Development of Microbodies in the yeast Kloeckera growing on methanol.

A number of microbodies appear regularly in methanol-grown yeast cells, but rarely in ethanol- or glucose-grown cells. When one of representative methanol-utilizing yeasts, Kloeckera sp.no. 2201 (also known as Candida bodinii), was cultured on glucose and then transferred into a methanol medium, microbodies of small size could be observed in 2-h old cells. The number of microbodies per sectioned cell reached five to six after 4 h of cultivation. Though the number of microbodies did not change during prolonged cultivation, their size became larger with the passage of cultivation time. The activities of catalase and alcohol oxidase were confirmed in the particulate fractions throughout the cultivation period, whereas the activities of formaldehyde dehydrogenase and formate dehydrogenase were not detected in the particles. The activity of isocitrate lyase was detected in the particulate fractions only at the early growth phase.     

Methanol metabolism in yeasts: Regulation of the synthesis of catabolic enzymes

The regulation of the synthesis of four dissimilatory enzymes involved in methanol metabolism, namely alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase and catalase was investigated in the yeasts Hansenula polymorpha and Kloeckera sp. 2201. Enzyme profiles in cell-free extracts of the two organisms grown under glucose limitation at various dilution rates, suggested that the synthesis of these enzymes is controlled by derepression — represion rather than by induction — repression. Except for alcohol oxidase, the extent to which catabolite repression of the catabolic enzymes was relieved at low dilution rates was similar in both organisms. In Hansenula polymorpha the level of alcohol oxidase in the cells gradually increased with decreasing dilution rate, whilst in Kloeckera sp. 2201 derepression of alcohol oxidase synthesis was only observed at dilution rates below 0.10 h^–1 and occurred to a much smaller extent than in Hansenula polymorpha.Derepression of alcohol oxidase and catalase in cells of Hansenula polymorpha was accompanied by synthesis of peroxisomes. Moreover, peroxisomes were degraded with a concurrent loss of alcohol oxidase and catalase activities when excess glucose was introduced into the culture. This process of catabolite inactivation of peroxisomal enzymes did not affect cytoplasmic formaldehyde dehydrogenase.     

Regulation of the synthesis of methanol oxidizing enzymes inKloeckera sp. 2201 andHansenula polymorpha,a comparison

A comparative study was made of the regulation of the synthesis of methanol dissimilating enzymes inkloeckera sp. 2201 andHansenula polymorpha using chemostat and batch growth conditions and methanol or glucose as carbon sources. During growth in methanol-limited chemostat cultures similar enzyme patterns for alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase in the two yeasts were found. When growing in batch culture with glucoseH. polymorpha, but notKloeckera sp. 2201, was found to produce ethanol which might affect the synthesis of these enzymes.     

Regulation of alcohol oxidase synthesis in Hansenula polymorpha: Oversynthesis during growth on mixed substrates and induction by methanol

The regulation of the synthesis of alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase was investigated in the methanol-utilizing yeast Hansenula polymorpha. The organism was found to synthesize immunologically identical alcohol oxidases during growth on glycerol and methanol. Growth on glycerol, however, was not dependent on the alcohol oxidase, as was shown with a mutant without alcohol oxidase protein. Similarly it was shown with a catalase activity negative mutant that high catalase activity during growth on glycerol was not a prerequisite for the utilization of this substrate, though absolutely required for growth on methanol.Experiments were conducted with mixed substrates to study the influence of methanol on alcohol oxidase synthesis. In batch cultures, growth on ribose plus methanol resulted in an enhanced rate of alcohol oxidase synthesis as compared to ribose alone. In continuous cultures, (D=0.1 h^-1) addition of methanol to glycerol-, glucose-, or sorbose-limited cultures gave rise to increased alcohol oxidase activity of up to 20 U/mg, which is about by 2 times higher than the specific activity used for growth on methanol alone. The increase in specific activity of the dissimilatory enzymes on the mixed substrates is partly due to methanol per se, as was shown by a mutant unable to dissimilate or assimilate methanol.     

Microbody of methanol-grown yeasts. Localization of catalase and flavin-dependent alcohol oxidase in the isolated microbody.

Profuse appearance of microbodies was observed in the cells of methanol-utilizing yeasts in connection with the enhanced catalase activity. These microbodies were isolated successfully by means of sucrose gradient centrifugation from the methanol-grown cells of Kloeckera sp. no. 2201. Localization of a flavin-dependent alcohol oxidase as well as characteristic microbody enzymes (catalase and D-amino acid oxidase) were ascertained in the isolated microbodies, whereas formaldehyde and formate dehydrogenases were detected in the cytoplasmic region. Localization of catalase in the isolated microbody was also demonstrated by the cytochemical technique with 3,3'-diaminobenzidine.     

Characterization of ψM1, a virulent phage of Methanobacterium thermoautotrophicum Marburg

We have studied the biogenesis and enzymic composition of microbodies in different yeasts during adaptation of cells to a new growth environment. After a shift of cells of Candida boidinii and Hansenula polymorpha from glucose to methanol/methylamine-containing media, newly synthesized alcohol oxidase and amine oxidase are imported in one and the same organelle together with catalase; as a consequence the cells contain one class of morphologically and enzymatically identical microbodies. Similar results were obtained when Candida utilis cells were transferred from glucose to ethanol/ethylamine-containing media upon which all cells formed microbodies containing amine oxidase and catalase.However, when methanol-limited cells of H. polymorpha were transferred from media containing ammonium sulphate to those with methylamine as the nitrogen source, newly synthesized amine oxidase was incorporated only in part of the microbodies present in these cells. This uptake was confined to the few smaller organelles generally present at the perimeter of the cells, which were considered not fully developed (immature) as judged by their size. Essentially similar results were obtained when stationary phase cells of C. boidinii or C. utilis — grown on methanol and ethanol plus ammonium sulphate, respectively — were shifted to media containing (m)ethylamine as the nitrogen source. These results indicate that mature microbodies may exist in yeasts which no longer are involved in the uptake of matrix proteins. Therefore, these yeasts may display heterogeneities in their microbody population.     

Immobilization of yeast microbodies by inclusion with photo-crosslinkable resins.

Yeast microbodies containing FAD-dependent alcohol oxidase, catalase and D-amino acid oxidase were isolated from methanol-grown cells of Kloeckera sp. 2201 and immobilized intact in matrices formed by a short-time illumination of photo-crosslinkable resin oligomers. The relative activities of catalase, alcohol oxidase and D-amino acid oxidase of the gel-entrapped microbodies were 36, 76 and 31% respectively as compared with those of free microbodies. Immobilization enhance d the stability of catalase to a certain degree, but not that of alcohol oxidase. The pH/activity profiles of catalase and alcohol oxidase of the entrapped organelles showed more narrow pH optima than those of the free counterparts. D-Amino acid oxidase in immobilized microbodies showed a somewhat higher Km value for D-alanine than that in free ones. Immobilized microbodies oxidized two moles of methanol to form two moles of formaldehyde with consumption of one mole of molecular oxygen. Addition of 3-amino-1,2,4-triazole, an inhibitor of catalase, reduced the formation of formaldehyde to half the amount without change in the amount of oxygen consumed, indicating the synergic action of alcohol oxidase and catalase in methanol oxidation in the microbodies of living yeast cells.     

Regulation of protein synthesis in methylotrophic yeasts: Repression of methanol dissimilating enzymes by nitrogen limitation

The influence of nitrogen limitation on the regulation of the methanol oxidizing enzymes alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase in the two methylotrophic yeastsHansenula polymorpha andKloeckera sp. 2201 was studied in continuous culture. When shifted from carbon-limited growth conditions (with a mixture of glucose and methanol as carbon sources) to a nitrogen-limited environment both cultures were found to go through a transition phase where neither enhanced residual concentrations of the nitrogen source nor of one of the two carbon sources could be detected in the supernatant. As soon as nitrogen became a limiting substrate an immediate reorganisation of the cell composition was initiated: protein content of the cells dropped to approximately 40% of its initial value, glycogen was synthesized and the enzyme composition of the cells was changed. The peroxisomal enzymes alcohol oxidase and catalase in both organisms and the two dehydrogenases for formaldehyde and formate in cells ofKloeckera sp. 2201 were subject to degradation (catabolite inactivation). The measured rates of inactivation indicated that in cells ofH. polymorpha this process might be limited to peroxisomes, whereas inKloeckera sp. 2201 the degradation was found to affect peroxisomal as well as cytoplasmic enzymes. In contrast to methanol dissimilating enzymes the net rate of synthesis of hexokinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was not affected by this process but those enzymes were synthesized with increased rates.     

Immobilization of yeast microbodies and the properties of immobilized microbody enzymes

Summary Yeast microbodies isolated from methanol-grown cells of Kloeckera sp. No. 2201 were immobilized by two types of entrapping techniques: photocrosslinking of liquid oligomers of suitable photosensitive resins and crosslinking of albumin molecules with glutaraldehyde. The apparent activities of catalase, alcohol oxidase, and D-amino acid oxidase in the gel-entrapped microbodies were 40–50, 70–80, and ca. 50% respectively as compared with those in the free microbodies. Alcohol oxidase in the immobilized microbodies, similarly to that in free ones, oxidized methanol, ethanol, n-propanol, n-butanol, n-amyl alcohol, and benzyl alcohol. Some properties of catalase and alcohol oxidase in the microbodies immobilized by the above-mentioned techniques were studied in comparison with those of the enzymes in the free microbodies.     

Development of microbodies in Candida tropicalis during incubation in a n-alkane medium

Development of microbodies in Candida tropicalis pK 233 was studied mainly by electron microscopical observation. The yeast cells, precultured on malt extract, scarcely contained microbodies and showed very low catalase activity. When the precultured cells were transferred to a n-alkane medium and incubated with shaking, the number of microbodies increased and concomitantly the activity of catalase was enhanced. That is, both the area ratio of microbodies in the cell and the ratio of microbodies to cytoplasm in area increased significantly during the utilization of n-alkanes for 8 hrs. Localization of catalase in the microbodies was demonstrated cytochemically by use of 3,3'-diaminobenzidine, but other organella in the cell, except for vacuoles appearing in the early growth phase and mitochondria, were not stained with this reagent. Microbodies seemed to grow by division. Biogenesis of microbodies in the yeast cells is also discussed.     

Biogenesis and metabolic significance of microbodies in urate-utilizing yeasts

Growth of Candida famata and Trichosporon cutaneum on uric acid as the sole source of carbon and nitrogen was associated with the development of a number of microbodies in the cells. Cytochemical staining experiments showed that the organelles contained urate oxidase, a key enzyme of uric acid metabolism, and catalase. Transfer of cells, precultured on glucose or glycerol, into uric acid-containing media indicated that these microbodies originated from the organelles, originally present in the inoculum cells, by growth and division. In urate-grown C. famata the microbodies were frequently observed in large clusters; in both organisms they existed in close association with mitochondria and strands of ER. The organelles lacked crystalline inclusions. In freeze-fractured cells their surrounding membranes showed smooth fracture faces.Exposure of urate-grown cells to glucose-excess conditions led to a rapid inactivation of urate oxidase activity but catalase was only slightly inactivated. Glucose-induced enzyme inactivation was not associated with the degradation of the microbodies present in the cells. Similarly, repression of urate oxidase synthesis by ammonium ions also did not lead to the degradation of peroxisomes.     

Derepression and partial insensitivity to carbon catabolite repression of the methanol dissimilating enzymes inHansenula polymorpha

Summary The regulation of the synthesis of alcohol oxidase, catalase, formaldehyde dehydrogenase, and formate dehydrogenase was investigated in the methanol-utilizing yeastHansenula polymorpha during growth on different carbon and energy sources. When cells were grown on glucose, the enzymes of the dissimilatory methanol metabolism were not detected during the exponential phase of growth, but were formed in the late stationary phase without addition of methanol. Moreover, the enzymes were synthesized during growth on sorbitol, glycerol, ribose, and xylose. It was shown that the carbon catabolite insensitivity of the synthesis of methanol-specific enzymes is not limited to substrates that are slowly metabolized.     

Metabolic regulation in the facultative methylotroph Arthrobacter P1. Growth on primary amines as carbon and energy sources

During growth of the facultative methylotroph Arthrobacter P1 on methylamine or ethylamine both substrates are metabolized initially in an identical fashion, via the respective aldehydes. The regulatory mechanisms governing the synthesis and activities of enzymes involved in amine and aldehyde utilization were studied in substrate transition experiments. Transfer of ethylamine-grown cells into a medium with methylamine resulted in immediate exeretion of low levels of formaldehyde (max. 0.5 mM) and formate. In the reverse experiment, transfer of methylaminegrown cells into a medium with ethylamine, excretion of much higher levels of acetaldehyde (max. 3.5 mM) occurred. These different levels of aldehyde accumulation were also observed in studies with mutants of Arthrobacter P1 blocked in the synthesis of hexulose phosphate synthase or acetaldehyde dehydrogenase. In wild type Arthrobacter P1, aldehyde production resulted in rapid induction of the synthesis of enzymes involved in their degradation but also in temporary inhibition of further amine utilization and growth. The latter aetivities only resumed at normal rates after the disappearance of the aldehydes from the cultures. Acetaldehyde utilization resulted in intermittent excretion of ethanol and acetate, whereas formaldehyde utilization resulted in further accumulation of formate.During growth of Arthrobacter P1 in the presence of methylamine accumulation of toxic levels of formaldehyde is prevented because of the rapid synthesis of hexulose phosphate synthase to high activities and, in transient state situations, by feedback inhibition of formaldehyde on the activities of the methylamine transport system and amine oxidase.Abbreviations DTNB 5,5-dithiobis-(2-nitrobenzoate) - HPS hexulosephosphate synthase - MS mineral salts - RuMP ribulose monophosphate     

Effect of increasing methanol concentrations on physiology and cytology ofCandida boidinii

Concentration of methanol in the medium strongly affected not only the physiology but also the cytology ofCandida boidinii strain 2 cells in a methanol-limited chemostat at a constant dilution rateD 0.1/h and at low pH 3.0. The formation of large cubic peroxisomes with high alcohol oxidase (AO) activity observed at low methanol concentration (S ₀ 3 g/L) disappeared on increasing the methanol concentration in the inflow medium. The AO activity in the cells sharply decreased, followed by accumulation of riboflavin phosphate and residual methanol in the medium. The activity of catalase was relatively stable. At methanol concentrationS ₀>K _I (K ₁ equal to 12 g methanol per L), which included a substantial increase in methanol dissimilation, documented by higher formaldehyde and formate dehydrogenase activities and by lower yield coefficient on methanol, the yeast cells contained large lobe-shaped peroxisomes and a smaller number of larger mitochondria. The cells formed pseudomycelium with a thick septum between the mother and daughter cells.     

Derepression of FAD Pyrophosphorylase and Flavin Changes during Growth of Kloeckera sp. No. 2201 on Methanol

A methanol-utilizing yeast Kloeckera sp. No. 2201, when grown with methanol as a sole carbon and energy source, accumulated about three times much flavin as those grown with glucose, ethanol, or glycerol. A high proportion of the total flavin was FAD in methanol-grown cells. A remarkable derepression of FAD pyrophosphorylase accompanied by an inducible formation of an FAD-dependent alcohol oxidase which catalyzes oxidation of methanol, the first step in the oxidation sequence, was observed during growth of the yeast on methanol. Significant elevations of riboflavin synthetase and flavokinase were also found. Formate, as well as methanol, effectively induced both FAD pyrophosphorylase and methanol-oxidizing enzymes (alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and catalase). Observations with other methanol-utilizing yeasts also gave essentially same results. These results led to the conclusion that cellular flavin level might be under control with level of flavoprotein physiologically required.     

Physiological role of microbodies in the yeast Trichosporon cutaneum during growth on ethylamine as the source of energy,carbon and nitrogen

Compartmentation of the metabolism of ethylamine in Trichosporon cutaneum X4 was studied in cells, grown on this compound as the sole source of energy, carbon, and nitrogen. Transfer experiments indicated that an amine oxidase is involved in the early metabolism of ethylamine. The synthesis of this enzyme was induced by primary amines and was subject to partial carbon catabolite repression. Repression by ammonium ions was not observed. Adaptation of glucose-grown cells to growth on ethylamine was associated with the development of many microbodies, which developed from already existing organelles present in the inoculum cells and multiplied by division. Cytochemical experiments indicated that the organelles contained amine oxidase and catalase. Therefore, they were considered to play a key role in the metabolism of ethylamine. The physiological significance of the microbodies was investigated by fractionation studies of homogenized protoplasts from ethylamine-grown cells by differential- and sucrose-gradient centrifugation of subcellular organelles. Intact microbodies were only obtained when the isolation procedure was performed at pH 5.8 in the absence of Mg²⁺-ions. Analysis of the different fractions indicated that the key enzymes of the glyoxylate cycle, namely isocitrate lyase and malate synthase, cosedimented together with catalase and amine oxidase. In addition, activities of malate dehydrogenase, glutamate:oxaloacetate aminotransferase (GOT) and (NAD-dependent) glutamate dehydrogenase were detected in these fractions. Electron microscopy revealed that they mainly contained microbodies. Cytochemical experiments indicated that the above enzymes were all present in the same organelle. These findings suggest that microbodies of ethylamine-grown T. cutaneum X4 produce aspartate, so allowing NADH generated in the oxidation of malate by malate dehydrogenase to be quantitatively reoxidized inside the organelles in a series of reactions involving GOT and glutamate dehydrogenase. Aspartase and fumarase were not detected in the microbodies; activities of these two enzymes were present in the cytoplasm.Abbreviations ABTS 2,2-Azino-di(3-ethylbenzthiazoline sulfonate [6]) - DTT dithiothreitol - GOT glutamate:oxaloacetate aminotransferase - DTNB 5,5-dithiobis-2-nitrobenzoate - DAB diaminobenzidine - BSPT 2-(2-benzothiazolyl)-3-(4-phthalhydrazidyl)-t-styryl-sH-tetrazolium chloride - PF convex fracture face - EF concave fracture face     

Isolation and characterization of mutants of the facultative methylotroph Arthrobacter P1 blocked in one-carbon metabolism

Among methylamine and/or ethylamine minus mutants of Arthrobacter P1 four different classes were identified, which were blocked either in the methylamine transport system, amine oxidase, hexulose phosphate synthase or acetaldehyde dehydrogenase. The results indicated that a common primary amine oxidase is involved in the metabolism of methylamine and ethylamine. Growth on ethylamine, however, was not dependent on the presence of the methylamine transport system. In mutants lacking amine oxidase, methylamine was unable to induce the synthesis of hexulose phosphate synthase, thus confirming the view that the actual inducer for the latter enzyme is not methylamine, but its oxidation product formaldehyde. Contrary to expectation, when the formaldehyde fixing enzyme hexulose phosphate synthase was deleted (mutant Art 11), accumulation of formaldehyde during growth on choline or on glucose plus methylamine as a nitrogen source did not occur. Evidence was obtained to indicate that under these conditions formaldehyde may be oxidized to carbon dioxide via formate, a sequence in which peroxidative reactions mediated by catalase are involved. In addition, a specific NAD-dependent formaldehyde dehydrogenase was detected in choline-grown cells of wild type Arthrobacter P1 and strain Art 11. This enzyme, however, does not play a role in methylamine or formaldehyde metabolism, apparently because these compounds do not induce its synthesis.Abbreviations RuMP ribulose monophosphate - HPS hexulose phosphate synthase - HPI hexulose phosphate isomerase     

Microbody of n-alkane-grown yeast

Microbodies appearing abundantly in n-alkane-grown cells of Candida tropicalis pK 233 were isolated by means of sucrose density gradient centrifugation. Electron microscopical observation showed that the microbodies isolated were intact. Localization of catalase and d-amino acid oxidase in the isolated microbodies was confirmed. Isocitrate lyase, malate synthase and NADP-linked isocitrate dehydrogenase were also located in the microbody, but malate dehydrogenase, citrate synthase, aconitase and NAD-linked isocitrate dehydrogenase were not. Neither cytochrome P-450 nor NADPH-cytochrome c reductase, the components involved in the n-alkane hydroxylation system of the yeast, were detected in the microbody fraction.     

Expression of multidrug transporter P-glycoprotein in Pichia pastoris affects the host's methanol metabolism

Pichia pastoris KM71H (Mut^S) is an efficient producer of hard-to-express proteins such as the membrane protein P-glycoprotein (Pgp), an ATP-powered efflux pump which is expressed properly, but at very low concentration, using the conventional induction strategy. Evaluation of different induction strategies indicated that it was possible to increase Pgp expression by inducing the culture with 20% media containing 2.5% methanol. By quantifying methanol, formaldehyde, hydrogen peroxide and formate, and by measuring alcohol oxidase, catalase, formaldehyde dehydrogenase, formate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenases, it was possible to correlate Pgp expression to the induction strategy. Inducing the culture by adding methanol with fresh media was associated with decreases in formaldehyde and hydrogen peroxide, and increases in formaldehyde dehydrogenase, formate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenases. At these conditions, Pgp expression was 1400-fold higher, an indication that Pgp expression is affected by increases in formaldehyde and hydrogen peroxide. It is possible that Pgp is responsible for this behaviour, since the increased metabolite concentrations and decreased enzymatic activities were not observed when parental Pichia was subjected to the same growth conditions. This report adds information on methanol metabolism during expression of Pgp from P. pastoris Mut^S strain and suggests an expression procedure for hard-to-express proteins from P. pastoris.