Existence of Cyanide-Insensitive Respiration in the Yeast Pichia stipitis and Its Possible Influence on Product Formation during Xylose Utilization

A cyanide-insensitive and salicyl hydroxamic acid-sensitive respiration (CIR) was found in the yeast Pichia stipitis in contrast to Candida utilis, Pachysolen tannophilus, and Saccharomyces cerevisiae. During xylose utilization in the presence of either salicyl hydroxamic acid or cyanide, P. stipitis formed xylitol, arabitol, and ribitol. The existence of CIR is discussed in terms of a redox sink preventing xylitol formation in P. stipitis.     

Activity of the key enzymes in xylose-assimilating yeasts at different rates of oxygen transfer to the fermentation medium

The activities of xylitol dehydrogenase and xylose reductase in the yeasts Candida shehatae, C. didensiae, C. intermediae, C. tropicalis, Kluyveromyces marxianus, Pichia stipitis, P. guillermondii, Pachysolen tannophilus, and Torulopsis molishiama were studied at different oxygen transfer rates (OTRs) to the fermentation medium (0, 5, and 140 mmol O2/(1 h)). The activities of these enzymes were maximum in the yeasts P. stipitis and C. shehatae. The xylitol dehydrogenase of all the yeasts was NAD-dependent, irrespective of the intensity of aeration. The xylose reductase of the yeasts C. didensiae, C. intermediae, C. tropicalis, Kl. marxianus, P. guillermondii, and T. molishiama was NADPH-dependent, whereas the xylose reductase of P. stipitis, C. shehatae, and Pa. tannophilus was specific for both NADPH and NADH. The effect of OTR on the activities of the different forms of xylitol dehydrogenase and xylose reductase in the xylose-assimilating yeasts is discussed.     

Induction of Xylose Reductase and Xylitol Dehydrogenase Activities in Pachysolen tannophilus and Pichia stipitis on Mixed Sugars

The induction of xylose reductase and xylitol dehydrogenase activities on mixed sugars was investigated in the yeasts Pachysolen tannophilus and Pichia stipitis. Enzyme activities induced on d-xylose served as the controls. In both yeasts, d-glucose, d-mannose, and 2-deoxyglucose inhibited enzyme induction by d-xylose to various degrees. Cellobiose, l-arabinose, and d-galactose were not inhibitory. In liquid batch culture, P. tannophilus utilized d-glucose and d-mannose rapidly and preferentially over d-xylose, while d-galactose consumption was poor and lagged behind that of the pentose sugar. In P. stipitis, all three hexoses were used preferentially over d-xylose. The results showed that the repressibility of xylose reductase and xylitol dehydrogenase may limit the potential of yeast fermentation of pentose sugars in hydrolysates of lignocellulosic substrates.     

Factors influencing the utilisation of l-malate by yeasts

The utilisation of L-malate and the effect of glucose concentration on malate utilisation under semi-anaerobic conditions were investigated in three yeasts unable to grow on malate as sole carbon source (Saccharomyces cerevisiae, Schizosaccharomyces malidevorans, Zygosaccharomyces bailii) and two yeasts able to utilise the TCA cycle intermediate as sole carbon source (Pichia stipitis and Pachysolen tannophilus). Utilisation of malate by both Schiz. malidevorans and Z. bailii was reduced at high and low levels of glucose. In the absence of glucose, P. stipitis and Pa. tannophilus utilised malate rapidly; however, their utilisation was drastically reduced in the presence of glucose, suggesting that malate utilisation is under catabolite repression.     

Fluorescence measurements under aerobic and anaerobic conditions on Pichia stipitis,Pachysolen tannophilus and Candida utilis grown on D-xylose

Batch growth of the yeasts Candida utilis, Pachysolen tannophilus and Pichia stipitis on 1% D-xylose was monitored using a commercial fluorosensor with an excitation wavelength of 340 nm and a detection wavelength of 460 nm. Step changes in oxygen concentration were made and in the presence of 0.3 g/l of xylose, step changes from aerobic to anaerobic conditions resulted in an increase of the fluorescence level by about 40% for the non-fermentative yeast C. utilis. However, the increases of the fluorescence levels for P. tannophilus and P. stipitis stayed below 10%. These measurements indicate better control of (or better redox balance for) intracellular NADH concentration in P. tannophilus and P. stipitis than in C. utilis.List of Symbols F NFU fluorescence - F ₀ NFU initial fluorescence - F NFU final fluorescence difference - t s time - s time constant     

Repression of xylose-specific enzymes by ethanol in Scheffersomyces (Pichia) stipitis and utility of repitching xylose-grown populations to eliminate diauxic lag

During the fermentation of lignocellulosic hydrolyzates to ethanol by native pentose-fermenting yeasts such as Scheffersomyces (Pichia) stipitis NRRL Y-7124 (CBS 5773) and Pachysolen tannophilus NRRL Y-2460, the switch from glucose to xylose uptake results in a diauxic lag unless process strategies to prevent this are applied. When yeast were grown on glucose and resuspended in mixed sugars, the length of this lag was observed to be a function of the glucose concentration consumed (and consequently, the ethanol concentration accumulated) prior to the switch from glucose to xylose fermentation. At glucose concentrations of 95 g/L, the switch to xylose utilization was severely stalled such that efficient xylose fermentation could not occur. Further investigation focused on the impact of ethanol on cellular xylose transport and the induction and maintenance of xylose reductase and xylitol dehydrogenase activities when large cell populations of S. stipitis NRRL Y-7124 were pre-grown on glucose or xylose and then presented mixtures of glucose and xylose for fermentation. Ethanol concentrations around 50 g/L fully repressed enzyme induction although xylose transport into the cells was observed to be occurring. Increasing degrees of repression were documented between 15 and 45 g/L ethanol. Repitched cell populations grown on xylose resulted in faster fermentation rates, particularly on xylose but also on glucose, and eliminated diauxic lag and stalling during mixed sugar conversion by P. tannophilus or S. stipitis, despite ethanol accumulations in the 60 or 70 g/L range, respectively. The process strategy of priming cells on xylose was key to the successful utilization of high mixed sugar concentrations because specific enzymes for xylose utilization could be induced before ethanol concentration accumulated to an inhibitory level.     

The activity of xylose reductase and xylitol dehydrogenase in yeasts

The activity and the cofactor specificity of xylose reductase and xylitol dehydrogenase were studied in extracts of yeasts from the genera Candida, Kluyveromyces, Pachysolen, Pichia, and Torulopsis grown under microaerobic conditions. It was found that xylitol dehydrogenase in all of the yeast species studied is specific for NAD+; xylose reductase in the xylitol-producing species C. didensiae, C. intermediae, C. parapsilosis, C. silvanorum, C. tropicalis, Kl. fragilis, Kl. marxianus, P. guillermondii, and T. molishiama is specific for NADPH; and xylose reductase in the ethanol-producing species P. stipitis, C. shehatae, and Pa. tannophilus is specific for both NADPH and NADH.     

Application of Saccharomyces cerevisiae and Pichia stipitis karyoductants to the production of ethanol from xylose

Karyoductants of Saccharomyces cerevisiae V30 and Pichia stipitis CCY 39501 with the ability to ferment D-xylose to ethanol were isolated. The ability of these isolates to assimilate different sugars, ethanol tolerance and ethanol production from D-xylose was investigated. Karyoductants didn't grow on starch, lactose and cellobiose, like S. cerevisiae, but showed good growth on xylose and L-arabinose, like P. stipitis. All isolates fermented xylose to ethanol slower than P. stipitis and with lower yields, 0.09 - 0.16 g/g. They secreted also about 3.4 - 7.1 g/dm3 of xylitol to the culture medium (P. stipitis only 0.06 g/dm3). The karyoductants showed an average tolerance to ethanol when compared with the parent strains and fermented glucose in the presence of 6% alcohol whereas parent strain S. cerevisiae and P. stipitis showed exogenic ethanol tolerance of 9% and 3%, respectively.     

Ethanol production from d-galactose and glycerol by Pachysolen tannophilus

Pachysolen tannophilus has recently been shown to be able to convert d-xylose, a pentose, to ethanol. Previously, d-xylose had been considered to be nonfermentable by yeasts. The present study shows that the organism can be used to obtain ethanol from other carbohydrates previously considered as nonfermentable, either by P. tannophilus in particular, d-galactose, or by yeasts in general, glycerol. Such identification for d-galactose allows P. tannophilus to be considered for fermentation of four of the five major plant monosaccharides: d-glucose, d-mannose, d-galactose and d-xylose. The ability to ferment glycerol is of potential use, in part, for the conversion of glycerol derived from algae into ethanol.     

Alcoholic Fermentation of d-Xylose by Yeasts

Type strains of 200 species of yeasts able to ferment glucose and grow on xylose were screened for fermentation of d-xylose. In most of the strains tested, ethanol production was negligible. Nineteen were found to produce between 0.1 and 1.0 g of ethanol per liter. Strains of the following species produce more than 1 g of ethanol per liter in the fermentation test with 2% xylose: Brettanomyces naardenensis, Candida shehatae, Candida tenuis, Pachysolen tannophilus, Pichia segobiensis, and Pichia stipitis. Subsequent screening of these yeasts for their capacity to ferment d-cellobiose revealed that only Candida tenuis CBS 4435 was a good fermenter of both xylose and cellobiose under the test conditions used.     

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.     

The D-xylose fermenting capacities of immobilizedPichia stipitis andPachysolen tannophilus

Summary The yeastsP. stipitis NRRL Y-7124 andP. tannophilus NRRL Y-2460 were entrapped in -carrageenan beads and used for repeated batch fermentation of D-xylose, in a series of four reactors. The operating conditions finally chosen gave an oxygen coefficient (K_La) of 0.83 min^–1, as measured by the sulphite method. Ethanol yields were 0.40 g/g forP. stipitis and 0.36 g/g forP. tannophilus (respectively 78.4% and 70.5% of the theoretical yields). In spite of its lower retention by the gel,P. stipitis exhibited greater fermenting capacities thanP. tannophilus.     

Dilute-acid hydrolysis for fermentation of the Bolivian straw material Paja Brava

Hydrolysis of the straw material Paja Brava, a sturdy grass characteristic for the high plains of Bolivia, was studied in order to find suitable conditions for hydrolysis of the hemicellulose and cellulose parts. Dried Paja Brava material was pre-steamed, impregnated with dilute sulfuric acid (0.5% or 1.0% by wt), and subsequently hydrolyzed in a reactor at temperatures between 170 and 230 degrees C for a reaction time between 3 and 10 min. The highest yield of xylose (indicating efficient hydrolysis of hemicellulose) were found at a temperature of 190 degrees C, and a reaction time of 5-10 min, whereas considerably higher temperatures (230 degrees C) were needed for hydrolysis of cellulose. Fermentability of hemicellulose hydrolyzates was tested using the xylose-fermenting yeast species Pichia stipitis, Candida shehatae and Pachysolen tannophilus. The fermentability of hydrolyzates decreased strongly for hydrolyzates produced at temperatures higher than 200 degrees C.     

Utilization of Xylan by Yeasts and Its Conversion to Ethanol by Pichia stipitis Strains

Yeasts able to grow on d-xylose were screened for the ability to hydrolyze xylan. Xylanase activity was found to be rare; a total of only 19 of more than 250 strains yielded a positive test result. The activity was localized largely in the genus Cryptococcus and in Pichia stipitis and its anamorph Candida shehatae. The ability to hydrolyze xylan was generally uncoupled from that to hydrolyze cellulose; only three of the xylan-positive strains also yielded a positive test for cellulolytic activity. Of the 19 xylanolytic strains, 2, P. stipitis CBS 5773 and CBS 5775, converted xylan into ethanol, with about 60% of a theoretical yield computed on the basis of the amount of d-xylose present originally that could be released by acid hydrolysis.     

Hybrid formation and copulation activity in the yeast Pachysolen tannophilus

The copulation activity and hybrid formation efficiency have been studied in the xylose-assimilating yeast Pachysolen tannophilus. It was shown that the presence of 2% D-glucose, 0.5% yeast extract, and 2% agarose in the growth medium provided for the highest frequencies of hybrid formation. Atypical hybrid cultures similar in morphophysiological characteristics to native haploid strains of P. tannophilus were revealed in the course of hybridization. The genesis mechanism of such cultures and the reasons for the restricted applicability of hybridological analysis to genetic studies of P. tannophilus are discussed.     

Phosphorus-31 and carbon-13 nuclear magnetic resonance studies of glucose and xylose metabolism in cell suspensions and agarose-immobilized cultures of Pichia stipitis and Saccharomyces cerevisiae.

The metabolism of glucose and xylose as a function of oxygenation in Pichia stipitis and Saccharomyces cerevisiae cell suspensions was studied by 31P and 13C nuclear magnetic resonance spectroscopy. The rate of both glucose and xylose metabolism was slightly higher and the production of ethanol was slightly lower in aerobic than in anoxic cell suspensions of P. stipitis. As well, the cytoplasmic pH of oxygenated cells was more alkaline than that of nonoxygenated cells. In contrast, in S. cerevisiae, the intracellular pH and the rate of glucose metabolism and ethanol production were the same under aerobic and anoxic conditions. Agarose-immobilized Pichia stipitis was able to metabolize xylose or glucose for 24 to 60 h at rates and with theoretical yields of ethanol similar to those obtained with anoxic cell suspensions. Cell growth within the beads, however, was severely compromised. The intracellular pH [pH(int)] of the entrapped cells fell to more acidic pH values in the course of the perfusions relative to corresponding cell suspensions. Of importance was the observation that no enhancement in the rate of carbohydrate metabolism occurred in response to changes in the pH(int) value. In contrast to P. stipitis, agarose-immobilized Saccharomyces cerevisiae showed a dramatic twofold increase in its ability to metabolize glucose in the immobilized state relative to cell suspensions. This strain was also able to grow within the beads, although the doubling time for the entrapped cells was longer, by a factor of 2, than the value obtained for log-phase batch cultures. Initially, the pH(int) of the immobilized cells was more alkaline than was observed with the corresponding S. cerevisiae cell suspensions; however, over time, the intracellular pH became increasingly acidic. As with immobilized P. stipitis, however, the pH(int) did not play a key role in controlling the rate of glucose metabolism.     

Genome sequence of the lignocellulose-bioconverting and xylose-fermenting yeast Pichia stipitis

Xylose is a major constituent of plant lignocellulose, and its fermentation is important for the bioconversion of plant biomass to fuels and chemicals. Pichia stipitis is a well-studied, native xylose-fermenting yeast. The mechanism and regulation of xylose metabolism in P. stipitis have been characterized and genes from P. stipitis have been used to engineer xylose metabolism in Saccharomyces cerevisiae. We have sequenced and assembled the complete genome of P. stipitis. The sequence data have revealed unusual aspects of genome organization, numerous genes for bioconversion, a preliminary insight into regulation of central metabolic pathways and several examples of colocalized genes with related functions. The genome sequence provides insight into how P. stipitis regulates its redox balance while very efficiently fermenting xylose under microaerobic conditions.     

Cre/LoxP重组系统的改造及在树干毕赤酵母中的应用

为了实现在P.stipitis中进行无痕基因敲除,以Cre/LoxP系统为研究对象,首先通过同源重组构建尿嘧啶营养缺陷型树干毕赤酵母(ura3-);同时通过定点突变pSH47-Hpt质粒的hpt基因和cre基因,将CDS区CTG突变为TTG;最后以乙醛脱氢酶基因为靶基因,验证突变后的Cre/LoxP系统在P.stipitis进行无痕基因敲除的可行性。结果表明:本文在P.stipitis中成功使用潮霉素B抗性标记,经过修饰后的Cre/LoxP敲除系统能够在P.stipitis中无痕敲除目的基因,为后续研究P.stipitis功能基因和改造代谢途径提供了一种试验方法和筛选标记。