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At least 19 recordsLinked to original sources
PubMed2025

Taxogenomic analysis of Pichia senei sp. nov. and new insights into hybridization events in the Pichia cactophila species complex.

Three strains of a novel yeast species were isolated from necrotic cactus tissues of Cereus saddianus and Micranthocereus dolichospermaticus and from phytotelmata of Bromelia karatas. DNA sequence analysis of the Internal Transcribed Spacer (ITS) region and D1/D2 domains of the large subunit ribosomal RNA, along with whole genome phylogenomic analysis, showed that this yeast is most closely related to Pichia insulana, Pichia cactophila, and Pichia inconspicua. The new species differs by 10-13 nucleotide substitutions from these species in D1/D2 sequences and exhibits <90% genome-wide average nucleotide identity to them. The name Pichia senei sp. nov. is proposed for the novel species, which is homothallic and produces asci with one to four hat-shaped ascospores. The holotype is CBS 16311 (MycoBank MB 858723). Taxogenomic analyses of the P. cactophila species complex, including P. senei, provide new insights about the hybridizations events that shaped this group. Pichia insulana and P. inconspicua are identified as the parental lineages that originated P. cactophila, and P. senei also appears closely related to one of the progenitors of P. inconspicua. We assess phylogeny, heterozygosity, and ploidy to explore the processes shaping diversity, showing how genomic data support yeast species delimitation and reveal complex hybridization.

Phylogeny↗
PubMed1976

Serological relationships among some Pichia species.

Antigenic analyses of five species of the genus Pichia were carried out for taxonomic study by the slide agglutination method using monospecific and absorbed antisera and the agglutinin absorption technique. Comparative studies were also performed with a few strains of each of the same species and their classifications are discussed with respect to the antigenic structures and the patterns of proton magnetic resonance (PMR) spectra of their cell wall polysaccharides. ichia delftensis and Pichia zaruensis possessed thermostable antigens 1,2,5 and 11, and the former had also thermoabile antigen m. Both species were closely related to Candida krusei. Pichia toletana possessed thermostable antigens 1,2,5,11,17 and 49. Pichia bovis contained thermostable antigens 1,2,14,15,16,20 and 21, and it was related to most species of the genus Hansenula, although assimilation of potassium nitrate was negative. Finally, Pichia etchellsii possessed thermostable antigens 1,2,3,4,9 and 14, and was closely related to Pichia vini. Patterns of PMR spectra of mannans of these species also supported their serological relationships. Therfore, P. delftensis, P. zaruensis and P. etchellsii are considered to be the synonyms of Pichia fluxuum, Pichia dispora and P. vini respectively, although P. toletanan and P. bovis are independent species.

Antigens, Fungal↗
PubMedDate not supplied

[A flavinogenic mutant of the yeast Pichia guilliermondii with impaired iron transport].

A mutant of the yeast Pichia guilliermondii was produced by means of UV; the mutant was capable of riboflavin overproduction in the presence of high concentrations of iron in the medium. The content of total and non-hemin iron and cytochrome c, and the activity of catalase, were lower in the cells of the mutant than in the parent cells, while the activity of riboflavin synthetase was higher. The content of iron in the cells increased when the mutant was cultivated on media with citric acid, siderochromes of Klebsiella aerogenes, Neurospora crassa, Rhodotorula glutinis, cultural broth of Pichia ohmeri, and autolysate of brewer's yeast, whereas the flavinogenous activity of the cells decreased. Rotenone inhibited respiration of the intact cells of the mutant producing elevated amounts of riboflavin; therefore, flavinogenesis was not regulated by non-hemin iron on the first segment of the respiratory chain. Overproduction of riboflavin in the mutant of Pichia guilliermondii was proved to be a recessive property.

Ascomycota↗
PubMed2026

Efficient scarless gene editing in Pichia pastoris via survival stress-based intramolecular homologous recombination.

To overcome low efficiency and/or genomic instability induced by DNA cleavage in current genome-editing approaches, a novel pop-in/pop-out-based editing system was developed for Pichia pastoris. An ingenious arrangement of components leads to a more efficient screening by permitting the only type of DNA recombination under defined pressure conditions, in terms of the overall efficiency of gene editing, the system virtually depends on the integration efficiency mediated by single-crossover recombination. It does not rely on exogenous recombinases or programmable nucleases such as Cas9, thereby avoiding nuclease induced double strand breaks and associated off target mutations or chromatin fatigue. This strategy preserves high editing efficiency with no modification to the host's inherent genetic properties. Relative to site-specific recombination methods, its dual MazF counterselection enables seamless editing, avoiding scar sequence-induced genomic instability. In this study, nearly 100% knockout efficiency and over 86.67% integration efficiency were achieved in the described experimental cases with this system, which provides a new gene-editing tool for synthetic biology in Pichia pastoris.

Efficient scarless editing↗
PubMedDate not supplied

[Comparative study of the protein makeup in diploid and haploid forms of Saccharomyces and Pichia].

The rates of growth, biomass accumulation, and electrophoretic spectra of mobile cytoplasmic proteins were studied with nonisogenous haploid and diploid cultures of Saccharomyces cerevisiae and Pichia guilliermondii as well as with isogenous haploid-diploid pairs of Saccharomyces cerevisiae and Pichia pinus. On a mineral medium with glucose, differences in these parameters in various yeast strains were found to be due to the genotype of a strain rather than to ploidy: nonisogenous haploid and diploid cultures displayed considerable and random variability of these properties while no differences were found in isogenous haploid-diploid pairs. Studies on solubility of protein fractions in various solvents made it possible to reveal differences connected with ploidy, namely: both in nonisogenous and isogenous haploid-diploid systems, the content of the water-soluble fraction decreased in diploid cultures.

Ascomycota↗
PubMedDate not supplied

[The activity of enzymes involved in synthesis and hydrolysis of flavin adenine dinucleotide is Pichia guilliermondii studies at different levels of flavinogenesis].

The activity of FAD-pyrophosphorylase and FAD-hydrolase (nucleotidepyrophosphatase) was studied in extracts of Pichia guilliermondii ATCC 9058 capable of riboflavin over-production. The specific activity of the enzymes was highest at the logarithmic growth phase (2.6 and 3.8 mcmoles of FAD per 1 min per 1 mg of protein X10(-5), respectively), and did not increase upon the induction of riboflavin overproduction. A decrease in the content of hemin compounds and a low content of flavins in the cells of Pichia guilliermondii mutants had no considerable effect on the activity of the two enzymes. When the yeast was cultivated on a medium containing hexadecane, an increase in the content of FAD in the cells was not accompanied with a rise in the activity of FAD-pyrophosphorylase. The activity of the enzyme did not change when succinate and lactate, the substrates of FAD-containing enzymes, were used as the source of carbon. The activity of FAD-pyrophosphorylase increased only when iron-deficient cells of the yeast were grown or incubated on a medium containing glycine; this stimulation was inhibited by cycloheximide.

Ascomycota↗
PubMed2024

Simulation and experimental study of a cold atmospheric pressure plasma and comparison of efficiency in boosting recombinant Endoglucanase II production in Pichia pastoris.

Recombinant proteins are essential in various industries, and scientists employ genetic engineering and synthetic biology to enhance the host cell's protein production capacity. Stress response pathways have been found effective in augmenting protein secretion. Cold atmospheric pressure plasma (CAP) can induce oxidative stress and enhance protein production. Previous studies have confirmed the applicability of CAP jets on Phytase and green fluorescent protein (GFP) production in Pichia pastoris hosts. This study investigates the effect of CAP treatment on another valuable recombinant protein, Endoglucanase II (EgII), integrated into the Pichia pastoris genome. The results demonstrated that plasma induction via two different ignition modes: sinusoidal alternating current (AC) and pulsed direct current (DC) for 120, 180, and 240 s has boosted protein secretion without affecting cell growth and viability. The AC-driven jet exhibited a higher percentage increase in secretion, up to 45%. Simulation of plasma function using COMSOL software provided a pattern of electron temperature (Te) and density distribution, which determine the plasma cocktail's chemistry and reactive species production. Furthermore, electron density (ne) and temperature were estimated from the recorded optical spectrum. The difference in electron properties may explain the moderately different impressions on expression capability. However, cell engineering to improve secretion often remains a trial-and-error approach, and improvements are, at least partially, specific to the protein produced.

Plasma Gases↗
PubMed2025

Comparison of stress tolerance mechanisms between Saccharomyces cerevisiae and the multistress-tolerant Pichia kudriavzevii.

Yeasts play a vital role in both research and industrial biomanufacturing. Saccharomyces cerevisiae has been extensively utilized as a model system. However, its application is often constrained by limited tolerance to the diverse stress conditions encountered in bioprocesses. These challenges have driven increasing interest in nonconventional, multistress-tolerant yeasts as alternative biomanufacturing hosts. This review highlights Pichia kudriavzevii as a promising nonconventional yeast for industrial applications. Unlike S. cerevisiae, P. kudriavzevii exhibits exceptional tolerance to high temperatures, elevated concentrations of furanic and phenolic inhibitors, osmotic stress, salinity, and extreme pH. These traits make it an attractive candidate for industrial processes without requiring extensive genetic modifications to enhance stress resistance. As a result, P. kudriavzevii has emerged as a flagship species for advancing bioeconomy. Despite its industrial potential, the molecular mechanisms underlying P. kudriavzevii's superior stress tolerance remain poorly understood. This review compiles current knowledge on P. kudriavzevii and compares its stress tolerance mechanisms with those of S. cerevisiae, providing insights into its innate resilience. By expanding our understanding of nonconventional yeasts, this review aims to facilitate their broader adoption as robust microbial platforms for industrial biomanufacturing.

Saccharomyces cerevisiae↗
PubMed1976

On the effect of glucono-delta-lactone on the yeast Pichia polymorpha.

Investigation has been made into the action of glucono-delta-lactone on living cells of Pichia polymorpha in relation to the uptake of D-(U-14C)glucose, and the incorporation of (2-14C) uracil and L-(U-14C) threonine into RNA and protein respectively. Other factors such as the action of glucono-delta-lactone on cell morphology and on enzymic synthesis have also been studied. The action of this compound on beta-glucanase has been found to take place in the hydrolytic power and not in the synthesis.

Ascomycota↗
PubMed1979

Production, purification and properties of a Pichia kluyveri killer toxin.

Production of the killer toxin of Pichia kluyveri 1002 was stimulated in the presence of yeast extract. In a minimal medium production was optimal at pH 3.8-4.0 and 22--25 degrees C. Addition of gelatin and nonionic detergents, like Brij-58 (polyoxyethylene 20 cetyl ether) and Triton-X-100, to this medium enhanced production significantly. The killer toxin was purified 140-fold by use of a stepwise ethanol precipitation and butyl Sepharose column chromatography. The purified killer toxin, which still contained some carbohydrates, appeared to be glycoprotein with a mol wt of about 19 000 and an isoelectric point of 4.3. It was stable between pH 2.5 and 4.7 and up to 40 degrees C.

Ascomycota↗
PubMed1976

Regulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis by riboflavin and iron in riboflavin-deficient mutants of Pichia guilliermondii yeast.

The effect of riboflavin and iron on 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate was investigated in the cultures of the yeast Pichia guilliermondii (rib2 mutants) with the blocked second reaction to flavinogenesis. It was shown that riboflavin inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-rich and iron-deficient cells of mutants with low riboflavin requirements. Cycloheximide did not prevent the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis caused by riboflavin starvation. 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)isoalloxazine strongly inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis, while 7-methyl-8-trifluoro-methyl-10-(beta-hydroxyethyl)izoalloxazine and galactoflavin exerted only a slight effect on this process. The 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-deficient cells was significantly higher than in iron-rich cells. The 2,2'-dipyridyl treatment of iron-rich cells caused the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis and cycloheximide abolished this effect. The results suggest that the activity of the first enzyme of flavinogenesis (guanylic cyclohydrolase) is under the control of feedback inhibition by flavins and the biosynthesis of this enzyme is regulated by iron.

2,2'-Dipyridyl↗
PubMed2024

The combined effect of the gene copy number and chaperone overexpression on the recombinant bovine chymosin production in Pichia pastoris, with mutant ADH2 promoter.

Chymosin is an enzyme used to coagulate milk, in the cheese industry. This study aimed to increase recombinant production of the chymosin in Pichia pastoris by determining the optimum copy number and overproduction of a Protein Disulfide Isomerase (PpPDI) chaperon protein. Bos taurus chymosin was expressed under the control of a mutant ADH2 promoter. The clones containing 1-4 gene copy numbers of the chymosin were constructed using the in vitro cloning method, and the effect of chaperone protein on chymosin secretion was investigated. The enzyme production levels are 4, 6.3, 4.5, and 3 IMCU/mL for 1, 2, 3, and 4-copy clones. The secreted chymosin levels increased up to two copies, and increasing the number of copies decreased the secretion level. Therefore, PpPDI was over-expressed in the clones regulated with the ADH2 promoter. The over-expression of PDI gene increased chymosin secretion in clones compared to the counterpart host. However, the highest chymosin level was obtained with C2 (2-copy chymosin containing clone; 6.3 IMCU/mL) and C2P2 (2-copy chymosin/2-copy PDI containing clone; 8.2 IMCU/mL). The maximum production was 39 IMCU/mL with the clone C2P2 in the fermenter scale production. The enzyme activity increased approximately 2-fold by adding two copies of the chaperone protein. The combined effect of gene copy number and chaperone overexpression on chymosin production was investigated. Two copies of the chymosin and PpPDI genes were the optimum among the tested clones.

Animals↗
PubMed1979

[Coordinate regulation of riboflavin permease and alpha-glucosidase synthesis in the yeast Pichia guilliermondii].

Riboflavin permease of the yeast Pichia guilliermondii appear to be inducible transport system. Its synthesis is induced by sucrose, maltose, alpha-methyl-D-glocoside, melizitose and raffinose, but not by D-glucose, trehalose or cellobiose. The synthesis of riboflavin permease in the presence of sucrose of maltose is depressed by cycloheximide, actinomycin D and 8-hydroxyquinoline. These results suggest that the synthesis of riboflavin permease is regulated on the transcription level. The inducers of riboflavin permease are also able to induce the synthesis of alpha-glucosidase. The mutants have been selected in which the synthesis of riboflavin permease occurs constitutively; the synthesis of alpha-glucosidase in the mutants is also constitutive. Growing of the yeast in a medium with high content of glucose results in a parallel decrease of the riboflavin permease and alpha -- glucosidase activities. These data are indicative of corrdinate regulation of riboflavin permease and alpha -- glucosidase in P. guilliermondii. Suboptimal or excessive content of vitamin B2 in the medium does not affect the level of riboflavin permease in this yeast species.

Ascomycota↗
PubMedDate not supplied

[Inhibition of alkaline phosphatase I of Pichia guilliermondii yeast in vitro and in vivo].

The rate of p-nitrophenyl phosphate and flavin mononucleotide (FMN) hydrolysis by the partially purified preparation of alkaline phosphatase I of Pichia guilliermondii flavinogenic yeast was studied as affected by different substrates and inorganic ions. Their Km was established to be 2.0 X 10(-4) m and 2.5 X 10(-4) M, respectively. Dephosphorylation of p-nitrophenylphosphate and FMN was inhibited competitively by beta-glycerophosphate (Ki = 3.1 X 10(-3) M, respectively). The presence of inorganic phosphate ions in the reaction mixture decreases or removes inhibition of these compounds hydrolysis by other substrates of alkaline phosphatase I. The activity of alkaline phosphatase I increases in the presence of Mg2+ and was strongly inhibited in the presence of Be2+, Cu2+, Zn2+, Cd2+ and inorganic phosphate, the mixture of Be2+ and F- being the most effective. This mixture inhibited the phosphatase activity of the partially purified preparation of alkaline phosphatase I of the cell-free extract as well as of intact cells in both the alkaline and acid zones of pH (8.6 and 5.5, respectively). Incubation of the washed iron-deficient P. guilliermondii cells in the presence of Be2+ and F- did not result in accumulation of FMN in the yeast culture. A possible role of nonspecific phosphomonoesterases in hydrolysis of FMN in vivo is discussed.

4-Nitrophenylphosphatase↗
PubMed1978

[Substrate and inhibitory specificity of riboflavin kinase from Pichia guilliermondii yeast].

The interaction of purified riboflavin kinase (EC 2.7.1.26) from Pichia guilliermondii with 44 structural vitamin B2 analogues is studied. The presence of D-ribityl lateral chain in an analogue structure is found to be necessary for the substrate activity. The substitution of CH3 groups in the 7 and 8 positions of isoalloxazine ring in the riboflavin molecule for CF3, Cl, H, NH2 and N(CH3)2 resulted in the decrease of the analogue affinity to riboflavin kinase as compared with the natural substrate, vitamin B2. The most efficient enzyme inhibitors of analogues without substrate properties turned to be trifluoromethylisoalloxazines, containing 2'-hydroxyethyl group at N10. The elongation of D-ribityl lateral chain, the elimination of change of CH3-groups in the 7 and 8 positions for CF3- Cl-, COOH-substitutors resulted in the decrease of the inhibitory effect of flavines. Modifications in the structure of isoalloxazine ring, etherification of OH-groups in the lateral D-ribityl chain, and the introduction of volume substitutors (N-piperidyl, D-ribitylamine, hydroxyethylamine) prevented the interaction of the analogue with riboflavin kinase. Flavin nucleotides (FMN and FAD) did not affect the rate of vitamin B2 phosphorylation.

Ascomycota↗
PubMed1978

[Effect of glucose and its derivatives on systems of riboflavin uptake and excretion in the yeast Pichia guilliermondii].

Riboflavin uptake by washed cells of riboflavin deficient mutant MS1-3 of Pichia guilliermondii yeast was strongly depressed by D-glucose, L-sorbose, alpha-methyl-D-glucoside, sucrose, trehalose, maltose and salicin but not by D-mannose, D-galactose, D-fructose or ribitol. Glucose decreased also the initial uptake rate of riboflavin analogue, 8-piperidyl-10-(1'-D-galactityl) isoalloxazine; the inhibition having a competitive character (Ki==5,7 mM). Apparently riboflavin permease is able to accept not only riboflavin and its analogues but also glucose and some of glucose derivates. Cells preloaded with riboflavin and transferred into riboflavin-free medium excreted vitamin B2 into the medium. This excretion was strongly stimulated by D-glucose, D-fructose, D-mannose but not by citrate or succinate. In contrast to riboflavin, 8-piperidyl-10-(1'-D-galactityl) isoalloxazine was not excreted into the medium even in the presence of glucose. The rate of riboflavin excretion depended on temperature and pH of incubation medium (pH optimum approximately 7.0) and was decreased in the presence of different inhibitors of energy metabolism. It seems that the exit of riboflavin from the cells is accomplished by energy-dependent specific system of excretion (excretase) which in some properties is different from that of riboflavin permease.

Ascomycota↗
PubMedDate not supplied

[Product of second-stage riboflavin biosynthesis in Pichia guilliermondii].

Dialyzed extracts of the mutants of Pichia guilliermondii RG80 (rib3, his-) and RA39 (rib3, ade-) in which the third step of flavinogenesis was genetically blocked converted GTP into 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine phosphate and 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine in the presence of NADPH2. Apparently, 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine phosphate is a true precursor of riboflavin whereas 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine is formed upon its hydrolysis.

Aminopyridines↗