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.     

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     

Biogenesis and turnover of peroxisomes involved in the concurrent oxidation of methanol and methylamine in Hansenula polymorpha

Growth of Hansenula polymorpha in shake flasks and chemostat cultures in the presence of methanol as the sole source of carbon and methylamine as the sole source of nitrogen was associated with the development of peroxisomes in the cells. The organelles were involved in the concurrent oxidation of these two compounds, since they contained both alcohol oxidase and amine oxidase, which are key enzymes in methanol and methylamine metabolism, respectively. In addition catalase was present. Peroxisomes with a completely crystalline substructure were observed in methanol-limited chemostat-grown cells. Amine oxidase probably formed an integral part of these crystalloids, whereas catalase was present in a freely diffusable form. Transfer of cells, grown in a methanol-limited chemostat in the presence of methylamine into glucose/ammonium sulphate media resulted in the loss of both alcohol oxidase and amine oxidase activity from the cells. This process was associated with degradation of the crystalline peroxisomes. However, when cells were transferred into glucose/methylamine media, amine oxidase activity only declined during 2 h after the transfer and thereafter increased again. This subsequent rise in amine oxidase activity was associated with the development of new peroxisomes in the cells in which degradation of the crystalline peroxisomes, originally present, continued. These newly formed organelles probably originated from peroxisomes which had not been affected by degradation. When in the methanollimited chemostat methylamine was replaced by ammonium sulphate, repression of the synthesis of amine oxidase was observed. However, inactivation of this enzyme or degradation of peroxisomes was not detected. The decrease of amine oxidase activity in the culture was accounted for by dilution of enzyme as a result of growth and washout.     

Degradation of microbodies in relation of activities of alcohol oxidase and catalase inCandida boidinii

Degradation of microbiodies in the methanolutilizing yeastCandida boidinii was mainly studies by electron microscopical observation. The yeast cells precultured on methanol medium contained five to six microbodies per section and showed high activities of alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase. When the precultured cells were transferred into an ethanol medium the number of microbodies and concomitantly the activities of alcohol oxidase and catalase decreased. After 6 h of cultivation microbodies were hardly detected. Also the activity of alcohol oxidase was not measurable and catalase activity was reduced to one tenth, whereas the activities of formaldehyde dehydrogenase and formate dehydrogenase decreased only to about 70%. Experiments with methanol-grown cells transferred into an ethanol medium without nitrogen source indicated that the inactivation of alcohol oxidase and catalase does not require protein synthesis. However, the reappearance of these enzymes is presumably due to de novo protein synthesis as shown by experiments with cycloheximide.     

Arthrobacter P 1, a fast growing versatile methylotroph with amine oxidase as a key enzyme in the metabolism of methylated amines

A facultative methylotrophic bacterium was isolated from enrichment cultures containing methylamine as the sole carbon source. It was tentatively identified as an Arthrobacter species. Extracts of cells grown on methylamine or ethylamine contained high levels of amine oxidase (E.C. 1.4.3.) activity. Glucose- or choline-grown cells lacked this enzyme. Oxidation of primary amines by the enzyme resulted in the formation of H₂O₂; as a consequence high levels of catalase were present in methylamine-and ethylamine-grown cells. The significance of catalase in vivo was demonstrated by addition of 20 mM aminotriazole (a catalase inhibitor) to exponentially growing cells. This completely blocked growth on methylamine whereas growth on glucose was hardly affected. Cytochemical studies showed that methylamine-dependent H₂O₂ production mainly occurred on invaginations of the cytoplasmic membrane. Assimilation of formaldehyde which is generated during methylamine oxidation was by the FBP variant of the RuMP cycle of formaldehyde fixation. The absence of NAD-dependent formaldehyde and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formal-dehyde via hexulose phosphate synthase. Enzyme profiles of the organism grown on various substrates suggested that the synthesis of amine oxidase, catalase and the enzymes of the RuMP cycle is not under coordinate control.     

Nitrogen metabolism in the facultative methylotroph Arthrobacter P1 grown with various amines or ammonia as nitrogen sources

The metabolism of trimethylamine (TMA) and dimethylamine (DMA) in Arthrobacter P1 involved the enzymes TMA monooxygenase and trimethylamine-N-oxide (TMA-NO) demethylase, and DMA monooxygenase, respectively. The methylamine and formaldehyde produced were further metabolized via a primary amine oxidase and the ribulose monophosphate (RuMP) cycle. The amine oxidase showed activity with various aliphatic primary amines and benzylamine. The organism was able to use methylamine, ethylamine and propylamine as carbon-and nitrogen sources for growth. Butylamine and benzylamine only functioned as nitrogen sources. Growth on glucose with ethylamine, propylamine, butylamine and benzylamine resulted in accumulation of the respective aldehydes. In case of ethylamine and propylamine this was due to repression by glucose of the synthesis of the aldehyde dehydrogenase(s) required for their further metabolism. Growth on glucose/methylamine did not result in repression of the RuMP cycle enzyme hexulose-6-phosphate synthase (HPS). High levels of this enzyme were present in the cells and as a result formaldehyde did not accumulate. Ammonia assimilation in Arthrobacter P1 involved NADP-dependent glutamate dehydrogenase (GDH), NAD-dependent alanine dehydrogenase (ADH) and glutamine synthetase (GS) as key enzymes. In batch cultures both GDH and GS displayed highest levels during growth on acetate with methylamine as the nitrogen source. A further increase in the levels of GS, but not GDH, was observed under ammonia-limited growth conditions in continuous cultures with acetate or glucose as carbon sources.Abbreviations HPS hexulose-6-phosphate synthase - RuMP ribulose monophosphate - DMA dimethylamine - TMA trimethylamine - TMA-NO trimethylamine-N-oxide - ICL isocitrate lyase - GS glutamine synthetase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase - GOGAT glutamate synthase     

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     

Regulation of methylotrophic and heterotrophic metabolism in Arthrobacter P1. Growth on mixtures of methylamine and acetate in batch and continuous cultures

Incubations of Arthrobacter P1 in batch culture in media with mixtures of acetate and methylamine resulted in sequential utilization of the two carbon substrates, but not in diauxic growth. Irrespective of the way cells were pregrown, acetate was the preferred substrate and subsequent studies showed that this is due to the fact that acetate is a strong inhibitor of the methylamine transport system and amine oxidase in Arthrobacter P1. An analysis of enzyme activities in cell-free extracts showed that synthesis of amine oxidase occurred already in the first growth phase with acetate, whereas rapid synthesis of hexulose phosphate synthase was only observed once methylamine utilization started. It is therefore concluded that in Arthrobacter P1 the synthesis of the enzymes specific for methylamine oxidation is not regulated co-ordinately with those involved in formaldehyde fixation, but induced sequentially by methylamine and formaldehyde, respectively.During growth of Arthrobacter P1 on the same mixture in carbon- and energy source-limited continuous cultures both substrates were used simultaneously and completely at dilution rates below the _max on either of these substrates. Addition of methylamine, in concentrations as low as 0.5 mM, to the medium reservoir of an acetate-limited continuous culture (D=0.10 h^-1) already resulted in synthesis of both amine oxidase and hexulose phosphate synthase. In the reverse experiment, addition of acetate to the medium reservoir of a methylamine-limited continuous culture (D=0.10 h^-1), acetate was initially only used as an energy source. Synthesis of the glyoxylate cycle enzymes, however, did occur at acetate concentration in the feed above 7.5–10 mM. This indicates that at acetate concentrations below 10 mM the metabolism of the C₁ substrate methylamine is able to cause a complete repression of the synthesis of the enzymes involved in carbon assimilation from the C₂ substrate acetate.Abbreviations HPS Hexulose phosphate synthase - MS mineral salts - RuMP ribulose monophosphate     

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.     

Cytochemical localization of glucose oxidase in peroxisomes of Aspergillus niger

Summary The subcellular localization of glucose oxidase (E.C. 1.1.3.4) in mycelia of Aspergillus niger has been investigated using cytochemical staining techniques. Mycelia from fermenter cultures, which produced gluconic acid from glucose, contained elevated levels of glucose oxidase and catalase. Both enzymes were located in microbodies. In addition, when the organism was grown on glucose with methylamine as a nitrogen source, amine oxidase activity was detected in the microbodies. These organelles can therefore be designated as peroxisomes.     

Functional expression of amine oxidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> (AO-I) in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The aim of this work was to prepare recombinant amine oxidase from Aspergillus niger after overexpressing in yeast. The yeast expression vector pDR197 that includes a constitutive PMA1 promoter was used for the expression in Saccharomyces cerevisiae. Recombinant amine oxidase was extracted from the growth medium of the yeast, purified to homogeneity and identified by activity assay and MALDI-TOF peptide mass fingerprinting. Similarity search in the newly published A. niger genome identified six genes coding for copper amine oxidase, two of them corresponding to the previously described enzymes AO-I a methylamine oxidase and three other genes coding for FAD amine oxidases. Thus, A. niger possesses an enormous metabolic gear to grow on amine compounds and thus support its saprophytic lifestyle.     

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     

Extension of Yeast Chronological Lifespan by Methylamine

Background

Chronological aging of yeast cells is commonly used as a model for aging of human post-mitotic cells. The yeast Saccharomyces cerevisiae grown on glucose in the presence of ammonium sulphate is mainly used in yeast aging research. We have analyzed chronological aging of the yeast Hansenula polymorpha grown at conditions that require primary peroxisome metabolism for growth.

Methodology/Principal Findings

The chronological lifespan of H. polymorpha is strongly enhanced when cells are grown on methanol or ethanol, metabolized by peroxisome enzymes, relative to growth on glucose that does not require peroxisomes. The short lifespan of H. polymorpha on glucose is mainly due to medium acidification, whereas most likely ROS do not play an important role. Growth of cells on methanol/methylamine instead of methanol/ammonium sulphate resulted in further lifespan enhancement. This was unrelated to medium acidification. We show that oxidation of methylamine by peroxisomal amine oxidase at carbon starvation conditions is responsible for lifespan extension. The methylamine oxidation product formaldehyde is further oxidized resulting in NADH generation, which contributes to increased ATP generation and reduction of ROS levels in the stationary phase.

Conclusion/Significance

We conclude that primary peroxisome metabolism enhanced chronological lifespan of H. polymorpha. Moreover, the possibility to generate NADH at carbon starvation conditions by an organic nitrogen source supports further extension of the lifespan of the cell. Consequently, the interpretation of CLS analyses in yeast should include possible effects on the energy status of the cell.     

Significance of yeast peroxisomes in the metabolism of choline and ethanolamine

The yeasts Candida utilis and Hansenula polymorpha were able to grow in media containing choline or ethanolamine as the sole nitrogen source. During growth in the presence of these substrates, large peroxisomes developed in the cells, and extracts of choline-grown C. utilis cells contained increased levels of amine oxidase and catalase. Incubation of whole cells with choline in the presence of the amine oxidase inhibitor aminoacetonitrile led to excretion of dimethylamine and methylamine. Cytochemical experiments in which spheroplasts prepared from choline-grown cells were incubated with CeCl₃ and choline, trimethylamine, dimethylamine or methylamine revealed positively stained peroxisomes, whereas in the presence of 1 mM aminoacetonitrile staining was not observed. This indicated that choline was degraded via methylated amines and that peroxisomes played a role in its metabolism. A similar involvement of peroxisomes in choline degradation was observed in H. polymorpha. Cell-free extracts of ethanolamine-grown C. utilis and H. polymorpha also contained increased levels of amine oxidase and catalase. Ethanolamine was oxidized by cell-free extracts of both organisms after growth in the presence of ethanolamine or choline. Incubation of spheroplasts of ethanolamine-or choline-grown C. utilis with CeCl₃ and ethanolamine resulted in positively stained peroxisomes. In this organism peroxisomes were therefore also involved in ethanolamine degradation.K. B. Zwart was supported by the Foundation for Fundamental Biological Research (BION) which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO).     

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.     

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.     

Characterization of the amine oxidase involved in the growth of Trichosporon cutaneum X4 on ethylamine as source of carbon,nitrogen and energy

The amine oxidase from Trichosporon cutaneum X₄ grown on ethylamine as carbon, nitrogen and energy source was purified to near homogeneity. The purified enzyme showed the highest resistance to heat of any amine oxidase hitherto characterized from a yeast (half-life at 62°C, 14 min). Measurement of kinetic parameters as a function of carbon chain length showed results typical of a benzylamine oxidase. Both non-denaturing- and sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed multiple bands, and dimethyl suberimidate cross-linking studies revealed that the enzyme consisted of multimers of two polypeptide chains of M_r respectively 19,000 and 26,000. The smallest structure to show activity probably contained two of each kind of subunit.Abbreviation SDS sodium dodecyl sulphate     

A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris

In methylotrophic yeasts, glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required for the metabolism of methanol as a carbon source and certain alkylated amines such as methylamine as nitrogen sources. We describe the isolation and characterization of the FLD1 gene from the yeast Pichia pastoris. The gene contains a single short intron with typical yeast-splicing signals near its 5′ end, the first intron to be demonstrated in this yeast. The predicted FLD1 product (Fld1p) is a protein of 379 amino acids (approx. 40 kDa) with 71% identity to the FLD protein sequence from the n-alkane-assimilating yeast Candida maltosa and 61–65% identity with dehydrogenase class III enzymes from humans and other higher eukaryotes. Using β-lactamase as a reporter, we show that the FLD1 promoter (P_FLD1) is strongly and independently induced by either methanol as sole carbon source (with ammonium sulfate as nitrogen source) or methylamine as sole nitrogen source (with glucose as carbon source). Furthermore, with either methanol or methylamine induction, levels of β-lactamase produced under control of P_FLD1 are comparable to those obtained with the commonly used alcohol oxidase I gene promoter (P_AOX1). Thus, P_FLD1 is an attractive alternative to P_AOX1 for expression of foreign genes in P. pastoris, allowing the investigator a choice of carbon (methanol) or nitrogen source (methylamine) regulation with the same expression strain.     

Control of benzylamine oxidase activity in Kluyveromyces fragilis grown on primary amines as nitrogen source

Abstract After growth on a mixture of ammonium and either methylamine or n -butylamine as nitrogen sources, benzylamine oxidase activity in yeasts from a number of different genera was found to be repressed to a lesser extent by ammonium than was methylamine oxidase. Catalase activity was better repressed by ammonium with methylamine as the nitrogen source than with n -butylamine. During growth of Kluyveromyces fragilis on equimolar mixtures of ammonium and an amine as nitrogen sources, benzylamine oxidase synthesis began during the period of exclusive growth on ammonium, and a period of simultaneous use of both nitrogen sources was observed just before the ammonium was exhausted. Addition of ammonium to cultures growing on n -butylamine as nitrogen source had no immediate repressive effect on benzylamine oxidase or catalase synthesis. However, growth on limiting ammonium in the absence of amines did give rise to low levels of amine oxidase and derepression of catalase activity. It is concluded that benzylamine oxidase in yeasts is induced strongly by amines as well as being less strongly repressed by ammonium than methylamine oxidase.     

Methylamine metabolism in Hansenula polymorpha: an in vivo 13C and 31P nuclear magnetic resonance study.

Methylamine uptake, oxidation, and assimilation were studied in Hansenula polymorpha, a methylotrophic yeast. The constitutive ammonia transport system was shown to be effective at accumulating methylamine within cells cultured with methylamine or ammonia as a nitrogen source. [13C]methylamine oxidation rates were measured in vivo in methylamine-adapted cells by 13C nuclear magnetic resonance and were found to be lower than its uptake rate into the cells. The 13C label of methylamine was found exclusively in trehalose and glycerol, and [13C]formaldehyde was also extensively assimilated, indicating the presence of an assimilation pathway for the methylamine carbon. In vivo 31P nuclear magnetic resonance analysis showed major differences in the endogenous polyphosphate levels and mean chain length during adaptation of the cells from ammonia to methylamine, indicating that methylamine accumulated in the vacuole in the same manner as basic amino acids and purines. [13C]glucose metabolism was drastically altered during adaptation of the cells from ammonia to methylamine as a nitrogen source. The total rate of glucose utilization and the rate of ethanol production fell. Direct trehalose synthesis from glucose increased, indicating a switch from carbon utilization for growth to that for storage. The rate of methylamine oxidation was sufficient to support a much higher flow of carbon into central biosynthetic pathways. These results suggest that this reduction in biosynthetic carbon flow, rather than nitrogen availability, was the main factor responsible for reducing the growth rate of the yeast when ammonia was replaced by methylamine as the nitrogen source.