Methylotrophic Yeast Candida Boidinii Research Articles

Autophagy as a Survival Strategy for Eukaryotic Microbes Living in the Phyllosphere.

Autophagy is an intracellular degradation process that is highly conserved among eukaryotes at the molecular level. The process was originally revealed in the budding yeast, but the physiological role of autophagy in yeast cells had remained unknown as autophagy-deficient yeast mutants did now show a clear growth phenotype in laboratory conditions. In this review, we introduce the role of autophagy in the methylotrophic yeast Candida boidinii grown on the leaf surface of Arabidopsis thaliana. Autophagy is shown to be required for proliferation in the phyllosphere, and selective autophagic pathways such as pexophagy and cytoplasm-to-vacuole targeting (Cvt) pathway are strictly regulated during both the daily cycle and the host plant life cycle. This review describes our current understanding of the role of autophagy as a survival strategy for phyllosphere fungi. Critical functions of autophagy for pathogen invasions are also discussed.

Yeast Hog1 proteins are sequestered in stress granules during high-temperature stress.

The yeast high-osmolarity glycerol (HOG) pathway plays a central role in stress responses. It is activated by various stresses, including hyperosmotic stress, oxidative stress, high-temperature stress and exposure to arsenite. Hog1, the crucial MAP kinase of the pathway, localizes to the nucleus in response to high osmotic concentrations, i.e. high osmolarity; but, otherwise, little is known about its intracellular dynamics and regulation. By using the methylotrophic yeast Candida boidinii, we found that CbHog1-Venus formed intracellular dot structures after high-temperature stress in a reversible manner. Microscopic observation revealed that CbHog1-mCherry colocalized with CbPab1-Venus, a marker protein of stress granules. Hog1 homologs in Pichia pastoris and Schizosaccharomyces pombe also exhibited similar dot formation under high-temperature stress, whereas Saccharomyces cerevisiae Hog1 (ScHog1)-GFP did not. Analysis of CbHog1-Venus in C. boidinii revealed that a β-sheet structure in the N-terminal region was necessary and sufficient for its localization to stress granules. Physiological studies revealed that sequestration of activated Hog1 proteins in stress granules was responsible for downregulation of Hog1 activity under high-temperature stress.This article has an associated First Person interview with the first author of the paper.

Biodecoloration of Reactive Black 5 by the methylotrophic yeast Candida boidinii MM 4035

Azo dyes are extensively used in textile dyeing and other industries. Effluents of dying industries are specially colored and could cause severe damage to the environment. The anaerobic treatment of textile dying effluents is nowadays the preferred option, but it could generate carcinogenic aromatic amines. Recently, yeasts have become a promising alternative, combining unicellular growth with oxidative mechanisms. This work reports the characterization of the first methylotrophic yeast with dye decolorizing ability, Candida boidinii MM 4035 and some insights into its decoloration mechanism. The analysis of two selected media revealed a possible two stages mechanism of Reactive Black 5 decoloration. In glucose poor media, decoloration is incomplete and only the first stage proceeds, leading to the accumulation of a purple compound. In media with higher glucose concentrations, the yeast is able to decolorize totally an initial concentration of 200mg/L. The entire process is co-metabolic, being largely dependent on glucose concentration but being able to proceed with several nitrogen sources. Manganese dependent peroxidase but not laccase activity could be detected during decoloration. Aromatic amines do not accumulate in culture media, supporting an oxidative decoloration mechanism of unknown ecophysiological relevance.

Unique C-terminal region of Hap3 is required for methanol-regulated gene expression in the methylotrophic yeast Candida boidinii

The Hap complex of the methylotrophic yeast Candida boidinii was found to be required for methanol-regulated gene expression. In this study, we performed functional characterization of CbHap3p, one of the Hap complex components in C. boidinii. Sequence alignment of Hap3 proteins revealed the presence of a unique extended C-terminal region, which is not present in Hap3p from Saccharomyces cerevisiae (ScHap3p), but is found in Hap3p proteins of methylotrophic yeasts. Deletion of the C-terminal region of CbHap3p (Δ256-292 or Δ107-237) diminished activation of methanol-regulated genes and abolished the ability to grow on methanol, but did not affect nuclear localization or DNA-binding ability. However, deletion of the N-terminal region of CbHap3p (Δ1-20) led to not only a growth defect on methanol and a decreased level of methanol-regulated gene expression, but also impaired nuclear localization and binding to methanol-regulated gene promoters. We also revealed that CbHap3p could complement the growth defect of the Schap3Δ strain on glycerol, although ScHap3p could not complement the growth defect of a Cbhap3Δ strain on methanol. We conclude that the unique C-terminal region of CbHap3p contributes to maximum activation of methanol-regulated genes, whilst the N-terminal region is required for nuclear localization and binding to DNA.

Regulation of nitrate and methylamine metabolism by multiple nitrogen sources in the methylotrophic yeastCandida boidinii

The methylotrophic yeast Candida boidinii, which is capable of growth on methanol as a sole carbon source, can proliferate on the leaf surface of Arabidopsis thaliana. Previously, we demonstrated that adaptation to a change in the major available nitrogen source from nitrate to methylamine during the host plant aging was crucial for yeast survival on the leaf environment. In this report, we investigated the regulatory profile of nitrate and methylamine metabolism in the presence of multiple nitrogen sources in C. boidinii. The transcript level of nitrate reductase (Ynr1) gene was induced by nitrate and nitrite, and was not repressed by the coexistence with other nitrogen sources. In contrast, the transcript level of amine oxidase (Amo1) gene, which was induced by methylamine, was significantly repressed by the coexistence with ammonium or glutamine. In addition, we investigated the intracellular dynamics of Ynr1 during the nitrogen source shift from nitrate to other compounds. Under these tested conditions, Ynr1 was effectively transported to the vacuole via selective autophagy only during the shift from nitrate to methylamine. Moreover, Ynr1 was subject to degradation after the shift from nitrate to nitrate plus methylamine medium even though nitrate was still available. These regulatory profiles may reflect life style of nitrogen utilization in this yeast living in the phyllosphere.

Yeast nitrogen utilization in the phyllosphere during plant lifespan under regulation of autophagy.

Recently, microbe-plant interactions at the above-ground parts have attracted great attention. Here we describe nitrogen metabolism and regulation of autophagy in the methylotrophic yeast Candida boidinii, proliferating and surviving on the leaves of Arabidopsis thaliana. After quantitative analyses of yeast growth on the leaves of A. thaliana with the wild-type and several mutant yeast strains, we showed that on young leaves, nitrate reductase (Ynr1) was necessary for yeast proliferation, and the yeast utilized nitrate as nitrogen source. On the other hand, a newly developed methylamine sensor revealed appearance of methylamine on older leaves, and methylamine metabolism was induced in C. boidinii, and Ynr1 was subjected to degradation. Biochemical and microscopic analysis of Ynr1 in vitro during a shift of nitrogen source from nitrate to methylamine revealed that Ynr1 was transported to the vacuole being the cargo for biosynthetic cytoplasm-to-vacuole targeting (Cvt) pathway, and degraded. Our results reveal changes in the nitrogen source composition for phyllospheric yeasts during plant aging, and subsequent adaptation of the yeasts to this environmental change mediated by regulation of autophagy.

Molecular characterization of hap complex components responsible for methanol-inducible gene expression in the methylotrophic yeast Candida boidinii.

We identified genes encoding components of the Hap complex, CbHAP2, CbHAP3, and CbHAP5, as transcription factors regulating methanol-inducible gene expression in the methylotrophic yeast Candida boidinii. We found that the Cbhap2Δ, Cbhap3Δ, and Cbhap5Δ gene-disrupted strains showed severe growth defects on methanol but not on glucose and nonfermentable carbon sources such as ethanol and glycerol. In these disruptants, the transcriptional activities of methanol-inducible promoters were significantly decreased compared to those of the wild-type strain, indicating that CbHap2p, CbHap3p, and CbHap5p play indispensable roles in methanol-inducible gene expression. Further molecular and biochemical analyses demonstrated that CbHap2p, CbHap3p, and CbHap5p localized to the nucleus and bound to the promoter regions of methanol-inducible genes regardless of the carbon source, and heterotrimer formation was suggested to be necessary for binding to DNA. Unexpectedly, distinct from Saccharomyces cerevisiae, the Hap complex functioned in methanol-specific induction rather than glucose derepression in C. boidinii. Our results shed light on a novel function of the Hap complex in methanol-inducible gene expression in methylotrophic yeasts.

Trm2p-dependent derepression is essential for methanol-specific gene activation in the methylotrophic yeast Candida boidinii

We identified a gene, designated TRM2, responsible for methanol-inducible gene expression in the methylotrophic yeast Candida boidinii. The encoded protein Trm2p contains two C(2)H(2)-type zinc finger motifs near the N terminus and shows high similarity to Saccharomyces cerevisiae Adr1p and Pichia pastoris Mxr1p. A C. boidinii gene-disrupted strain (trm2Delta) could not grow on methanol or oleate, but could grow on glucose or ethanol. Trm2p was necessary for the activation of five methanol-inducible promoters tested. Trm2p was localized to the nucleus during growth on nonfermentable carbon sources, but to the cytosol during growth on glucose. A chromatin immunoprecipitation assay revealed that Trm2p specifically bound to the promoters of the alcohol oxidase gene (AOD1) and the dihydroxyacetone synthase gene in cells grown on methanol or oleate, but did not bind to these promoters in cells grown on glucose. The derepressed level of expression of AOD1, which was observed in the trm1Delta strain (the TRM1 gene encodes a transcription factor responsible for methanol-specific gene activation), was decreased in the trm1Deltatrm2Delta strain to a level similar to that observed in the trm2Delta strain. These results suggest that Trm2p-dependent derepression is essential for the Trm1p-dependent methanol-specific gene activation in C. boidinii.

Gene-tagging mutagenesis in the methylotrophic yeast Candida boidinii

A gene-tagging mutagenesis method by random integration of linear DNA fragments was developed and used in the methylotrophic yeast Candida boidinii to isolate mutants defective in methanol-inducible gene expression. A large number of mutants were obtained, indicating that this method is a powerful tool for random mutagenesis in C. boidinii.

Alcohol dehydrogenases that catalyse methyl formate synthesis participate in formaldehyde detoxification in the methylotrophic yeast Candida boidinii.

Methyl formate synthesis during growth on methanol by methylotrophic yeasts has been considered to play a role in formaldehyde detoxification. An enzyme that catalyses methyl formate synthesis was purified from methylotrophic yeasts, and was suggested to belong to a family of alcohol dehydrogenases (ADHs). In this study we report the gene cloning and gene disruption analysis of three ADH-encoding genes in the methylotrophic yeast Candida boidinii (CbADH1, CbADH2 and CbADH3) in order to clarify the physiological role of methyl formate synthesis. From the primary structures of these three genes, CbAdh1 was shown to be cytosolic and CbAdh2 and CbAdh3 were mitochondrial enzymes. Gene products of CbADH1, CbADH2 and CbADH3 expressed in Escherichia coli showed both ADH- and methyl formate-synthesizing activities. The results of gene-disruption analyses suggested that methyl formate synthesis was mainly catalysed by a cytosolic ADH (CbAdh1), and this enzyme contributed to formaldehyde detoxification through glutathione-independent formaldehyde oxidation during growth on methanol by methylotrophic yeasts.

Cis-acting elements sufficient for induction of FDH1 expression by formate in the methylotrophic yeast Candida boidinii.

The FDH1 gene of Candida boidinii encodes an NAD+-dependent formate dehydrogenase, which catalyzes the last reaction in the methanol dissimilation pathway. FDH1 expression is strongly induced by methanol, as are the promoters of the genes AOD1 (alcohol oxidase) and DAS1 (dihydroxyacetone synthase). FDH1 expression can be induced by formate when cells are grown on a medium containing glucose as a carbon source, whereas expression of AOD1 and DAS1 is completely repressed in the presence of glucose. Using deletion analyses, we identified two cis-acting regulatory elements, termed UAS-FM and UAS-M, respectively, in the 5' non-coding region of the FDH1 gene. Both elements were necessary for full induction of the FDH1 promoter by methanol, while only the UAS-FM element was required for full induction by formate. Irrespective of whether induction was achieved with methanol or formate, the UAS-FM element enhanced the level of induction of the FDH1 promoter in a manner dependent on the number of copies, but independent of their orientation, and also converted the ACT1 promoter from a constitutive into an inducible element. Our results not only provide a powerful promoter for heterologous gene expression, but also yield insights into the mechanism of regulation of FDH1 expression at the molecular level.

Physiological role of S-formylglutathione hydrolase in C1 metabolism of the methylotrophic yeast Candida boidinii

The methylotrophic yeast Candida boidinii exhibits S-formylglutathione hydrolase activity (FGH, EC 3.1.2.12), which is involved in the glutathione-dependent formaldehyde oxidation pathway during growth on methanol as the sole carbon source. The structural gene, FGH1, was cloned from C. boidinii, and its predicted amino acid sequence showed more than 60 % similarity to those of FGHs from Paracoccus denitrificans and Saccharomyces cerevisiae, and human esterase D. FGH from C. boidinii contained a C-terminal tripeptide, SKL, which is a type I peroxisome-targeting signal, and a bimodal distribution of FGH between peroxisomes and the cytosol was demonstrated. The FGH1 gene was disrupted in the C. boidinii genome by one-step gene disruption. The fgh1Delta strain was still able to grow on methanol as a carbon source under methanol-limited chemostat conditions with low dilution rates (D<0.05 h(-1)), conditions under which a strain with disruption of the gene for formaldehyde dehydrogenase (another enzyme involved in the formaldehyde oxidation pathway) could not survive. These results suggested that FGH is not essential but necessary for optimal growth on methanol. This is believed to be the first report of detailed analyses of the FGH1 gene in a methylotrophic yeast strain.

Physiological role of the glutathione-dependent formaldehyde dehydrogenase in the methylotrophic yeast Candida boidinii.

The methylotrophic yeast Candida boidinii exhibits formaldehyde dehydrogenase activity (FLD, EC 1.2.1.1) during growth on methanol as a sole carbon source. The structural gene, FLD1, was cloned from a genomic library of C. boidinii. The 1263 bp FLD1 gene contained a 123 bp intron and its exon encoded a gene product of 380 amino acids, whose predicted amino acid sequence showed high similarity to the sequences of FLDs from other organisms. The FLD1 gene was disrupted in the C. boidinii genome by one-step gene disruption. The fld1Delta strain could not grow on methanol as a carbon source under methanol-limited chemostat culture conditions, even with low dilution rates (D<0.05 x h(-1)), whereas a strain with a disruption in the gene for formate dehydrogenase (FDH; another NADH-generating dehydrogenase involved in the formaldehyde oxidation pathway) could survive. These results indicated that FLD, but not FDH, is essential for growth of C. boidinii on methanol.

Production of active bovine cathepsin C (dipeptidyl aminopeptidase I) in the methylotrophic yeast Candida boidinii.

The heterologous production of active bovine cathepsin C (CTC; dipeptidyl aminopeptidase I) was investigated. Attempts to express CTC in Escherichia coli were hampered by formation of inclusion bodies that were partially degraded. To overcome this impediment, secretion of recombinant CTC was attempted in the methylotrophic yeast Candida boidinii. A DNA fragment encoding bovine procathepsin C was synthesized based on preferred codon usage in C. boidinii and placed downstream of the C. boidinii proteinase A signal sequence resulting in secretion of active CTC into the culture medium. The gene was expressed under the control of the methanol-inducible formate dehydrogenase gene promoter. Production levels were significantly improved by using a protease-deficient strain, changing medium composition, and by lowering the temperature of induction. When the recombinant C. boidinii was grown for 90 h in a jar-fermenter, active CTC was secreted with a yield of up to approximately 12 mg/l.

Copper adaptation and methylotrophic metabolism in Candida boidinii.

The importance of the antioxidant enzyme superoxide dismutase (CuZnSOD) in the metabolic switch from normotrophic to methylotrophic conditions was studied in the facultative methylotrophic yeast Candida boidinii. Copper adaptation was performed to qualify C. boidinii as a suitable cellular system to study the effect of induction of CuZnSOD, and other biochemical components along the copper detoxification system, on methanol adaptation. Copper adaptation results in the induction of CuZnSOD peroxidase activity as well as of glutathione. The effects at the metabolic level of exposure to both copper and methanol were also studied: the results suggest that the effect on antioxidant enzyme levels as a function of the change of trophic condition are predominant with respect to the effects of copper administration. Thus, the methanol-dependent induction of such enzymes is likely to provide a sufficient protection for the cells against toxic effects depending on copper administration. Administration of copper under methylotrophic conditions decreases the growth rate in spite of the high levels of antioxidant enzymes that are elicited by copper treatment. The adaptation to methanol metabolism was studied alsoafter methanol-independent induction of CuZnSOD, glutathione and catalase levels, obtained by exposure to high copper concentrations in glucose-containing medium. The metabolic changes induced by copper are persistent over several re-inoculations in normo-cupric glucose medium, thus allowing the study of the glucose-to-methanol switch on cells exhibiting high levels of antioxidant enzyme activities. Under such conditions the lag time observed during the transition from normotrophic to methylotrophic conditions is strongly reduced.

Efficient Production of Recombinant Bovine Cathepsin C by High Cell Density Culture of Methylotrophic Yeast Candida boidinii.

Bovine cathepsin C production from recombinant methylotrophic yeast, Candida boidinii, was conducted in a jar-fermentor culture of fed-batch mode. Changing the carbon source from glucose to methanol induced cathepsin C expression. After changing the carbon source, intermittent addition of methanol was effective for the production of cathepsin C. During the growth phase, the level of dissolved oxygen was kept between 40 and 80% of the saturated DO level. With this control, the lack of oxygen could be avoided and cells were grown up to ca. 50 g dry cell weight/l, approximately two fold higher than that without the DO control. However, the expression of cathepsin C in the induction phase was reduced. Half volume of the medium was replaced with the same volume of the fresh medium just before the induction. The production was improved by the medium exchange and the activity of cathepsin C in the supernatant reached over 370 U/l after 16 days of induction, corresponding to a two-fold higher value than that obtained without medium exchange.

Monomeric alcohol oxidase is preferentially digested by a novel protease from Candida boidinii

A protease activity has been partially purified from peroxisomal matrix fractions of the methylotrophic yeast Candida boidinii. The enzyme migrates as a single peak on a sucrose velocity gradient with an apparent native molecular mass of ∼80–90 kDa. Activity can be recovered from nonreducing sodium dodecyl sulfate gels as a ∼20 kDa species, suggesting it is an oligomer. The protein exhibits chymotrypsin-like activity and cleaves the model compound suc-L-L-V-Y-AMC. Additionally, monomers of alcohol oxidase (AO), an abundant protein of C. boidinii peroxisomes, generated in vitro or in pulse-radiolabeled cells, are preferentially sensitive to degradation by the protease. Sensitivity is lost over time in vivo as AO folds and matures into octamers, suggesting that the protease may be involved in these processes.

Peroxisomal catalase in the methylotrophic yeast Candida boidinii: transport efficiency and metabolic significance.

In this study we cloned CTA1, the gene encoding peroxisomal catalase, from the methylotrophic yeast Candida boidinii and studied targeting of the gene product, Cta1p, into peroxisomes by using green fluorescent protein (GFP) fusion proteins. A strain from which CTA1 was deleted (cta1Delta strain) showed marked growth inhibition when it was grown on the peroxisome-inducing carbon sources methanol, oleate, and D-alanine, indicating that peroxisomal catalase plays an important nonspecific role in peroxisomal metabolism. Cta1p carries a peroxisomal targeting signal type 1 (PTS1) motif, -NKF, in its carboxyl terminus. Using GFP fusion proteins, we found that (i) Cta1p is transported to peroxisomes via its PTS1 motif, -NKF; (ii) peroxisomal localization is necessary for Cta1p to function physiologically; and (iii) Cta1p is bimodally distributed between the cytosol and peroxisomes in methanol-grown cells but is localized exclusively in peroxisomes in oleate- and D-alanine-grown cells. In contrast, the fusion protein GFP-AKL (GFP fused to another typical PTS1 sequence, -AKL), in the context of CbPmp20 and D-amino acid oxidase, was found to localize exclusively in peroxisomes. A yeast two-hybrid system analysis suggested that the low transport efficiency of the -NKF sequence is due to a level of interaction between the -NKF sequence and the PTS1 receptor that is lower than the level of interaction with the AKL sequence. Furthermore, GFP-Cta1pDeltankf coexpressed with Cta1p was successfully localized in peroxisomes, suggesting that the oligomer was formed prior to peroxisome import and that it is not necessary for all four subunits to possess a PTS motif. Since the main physiological function of catalase is degradation of H2O2, suboptimal efficiency of catalase import may confer an evolutionary advantage. We suggest that the PTS1 sequence, which is found in peroxisomal catalases, has evolved in such a way as to give a higher priority for peroxisomal transport to peroxisomal enzymes other than to catalases (e.g., oxidases), which require a higher level of peroxisomal transport efficiency.

Isolation, purification, and characterization of catalase from the methylotrophic yeast Pichia pastoris.

Catalase (CATpp) with molecular weight 223 kD was isolated from the methylotrophic yeast Pichia pastoris and purified 90-fold by ion-exchange chromatography and gel filtration. Quantitative parameters of absorption and CD spectra of CATpp solutions and of its membrane-concentrated form (CATpp-conc) were studied. Rates of H2O2 decomposition and kinetic characteristics Km and kcat of CATpp and CATpp-conc were determined in 10 mM phosphate buffer (pH 7.4) at 30 degrees C, as well as the effective constant kin of the enzyme inactivation rate during the catalysis and the constant k2 of the interaction rate of the Complex I catalases with H2O2. Thermal inactivation of CATpp in solutions at 45 degrees C was characterized by the effective rate constant kin*, and the low-frequency (27 kHz) ultrasonic inactivation of CATpp at 20 degrees C was characterized by the first-order rate constant kin(US). All spectral and kinetic characteristics of CATpp and CATpp-conc were compared with the corresponding values for catalase from bovine liver (CAT) and for catalase from the methylotrophic yeast Candida boidinii (CATcb). All three catalases were rather similar in their spectral properties but strongly varied in their kinetic parameters, and their comparison suggests that CATpp should be the best enzyme in its overall properties as it displayed the maximal efficiency in terms of kcat/Km, thermal stability comparable with the thermal stability of CAT in terms of kin*, the minimal kin, and high stability in the ultrasonic cavitation field at the US power of 60 W/cm2.

Optimization of Bovine Cathepsin C Production by Cultivation of Recombinant Methylotrophic Yeast Candida boidinii.

Bovine cathepsin C (CTC) production from a recombinant methylotrophic yeast Candida boidinii CT714 was investigated, in which CTC gene was driven by formate dehydrogenase promoter and strongly induced in the presence of methanol. Two step fermentation using DYS medium containing 1.0% (w/v) glucose for cell growth and MYS medium containing 2.5% (v/v) methanol for inducible production of CTC was suitable for efficient production. Methanol concentration within 1.5 to 3.5% (v/v) was preferable for CTC induction. After 24 h of cell growth and 72 h of induction, CTC production reached approximately 12 U/I (this corresponded to 2.15 U/g-dry cells). When vitamins were enriched in growth medium, CTC productivity was improved more than 3 times higher, and it was found that the effective vitamins were biotin, p-aminobenzoic acid and thioctic acid.