Enhanced utilization-rate of methanol during growth on a mixed substrate: A continuous culture study with Hansenula polymorpha

The yeast Hansenula polymorpha was grown in a chemostat using either methanol or sorbitol as substrate or a mixture of both. Methanol alone could be utilized up to a dilution rate (D) of 0.18 h^-1, and sorbitol allowed growth at D's higher than 0.52 h^-1. In combination with sorbitol, methanol was completely utilized in the mixture even up to a D of 0.3 h^-1, and partially utilized at higher D's, To elucidate the basis of methanol utilization at high D's, enzyme activities on the single substrates and on the substrate mixture were compared. At D's above 0.3 h^-1 an increase of formate dehydrogenase activity was evident, an enzyme involved in the oxidation of methanol to carbon dioxide. It was concluded that at high D's large amounts of methanol were oxidized to generate energy. This was proved with ¹⁴C-methanol, and it was found that in the range of partial methanol utilization approximately 75% of methanol was converted to carbon dioxide and 25% incorporated into cell material.Abbreviation D dilution rate     

Regulation of methanol metabolism in the yeast Hansenula polymorpha

A study of enzyme profiles in Hansenula polymorpha grown on various carbon substrates revealed that the synthesis of the methanol dissimilatory and assimilatory enzymes is regulated in the same way, namely by catabolite repression and induction by methanol. Mutants of H. polymorpha blocked in dihydroxyacetone (DHA) synthase (strain 70 M) or DHA kinase (strain 17 B) were unable to grow on methanol which confirmed the important role attributed to these enzymes in the biosynthetic xylulose monophosphate (XuMP) cycle. Both mutant strains were still able to metabolize methanol. In the DNA kinase-negative strain 17 B this resulted in accumulation of DHA. Although DHA kinase is thought to be involved in DHA and glycerol metabolism in methylotrophic yeasts, strain 17 B was still able to grow on glycerol at a rate similar to that of the wild type. DHA on the other hand only supported slow growth of this mutant when relatively high concentrations of this compound were provided in the medium. This slow but definite growth of strain 17 B on DHA was not based on the reversible DHA synthase reaction but on conversion of DHA into glycerol, a reaction catalyzed by DNA reductase. The subsequent metabolism of glycerol in strain 17 B and in wild type H. polymorpha, however, remains to be elucidated.Abbreviations XuMP xylulose monophosphate - DHA dihydroxyacetone - EMS ethyl methanesulphonate     

In vivo inactivation of peroxisomal alcohol oxidase in Hansenula polymorpha by KCN is an irreversible process

The fate of alcohol oxidase (AO) in chemostatgrown cells of Hansenula polymorpha, after its inactivation by KCN, was studied during subsequent cultivation of the cyanide-treated cells in fresh methanol media. Biochemical experiments showed that the cyanide-induced inactivation of AO was due to the release of flavin adenine dinucleotide (FAD) from the holo enzyme. However, dissociation of octameric AO into subunits was not observed. Subsequent growth of intact cyanide-treated cells in fresh methanol media was paralelled by proteolytic degradation of part of the peroxisomes present in the cells. The recovery of AO activity, concurrently observed in these cultures, was accounted for by synthesis of new enzyme protein. Reactivation of previously inactivated AO was not observed, even in the presence of FAD in such cultures. Newly synthesized AO protein was incorporated in only few of the peroxisomes present in the cells. ³¹P nuclear magnetic resonance (NMR) studies showed that cyanide-treatment of the cells led to a dissipation of the pH gradient across the peroxisomal membrane. However, restoration of this pH gradient was fast when cells were incubated in fresh methanol medium after removal of the cyanide.Abbreviations AO alcohol oxidase - FAD flavin adenine dinucleotide - CHI cycloheximide - NMR nuclear magnetic resonance - FPLC fast protein liquid chromatography - RIE rocket immuno electrophoresis     

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.     

Growth of Hansenula polymorpha in a methanol-limited chemostat

Hansenula polymorpha has been grown in a methanol-limited continuous culture at a variety of dilution rates. Cell suspensions of the yeast grown at a dilution rate of 0.16 h^-1 showed a maximal capacity to oxidize excess methanol (QO ₂ ^max ) which was 1.6 times higher than the rate required to sustain the growth rate (Q _O2). When the dilution rate was decreased to 0.03 h^-1, QO ₂ ^max of the cells increased to a value of more than 20 times that of Q _O2. The enzymatic basis for this tremendous overcapacity for the oxidation of excess methanol at low growth rates was found to be the methanol oxidase content of the cells. The level of this enzyme increased from 7% to approximately 20% of the soluble protein when the growth rate was decreased from 0.16 to 0.03 h^-1. These results were explained on the basis of the poor affinity of methanol oxidase for its substrates. Methanol oxidase purified from Hansenula polymorpha showed an apparent K _mfor methanol of 1.3 mM in air saturated reaction mixtures and the apparent K _mof the enzyme for oxygen was 0.4 mM at a methanol concentration of 100 mM.The involvement of an oxygen dependent methanol oxidase in the dissimilation of methanol in Hansenula polymorpha was also reflected in the growth yield of the organism. The maximal yield of the yeast was found to be low (0.38 g cells/g methanol). This was not due to a very high maintenance energy requirement which was estimated to be 17 mg methanol/g cells x h.     

Regulation of flavin biosynthesis in the methylotrophic yeast Hansenula polymorpha

Under various conditions of growth of the methylotrophic yeast Hansenula polymorpha, a tight correlation was observed between the levels of flavin adenine dinucleotide (FAD)-containing alcohol oxidase, and the levels of intracellularly bound FAD and flavin biosynthetic enzymes. Adaptation of the organism to changes in the physiological requirement for FAD was by adjustment of the levels of the enzymes catalyzing the last three steps in flavin biosynthesis, riboflavin synthetase, riboflavin kinase and flavin mononucleotide adenylyltransferase. The regulation of the synthesis of the latter enzymes in relation to that of alcohol oxidase synthesis was studied in experiments involving addition of glucose to cells of H. polymorpha growing on methanol in batch cultures or in carbon-limited continuous cultures. This resulted not only in selective inactivation of alcohol oxidase and release of FAD, as previously reported, but invariably also in repression/inactivation of the flavin biosynthetic enzymes. In further experiments involving addition of FAD to the same type of cultures it became clear that inactivation of the latter enzymes was not caused directly by glucose, but rather by free FAD that accumulated intracellularly. In these experiments no repression or inactivation of alcohol oxidase occurred and it is therefore concluded that the synthesis of this enzyme and the flavin biosynthetic enzymes is under separate control, the former by glucose (and possibly methanol) and the latter by intracellular levels of free FAD.Abbreviations FAD Flavin adenine dinucleotide - FMN riboflavin-5-phosphate; flavin mononucleotide - Rf riboflavin     

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.     

Methanol metabolism in a peroxisome-deficient mutant of Hansenula polymorpha: a physiological study

We have studied methanol-utilization in a peroxisome-deficient (PER) mutant of Hansenula polymorphoa. In spite of the fact that in carbon-limited chemostat cultures under induced conditions the enzymes involved in methanol metabolism were present at wild-type (WT) levels, this mutant is unable to grow on methanol as a sole carbon and energy source. Addition of methanol to glucose-limited (S_R=12.5mM) chemostat cultures of the PER mutant only resulted in an increase in yield when small amounts were used (up to 22.5 mM). At increasing amounts however, a gradual decrease in cell density was observed which, at 80 mM methanol in the feed, had dropped below the original value of the glucose-limited culture. This reduction in yield was not observed when increasing amounts of formate instead of methanol were used as supplements for the glucose-limited mutant culture and also not in WT cells, used as control in these experiments. The effect of addition of methanol to a glucose-limited PER culture was also studied in the transient state during adaptation of the cells to methanol. The enzyme patterns obtained suggested that the ultimate decrease in yield observed at enhanced methanol concentrations was due to an inefficient methanolmetabolism as a consequence of the absence of peroxisomes. The absence of intact peroxisomes results in two major problems namely i) in H₂O₂-metabolism, which most probably is no longer mediated by catalase and ii) the inability of the cell to control the fluxes of formaldehyde, generated from methanol. The energetic consequences of this metabolism, compared to the WT situation with intact peroxisomes, are discussed.Abbreviations AO alcohol oxidase - DHAS dihydroxyacetone synthase - WT wild-type - PER peroxisome-deficient - GSH reduced glutathione - GSSG glutathione disulphide     

Cytochemical studies on the localization of methanol oxidase and other oxidases in peroxisomes of methanol-grown Hansenula polymorpha

The localization of methanol oxidase activity in cells of methanol-limited chemostat cultures of the yeast Hansenula polymorpha has been studied with different cytochemical staining techniques. The methods were based on enzymatic or chemical trapping of the hydrogen peroxide produced by the enzyme during aerobic incubations of whole cells in methanol-containing media. The results showed that methanol-dependent hydrogen peroxide production in either fixed or unfixed cells exclusively occurred in peroxisomes, which characteristically develop during growth of this yeast on methanol. Apart from methanol oxidase and catalase, the typical peroxisomal enzymes d-aminoacid oxidase and l--hydroxyacid oxidase were also found to be located in the peroxisomes. Urate oxidase was not detected in these organelles. Phase-contrast microscopy of living cells revealed the occurrence of peroxisomes which were cubic of form. This unusual shape was also observed in thin sections examined by electron microscopy. The contents of the peroxisomes showed, after various fixation procedures, a completely crystalline or striated substructure. It is suggested that this substructure might represent the in vivo organization structure of the peroxisomal enzymes.     

Development of a set of expression vectors in Hansenula polymorpha

Four expression vectors based on formate dehydrogenase promoter (FMDp) and methanol oxidase promoter (MOXp) from Hansenula polymorpha were developed to express heterologous genes in Hansenula polymorpha. A secretion signal sequence of the mating factor-alpha from Saccharomyces cerevisiae was inserted in the secretory expression plasmids for efficient secretion. A modified green fluorescent protein (mGFP5) was used as the marker of expression for the first time in H. polymorpha NCYC495 (leu 1.1) to determine the expression ability of these plasmids. The mGFP5 thus expressed retained its biochemical and physiological properties, such as accumulation inside cells and efficient secretion into the culture media. These results indicated that the four integrative vectors are useful expression systems which could be directly applied for production of heterologous proteins of interests in H. polymorpha.     

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     

Development of crystalline peroxisomes in methanol-grown cells of the yeast Hansenula polymorpha and its relation to environmental conditions

The development of peroxisomes has been studied in cells of the yeast Hansenula polymorpha during growth on methanol in batch and chemostat cultures. During bud formation, new peroxisomes were generated by the separation of small peroxisomes from mature organelles in the mother cells. The number of peroxisomes migrating to the buds was dependent upon environmental conditions. Aging of cells was accompanied by an increase in size of the peroxisomes and a subsequent increase in their numbers per cell. Their ultimate shape and substructure as well as their number per cell was dependent upon the physiological state of the culture. The change in number and volume density of peroxisomes was related to the level of alcohol oxidase in the cells. Development of peroxisomes in cells of batch cultures was accompanied by an increase in size of the crystalline inclusions in the organelles; they had become completely crystalline when the cells were in the stationary phase. Peroxisomes in cells from methanol-limited chemostat cultures were completely crystalline, irrespective of growth rate. Results of biochemical and cytochemical experiments suggested that alcohol oxidase is a major component of the crystalline inclusions in the peroxisomes of methanol-grown Hansenula polymorpha. Possible mechanisms involved in the ultrastructural changes in peroxisomes during their development have been discussed.Abbreviations DAB 3,3-diaminobenzidine - OD optical density (663 nm)     

Dihydroxyacetone synthase is localized in the peroxisomal matrix of methanol-grown Hansenula polymorpha

The subcellular localization of dihydroxyacetone synthase (DHAS) in the methylotrophic yeast Hansenula polymorpha was studied by various biochemical and immunocytochemical methods. After cell fractionation involving differential and sucrose gradient centrifugation of protoplast homogenates prepared from methanol-grown cells, DHAS cosedimented with the peroxisomal enzymes alcohol oxidase and catalase. Electron microscopy of this fraction showed that it contained mainly intact peroxisomes, whereas SDS-polyacrylamide gel electrophoresis revealed two major protein bands (75 and 78 kDa) which were identified as alcohol oxidase and DHAS, respectively. The localization of DHAS in peroxisomes was further established by immunocytochemistry. After immuno-gold staining carried out on ultrathin sections of methanol-grown H. polymorpha using DHAS-specific antibodies, labelling was confined to the peroxisomal matrix.Abbreviations MES 2-(N-morpholino)ethanesulfonic acid - DTT dithiothreitol - SDS sodium dodecyl sulphate - TPP thiamin pyrophosphate - DHAS dihydroxyacetone synthase - GSH reduced glutathione     

Characterization of peroxisomes in glucose-grown Hansenula polymorpha and their development after the transfer of cells into methanol-containing media

Cells of Hansenula polymorpha growing exponentially on glucose generally contained a single peroxisome of small dimension, irregular in shape and located in close proximity to the cell wall. Crystalline inclusions in the peroxisomal matrix were not observed. Associations of the organelles with one or more strands of endoplasmic reticulum were evident. In stationary phase cells the size of the peroxisomes had increased considerably. They were more cubical in form and showed a partly or completely crystalline matrix.After the transfer of cells growing exponentially on glucose into media containing methanol, large peroxisomes with a partly crystalline matrix developed in the cells within 6 h. These organelles originated from the small peroxisomes in the glucose-grown cells. De novo synthesis of peroxisomes was not observed. Prolonged cultivation in the presence of methanol resulted in a gradual increase in the number of peroxisomes by means of separation of small peroxisomes from mature organelles. During growth of peroxisomes associations with the endoplasmic reticulum remained evident.The increase in volume density of peroxisomes in stationary phase cells grown on glucose and in methanol-grown cells was accompanied by the synthesis of the peroxisomal enzymes alcohol oxidase and catalase. Cytochemical staining techniques revealed that alcohol oxidase activity was only detected when the peroxisomes contained a crystalloid inclusion. Since in peroxisomes of an alcohol oxidase-negative mutant of Hansenula polymorpha crystalline inclusions were never detected, it is concluded that the development of crystalloids inside peroxisomes is due to the accumulation of alcohol oxidase in these organelles.     

Catabolite inactivation,cyclic AMP and protein phosphorylation in the methylotrophic yeastHansenula polymorpha

The inactivation of the peroxisomal enzyme alcohol oxidase and the cytoplasmic enzymes fructose-1,6-bisphosphatase, malate dehydrogenase and phosphoenolpyruvate carboxykinase was found to occur after addition of glucose to methanol-grown cells of the yeastHansenula polymorpha. The concentration of cyclic AMP increased nearly twofold within 3 min under the same conditions. In crude extracts ofH. polymorpha about 20 proteins are phosphorylated by cyclic AMP dependent protein kinases, among them also fructose-1,6-bisphosphatase. No phosphorylation of the alcohol oxidase protein could be detected. From this fact, it was concluded that the inactivation of the peroxisomal alcohol oxidase is independent of cyclic AMP-dependent protein phosphorylation.     

Glycerol metabolism in the methylotrophic yeast Hansenula polymorpha: phosphorylation as the initial step

In hansenula polymorpha glycerol is metabolized via glycerol kinase and NAD(P)-independent glycerol-3-phosphate (G3P) dehydrogenase, enzymes which hitherto were reported to be absent in this methylotrophic yeast. Activity of glycerol kinase was readily detectable when cell-free extracts were incubated at pH 7–8 with glycerol/ATP/Mg²⁺ and a discontinuous assay for G3P formation was used. This glycerol kinase activity could be separated from dihydroxyacetone (DHA) kinase activity by ion exchange chromatography. Glycerol kinase showed relatively low affinities for glycerol (apparent K _m=1.0 mM) and ATP (apparent K _m=0.5 mM) and was not active with other substrates tested. No inhibition by fructose-1,6-bisphosphate (FBP) was observed. Both NAD-dependent and NAD(P)-independent G3P dehydrogenases were present. The latter enzyme could be assayed with PMS/MTT and cosedimented with the mitochondrial fraction. Glucose partly repressed synthesis of glycerol kinase and NAD(P)-independent G3P dehydrogenase, but compared to several other non-repressing carbon sources no clear induction of these enzymes by glycerol was apparent. Amongst glycerolnegative mutants of H. polymorpha strain 17B (a DHA kinase-negative mutant), strains blocked in either glycerol kinase or membrane-bound G3P dehydrogenase were identified. Crosses between representatives of the latter mutants and wild type resulted in the isolation of, amongst others, segregants which had regained DHA kinase but were still blocked in the membrane-bound G3P dehydrogenase. These strains, employing the oxidative pathway, were only able to grow very slowly in glycerol mineral medium.Abbreviations DHA dihydroxyacetone - G3P glycerol-3-phosphate - EMS ethyl methanesulphonate - MTT 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide - PMS phenazine methosulphate - FBP fructose-1,6-bisphosphate     

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.     

Production of recombinant hirudin in Hansenula polymorpha: variation of gene expression level depends on methanol oxidase and fermentation strategies

Various recombinant Hansenula polymorpha strains were developed and compared for their level of expression of the anticoagulant hirudin. H. polymorpha DL1-57 harboring an autonomously replicating sequence, HARS36, efficiently expressed the gene for recombinant hirudin. The effect of methanol oxidase (MOX) on the expression of the hirudin gene in H. polymorpha DL1-57 was studied, and the fermentation strategies coupled with the MOX activity and an antioxidant, tocopherol, were also examined. Received 4 February 1998/ Accepted in revised form 24 June 1998     

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.     

Regulatory flexibility of methylotrophic yeasts in chemostat cultures: Simultaneous assimilation of glucose and methanol at a fixed dilution rate

The influence of the composition of methanol/glucose-mixtures as only sources of carbon and energy on growth and regulation of the synthesis of enzymes involved in methanol-dissimilation was studied under chemostat conditions at a fixed dilution rate with the methylotrophic yeasts Hansenula polymorpha and Kloeckera sp. 2201. Both carbon sources were found to be utilized completely independently of the composition of the C₁/C₆ mixture. Using mixtures of ¹⁴C-labelled methanol and glucose the growth yield for glucose was found to be constant for all C₁/C₆-mixtures tested and both yeasts. The growth yield for methanol, however, was reduced by up to 25% when the proportion of methanol in the inflowing medium was lower than 20% (w/w with respect to glucose) for H. polymorpha and 50% (w/w with respect to glucose) for Kloeckera sp. 2201 respectively. During growth with C₁/C₆-mixtures containing higher C₁-proportions of methanol regular growth yields for methanol were recorded which corresponded to the growth yields found with methanol as the only carbon source.The regulation of the synthesis of the enzymes of the dissimilatory pathway for methanol was found to be under multiple control. Although glucose was present in the medium methanol had a positive effect on the synthesis of these enzymes. Thus, in addition to derepression induction by methanol was also observed. This inductive effect was found to increase with increasing proportions of methanol in the mixture. Depending on the enzyme, 10–40% methanol in the mixture resulted in a maximal induction with enzyme specific activities equal to those found in cells grown with methanol as the only carbon source. No further enhancements in enzyme specific activities were observed during growth on mixtures containing more than 40% methanol.Abbreviations and terms C₁ Methanol - C₆ glucose - C₁/C₆ mixture compositions are given in % (w/w) - C₀ concentration of ¹⁴C in the inflowing medium (DPM ml^-1) - C(t) concentration of ¹⁴C incorporated in cells as a function of time t (DPM ml^-1) - d dilution rate (h^-1) - DPM disintegrations per minute - q _s q _C1 and q _C6 are specific rates of consumption of substrate, methanol and glucose respectively [g (g cell dry weight)^-1 h^-1] - q _O2 and q _CO2 are the specific rates of oxygen consumption and carbon dioxide release [mmol (g cell dry weight)^-1 h^-1] - RQ respiration quotient (q _CO2 q _O2 ^-1) - s _C1 and s _C6 are the residual concentrations of methanol and glucose in the culture liquid (g l^-1) - s _O/C1 and s _O/C6 are the concentrations of methanol and glucose in the inflowing medium (g l^-1) - Sp.A. enzyme specific activity - x cell dry weight concentration (g l^-1) - Y _X/C1 and Y _X/C6 are growth yields on methanol and glucose respectively (g cell dry weight (g substrate)^-1 - Y _C/C1 growth yield with methanol with respect to carbon (g carbon assimilated (g carbon supplied)^-1 - _m maximum specific growth rate (h^-1)