The relation between the assimilation of methanol and glycerol in yeasts

Fifteen yeast strains of the genera Candida, Lodderomyces, Endomycopsis, Saccharomyces, Hansenula, Pichia and Torulopsis were investigated with respect to their ability to grow on methanol, glycerol and glucose as sole carbon and energy source. Eight of them can grow on both methanol and glycerol.Methanol is assimilated via triosephosphate (dihydroxyacetone) pathway. The dihydroxyacetone kinase is a key enzyme in methanol metabolism.The assimilation of glycerol can take place in bacteria via a phosphorylative or/and oxidative pathways. In general, the phosphorylative pathway is found in eucaryotes. In the present paper it is shown that in yeasts, which can utilize methanol and glycerol, too, glycerol is assimilated via an oxidative pathway, Dihydroxyacetone is a central intermediate in the assimilation of methanol as well as glycerol. It is metabolized by means of the dihydroxyacetone kinase.The enzyme formed during growth of Candida methylica on methanol does not differ from that of Candida valida H 122 after growing on glycerol as far as the regulatory properties are concerned.     

Regulation of methanol metabolism in the facultative methylotroph Nocardia sp. 239 during growth on mixed substrates in batch- and continuous cultures

The regulation of methanol metabolism in Nocardia sp. 239 was investigated. Growth on mixtures of glucose or acetate plus methanol in batch cultures resulted in simultaneous utilization of the substrates. The presence of glucose, but not of acetate, repressed synthesis of the ribulose monophosphate (RuMP) cycle enzymes hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), and methanol was used as an energy source only. Comparable results were obtained following addition of formaldehyde (fed-batch system) to a culture growing on glucose. The synthesis of the methanol dissimilatory and assimilatory enzymes in Nocardia sp. 239 thus appears to be controlled differently. Methanol and/or formaldehyde induce the synthesis of these enzymes, but under carbon-excess conditions their inducing effect on HPS and HPI synthesis is completely overruled by glucose, or metabolites derived from it. Repression of the synthesis of these RuMP cycle enzymes was of minor importance under carbon- and energy-limiting conditions in chemostat cultures. Addition of a pulse of glucose to a formaldehyde-limited (2.5 mmol l^–1 h^–1) fed-batch culture resulted in a decrease in the levels of several enzymes of methanol metabolism (including HPI), whereas the HPS levels remained relatively constant. Increasing HPS/HPI activity ratios were also observed with increasing growth rates in formaldehyde-limited chemostat cultures. The data indicate that additional mechanisms, the identity of which remains to be elucidated, are involved in controlling the levels of these C₁-specific enzymes in Nocardia sp. 239.Abbreviations HPS hexulose-6-phosphate synthase - HPI hexulose-6-phosphate isomerase - RuMP ribulose monophosphate - FBP fructose-1,6-bisphosphate - PFK 6-phosphofructokinase     

A Novel Class of Fungicides: α-Benzylidene-γ-valerolactones

Enzyme activities involved in the initial step of glycerol metabolism were determined in cells of methylotrophic yeasts grown on glycerol, methanol or glucose. In Candida boidinii (Kloeckera sp.) No. 2201, the activities of glycerol kinase and dihydroxyacetone kinase were detected in cells grown on glycerol and methanol, respectively. The activity of NAD⁺-linked glycerol dehydrogenase of Hansenula polymorpha dl-1 was induced by glycerol and methanol, while that of Hansenula ofunaensis was induced by glycerol. The enzymes of both strains were subject to catabolite repression by glucose.

The yeasts tested were divided into three groups as to the glycerol dissimilation patterns. Strains of the genera Candida, Saccharomyces, Pichia and Torulopsis had the phosphorylative pathway, in which glycerol is first phosphorylated. H. ofunaensis had the oxidative pathway, in which glycerol is first oxidized. H. polymorpha dl-1 had both the phosphorylative and oxidative pathways.     

Mikrobielle Verwertung von Methanol

Summary The oxidation of formaldehyde to carbon dioxide in cell-free extracts of methanol-grown Candida boidinii has been investigated. A specific NAD-dependent formaldehyde dehydrogenase requiring reduced glutathione has been partially purified. Furthermore, a NAD-linked formate dehydrogenase was found in cell-free extracts. The synthesis of these two enzymes is induced by methanol and repressed by glucose. The possible significance of these enzymes in the energy-generating system is discussed.     

Metabolic changes during substrate shifts in continuous cultures of the yeast Candida boidinii variant 60

Summary Substrate shift experiments in chemostat cultures with either methanol or glucose as carbon source were performed with the yeast Candida boidinii variant 60. At low dilution rates of 0.064 h^–1 the culture may be easily shifted from methanol to glucose medium and back again to methanol. From these experiments it can be seen that glucose does not give rise to any catabolite inhibition of alcohol oxidase. Alcohol oxidase and formaldehyde dehydrogenase seem to be regulated by a repression-derepression mechanism, as small basal activities of both these enzymes can still be measured during growth on glucose. On the other hand, formate dehydrogenase activity is completely absent in the presence of glucose. This kind of regulation seems to favor the smooth switch from growth on glucose to methanol metabolism.With methanol or glucose, growth yields (Y_S) of 0.3 and 0.35, respectively may be obtained, and oxygen consumption (Q_{O 2}) is much higher in methanol cultures than in glucose-grown cells. Accordingly, the RQ values during growth on methanol decrease to about 0.5. Based on the yield coefficient of 0.3, it is possible to calculate that 38% of the methanol consumed must be incorporated into biomass, whereas 62% of the methanol is oxidized to CO₂. The corresponding RQ of 0.56 could not be experimentally ascertained.The activities of three mitochondrial enzymes were found to be higher in methanol-grown cells than in cells from glucose cultures. The low activites of enzymes for the phosphogluconate route in methanol-grown cells indicates that a cyclic oxidation of formaldehyde via hexose phosphate to CO₂ cannot be of great importance for methanol metabolism.List of Symbols D 1/h Dilution rate - 1/h Specific growth rate - Q_{CO 2} mmol/g·h Specific CO₂ production rate - Q_{O 2} mmol/g·h Specific O₂ comsumption rate - Q_S g/g·h Specific substrate consumption rate - RQ ./. Respiratory quotient (Q_{CO 2}/Q_{O 2}) - S_O g/l Substrate concentration in the feeding medium - $#x0073;$#x0304 g/l Substrate concentration in the fermentor - $#x0078;$#x0304 g/l Biomass in the fermentor - Y_{O 2} g/mmol O₂ Biomass yield on oxygen - Y_S g/g Biomass yield on carbon source     

Effect of formaldehyde on the level of cytochrome c in methylotrophic yeasts

The effect of formaldehyde on the cytochrome system of the methylotrophic yeasts Candida tropicalis, Candida boidinii, Candida methylica, Hansenula capsulata, Hansenula polymorpha, Pichia pastoris and nonmethylotrophic yeasts Saccharomyces cerevisiae was studied. In the investigated concentration range of 0.58 – 2.5 mol · 1^?1, formaldehyde decreases the level of the reduced cytochrome c in the above-mentioned microorganisms. The kinetics of the change in the reduced cytochrome c content has two phases: a phase of slow decrease and a phase of complete disappearance of the reduced cytochrome c in the cells. The duration of the first phase shortens with increasing concentrations of formaldehyde.     

Constitutive appearance of peroxisomes in a regulatory mutant of the methylotrophic yeast Hansenula polymorpha

Summary A selection by glucosamine for mutants of Hansenula polymorpha insensitive to glucose repression of methanol assimilation is described. Constitutive synthesis of enzymes is established in standard batch cultures of glucosegrown cells. Upon prolonged glucose metabolism the phenotype is masked by catabolite inactivation and degradation of enzymes. Addition of the substrate methanol remarkably improves constitutive synthesis by preventing catabolite inactivation and delaying degradation. Regular peroxisomes of reduced number are formed in mutant cells under repressed conditions. No constitutive synthesis is detectable using ethanol as a carbon source. In addition, this alcohol is detrimental to growth of the mutants, indicating that H. polymorpha is constrained to repress synthesis of enzymes involved in the C₁-metabolism when ethanol is present as a substrate.     

Peroxisomal Localization of Enzymes Related to Fatty Acid β-Oxidation in an n-Alkane-grown Yeast,Candida tropicalis

In Candida tropicalis cells grown on n-alkanes (C₁₀-C₁₃), the levels of the activities of the enzymes related to fatty acid β—oxidation—acyl-CoA oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase—were found to be higher than those in cells grown on glucose, indicating that these enzymes were induced by alkanes. The enzymes were first confirmed to be localized only in peroxisomes, while none of these enzymes nor acyl-CoA dehydrogenase, which is known to participate in the initial step of mitochondrial β-oxidation in mammalian cells, were detected in yeast mitochondria under the conditions employed.

The significance of the peroxisomal β-oxidation system in the metabolism of alkanes by the yeast was also discussed.     

A single mutation in the NAD-specific formate dehydrogenase from Candida methylica allows the enzyme to use NADP

An homology model of Candida methylica formate dehydrogenase (cmFDH) was constructed based on the Pseudomonas sp. 101 formate dehydrogenase (psFDH) structure. An aspartic acid residue in the model was predicted to interact with the adenine ribose of the NAD cofactor, in common with many NAD-dependent oxoreductases. Replacement of this aspartic acid residue by serine in cmFDH removed the absolute requirement for NAD over NADP shown by the wild type enzyme. Taken with similar results shown by d- and l-lactate dehydrogenases, this suggests that an aspartic acid in this position is a major determinant of coenzyme specificity in NAD/NADP-dependent dehydrogenases.     

Fructose-1,6-bisphosphatase degradation in the methylotrophic yeast Komagataella phaffii occurs in autophagy pathway

Many enzymes of methanol metabolism of methylotrophic yeasts are located in peroxisomes whereas some of them have the cytosolic localization. After shift of methanol-grown cells of methylotrophic yeasts to glucose medium, a decrease in the activity of cytosolic (formaldehyde dehydrogenase, formate dehydrogenase, and fructose-1,6-bisphosphatase [FBP]) along with peroxisomal enzymes of methanol metabolism is observed. Mechanisms of inactivation of cytosolic enzymes remain unknown. To study the mechanism of FBP inactivation, the changes in its specific activity of the wild type strain GS200, the strain with the deletion of the GSS1 hexose sensor gene and strain defected in autophagy pathway SMD1163 of Komagataella phaffii with or without the addition of the MG132 (proteasome degradation inhibitor) were investigated after shift of methanol-grown cells in glucose medium. Western blot analysis showed that inactivation of FBP in GS200 occurred due to protein degradation whereas inactivation in the strains SMD1163 and gss1Δ was negligible in such conditions. The effect of the proteasome inhibitor MG132 on FBP inactivation was insignificant. To confirm FBP degradation pathway, the recombinant strains with GFP-labeled Fbp1 of K. phaffii and red fluorescent protein-labeled peroxisomes were constructed on the background of GS200 and SMD1163. The fluorescent microscopy analysis of the constructed strains was performed using the vacuolar membrane dye FM4-64. Microscopic data confirmed that Fbp1 degrades by autophagy pathway in K. phaffii. K. phaffii transformants, which express heterologous β-galactosidase under FLD promoter, have been constructed.     

C<Subscript>1</Subscript> compounds as auxiliary substrate for engineered <Emphasis Type="Italic">Pseudomonas putida</Emphasis> S12

The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to efficiently utilize the C₁ compounds methanol and formaldehyde as auxiliary substrate. The hps and phi genes of Bacillus brevis, encoding two key steps of the ribulose monophosphate (RuMP) pathway, were introduced to construct a pathway for the metabolism of the toxic methanol oxidation intermediate formaldehyde. This approach resulted in a remarkably increased biomass yield on the primary substrate glucose when cultured in C-limited chemostats fed with a mixture of glucose and formaldehyde. With increasing relative formaldehyde feed concentrations, the biomass yield increased from 35% (C-mol biomass/C-mol glucose) without formaldehyde to 91% at 60% relative formaldehyde concentration. The RuMP-pathway expressing strain was also capable of growing to higher relative formaldehyde concentrations than the control strain. The presence of an endogenous methanol oxidizing enzyme activity in P. putida S12 allowed the replacement of formaldehyde with the less toxic methanol, resulting in an 84% (C-mol/C-mol) biomass yield. Thus, by introducing two enzymes of the RuMP pathway, co-utilization of the cheap and renewable substrate methanol was achieved, making an important contribution to the efficient use of P. putida S12 as a bioconversion platform host.     

Characterization of catalase-negative mutants of methylotrophic yeastHansenula polymorpha

Three recently isolated catalase-negative mutants ofHansenula polymorpha lost the ability to grow on methanol but grew in media containing glucose, ethanol or glycerol. Their incubation in a medium with methanol resulted in an accumulation of hydrogen peroxide and cell death. During growth of a catalase-negative mutant in chemostat on a mixture of methanol and glucose, neither H₂O₂ accumulation nor cell death were observed up to the molar ratio of 10:1 of the two substrates. Cytochrome-c peroxidase and NADH-peroxidase activities were detected in the cells. In methylotrophic yeasts, catalase seems to be an enzyme characteristic of the metabolism of methanol but not needed for the metabolism of multicarbon substrates. The hydrogen peroxide produced during growth of the mutants on mixed substrates is detoxified by cytochrome-c peroxidase and other peroxidases. Translated by Č. Novotny     

Investigations of mannan and glucan in the cell wall ofCandida boidinii cultivated on methanol

The polysaccharide components (mannan and glucan) in the cell wall ofCandida boidinii M 363 grown on methanol and glucose as control were investigated using electron microscopy, cytochemical and biochemical methods. An ultrastructural rearrangement of the polymers in the cell wall of yeasts cultivated on methanol in comparison to those cultivated on glucose was established. The morphological changes correlate to the quantitative changes in the polysaccharide constituents of the cell wall. The forming and the role of thiosemihydrocarbazide (TSHC) — negative zones in theCandida boidinii cell wall cultivated on methanol media are discussed.     

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.     

Catalase Activities of Hydrocarbon-utilizing Candida Yeasts

The catalase activities of the Candida cells grown on hydrocarbons were generally much higher than those of the cells grown on Iauryl alcohol, glucose or ethanol. Km values for hydrogen peroxide of the enzymes from the glucose- and the hydrocarbon-grown cells of Candida tropicalis were the same level. The enzyme activities of the yeasts were higher at the exponential growth phase, especially of the hydrocarbon-grown cells, than at the stationary phase. Profuse appearance of microbodies having homogeneous matrix surrounded by a single-layer membrane has also been observed electronmicroscopically in the hydrocarbon- grown cells of several Candida yeasts. Cytochemical studies using 3,3′-diaminobenzidine (DAB) revealed that the catalase activity was located in microbodies. These facts suggest that the catalase activities would be related to the hydrocarbon metabolism in the yeasts.     

Mikrobielle Verwertung von Methanol

Zusammenfassung Aus einer Bodenprobe wurde eine Hefe, Candida boidinii, isoliert, welche auf Methanol als einziger C-Quelle wachsen kann. Als Wachstumsfaktor benötigt dieser Hefestamm Biotin in sehr geringer Konzentration.Unter günstigen Kulturbedingungen beträgt die Zellausbeute pro 1000 ml Kulturmedium 2,3g Trockenmasse bei Zugabe von 1% (v/v) Methanol, und 8,3g bei 4% Methanol. Das Wachstum wird bei Zusatz von 5% Methanol zum Minimalmedium vollständig gehemmt. Der Stamm verwertet Kohlenhydrate und Äthanol schneller als Methanol oder Milchsäure. Die Enzyme für den Methanol-Stoffwechsel scheinen jedoch konstitutiv zu sein.Die optimalen Kulturbedingungen für kurze Generationszeiten und hohe Zellausbeuten auf Methanol sind: 28°C, NH₄ ⁺ als Stickstoffquelle und pH 5,0. Die Elementaranalyse ergab folgende Werte für die Zusammensetzung der Hefezellen: 42,81% C, 7,23% H und 5,54% N. Die Aminosäuren (in den Zellen) wurden mit dem Aminosäure-Analysator quantitativ bestimmt.

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Microbial assimilation of methanolIsolation and characterization of the yeast Candida boidinii

Summary A yeast, Candida boidinii, isolated from soil, capable of growing on a medium containing methanol as the only carbon source is described. Biotin is required in very low concentration as a growth factor.In a study on the effect of the methanol concentration on the cell growth under favorable conditions, the cell yield was 2.3 g (dry weight) with 1% (v/v) methanol and 8.3 g with 4% methanol per 1000 ml of culture medium. However, the growth was inhibited by 5% methanol. The strain assimilated carbohydrate and ethanol faster than methanol or lactate. The enzymes for the methanol metabolism are probably constitutive.Optimal conditions for rapid growth and high cell yeild from methanol were found to be: 28°C, NH₄ ⁺ as nitrogen source and pH 5.0.The cell composition was as follows: 42.81% C, 7.23% H and 5.54% N. Amino acids in the cells were analyzed by the amino acid autoanalyzer.

     

Mixed substrate growth of methylotrophic yeasts in chemostat culture: Influence of the dilution rate on the utilisation of a mixture of glucose and methanol

The growth of Hansenula polymorpha and Kloeckera sp. 2201 with a mixture of glucose and methanol (38.8%/61.2%, w/w) and the regulation of the methanol dissimilating enzymes alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase were studied in chemostat culture, as a function of the dilution rate. Both organisms utilized and assimilated glucose and methanol simultaneously up to dilution rates of 0.30 h^-1 (H. polymorpha) and 0.26h^-1, respectively (Kloeckera sp. 2201) which significantly exceeded _max found for the two yeasts with methanol as the only source of carbon. At higher dilution rates methanol utilisation ceased and only glucose was assimilated. Over the whole range of mixed-substrate growth both carbon sources were assimilated with the same efficiency as during growth with glucose or methanol alone.In cultures of H. polymorpha, however, the growth yield for glucose was lowered by the unmetabolized methanol at high dilution rates. During growth on both carbon sources the repression of the synthesis of all catabolic methanol enzymes which is normally caused by glucose was overcome by the inductive effect of the simultaneously fed methanol. In both organisms the synthesis of alcohol oxidase was found to be regulated differently as compared to catalase, formaldehyde and formate dehydrogenase. Whereas increasing repression of the synthesis of alcohol oxidase was found with increasing dilution rates as indicated by gradually decreasing specific activities of this enzyme in cell-free extracts, the specific activities of this enzyme in cell-free extracts, the specific activities of catalase and the dehydrogenases increased with increasing growth rates until repression started. The results indicate similar patterns of the regulation of the synthesis of methanol dissimilating enzymes in different methylotrophic yeasts.Abbreviations and Terms C₁ Methanol - C₆ glucose; D dilution rate (h^-1) - D _c critical dilution rate (h^-1) - q _s specific, rate of substrate consumption (g substrate [g cell dry weight]^-1 h^-1) - q _CO2 and q _O2 are the specific rates of carbon dioxide release and oxygen consumption (mmol [g cell dry weight]^-1 h^-1) - RQ respiration quotient (q _CO2 q _O2 ¹ ) - s ₀(C₁) and s ₀(C₆) are the concentrations of methanol and glucose in the inflowing medium (g l^-1) - s residual substrate concentration in the culture liquid (g l^-1) - Sp. A. enzyme specific activity - x cell dry weight concentration (gl^-1) - Y _X/C6 growth yield on glucose (g cell dry weight [g substrate]^-1     

Comparative studies on citric acid production by Aspergillus niger and Candida lipolytica using molasses and glucose

Citric acid production by Aspergillus niger NCIM 548 and Candida lipolytica NCIM 3472 has been studied in shake culture using glucose and molasses as carbon sources. Methanol addition (3% v/v) at 40 h of fermentation enhanced the production of citric acid by Aspergillus niger whereas a reduction in citric acid production by Candida lipolytica was observed with addition of methanol. Maximum citric acid concentration of 12 kg/m³ was obtained with Aspergillus niger using molasses in the presence of methanol, while maximum citric acid concentration of 8.4 kg/m³ was obtained with Candida lipolytica using glucose without methanol. It appears that product formation by Aspergillus niger is either non-growth associated or partially growth associated depending on the substrate. Methanol addition changes the nature of product formation in case of Candida lipolytica.     

Heterologous protein production in methylotrophic yeasts

The facultative methylotrophic yeasts Candida boidinii, Pichia methanolica, Pichia pastoris and Hansenula polymorpha have been developed as systems for heterologous gene expression. They are based on strong and regulatable promoters for expression control derived from methanol metabolism pathway genes. An increasing number of biotechnological applications attest to their status as preferred options among the various gene expression hosts. The well-established P. pastoris and H. polymorpha systems have been utilized in especially competitive and consistent industrial-scale production processes. Pharmaceuticals and technical enzymes produced in these methylotrophs have either already entered the market or are expected to do so in the near future. The article describes the present status of the methylotrophic yeasts as expression systems, focusing on applied examples of the recent past. Received: 9 May 2000 / Received revision: 20 June 2000 / Accepted: 23 June 2000