Isolation and characterization of the positive regulatory gene ADR1 from Saccharomyces cerevisiae.

The DNA segments containing the ADR1 gene and a mutant allele, ADR1-5c, have been isolated by complementation of function in Saccharomyces cerevisiae. The ADR1 gene is required for synthesis of the glucose-repressible alcohol dehydrogenase (ADHII) when S. cerevisiae cells are grown on a nonfermentable carbon source, whereas the ADR1-5c allele allows ADHII synthesis even during glucose repression. A plasmid pool consisting of yeast DNA fragments isolated from a strain carrying the ADR1-5c allele was used to transform a strain containing the adr1-1 allele, which prevents ADHII depression. Transformants were isolated which expressed ADHII during glucose repression. A plasmid isolated from one of these transformants was shown to carry the ADR1-5c allele by its ability to integrate at the chromosomal adr1-1 locus. The wild-type ADR1 gene was isolated by colony hybridization, using the cloned ADR1-5c gene as a probe. The ADR1-5c and ADR1 DNA segments were indistinguishable by restriction site mapping. A partial ADR1 phenotype could be conferred by a 1.9-kilobase region, but DNA outside of this region appeared to be necessary for normal activation of ADHII by the ADR1 gene.     

Regulation of expression and activity of the yeast transcription factor ADR1.

Disruption of ADR1, a positive regulatory gene in the yeast Saccharomyces cerevisiae, abolished derepression of ADH2 but did not affect glucose repression of ADH2 or cell viability. The ADR1 mRNA was 5 kilobases long and had an unusually long leader containing 509 nucleotides. ADR1 mRNA levels were regulated by the carbon source in a strain-dependent fashion. beta-Galactosidase levels measured in strains carrying an ADR1-lacZ gene fusion paralleled ADR1 and ADR1-lacZ mRNA levels, indicating a lack of translational regulation of ADR1 mRNA. ADH2 was regulated by the carbon source to the same extent in all strains examined and showed complete dependence on ADR1 as well. The expression of ADR1 mRNA and an ADR1-beta-galactosidase fusion protein during glucose repression suggested that the activity of the ADR1 protein is regulated at the posttranslational level to properly regulate ADH2 expression. The ADR1-beta-galactosidase fusion protein was able to activate ADH2 expression during glucose repression but showed significantly higher levels of activation upon derepression. A similar result was obtained when ADR1 was present on a multicopy plasmid. These results suggest that low-level expression of ADR1 is required to maintain glucose repression of ADH2 and are consistent with the hypothesis that ADR1 is regulated at the posttranslational level.     

Overproduction of mycobacterial ribosomal protein S13 induces catalase/peroxidase activity and hypersensitivity to isoniazid in Mycobacterium smegmatis

A Bacillus Calmette Guerlin (BCG) DNA fragment was identified which conferred hypersensitivity to isoniazid (INH) upon Mycobacterium smegmatis (Ms) when present on a multicopy plasmid. The gene cluster present on this fragment contains the genes encoding ribosomal proteins L36 (rpmJ), S13 (rpsM), S11 (rpsK) and S4 (rpsD), as well as the gene encoding initiation factor-1 (infA), an open reading frame of unknown function (ORFX) and a putative promoter region. The rpsM gene, from either BCG or Ms is necessary and sufficient to produce the INH-hypersensitive phenotype in Ms, but the gene cluster has no effect on INH sensitivity when introduced into BCG on a multicopy plasmid. The presence of rpsM on a multicopy plasmid also causes an increase in catalase/peroxidase (Kat/Prx) activity in Ms. The overproduction of S13 may induce a stress response, resulting in increased expression of katG (encoding Kat/Prx) in Ms, thereby causing hypersensitivity to INH     

Peroxisomal catalase in the methylotrophic yeast Candida boidinii: transport efficiency and metabolic significance

In this study we cloned CTA1, the gene encoding peroxisomal catalase, from the methylotrophic yeast Candida boidinii and studied targeting of the gene product, Cta1p, into peroxisomes by using green fluorescent protein (GFP) fusion proteins. A strain from which CTA1 was deleted (cta1Delta strain) showed marked growth inhibition when it was grown on the peroxisome-inducing carbon sources methanol, oleate, and D-alanine, indicating that peroxisomal catalase plays an important nonspecific role in peroxisomal metabolism. Cta1p carries a peroxisomal targeting signal type 1 (PTS1) motif, -NKF, in its carboxyl terminus. Using GFP fusion proteins, we found that (i) Cta1p is transported to peroxisomes via its PTS1 motif, -NKF; (ii) peroxisomal localization is necessary for Cta1p to function physiologically; and (iii) Cta1p is bimodally distributed between the cytosol and peroxisomes in methanol-grown cells but is localized exclusively in peroxisomes in oleate- and D-alanine-grown cells. In contrast, the fusion protein GFP-AKL (GFP fused to another typical PTS1 sequence, -AKL), in the context of CbPmp20 and D-amino acid oxidase, was found to localize exclusively in peroxisomes. A yeast two-hybrid system analysis suggested that the low transport efficiency of the -NKF sequence is due to a level of interaction between the -NKF sequence and the PTS1 receptor that is lower than the level of interaction with the AKL sequence. Furthermore, GFP-Cta1pDeltankf coexpressed with Cta1p was successfully localized in peroxisomes, suggesting that the oligomer was formed prior to peroxisome import and that it is not necessary for all four subunits to possess a PTS motif. Since the main physiological function of catalase is degradation of H2O2, suboptimal efficiency of catalase import may confer an evolutionary advantage. We suggest that the PTS1 sequence, which is found in peroxisomal catalases, has evolved in such a way as to give a higher priority for peroxisomal transport to peroxisomal enzymes other than to catalases (e.g., oxidases), which require a higher level of peroxisomal transport efficiency.     

The Saccharomyces Cerevisiae Rtg2 Gene Is a Regulator of Aconitase Expression under Catabolite Repression Conditions

The ACO1 gene, encoding mitochondrial aconitase of Saccharomyces cerevisiae, is required both for oxidative metabolism and for glutamate prototrophy. This gene is subject to catabolite repression; the ACO1 mRNA level is further reduced when glutamate is supplied with glucose. To further explore regulation of ACO1 expression, we have screened for mutations that reduce expression of an ACO1-lacZ fusion borne on a multicopy vector. We identified a gene required for wild-type expression of ACO1 only under catabolite repression conditions. Sequencing of the corresponding cloned gene revealed that it is identical to RTG2 previously cloned as a pivotal gene in controlling interorganelle retrograde communication. Cells containing either the original rtg2-2 mutation or a null rtg2 allele are not petite but show a residual growth on minimum glucose medium with ammonium sulfate as the sole nitrogen source. This growth defect is partially restored by supplying aspartate or threonine, and fully with glutamate or proline supplement. Surprisingly, this phenotype is not observed on complete medium lacking either of these amino acids. In addition, a genetic analysis revealed an interaction between RTG2 and ASP5 (encoding aspartate amino transferase), thus supporting our hypothesis that RTG2 may be involved in the control of several anaplerotic pathways.     

Construction of flavocytochrome b2-overproducing strains of the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b2, FC b2) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker's yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b2 producers with overexpression of the H. polymorpha CYB2 gene, encoding FC b2. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (gcr1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b2 activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b2 producer characterized by a sixfold increased (to 3 micromol min(-1) mg(-1) protein in cell-free extract) activity of the enzyme.     

Cloning and characterization of glucokinase from a methylotrophic yeast Hansenula polymorpha: different effects on glucose repression in H. polymorpha and Saccharomyces cerevisiae

Glucokinase gene (HPGLK1) was cloned from a methylotrophic yeast Hansenula polymorpha by complementation of glucose-phosphorylation deficiency in a H. polymorpha double kinase-negative mutant A31-10 by a genomic library. An open reading frame of 1416 nt encoding a 471-amino-acid protein with calculated molecular weight 51.6 kDa was characterized in the genomic insert of the plasmid pH3. The protein sequence deduced from HPGLK1 exhibited 55 and 46% identity with glucokinases from Saccharomyces cerevisiae and Aspergillus niger, respectively. The enzyme phosphorylated glucose, mannose and 2-deoxyglucose, but not fructose. Transformation of HPGLK1 into A31-10 restored glucose repression of alcohol oxidase and catalase in the mutant. Transformation of HPGLK1 into S. cerevisiae triple kinase-negative mutant DFY632 showed that H. polymorpha glucokinase cannot transmit the glucose repression signal in S. CEREVSIAE: synthesis of invertase and maltase in respective transformants was insensitive to glucose repression similarly to S. cerevisiae DFY568 possessing only glucokinase.     

The role of peroxisomes in xylose alcoholic fermentation in the engineered Saccharomyces cerevisiae

Xylose is a second‐most abounded sugar after glucose in lignocellulosic hydrolysates and should be efficiently fermented for economically viable second‐generation ethanol production. Despite significant progress in metabolic and evolutionary engineering, xylose fermentation rate of recombinant Saccharomyces cerevisiae remains lower than that for glucose. Our recent study demonstrated that peroxisome‐deficient cells of yeast Ogataea polymorpha showed a decrease in ethanol production from xylose. In this work, we have studied the role of peroxisomes in xylose alcoholic fermentation in the engineered xylose‐utilizing strain of S. cerevisiae. It was shown that peroxisome‐less pex3Δ mutant possessed 1.5‐fold decrease of ethanol production from xylose. We hypothesized that peroxisomal catalase Cta1 may have importance for hydrogen peroxide, the important component of reactive oxygen species, detoxification during xylose alcoholic fermentation. It was clearly shown that CTA1 deletion impaired ethanol production from xylose. It was found that enhancing the peroxisome population by modulation the peroxisomal biogenesis by overexpression of PEX34 activates xylose alcoholic fermentation.     

Sequence of the Saccharomyces cerevisiae CTA1 gene and amino acid sequence of catalase A derived from it

The nucleotide sequence of a 2785-base-pair stretch of DNA containing the Saccharomyces cerevisiae catalase A (CTA1) gene has been determined. This gene contains an uninterrupted open reading frame encoding a protein of 515 amino acids (relative molecular mass 58,490). Catalase A, the peroxisomal catalase of S. cerevisiae was compared to the peroxisomal catalases from bovine liver and from Candida tropicalis and to the non-peroxisomal, presumably cytoplasmic, catalase T of S. cerevisiae. Whereas the peroxisomal catalases are almost colinear, three major insertions have to be introduced in the catalase T sequence to obtain an optimal fit with the other proteins. Catalase A is most closely related to the C. tropicalis enzyme. It is also more similar to the bovine liver catalase than to the second S. cerevisiae catalase. The differences between the two S. cerevisiae enzymes are most striking within four blocks of amino acids consisting of a total of 37 residues with high homology between the three peroxisomal, but low conservation between the S. cerevisiae catalases. The results obtained indicate that the peroxisomal catalases compared have very similar three-dimensional structures and might have similar targeting signals.