Comparison of Scheffersomyces stipitis strains CBS 5773 and CBS 6054 with regard to their xylose metabolism: implications for xylose fermentation.
ABSTRACT: The various strains of Scheffersomyces stipitis (Pichia stipitis) differ substantially with respect to their ability to ferment xylose into ethanol. Two P. stipitis strains CBS 5773 and CBS 6054 have been most often used in literature but comparison of their performance in xylose fermentation under identical conditions has not been reported so far. Conversion of xylose (22 g/L) by each of these P. stipitis strain was analyzed under anaerobic and microaerobic conditions. Ethanol yields of ?0.41 g/g were independent of strain and conditions used. Glycerol and acetate were formed in constant yields of 0.006 g/g and 0.02 g/g, respectively. Xylitol formation decreased from ?0.08 g/g to ?0.05 g/g upon switch from anaerobic to microaerobic conditions. Specific activities of enzymes of the two-step oxidoreductive xylose conversion pathway (xylose reductase and xylitol dehydrogenase) matched for both strains within limits of error. When xylose was offered at 76 g/L under microaerobic reaction conditions, ethanol yields were still high (0.37-0.39 g/g) for both strains even though the xylitol yields (0.12-0.13 g/g) were increased as compared to the conditions of low xylose concentration. P. stipitis strains CBS 5773 and CBS 6054 are therefore identical by the criteria selected and show useful performance during conversion of xylose into ethanol, irrespective of the supply of oxygen.
Project description:XYL3, which encodes a D-xylulokinase (EC 184.108.40.206), was isolated from Pichia stipitis CBS 6054 genomic DNA by using primers designed against conserved motifs. Disruption of XYL3 eliminated D-xylulokinase activity, but D-ribulokinase activity was still present. Southern analysis of P. stipitis genomic DNA with XYL3 as a probe confirmed the disruption and did not reveal additional related genes. Disruption of XYL3 stopped ethanol production from xylose, but the resulting mutant still assimilated xylose slowly and formed xylitol and arabinitol. These results indicate that XYL3 is critical for ethanol production from xylose but that P. stipitis has another pathway for xylose assimilation. Expression of XYL3 using its P. stipitis promoter increased Saccharomyces cerevisiae D-xylulose consumption threefold and enabled the transformants to produce ethanol from a mixture of xylose and xylulose, whereas the parental strain only accumulated xylitol. In vitro, D-xylulokinase activity in recombinant S. cerevisiae was sixfold higher with a multicopy than with a single-copy XYL3 plasmid, but ethanol production decreased with increased copy number. These results confirmed the function of XYL3 in S. cerevisiae.
Project description:Two genes coding for isozymes of alcohol dehydrogenase (ADH); designated PsADH1 and PsADH2, have been identified and isolated from Pichia stipitis CBS 6054 genomic DNA by Southern hybridization to Saccharomyces cerevisiae ADH genes, and their physiological roles have been characterized through disruption. The amino acid sequences of the PsADH1 and PsADH2 isozymes are 80.5% identical to one another and are 71.9 and 74.7% identical to the S. cerevisiae ADH1 protein. They also show a high level identity with the group I ADH proteins from Kluyveromyces lactis. The PsADH isozymes are presumably localized in the cytoplasm, as they do not possess the amino-terminal extension of mitochondrion-targeted ADHs. Gene disruption studies suggest that PsADH1 plays a major role in xylose fermentation because PsADH1 disruption results in a lower growth rate and profoundly greater accumulation of xylitol. Disruption of PsADH2 does not significantly affect ethanol production or aerobic growth on ethanol as long as PsADH1 is present. The PsADH1 and PsADH2 isozymes appear to be equivalent in the ability to convert ethanol to acetaldehyde, and either is sufficient to allow cell growth on ethanol. However, disruption of both genes blocks growth on ethanol. P. stipitis strains disrupted in either PsADH1 or PsADH2 still accumulate ethanol, although in different amounts, when grown on xylose under oxygen-limited conditions. The PsADH double disruptant, which is unable to grow on ethanol, still produces ethanol from xylose at about 13% of the rate seen in the parental strain. Thus, deletion of both PsADH1 and PsADH2 blocks ethanol respiration but not production, implying a separate path for fermentation.
Project description:Investigation of whole genome gene expression level changes in Pichia stipitis CBS 6054 grown aerobically in xylose, compared to the same strain grown aerobically in glucose. Overall design: A six array study using total RNA recovered from three separate cultures of Pichia stipitis CBS 6054 grown in glucose and three separate cultures of Pichia stipitis CBS 6054 grown in xylose. Each array measures the expression level of 374,100 probes (average probe length 53.6 +/- 4.1 nt) tiled across the Pichia stipitis CBS 6054 genome with a median spacing distance of 33 nt. During data processing, probes are filtered to include only those probes corresponding to annotated protein-coding genes.
Project description:<h4>Background</h4>This study is the first to investigate the Brazilian Amazonian Forest to identify new D-xylose-fermenting yeasts that might potentially be used in the production of ethanol from sugarcane bagasse hemicellulosic hydrolysates.<h4>Methodology/principal findings</h4>A total of 224 yeast strains were isolated from rotting wood samples collected in two Amazonian forest reserve sites. These samples were cultured in yeast nitrogen base (YNB)-D-xylose or YNB-xylan media. Candida tropicalis, Asterotremella humicola, Candida boidinii and Debaryomyces hansenii were the most frequently isolated yeasts. Among D-xylose-fermenting yeasts, six strains of Spathaspora passalidarum, two of Scheffersomyces stipitis, and representatives of five new species were identified. The new species included Candida amazonensis of the Scheffersomyces clade and Spathaspora sp. 1, Spathaspora sp. 2, Spathaspora sp. 3, and Candida sp. 1 of the Spathaspora clade. In fermentation assays using D-xylose (50 g/L) culture medium, S. passalidarum strains showed the highest ethanol yields (0.31 g/g to 0.37 g/g) and productivities (0.62 g/L · h to 0.75 g/L · h). Candida amazonensis exhibited a virtually complete D-xylose consumption and the highest xylitol yields (0.55 g/g to 0.59 g/g), with concentrations up to 25.2 g/L. The new Spathaspora species produced ethanol and/or xylitol in different concentrations as the main fermentation products. In sugarcane bagasse hemicellulosic fermentation assays, S. stipitis UFMG-XMD-15.2 generated the highest ethanol yield (0.34 g/g) and productivity (0.2 g/L · h), while the new species Spathaspora sp. 1 UFMG-XMD-16.2 and Spathaspora sp. 2 UFMG-XMD-23.2 were very good xylitol producers.<h4>Conclusions/significance</h4>This study demonstrates the promise of using new D-xylose-fermenting yeast strains from the Brazilian Amazonian Forest for ethanol or xylitol production from sugarcane bagasse hemicellulosic hydrolysates.
Project description:Investigation of whole genome gene expression level changes in Pichia stipitis CBS 6054 grown aerobically in xylose, compared to the same strain grown aerobically in glucose. A six array study using total RNA recovered from three separate cultures of Pichia stipitis CBS 6054 grown in glucose and three separate cultures of Pichia stipitis CBS 6054 grown in xylose. Each array measures the expression level of 374,100 probes (average probe length 53.6 +/- 4.1 nt) tiled across the Pichia stipitis CBS 6054 genome with a median spacing distance of 33 nt. During data processing, probes are filtered to include only those probes corresponding to annotated protein-coding genes.
Project description:Xylitol production was compared in fed batch fermentation by Saccharomyces cerevisiae strains overexpressing xylose reductase (XR) genes from Candida tropicalis, Pichia stipitis, Neurospora crassa, and an endogenous gene GRE3. The gene encoding a xylose specific transporter (SUT1) from P. stipitis was cloned to improve xylose transport and fed batch fermentation was used with glucose as a cosubstrate to regenerate NADPH. Xylitol yield was near theoretical for all the strains in fed batch fermentation. The highest volumetric (0.28 gL-1 h-1) and specific (34 mgg-1 h-1) xylitol productivities were obtained by the strain overexpressing GRE3 gene, while the control strain showed 7.2 mgg-1 h-1 specific productivity. The recombinant strains carrying XR from C. tropicalis, P. stipitis, and N. crassa produced xylitol with lower specific productivity of 14.3, 6.8, and 6.3 mgg-1 h-1, respectively, than GRE3 overexpressing strain. The glucose fed as cosubstrate was converted to biomass and ethanol, while xylose was only converted to xylitol. The efficiency of ethanol production was in the range of 38-45 % of the theoretical maximum for all the strains. Xylitol production from the non-detoxified corncob hemicellulosic hydrolysate by recombinant S. cerevisiae was reported for the first time. Xylitol productivity was found to be equivalent in the synthetic xylose as well as hemicellulosic hydrolysate-based media showing no inhibition on the S. cerevisiae due to the inhibitors present in the hydrolysate. A systematic evaluation of heterologous XRs and endogenous GRE3 genes was performed, and the strain overexpressing the endogenous GRE3 gene showed the best xylitol productivity.
Project description:The production of ethanol and other fuels and chemicals from lignocellulosic materials is dependent of efficient xylose conversion. Xylose fermentation capacity in yeasts is usually linked to xylose reductase (XR) accepting NADH as cofactor. The XR from Scheffersomyces stipitis, which is able to use NADH as cofactor but still prefers NADPH, has been used to generate recombinant xylose-fermenting Saccharomyces cerevisiae. Novel xylose-fermenting yeasts species, as those from the Spathaspora clade, have been described and are potential sources of novel genes to improve xylose fermentation in S. cerevisiae.Xylose fermentation by six strains from different Spathaspora species isolated in Brazil, plus the Sp. passalidarum type strain (CBS 10155(T)), was characterized under two oxygen-limited conditions. The best xylose-fermenting strains belong to the Sp. passalidarum species, and their highest ethanol titers, yields, and productivities were correlated to higher XR activity with NADH than with NADPH. Among the different Spathaspora species, Sp. passalidarum appears to be the sole harboring two XYL1 genes: XYL1.1, similar to the XYL1 found in other Spathaspora and yeast species and XYL1.2, with relatively higher expression level. XYL1.1p and XYL1.2p from Sp. passalidarum were expressed in S. cerevisiae TMB 3044 and XYL1.1p was confirmed to be strictly NADPH-dependent, while XYL1.2p to use both NADPH and NADH, with higher activity with the later. Recombinant S. cerevisiae strains expressing XYL1.1p did not show anaerobic growth in xylose medium. Under anaerobic xylose fermentation, S. cerevisiae TMB 3504, which expresses XYL1.2p from Sp. passalidarum, revealed significant higher ethanol yield and productivity than S. cerevisiae TMB 3422, which harbors XYL1p N272D from Sc. stipitis in the same isogenic background (0.40 vs 0.34 g gCDW (-1) and 0.33 vs 0.18 g gCDW (-1) h(-1), respectively).This work explored a new clade of xylose-fermenting yeasts (Spathaspora species) towards the engineering of S. cerevisiae for improved xylose fermentation. The new S. cerevisiae TMB 3504 displays higher XR activity with NADH than with NADPH, with consequent improved ethanol yield and productivity and low xylitol production. This meaningful advance in anaerobic xylose fermentation by recombinant S. cerevisiae (using the XR/XDH pathway) paves the way for the development of novel industrial pentose-fermenting strains.
Project description:As one of the best xylose utilization microorganisms, Scheffersomyces stipitis exhibits great potential for the efficient lignocellulosic biomass fermentation. Therefore, a comprehensive understanding of its unique physiological and metabolic characteristics is required to further improve its performance on cellulosic ethanol production.A constraint-based genome-scale metabolic model for S. stipitis CBS 6054 was developed on the basis of its genomic, transcriptomic and literature information. The model iTL885 consists of 885 genes, 870 metabolites, and 1240 reactions. During the reconstruction process, 36 putative sugar transporters were reannotated and the metabolisms of 7 sugars were illuminated. Essentiality study was conducted to predict essential genes on different growth media. Key factors affecting cell growth and ethanol formation were investigated by the use of constraint-based analysis. Furthermore, the uptake systems and metabolic routes of xylose were elucidated, and the optimization strategies for the overproduction of ethanol were proposed from both genetic and environmental perspectives.Systems biology modelling has proven to be a powerful tool for targeting metabolic changes. Thus, this systematic investigation of the metabolism of S. stipitis could be used as a starting point for future experiment designs aimed at identifying the metabolic bottlenecks of this important yeast.
Project description:The ascomycetes Candida albicans, Saccharomyces cerevisiae and Scheffersomyces stipitis metabolize the pentose sugar xylose very differently. S. cerevisiae fails to grow on xylose, while C. albicans can grow, and S. stipitis can both grow and ferment xylose to ethanol. However, all three species contain highly similar genes that encode potential xylose reductases and xylitol dehydrogenases required to convert xylose to xylulose, and xylulose supports the growth of all three fungi. We have created C. albicans strains deleted for the xylose reductase gene GRE3, the xylitol dehydrogenase gene XYL2, as well as the gre3 xyl2 double mutant. As expected, all the mutant strains cannot grow on xylose, while the single gre3 mutant can grow on xylitol. The gre3 and xyl2 mutants are efficiently complemented by the XYL1 and XYL2 from S. stipitis. Intriguingly, the S. cerevisiae GRE3 gene can complement the Cagre3 mutant, while the ScSOR1 gene can complement the Caxyl2 mutant, showing that S. cerevisiae contains the enzymatic capacity for converting xylose to xylulose. In addition, the gre3 xyl2 double mutant of C. albicans is effectively rescued by the xylose isomerase (XI) gene of either Piromyces or Orpinomyces, suggesting that the XI provides an alternative to the missing oxido-reductase functions in the mutant required for the xylose-xylulose conversion. Overall this work suggests that C. albicans strains engineered to lack essential steps for xylose metabolism can provide a platform for the analysis of xylose metabolism enzymes from a variety of species, and confirms that S. cerevisiae has the genetic potential to convert xylose to xylulose, although non-engineered strains cannot proliferate on xylose as the sole carbon source.
Project description: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 ?-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).