Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying XYL1, XYL2, and XKS1 in mineral medium chemostat cultures

For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. Anaerobic ethanol formation from xylose by recombinant S. cerevisiae was demonstrated for the first time. However, the strain grew on xylose only in the presence of oxygen. Ethanol yields of 0.45 to 0.50 mmol of C/mmol of C (0.35 to 0.38 g/g) and productivities of 9.7 to 13.2 mmol of C h(-1) g (dry weight) of cells(-1) (0.24 to 0.30 g h(-1) g [dry weight] of cells(-1)) were obtained from xylose-glucose mixtures in anaerobic chemostat cultures, with a dilution rate of 0.06 h(-1). The anaerobic ethanol yield on xylose was estimated at 0.27 mol of C/(mol of C of xylose) (0.21 g/g), assuming a constant ethanol yield on glucose. The xylose uptake rate increased with increasing xylose concentration in the feed, from 3.3 mmol of C h(-1) g (dry weight) of cells(-1) when the xylose-to-glucose ratio in the feed was 1:3 to 6.8 mmol of C h(-1) g (dry weight) of cells(-1) when the feed ratio was 3:1. With a feed content of 15 g of xylose/liter and 5 g of glucose/liter, the xylose flux was 2.2 times lower than the glucose flux, indicating that transport limits the xylose flux.     

Parametric studies of ethanol production form xylose and other sugars by recombinant Escherichia coli

The conversion of xylose to ethanol by recombinant Escherichia coli has been investigated in pH-controlled batch fermentations. Chemical and environmental parameters were varied to determine tolerance and to define optimal conditions. Relatively high concentrations of ethanol (56 g/L) were produced from xylose with excellent efficiencies. Volumetric productivities of up to 1.4 g ethanol/L h were obtained. Productivities, yields, and final ethanol concentrations achieved from xylose with recombinant E. coli exceeded the reported values with other organisms. In addition to xylose, all other sugar constituents of biomass (glucose, mannose, arabinose, and galactose) were efficiently converted to ethanol by recombinant E. coli. Unusually low inocula equivalent to 0.033 mg of dry cell weight/L were adequate for batch fermentations. The addition of small amounts of calcium, magnesium, and ferrous ions stimulated fermentation. The inhibitory effects of toxic compounds (salts, furfural, and acetate) which are present in hemicellulose hydrolysates were also examined.     

Ethanolic fermentation of acid pre-treated starch industry effluents by recombinant Saccharomyces cerevisiae strains

Two industrial effluents, a pre-fermentation effluent and a post-fermentation effluent from a wheat starch production plant, were used as substrates for fuel ethanol production in anaerobic batch cultures using minimal nutritional amendment. The performances of three metabolically engineered xylose-utilizing Saccharomyces cerevisiae strains: TMB 3001 expressing XYL1, XYL2 and XKS1, redox metabolism modulated CPB.CR1 and glucose de-repressed CPB.CR2, as well as a reference strain CEN.PK 113-7D not fermenting xylose, were evaluated. For the recombinant strains a glucose consumption phase preceded the xylose consumption phase. In both effluents, biomass and ethanol production occurred predominantly during the glucose consumption phase, whereas xylitol and glycerol formation were predominant in the xylose consumption phase. Total specific ethanol productivities on glucose were 6-fold higher than on xylose in the pre-fermentation effluent and 15-fold higher than on xylose in the post-fermentation effluent. CPB.CR1 showed impaired growth compared to the two other xylose-utilizing strains, but displayed 18% increased ethanol yield in the post-fermentation effluent.     

Xylulokinase overexpression in two strains of Saccharomyces cerevisiae also expressing xylose reductase and xylitol dehydrogenase and its effect on fermentation of xylose and lignocellulosic hydrolysate

Fermentation of the pentose sugar xylose to ethanol in lignocellulosic biomass would make bioethanol production economically more competitive. Saccharomyces cerevisiae, an efficient ethanol producer, can utilize xylose only when expressing the heterologous genes XYL1 (xylose reductase) and XYL2 (xylitol dehydrogenase). Xylose reductase and xylitol dehydrogenase convert xylose to its isomer xylulose. The gene XKS1 encodes the xylulose-phosphorylating enzyme xylulokinase. In this study, we determined the effect of XKS1 overexpression on two different S. cerevisiae host strains, H158 and CEN.PK, also expressing XYL1 and XYL2. H158 has been previously used as a host strain for the construction of recombinant xylose-utilizing S. cerevisiae strains. CEN.PK is a new strain specifically developed to serve as a host strain for the development of metabolic engineering strategies. Fermentation was carried out in defined and complex media containing a hexose and pentose sugar mixture or a birch wood lignocellulosic hydrolysate. XKS1 overexpression increased the ethanol yield by a factor of 2 and reduced the xylitol yield by 70 to 100% and the final acetate concentrations by 50 to 100%. However, XKS1 overexpression reduced the total xylose consumption by half for CEN.PK and to as little as one-fifth for H158. Yeast extract and peptone partly restored sugar consumption in hydrolysate medium. CEN.PK consumed more xylose but produced more xylitol than H158 and thus gave lower ethanol yields on consumed xylose. The results demonstrate that strain background and modulation of XKS1 expression are important for generating an efficient xylose-fermenting recombinant strain of S. cerevisiae.     

Optimal growth and ethanol production from xylose by recombinant Saccharomyces cerevisiae require moderate D-xylulokinase activity

D-Xylulokinase (XK) is essential for the metabolism of D-xylose in yeasts. However, overexpression of genes for XK, such as the Pichia stipitis XYL3 gene and the Saccharomyces cerevisiae XKS gene, can inhibit growth of S. cerevisiae on xylose. We varied the copy number and promoter strength of XYL3 or XKS1 to see how XK activity can affect xylose metabolism in S. cerevisiae. The S. cerevisiae genetic background included single integrated copies of P. stipitis XYL1 and XYL2 driven by the S. cerevisiae TDH1 promoter. Multicopy and single-copy constructs with either XYL3 or XKS1, likewise under control of the TDH1 promoter, or with the native P. stipitis promoter were introduced into the recombinant S. cerevisiae. In vitro enzymatic activity of XK increased with copy number and promoter strength. Overexpression of XYL3 and XKS1 inhibited growth on xylose but did not affect growth on glucose even though XK activities were three times higher in glucose-grown cells. Growth inhibition increased and ethanol yields from xylose decreased with increasing XK activity. Uncontrolled XK expression in recombinant S. cerevisiae is inhibitory in a manner analogous to the substrate-accelerated cell death observed with an S. cerevisiae tps1 mutant during glucose metabolism. To bypass this effect, we transformed cells with a tunable expression vector containing XYL3 under the control of its native promoter into the FPL-YS1020 strain and screened the transformants for growth on, and ethanol production from, xylose. The selected transformant had approximately four copies of XYL3 per haploid genome and had moderate XK activity. It converted xylose into ethanol efficiently.     

High expression of XYL2 coding for xylitol dehydrogenase is necessary for efficient xylose fermentation by engineered Saccharomyces cerevisiae

The traditional ethanologenic yeast Saccharomyces cerevisiae cannot metabolize xylose, which is an abundant sugar in non-crop plants. Engineering this yeast for a practicable fermentation of xylose will therefore improve the economics of bioconversion for the production of fuels and chemicals such as ethanol. One of the most widely employed strategies is to express XYL1, XYL2, and XYL3 genes derived from Scheffersomyces stipitis (formerly Pichia stiptis) in S. cerevisiae. However, the resulting engineered strains have been reported to exhibit large variations in xylitol accumulation and ethanol yields, generating many hypotheses and arguments for elucidating these phenomena. Here we demonstrate that low expression levels of the XYL2 gene, coding for xylitol dehydrogenase (XDH), is a major bottleneck in efficient xylose fermentation. Through an inverse metabolic engineering approach using a genomic library of S. cerevisiae, XYL2 was identified as an overexpression target for improving xylose metabolism. Specifically, we performed serial subculture experiments after transforming a genomic library of wild type S. cerevisiae into an engineered strain harboring integrated copies of XYL1, XYL2 and XYL3. Interestingly, the isolated plasmids from efficient xylose-fermenting transformants contained XYL2. This suggests that the integrated XYL2 migrated into a multi-copy plasmid through homologous recombination. It was also found that additional overexpression of XYL2 under the control of strong constitutive promoters in a xylose-fermenting strain not only reduced xylitol accumulation, but also increased ethanol yields. As the expression levels of XYL2 increased, the ethanol yields gradually improved from 0.1 to 0.3g ethanol/g xylose, while the xylitol yields significantly decreased from 0.4 to 0.1g xylitol/g xylose. These results suggest that strong expression of XYL2 is a necessary condition for developing efficient xylose-fermenting strains.     

A UV-induced mutant of Pichia stipitis with increased ethanol production from xylose and selection of a spontaneous mutant with increased ethanol tolerance

In the fermentation process of lignocellulosic biomass (such as wood and rice straw), efficient conversion of pentose (mainly xylose) into ethanol is important. Mutants of Pichia stipitis NBRC1687 were obtained after UV mutagenesis and selection of large colonies on ethanol-containing medium. One mutant, PXF58, produced 4.3% ethanol from 11.4% xylose while the parent strain only produced 3.1%. The ethanol productivities of PXF58 from glucose and fructose were about were about 1.4-fold higher than those of the parent strain. After continuous cultivation of PXF58 in YNB (yeast nitrogen base) medium containing 2% xylose and 5-7% ethanol, an ethanol-tolerant mutant, PET41, was obtained. Strain PET41 was able to produce 4.4% ethanol when first supplied with xylose then with glucose. This isolate might be thus useful for two-phase fermentation in which xylan is saccharified by xylanase to produce xylose, and glucan is saccharified later by cellulase and β-glucosidase to produce glucose.     

Genetic improvement of Saccharomyces cerevisiae for xylose fermentation

There is considerable interest in recent years in the bioconversion of forestry and agricultural residues into ethanol and value-added chemicals. High ethanol yields from lignocellulosic residues are dependent on efficient use of all the available sugars including glucose and xylose. The well-known fermentative yeast Saccharomyces cerevisiae is the preferred microorganism for ethanol production, but unfortunately, this yeast is unable to ferment xylose. Over the last 15 years, this yeast has been the subject of various research efforts aimed at improving its ability to utilize xylose and ferment it to ethanol. This review examines the research on S. cerevisiae strains that have been genetically modified or adapted to ferment xylose to ethanol. The current state of these efforts and areas where further research is required are identified and discussed.     

A C6/C5 co‐fermenting Saccharomyces cerevisiae strain with the alleviation of antagonism between xylose utilization and robustness

During second‐generation bioethanol production from lignocellulosic biomass, the desired traits for fermenting microorganisms, such as Saccharomyces cerevisiae, are high xylose utilization and high robustness to inhibitors in lignocellulosic hydrolysates. However, as observed previously, these two traits easily showed the antagonism, one rising and the other falling, in the C6/C5 co‐fermenting S. cerevisiae strain. In this study, LF1 obtained in our previous study is an engineered budding yeast strain with a superior co‐fermentation capacity of glucose and xylose, and was then mutated by atmospheric and room temperature plasma (ARTP) mutagenesis to improve its robustness. The ARTP‐treated cells were grown in 50% (v/v) leachate from lignocellulose pretreatment with high inhibitors content for adaptive evolution. After 30 days, the generated mutant LF1‐6 showed significantly enhanced tolerance, with a six‐fold increase in cell density in the above leachate. Unfortunately, its xylose utilization dropped markedly, indicating the recurrence of the negative correlation between xylose utilization and robustness. To alleviate this antagonism, LF1‐6 cells were iteratively mutated with ARTP mutagenesis and then anaerobically grown using xylose as the sole carbon source, and xylose utilization was restored in the resulting strain 6M‐15. 6M‐15 also exhibited increased co‐fermentation performance of xylose and glucose with the highest ethanol productivity reported to date (0.525 g g^?1 h^?1) in high‐level mixed sugars (80 g L^?1 glucose and 40 g L^?1 xylose) with no inhibitors. Meanwhile, its fermentation time was shortened by 8 h compared to that of LF1. During the fermentation of non‐detoxified lignocellulosic hydrolysate with high inhibitor concentrations at pH ~3.5, 6M‐15 can efficiently convert glucose and xylose with an ethanol yield of 0.43 g g^?1. 6M‐15 is also regarded as a potential chassis cell for further design of a customized strain suitable for production of second‐generation bioethanol or other high value‐added products from lignocellulosic biomass.     

The effect of xylose reductase genes on xylitol production by industrial Saccharomyces cerevisiae in fermentation of glucose and xylose

The co-production of xylitol and ethanol from agricultural straw has more economic advantages than the production of ethanol only. Saccharomyces cerevisiae, the most widely used ethanol-producing yeast, can be genetically engineered to ferment xylose to xylitol. In the present study, the effects of xylose-specificity, cofactor preference, and the gene copy number of xylose reductase (XR; encoding by XYL1 gene) on xylitol production of S. cerevisiae were investigated. The results showed that overexpression of XYL1 gene with a lower xylose-specificity and a higher NADPH preference favored the xylitol production. The copy number of XYL1 had a positive correlation with the XR activity but did not show a good correlation with the xylitol productivity. The overexpression of XYL1 from Candida tropicalis (CtXYL1) achieved a xylitol productivity of 0.83 g/L/h and a yield of 0.99 g/g-consumed xylose during batch fermentation with 43.5 g/L xylose and 17.0 g/L glucose. During simultaneous saccharification and fermentation (SSF) of pretreated corn stover, the strain overexpressing CtXYL1 produced 45.41 g/L xylitol and 50.19 g/L ethanol, suggesting its application potential for xylitol and ethanol co-production from straw feedstocks.     

Optimum pH and temperature conditions for xylose fermentation by Pichia stipitis

Pichia stipitis NRRL Y-7124 is a xylose-fermenting yeast able to accumulate ca. 57 g/L ethanol. Because optimum process conditions are important, data were collected to determine the effects of temperature and pH on growth and fermentation rates and product accumulations. Temperatures (26-35 degrees C) providing optimum biomass and ethanol productivities did not necessarily provide maximum ethanol accumulation. Xylitol and residual xylose concentrations increased with temperature. Maximum ethanol selectivity was achieved at 25-26 degrees C with minimal sacrifice to production rates. The temperature optimum for xylose could not be generalized to glucose fermentations, in which ethanol productivity and accumulation were optimum at 34 degrees C. The optimum pH range for growth and fermentation on xylose was 4-7 at 25 degrees C.     

Effects of NADH-preferring xylose reductase expression on ethanol production from xylose in xylose-metabolizing recombinant Saccharomyces cerevisiae

Efficient conversion of xylose to ethanol is an essential factor for commercialization of lignocellulosic ethanol. To minimize production of xylitol, a major by-product in xylose metabolism and concomitantly improve ethanol production, Saccharomyces cerevisiae D452-2 was engineered to overexpress NADH-preferable xylose reductase mutant (XR(MUT)) and NAD?-dependent xylitol dehydrogenase (XDH) from Pichia stipitis and endogenous xylulokinase (XK). In vitro enzyme assay confirmed the functional expression of XR(MUT), XDH and XK in recombinant S. cerevisiae strains. The change of wild type XR to XR(MUT) along with XK overexpression led to reduction of xylitol accumulation in microaerobic culture. More modulation of the xylose metabolism including overexpression of XR(MUT) and transaldolase, and disruption of the chromosomal ALD6 gene encoding aldehyde dehydrogenase (SX6(MUT)) improved the performance of ethanol production from xylose remarkably. Finally, oxygen-limited fermentation of S. cerevisiae SX6(MUT) resulted in 0.64 g l?1 h?1 xylose consumption rate, 0.25 g l?1 h?1 ethanol productivity and 39% ethanol yield based on the xylose consumed, which were 1.8, 4.2 and 2.2 times higher than the corresponding values of recombinant S. cerevisiae expressing XR(MUT), XDH and XK only.     

Efficient xylose fermentation by the brown rot fungus Neolentinus lepideus

The efficient production of bioethanol on an industrial scale requires the use of renewable lignocellulosic biomass as a starting material. A limiting factor in developing efficient processes is identifying microorganisms that are able to effectively ferment xylose, the major pentose sugar found in hemicellulose, and break down carbohydrate polymers without pre-treatment steps. Here, a basidiomycete brown rot fungus was isolated as a new biocatalyst with unprecedented fermentability, as it was capable of converting not only the 6-carbon sugars constituting cellulose, but also the major 5-carbon sugar xylose in hemicelluloses, to ethanol. The fungus was identified as Neolentinus lepideus and was capable of assimilating and fermenting xylose to ethanol in yields of 0.30, 0.33, and 0.34 g of ethanol per g of xylose consumed under aerobic, oxygen-limited, and anaerobic conditions, respectively. A small amount of xylitol was detected as the major by-product of xylose metabolism. N. lepideus produced ethanol from glucose, mannose, galactose, cellobiose, maltose, and lactose with yields ranging from 0.34 to 0.38 g ethanol per g sugar consumed, and also exhibited relatively favorable conversion of non-pretreated starch, xylan, and wheat bran. These results suggest that N. lepideus is a promising candidate for cost-effective and environmentally friendly ethanol production from lignocellulosic biomass. To our knowledge, this is the first report on efficient ethanol fermentation from various carbohydrates, including xylose, by a naturally occurring brown rot fungus.     

Efficient production of L-lactic acid from xylose by Pichia stipitis

Microbial conversion of renewable raw materials to useful products is an important objective in industrial biotechnology. Pichia stipitis, a yeast that naturally ferments xylose, was genetically engineered for l-(+)-lactate production. We constructed a P. stipitis strain that expressed the l-lactate dehydrogenase (LDH) from Lactobacillus helveticus under the control of the P. stipitis fermentative ADH1 promoter. Xylose, glucose, or a mixture of the two sugars was used as the carbon source for lactate production. The constructed P. stipitis strain produced a higher level of lactate and a higher yield on xylose than on glucose. Lactate accumulated as the main product in xylose-containing medium, with 58 g/liter lactate produced from 100 g/liter xylose. Relatively efficient lactate production also occurred on glucose medium, with 41 g/liter lactate produced from 94 g/liter glucose. In the presence of both sugars, xylose and glucose were consumed simultaneously and converted predominantly to lactate. Lactate was produced at the expense of ethanol, whose production decreased to approximately 15 to 30% of the wild-type level on xylose-containing medium and to 70 to 80% of the wild-type level on glucose-containing medium. Thus, LDH competed efficiently with the ethanol pathway for pyruvate, even though the pathway from pyruvate to ethanol was intact. Our results show, for the first time, that lactate production from xylose by a yeast species is feasible and efficient. This is encouraging for further development of yeast-based bioprocesses to produce lactate from lignocellulosic raw material.     

Development and application of co-culture for ethanol production by co-fermentation of glucose and xylose: a systematic review

This article reviews current co-culture systems for fermenting mixtures of glucose and xylose to ethanol. Thirty-five co-culture systems that ferment either synthetic glucose and xylose mixture or various biomass hydrolysates are examined. Strain combinations, fermentation modes and conditions, and fermentation performance for these co-culture systems are compared and discussed. It is noted that the combination of Pichia stipitis with Saccharomyces cerevisiae or its respiratory-deficient mutant is most commonly used. One of the best results for fermentation of glucose and xylose mixture is achieved by using co-culture of immobilized Zymomonas mobilis and free cells of P. stipitis, giving volumetric ethanol production of 1.277 g/l/h and ethanol yield of 0.49–0.50 g/g. The review discloses that, as a strategy for efficient conversion of glucose and xylose, co-culture fermentation for ethanol production from lignocellulosic biomass can increase ethanol yield and production rate, shorten fermentation time, and reduce process costs, and it is a promising technology although immature.     

Continuous Ethanol Fermentation of Pretreated Lignocellulosic Biomasses,Waste Biomasses,Molasses and Syrup Using the Anaerobic,Thermophilic Bacterium Thermoanaerobacter italicus Pentocrobe 411

Lignocellosic ethanol production is now at a stage where commercial or semi-commercial plants are coming online and, provided cost effective production can be achieved, lignocellulosic ethanol will become an important part of the world bio economy. However, challenges are still to be overcome throughout the process and particularly for the fermentation of the complex sugar mixtures resulting from the hydrolysis of hemicellulose. Here we describe the continuous fermentation of glucose, xylose and arabinose from non-detoxified pretreated wheat straw, birch, corn cob, sugar cane bagasse, cardboard, mixed bio waste, oil palm empty fruit bunch and frond, sugar cane syrup and sugar cane molasses using the anaerobic, thermophilic bacterium Thermoanaerobacter Pentocrobe 411. All fermentations resulted in close to maximum theoretical ethanol yields of 0.47–0.49 g/g (based on glucose, xylose, and arabinose), volumetric ethanol productivities of 1.2–2.7 g/L/h and a total sugar conversion of 90–99% including glucose, xylose and arabinose. The results solidify the potential of Thermoanaerobacter strains as candidates for lignocellulose bioconversion.     

Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h(-1) g [dry weight](-1)) and ethanol production (0.29 g h(-1) g [dry weight](-1)) and a high ethanol yield (0.43 g g(-1)) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.     

Cofermentation of glucose, xylose, and cellobiose by the beetle-associated yeast Spathaspora passalidarum

Fermentation of cellulosic and hemicellulosic sugars from biomass could resolve food-versus-fuel conflicts inherent in the bioconversion of grains. However, the inability to coferment glucose and xylose is a major challenge to the economical use of lignocellulose as a feedstock. Simultaneous cofermentation of glucose, xylose, and cellobiose is problematic for most microbes because glucose represses utilization of the other saccharides. Surprisingly, the ascomycetous, beetle-associated yeast Spathaspora passalidarum, which ferments xylose and cellobiose natively, can also coferment these two sugars in the presence of 30 g/liter glucose. S. passalidarum simultaneously assimilates glucose and xylose aerobically, it simultaneously coferments glucose, cellobiose, and xylose with an ethanol yield of 0.42 g/g, and it has a specific ethanol production rate on xylose more than 3 times that of the corresponding rate on glucose. Moreover, an adapted strain of S. passalidarum produced 39 g/liter ethanol with a yield of 0.37 g/g sugars from a hardwood hydrolysate. Metabolome analysis of S. passalidarum before onset and during the fermentations of glucose and xylose showed that the flux of glycolytic intermediates is significantly higher on xylose than on glucose. The high affinity of its xylose reductase activities for NADH and xylose combined with allosteric activation of glycolysis probably accounts in part for its unusual capacities. These features make S. passalidarum very attractive for studying regulatory mechanisms enabling bioconversion of lignocellulosic materials by yeasts.     

Optimization of CDT-1 and XYL1 Expression for Balanced Co-Production of Ethanol and Xylitol from Cellobiose and Xylose by Engineered Saccharomyces cerevisiae

Production of ethanol and xylitol from lignocellulosic hydrolysates is an alternative to the traditional production of ethanol in utilizing biomass. However, the conversion efficiency of xylose to xylitol is restricted by glucose repression, causing a low xylitol titer. To this end, we cloned genes CDT-1 (encoding a cellodextrin transporter) and gh1-1 (encoding an intracellular β-glucosidase) from Neurospora crassa and XYL1 (encoding a xylose reductase that converts xylose into xylitol) from Scheffersomyces stipitis into Saccharomyces cerevisiae, enabling simultaneous production of ethanol and xylitol from a mixture of cellobiose and xylose (main components of lignocellulosic hydrolysates). We further optimized the expression levels of CDT-1 and XYL1 by manipulating their promoters and copy-numbers, and constructed an engineered S. cerevisiae strain (carrying one copy of PGK1p-CDT1 and two copies of TDH3p-XYL1), which showed an 85.7% increase in xylitol production from the mixture of cellobiose and xylose than that from the mixture of glucose and xylose. Thus, we achieved a balanced co-fermentation of cellobiose (0.165 g/L/h) and xylose (0.162 g/L/h) at similar rates to co-produce ethanol (0.36 g/g) and xylitol (1.00 g/g).