Expression level of methanol-inducible peroxisomal proteins and peroxisome morphology are affected by oxygen conditions and mitochondrial respiratory pathway function in the methylotrophic yeast Candida boidinii.

In the methylotrophic yeast, Candida boidinii, methanol-inducible peroxisomal proteins, for example alcohol oxidase (AOD), dihydroxyacetone synthase (DAS), and peroxisomal glutathione peroxidase (Pmp20), were induced only under aerobic conditions, while expression of PMP47 encoding peroxisomal integral membrane protein Pmp47 was independent of oxygen conditions. Expression of the methanol-inducible peroxisomal enzymes was repressed by inhibition of the mitochondrial respiratory chain. In the respiratory-deficient (ρ0) mutant strain, their induction was at very low levels despite the presence of oxygen, whereas the expression of PMP47 was unaffected. Taken together, these facts indicate that C. boidinii can sense oxygen conditions, and that mitochondrial respiratory function may have a profound effect on induction of methanol-inducible gene expression of peroxisomal proteins. Peroxisome morphology was also affected by oxygen conditions and respiratory function. Under hypoxic conditions or respiration-inhibited conditions, cells induced by methanol contained small peroxisomes, indicating that peroxisome biogenesis and the protein import machinery were not affected by oxygen conditions but that peroxisome morphology was dependent on induction of peroxisomal matrix proteins.

The peroxisomal catalase gene in the methylotrophic yeast Pichia methanolica.

In this paper, we describe the CTA1 gene, which encodes a peroxisomal catalase in the methylotrophic yeast Pichia methanolica. The P. methanolica CTA1 gene (PmCTA1) comprises a 1,530-bp open reading frame corresponding to a protein of 510 amino acid residues, and its deduced amino acid sequence shows high similarity to those of Cta1ps from other methylotrophic yeasts (about 79%). Expression of PmCTA1 in a peroxisomal catalase-depleted (Cbcta1Delta) Candida boidinii strain restored the methylotrophic growth of the host strain, while the expression of PmCTA1-DeltaSRL, which lacks peroxisome targeting signal type 1, did not. In P. methanolica, expression of PmCTA1 was induced when cells were grown on peroxisome-inducing carbon sources, viz., methanol, oleate, and D-alanine. Taken together, these results indicate that PmCTA1 encodes a functional peroxisomal catalase in P. methanolica.

Molecular characterization of two genes with high similarity to the dihydroxyacetone synthase gene in the methylotrophic yeast Pichia methanolica.

The methylotrophic yeast Pichia methanolica possesses two genes, PmDAS1 and PmDLP1, whose amino acid sequences show high similarity to dihydroxyacetone synthase (DAS), the formaldehyde-fixing enzyme for methanol metabolism within the peroxisome. The PmDAS1 and PmDLP1 genes encode 709 and 707 amino acid residues respectively, and PmDas1p contains a type-1 peroxisomal targeting signal (PTS1), while PmDlp1p does not. Upon phylogenetic analysis, PmDas1p fit into the DAS group with other DASs, while PmDlp1p was grouped with the DAS-like proteins (DLP) of non-methylotrophic yeasts and fungi, a branch of the phylogenetic tree independent of the DAS and transketolase (TK) groups. While expression of PmDAS1 restored the methylotrophic growth of the Candida boidinii das1Delta strain, the PmDLP1 and PmDAS1-DeltaPTS1 genes did not. Taken together, these results indicate that PmDAS1 encodes a functional DAS and has an indispensable role in methanol metabolism, and that PmDlp1p share a common, as yet uncharacterized function in P. methanolica as well as in non-methylotrophic yeasts and fungi.

Acetaldehyde tolerance in Saccharomyces cerevisiae involves the pentose phosphate pathway and oleic acid biosynthesis.

To identify genes responsible for acetaldehyde tolerance, genome-wide screening was performed using a collection of haploid Saccharomyces cerevisiae strains deleted in single genes. The screen identified 49 genes whose deletion conferred acetaldehyde sensitivity, and these were termed the genes required for acetaldehyde tolerance. We focused on six of these genes required for acetaldehyde tolerance, ZWF1, GND1, RPE1, TKL1 and TAL1, which encode enzymes in the pentose phosphate pathway (PPP), and OAR1, which encodes for NADPH-dependent 3-oxoacyl-(acyl-carrier-protein) reductase. These genes were not only responsible for acetaldehyde tolerance but also turned out to be induced by acetaldehyde. Moreover, the content of oleic acid was remarkably increased in yeast cells under acetaldehyde stress, and supplementation of oleic acid into the media partially alleviated acetaldehyde stress-induced growth inhibition of strains disrupted in the genes required for acetaldehyde tolerance and OLE1. Taken together, our data suggest that the supply of NADPH and the process of fatty acid biosynthesis are the key factors in acetaldehyde tolerance in the yeast, and that oleic acid plays an important role in acetaldehyde tolerance.

Assessment of the microbial contribution to the processing of salted salmon roe (Sujiko).

As the microbial contributions to the processing of salted foods have been little investigated, there remains a possibility that excess sterilization of raw materials for salted foods leads to deterioration in food quality and safety. At a salmon roe (sujiko) processing company, we investigated salted sujiko made identically to commercial products, but that had been processed with or without antibiotics. The antibiotics caused no significant difference in the content of free amino acids, lactic acid or acetic acid. These results show that general aerobic bacteria have no impact on the formation of these flavor compounds.

Cold-active acid beta-galactosidase activity of isolated psychrophilic-basidiomycetous yeast Guehomyces pullulans.

In the present study, psychrophilic yeasts, which grow on lactose as a sole carbon source at low temperature and under acidic conditions, were isolated from soil from Hokkaido, Japan. The phenotypes and sequences of 28S rDNA of the isolated strains indicated a taxonomic affiliation to Guehomyces pullulans. The isolated strains were able to grow on lactose at below 5 degrees C, and showed cold-active acid beta-galactosidase activity even at 0 degrees C and pH 4.0 in the extracellular fractions. Moreover, K(m) of beta-galactosidase activity for lactose in the extracellular fraction from strain R1 was found to be 50.5 mM at 10 degrees C, and the activity could hydrolyze lactose in milk at 10 degrees C. The findings in this study indicate the possibility that the isolated strains produce novel acid beta-galactosidases that are able to hydrolyze lactose at low temperature.

Regulation of intracellular formaldehyde toxicity during methanol metabolism of the methylotrophic yeast Pichia methanolica.

In this study we found that the methylotrophic yeast Pichia methanolica showed impaired growth on high methanol medium (>5%, or 1.56 M, methanol). In contrast, P. methanolica grew well on glucose medium containing 5% methanol, but the growth defects reappeared on glucose medium supplemented with 5 mM formaldehyde. During methanol growth of P. methanolica, formaldehyde accumulated in the medium up to 0.3 mM before it was consumed rapidly based on cell growth. These findings indicate that the growth defect of P. methanolica on high methanol media is not caused directly by methanol toxicity, but rather by formaldehyde, which is a key toxic intermediate of methanol metabolism. Moreover, during methanol growth of P. methanolica, expression of enzymes in the methanol-oxidation pathway were induced before the alcohol oxidase isozymes Mod1p and Mod2p, and Mod1p expression was induced before Mod2p. These results suggest that to avoid excess accumulation of formaldehyde-the toxic intermediate of methanol metabolism-P. methanolica grown on methanol strictly regulates the order in which methanol-metabolizing enzymes are expressed.

Cold-active pectinolytic activity of psychrophilic-basidiomycetous yeast Cystofilobasidium capitatum strain PPY-1.

A pectinolytic and psychrophilic yeast was isolated from soil from Abashiri, Hokkaido, Japan. The phenotype and sequencing of the 28S rDNA of the isolated strain (PPY-1) indicated a taxonomic affiliation to the basidiomycetous yeast Cystofilobasidium capitatum. C. capitatum strain PPY-1 was able to grow on two pectic compounds, polygalacturonate and pectin, at below 5 degrees C. Moreover, the extracellular fraction of the strain exhibited pectin methylesterase, pectin lyase and polygalacturonase activities at 5 degrees C. Thus strain PPY-1 may produce novel enzymes that are able to degrade pectin at low temperature, although the strain has isozymes of these enzymes.

Molecular characterization of the PEX14 gene from the methylotrophic yeast Pichia methanolica.

In this study, we describe the molecular characterization of the PmPEX14 gene encoding the peroxisomal membrane protein from the methylotrophic yeast Pichia methanolica. The pex14Δ strain of P. methanolica lost its ability to grow on methanol and oleate but grew normally on glucose. Disruption of the PmPEX14 caused a decrease in the activities of peroxisomal methanol-metabolizing enzymes and mislocalization of those proteins into the cytosol and vacuole. Taken together, these findings show that PmPex14p has an essential physiological role in methanol metabolism in P. methanolica.

Flavobacterium algicola sp. nov., isolated from marine algae.

A rod-shaped Gram-staining-negative, non-motile, aerobic and fucoidan-digesting strain, designated TC2(T), was isolated from marine algae collected from the coast of the Sea of Okhotsk at Abashiri, Hokkaido, Japan. The bacterium formed yellow, translucent, circular and convex colonies. Comparative 16S rRNA gene sequence analysis indicated that the strain belonged to the genus Flavobacterium, with the highest sequence similarities of 97.1 to 97.3 % to the type strains of Flavobacterium frigidarium, Flavobacterium frigoris, Flavobacterium limicola and Flavobacterium psychrolimnae. DNA-DNA relatedness values between strain TC2(T) and the above-mentioned species were lower than 28 %. The genomic DNA G+C content was 33.9 mol%. The major respiratory quinone was menaquinone-6 and the predominant fatty acids were iso-C(15 : 1) G, iso-C(15 : 0), iso-C(15 : 0) 3-OH and summed feature 3 (which comprises iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c). Strain TC2(T) could be differentiated from related species by several phenotypic characteristics. Thus, on the basis of these results, strain TC2(T) represents a novel species of the genus Flavobacterium, for which the name Flavobacterium algicola sp. nov. is proposed. The type strain is TC2(T) (=NBRC 102673(T) =CIP 109574(T)).

Degradation of dimethyl disulfide by pseudomonas fluorescens strain 76.

Strain 76, which was able to utilize dimethyl disulfide (DMDS) as a sole sulfur source, was screened from our microbial collection. It was identified as Pseudomonas fluorescens by taxonomical characterization and 16S rDNA sequence analysis. It does not belong to the methylotrophs, because it did not grow on DMDS or other C1 compounds as sole carbon source, and DMDS degradation was not repressed in the presence of glucose, Na(2)SO(4), or nutrient broth. Moreover, it showed high resistance to DMDS by growing in DMDS at concentrations up to 9.04 mM. Based on these findings, strain 76 metabolizes DMDS and has dual physiological roles: sulfur assimilation and degradation. Thus it has advantages as a biological scavenger of DMDS.

Overexpression and functional analysis of cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2.

Cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2 was overexpressed in Escherichia coli using the Cold expression system and the recombinant enzyme, rBglAp, was characterized. The purified rBglAp exhibited similar enzymatic properties to the native enzyme, e.g., (i) it had high activity at 0 degrees C, (ii) its optimum temperature and pH were 10 degrees C and 8.0, respectively, and (iii) it was possible to rapidly inactivate the rBglAp at 50 degrees C in 5 min. Moreover, rBglAp was able to hydrolyze both ONPG and lactose with K(m) values of 2.7 and 42.1mM, respectively, at 10 degrees C. One U of rBglAp could hydrolyze about 70% of the lactose in 1 ml of milk in 24h, and the enzyme produced trisaccharide from lactose. We conclude that rBglAp is a cold-active enzyme that is extremely heat labile and has significant potential application to the food industry.

Peroxisomal metabolism is regulated by an oxygen-recognition system through organelle crosstalk between the mitochondria and peroxisomes.

In the present study using Pichia methanolica, it was found that expressions of methanol-metabolic enzymes were strictly regulated by the presence of oxygen, and that induction of alcohol oxidase (AOD) isozymes was completely dependent on oxygen concentrations. A proportion of AOD-isozyme species responded to oxygen conditions, e.g. in a low oxygen condition, Mod1p was dominant, but with an increase in the oxygen concentration, the ratio of Mod2p increased. The K(m) value of Mod1p for oxygen was ca. one-seventh lower than that of Mod2p (0.47 and 3.51 mM, respectively). This shows that Mod1p is suitable at low oxygen concentrations and Mod2p at high oxygen concentrations. Also, zymogram changes for AOD isozymes were observed by inhibition of respiratory chain activity. These indicated that P. methanolica has the ability to recognize oxygen conditions and the respiratory chain should participate in the sensor for available oxygen. These facts indicate that there is organelle crosstalk between mitochondria and peroxisomes through nucleus gene regulation in order to control the consumption balance of available oxygen between the mitochondrial respiratory chain and peroxisomal AODs.

Molecular characterization of the PEX5 gene encoding peroxisomal targeting signal 1 receptor from the methylotrophic yeast Pichia methanolica.

In this study, we describe the molecular characterization of the PEX5 gene encoding the peroxisomal targeting signal 1 (PTS1) receptor from the methylotrophic yeast Pichia methanolica. The P. methanolica PEX5 (PmPEX5) gene contains a open reading frame corresponding to a gene product of 646 amino acid residues, and its deduced amino acid sequence shows a high similarity to those of Pex5ps from other methylotrophic yeasts. Like other Pex5ps, the PmPex5p possesses seven repeats of the TPR motif in the C-terminal region and three WXXXF/Y motifs. A strain with the disrupted PEX5 gene (pex5Delta) lost its ability to grow on peroxisome-inducible carbon sources, methanol and oleate, but grew normally on glucose and glycerol. Disruption of PmPEX5 caused a drastic decrease in peroxisomal enzyme activities and mislocalization of GFP-PTS1 and some peroxisomal methanol-metabolizing enzymes in the cytosol. Expression of the PmPEX5 gene was regulated by carbon sources, and it was strongly expressed by peroxisome-inducible carbon sources, especially methanol. Taken together, these findings show that PmPex5p has an essential physiological role in peroxisomal metabolism of P. methanolica, including methanol metabolism, and in peroxisomal localization and activation of methanol-metabolizing enzymes, e.g. AOD isozymes, DHAS and CTA.

Distribution, diversity and regulation of alcohol oxidase isozymes, and phylogenetic relationships of methylotrophic yeasts.

In this study, we attempted to classify the methylotrophic yeasts based on diversities of alcohol oxidase (AOD), i.e. zymogram patterns and partial amino acid sequences. According to zymogram patterns for AOD, members of the methylotrophic yeasts separate into two major lineages, one group involving strains having a single AOD and the other group, including Pichia methanolica, Candida pignaliae and C. sonorensis, showing nine AOD isozymes. Based on partial amino acid sequences of AOD, the methylotrophic yeasts could be divided into five groups, and this classification agrees mostly with grouping based on 26S domain D1/D2 rDNA nucleotide sequences, except for some strains. Moreover, the strains having AOD isozymes constitute one group with P. trehalophila, P. glucozyma and Pichia sp. strain BZ159, although these strains are divided into two types, based on amino acid sequences of second AODs. On the other hand, these AOD isozymes consist of two subunits; the first subunits are induced not only by methanol but also by glycerol and pectin, although the second subunits are mainly induced by methanol. These data indicate that AOD isozymes and second AOD genes distribute widely in several methylotrophic yeasts in the natural environment, and second AOD genes may have evolved as methylotrophic genes that can adapt to the environmental conditions of higher methanol concentrations.

Purification and molecular characterization of cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2.

In this study, we purified and molecularly characterized a cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2. The purified beta-galactosidase from strain F2 exhibited high activity at 0 degrees C, and its optimum temperature and pH were 10 degrees C and 8.0, respectively. It was possible to inactivate the beta-galactosidase rapidly at 45 degrees C in 5 min. The enzyme was able to hydrolyze lactose as a substrate, as well as o-nitrophenyl-beta-D-galactopyranoside (ONPG), the Km values with ONPG and lactose being calculated to be 2.8 mM and 50 mM, respectively, at 10 degrees C. Moreover, the bglA gene encoding the beta-galactosidase of strain F2 was cloned and analyzed. The bglA gene consists of a 3,084-bp open reading frame corresponding to a protein of 1,028 amino acid residues. BglAp, the gene product derived from bglA, had several conserved regions for glycosyl hydrolase family 2, e.g., the glycosyl hydrolase 2 (GH2) sugar binding domain, GH2 acid-base catalyst, GH2 triosephosphate isomerase barrel domain, GH2 signature 1, and several other GH2 conserved regions. From these facts, we conclude that the beta-galactosidase from A. psychrolactophilus strain F2, which is a new member of glycosyl hydrolase family 2, is a cold-active enzyme that is extremely heat labile and could have advantageous applications in the food industry.

Molecular characterization of the Tim9 homologue from the methylotrophic yeast Pichia methanolica.

In this paper we describe molecular characterization of the TIM9 gene encoding the essential mitochondrial inner-membrane protein in the methylotrophic yeast Pichia methanolica. PmTIM9 contains two exons corresponding to a gene product of 89 amino acid residues and a 140 bp intron. The deduced amino acid sequence exhibited high identity to those of other yeast Tim9ps, and possessed two CX(3)C motifs that contained two cysteine residues conserved among small Tim family proteins. Moreover, PmTIM9 had the ability to partially suppress the temperature sensitivity of Saccharomyces cerevisiae strain tim9-3, suggesting that PmTIM9 is a functional homologue of the ScTIM9 gene.

Regulation of two distinct alcohol oxidase promoters in the methylotrophic yeast Pichia methanolica.

In this study, two Pichia methanolica alcohol oxidase (AOD) promoters, P(MOD1) and P(MOD2), were evaluated in a promoter assay system utilizing the acid phosphatase (AP) gene from Saccharomyces cerevisiae (ScPHO5) as a reporter. Heterologous gene expression driven by the P(MOD1) and P(MOD2) promoters was found to be strong and tightly regulated by carbon source at the transcriptional level. P(MOD1) was induced not only by methanol but also by glycerol. P(MOD2) was induced only by methanol, although it was not repressed on the addition of glycerol to a methanol medium, suggesting that P(MOD2) is regulated in a manner distinct from that of other AOD-gene promoters. On the other hand, methanol and oxygen level-influenced gene expression mediated by P(MOD1) and P(MOD2). P(MOD1) expression was optimal at low methanol concentrations, whereas P(MOD2) was predominantly expressed at high methanol and high oxygen concentrations. Based on these results, both P(MOD2) and P(MOD1) should be useful tools for controlling heterologous gene expression in P. methanolica. In particular, it should be possible to differentially control the production phases of two heterologous proteins, using P(MOD1) and P(MOD2) in the same host cell and in the same flask.

Cold-active polygalacturonase from psychrophilic-basidiomycetous yeast Cystofilobasidium capitatum strain PPY-1.

We purified and characterized a cold-active polygalacturonase (PG) from the extracellular fraction of Cystofilobasidium capitatum strain PPY-1. The purified PG from strain PPY-1 has a molecular mass of about 44 kDa, and exhibited high activity at 0 degrees C, although its optimum temperature was 45 degrees C. Although the Km value for polygalacturonate as a substrate at 45 degrees C was found to be 11.2 mg/ml, it decreased gradually with decreasing temperature, and it was 0.66 mg/ml at 0 degrees C. Moreover, its cleavage pattern was of the endo-type. These findings might indicate that PG from strain PPY-1 is a novel type of cold-active endo-PG that is able to degrade pectin compounds at low temperatures.

A cold-active pectin lyase from the psychrophilic and basidiomycetous yeast Cystofilobasidium capitatum strain PPY-1.

In the present study we purified a cold-active PNL (pectin lyase) from the extracellular fraction of the PPY (pectinolytic and psychrophilic yeast) Cystofilobasidium capitatum strain PPY-1. The purified PNL has a molecular mass of approx. 42 kDa, and its N-terminal amino acid sequence is ATGVTGSAYGFATGTTGGGSATPAY, which exhibits 72% identity with that of PNL F from Aspergillus niger. The purified PNL exhibited high activity at 10 degrees C, although its optimum temperature was 40 degrees C. Moreover, Km and Vmax for pectin as a substrate were found to have values 36.6 mg/ml and 3000 units/mg respectively. These findings may indicate that this enzyme from strain PPY-1 is a cold-active PNL that is able to degrade pectin compounds at low temperature.