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

Abstract

dFusel 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. In Saccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine pyrophosphate-dependent 2-oxoacid decarboxylases (2ODCs). PDC1, PDC5, and PDC6 encode differentially regulated pyruvate decarboxylase isoenzymes; ARO10 encodes a 2-oxo-acid decarboxylase with broad substrate specificity, and THI3 has not yet been shown to encode an active decarboxylase. Despite the importance of fusel alcohol production in S. cerevisiae, the substrate specificities of these five 2ODCs have not been systematically compared. When the five 2ODCs were individually overexpressed in a pdc1⌬ pdc5⌬ pdc6⌬ aro10⌬ thi3⌬ strain, only Pdc1, Pdc5, and Pdc6 catalyzed the decarboxylation of the linear-chain 2-oxo acids pyruvate, 2-oxobutanoate, and 2-oxo-pentanoate in cell extracts. The presence of a Pdc isoenzyme was also required for the production of n-propanol and n-butanol in cultures grown on threonine and norvaline, respectively, as nitrogen sources. These results demonstrate the importance of pyruvate decarboxylases in the natural production of n-propanol and n-butanol by S. 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. P yruvate decarboxylase (PDC) catalyzes the thiamine pyrophosphate (TPP)-dependent decarboxylation of pyruvate to acetaldehyde. In Saccharomyces cerevisiae, PDC is not only a key enzyme of alcoholic fermentation but is also required for the synthesis of cytosolic acetyl coenzyme A (acetyl-CoA) (15, 16), a key precursor for the synthesis of lipids and lysine. Three PDC isoenzymes are encoded by the S. cerevisiae PDC1, PDC5, and PDC6 genes (25). Pdc1, the main isoenzyme, is highly expressed under most conditions, while PDC5 exhibits tight transcriptional control, with high expression levels in the absence of a functional PDC1 gene (48), under nitrogen-limited conditions (3-5, 53), and under conditions of thiamine limitation (40). PDC1 and PDC5 expressions are subject to autoregulation (24) and require the transcription factor Pdc2 (23). The third PDC isoenzyme, Pdc6, has a very low content of sulfur-containing amino acids and is highly expressed in sulfur-limited cultures (4, 53). This transcriptional regulation of PDC6 has been interpreted as a "sulfur economy" response (4, 14, 53).The ARO10 and THI3 genes show sequence homology to the PDC genes but do not encode pyruva...

Transcriptional response of Saccharomyces cerevisiae to low temperature during wine fermentation

“…For example, while ARO9 and ARO10 transcripts increased in the cold at exp, they had reduced at stat to be unchanged from 25°C. In contrast, the CHA1 transcript [implicated in threonine degradation at the same step as Aro9p (Romagnoli et al 2012)] was lower in the cold at exp, and remained depressed even at stat.…”

“…Furthermore, the NCR-regulated glutamate/aspartate permease, DIP5, was 3.3-fold down-regulated in the cold at exp. Conversely, the ARO9 [catabolism of phenylalanine and methionine; (Romagnoli et al 2012)] and ARO10 transcripts [catalysing the subsequent step in the catabolism of multiple amino acids; (Romagnoli et al 2012)] were both induced, consistent with elevated amino acid catabolism at low temperature during exponential growth. The differential expression of amino acid biosynthesis and degradation genes occurred in both directions, and was not always consistent at the two different time points, suggesting that there was not a simple shift in amino acid metabolism with temperature.…”

De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae

“…Decarboxylation of phenylpyruvate, the 2-oxo acid associated to phenylalanine, yields phenylacetaldehyde, which is subsequently reduced into phenylethanol and/or oxidized to phenylacetate [41-43]. Decarboxylation of phenylpyruvate can be catalyzed by four different thiamine pyrophosphate-dependent 2-oxo acid decarboxylases encoded by ARO10, PDC1, PDC5, and PDC6 [43,44]. Recent work in our group has demonstrated that, among the four decarboxylases capable of phenylpyruvate decarboxylase, Pdc1 and Pdc6 showed a much lower affinity and decarboxylation rate of phenylpyruvate than Pdc5 and, in particular, Aro10 [44].…”

“…Decarboxylation of phenylpyruvate can be catalyzed by four different thiamine pyrophosphate-dependent 2-oxo acid decarboxylases encoded by ARO10, PDC1, PDC5, and PDC6 [43,44]. Recent work in our group has demonstrated that, among the four decarboxylases capable of phenylpyruvate decarboxylase, Pdc1 and Pdc6 showed a much lower affinity and decarboxylation rate of phenylpyruvate than Pdc5 and, in particular, Aro10 [44]. Since absence of all three pyruvate decarboxylase genes ( PDC1 , PDC 5 and PDC6 ) abolishes growth on glucose in synthetic media, strains only retaining PDC1 ( aro10Δ, pcd5Δ, pdc6Δ ) were constructed [45].…”

An Efficient Method Using Gluconacetobacter europaeus To Reduce an Unfavorable Flavor Compound, Acetoin, in Rice Vinegar Production

“…The flavor of fermented foods and beverages is characterized by many low-molecular-weight flavor compounds (e.g., short-chain alcohols, aldehydes, organic acids, and esters) (28,29). These compounds are produced by several microorganisms that are involved in fermentation, and they have a strong impact on product quality.…”