Hansenula Henricii Research Articles

Occurrence of the general control of amino acid biosynthesis in yeasts

The response of three amino acid biosynthetic enzymes, threonine dehydratase, tyrosine aminotransferase and saccharopine dehydrogenase, to conditions of histidine, tryptophan or lysine limitation was investigated in 15 yeast species. The activities of all these enzymes increased about two- to fourfold as a result of action of the general control of amino acid biosynthesis in Brettanomyces anomalus, Candida maltosa, Hansenula polymorpha, Rhodosporidium toruloides, Saccharomyces cerevisiae and Yarrowia lipolytica. No evidence for the existence of the general control was found in Candida brumptii, Candida utilis, Hansenula anomala, Hansenula henricii, Kluyveromyces marxianus, Pichia guilliermondii, Saccharomycopsis capsularis, Trichosporon adeninovorans and Trigonopsis variabilis.

Untersuchungen zur Überproduktion von Tryptophan und anderer Indolderivate durch Hansenula henricii

AbstractThe cell pools of tryptophan and anthranilate, the excretion of indole‐containing metabolites, and the levels of the enzymes of aromatic amino acid biosynthesis have been determined in regulatory mutants of Hansenula henricii. The strain Hg 48‐2‐M8 produces indoles with a maximum specific productivity of 0.37 mg/g · h at a maximum specific production value of 21 mg/g dry cell weight. This methyl‐tryptophan resistant mutant possess an anthranilate synthase, whose inhibition by tryptophan is reduced. The best conditions for production of indoles are the following: 1% glucose as C‐source; ammonium as N‐source; pH value smaller than 4. We found that under various growth conditions 25–60% synthesized indole‐containing metabolites consists of tryptophan.

Säureausscheidung und Kaliumaufnahme der Hefe Hansenula henricii Hg 48‐2 in Chemostat‐Kulturen bei verschiedenen pH‐Werten

AbstractProton release and K+‐content of biomass in chemostat cultures of the yeast Hansenula henricii Hg 48‐2 were measured in 12 steady states at different K+‐concentrations in the medium reservoire (sR) and at different pH‐values in the culture. Competitive inhibition of K+‐uptake by H+‐ions, described for non‐growing yeast cultures, could not be found. The growing cultures show H+/K+ exchange quotients between 8:1 and 40:1. The actual value of the H+/K+ exchange quotient is determined by the relation between the NH+4‐and the K+‐content in the medium reservoire. The exchange quotient is not influenced by the concentration of biomass and slightly modulated by the pH‐value in the culture. The difference between the H+ excretion and the K+ uptake is caused mainly by the growth‐dependent NH+4 demand. The K+ content of the biomass increases non‐linear with increasing K+ concentrations sR constant pH values. There exists a linear relationship between the yield coefficient YH+ and the biomass concentration in the steady state χ. These findings are discussed as a result of interactions between potassium‐, nitrogen‐and oxygen‐limitation.

Untersuchungen der Tryptophan- und Anthranilsäure-Biosynthese in Hansenula henricii

Summary The cell pools of tryptophan and anthranilic acid and the levels of the enzymes of aromatic amino acid biosynthesis have been determined in Hansenula henricii Hg 48-2. The in vivo biosynthesis of tryptophan and anthranilic acid appears to be controlled primarily by feedback inhibition of the anthranilate synthase by tryptophan. The flux through the pathway is adjusted also by the lowering of concentration of anthranilate synthase and tryptophan synthase during the growth. The relative contributions of these 2 mechanisms to the control in vivo were analyzed. The other enzymes are present in excess. These exceeded the rate of consumption of the aromatic amino acids on minimal medium by a factor of 2-11. Our results suggest that the presence of required metabolites may be also an instrument for the specific regulation of the flux through the tryptophan pathway.

Regulation der Anthranilsäure-Akkumulation in Hansenula henricii Hg 48-2

Summary In the anthranilate excreting mutant Hg 48-2 of Hansenula henricii the parameters influencing the production of anthranilate were investigated. This strain accumulates anthranilate only under certain conditions: glucose as C-source in a concentration smaller than 4%; ammonium as N-source; p H value smaller than 4. Other growth conditions lead to decreased excretion, but the accumulation of anthranilic acid can be increased by adding arginine, ornithine, citrulline, histidine, tyrosine, methionine, adenine, hypoxanthine, or cytosine. Measuring the metabolite concentration in cells grown in different media we found changes in the intracellular pool of ATP and phosphoribosylpyrophosphate (PRPP). Both these substances are very important in the regulation of the tryptophan biosyntheses in Hansenula henricii Hg 48-2. The ability for overproduction of anthranilic acid always is connected with a low concentration of PRPP and the PRPP pool for itself can be influenced apparently by the ATP concentration.

Arogenate (pretyrosine) as an obligatory intermediate of the biosynthesis of l-tyrosine in chloridazon-degrading bacteria

The biosynthesis of the aromatic amino acids L-phenylalanine and c-tyrosine in microorganisms is extensively studied [1-3]. The textbook route utilizes the intermediates phenylpyruvate for phenylalanine and 4-hydroxyphenylpyruvate for tyrosine biosynthesis. Cyanobacteria were the first group of prokaryotes shown to depend upon a different sequence requiring arogenate as a tyrosine precursor [4]. Subsequently, the obligatory use of the arogenate for tyrosine biosynthesis in coryneform bacteria [5] and the yeast Hansenula henricii [6], and the dual enzyme sequences to tyrosine via 4 -hydroxyphenylpyruva te and arogenate in Pseudomonas aeruginosa [7] were demonstrated. Studying the biosynthesis of phenylalanine and tyrosine in chloridazon-degrading bacteria which are able to grow on the synthetic compound chloridazon (5 amino 4 chloro2 phenyl3(2H)pyridazinone, the active ingredient of the herbicide Pyramin ~) as sole carbon source, only a petty prephenate dehydrogenase activity could be detected [8]. The taxonomic classification of the chloridazon-degrading bacteria is very difficult, and they could not be unequivocally classified according to Bergey's Manual [9]. This paper concerns the evidence of the arogenate dehydrogenase in chloridazon-degrading bacteria, and we discuss the approach to confine the relatedness of this bacterium to other groups of bacteria by an enzymological patterning.

Allgemeine Regulation der Aminosäurebiosynthese in Hansenula henricii

AbstractThe general control of amino acid biosynthesis was investigated in Hansenula henricii. By limitation for single amino acids in wild type strain and mutants no derepression of enzymes was caused. In prototrophic revertants however, obtained from auxotrophic mutants (his, pdx) enzyme activities were 2 – 17 times higher than in the wild type. In these revertants enzymes of the biosynthetic pathways for tryptophan, phenylalanine, tyrosine, arginine, histidine, cysteine, methionine, asparagine, threonine, and isoleucine were derepressed. The amino acid pool pattern of the revertants is completely different from that in the wild type strain. A discussion of possible mechanisms of general control of amino acid biosynthesis in H. henricii is presented.

General control of amino acid biosynthesis in Hansenula henricii

The general control of amino acid biosynthesis was investigated in Hansenula henricii. By limitation for single amino acids in wild type strain and mutants no derepression of enzymes was caused. In prototrophic revertants however, obtained from auxotrophic mutants (his, pdx) enzyme activities were 2 - 17 times higher than in the wild type. In these revertants enzymes of the biosynthetic pathways for tryptophan, phenylalanine, tyrosine, arginine, histidine, cysteine, methionine, asparagine, threonine, and isoleucine were derepressed. The amino acid pool pattern of the revertants if completely different from that in the wild type strain. A discussion of possible mechanisms of general control of amino acid biosynthesis in H. henricii is presented.

Aggregation und Trennbarkeit der Enzyme des Shikimat‐Pathway bei Hefen

AbstractGel filtration was employed to estimate the molecular weights and to determine possible physical aggregation of enzymes [5‐dehydroquinate synthase (DHQ synthase), 5‐dehydroquinase (DHQase, EC 4.2.1.10), shikimate: NADP oxidoreductase (EC 1.1.1.25), shikimate kinase (EC 2.7.1.71), 3‐enolpyruvylshikimate 5‐phosphate synthase (EPSP synthase)] in the shikimate pathway in eleven species of yeasts. The five enzymes were not aggregated in extracts of Hansenula henricii, H. fabianii, H. anomala, Candida utilis, Pichia guilliermondii, and Lodderomyces elongisporus. Two enzymes (DHQase and shikimate:NADP oxidoreductase) were not separable by this method and by ion exchange chromatography, and we conclude that they exist as an aggregate in these yeasts. Evidence is presented for an enzyme aggregate containing five activities, with a molecular weight of approximately 280,000 in Rhodosporidium spaerocarpum, Rh. toruloides, Rhodotorula rubra, Saccharomycopsis lipolytica, and Saccharomyces cerevisiae. Similarities between the enzymes in the shikimate pathway of plants, bacteria, and other fungi and those of investigated yeasts are discussed.

Eigenschaften und Regulation der Enzyme des Shikimat-Pathway von Hansenula henricii: Properties and Regulation of Enzymes in the Shikimate Pathway of Hansenula henricii

Summary The following enzymes has been isolated of the yeast Hansenula henricii by means of ammonium sulfate fractionation, DEAE-Sephadex A-50 and Sepharose 6B column chromatography: 3-dehydroquinate synthase, 3-dehydroquinase-shikimate: NADP oxido-reductase complex, shikimate kinase, 5-enolpyruvyl shikimate-3-phosphate synthase, and quinate: NAD oxido-reductase. The approximate molecular weights of these enzymes have been estimated using gel filtration. Some properties of purified enzymes have been studied. Kinetic parameters and regulation of activity of enzymes were similar to those found in higher plants and bacteria. Aromatic amino acids do not regulate activity and level of the enzymes studied.

Charakterisierung von 5-Fluor- und 5-Methyltryptophan-resistenten Mutanten von Hansenula henricii

Strains of Hansenula henricii resistant to growth inhibition by various tryptophan analogs were selected. DL-5-methyltryptophan resistant cells appeared with a frequency of about 5 x 10- 6 (2 X 10- 7 for DL-5-fluoro-tryptophan) resistance. All strains resistant to fluoro-tryptophan have an defect in permeation for this tryptophan analogue. The Hansenula mutants resistant to methyl-tryptophan can be devided into four groups. To the first group belong mutants, which are permeation-resistant for 5-methyl-tryptophan. Strains of group II posess an anthranilate synthase, whose inhibition by tryptophan is decreased. Mutants of the third group show anthranilate synthase level increased to about 200 per cent; specific activity of the anthranilate phosphoribosylpyrophosphate transferase and of the indoleglycerolphosphate synthase were not altered. To the fourth group belong mutants, which contain anthranilate synthase derepressed by added tryptophan antimetabolites, L-tryptophan and several other derivatives of indole. An increase of the activity of the other tryptophan synthesizing enzymes was not observed in this case.

Aggregation and separability of the shikimate pathway enzymes in yeasts

Gel filtration was employed to estimate the molecular weights and to determine possible physical aggregation of enzymes [5-dehydroquinate synthase (DHQ synthase), 5-dehydroquinase (DHQase, EC 4.2.1.10), shikimate: NADP oxidoreductase (EC 1.1.1.25), shikimate kinase (EC 2.7.1.71), 3-enolpyruvylshikimate 5-phosphate synthase (EPSP synthase)] in the shikimate pathway in eleven species of yeasts. The five enzymes were not aggregated in extracts of Hansenula henricii, H. fabianii, H. anomala, Candida utilis, Pichia guilliermondii, and Lodderomyces elongisporus. Two enzymes (DHQase and shikimate:NADP oxidoreductase) were not separable by this method and by ion exchange chromatography, and we conclude that they exist as an aggregate in these yeasts. Evidence is presented for an enzyme aggregate containing five activities, with a molecular weight of approximately 280,000 in Rhodosporidium spaerocarpum, Rh. toruloides, Rhodotorula rubra, Saccharomycopsis lipolytica, and Saccharomyces cerevisiae. Similarities between the enzymes in the shikimate pathway of plants, bacteria, and other fungi and those of investigated yeasts are discussed.

Die Formamidase von Hansenula henricii: Isolierung, Charakterisierung und Regulation multipler Formen

Two active formamidases which hydrolyze N-formylkynurenine and N-formylanthranilic acid into formic acid and kynurenine or anthranilic acid, respectively, have been isolated from the yeast Hansenula henricii CCY 38-10-2. The molecular weight of the formamidase I is about 56,000 D calculated from gel filtration (Sephadex G-100) and 54,00 D by SDS polyacrylamide gel electrophoresis, respectively. Formamidase II has an molecular weight of about 28,000 D, estimated by both methods. After boiling of the formamidase I for 5 min in the presence of SDS (1%) and 2-mercaptoethanol (0.1 %) the enzyme is split into a smaller protein, which in SDS polyacrylamide gel electrophoresis as formamidase II. The p H optimum is different for both enzyme forms: 7.0–8.0 for formamidase I and 5.5 –7.0 for formamidase II. Enzyme activities of the two enzymes are inhibited by Zn ++ , Hg ++ , Cu ++ , NaHSO 4 , p-chloromercuribenzoate, iodacetamide, and NaF. Formamidase II was more strongly inhibited by Zn ++ , but formamidase I more by Cu ++ , whereas the other inhibitors did not show differences. The stability against high temperatures of formamidase I is greater than that of formamidase II. The latter is completely inactivated after 40 min at 45°C, while the smaller enzyme shows still 50% of the initial activity under the same conditions. The K m -values for both substrates are nearly identical 1.9 mM and 2.2 mM for formamidase I and formamidase II, respectively. N-formylkynurenine is hydrolyzed 10 times faster than N-formylanthranilic acid by formamidase I, whereas this factor is 3.6 for formamidase II. If the Hansenula yeasts are grown on minimal medium at p H 4.5 with glucose as carbon source in the presence of anthranilic acid, p-aminobenzoic acid, 3-hydroxyanthranilic acid, kynurenine or 3-hydroxykynurenine, the amount of active formamidase decrease markedly. A repression of enzyme synthesis by these substances is not observed if the p H of the glucose medium is maintained at 6.6, if glucose in the medium is replaced by glycerol, or if ammonia in the medium is replaced by asparagine. Although the ratio of formamidase I to formamidase II varied strongly under the different growth conditions it remains constant during repressed enzyme synthesis.

Enzymes of the aromatic amino acid biosynthesis in Hansenula henricii: phosphoribosyl-anthranilate isomerase and indole-glycerol-phosphate synthase (E.C.4.1.1.48)

Enzymes of the aromatic amino acid biosynthesis in Hansenula henricii: phosphoribosyl-anthranilate isomerase and indole-glycerol-phosphate synthase (E.C.4.1.1.48)

Die Enzyme der Biosynthese aromatischer Aminosäuren bei Hansenida henricii: 3-Desoxy-D-arabino-heptulonsäure-7-phosphat-(DAHP-) Synthase

DAHP synthase (EG 4.1.2.15) of the yeast Hansenula henricii CCY 38-10-2 could be separated by chromatography on DEAE-Sephadex A-50 in two isoenzymes. One of these was inhibited by L-phenylalanine, the other by L-tyrosin. The molecular weight of both isoenzymes as determined by gel filtration on Sephadex G-100 was about 21,000 dalton. From this result and from the fact, that we found only one binding site (Hill equation) for both substrates and for the inhibitor amino acids we concluded, that the isoenzymes in Hansenula henricii are present as monomers. The Km-values of the DAHP synthase (phe) are 0.24 ml for E-4-P and 0.31 mM for PEP and of the DAHP synthase (tyr) 0.35 mM for E-4-P and 0.36 mM for PEP. From the Lineweaver-Burk plots it is apparent, that the reaction mechanism is of nonsequential type (ping-pong mechanism). The isoenzymes are inhibited noncompetitively by the inhibitor amino acids with respect to both substrates. EDTA and Zn2+ inhibit the DAHP synthase completely, while other metalions have no or only weak effect.

Die Enzyme der Biosynthese aromatischer Aminosäuren bei Hansenula henricii : Die Aminotransferasen

In the yeast Hansenula henricii CCY 38-10-2 the tryptophan aminotransferase (EC 2.6.1.27), the tyrosine aminotransferase (EC 2.6.1.5.), the phenylalanine aminotransferase, and the prephenate aminotransferase, an enzyme of the pretyrosine pathway were studied. Pyridoxal-5-phosphate was used as coenzyme by the four aminotransferases. 2-oxo-glutarate was the amino group acceptor for the first three enzymes, while L-glutamate was found to be the best amino group donator for prephenate aminotransferase. The results of the regulation on enzyme level and of the regulation of enzyme synthesis are discussed. It is concluded, that the tyrosine and phenylalanine aminotransferase reaction is catalyzed by the same enzyme, while tryptophan aminotransferase and prephenate aminotransferase are specific enzymes.