Xylulose 5-phosphate-dependent fixation of formaldehyde in a methanol-utilizing yeast, Kloeckera sp. No. 2201.

The methylotrophic yeast Kloeckera sp. 2201 was grown in double carbon (glucose/methanol) limitation in a chemostat at a constant dilution rate and the regulation of the synthesis of enzymes involved in the assimilation of methanol and the breakdown of glucose was studied as a function of the composition of the glucose/methanol mixture in the inflowing medium. Enhanced synthesis of most of the enzymes taking part in the assimilation of C, units took place despite the presence of glucose in the medium. Depending on the enzyme, 40 to 60% (w/w of total substrate) methanol in the mixture was enough to cause maximal induction and no further enhancement of their specific activities was observed during growth on mixtures containing higher methanol/glucose ratios. The only two enzymes involved in the assimilation of methanol not showing these characteristic patterns were 'classical' transketolase and transaldolase. Substrate fluxes through the individual steps of glucose and methanol metabolism have been calculated and compared with the experimentally determined specific activities of the enzymes concerned. The three enzymes whose specific activities were least in excess of the substrate flux calculated to be catalysed by them were transketolase, transaldolase and (possibly) di hydroxyacetone synthase.The residual concentration of methanol was measured in the culture as a function of the composition of the supplied glucose/methanol mixture. During growth with mixtures the concentration of methanol was always lower than during growth with this substrate as the only carbon source. Possible ecological advantages accruing to a facultative methylotroph as a consequence of this behaviour are discussed.

Section: Introductionmentioning

confidence: 99%

Regulation of Enzyme Synthesis and Variation of Residual Methanol Concentration during Carbon-limited Growth of Kloeckera sp. 2201 on Mixtures of Methanol and Glucose

Egli¹,

Lindley²,

Quayle³

1983

“…Growth of methylotrophic yeasts with methanol is characterized by low growth yields and long generation times. The detection of the energetically expensive xylulose monophosphate cycle of formaldehyde fixation (van Dijken et al, 1978), which seems to be a feature of all methylotrophic yeasts (Babel & Lomagen, 1979; Kato et al, 1979), partly explained these growth characteristics and confirmed the theoretically predicted influence of the assimilatory pathway on the growth yield in methylotrophs Anthony, 1978; t Present address: Swiss Federal Institute for Water Research and Water PoiIution Control, Swiss Federal On: Sat, 22 Dec 2018 08:32:40 Harder et al, 1981). Although these authors also stressed the importance of the efficiency of energy conservation on these parameters, little is known about the influence of the substrate methanol on mitochondria in general, and the composition of the respiratory chain in particular, in methylotrophic yeasts.…”

Section: Introductionmentioning

confidence: 99%

Mitochondrial Activities in the Methylotrophic Yeast Kloeckera sp. 2201 During Growth with Glucose and/or Methanol

Egli

Lindley

1984

The methylotrophic yeast Kloeckera sp. 2201 was grown in carbon-limited chemostat cultures with glucose and/or methanol as the carbon sources, to investigate the regulation of the synthesis of enzymes involved in the tricarboxylic acid (TCA) cycle and the electron transport chain. Except for malate dehydrogenase, enzymes of the TCA cycle were repressed during growth with methanol to specific activities of 20-40% of those found in cells growing under identical growth conditions but with glucose as the carbon source. In methanol-grown cells, the specific activities of the enzymes of the respiratory chain were also repressed compared to cells grown with glucose [60-70% for NADH:cytochrome c (cyt c) oxidoreductase and cyt c oxidase; approximately 40 % for succinate : cyt c oxidoreductase]. The measurement of cytochrome oxidized-minus-reduced spectra was impossible in methanol-grown cells because of interference of the chromophoric group of the peroxisomal enzyme alcohol oxidase under these growth conditions. The relative cytochrome c content of mitochondria from methanol-grown cells, separated from peroxisomes by density gradient centrifugation, was significantly lower (cyt aa3 : cyt b : cyt c = 1 : 1.24 : 0436) than in mitochondria from glucose-grown cells (cyt aa3 : cyt b : cyt c = 1 : 1-27 : 2-20). The results are discussed with respect to the generation of energy for biosynthesis during growth with glucose and/or methanol.

“…A key step in the xylulose monophosphate (XuMP) cycle of formaldehyde fixation which operates in methylotrophic yeasts during growth on methanol is a transketolase-type reaction involving the transfer of a glycolaldehyde fragment from XuMP to formaldehyde, producing dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (Kato et al, 1979 ;O'Connor & Quayle, 1980;Waites & Quayle, 1980). During growth on methanol two transketolases are synthesized which can be separated by ion-exchange or hydroxylapatite chromatography.…”

Section: Introductionmentioning

confidence: 99%

Dihydroxyacetone Synthase: a Special Transketolase for Formaldehyde Fixation from the Methylotrophic Yeast Candida boidinii CBS 5777

Waites

Quayle

1983

The transketolase synthesized by Candida boidinii CBS 5777 during growth on non-C, substrates has been purified and shown to have a molecular weight of approximately 135 000 and to consist of two subunits of approximate molecular weight 68000. The enzyme is active with xylulose 5phosphate as glycolyl donor and ribose 5-phosphate as acceptor; under the conditions of assay formaldehyde was inactive as acceptor. Candida boidinii CBS 5777 also synthesizes a second transketolase during growth on methanol. This enzyme, which is unstable, has been purified to homogeneity. It has a wide substrate specificity towards glycolyl donors and acceptors; formaldehyde is a good acceptor and in terms of its physiological role during growth on methanol the enzyme can be described as a dihydroxyacetone synthase. This enzyme has a molecular weight of 105000-1 10000, with two subunits of molecular weight 62000-67000. Its properties have been compared with analogous enzymes purified elsewhere from methanolgrown Candida boidinii KD1 and Candida boidinii (Kloeckera sp.) 2201.