Improvement of xylose utilization in Clostridium acetobutylicum via expression of the talA gene encoding transaldolase from Escherichia coli

Clostridium acetobutylicum ATCC 824 was metabolically engineered for improved xylose utilization. The gene talA, which encodes transaldolase from Escherichia coli K-12, was cloned and overexpressed in C. acetobutylicum ATCC 824. Compared with C. acetobutylicum ATCC 824 (824-WT), the transformant bearing the E. coli talA gene (824-TAL) showed improved ability on xylose utilization and solvents production using xylose as the sole carbon source. During the fermentation of xylose and glucose mixtures with three xylose/glucose ratios (approximately 1:2, 1:1 and 2:1), the rate of xylose consumption and final solvents titers of 824-TAL were all higher than those of 824-WT, despite glucose repression on xylose uptake still existing. These results suggest that the insufficiency of transaldolase in the pentose phosphate pathway (PPP) of C. acetobutylicum is one of the bottlenecks for xylose metabolism and therefore, overexpressing the gene encoding transaldolase is able to improve xylose utilization and solvent production.     

Effect of the carbon source on the carotenoid profiles of Phaffia rhodozyma strains

Phaffia rhodozyma strains ATCC 24202, ATCC 24203, ATCC 24228, ATCC 24229, ATCC 24261, NRRL Y-10921, NRRL Y-10922 and NRRL Y-17268 were grown on culture media containing glucose, sucrose or xylose as carbon sources. Carotenoids were extracted from biomass and analyzed by HPLC with diode-array detection. The carotenoid profiles depended on both the strain considered and the carbon source employed. Astaxanthin, the main pigment found in P. rhodozyma, accounted for 42–91% of total carotenoids. Other carotenoids such as canthaxanthin, echinenone, 3-hydroxyechinenone, lycopene, 4-hydroxy-3′, 4′-didehydro-β-ψ-carotene and phoenicoxanthin were detected. The highest volumetric carotenoid concentration (3.60 mg L⁻¹) was obtained with strain NRRL Y-17268 growing on xylose. In this case, astaxanthin accounted for 82% of total carotenoids. Received 29 May 1997/ Accepted in revised form 08 August 1997     

Oxidative d-xylose metabolism of Gluconobacter oxydans

Summary Gluconobacter oxydans subsp. suboxydans ATCC 621 oxidizes d-xylose to xylonic acid very efficiently, although it cannot grow on xylose as sole carbon source. The oxidation of xylose was found to be catalyzed by a membrane-bound xylose dehydrogenase. The xylono--lactone formed in the oxidation reaction is subsequently hydrolyzed to xylonic acid by a -lactonase. The complete oxidation pathway of d-xylose in G. oxydans is evidently located in the periplasmic space.     

The utilization of metabolic inhibitors for shifting product formation from xylitol to ethanol in pentose fermentations using Candida tropicalis

Summary Xylose, glucose and xylose/glucose mixtures were fermented with Candida tropicalis ATCC 32113 under aerobic, oxygen limited and anaerobic conditions. Ethanol yields were highest under oxygen limited conditions with xylose and xylose/glucose. Anaerobic conditions were best for glucose fermentations.The effect of four metabolic inhibitors (azide, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), oligomycin A and valinomycin-K⁺) were then studied under oxygen limited conditions. Only azide had a significant influence on ethanol production. At 2¢10^-4 M concentrations, ethanol yield increased up to two times and xylitol levels were repressed by 90% for xylose and glucose/xylose fermentations. 4.2×10^-3 M azide gave highest ethanol yields in glucose fermentations. At this concentration of azide, however, cell growth was inhibited, which seemed to prevent ethanol production in xylose fermentations. The effect of azide is discussed in terms of fine-tuning the respiratory activity necessary for metabolism.     

Optimization of the pretreatment of rice straw hemicellulosic hydrolyzates for microbial production of xylitol

Hemicellulosic hydrolyzate obtained from rice straw was evaluated to determine if it was a suitable fementation medium for the production of xylitol byCandida mogii ATCC 18364. To obtain xylose selectively from rice straw, it is important to establish rapid hydrolysis conditions that yield xylose-rich substrates. The results of hydrolysis experiments indicated that the optimal reaction conditions for the recovery of xylose from rice straw hemicellulose were obtained using a sulfuric acid concentration of 1.5%, a reaction temperature of 130°C, a reaction time of 20 min and a solid to liquid ratio of 1∶10. Because the fermentation of concentrated acid hydrolyzates can be inhibited by compounds present in the raw material or produced during the hydrolysis process, various methods were tested to determine if they could detoxify the hydrolyzates and thus improve xylitol production. The greatest xylitol yield (0.53 g/g) and volumetric productivity (0.38 g/L·h) were obtained when an overlimed hydrolyzate was treated with activated charcoal.     

Identification of required nutrient components of yeast nitrogen base for Candida shehatae ATTCC 22984 fermenting xylose to ethanol

Summary Five components of Yeast Nutrient Base (YNB) of Difco have been identified as required nutrients for Candida shehatae ATCC 22984 in fermenting xylose to ethanol with ammonium sulfate as the nitrogen source. They are potassium phosphate monobasic, magnesium sulfate, zinc sulfate, thiamine hydrochloride, and biotin. The fermentation results in the minimum medium containing only the five required nutrient components plus xylose and ammonium sulfate have been shown to be comparable to those in the full YNB plus xylose.     

Comparison of Pseudomonas fragi and Gluconobacter oxydans for production of xylonic acid from hemicellulose hydrolyzates

Summary Xylonic acid was produced efficiently from pure xylose by Pseudomonas fragi ATCC 4973 and Gluconobacter oxydans subsp. suboxydans ATCC 621. The yield from 10% xylose was in both cases over 95% of the theoretical. However, the sensitivities of the strains towards the major inhibitors found in hemicellulose hydrolyzates, ie. acetic acid, furfural and two lignin-derived compounds, varied. G. oxydans tolerated all these inhibitors better than P. fragi. In tests using steamed hemicellulose hydrolyzate, G. oxydans was able to utilize the substrate only at dilute xylose concentrations. After ether extraction or mixed bed resin pretreatment, the fermentability of the hydrolyzate was increased significantly.     

Ethanol production from xylose by Thermoanaerobacter ethanolicus in batch and continuous culture

The fermentation of xylose by Thermoanaerobacter ethanolicus ATCC 31938 was studied in pH-controlled batch and continuous cultures. In batch culture, a dependency of growth rate, product yield, and product distribution upon xylose concentration was observed. With 27 mM xylose media, an ethanol yield of 1.3 mol ethanol/mol xylose (78% of maximum theoretical yield) was typically obtained. With the same media, xylose-limited growth in continuous culture could be achieved with a volumetric productivity of 0.50 g ethanol/liter h and a yield of 0.42 g ethanol/g xylose (1.37 mol ethanol/mol xylose). With extended operation of the chemostat, variation in xylose uptake and a decline in ethanol yield was seen. Instability with respect to fermentation performance was attributed to a selection for mutant populations with different metabolic characteristics. Ethanol production in these T. ethanolicus systems was compared with xylose-to-ethanol conversions of other organisms. Relative to the other systems, T. ethanolicus offers the advantages of a high ethanol yield at low xylose concentrations in batch culture and of a rapid growth rate. Its disadvantages include a lower ethanol yield at higher xylose concentrations in batch culture and an instability of fermentation characteristics in continuous culture.     

Metabolic analysis of acetate accumulation during xylose consumption by<Emphasis Type="Italic"> Paenibacillus polymyxa</Emphasis>

Paenibacillus polymyxa ATCC 12321 produced more acetic acid and less butanediol from xylose than from glucose. The product yields from xylose were ethanol (0.72 mol/mol sugar), (R,R)-2,3-butanediol (0.31 mol/mol sugar), and acetate (0.38 mol/mol sugar) while those from glucose were ethanol (0.74 mol/mol sugar), (R,R)-2,3-butanediol (0.46 mol/mol sugar), and acetate (0.05 mol/mol sugar). Higher acetate kinase activity and lower acetate uptake ability were found in xylose-grown cells than in glucose-grown cells. Furthermore, phosphoketolase activity was higher in xylose-grown cells than in glucose-grown cells. In fed-batch culture on xylose, glucose feeding raised the butanediol yield to 0.56 mol/mol sugar and reduced acetate accumulation to 0.04 mol/mol sugar.     

Isolation and characterization of xylose- and xylan-utilizing anaerobic bacteria

By enrichment with xylose, nine mesophilic strains of anaerobic bacteria were obtained from various sources. Two isolates appear to belong to the genus Eubacterium. Six other strains belong to the genus Clostridium. Three of the isolated strains utilized larch wood xylan. The percentage of utilization of xylose and xylan and the yield of fermentation end products — viz. acetic acid and butyric acid-are equivalent to that of Clostridium acetobutylicum (ATCC 824) and reported thermophilic strains.     

Integration and expression of theEscherichia coli xylose isomerase gene inSchizosaccharomyces pombe

Summary The xyclose isomerase gene inEscherichia coli was cloned complementarily into a Leu2-negativeSchizosaccharomyces pombe mutant (ATCC 38399). The subsequent integration of the plasmid into the chromosomal DNA of the host yeast was verified by using the dot blot and southern blot techniques. The expressed xylose isomerase showed activity on a nondenaturing polyacrylamide gel. The expression of xylose isomerase gene was influenced by the concentration of nutrients in the fermentation broth. The yeast possessed a xylose isomerase activity of 20 nmol/min/mg by growing in an enriched medium containing yeast extract-malt extract-peptone (YMP) andd-xylose. The conversion ofd-xylose tod-xylulose catalyzed by xylose isomerase in the transformed yeast cells makes it possible to fermentd-xylose with ethanol as a major product. When the fermentation broth contained YMP and 5% (w/v)d-xylose, the maximal ethanol yield and productivity reached 0.42 g/g and 0.19 g/l/h, respectively.     

Combined alcoholic fermentation of D-xylose and D-glucose by four selected microbial strains: Process considerations in relation to ethanol tolerance

Summary As components of combined fermentation of both glucose and xylose to ethanol by separated or coculture processes, the effects of initial sugar concentrations on the fermentative performances ofPichia stipitis Y7124,Candida shehatae ATCC 22984,Saccharomyces cerevisiae CBS1200 andZymomonas mobilis ATCC10988 were investigated. From the characteristics of sugar and produced ethanol tolerances the most suitable microorganisms for the achievement of glucose and xylose fermentations have been selected with respect to different fermentation schemes.Nomenclature Tf fermentation time (hours) - Ef ethanol concentration (g/l) - Y_P/S ethanol yield (g of ethanol produced/g of sugar used) - qp average specific productivity of ethanol (g ethanol/g of cells per hour) - max maximum specific growth rate (h^–1)     

A model of xylitol production by the yeast Candida mogii

Production of xylitol from xylose in batch fermentations of Candida mogii ATCC 18364 is discussed in the presence of glucose as the cosubstrate. Various initial ratios of glucose and xylose concentrations are assessed for their impact on yield and rate of production of xylitol. Supplementation with glucose at the beginning of the fermentation increased the specific growth rate, biomass yield and volumetric productivity of xylitol compared with fermentation that used xylose as the sole carbon source. A mathematical model is developed for eventual use in predicting the product formation rate and yield. The model parameters were estimated from experimental observations, using a genetic algorithm. Batch fermentations, which were carried out with xylose alone and a mixture of xylose and glucose, were used to validate the model. The model fitted well with the experimental data of cell growth, substrate consumption and xylitol production.     

Kinetic modeling of Candida shehatae ATCC 22984 on xylose and glucose for ethanol production

Candida shehatae ATCC 22984, a xylose-fermenting yeast, showed an ability to produce ethanol in both glucose and xylose medium. Maximum ethanol produced by the yeast was 48.8?g/L in xylose and 52.6?g/L in glucose medium with ethanol yields that varied between 0.3 and 0.4?g/g depended on initial sugar concentrations. Xylitol was a coproduct of ethanol production using xylose as substrate, and glycerol was detected in both glucose and xylose media. Kinetic model equations indicated that growth, substrate consumption, and product formation of C. shehatae were governed by substrate limitation and inhibition by ethanol. The model suggested that cell growth was totally inhibited at 40?g/L of ethanol and ethanol production capacity of the yeast was 52?g/L, which were in good agreement with experimental results. The developed model could be used to explain C. shehatae fermentation in glucose and xylose media from 20 to 170?g/L sugar concentrations.     

Cloning and expression of Candida guilliermondii xylose reductase gene (xyl1) in Pichia pastoris

A xylose reductase gene (xyl1) of Candida guilliermondii ATCC 20118 was cloned and characterized. The open reading frame of xyl1 contained 954 nucleotides encoding a protein of 317 amino acids with a predicted molecular mass of 36 kDa. The derived amino acid sequence of C. guilliermondii xylose reductase was 70.4% homologous to that of Pichia stipitis. The gene was placed under the control of an alcohol oxidase promoter (AOX1) and integrated into the genome of a methylotrophic yeast, Pichia pastoris. Methanol induced the expression of the 36-kDa xylose reductase in both intracellular and secreted expression systems. The expressed enzyme preferentially utilized NADPH as a cofactor and was functional both in vitro and in vivo. The different cofactor specificity between P. pastoris and C. guilliermondii xylose reductases might be due to the difference in the numbers of histidine residues and their locations between the two proteins. The recombinant was able to ferment xylose, and the maximum xylitol accumulation (7.8 g/l) was observed when the organism was grown under aerobic conditions. Received: 26 August 1997 / Received revision: 6 November 1997 / Accepted: 21 November 1997     

Improved inhibitor tolerance in xylose-fermenting yeast <Emphasis Type="BoldItalic">Spathaspora passalidarum</Emphasis> by mutagenesis and protoplast fusion

The xylose-fermenting yeast Spathaspora passalidarum showed excellent fermentation performance utilizing glucose and xylose under anaerobic conditions. But this yeast is highly sensitive to the inhibitors such as furfural present in the pretreated lignocellulosic biomass. In order to improve the inhibitor tolerance of this yeast, a combination of UV mutagenesis and protoplast fusion was used to construct strains with improved performance. Firstly, UV-induced mutants were screened and selected for improved tolerance towards furfural. The most promised mutant, S. passalidarum M7, produced 50% more final ethanol than the wild-type strain in a synthetic xylose medium containing 2 g/l furfural. However, this mutant was unable to grow in a medium containing 75% liquid fraction of pretreated wheat straw (WSLQ), in which furfural and many other inhibitors were present. Hybrid yeast strains, obtained from fusion of the protoplasts of S. passalidarum M7 and a robust yeast, Saccharomyces cerevisiae ATCC 96581, were able to grow in 75% WSLQ and produce around 0.4 g ethanol/g consumed xylose. Among the selected hybrid strains, the hybrid FS22 showed the best fermentation capacity in 75% WSLQ. Phenotypic and partial molecular analysis indicated that S. passalidarum M7 was the dominant parental contributor to the hybrid. In summary, the hybrids are characterized by desired phenotypes derived from both parents, namely the ability to ferment xylose from S. passalidarum and an increased tolerance to inhibitors from S. cerevisiae ATCC 96581.     

Acetone-butanol fermentation of xylose and sugar mixtures

Summary Three strains ofCl. acetobutylicum and one ofCl. butyricum have been tested for their ability to ferment xylose to butanol. ATCC 824 and NRRL 527 produced 0.28 g solvents/g xylose, while ATCC 8260 and NRRL 594 produced much butyric acid. In 2-stage fermentations in which ATCC 8260 or NRRL 594 acted upon xylose for 12 to 20 h, followed by NRRL 527 for a total of 3 days, yields of solvent were better, 0.32 g/g xylose. Upon fermenting a mixture of sugars simulating sulphite waste liquor 0.36 g solvents/g sugar were obtained. Sugar consumption in both cases was about 96%.     

A genome shuffling-generated <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> isolate that ferments xylose and glucose to produce high levels of ethanol

Genome shuffling is an efficient approach for the rapid improvement of industrially important microbial phenotypes. This report describes optimized conditions for protoplast preparation, regeneration, inactivation, and fusion using the Saccharomyces cerevisiae W5 strain. Ethanol production was confirmed by TTC (triphenyl tetrazolium chloride) screening and high-performance liquid chromatography (HPLC). A genetically stable, high ethanol-producing strain that fermented xylose and glucose was obtained following three rounds of genome shuffling. After fermentation for 84 h, the high ethanol-producing S. cerevisiae GS3-10 strain (which utilized 69.48 and 100% of the xylose and glucose stores, respectively) produced 26.65 g/L ethanol, i.e., 47.08% higher than ethanol production by S. cerevisiae W5 (18.12 g/L). The utilization ratios of xylose and glucose were 69.48 and 100%, compared to 14.83 and 100% for W5, respectively. The ethanol yield was 0.40 g/g (ethanol/consumed glucose and xylose), i.e., 17.65% higher than the yield by S. cerevisiae W5 (0.34 g/g).     

Characterization of recombinantE. coli ATCC 11303 (pLOI 297) in the conversion of cellulose and xylose to ethanol

This work describes the characterization of recombinantEsherichia coli ATCC 11303 (pLOI 297) in the production of ethanol from cellulose and xylose. We have examined the fermentation of glucose and xylose, both individually and in mixtures, and the selectivity of ethanol production under various conditions of operation. Xylose metabolism was strongly inhibited by the presence of glucose. Ethanol was a strong inhibitor of both glucose and xylose fermentations; the maximum ethanol levels achieved at 37°C and 42°C were about 50 g/l and 25 g/l respectively. Simmultaneous sacharification and fermentation of cellulose with recombinantE. coli and exogenous cellulose showed a high ethanol yield (84% of theoretical) in the hydrolysis regime of pH 5.0 and 37°C. The selectivity of organic acid formation relative to that of ethanol increased at extreme levels of initial glucose concentration; production of succinic and acetic acids increased at low levels of glucose ( <1 g/l), and lactic acid production increased when initial glucose was higher than 100 g/l.     

Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli

Lactose and all of the major sugars (glucose, xylose, arabinose, galactose, and mannose) present in cellulose and hemicellulose were converted to ethanol by recombinant Escherichia coli containing plasmid-borne genes encoding the enzymes for the ethanol pathway from Zymomonas mobilis. Environmental tolerances, plasmid stability, expression of Z. mobilis pyruvate decarboxylase, substrate range, and ethanol production (from glucose, lactose, and xylose) were compared among eight American Type Culture Collection strains. E. coli ATCC 9637(pLO1297), ATCC 11303(pLO1297), and ATCC 15224(pLO1297) were selected for further development on the basis of environmental hardiness and ethanol production. Volumetric ethanol productivities per hour in batch culture were 1.4 g/liter for glucose (12%), 1.3 g/liter for lactose (12%), and 0.64 g/liter for xylose (8%). Ethanol productivities per hour ranged from 2.1 g/g of cell dry weight with 12% glucose to 1.3 g/g of cell dry weight with 8% xylose. The ethanol yield per gram of xylose was higher for recombinant E. coli than commonly reported for Saccharomyces cerevisiae with glucose. Glucose (12%), lactose (12%), and xylose (8%) were converted to (by volume) 7.2% ethanol, 6.5% ethanol, and 5.2% ethanol, respectively.