converting enzymes contributed to the improved phenotype.

Identification and inactivation of pleiotropic regulator CcpA to eliminate glucose repression of xylose utilization in Clostridium acetobutylicum

d-xylose utilization is a key issue for lignocellulosic biomass fermentation, and a major problem in this process is carbon catabolite repression (CCR). In this investigation, solvent-producing bacterium Clostridium acetobutylicum ATCC 824 was metabolically engineered to eliminate d-glucose repression of d-xylose utilization. The ccpA gene, encoding the pleiotropic regulator CcpA, was experimentally characterized and then disrupted . Under pH-controlled conditions, the ccpA-disrupted mutant (824ccpA) can use a mixture of d-xylose and d-glucose simultaneously without CCR. Moreover, this engineered strain produced acetone, butanol and ethanol (ABE) at a maximal titer of 4.94, 12.05 and 1.04 g/L, respectively, which was close to the solvent level of maize- or molasses-based fermentation by wild type C. acetobutylicum. Molar balance analysis for improved process of mixed sugars utilization also revealed less acid accumulation and more butanol yield by the engineered strain as compared to the wild type. This study offers a genetic modification strategy for improving simultaneous utilization of mixed sugars by Clostridium, which is essential for commercial exploitation of lignocellulose for the production of solvents and biofuels.

Engineering of pentose transport in Corynebacterium glutamicum to improve simultaneous utilization of mixed sugars

Corynebacterium glutamicum strains CRA1 and CRX2 are able to grow on L-arabinose and D-xylose, respectively, as sole carbon sources. Nevertheless, they exhibit the major shortcoming that their sugar consumption appreciably declines at lower concentrations of these substrates. To address this, the C. glutamicum ATCC31831 L-arabinose transporter gene, araE, was independently integrated into both strains. Unlike its parental strain, resultant CRA1-araE was able to aerobically grow at low (3.6 g.l(-1)) L-arabinose concentrations. Interestingly, strain CRX2-araE grew 2.9-fold faster than parental CRX2 at low (3.6 g.l(-1)) D-xylose concentrations. The corresponding substrate consumption rates of CRA1-araE and CRX2-araE under oxygen-deprived conditions were 2.8- and 2.7-fold, respectively, higher than those of their respective parental strains. Moreover, CRA1-araE and CRX2-araE utilized their respective substrates simultaneously with D-glucose under both aerobic and oxygen-deprived conditions. Based on these observations, a platform strain, ACX-araE, for C. glutamicum-based mixed sugar utilization was designed. It harbored araBAD for L-arabinose metabolism, xylAB for D-xylose metabolism, D-cellobiose permease-encoding bglF317A, beta-glucosidase-encoding bglA and araE in its chromosomal DNA. In mineral medium containing a sugar mixture of D-glucose, D-xylose, L-arabinose, and D-cellobiose under oxygen-deprived conditions, strain ACX-araE simultaneously and completely consumed all sugars.

A substrate‐selective co‐fermentation strategy with Escherichia coli produces lactate by simultaneously consuming xylose and glucose

We describe a new approach for the simultaneous conversion of xylose and glucose sugar mixtures which potentially could be used for lignocellulosic biomass hydrolysate. In this study we used this approach to demonstrate the production of lactic acid. This process uses two substrate-selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. In addition to knockouts in pflB encoding for pyruvate formate lyase, the xylose-selective (glucose deficient) strain E. coli ALS1073 has deletions of the glk, ptsG, and manZ genes while the glucose-selective (xylose deficient) strain E. coli ALS1074 has a xylA deletion. By combining these two strains in a single process the xylose and glucose in a mixed sugar solution are simultaneously converted to lactate. Furthermore, the biomass concentrations of each strain can readily be adjusted in order to optimize the overall product formation. This approach to the utilization of mixed sugars eliminates the problem of diauxic growth, and provides great operational flexibility.

Developing a thermophilic hydrogen-producing co-culture for efficient utilization of mixed sugars

Previous studies on the extreme thermophile Caldicellulosiruptor saccharolyticus revealed that the organism produces high yields of hydrogen on glucose and xylose, the major components of lignocellulosic hydrolysates. Preliminary experiments on mixed sugar substrates, however, indicated that xylose was preferred over glucose. The sugar preference of some other extreme thermophiles, including Caldicellulosiruptor owensensis, Caldicellulosiruptor kristjanssonii and newly enriched, thermophilic compost sludge microflora, was investigated in an attempt to find complementary organisms to C. saccharolyticus for rapid and efficient utilization of lignocellulosic sugars. The behavior of C. owensensis and C. kristjanssonii appeared to be similar to that of C. saccharolyticus, either in pure cultures or in co-cultures with the latter. Co-culturing C. saccharolyticus with the enriched compost microflora resulted in fast, simultaneous consumption of both glucose and xylose in the medium with a relatively high specific hydrogen production rate, 40 mmol 1/(gCDW).h, and high volumetric productivity, 22.5 mmol/l.h. (Less)

Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting 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.

Use of catabolite repression mutants for fermentation of sugar mixtures to ethanol

Use of agricultural biomass, other than corn-starch, to produce fuel ethanol requires a microorganism that can ferment the mixture of sugars derived from hemicellulose. Escherichia coli metabolizes a wide range of substrates and has been engineered to produce ethanol in high yield from sugar mixtures. E. coli metabolizes glucose in preference to other sugars and, as a result, utilization of the pentoses in hemicellulose-derived sugar mixtures is delayed and may be incomplete. Residual sugar lowers the ethanol yield and is problematic for downstream processing of fermentation products. Therefore, a catabolite repression mutant that simultaneously utilizes glucose and pentoses would be useful for fermentation of complex substrate mixtures. We constructed ethanologenic E. coli strains with a glucose phosphotransferase (ptsG) mutation and used the mutants to ferment glucose, arabinose, and xylose, singly and in mixtures, to ethanol. Yields were 87-94% of theoretical for both the wild type and mutants, but the mutants had an altered pattern of mixed sugar utilization. Phosphotransferase mutants metabolized the pentoses simultaneously with glucose, rather than sequentially. Based upon fermentations of sugar mixtures, a catabolite-repression mutant of ethanologenic E. coli is expected to provide more efficient fermentation of hemicellulose hydrolysates by allowing direct utilization of pentoses.

Induction of Xylose Reductase and Xylitol Dehydrogenase Activities on Mixed Sugars in Candida guilliermondii

The ability of various sugars to induce xylose reductase (aldose reductase, EC 1.1.1.21) and xylitol dehydrogenase (D-xylulose reductase, EC 1.1.1.9) activities in Candida guilliermondii was studied. D-Xylose was found to be the best inducer of activities of both enzymes, followed closely by L-arabinose. Very low xylose reductase activity was induced by cellobiose, D-mannose, D-glucose, D-galactose, D-fructose or glycerol. With xylitol dehydrogenase, cellobiose and D-fructose caused partial induction of enzyme activity, while negligible activity was induced by D-mannose, D-glucose, D-galactose or glycerol. Several sugars were tested for the ability to repress the induction by D-xylose of xylose reductase and xylitol dehydrogenase activities in C. guilliermondii. Enzyme activities induced on D-xylose served as controls. L-Arabinose, cellobiose and D-galactose did not repress enzyme induction by D-xylose, while D-mannose, D-glucose and D-fructose repressed enzyme induction to varying extents. Results from enzyme induction generally correlated with patterns of mixed sugar utilization, with some anomalies associated with the utilization of D-galactose and D-fructose in the presence of D-xylose. The results show that the utilization of D-xylose by C. guilliermondii is subject to regulation by induction and catabolite repression.

Correlation of lags in the utilization of mixed sugars in continuous fermentation.

Correlation of lags in the utilization of mixed sugars in continuous fermentation.

The utilization of mixed sugars in continuous fermentation. I.

AbstractA strain of Klebsiella aerogenes was selected which gave marked diauxic growth in a batch system on a mixture of glucose and lactose in a simple salts medium; the diauxic lag was 15–20 hr. at 30°C. The growth of this organism on glucose and lactose was studied in a single‐stream two‐stage continuous‐stirred fermentor system over a wide range of flow rates. Glucose was metabolized instantaneously to give very low reactor concentrations at all flow rates, but the time lag before lactose was attacked, when present for the first time, was never less than 40 hr. at low feed rates, rising to 60 hr. at higher rates. The adaptation to lactose of cells in the first vessel lagged behind that in the second vessel but eventually both sugars were completely utilized in the first vessel except at very high dilution rates. At these feed rates, lactose utilization was not only prevented completely in the first vessel but also could be delayed almost indefinitely in the second vessel at the highest dilution rates; thus the lactose passed unchanged through both vessels. Once the enzymes required for lactose utilization had been induced, this ability to use lactose was retained, even in the absence of lactose, for very long periods of time under continuous conditions. Thus on presenting lactose for the second and subsequent occasions it was immediately metabolized. The significance of these results is discussed.

Utilization of mixed sugars in continuous fermentation. II.

AbstractBatch growth characteristics of various organisms were determined on a number of pairs of sugars to find a stable system showing clear‐cut classical diauxie. The system selected for further study was a strain of Klebsiella (Acrobacter) aerogenes, NCIB 8021 growing on a mixture of glucose and maltose in minimal salts medium at 30°C. This showed a specific growth rate (μ) of 1.19 ± 0.03 hr.−1 on 0.01% (w/v) glucose, followed by a diauxie lag of 0.73 ± 0.04 hr and then further growth on 0.01% (w/v) maltose at μ = 0.60 ± 0.03 hr−1. This system was applied to a two‐stage continuous, stirred, aerated fermentor system, with working volumes of 1.85 and 2.77 liters, respectively, and growth was followed (mainly by optical density, referred to dry weights and viable counts) and also the concentrations of the sugars were measured. Except at the very highest flow rates, glucose was immediately and virtually completely consumed, but the utilization of maltose showed interesting variations: (a) At low feed rates between 0.09 and 0.4 vol./hr. exactly the same response was found with mixed sugars as with double concentration glucose, showing that the organism was able to metabolize maltose as well and as quickly as glucose. (b) At medium feed rates of 0.46 to 1.03 vol./hr. two deviations were observed, both of which increased as the dilution rate increased: the system showed a time lag on maltose before the cell population began to rise and the volume of medium used before the steady state was established was greater than predicted, (c) At fast feed rates, approaching “washout” condition of 1.055 to 1.135 vol./hr. the first culture vessel showed no reaction to a step change which included maltose, although, of course, with doubled glucose it responded immediately. The second vessel, however, quickly metabolized the overflow maltose, and showed a steady increase of cell population to the theoretical steady state. These results may have significance for industrial systems using complex commercial substrates.