scholarly journals Identification and Characterization of Phenylpyruvate Decarboxylase Genes in Saccharomyces cerevisiae

2003 ◽  
Vol 69 (8) ◽  
pp. 4534-4541 ◽  
Author(s):  
Zeynep Vuralhan ◽  
Marcos A. Morais ◽  
Siew-Leng Tai ◽  
Matthew D. W. Piper ◽  
Jack T. Pronk
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candidate Gene ◽  
Nitrogen Source ◽  
Candidate Genes ◽  
Sequence Similarity ◽  
Transcript Level ◽  
Decarboxylase Activity ◽  
Sole Nitrogen Source ◽  
Ehrlich Pathway ◽  
Phenylpyruvate Decarboxylase

ABSTRACT Catabolism of amino acids via the Ehrlich pathway involves transamination to the corresponding α-keto acids, followed by decarboxylation to an aldehyde and then reduction to an alcohol. Alternatively, the aldehyde may be oxidized to an acid. This pathway is functional in Saccharomyces cerevisiae, since during growth in glucose-limited chemostat cultures with phenylalanine as the sole nitrogen source, phenylethanol and phenylacetate were produced in quantities that accounted for all of the phenylalanine consumed. Our objective was to identify the structural gene(s) required for the decarboxylation of phenylpyruvate to phenylacetaldehyde, the first specific step in the Ehrlich pathway. S. cerevisiae possesses five candidate genes with sequence similarity to genes encoding thiamine diphosphate-dependent decarboxylases that could encode this activity: YDR380w/ARO10, YDL080C/THI3, PDC1, PDC5, and PDC6. Phenylpyruvate decarboxylase activity was present in cultures grown with phenylalanine as the sole nitrogen source but was absent from ammonia-grown cultures. Furthermore, the transcript level of one candidate gene (ARO10) increased 30-fold when phenylalanine replaced ammonia as the sole nitrogen source. Analyses of phenylalanine catabolite production and phenylpyruvate decarboxylase enzyme assays indicated that ARO10 was sufficient to encode phenylpyruvate decarboxylase activity in the absence of the four other candidate genes. There was also an alternative activity with a higher capacity but lower affinity for phenylpyruvate. The candidate gene THI3 did not itself encode an active phenylpyruvate decarboxylase but was required along with one or more pyruvate decarboxylase genes (PDC1, PDC5, and PDC6) for the alternative activity. The Km and V max values of the two activities differed, showing that Aro10p is the physiologically relevant phenylpyruvate decarboxylase in wild-type cells. Modifications to this gene could therefore be important for metabolic engineering of the Ehrlich pathway.

scholarly journals Purification of arginase from Aspergillus nidulans.

1994 ◽  
Vol 41 (4) ◽  
pp. 467-471 ◽  
Author(s):  
A Dzikowska ◽  
J P Le Caer ◽  
P Jonczyk ◽  
P Wëgleński
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Neurospora Crassa ◽  
Molecular Mass ◽  
Nitrogen Source ◽  
Aspergillus Nidulans ◽  
Sole Nitrogen Source ◽  
Mass Ratio ◽  
Conserved Domains ◽  
Native Molecular Mass ◽  
High Degree

Arginase (EC 3.5.3.1) of Aspergillus nidulans, the enzyme which enables the fungus to use arginine as the sole nitrogen source was purified to homogeneity. Molecular mass of the purified arginase subunit is 40 kDa and is similar to that reported for the Neurospora crassa (38.3 kDa) and Saccharomyces cerevisiae (39 kDa) enzymes. The native molecular mass of arginase is 125 kDa. The subunit/native molecular mass ratio suggests a trimeric form of the protein. The arginase protein was cleaved and partially sequenced. Two out of the six polypeptides sequenced show a high degree of homology to conserved domains in arginases from other species.

scholarly journals Improved Anaerobic Use of Arginine by Saccharomyces cerevisiae

2003 ◽  
Vol 69 (3) ◽  
pp. 1623-1628 ◽  
Author(s):  
Olga Martin ◽  
Marjorie C. Brandriss ◽  
Gisbert Schneider ◽  
Alan T. Bakalinsky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nitrogen Source ◽  
Genetically Modified ◽  
Cell Mass ◽  
Sole Nitrogen Source ◽  
Mitochondrial Targeting ◽  
Arginine Catabolism

ABSTRACT Anaerobic arginine catabolism in Saccharomyces cerevisiae was genetically modified to allow assimilation of all four rather than just three of the nitrogen atoms in arginine. This was accomplished by bypassing normal formation of proline, an unusable nitrogen source in the absence of oxygen, and causing formation of glutamate instead. A pro3 ure2 strain expressing a PGK1 promoter-driven PUT2 allele encoding Δ1-pyrroline-5-carboxylate dehydrogenase lacking a mitochondrial targeting sequence produced significant cytoplasmic activity, accumulated twice as much intracellular glutamate, and produced twice as much cell mass as the parent when grown anaerobically on limiting arginine as sole nitrogen source.

scholarly journals Physiological Characterization of the ARO10-Dependent, Broad-Substrate-Specificity 2-Oxo Acid Decarboxylase Activity of Saccharomyces cerevisiae

2005 ◽  
Vol 71 (6) ◽  
pp. 3276-3284 ◽  
Author(s):  
Zeynep Vuralhan ◽  
Marijke A. H. Luttik ◽  
Siew Leng Tai ◽  
Viktor M. Boer ◽  
Marcos A. Morais ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Substrate Specificity ◽  
Nitrogen Source ◽  
Constitutive Expression ◽  
Thiamine Pyrophosphate ◽  
Acid Decarboxylase ◽  
Decarboxylase Activity ◽  
Broad Substrate Specificity ◽  
Fusel Alcohols ◽  
Chemostat Cultures

ABSTRACT Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK113-7D were grown with different nitrogen sources. Cultures grown with phenylalanine, leucine, or methionine as a nitrogen source contained high levels of the corresponding fusel alcohols and organic acids, indicating activity of the Ehrlich pathway. Also, fusel alcohols derived from the other two amino acids were detected in the supernatant, suggesting the involvement of a common enzyme activity. Transcript level analysis revealed that among the five thiamine-pyrophospate-dependent decarboxylases (PDC1, PDC5, PDC6, ARO10, and THI3), only ARO10 was transcriptionally up-regulated when phenylalanine, leucine, or methionine was used as a nitrogen source compared to growth on ammonia, proline, and asparagine. Moreover, 2-oxo acid decarboxylase activity measured in cell extract from CEN.PK113-7D grown with phenylalanine, methionine, or leucine displayed similar broad-substrate 2-oxo acid decarboxylase activity. Constitutive expression of ARO10 in ethanol-limited chemostat cultures in a strain lacking the five thiamine-pyrophosphate-dependent decarboxylases, grown with ammonia as a nitrogen source, led to a measurable decarboxylase activity with phenylalanine-, leucine-, and methionine-derived 2-oxo acids. Moreover, even with ammonia as the nitrogen source, these cultures produced significant amounts of the corresponding fusel alcohols. Nonetheless, the constitutive expression of ARO10 in an isogenic wild-type strain grown in a glucose-limited chemostat with ammonia did not lead to any 2-oxo acid decarboxylase activity. Furthermore, even when ARO10 was constitutively expressed, growth with phenylalanine as the nitrogen source led to increased decarboxylase activities in cell extracts. The results reported here indicate the involvement of posttranscriptional regulation and/or a second protein in the ARO10-dependent, broad-substrate-specificity decarboxylase activity.

scholarly journals Genetics of the synthesis of serine from glycine and the utilization of glycine as sole nitrogen source by Saccharomyces cerevisiae.

Genetics ◽  
1995 ◽  
Vol 140 (4) ◽  
pp. 1213-1222 ◽  
Author(s):  
D A Sinclair ◽  
I W Dawes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nitrogen Source ◽  
Single Gene ◽  
Glycine Decarboxylase ◽  
Multienzyme Complex ◽  
Sole Nitrogen Source ◽  
Yeast Strains ◽  
Complementation Groups ◽  
Lipoamide Dehydrogenase ◽  
Serine Biosynthesis

Abstract Saccharomyces cerevisiae can grow on glycine as sole nitrogen source and can convert glycine to serine via the reaction catalyzed by the glycine decarboxylase multienzyme complex (GDC). Yeast strains with mutations in the single gene for lipoamide dehydrogenase (lpd1) lack GDC activity, as well as the other three 2-oxoacid dehydrogenases dependent on this enzyme. The LPD1 gene product is also required for cells to utilize glycine as sole nitrogen source. The effect of mutations in LPD1 (L-subunit of GDC), SER1 (synthesis of serine from 3-phosphoglycerate), ADE3 (cytoplasmic synthesis of one-carbon units for the serine synthesis from glycine), and all combinations of each has been determined. The results were used to devise methods for isolating mutants affected either in the generation of one-carbon units from glycine (via GDC) or subsequent steps in serine biosynthesis. The mutants fell into six complementation groups (gsd1-6 for defects in conversion of glycine to serine). Representatives from three complementation groups were also unable to grow on glycine as sole nitrogen source (gsd1-3). Assays of the rate of glycine uptake and decarboxylation have provided insights into the nature of the mutations.

scholarly journals Mechanisms of copper loading on the Schizosaccharomyces pombe copper amine oxidase 1 expressed in Saccharomyces cerevisiae

Microbiology ◽  
2006 ◽  
Vol 152 (9) ◽  
pp. 2819-2830 ◽  
Author(s):  
Julie Laliberté ◽  
Simon Labbé
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Schizosaccharomyces Pombe ◽  
Nitrogen Source ◽  
Oxidase Activity ◽  
Amine Oxidase ◽  
Yeast Cells ◽  
Primary Amines ◽  
Sole Nitrogen Source ◽  
Amine Oxidase Activity ◽  
Copper Amine

Copper amine oxidases (CAOs) are found in almost every living kingdom. Although Saccharomyces cerevisiae is one of the few yeast species that lacks an endogenous CAO, heterologous gene expression of CAOs from other organisms produces a functional enzyme. To begin to characterize their function and mechanisms of copper acquisition, two putative cao + genes from Schizosaccharomyces pombe were expressed in S. cerevisiae. Expression of spao1 + resulted in the production of an active enzyme capable of catalysing the oxidative deamination of primary amines. On the other hand, expression of spao2 + failed to produce an active CAO. Using a functional spao1 +–GFP fusion allele, the SPAO1 protein was localized in the cytosol. Under copper-limiting conditions, yeast cells harbouring deletions of the MAC1, CTR1 and CTR3 genes were defective in amine oxidase activity. Likewise, atx1Δ null cells exhibited no CAO activity, while ccc2Δ mutant cells exhibited decreased levels of amine oxidase activity, and mutations in cox17Δ and ccs1Δ did not cause any defects in this activity. Copper-deprived S. cerevisiae cells expressing spao1 + required a functional atx1 + gene for growth on minimal medium containing ethylamine as the sole nitrogen source. Under these conditions, the inability of the atx1Δ cells to utilize ethylamine correlated with the lack of SPAO1 activity, in spite of the efficient expression of the protein. Cells carrying a disrupted ccc2Δ allele exhibited only weak growth on ethylamine medium containing a copper chelator. The results of these studies reveal that expression of the heterologous spao1 + gene in S. cerevisiae is required for its growth in medium containing ethylamine as the sole nitrogen source, and that expression of an active Schiz. pombe SPAO1 protein in S. cerevisiae depends on the acquisition of copper through the high-affinity copper transporters Ctr1 and Ctr3, and the copper chaperone Atx1.

Investigation by 13C-NMR and tricarboxylic acid (TCA) deletion mutant analysis of pathways for succinate formation in Saccharomyces cerevisiae during anaerobic fermentation

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2669-2678 ◽  
Author(s):  
Carole Camarasa ◽  
Jean-Philippe Grivet ◽  
Sylvie Dequin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nitrogen Source ◽  
Anaerobic Fermentation ◽  
Metabolite Profile ◽  
Tricarboxylic Acid ◽  
Yeast Fermentation ◽  
Oxidative Decarboxylation ◽  
Sole Nitrogen Source ◽  
Glucose Fermentation ◽  
Succinate Production

NMR isotopic filiation of 13C-labelled aspartate and glutamate was used to explore the tricarboxylic acid (TCA) pathway in Saccharomyces cerevisiae during anaerobic glucose fermentation. The assimilation of [3-13C]aspartate led to the formation of [2,3-13C]malate and [2,3-13C]succinate, with equal levels of 13C incorporation, whereas site-specific enrichment on C-2 and C-3 of succinate was detected only with [3-13C]glutamate. The non-random distribution of 13C labelling in malate and succinate demonstrates that the TCA pathway operates during yeast fermentation as both an oxidative and a reductive branch. The observed 13C distribution suggests that the succinate dehydrogenase (SDH) complex is not active during glucose fermentation. This hypothesis was tested by deleting the SDH1 gene encoding the flavoprotein subunit of the SDH complex. The growth, fermentation rate and metabolite profile of the sdh1 mutant were similar to those of the parental strain, demonstrating that SDH was indeed not active. Filiation experiments indicated the reductive branch of the TCA pathway was the main pathway for succinate production if aspartate was used as the nitrogen source, and that a surplus of succinate was produced by oxidative decarboxylation of 2-oxoglutarate if glutamate was the sole nitrogen source. Consistent with this finding, a kgd1 mutant displayed lower levels of succinate production on glutamate than on other nitrogen sources, and higher levels of oxoglutarate dehydrogenase activity were observed on glutamate. Thus, the reductive branch generating succinate via fumarate reductase operates independently of the nitrogen source. This pathway is the main source of succinate during fermentation, unless glutamate is the sole nitrogen source, in which case the oxidative decarboxylation of 2-oxoglutarate generates additional succinate.

scholarly journals Molecular genetics of serine and threonine catabolism in Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 119 (3) ◽  
pp. 527-534
Author(s):  
J G Petersen ◽  
M C Kielland-Brandt ◽  
T Nilsson-Tillgren ◽  
C Bornaes ◽  
S Holmberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzyme Activity ◽  
Nitrogen Source ◽  
Regulatory Protein ◽  
Growth Conditions ◽  
Northern Analysis ◽  
Recessive Mutation ◽  
Sole Nitrogen Source ◽  
Threonine Deaminase ◽  
Hydroxyamino Acids

Abstract The catabolic L-serine (L-threonine) deaminase of Saccharomyces cerevisiae allows the yeast to grow on media with L-serine or L-threonine as sole nitrogen source. A mutant, cha1 (catabolism of hydroxyamino acids), lacking this enzyme activity has been isolated. We have cloned the CHA1 gene by complementation of a cha1 mutation. Northern analysis showed that CHA1 mRNA has a size of about 1200 ribonucleotides. CHA1 is probably the structural gene for the enzyme; it is an abundant RNA in cells grown with serine and threonine as nitrogen source, whereas it is not detected when cells are grown on ammonium or proline, i.e., the transcription of the CHA1 gene is induced by serine or threonine. Under induced growth conditions haploid ilv1 CHA1 strains do not require isoleucine, i.e., the catabolic deaminase is able to substitute for the biosynthetic threnonine deaminase encoded by the ILV1 gene. We have identified a nuclear, recessive mutation, sil1, that suppresses ilv1 mutations by increased transcription of the CHA1 gene under growth conditions leading to partial induction. The sil1 mutation could exert its effect by increasing the effective pools of the hydroxyamino acids. Alternatively SIL1 may encode a negatively acting regulatory protein for CHA1.

PRODUCTION OF CYTASES ACTIVE ON BARLEY GUM BY BACTERIA OF THE GENUS BACILLUS

1953 ◽  
Vol 31 (1) ◽  
pp. 28-32 ◽  
Author(s):  
A. C. Blackwood
Keyword(s):  
Bacillus Subtilis ◽  
Enzymatic Activity ◽  
Nitrogen Source ◽  
Freeze Drying ◽  
Shake Flask ◽  
Sole Nitrogen Source ◽  
Genus Bacillus ◽  
Original Activity ◽  
Bacterial Cultures ◽  
Bacillus Genus

One hundred and fourteen bacterial cultures representing most of the species in the Bacillus genus were tested for the production of extracellular barley gum cytase. Assays were made on shake-flask cultures grown on a medium containing glucose and yeast extract. Although all the organisms had some enzymatic activity, certain strains of Bacillus subtilis gave the best yields of cytase. On a medium with asparagine as the sole nitrogen source even higher yields were obtained. The crude cytase preparations were stable and after freeze-drying most of the original activity remained.

scholarly journals Expression of an ATP-binding cassette transporter-encoding gene (YOR1) is required for oligomycin resistance in Saccharomyces cerevisiae.

1995 ◽  
Vol 15 (12) ◽  
pp. 6875-6883 ◽  
Author(s):  
D J Katzmann ◽  
T C Hallstrom ◽  
M Voet ◽  
W Wysock ◽  
J Golin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Gene ◽  
Sequence Similarity ◽  
Yeast Cells ◽  
Atp Binding ◽  
Zinc Cluster ◽  
Atp Binding Cassette Transporter ◽  
Transporter Family ◽  
Encoding Gene ◽  
Atp Binding Cassette

Semidominant mutations in the PDR1 or PDR3 gene lead to elevated resistance to cycloheximide and oligomycin. PDR1 and PDR3 have been demonstrated to encode zinc cluster transcription factors. Cycloheximide resistance mediated by PDR1 and PDR3 requires the presence of the PDR5 membrane transporter-encoding gene. However, PDR5 is not required for oligomycin resistance. Here, we isolated a gene that is necessary for PDR1- and PDR3-mediated oligomycin resistance. This locus, designated YOR1, causes a dramatic elevation in oligomycin resistance when present in multiple copies. A yor1 strain exhibits oligomycin hypersensitivity relative to an isogenic wild-type strain. In addition, loss of the YOR1 gene blocks the elevation in oligomycin resistance normally conferred by mutant forms of PDR1 or PDR3. The YOR1 gene product is predicted to be a member of the ATP-binding cassette transporter family of membrane proteins. Computer alignment indicates that Yor1p shows striking sequence similarity with multidrug resistance-associated protein, Saccharomyces cerevisiae Ycf1p, and the cystic fibrosis transmembrane conductance regulator. Use of a YOR1-lacZ fusion gene indicates that YOR1 expression is responsive to PDR1 and PDR3. While PDR5 expression is strictly dependent on the presence of PDR1 or PDR3, control of YOR1 expression has a significant PDR1/PDR3-independent component. Taken together, these data indicate that YOR1 provides the link between transcriptional regulation by PDR1 and PDR3 and oligomycin resistance of yeast cells.

Sign in / Sign up

Export Citation Format

Share Document