Xylose-fermenting Yeast Pichia Stipitis Research Articles

Optimization of the Protoplast Fusion Conditions of Saccharomyces cerevisiae and Pichia stipitis for Improvement of Bioethanol Production from Biomass

Bioethanol production using lignocellulosic biomass has gained increased attention because of the abundant supply of this biomass. Saccharomyces cerevisiae is a commonly used microorganism for ethanol production. Nevertheless, S. cerevisiae can not ferment xylose, the second most abundant sugar in plant tissues. In this study, protoplast fusion with the xylose-fermenting yeast Pichia stipitis was performed to improve bioethanol production from biomass. The protoplast formation of S. cerevisiae and P. stipitis (ATCC 58785) cells was achieved using zymolase 20T. The effects of zymolase concentration, enzymatic treatment time and osmotic stabilizers were further investigated. The optimal parameter for the protoplast release of S. cerevisiae and P. stipitis included 500 μg μL–1 zymolase for 60 min and 750 μg μL–1 zymolase for 120 min, respectively. The maximum protoplast formation ratios were 98.48 and 84.42% for S. cerevisiae and P. stipitis, respectively, with 1 mol L–1 sorbitol as the osmotic stabilizer. About 4×106 mL–1 protoplasts from S. cerevisiae and P. stipitis were isolated. Protoplast fusion frequency was determined using polyethylene glycol (PEG) as fusogen. The optimized fusion conditions of S. cerevisiae protoplasts with P. stipitis required 35% (w/v) PEG 6000, 10 mM CaCl2 level and 30 min of fusion time. The protoplast fusion rate was 52.21% under the optimized fusion condition.

Fermentation of the Straw Material Paja Brava by the Yeast <i>Pichia stipitis</i> in a Simultaneous Saccharification and Fermentation Process

Paja Brava is a native South American grass with a high carbohydrate content. In the current work, the potential of using this feedstock for ethanol production using a simultaneous saccharification and fermentation (SSF) process with the xylose-fermenting yeast Pichia stipitis (Scheffersomyces stipitis) CBS6054 was investigated. The straw material was subjected to SO2 catalyzed steam pretreatment at 200°C and 5 min residence time, which resulted in a solubilization of pentose sugars (mainly xylose) of 64% with only minor amounts of degradation products. The obtained material, including the pretreatment liquid, was subsequently hydrolyzed and fermented in an SSF process at microaerobic conditions using either a batch or a fed-batch process at a total water-insoluble solids loading of 10%. Overall yields of ethanol based on all available sugars of 0.24 g/g and 0.27 g/g were obtained for batch and fed-batch mode of operation, respectively. The higher yield in the fed-batch process coincided with a higher degree of conversion of the sugars in the liquid medium, in particular of arabinose, for which the conversion was doubled (from 48% to 97%).

Inverting character of family GH115 α-glucuronidases

α-Glucuronidases of glycoside hydrolase family 115 of the xylose-fermenting yeast Pichia stipitis and wood-destroying fungus Schizophyllum commune liberate 4- O-methyl- d-glucuronic acid residues from aldouronic acids and glucuronoxylan. The specific activities of both enzymes depended on polymerization degree of the acidic xylooligosaccharides and were inhibited by linear β-1,4-xylooligosaccharides. These results suggest interaction of the enzyme with several xylopyranosyl residues of the xylan main chain. Using 1H NMR spectroscopy and reduced aldopentaouronic acid (MeGlcA 3Xyl 4-ol) as a substrate, it was found that both enzymes are inverting glycoside hydrolases releasing 4- O-methyl- d-glucuronic acid (MeGlcA) as its β-anomer.

A novel family of hemicellulolytic α-glucuronidase

Investigation of the xylanolytic enzyme system of the xylose-fermenting yeast Pichia stipitis resulted in the discovery of an extracellular α-glucuronidase efficiently debranching hardwood glucuronoxylan. This activity is not exhibited by more extensively investigated α-glucuronidases of glycoside hydrolase (GH) family 67, operating on substrates in which the uronic acid is linked to the non-reducing xylopyranosyl residues of main chain fragments. The N-terminus of the purified enzyme corresponded exactly to the P. stipitis gene ABN67901 coding for a protein of unknown function. BLAST search revealed the presence of similar genes in genomes of other microorganisms. These results lead to the emergence of a new family of α-glucuronidases.

Reconstruction of genome scale metabolic model for naturally xylose fermenting yeast Pichia stipitis

Reconstruction of genome scale metabolic model for naturally xylose fermenting yeast Pichia stipitis

Xylose and cellobiose fermentation to ethanol by the thermotolerant methylotrophic yeast

Wild-type strains of the thermotolerant methylotrophic yeast Hansenula polymorpha are able to ferment glucose, cellobiose and xylose to ethanol. H. polymorpha most actively fermented sugars to ethanol at 37 degrees C, whereas the well-known xylose-fermenting yeast Pichia stipitis could not effectively ferment carbon substrates at this temperature. H. polymorpha even could ferment both glucose and xylose up to 45 degrees C. This species appeared to be more ethanol tolerant than P. stipitis but more susceptible than Saccharomyces cerevisiae. A riboflavin-deficient mutant of H. polymorpha increased its ethanol productivity from glucose and xylose under suboptimal supply with riboflavin. Mutants of H. polymorpha defective in alcohol dehydrogenase activity produced lower amounts of ethanol from glucose, whereas levels of ethanol production from xylose were identical for the wild-type strain and the alcohol dehydrogenase-defective mutant.

Differential expression of the Trichoderma reesei beta-xylanase II (xyn2) gene in the xylose-fermenting yeast Pichia stipitis.

The transcriptional control of two native promoters and one heterologous promoter and the production of a heterologous protein from these promoters were evaluated in the xylose-fermenting yeast Pichia stipitis cultivated on xylose and glucose as carbon sources, using the beta-xylanase II xyn2 gene of Trichoderma reesei. The xyn2 gene open reading frame was fused to the P. stipitis xylose reductase gene (XYL1) promoter, the P. stipitis transketolase gene (TKL) promoter and the Saccharomyces cerevisiae phosphoglycerate kinase gene (PGKI) promoter DNA sequences on episomal plasmids. The plasmids were transformed into Pichia stipitis and gene expression and beta-xylanase production monitored. The XYL1 promoter was shown to be inducible in the presence of xylose, as xyn2 transcription and beta-xylanase activity could be measured when the recombinant strain was cultivated on xylose but not when it was cultivated on glucose. TKL promoter expression was found to be constitutive when either glucose or xylose was used as sole carbon source. The PGK1 promoter did not promote xyn2 transcription in P. stipitis. The molecular size of the recombinant Xyn2 protein produced by P. stipitis was 20.7 kDa, which is similar to that of the native T. reesei Xyn2 protein. This indicates no or minimal glycosylation of the recombinant protein. The recombinant xyn2-expressing strain also yielded twice the amount of biomass yielded by the control strain when cultivated in medium containing 1% birchwood xylan as sole carbon source.

Development of xylose-fermenting yeast Pichia stipitis for ethanol production through adaptation on hardwood hemicellulose acid prehydrolysate.

The objective of this study was to develop a mutant from Pichia stipitis NRRL Y-7124, tolerant of high concentrations of acetic acid and other inhibitory components present in acid hydrolysates, to improve ethanol yield and productivity. The mutant was developed through adaptation in acid hydrolysate supplemented with nutrients and minerals at 30 +/- 0.5 degrees C. When it was tested for its ability to ferment acid hydrolysate, it showed shorter fermentation time, better tolerance to acid and could ferment at lower pH. The ethanol yield (Yp/s) and productivity (Qp) were increased 1.6- and 2.1-fold, respectively. The development of a mutant and its tolerance to acetic acid present in hydrolysates is described. The selected mutant is capable of fermenting both hexoses and pentoses present in hydrolysate at lower pH in comparison with the parent strain. The mutant could play a significant role in reducing environmental pollution by using sugars present in pulp mill effluent and, at the same time, could produce a marketable liquid fuel ethanol.

Transcriptional control of ADH genes in the xylose-fermenting yeast Pichia stipitis.

We studied the expression of the genes encoding group I alcohol dehydrogenases (PsADH1 and PsADH2) in the xylose-fermenting yeast Pichia stipitis CBS 6054. The cells expressed PsADH1 approximately 10 times higher under oxygen-limited conditions than under fully aerobic conditions when cultivated on xylose. Transcripts of PsADH2 were not detectable under either aeration condition. We used a PsADH1::lacZ fusion to monitor PsADH1 expression and found that expression increased as oxygen decreased. The level of PsADH1 transcript was repressed about 10-fold in cells grown in the presence of heme under oxygen-limited conditions. Concomitantly with the induction of PsADH1, PsCYC1 expression was repressed. These results indicate that oxygen availability regulates PsADH1 expression and that regulation may be mediated by heme. The regulation of PsADH2 expression was also examined in other genetic backgrounds. Disruption of PsADH1 dramatically increased PsADH2 expression on nonfermentable carbon sources under fully aerobic conditions, indicating that the expression of PsADH2 is subject to feedback regulation under these conditions.

The molecular genetics of hexose transport in yeasts.

Transport across the plasma membrane is the first, obligatory step of hexose utilization. In yeast cells the uptake of hexoses is mediated by a large family of related transporter proteins. In baker's yeast Saccharomyces cerevisiae the genes of 20 different hexose transporter-related proteins have been identified. Six of these transmembrane proteins mediate the metabolically relevant uptake of glucose, fructose and mannose for growth, two others catalyze the transport of only small amounts of these sugars, one protein is a galactose transporter but also able to transport glucose, two transporters act as glucose sensors, two others are involved in the pleiotropic drug resistance process, and the functions of the remaining hexose transporter-related proteins are not yet known. The catabolic hexose transporters exhibit different affinities for their substrates, and expression of their corresponding genes is controlled by the glucose sensors according to the availability of carbon sources. In contrast, milk yeast Kluyveromyces lactis contains only a few different hexose transporters. Genes of other monosaccharide transporter-related proteins have been found in fission yeast Schizosaccharomyces pombe and in the xylose-fermenting yeast Pichia stipitis. However, the molecular genetics of hexose transport in many other yeasts remains to be established. The further characterization of this multigene family of hexose transporters should help to elucidate the role of transport in yeast sugar metabolism.

Fusion between protoplasts of Pichia stipitis and isolated filamentous fungi nuclei

Nuclei were isolated from mycelium of the filamentous fungi Fusarium moniliforme and Trichoderma reesei, and fused with protoplasts of the xylose-fermenting yeast Pichia stipitis using PEG as the fusogenic agent. Hybrids which were morphologically yeastlike and able to hydrolyze xylan showed a similar chromosomal pattern to the parental yeast when clamped homogeneous electric field (CHEF) electrophoresis was done. These hybrids were isolated and their xylanase activity determined quantitatively. Xylanase activity in the parental fungal species was maximum at 96 h growth; it reached 796 nkat ml −1 in the culture medium for F. moniliforme and 898 nkat ml −1 at 120 h growth for T. reesei. Xylanase activity in one of the hybrids reached 350 nkat ml −1 in 9 h. P. stipitis itself showed no xylanase activity.

Characterization of the Genetic System of the Xylose-Fermenting Yeast Pichia stipitis

High mutant frequencies indicated that the wild-type strains of Pichia stipitis are haploid. Sporulation ability of these clones pointed to a homothallic life cycle. Mating was induced by cultivation under nutritionally poor conditions on malt extract medium. Conjugation was followed immediately by sporulation. However, hybrids could be rescued by transferring the nascent zygotes to complete medium before meiosis had started. Under rich nutritional conditions, hybrids were mitotically stable and did not sporulate. The segregation pattern of auxotrophic markers of diploid zygotes indicated regular meiosis, although asci contained preferentially spore dyads.

Peculiarities of the regulation of fermentation and respiration in the crabtree-negative, xylose-fermenting yeast Pichia stipitis.

The respiration of Pichia stipitis was not repressed by either high concentrations of fermentable sugars or oxygen limitation. Fermentation was not induced by high sugar concentrations, but was inactivated by aerobic conditions. The activity of pyruvate dehydrogenase was constitutive. In contrast, pyruvate decarboxylase, alcohol dehydrogenase, and aldehyde dehydrogenase were induced by a reduction in the oxygen tension. It was demonstrated that in P. stipitis, the pyruvate decarboxylase is not induced by a signal from glycolysis. Contrary to Saccharomyces cerevisiae, the pyruvate decarboxylase was not inhibited by phosphate.

Increased xylose reductase activity in the xylose-fermenting yeastPichia stipitis by overexpression ofXYL1

Increased xylose reductase activity in the xylose-fermenting yeastPichia stipitis by overexpression ofXYL1

High-efficiency transformation of Pichia stipitis based on its URA3 gene and a homologous autonomous replication sequence, ARS2

This paper describes the first high-efficiency transformation system for the xylose-fermenting yeast Pichia stipitis. The system includes integrating and autonomously replicating plasmids based on the gene for orotidine-5'-phosphate decarboxylase (URA3) and an autonomous replicating sequence (ARS) element (ARS2) isolated from P. stipitis CBS 6054. Ura- auxotrophs were obtained by selecting for resistance to 5-fluoroorotic acid and were identified as ura3 mutants by transformation with P. stipitis URA3. P. stipitis URA3 was cloned by its homology to Saccharomyces cerevisiae URA3, with which it is 69% identical in the coding region. P. stipitis ARS elements were cloned functionally through plasmid rescue. These sequences confer autonomous replication when cloned into vectors bearing the P. stipitis URA3 gene. P. stipitis ARS2 has features similar to those of the consensus ARS of S. cerevisiae and other ARS elements. Circular plasmids bearing the P. stipitis URA3 gene with various amounts of flanking sequences produced 600 to 8,600 Ura+ transformants per micrograms of DNA by electroporation. Most transformants obtained with circular vectors arose without integration of vector sequences. One vector yielded 5,200 to 12,500 Ura+ transformants per micrograms of DNA after it was linearized at various restriction enzyme sites within the P. stipitis URA3 insert. Transformants arising from linearized vectors produced stable integrants, and integration events were site specific for the genomic ura3 in 20% of the transformants examined. Plasmids bearing the P. stipitis URA3 gene and ARS2 element produced more than 30,000 transformants per micrograms of plasmid DNA. Autonomously replicating plasmids were stable for at least 50 generations in selection medium and were present at an average of 10 copies per nucleus.

Fermentation of lignocellulosic sugars to ethanol: Selection of mutants of Pichia stipitis affected for d-glucose utilization

Mutants of the xylose-fermenting yeast Pichia stipitis exhibiting a decreased ability to convert glucose to ethanol were obtained by mutagenesis with UV light. Mutants were isolated by replica-plating onto complete medium containing either d- galactose or d- glucose as carbon source. Mutants showing a reduction of growth on glucose were further tested for their ability to still use xylose. Finally, the kinetic behavior of six mutant strains grown on glucose and xylose is presented.

Genetic transformation of xylose-fermenting yeastPichia stipitis

A plasmid-mediated transformation system has been developed for the xylose-fermenting yeast Pichia stipitis. We found that plasmid vectors containing the Saccharomyces cerevisiae 2 mu replicon and the kanamycin resistance gene (KmR) could be introduced into the Pichia cells and maintained as extrachromosomal elements. Pichia transformants containing such vectors will be resistant to the antibiotic geneticin that can be inactivated by the protein product of KmR. Plasmids identical to those used for transformation can be recovered from the Pichia transformants. Protocols for transformation of P. stipitis by the CaCl2-polyethylene glycol-protoplast process or by direct electroporation of intact Pichia cells have both been developed.

Purification and properties of the NAD +-xylitol-dehydrogenase from the yeast Pichia stipitis

Cell-free extracts of the xylose fermenting yeast Pichia stipitis exhibited xylitol dehydrogenase activity with NAD + and NADP +. During the purification step on DEAE-sephadex A-50 a NAD +-dependent xylitol dehydrogenase could be separated from a NADP +-dependent. The NAD +-xylitol dehydrogenase was further purified to electrophoretic homogeneity via gel and affinity chromatography. The purified enzyme was most active at pH 9 and 35°C. Its molecular weight was determined to be 63,000 dalton by Sephadex G-200 column chromatography, and that of its subunit was 32,000 dalton by sodium dodecyl sulphate polyacrylamide gel electrophoresis. From the results of substrate specificity, the enzyme should be named l-iditol:NAD +-5-oxidoreductase (EC 1.1.1.14, sorbitol dehydrogenase).

Transport of xylose and glucose in the xylose-fermenting yeast Pichia stipitis

A low-affinity and a high-affinity sylose proton symport operated simultaneously in both starved and non-starved cells of Pichia stipitis. Glucose competed with xylose for transport by the low-affinity system and inhibited xylose transport by the high-affinity system non-competitively. The low affinity system was subject to substrate inhibition when glucose but not when xylose was the substrate. The differences between the characteristics of monosaccharide transport by Pichia stipitis and its imperfect state, Candida shehatae, are discussed.

Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis.

Xylose reductase from the xylose-fermenting yeast Pichia stipitis was purified to electrophoretic and spectral homogeneity via ion-exchange, affinity and high-performance gel chromatography. The enzyme was active with various aldose substrates, such as DL-glyceraldehyde, L-arabinose, D-xylose, D-ribose, D-galactose and D-glucose. Hence the xylose reductase of Pichia stipitis is an aldose reductase (EC 1.1.1.21). Unlike all aldose reductases characterized so far, the enzyme from this yeast was active with both NADPH and NADH as coenzyme. The activity with NADH was approx. 70% of that with NADPH for the various aldose substrates. NADP+ was a potent inhibitor of both the NADPH- and NADH-linked xylose reduction, whereas NAD+ showed strong inhibition only with the NADH-linked reaction. These results are discussed in the context of the possible use of Pichia stipitis and similar yeasts for the anaerobic conversion of xylose into ethanol.