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.

Pyruvate Decarboxylase Catalyzes Decarboxylation of Branched-Chain 2-Oxo Acids but Is Not Essential for Fusel Alcohol Production by Saccharomyces cerevisiae

1998 ◽  
Vol 64 (4) ◽  
pp. 1303-1307 ◽  
Author(s):  
Eelko G. ter Schure ◽  
Marcel T. Flikweert ◽  
Johannes P. van Dijken ◽  
Jack T. Pronk ◽  
C. Theo Verrips
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Alcohol Dehydrogenase ◽  
Pyruvate Decarboxylase ◽  
Wild Type ◽  
Alcohol Production ◽  
Whole Cells ◽  
Fusel Alcohols ◽  
Cell Extracts ◽  
Fusel Alcohol ◽  
Branched Chain

ABSTRACT The fusel alcohols 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-propanol are important flavor compounds in yeast-derived food products and beverages. The formation of these compounds from branched-chain amino acids is generally assumed to occur via the Ehrlich pathway, which involves the concerted action of a branched-chain transaminase, a decarboxylase, and an alcohol dehydrogenase. Partially purified preparations of pyruvate decarboxylase (EC 4.1.1.1 ) have been reported to catalyze the decarboxylation of the branched-chain 2-oxo acids formed upon transamination of leucine, isoleucine, and valine. Indeed, in a coupled enzymatic assay with horse liver alcohol dehydrogenase, cell extracts of a wild-type Saccharomyces cerevisiae strain exhibited significant decarboxylation rates with these branched-chain 2-oxo acids. Decarboxylation of branched-chain 2-oxo acids was not detectable in cell extracts of an isogenic strain in which all threePDC genes had been disrupted. Experiments with cell extracts from S. cerevisiae mutants expressing a singlePDC gene demonstrated that both PDC1- andPDC5-encoded isoenzymes can decarboxylate branched-chain 2-oxo acids. To investigate whether pyruvate decarboxylase is essential for fusel alcohol production by whole cells, wild-type S. cerevisiae and an isogenic pyruvate decarboxylase-negative strain were grown on ethanol with a mixture of leucine, isoleucine, and valine as the nitrogen source. Surprisingly, the three corresponding fusel alcohols were produced in both strains. This result proves that decarboxylation of branched-chain 2-oxo acids via pyruvate decarboxylase is not an essential step in fusel alcohol production.

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 Characterization of the Efflux Capability and Substrate Specificity of Aspergillus fumigatus PDR5-like ABC Transporters Expressed in Saccharomyces cerevisiae

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Brooke D. Esquivel ◽  
Jeffrey M. Rybak ◽  
Katherine S. Barker ◽  
Jarrod R. Fortwendel ◽  
P. David Rogers ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Substrate Specificity ◽  
Abc Transporter ◽  
Drug Susceptibility ◽  
Antifungal Drugs ◽  
Efflux Transporters ◽  
Content Type ◽  
Background Strain

ABSTRACT This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. The A. fumigatus genes abcA, abcC, abcF, abcG, abcH, and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active endogenous ABC transporter gene PDR5 was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine β-naphthylamide. The transporters AbcA, AbcC, and AbcF had the strongest efflux activities of these compounds. All of the strains with plasmid-expressed transporters had more efflux activity than did the PDR5-deleted background strain. We performed broth microdilution drug susceptibility testing and agar spot assays using an array of compounds and antifungal drugs to determine the transporter specificity and drug susceptibility of the strains. The transporters AbcC and AbcF showed the broadest range of substrate specificity, while AbcG and AbcH had the narrowest range of substrates. Strains expressing the AbcA, AbcC, AbcF, or AbcI transporter were more resistant to fluconazole than was the PDR5-deleted background strain. Strains expressing AbcC and AbcF were additionally more resistant to clotrimazole, itraconazole, ketoconazole, and posaconazole than was the background strain. Finally, we analyzed the expression levels of the genes by reverse transcription-quantitative PCR (RT-qPCR) in triazole-susceptible and -resistant A. fumigatus clinical isolates. All of these transporters are expressed at a measurable level, and transporter expression varied significantly between strains, demonstrating the high degree of phenotypic variation, plasticity, and divergence of which this species is capable. IMPORTANCE One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus. Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance. Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus. Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs.

scholarly journals The Ehrlich Pathway for Fusel Alcohol Production: a Century of Research on Saccharomyces cerevisiae Metabolism

2008 ◽  
Vol 74 (12) ◽  
pp. 3920-3920 ◽  
Author(s):  
Lucie A. Hazelwood ◽  
Jean-Marc Daran ◽  
Antonius J. A. van Maris ◽  
Jack T. Pronk ◽  
J. Richard Dickinson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ehrlich Pathway ◽  
Alcohol Production ◽  
Fusel Alcohol

scholarly journals Genomic Sequence of Saccharomyces cerevisiae BAW-6, a Yeast Strain Optimal for Brewing Barley Shochu

2018 ◽  
Vol 6 (14) ◽  
Author(s):  
Yasuhiro Kajiwara ◽  
Kazuki Mori ◽  
Kosuke Tashiro ◽  
Yujiro Higuchi ◽  
Kaoru Takegawa ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Basis ◽  
Genomic Sequence ◽  
Draft Genome ◽  
Production Capacity ◽  
Acid Tolerance ◽  
High Alcohol ◽  
Genomic Information ◽  
Content Type ◽  
Alcohol Production

ABSTRACT Here, we report the draft genome sequence of Saccharomyces cerevisiae strain BAW-6, which is used for the production of barley shochu, a traditional Japanese spirit. This genomic information can be used to elucidate the genetic basis underlying the high alcohol production capacity and citric acid tolerance of shochu yeast.

scholarly journals The Ehrlich Pathway for Fusel Alcohol Production: a Century of Research on Saccharomyces cerevisiae Metabolism

2008 ◽  
Vol 74 (8) ◽  
pp. 2259-2266 ◽  
Author(s):  
Lucie A. Hazelwood ◽  
Jean-Marc Daran ◽  
Antonius J. A. van Maris ◽  
Jack T. Pronk ◽  
J. Richard Dickinson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ehrlich Pathway ◽  
Alcohol Production ◽  
Fusel Alcohol

scholarly journals Serine-Type Carboxypeptidase KexA of Aspergillus oryzae Has Broader Substrate Specificity than Saccharomyces cerevisiae Kex1 and Is Required for Normal Hyphal Growth and Conidiation

2012 ◽  
Vol 78 (22) ◽  
pp. 8154-8157 ◽  
Author(s):  
Hiroto Morita ◽  
Sayako Tomita ◽  
Hiroshi Maeda ◽  
Ayako Okamoto ◽  
Youhei Yamagata ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Substrate Specificity ◽  
Aspergillus Oryzae ◽  
Normal Growth ◽  
Hyphal Growth ◽  
Truncated Form ◽  
Hyphal Branching ◽  
Content Type

ABSTRACTAspergillus oryzaehas an ortholog ofSaccharomyces cerevisiae KEX1, termedkexA. A truncated form of KexA protein showed serine-type carboxypeptidase activity and somewhat broader substrate specificity than Kex1 protease. Furthermore, our results indicated that KexA is required for normal growth ofA. oryzaeand that it might be involved in hyphal branching.

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