Induction of aldose reductase activity inCandida guilliermondii by pentose sugars

Summary Cell extracts ofCandida guilliermondii grown ind-xylose,l-arabinose,d-galactose,d-glucose,d-mannose and glycerol as sole carbon sources possessed NADPH-dependent aldose reductase activity, but no NADH-dependent activity was detected.d-xylose andl-arabinose were the best inducers of aldose reductase activity. The highest enzyme activity ind-xylose orl-arabinose-grown cells was observed first withl-arabinose followed byd-xylose as substrates of the enzymatic reaction. However, only low activity was found ind-glucose,d-mannose andd-galactose-grown cells, indicating that these carbon sources cause catabolite repression. Enzyme activities induced ind-xylose-grown cells were twice as high as those obtained from the cells under resting conditions. Furthermore, the level of induction of aldose reductase activity depended on the initial concentration ofd-xylose. The present study shows that aldose reductase activity may be efficiently induced by pentose sugars of hemicellulosic hydrolysates and weakly by hemicellulosic hexoses.     

L-Arabinose metabolism in Candida arabinofermentans PYCC 5603T and Pichia guilliermondii PYCC 3012: influence of sugar and oxygen on product formation

l-Arabinose utilization by the yeasts Candida arabinofermentans PYCC 5603^T and Pichia guilliermondii PYCC 3012 was investigated in aerobic batch cultures and compared, under similar conditions, to d-glucose and d-xylose metabolism. At high aeration levels, only biomass was formed from all the three sugars. When oxygen became limited, ethanol was produced from d-glucose, demonstrating a fermentative pathway in these yeasts. However, pentoses were essentially respired and, under oxygen limitation, the respective polyols accumulated—arabitol from l-arabinose and xylitol from d-xylose. Different l-arabinose concentrations and oxygen conditions were tested to better understand l-arabinose metabolism. P. guilliermondii PYCC 3012 excreted considerably more arabitol from l-arabinose (and also xylitol from d-xylose) than C. arabinofermentans PYCC 5603^T. In contrast to the latter, P. guilliermondii PYCC 3012 did not produce any traces of ethanol in complex l-arabinose (80 g/l) medium under oxygen-limited conditions. Neither sustained growth nor active metabolism was observed under anaerobiosis. This study demonstrates, for the first time, the oxygen dependence of metabolite and product formation in l-arabinose-assimilating yeasts.     

Induction of aldose reductase and polyol dehydrogenase activities in Aureobasidium pullulans by d-xylose,l-arabinose and d-galactose

Summary The induction of aldose reductase and polyol dehydrogenase activities by d-xylose, l-arabinose, d-galactose and d-glucose was studied in the yeast-like organism Aureobasidium pullulans CCY 27-1-26. d-xylose and l-arabinose induced two distinct NADPH-dependent aldose reductases and the inducing saccharide was simultaneously the most efficient substrate for the corresponding enzymatic reaction. Polyol dehydrogenase induced by d-xylose, l-arabinose and d-galactose was strictly NAD⁺-dependent and required only xylitol as a substrate of the enzymatic reaction. l-Arabitol did not act as a substrate for l-arabinose-induced polyol dehydrogenase either in the presence of NAD⁺ or NADP⁺.     

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 bglF ^317A , β-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.     

Engineering of an <Emphasis Type="SmallCaps">l</Emphasis>-arabinose metabolic pathway in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar l-arabinose, a product of the degradation of lignocellulosic biomass. The resultant CRA1 recombinant strain expressed the Escherichia coli genes araA, araB, and araD encoding l-arabinose isomerase, l-ribulokinase, and l-ribulose-5-phosphate 4-epimerase, respectively, under the control of a constitutive promoter. Unlike the wild-type strain, CRA1 was able to grow on mineral salts medium containing l-arabinose as the sole carbon and energy source. The three cloned genes were expressed to the same levels whether cells were cultured in the presence of d-glucose or l-arabinose. Under oxygen deprivation and with l-arabinose as the sole carbon and energy source, strain CRA1 carbon flow was redirected to produce up to 40, 37, and 11%, respectively, of the theoretical yields of succinic, lactic, and acetic acids. Using a sugar mixture containing 5% d-glucose and 1% l-arabinose under oxygen deprivation, CRA1 cells metabolized l-arabinose at a constant rate, resulting in combined organic acids yield based on the amount of sugar mixture consumed after d-glucose depletion (83%) that was comparable to that before d-glucose depletion (89%). Strain CRA1 is, therefore, able to utilize l-arabinose as a substrate for organic acid production even in the presence of d-glucose.     

Improved xylanase production by<Emphasis Type="Italic"> Trichoderma reesei</Emphasis> grown on <Emphasis Type="SmallCaps">l</Emphasis>-arabinose and lactose or <Emphasis Type="SmallCaps">d</Emphasis>-glucose mixtures

Trichoderma reesei Rut C-30 was grown on eight different natural or rare aldopentoses as the main carbon source and on mixtures of an aldopentose with d-glucose or lactose. The fungal cells consumed all aldopentoses tested, except l-xylose and l-ribose. The highest total xylanase and cellulase activities were achieved when cells were grown on l-arabinose as the main carbon source. The total xylanase activity produced by cells grown on l-arabinose was even higher than that produced by cells grown on an equal amount of lactose. In co-metabolism of d-glucose (15 g l^–1) and l-arabinose (5 g l^–1), the total volumetric and specific xylanase productivities were improved (derepressed) approximately 23- and 18-fold, respectively, compared to a cultivation on only d-glucose (20 g l^–1). In a similar experiment, in which cells were grown on a mixture of lactose and l-arabinose, the xylanase productivity was approximately doubled, compared to a cultivation on only lactose. The cellulase productivities, however, were not improved by the addition of l-arabinose. Compared with a typical industrial fungal enzyme production process with lactose as the main carbon source, better volumetric and specific xylanase productivities were achieved both on a lactose/arabinose mixture and on a glucose/arabinose mixture.     

Cultivation ofCandida blankii in simulated bagasse hemicellulose hydrolysate

Summary All fourCandida blankii isolates evaluated for growth in simulated bagasse hemicellulose hydrolysate utilized the sugars and acetic acid completely. The utilization ofd-xylose,l-arabinose and acetic acid were delayed by the presence ofd-glucose, but after glucose depletion the other carbon sources were utilized simultaneously. The maximum specific growth rate of 0.36 h^–1 and cell yield of 0.47 g cells/g carbon source assimilate compared with published results obtained withC. utilis. C. blankii appeared superior toC. utilis for biomass production from hemicellulose hydrolysate in that it utilizedl-arabinose and was capable of growth at higher temperatures.     

Identification and characterization of d-arabinose reductase and d-arabinose transporters from Pichia stipitis

d-xylose and l-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. Although Pichia stipitis CBS 6054 grows using d-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependent d-arabinose reductase from cells grown on d-arabinose, and found that the enzyme was identical to the known d-xylose reductase (XR). The enzyme activity of XR with d-arabinose was previously reported to be only 1% that with d-xylose. The k_cat/K_m value with d-arabinose (1.27 min^?1 mM^?1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those with l-arabinose and d-xylose, respectively. Among the 34 putative sugar transporters from P. stipitis, only seven genes exhibited uptake ability not only for d-arabinose, but also for d-glucose and other pentose sugars including d-xylose and l-arabinose in Saccharomyces cerevisiae.     

Fermentation of seaweed sugars by Lactobacillus species and the potential of seaweed as a biomass feedstock

It is known that seaweeds differ greatly from land plants in their sugar composition. The current research on the L-lactic acid fermentation process focuses on land plant sugars as a carbon source, with the potential of seaweed sugars being largely ignored. This study examined the feasibility of seaweed biomass as a possible carbon source for the production of l-lactic acid, by comparing the fermentation of seaweed sugars (d-galactose, d-mannitol, l-rhamnose, d-glucuronic acid, and l-fucose) and land plant sugars (d-glucose, d-xylose, d-mannose, and l-arabinose). The experiments were repeated with 2 sugar acids (d-gluconic acid, d-glucaric acid) in order to investigate the effect of the degree of reduction of carbon source on the fermentation yield. This research also examined the effect of bacterial strain on the characteristics of fermentation reactions, by conducting l-lactic acid fermentation with 7 different Lactobacillus species. Taking into account the sugar composition of seaweed and the levels of lactic acid production from each pure sugar, it was possible to predict the lactic acid production yield of various seaweeds and land plants. From comparative analysis of the predicted lactic acid production yield, it was found that seaweeds are already comparable to lignocellulosics at the current stage of technology. If new technologies for the utilization of non-fermentable seaweed sugars are developed, seaweeds show promise as an even more useful biomass feedstock than lignocellulosics.     

Characterization of Candida sp. NY7122, a novel pentose-fermenting soil yeast

Yeasts that ferment both hexose and pentose are important for cost-effective ethanol production. We found that the soil yeast strain NY7122 isolated from a blueberry field in Tsukuba (East Japan) could ferment both hexose and pentose (d-xylose and l-arabinose). NY7122 was closely related to Candida subhashii on the basis of the results of molecular identification using the sequence in the D1/D2 domains of 26S rDNA and 5.8S-internal transcribed spacer region. NY7122 produced at least 7.40 and 3.86 g l⁻¹ ethanol from 20 g l⁻¹ d-xylose and l-arabinose within 24 h. NY7122 could produce ethanol from pentose and hexose sugars at 37°C. The highest ethanol productivity of NY7122 was achieved under a low pH condition (pH 3.5). Fermentation of mixed sugars (50 g l⁻¹ glucose, 20 g l⁻¹ d-xylose, and 10 g l⁻¹ l-arabinose) resulted in a maximum ethanol concentration of 27.3 g l⁻¹ for the NY7122 strain versus 25.1 g l⁻¹ for Scheffersomyces stipitis. This is the first study to report that Candida sp. NY7122 from a soil environment could produce ethanol from both d-xylose and l-arabinose.     

Simultaneous utilization of D-cellobiose, D-glucose, and D-xylose by recombinant Corynebacterium glutamicum under oxygen-deprived conditions

Corynebacterium glutamicum R was metabolically engineered to broaden its sugar utilization range to d-xylose and d-cellobiose contained in lignocellulose hydrolysates. The resultant recombinants expressed Escherichia coli xylA and xylB genes, encoding d-xylose isomerase and xylulokinase, respectively, for d-xylose utilization and expressed C. glutamicum R bglF ^317A and bglA genes, encoding phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) β-glucoside-specific enzyme IIBCA component and phospho-β-glucosidase, respectively, for d-cellobiose utilization. The genes were fused to the non-essential genomic regions distributed around the C. glutamicum R chromosome and were under the control of their respective constitutive promoter trc and tac that permitted their expression even in the presence of d-glucose. The enzyme activities of resulting recombinants increased with the increase in the number of respective integrated genes. Maximal sugar utilization was realized with strain X5C1 harboring five xylA–xylB clusters and one bglF ^317A –bglA cluster. In both d-cellobiose and d-xylose utilization, the sugar consumption rates by genomic DNA-integrated strain were faster than those by plasmid-bearing strain, respectively. In mineral medium containing 40 g l⁻¹ d-glucose, 20 g l⁻¹ d-xylose, and 10 g l⁻¹ d-cellobiose, strain X5C1 simultaneously and completely consumed these sugars within 12 h and produced predominantly lactic and succinic acids under growth-arrested conditions.     

Pentose metabolism byBacteroides ruminicola subsp.brevis strain B14

The fermentation ofd-arabinose byBacteroides ruminicola strain B₁4 occurs in a manner similar to or identical with that shown previously forl-arabinose metabolism by the organism, a combination of hexose resynthesis and the Embden-Meyerhof sequence. The use ofd-arabinose by strain B₁4 was repressed by prior growth in medium containingd-glucose and induced by prior growth in the presence ofl-arabinose ord-xylose. The use ofd-ribose andd-xylose by strain B₁4 is different from that ford-arabinose. During growth in the presence of 1-14C-d-arabinose, labeled acetate, propionate, and succinate were formed, whereas during 1-14C-d-ribose growth only labeled acetate and propionate were obtained. Under the conditions used,d-xylose growth failed to allow formation of acetate, propionate, or succinate. Strain B₁4 incorporates label from 1- or 2-labeled glycine into acetate, propionate, and succinate by a mechanism involving the cleavage of glycine and equilibration of glycine carbons 1 and 2 with different metabolic pools.     

Fungal arabinan and <Emphasis Type="SmallCaps">l</Emphasis>-arabinose metabolism

l-Arabinose is the second most abundant pentose beside d-xylose and is found in the plant polysaccharides, hemicellulose and pectin. The need to find renewable carbon and energy sources has accelerated research to investigate the potential of l-arabinose for the development and production of biofuels and other bioproducts. Fungi produce a number of extracellular arabinanases, including α-l-arabinofuranosidases and endo-arabinanases, to specifically release l-arabinose from the plant polymers. Following uptake of l-arabinose, its intracellular catabolism follows a four-step alternating reduction and oxidation path, which is concluded by a phosphorylation, resulting in d-xylulose 5-phosphate, an intermediate of the pentose phosphate pathway. The genes and encoding enzymes l-arabinose reductase, l-arabinitol dehydrogenase, l-xylulose reductase, xylitol dehydrogenase, and xylulokinase of this pathway were mainly characterized in the two biotechnological important fungi Aspergillus niger and Trichoderma reesei. Analysis of the components of the l-arabinose pathway revealed a number of specific adaptations in the enzymatic and regulatory machinery towards the utilization of l-arabinose. Further genetic and biochemical analysis provided evidence that l-arabinose and the interconnected d-xylose pathway are also involved in the oxidoreductive degradation of the hexose d-galactose.     

Uptake of amino acids by actidione-treated yeast cells

The steady-state levels of distribution of glycine,l-aspartic acid,l-leucine and, to a lesser extent, ofl-lysine andl-methionine, in actidione-treated baker’s yeast cells are significantly altered (usually decreased) in the presence ofd-glucose,d-mannose,d-fructose, 2-deoxy-d-glucose, maltose, sucrose and, after induction,d-galactose. Stimulatory effects ofd-ribose,l-sorbose andd-xylose are not highly significant. Pronounced effects of sugars were also found anaerobically. No effect of amino acids on sugar uptake was observed. Three types of interaction appear to be present: (1) increase of energy reserves by metabolized sugars; (2) increased rate of carrier breakdown in the presence of metabolized sugars; (3) interaction at the carrier level in a “heteropolyvalent” membrane complex.     

Production of L-arabitol from L-arabinose by Candida entomaea and Pichia guilliermondii

 Lignocellulosic biomass, particularly corn fiber, represents a renewable resource that is available in sufficient quantities from the corn wet milling industry to serve as a low cost feedstock for production of fuel alcohol and valuable coproducts. Several enzymatic and chemical processes have potential for the conversion of cellulose and hemicellulose to fermentable sugars. The hydrolyzates are generally rich in pentoses (D-xylose and L-arabinose) and D-glucose. Yeasts produce a variety of polyalcohols from pentose and hexose sugars. Many of these sugar alcohols have food applications as low-calorie bulking agents. During the screening of 49 yeast strains capable of growing on L-arabinose, we observed that two strains were superior secretors of L-arabitol as a major extracellular product of L-arabinose. Candida entomaea NRRL Y-7785 and Pichia guilliermondii NRRL Y-2075 produced L-arabitol (0.70 g/g) from L-arabinose (50 g/l) at 34°C and pH 5.0 and 4.0, respectively. Both yeasts produced ethanol (0.32–0.33 g/g) from D-glucose (50 g/l) and only xylitol (0.43–0.51 g/g) from D-xylose (50 g/l). Both strains preferentially utilized D-glucose>D-xylose>L-arabinose from mixed substrate (D-glucose, D-xylose and L-arabinose, 1:1:1, 50 g/l, total) and produced ethanol (0.36–0.38 g/g D-glucose), xylitol (0.02–0.08 g/g D-xylose) and L-arabitol (0.70–0.81 g/g L-arabinose). The yeasts co-utilized D-xylose (6.2–6.5 g/l) and L-arabinose (4.9–5.0 g/l) from corn fiber acid hydrolyzate simultaneously and produced xylitol (0.10 g/g D-xylose) and L-arabitol (0.53–0.54 g/g L-arabinose). Received: 24 April 1995/Received revision: 9 August 1995/Accepted: 7 September 1995     

Xylose reductase activity in <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis> UFV-170 cultivated in semi-synthetic medium and cotton husk hemicellulose hydrolyzate

To develop a new enzymatic xylose-to-xylitol conversion, deeper knowledge on the regulation of xylose reductase (XR) is needed. To this purpose, a new strain of Debaryomyces hansenii (UFV-170), which proved a promising xylitol producer, was cultivated in semi-synthetic media containing different carbon sources, specifically three aldo-hexoses (d-glucose, d-galactose and d-mannose), a keto-hexose (d-fructose), a keto-pentose (d-xylose), three aldo-pentoses (d-arabinose, l-arabinose and d-ribose), three disaccharides (maltose, lactose and sucrose) and a pentitol (xylitol). The best substrate was lactose on which cell concentration reached about 20 g l⁻¹ dry weight (DW), while the highest specific growth rates (0.58–0.61 h⁻¹) were detected on lactose, d-mannose, d-glucose and d-galactose. The highest specific activity of XR (0.24 U mg⁻¹) was obtained in raw extracts of cells grown on d-xylose and harvested in the stationary growth phase. When grown on cotton husk hemicellulose hydrolyzates, cells exhibited XR activities five to seven times higher than on semi-synthetic media.     

Endogenous respiration reflects the energy load imposed by transport of nonmetabolizable substrates and by induced de novo protein synthesis in Rhodotorula glutinis

Uptake of the nonmetabolizable sugars 6-deoxy-d-glucose, l-rhamnose and l-xylose, which are taken up by a common carrier, stimulated significantly cell respiration in Rhodotorula glutinis. The extra oxygen consumption for uptake (0.5–0.7 equivalents O₂/mol transported sugar) was proportional to the uptake rate and was independent of the K _tvalue of the transport system. Sugars that become metabolized after induction, d-arabinose and methyl--d-glucoside, caused a higher stimulation, 1.4 and 3.6 equivalents O₂/mol respectively, which was reduced to 0.6 equivalents O₂/mol when de novo protein synthesis was blocked by cycloheximide. The stimulation of respiration thus includes a fraction related purely to the energy demand for uptake and another one related to the induced de novo protein synthesis. The net uptake-induced respiration boost was similar with all sugars under study irrespective of their transport systems. The estimated energy demand was equivalent to about 2 ATP/sugar molecule. For comparison, the amino acid analogue -aminoisobutyric acid (AIB) was also investigated; the overall energy demand for its uptake corresponded to the equivalent of about 4 ATP/molecule.Abbreviation AIB -aminoisobutyric acid     

The Production of Xylitol,l-Arabinitol and Ribitol by Yeasts

The polyalcohol production from the pentoses such as d-xylose, l-arabinose and d-ribose by various genera and species of yeasts was examined. Candida polymorpha dissimilated aerobically these three pentoses and produced xylitol from d-xylose, l-arabinitol from l-arabinose and ribitol from d-ribose at good yield of 30~40% of sugar consumed. The result suggests that these polyalcohols would be major products from pentoses by yeasts, but some unidentified minor polyalcohols were also produced.     

Establishment of l-arabinose fermentation in glucose/xylose co-fermenting recombinant Saccharomyces cerevisiae 424A(LNH-ST) by genetic engineering

Cost-effective and efficient ethanol production from lignocellulosic materials requires the fermentation of all sugars recovered from such materials including glucose, xylose, mannose, galactose, and l-arabinose. Wild-type strains of Saccharomyces cerevisiae used in industrial ethanol production cannot ferment d-xylose and l-arabinose. Our genetically engineered recombinant S. cerevisiae yeast 424A(LNH-ST) has been made able to efficiently ferment xylose to ethanol, which was achieved by integrating multiple copies of three xylose-metabolizing genes. This study reports the efficient anaerobic fermentation of l-arabinose by the derivative of 424A(LNH-ST). The new strain was constructed by over-expression of two additional genes from fungi l-arabinose utilization pathways. The resulting new 424A(LNH-ST) strain exhibited production of ethanol from l-arabinose, and the yield was more than 40%. An efficient ethanol production, about 72.5% yield from five-sugar mixtures containing glucose, galactose, mannose, xylose, and arabinose was also achieved. This co-fermentation of five-sugar mixture is important and crucial for application in industrial economical ethanol production using lignocellulosic biomass as the feedstock.     

Phylogenetic and biochemical characterization of the oil-producing yeast Lipomyces starkeyi

Lipomyces starkeyi is an oleaginous yeast, and has been classified in four distinct groups, i.e., sensu stricto and custers α, β, and γ. Recently, L. starkeyi clusters α, β, and γ were recognized independent species, Lipomyces mesembrius, Lipomyces doorenjongii, and Lipomyces kockii, respectively. In this study, we investigated phylogenetic relationships within L. starkeyi, including 18 Japanese wild strains, and its related species, based on internal transcribed spacer sequences and evaluated biochemical characters which reflected the phylogenetic tree. Phylogenetic analysis showed that most of Japanese wild strains formed one clade and this clade is more closely related to L. starkeyi s.s. clade including one Japanese wild strain than other clades. Only three Japanese wild strains were genetically distinct from L. starkeyi. Lipomyces mesembrius and L. doorenjongii shared one clade, while L. kockii was genetically distinct from the other three species. Strains in L. starkeyi s.s. clade converted six sugars, d-glucose, d-xylose, l-arabinose, d-galactose, d-mannose, and d-cellobiose to produce high total lipid yields. The Japanese wild strains in subclades B, C, and D converted d-glucose, d-galactose, and d-mannose to produce high total lipid yields. Lipomyces mesembrius was divided into two subclades. Lipomyces mesembrius CBS 7737 converted d-xylose, l-arabinose, d-galactose, and d-cellobiose, while the other L. mesembrius strains did not. Lipomyces doorenjongii converted all the sugars except d-cellobiose. In comparison to L. starkeyi, L. mesembrius, and L. doorenjongii, L. kockii produced higher total lipid yields from d-glucose, d-galactose, and d-mannose. The type of sugar converted depended on the subclade classification elucidated in this study.