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 Substrate Specificity of Thiamine Pyrophosphate-Dependent 2-Oxo-Acid Decarboxylases in Saccharomyces cerevisiae

2012 ◽  
Vol 78 (21) ◽  
pp. 7538-7548 ◽  
Author(s):  
Gabriele Romagnoli ◽  
Marijke A. H. Luttik ◽  
Peter Kötter ◽  
Jack T. Pronk ◽  
Jean-Marc Daran
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Substrate Specificity ◽  
Thiamine Pyrophosphate ◽  
Content Type ◽  
Alcohol Production ◽  
Fusel Alcohols ◽  
Cell Extracts ◽  
Fusel Alcohol ◽  
Branched Chain ◽  
Sulfur Containing

ABSTRACTFusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. InSaccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases (2ODCs).PDC1,PDC5, andPDC6encode differentially regulated pyruvate decarboxylase isoenzymes;ARO10encodes a 2-oxo-acid decarboxylase with broad substrate specificity, andTHI3has not yet been shown to encode an active decarboxylase. Despite the importance of fusel alcohol production inS. cerevisiae, the substrate specificities of these five 2ODCs have not been systematically compared. When the five 2ODCs were individually overexpressed in apdc1Δpdc5Δpdc6Δaro10Δthi3Δ strain, only Pdc1, Pdc5, and Pdc6 catalyzed the decarboxylation of the linear-chain 2-oxo acids pyruvate, 2-oxo-butanoate, and 2-oxo-pentanoate in cell extracts. The presence of a Pdc isoenzyme was also required for the production ofn-propanol andn-butanol in cultures grown on threonine and norvaline, respectively, as nitrogen sources. These results demonstrate the importance of pyruvate decarboxylases in the natural production ofn-propanol andn-butanol byS. cerevisiae. No decarboxylation activity was found for Thi3 with any of the substrates tested. Only Aro10 and Pdc5 catalyzed the decarboxylation of the aromatic substrate phenylpyruvate, with Aro10 showing superior kinetic properties. Aro10, Pdc1, Pdc5, and Pdc6 exhibited activity with all branched-chain and sulfur-containing 2-oxo acids tested but with markedly different decarboxylation kinetics. The high affinity of Aro10 identified it as a key contributor to the production of branched-chain and sulfur-containing fusel alcohols.

scholarly journals Amino Acid Catabolism by an areA-Regulated Gene Encoding an l-Amino Acid Oxidase with Broad Substrate Specificity in Aspergillus nidulans

2005 ◽  
Vol 71 (7) ◽  
pp. 3551-3555 ◽  
Author(s):  
Meryl A. Davis ◽  
Marion C. Askin ◽  
Michael J. Hynes
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Nitrogen Source ◽  
Aspergillus Nidulans ◽  
Nitrogen Sources ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Broad Substrate Specificity ◽  
Strong Growth

ABSTRACT The filamentous fungus Aspergillus nidulans can use a wide range of compounds as nitrogen sources. The synthesis of the various catabolic enzymes needed to breakdown these nitrogen sources is regulated by the areA gene, which encodes a GATA transcription factor required to activate gene expression under nitrogen-limiting conditions. The areA102 mutation results in pleiotropic effects on nitrogen source utilization, including better growth on certain amino acids as nitrogen sources. Mutations in the sarA gene were previously isolated as suppressors of the strong growth of an areA102 strain on l-histidine as a sole nitrogen source. We cloned the sarA gene by complementation of a sarA mutant and showed that it encodes an l-amino acid oxidase enzyme with broad substrate specificity. Elevated expression of this enzyme activity in an areA102 background accounts for the strong growth of these strains on amino acids that are substrates for this enzyme. Loss of function sarA mutations, which abolish the l-amino acid oxidase activity, reverse the areA102 phenotype. Growth tests with areA102 and sarA mutants show that this enzyme is the primary route of catabolism for some amino acids, while other amino acids are metabolized through alternative pathways that yield either ammonium or glutamate for growth.

scholarly journals Resolving Phenylalanine Metabolism Sheds Light on Natural Synthesis of Penicillin G in Penicillium chrysogenum

2011 ◽  
Vol 11 (2) ◽  
pp. 238-249 ◽  
Author(s):  
Tânia Veiga ◽  
Daniel Solis-Escalante ◽  
Gabriele Romagnoli ◽  
Angela ten Pierick ◽  
Mark Hanemaaijer ◽  
...  
Keyword(s):  
Nitrogen Source ◽  
Transcriptome Analysis ◽  
Penicillium Chrysogenum ◽  
Penicillin G ◽  
Side Chain ◽  
Acid Decarboxylase ◽  
Content Type ◽  
Chemostat Cultures ◽  
Phenylpyruvate Decarboxylase ◽  
Dual Substrate

ABSTRACTThe industrial production of penicillin G byPenicillium chrysogenumrequires the supplementation of the growth medium with the side chain precursor phenylacetate. The growth ofP. chrysogenumwith phenylalanine as the sole nitrogen source resulted in the extracellular production of phenylacetate and penicillin G. To analyze this natural pathway for penicillin G production, chemostat cultures were switched to [U-13C]phenylalanine as the nitrogen source. The quantification and modeling of the dynamics of labeled metabolites indicated that phenylalanine was (i) incorporated in nascent protein, (ii) transaminated to phenylpyruvate and further converted by oxidation or by decarboxylation, and (iii) hydroxylated to tyrosine and subsequently metabolized via the homogentisate pathway. The involvement of the homogentisate pathway was supported by the comparative transcriptome analysis ofP. chrysogenumcultures grown with phenylalanine and with (NH4)2SO4as the nitrogen source. This transcriptome analysis also enabled the identification of two putative 2-oxo acid decarboxylase genes (Pc13g9300 and Pc18g01490). cDNAs of both genes were cloned and expressed in the 2-oxo-acid-decarboxylase-freeSaccharomyces cerevisiaestrain CEN.PK711-7C (pdc1 pdc5 pdc6Δ aro10Δ thi3Δ). The introduction of Pc13g09300 restored the growth of thisS. cerevisiaemutant on glucose and phenylalanine, thereby demonstrating that Pc13g09300 encodes a dual-substrate pyruvate and phenylpyruvate decarboxylase, which plays a key role in an Ehrlich-type pathway for the production of phenylacetate inP. chrysogenum. These results provide a basis for the metabolic engineering ofP. chrysogenumfor the production of the penicillin G side chain precursor phenylacetate.

scholarly journals L-Phenylalanine Transport in Saccharomyces cerevisiae: Participation of GAP1, BAP2, and AGP1

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Daniel A. Sáenz ◽  
Mónica S. Chianelli ◽  
Carlos A. Stella
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Substrate Specificity ◽  
Nitrogen Source ◽  
Type Strain ◽  
Wild Type Strain ◽  
Ammonium Ion ◽  
Wild Type ◽  
Synthetic Media ◽  
Phenylalanine Transport

We focused on the participation of GAP1, BAP2, and AGP1 in L-phenylalanine transport in yeast. In order to study the physiological functions of GAP1, BAP2, and AGP1 in L-phenylalanine transport, we examined the kinetics, substrate specificity, and regulation of these systems, employing isogenic haploid strains with the respective genes disrupted individually and in combination. During the characterization of phenylalanine transport, we noted important regulatory phenomena associated with these systems. Our results show that Agp1p is the major transporter of the phenylalanine in a gap1 strain growing in synthetic media with leucine present as an inducer. In a wild type strain grown in the presence of leucine, when ammonium ion was the nitrogen source, Bap2p is the principal phenylalanine carrier.

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.

Modification of thiamine pyrophosphate dependent enzyme activity by oxythiamine in Saccharomyces cerevisiae cells

2005 ◽  
Vol 51 (10) ◽  
pp. 833-839 ◽  
Author(s):  
Adam Tylicki ◽  
Jan Czerniecki ◽  
Pawel Dobrzyn ◽  
Agnieszka Matanowska ◽  
Anna Olechno ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pyruvate Dehydrogenase ◽  
Specific Activity ◽  
Pyruvate Decarboxylase ◽  
Yeast Cells ◽  
Thiamine Pyrophosphate ◽  
Decarboxylase Activity ◽  
Mitochondrial Enzymes ◽  
Oxoglutarate Dehydrogenase ◽  
Cytosolic Enzymes

Oxythiamine is an antivitamin derivative of thiamine that after phosphorylation to oxythiamine pyro phos phate can bind to the active centres of thiamine-dependent enzymes. In the present study, the effect of oxythiamine on the viability of Saccharomyces cerevisiae and the activity of thiamine pyrophosphate dependent enzymes in yeast cells has been investigated. We observed a decrease in pyruvate decarboxylase specific activity on both a control and an oxythiamine medium after the first 6 h of culture. The cytosolic enzymes transketolase and pyruvate decarboxylase decreased their specific activity in the presence of oxythiamine but only during the beginning of the cultivation. However, after 12 h of cultivation, oxythiamine-treated cells showed higher specific activity of cytosolic enzymes. More over, it was established by SDS–PAGE that the high specific activity of pyruvate decarboxylase was followed by an increase in the amount of the enzyme protein. In contrast, the mitochondrial enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, were inhibited by oxythiamine during the entire experiment. Our results suggest that the observed strong decrease in growth rate and viability of yeast on medium with oxythiamine may be due to stronger in hibition of mitochondrial pyruvate dehydrogenase than of cytosolic enzymes.Key words: pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, transketolase, pyruvate decarboxylase, activity, oxythiamine, inhibition.

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