Identification of species in the genus Pichia by restriction of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal DNA gene

In this study, the variability within the ribosomal DNA region spanning the internal transcribed spacers ITS1 and ITS2 and the 5.8S gene (5.8S-ITS rDNA) was used to differentiate species in the genus Pichia. The 5.8S-ITS rDNA region was PCR-amplified and the PCR product digested with the enzymes CfoI, HinfI, and HaeIII. The variability in the size of the amplified product and in the restriction patterns enabled differentiation between species in the genus Pichia, and between Pichia species and yeast species from other genera in the Yeast-id database (). Moreover, the restriction fragment length polymorphism (RFLP) patterns of the 5.8S-ITS enabled misidentified strains to be detected and revealed genetic heterogeneity between strains within the Pichia membranifaciens and Pichia nakazawae species. Ultimately, the RFLP patterns of the 5.8S-ITS rDNA failed to differentiate between some Pichia and Candida species that could be distinguished on the basis of the sequence of the 5.8S-ITS rRNA region or the sequence of the D1/D2 domain of the 26S rDNA gene.     

Toward systems metabolic engineering of Aspergillus and Pichia species for the production of chemicals and biofuels

Recently genome sequence data have become available for Aspergillus and Pichia species of industrial interest. This has stimulated the use of systems biology approaches for large-scale analysis of the molecular and metabolic responses of Aspergillus and Pichia under defined conditions, which has resulted in much new biological information. Case-specific contextualization of this information has been performed using comparative and functional genomic tools. Genomics data are also the basis for constructing genome-scale metabolic models, and these models have helped in the contextualization of knowledge on the fundamental biology of Aspergillus and Pichia species. Furthermore, with the availability of these models, the engineering of Aspergillus and Pichia is moving from traditional approaches, such as random mutagenesis, to a systems metabolic engineering approach. Here we review the recent trends in systems biology of Aspergillus and Pichia species, highlighting the relevance of these developments for systems metabolic engineering of these organisms for the production of hydrolytic enzymes, biofuels and chemicals from biomass.     

Comparative analysis of glycogen and trehalose accumulation in methylotrophic and nonmethylotrophic yeasts

Trehalose and glycogen accumulate in certain yeast species when they are exposed to unfavorable growth conditions. Accumulations of these reserve carbohydrates in yeasts provide resistance to stress conditions. The results of this study indicate that certain Pichia species do not accumulate high levels of glycogen and trehalose under normal growth conditions. However, depending on the Pichia species, both saccharides accumulate at high levels when the Pichia cells are exposed to unfavorable or stress-inducing growth conditions. Growth in glycerol or methanol medium mostly led to trehalose accumulation in Pichia species tested in this study. It was shown that the metabolic pathways for glycogen and trehalose biosynthesis are present in Pichia species. However, it appears that the biosynthesis of trehalose and glycogen may be regulated in different manners in Pichia species than in the yeast S. cerevisiae. Published in Russian in Mikrobiologiya, 2006, Vol. 75, No. 6, pp. 737–741. The text was submitted by the author in English.     

Contribution à la systématique du genre Pichia

The presence of intracellular osidases, nitrite and nitrate reductases, vitamin requirements and GC content in the DNA, have been investigated for in 18 species of Pichia.According to their typical characters and their biocaracters, a pattern is suggested for the integration of these new species into the formerly existing groups of the genus Pichia. A new way of classifying the species of the genus Pichia is proposed.     

Pichia spartinae sp. n. from Louisiana marshland habitats

A new species ofPichia has been described.Pichia spartinae was one of the predominant yeasts isolated from the rhizosphere and tissue of oyster grass,Spartina alterniflora, in the Louisiana marshland. The species occurs in the environment in both the homo- and heterothallic state. This is the first species ofPichia in high densities in an estuarine locality.We wish to express our gratitude to Mr. J. C. Raadschelders for correcting the latin diagnosis.     

DNA relatedness among saturn-spored yeasts assigned to the generaWilliopsis andPichia

Saturn-spored species assigned to the generaWilliopsis andPichia were compared from extent of nuclear DNA complementarity. Of thePichia spp., four were recognized as distinct taxa:P. dispora, P. saitoi, P. zaruensis andPichia sp. nov. AmongWilliopsis spp., the following were accepted:W. californica, W. mucosa comb. nov.,W. pratensis, W. saturnus var.saturnus, W. saturnus var.mrakii comb. nov.,W. saturnus var.sargentensis comb. nov.,W. saturnus var.subsufficiens comb. nov. andWilliopsis sp. nov. The newPichia andWilliopsis species are described elsewhere. Moderate (36–68%) DNA relatedness was detected between the formerPichia sargentensis and varieties ofW. saturnus again demonstrating that nitrate assimilation is not a reliable criterion for separating yeast species.     

QUANTUM AND CONTINUOUS EVOLUTION OF DNA BASE COMPOSITION IN THE YEAST GENUS PICHIA

This paper investigates the noncontinuous nature and evolution of the base composition of nuclear DNA (expressed as mol% guanine + cytosine) in species of the yeast genus Pichia (sensu Kurtzman, 1984b). The pattern of change in the G + C contents in species of this genus, which range from about 27 to 52 mol%, was evaluated. When specifically those species of Pichia were analyzed that have evolved in necroses of cactus species and associated Drosophila, a periodic change in the G + C contents of approximately 3.0–3.2 mol% was detected by a “bootstrapping” method, Fourier analysis, and a nonlinear trigonometric model. Pichia species occurring in exudates of broad-leaved deciduous trees or associated Drosophila and substrates such as soil and water (“other”) showed a periodicity of 2.5–2.6 mol%, whereas species associated with conifers and associated bark beetles showed no significant periodicity. Periodicity in the most recent association (cactus and resident Drosophila) as compared to the lack of periodicity in the oldest association (conifer-beetle) may indicate mixed evolutionary processes. Low mol% G + C values appear more frequently in the relatively recent cactus and Drosophila-associated yeast species. In addition, low mol% G + C species do not display the ancestral bud-meiosis mode of sexual reproduction which occurs frequently in medium to high mol% G + C yeasts. It was found that the mol% G + C content of the Drosophila- and cactus-associated Pichia species is positively correlated with the number of compounds fermented or respired by these yeast species. Possible reasons for the periodic changes in mol% G + C content accompanying speciation include aneuploidy, allopolyploidy, the presence of nuclear plasmids, and regular differences in moderately repetitive portions of DNA. Since significant DNA complementarity is virtually limited to species within a relatively narrow G + C group, this suggests that there are at least two processes which alter the G + C content between species, one saltational and one continuous.     

Phylogenetic relationships among species ofWilliopsis andSaturnospora gen. nov. as determined from partial rRNA sequences

Phylogenetic relationships among those yeast species that form saturn-shaped ascospores and which are assigned to the generaWilliopsis andPichia were estimated from their extent of nucleotide sequence divergence in three regions of ribosomal RNA. ThePichia species (P. dispora, P. saitoi, P. zaruensis andP. sp. nov.) are a closely clustered group only distantly related toWilliopsis, and it is proposed that they be reassigned toSaturnospora gen. nov. The extent of divergence amongWilliopsis species (W. californica, W. mucosa, W. pratensis, W. saturnus andW. sp. nov.) is greater than that previously observed within other ascomycetous yeast genera.     

Pichia sorbitophila sp nov.

A new species of the genus Pichia has been described. The species was isolated from a 70 percent solution of d-sorbitol and is distinctly osmotophilic.     

Synonomy of the yeast generaHansenula andPichia demonstrated through comparisons of deoxyribonucleic acid relatedness

The relationship between the generaHansenula andPichia was examined through comparisons of DNA relatedness among phenotypically similar species.Hansenula minuta andPichia lindneri showed 75% DNA base sequence complementarity. In other comparisons,H. nonfermentans was found to share nearly 50% of its DNA sequences with bothH. minuta andP. lindneri. Because of the high degree of relatedness observed, it is proposed that ability to assimilate nitrate, the sole distinction betweenHansenula andPichia, is of insufficient taxonomic value for the reliable separation of either species or genera. Hat-spored species ofHansenula H. et P. Sydow 1919 are being transferred toPichia Hansen 1904. Species ofHansenula andPichia with Saturn-shaped ascospores will be transferred to the genusWilliopsis. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Two new species ofPichia isolated from slime fluxes of deciduous trees

Two new homothallic species ofPichia, associated with exudates ofPopulus trichocarpa andSalix sp. have been described.Pichia trehalophila sp.n. was isolated from slime exudates in two widely separatedPopulus trichocarpa trees andPichia salictaria sp.n. was isolated from two exudates ofSalix sp. The latter species was previously present as an unidentified strain ofPichia in the culture collection of the Centraalbureau voor Schimmelcultures and had been isolated from a patient in Germany. The natural habitat ofP. salictaria is considered to be in association with willow trees.     

The taxonomy of yeasts found in exudates of certain trees and other natural breeding sites of some species ofDrosophila

Summary A survey was made of the yeasts occurring in slime fluxes ofQuercus kellogii (black oak),Abies concolor (red fir) and in certain mushrooms and dead logs in the mountains of the Yosemite region of California. Most of the 134 isolates identified were found to be non-fermentative or poorly fermentative (fermenting glucose only and usually weakly). The isolates were placed in the following genera:Pichia (34),Debaryomyces (35), imperfect forms ofHansenula (39),Endomyces (3),Saccharomyces (2),Candida (11),Trichosporon (7),Torulopsis (1),Cryptococcus (1) andSporobolomyces (1). Four new species have been described,Pichia silvestris, Pichia quercibus, Pichia carsonii andDebaryomyces fluxorum. P. quercibus andP. carsonii are unusual representatives of the genusPichia in that they form a very primitive pseudomycelium and lack pellicles on malt extract. To accommodate such species an amended diagnosis of the genusPichia has been proposed byPhaff in an accompanying paper.Saccharomyces pastori, of which 12 isolates were obtained, has been transferred to the amended genusPichia asP. pastori (Guilliermond) nov. comb.     

Pichia besseyi sp. n.

A new species ofPichia has been described.Pichia besseyi was isolated from a marsh near the town of Bonaventure, Gaspé Peninsula, Quebec, Canada. The hemispheroidal shape of the ascospores of this species is particularly noteworthy.     

Differential toxinogenesis in the genusPichia detected by an anti-yeast killer toxin monoclonal antibody

The differential toxinogenesis of 25 isolates belonging to species of the potential yeast killer genusPichia that were previously classified in the genusHansenula was comparatively demonstrated by two serologic techniques (indirect immunofluorescence and double immunodiffusion) by using a monoclonal antibody against a yeast killer toxin produced by a selected strain ofPichia anomala (UCSC 25F). The killer phenotypes of thePichia isolates were evaluated by their ability to kill each other. The results, although of insufficient taxonomic value for a reliable separation of either species or genera, attest to the genomic heterogeneity for the killer character in the genusPichia as well as the presumptive dual killer/sensitive identity for each single isolate.     

Cloning and Expression of Fructosyl-amine Oxidase from Marine Yeast <Emphasis Type="Italic">Pichia</Emphasis> Species N1-1

The gene encoding the fructosyl-amine oxidase (FAOD) from the marine yeast Pichia sp. N1-1 was cloned and expressed in Escherichia coli. Partial amino acid sequence analysis of the Pichia sp. N1-1 FAOD allowed the design of oligonucleotide primers for the amplification of the gene by inverse polymerase chain reaction. The FAOD gene was found to be devoid of introns and to encode a 48-kDa protein composed of 429 amino acid residues. The FAD-binding consensus sequence GXGXXG and the FAD covalent attachment-site cysteine residue have been identified within the predicted amino acid sequence. Comparisons with the amino acid sequences of other eukaryotic FAODs showed only 30% to 40% identities, establishing that the isolated Pichia N1-1 gene encodes a unique FAOD. Recombinant FAOD expression levels in E. coli reached 0.48 U/mg of soluble protein, which is considerably greater than native expression levels by inducing Pichia sp. N1-1 with fructosyl-valine (f-Val). The kinetic properties of the recombinant enzyme were almost indistinguishable from those of the native enzyme. We previously reported on the construction of a number of effective Pichia sp. N1-1 FAOD-based biosensors for measuring f-Val, a model compound for glycated hemoglobin. The further development of these biosensor systems can now greatly benefit from protein engineering and recombinant expression of the FAOD from Pichia N1-1.Note: The previous online version (January 20, 2005) of this article appeared with the legends of Figures 1 and 2 transposed. This version contains the figures with their appropriate legends.     

Genomic studies on killer yeasts belonging to the genusPichia

Twenty-four species belonging to the genusPichia were investigated using restriction fragment length polymorphism (RFLP) and Southern blot hybridization of their genomic DNA.Saccharomyces cerevisiae, Kluyveromyces lactis, Williopsis mrakii andCandida albicans were also included in this study. The RFLP patterns were obtained from digestion of yeast DNA with several restriction endonuclease enzymes, and showed various bands with different mobility; in most isolates, the more deeply stained bands were species-specific. This observation was confirmed by the results obtained from Southern blot hybridization of theEcoRI andXhoI RFLP patterns withP. anomala UCSC 25F DNA, digested with the same enzymes, used as probes. These bands are likely to be ribosomal DNA as shown by hybridization of digested DNA from unrelated yeast species (S. cerevisiae, K. lactis andC. albicans). However, one hybridized band, located at 3.9–4.1 Kb, seems to be peculiar to thePichia species. Our study confirms the usefulness of molecular tools in studying genetic relatedness among yeasts.     

A proposal for amendment of the diagnosis of the genusPichia hansen

Summary An amended diagnosis has been given for the genusPichia Hansen to allow inclusion of species characterized by a lack of pellicle formation, by the absence or a strongly reduced pseudomycelium or by a combination of both properties. The following species fit the amended diagnosis ofPichia: P haplophila Shifrine et Phaff,P. carsonii Phaff et Knapp,P. quercibus Phaff et Knapp andP. xylosa Phaff, Miller et Shifrine. Furthermore, it has been proposed to transferSaccharomyces pastori Guilliermond,Saccharomyces pini Holst andDebaryomyces vini Zimmermann to the amended genusPichia, where they fit well rather than occupying and exceptional position in their present genera.     

Phylogenetic analysis of ascomycete yeasts that form coenzyme Q-9 and the proposal of the new genera Babjeviella, Meyerozyma, Millerozyma, Priceomyces, and Scheffersomyces

Species assigned to the genera Debaryomyces, Lodderomyces, Spathaspora, and Yamadazyma, as well as selected species of Pichia and Candida that also form coenzyme Q-9, were phylogenetically analyzed from the combined sequences of the D1/D2 domains of the large subunit and the nearly complete small subunit rRNA genes. Species assigned to Debaryomyces partitioned into three clades and species assigned to Pichia were distributed among six clades. These well-supported clades were interpreted as genera, and from this analysis, the following new genera are proposed: Babjeviella, Meyerozyma, Millerozyma, Priceomyces, and Scheffersomyces. The genus Schwanniomyces was reinstated and emended, and the genus Yamadazyma was phylogenetically defined. From this study, 23 new combinations and 3 new ranks are proposed. The preceding genera are members of a single, large clade, and it is proposed to delineate this clade as the new family Debaryomycetaceae.     

Pichia angophorae,sp.n. from the exudate of an Australian Red Gum tree

A new species ofPichia has been described.Pichia angophorae sp.n. was the only yeast present in an exudate ofAngophora costata collected near North Ryde, N. S. W., Australia. The new species exists in both the homo- and heterothallic state. Heterothallic mating types have been isolated as segregants from originally homothallic cultures.