Bioconversion of xylose, hexoses and biomass to ethanol by a new isolate of the white rot basidiomycete Trametes versicolor.
ABSTRACT: Second-generation bioethanol production requires the development of economically feasible and sustainable processes that use renewable lignocellulosic biomass as a starting material. However, the microbial fermentation of xylose, which is the principal pentose sugar in hemicellulose, is a limiting factor in developing such processes. Here, a strain of the white rot basidiomycete Trametes versicolor that was capable of efficiently fermenting xylose was newly isolated and characterized. This strain, designated KT9427, was capable of assimilating and converting xylose to ethanol under anaerobic conditions with a yield of 0.44 g ethanol per 1 g of sugar consumed. In culture medium containing low yeast extract concentrations, xylose consumption and ethanol productivity were enhanced. Adjusting the initial pH between 3.0 and 5.0 did not markedly influence xylose fermentation. T. versicolor KT9427 also produced ethanol from glucose, mannose, fructose, cellobiose and maltose at yields ranging from 0.45 to 0.49 g ethanol per 1 g of sugar consumed. In addition, strain KT9427 exhibited favourable conversion of non-pretreated starch, cellulose, xylan, wheat bran and rice straw into ethanol compared to common recombinant yeast strains. Taken together, the present findings suggest that T. versicolor KT9427 is a promising candidate for environmentally friendly ethanol production directly from lignocellulosic biomass.
Project description: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.
Project description:Economics of ethanol production from lignocellulosic biomass depends on complete utilization of constituent carbohydrates and efficient fermentation of mixed sugars present in biomass hydrolysates. Saccharomyces cerevisiae, the commercial strain for ethanol production uses only glucose while pentoses remain unused. Recombinant strains capable of utilizing pentoses have been engineered but with limited success. Recently, presence of endogenous pentose assimilation pathway in S. cerevisiae was reported. On the contrary, evolutionary engineering of native xylose assimilating strains is promising approach. In this study, a native strain S. cerevisiae LN, isolated from fruit juice, was found to be capable of xylose assimilation and mixed sugar fermentation. Upon supplementation with yeast extract and peptone, glucose (10%) fermentation efficiency was 78% with ~90% sugar consumption. Medium engineering augmented mixed sugars (5% glucose + 5% xylose) fermentation efficiency to ~50 and 1.6% ethanol yield was obtained with concomitant sugar consumption ~60%. Statistical optimization of input variables Glucose (5.36%), Xylose (3.30%), YE (0.36%), and peptone (0.25%) with Response surface methodology led to improved sugar consumption (74.33%) and 2.36% ethanol within 84 h. Specific activities of Xylose Reductase and Xylitol Dehydrogenase exhibited by S. cerevisiae LN were relatively low. Their ratio indicated metabolism diverted toward ethanol than xylitol and other byproducts. Strain was tolerant to concentrations of HMF, furfural and acetic acid commonly encountered in biomass hydrolysates. Thus, genetic setup for xylose assimilation in S. cerevisiae LN is not merely artifact of xylose metabolizing pathway and can be augmented by adaptive evolution. This strain showed potential for commercial exploitation.
Project description:The development of robust microorganisms that can efficiently ferment both glucose and xylose represents one of the major challenges in achieving a cost-effective lignocellulosic bioethanol production. Candida intermedia is a non-conventional, xylose-utilizing yeast species with a high-capacity xylose transport system. The natural ability of C. intermedia to produce ethanol from xylose makes it attractive as a non-GMO alternative for lignocellulosic biomass conversion in biorefineries. We have evaluated the fermentation capacity and the tolerance to lignocellulose-derived inhibitors and the end product, ethanol, of the C. intermedia strain CBS 141442 isolated from steam-exploded wheat straw hydrolysate. In a mixed sugar fermentation medium, C. intermedia CBS 141442 co-fermented glucose and xylose, although with a preference for glucose over xylose. The strain was clearly more sensitive to inhibitors and ethanol when consuming xylose than glucose. C. intermedia CBS 141442 was also subjected to evolutionary engineering with the aim of increasing its tolerance to inhibitors and ethanol, and thus improving its fermentation capacity under harsh conditions. The resulting evolved population was able to ferment a 50% (v/v) steam-exploded wheat straw hydrolysate (which was completely inhibitory to the parental strain), improving the sugar consumption and the final ethanol concentration. The evolved population also exhibited a better tolerance to ethanol when growing in a xylose medium supplemented with 35.5 g/L ethanol. These results highlight the potential of C. intermedia CBS 141442 to become a robust yeast for the conversion of lignocellulose to ethanol.
Project description:BACKGROUND: Sustainable and economically viable manufacturing of bioethanol from lignocellulose raw material is dependent on the availability of a robust ethanol producing microorganism, able to ferment all sugars present in the feedstock, including the pentose sugars L-arabinose and D-xylose. Saccharomyces cerevisiae is a robust ethanol producer, but needs to be engineered to achieve pentose sugar fermentation. RESULTS: A new recombinant S. cerevisiae strain expressing an improved fungal pathway for the utilization of L-arabinose and D-xylose was constructed and characterized. The new strain grew aerobically on L-arabinose and D-xylose as sole carbon sources. The activities of the enzymes constituting the pentose utilization pathway(s) and product formation during anaerobic mixed sugar fermentation were characterized. CONCLUSION: Pentose fermenting recombinant S. cerevisiae strains were obtained by the expression of a pentose utilization pathway of entirely fungal origin. During anaerobic fermentation the strain produced biomass and ethanol. L-arabitol yield was 0.48 g per gram of consumed pentose sugar, which is considerably less than previously reported for D-xylose reductase expressing strains co-fermenting L-arabinose and D-xylose, and the xylitol yield was 0.07 g per gram of consumed pentose sugar.
Project description:BACKGROUND: It remains a challenge for recombinant S. cerevisiae to convert xylose in lignocellulosic biomass hydrolysates to ethanol. Although industrial diploid strains are more robust compared to laboratory haploid strains, however, industrial diploid S. cerevisiae strains have been less pursued in previous studies. This work aims to construct fast xylose-fermenting yeast using an industrial ethanol-producing diploid S. cerevisiae strain as a host. RESULTS: Fast xylose-fermenting yeast was constructed by genome integration of xylose-utilizing genes and adaptive evolution, including 1) Piromyces XYLA was introduced to enable the host strain to convert xylose to xylulose; 2) endogenous genes (XKS1, RKI1, RPE1, TKL1, and TAL1) were overexpressed to accelerate conversion of xylulose to ethanol; 3) Candida intermedia GXF1, which encodes a xylose transporter, was introduced at the GRE3 locus to improve xylose uptake; 4) aerobic evolution in rich xylose media was carried out to increase growth and xylose consumption rates. The best evolved strain CIBTS0735 consumed 80 g/l glucose and 40 g/l xylose in rich media within 24 hours at an initial OD600 of 1.0 (0.63 g DCW/l) and produced 53 g/l ethanol. CONCLUSIONS: Based on the above fermentation performance, we conclude that CIBTS0735 shows great potential for ethanol production from lignocellulosic biomass.
Project description:A thermo-tolerant NADP(H)-preferring xylose pathway was constructed in Kluyveromyces marxianus for ethanol production with xylose at elevated temperatures (Zhang et al., 2015 ). Ethanol production yield and efficiency was enhanced by pathway engineering in the engineered strains. The constructed strain, YZJ088, has the ability to co-ferment glucose and xylose for ethanol and xylitol production, which is a critical step toward enabling economic biofuel production from lignocellulosic biomass. This study contains the fermentation results of strains using the metabolic pathway engineering procedure. The ethanol-producing abilities of various yeast strains under various conditions were compared, and strain YZJ088 showed the highest production and fastest productivity at elevated temperatures. The YZJ088 xylose fermentation results indicate that it fermented well with xylose at either low or high inoculum size. When fermented with an initial cell concentration of OD600=15 at 37 °C, YZJ088 consumed 200 g/L xylose and produced 60.07 g/L ethanol; when the initial cell concentration was OD600=1 at 37 °C, YZJ088 consumed 98.96 g/L xylose and produced 33.55 g/L ethanol with a productivity of 0.47 g/L/h. When fermented with 100 g/L xylose at 42 °C, YZJ088 produced 30.99 g/L ethanol with a productivity of 0.65 g/L/h, which was higher than that produced at 37 °C.
Project description:Lignocellulosic biomass is a vast and underutilized resource for the production of sugars and biofuels. However, the structural complexity of lignocellulosic biomass and the need for multiple pretreatment and enzymatic steps for sugar release renders this process economically challenging. Here, we report a novel approach for direct, single container, exogenous enzyme-free conversion of lignocellulosic biomass to sugars and biofuels using the anaerobic fungal isolate strain C1A. This approach utilizes simple physiological manipulations for timely inhibition and uncoupling of saccharolytic and fermentative capabilities of strain C1A, leading to the accumulation of sugar monomers (glucose and xylose) in the culture medium. The produced sugars, in addition to fungal hyphal lysate, are subsequently converted by Escherichia coli strain K011 to ethanol. Using this approach, we successfully recovered 17.0% (w/w) of alkali-pretreated corn stover (20.0% of its glucan and xylan content) as sugar monomers in the culture media. More importantly, 14.1% of pretreated corn stover (17.1% of glucan and xylan content) was recovered as ethanol at a final concentration of 28.16 mM after the addition of the ethanologenic strain K011. The high ethanol yield obtained is due to its accumulation as a minor fermentation end product by strain C1A during its initial growth phase, the complete conversion of sugars to ethanol by strain K011, and the possible conversion of unspecified substrates in the hyphal lysate of strain C1A to ethanol by strain K011. This study presents a novel, versatile, and exogenous enzyme-free strategy that utilizes a relatively unexplored group of organisms (anaerobic fungi) for direct biofuel production from lignocellulosic biomass.
Project description:Background:Lignocellulosic biorefinery offers economical and sustainable production of fuels and chemicals. Saccharomyces cerevisiae, a promising industrial host for biorefinery, has been intensively developed to expand its product profile. However, the sequential and slow conversion of xylose into target products remains one of the main challenges for realizing efficient industrial lignocellulosic biorefinery. Results:In this study, we developed a powerful mixed-sugar co-fermenting strain of S. cerevisiae, XUSEA, with improved xylose conversion capacity during simultaneous glucose/xylose co-fermentation. To reinforce xylose catabolism, the overexpression target in the pentose phosphate pathway was selected using a DNA assembler method and overexpressed increasing xylose consumption and ethanol production by twofold. The performance of the newly engineered strain with improved xylose catabolism was further boosted by elevating fermentation temperature and thus significantly reduced the co-fermentation time by half. Through combined efforts of reinforcing the pathway of xylose catabolism and elevating the fermentation temperature, XUSEA achieved simultaneous co-fermentation of lignocellulosic hydrolysates, composed of 39.6 g L-1 glucose and 23.1 g L-1 xylose, within 24 h producing 30.1 g L-1 ethanol with a yield of 0.48 g g-1. Conclusions:Owing to its superior co-fermentation performance and ability for further engineering, XUSEA has potential as a platform in a lignocellulosic biorefinery toward realizing a more economical and sustainable process for large-scale bioethanol production.
Project description:The development of biocatalysts capable of fermenting xylose, a five-carbon sugar abundant in lignocellulosic biomass, is a key step to achieve a viable production of second-generation ethanol. In this work, a robust industrial strain of Saccharomyces cerevisiae was modified by the addition of essential genes for pentose metabolism. Subsequently, taken through cycles of adaptive evolution with selection for optimal xylose utilization, strains could efficiently convert xylose to ethanol with a yield of about 0.46 g ethanol/g xylose. Though evolved independently, two strains carried shared mutations: amplification of the xylose isomerase gene and inactivation of ISU1, a gene encoding a scaffold protein involved in the assembly of iron-sulfur clusters. In addition, one of evolved strains carried a mutation in SSK2, a member of MAPKKK signaling pathway. In validation experiments, mutating ISU1 or SSK2 improved the ability to metabolize xylose of yeast cells without adaptive evolution, suggesting that these genes are key players in a regulatory network for xylose fermentation. Furthermore, addition of iron ion to the growth media improved xylose fermentation even by non-evolved cells. Our results provide promising new targets for metabolic engineering of C5-yeasts and point to iron as a potential new additive for improvement of second-generation ethanol production.
Project description:Microbial production of fuels and chemicals from lignocellulosic biomass provides promising biorenewable alternatives to the conventional petroleum-based products. However, heterogeneous sugar composition of lignocellulosic biomass hinders efficient microbial conversion due to carbon catabolite repression. The most abundant sugar monomers in lignocellulosic biomass materials are glucose and xylose. Although industrial Escherichia coli strains efficiently use glucose, their ability to use xylose is often repressed in the presence of glucose. Here we independently evolved three E. coli strains from the same ancestor to achieve high efficiency for xylose fermentation. Each evolved strain has a point mutation in a transcriptional activator for xylose catabolic operons, either CRP or XylR, and these mutations are demonstrated to enhance xylose fermentation by allelic replacements. Identified XylR variants (R121C and P363S) have a higher affinity to their DNA binding sites, leading to a xylose catabolic activation independent of catabolite repression control. Upon introducing these amino acid substitutions into the E. coli D-lactate producer TG114, 94% of a glucose-xylose mixture (50 g?L-1 each) was used in mineral salt media that led to a 50% increase in product titer after 96 h of fermentation. The two amino acid substitutions in XylR enhance xylose utilization and release glucose-induced repression in different E. coli hosts, including wild type, suggesting its potential wide application in industrial E. coli biocatalysts.