D. Birnbaum

Mode of action of glyphosate in Candida maltosa

The broad-spectrum herbicide glyphosate inhibits the growth of Candida maltosa and causes the accumulation of shikimic acid and shikimate-3-phosphate. Glyphosate is a potent inhibitor of three enzymes of aromatic amino acid biosynthesis in this yeast. In relation to tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and dehydroquinate synthase, the inhibitory effect appears at concentrations in the mM range, but 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase is inhibited by micromolar concentrations of glyphosate. Inhibition of partially purified EPSP synthase reaction by glyphosate is competitive with respect to phosphoenolpyruvate (PEP) with a Ki-value of 12 μM. The app. Km for PEP is about 5-fold higher and was 62 μM. Furthermore, the presence of glyphosate leads to derepression of many amino acid biosynthetic enzymes.

Identification of a lys2 mutant of Candida maltosa by means of transformation

SummaryWe have isolated five mutants of Candida maltos, which lack the 2-aminoadipate reductase activity, an enzyme involved in the lysine biosynthesis. By means of complementation analysis using protoplast fusion, the isolated mutants were divided into two complementation groups. Thereof the C. maltosa strain G457 could be transformed by the plasmids pDP12 and pDP13, which contain the L YS2-coding gene of Saccharomyces cerevisiae. On the basis of our presented results obtained by studies on hybridization, stability, and recovery of plasmids from C. maltosa transformants, we suggest that transformation does proceed integratively.

Transformation of Candida maltosa and Pichia guilliermondii by a plasmid containing Saccharomyces cerevisiae ARG4 DNA

SummarySaccharomyces cerevisiae, Candida maltosa and Pichia guilliermondii have been transformed by the plasmid pYe(ARG4)411, which contains the S. cerevisiae ARG4 gene inserted into pBR322. In all transformants argininosuccinate lyase as well as β-lactamase were detected. The ARG+ phenotype of transformants is mitotically unstable. Closed circular pYe(ARG4)411, DNA was detected in transformant DNA preparations by hybridization to pBR322 DNA and by transformation of E. coli to ampicillin resistance.

Expression in yeast of a Bacillus alpha-amylase gene by the ADH1 promoter

Abstract A DNA fragment of Bacillus amyloliquefaciens encoding alpha-amylase was cloned and combined with the yeast ADH1 promoter on Escherichia coli/Saccharomyces cerevisiae shuttle vectors and transformed into S. cerevisiae laboratory strains. The Bacillus gene expression in yeast was found essentially to depend on activity of the yeast ADH1 promoter in plasmid pAAH5, which also contained the ADH1 terminator sequence. The yeast transformants were characterized as to the presence of the recombinant plasmid, identity of the cloned Bacillus DNA fragment, mitotic stability and significance of the alpha-amylase activity. By Northern blotting of total RNA isolated either from E. coli or yeast transformants, 1 or 3 transcripts, respectively, were identified by hybridization with the ClaI/BamHI Bacillus DNA fragment containing the amylase gene. Variable length of amylase gene transcripts is suggested to be due to not strictly determined transcription (probably termination) within Bacillus DNA. Alpha-amylase was determined by enzymographic techniques after isoelectric focusing of yeast cellular proteins and found to differ in its isoelectric point from alpha-amylase of B. amyloliquefaciens. Incubation of pAAH5-amy transformed yeast cells on starch media showed, after a prolonged period of time, hydrolytic activities as well as cell division, thus indicating substrate utilization.

Lysine degradation in Candida maltosa: occurrence of a novel enzyme, acetyl-CoA: L-lysine N-acetyltransferase

The yeast Candida maltosa can utilize L-lysine as sole nitrogen and sole carbon source accompanied by accumulation of ε-N-acetyl-L-lysine, indicating that lysine is metabolized by way of N-acetylated intermediates. A novel lysine acetyltransferase catalyzing the first step in this pathway, the N-acetylation of the ε-amino group of L-lysine, was found in this yeast. The enzyme, acetyl-CoA:L-lysine N-acetyltransferase, is strongly induced in cells grown on L-lysine as sole carbon source. The enzyme is specific for both L-lysine and acetyl-CoA. The Km values are 10 mM for L-lysine and 0.33 mM for acetyl-CoA. The enzyme has a maximum activity at pH 8.1.

Occurrence of a novel yeast enzyme, L-lysine ε-dehydrogenase, which catalyses the first step of lysine catabolism in Candida albicans

The yeast Candida albicans is able to utilize L-lysine as the sole nitrogen and carbon source accompanied by intracellular accumulation of alpha-aminoadipate-delta-semialdehyde. A novel yeast amino acid dehydrogenase catalysing the oxidative deamination of the epsilon-group of L-lysine was found in this yeast. The enzyme, L-lysine epsilon-dehydrogenase, is strongly induced in cells grown on L-lysine as the sole nitrogen source. The enzyme is specific for both L-lysine and NADP+. The Km values were determined to be 0.87 mM for L-lysine and 0.071 mM for NADP+. An apparent Mr of 87,000 was estimated by gel filtration. The enzyme has maximum activity at pH 9.5 and a temperature optimum of 32 degrees C under our assay conditions.

Comparative studies on the enzymological basis for growth inhibition by glyphosate in some yeast species

Summary We examined the mode of action of the herbicide glyphosate (N-[phosphonomethyl]-glycine) on 19 strains of ascomycetous and basidiomycetous yeasts. In all yeast species we observed the following physiological and enzymological effects: (i) aromatic amino acids reverse the glyphosate-induced growth inhibition, (ii) yeast cells excrete shikimic acid in the presence of glyphosate, (iii) the tyrosinesensitive enzyme was the only one of the 3-deoxy-D-arabisto-heptulosonate 7-phosphate synthase isoenzymes which was inhibited by glyphosate at concentrations in the millimolar range, (iv) 5-cnolpyruvylshikimate 3-phosphate (EPSP) synthase activity was inhibited by micromolar concentrations of glyphosate. Yarrowia lipolytica was extremely sensitive, whereas a similar growth inhibition of Candida albicans, C. utilis and Hansenula polymorpha was only achieved by about 400-fold concentration of the herbicide. Our in vivo and in vitro results show that the presumable primary site of the glyphosate action in all yeast strains is the effective inhibition of the EPSP synthase activity.

Regulation of the lysine biosynthesis in Pichia guilliermondii

The regulatory properties of four enzymes (homocitrate synthase, α-aminoadipate reductase, saccharopine reductase, saccharopine dehydrogenase) involved in the lysine biosynthesis of Pichia guilliermondii were investigated and compared with the regulatory patterns found in other yeast species. The first enzyme of the pathway, homocitrate synthase, is feedback-inhibited by L-lysine. Some other amino acids (α-aminoadipate, glutamate, tryptophan, leucine) and lysine analogues are also inhibitors of one or more enzymes. It is shown that only the synthesis of homocitrate synthase is weakly repressed by L-lysine.

Regulation of chorismate mutase activity of various yeast species by aromatic amino acids

The regulatory properties of chorismate mutase, its cellular localization and isoenzyme pattern were investigated in 23 yeast species. All yeasts contained only a single form of the enzyme, which is localized exclusively in the cytosol. The enzyme activity from all sources was activated 3-(Rhodotorula aurantiaca) to 185-fold (Candida maltosa) by tryptophan. The tryphtophan concentration, which was necessary to obtain half maximum velocity was determined to be between 2 (Pichia guilliermondii) and 95 μM (Yarrowia lipolytica). Ten yeast species possessed an enzyme that was inhibited by both phenylalanine and tyrosine. The chorismate mutase from four strains was inhibited only by tyrosine and the enzyme from two species was inhibited by phenylalanine alone. The enzyme inhibition by phenylalanine and tyrosine was completely reversed by tryptophan. Six enzyme sources were not inhibited and theY. lipolytica chorismate mutase was slightly activated by both amino acids.

Physical mapping and genome organization of mitochondrial DNA from Candida maltosa

SummaryMitochondrial (mt) DNA of the ascomycetous yeast Candida maltosa was isolated and characterized. The mtDNA is circular and the size estimated from restriction analysis performed with 7 endonucleases was 52 kb pairs. A restriction map was constructed, using the cleavage data of four endonucleases. Using mt genes from Saccharomyces cerevisiae, six structural genes (large rRNA, apocytochrome b, cytochrome c oxidase subunit I and subunit 11, ATPase subunit 6 and subunit 9) were located on the C. maltosa chondriome by cross hybridization experiments. The comparison between the mt genomes of C. maltosa and six other yeasts showed differences in the overall genome organization.