A 13C Nuclear Magnetic Resonance Investigation of the Metabolism of Leucine to Isoamyl Alcohol in Saccharomyces cerevisiae

Abstract

The metabolism of leucine to isoamyl alcohol in yeast was examined by 13C nuclear magnetic resonance spectroscopy. The product of leucine transamination, α-ketoisocaproate had four potential routes to isoamyl alcohol. The first, via branched-chain α-keto acid dehydrogenase to isovaleryl-CoA with subsequent conversion to isovalerate by acyl-CoA hydrolase operates in wild-type cells where isovalerate appears to be an end product. This pathway is not required for the synthesis of isoamyl alcohol because abolition of branched-chain α-keto acid dehydrogenase activity in an lpd1 disruption mutant did not prevent the formation of isoamyl alcohol. A second possible route was via pyruvate decarboxylase; however, elimination of pyruvate decarboxylase activity in a pdc1 pdc5 pdc6 triple mutant did not decrease the levels of isoamyl alcohol produced. A third route utilizes α-ketoisocaproate reductase (a novel activity in Saccharomyces cerevisiae) but with no role in the formation of isoamyl alcohol from α-hydroxyisocaproate because cell homogenates could not convert α-hydroxyisocaproate to isoamyl alcohol. The final possibility was that a pyruvate decarboxylase-like enzyme encoded byYDL080c appears to be the major route of decarboxylation of α-ketoisocaproate to isoamyl alcohol although disruption of this gene reveals that at least one other unidentified decarboxylase can substitute to a minor extent. The metabolism of leucine to isoamyl alcohol in yeast was examined by 13C nuclear magnetic resonance spectroscopy. The product of leucine transamination, α-ketoisocaproate had four potential routes to isoamyl alcohol. The first, via branched-chain α-keto acid dehydrogenase to isovaleryl-CoA with subsequent conversion to isovalerate by acyl-CoA hydrolase operates in wild-type cells where isovalerate appears to be an end product. This pathway is not required for the synthesis of isoamyl alcohol because abolition of branched-chain α-keto acid dehydrogenase activity in an lpd1 disruption mutant did not prevent the formation of isoamyl alcohol. A second possible route was via pyruvate decarboxylase; however, elimination of pyruvate decarboxylase activity in a pdc1 pdc5 pdc6 triple mutant did not decrease the levels of isoamyl alcohol produced. A third route utilizes α-ketoisocaproate reductase (a novel activity in Saccharomyces cerevisiae) but with no role in the formation of isoamyl alcohol from α-hydroxyisocaproate because cell homogenates could not convert α-hydroxyisocaproate to isoamyl alcohol. The final possibility was that a pyruvate decarboxylase-like enzyme encoded byYDL080c appears to be the major route of decarboxylation of α-ketoisocaproate to isoamyl alcohol although disruption of this gene reveals that at least one other unidentified decarboxylase can substitute to a minor extent. In most eukaryotes, the catabolism of the branched-chain amino acids leucine, isoleucine, and valine has been well understood for many years (1Bender D.A. Amino Acid Metabolism. John Wiley & Sons, New York, NY1975Google Scholar). The first step is a transamination in which α-ketoglutarate accepts the amino group (from leucine, isoleucine, and valine) producing glutamate and α-ketoisocaproic acid, α-keto-ॆ-methylvaleric acid and α-ketoisovaleric acid, respectively. Next is oxidative decarboxylation of the keto acids by branched-chain α-keto acid dehydrogenase to the corresponding acyl-CoA derivatives. Further steps yield, ultimately, acetyl-CoA and acetoacetate (from leucine), acetyl-CoA and propionyl-CoA (from isoleucine), and succinyl-CoA (from valine). All of these metabolites can enter the tricarboxylic acid (TCA) 1The abbreviations used are: TCA, tricarboxylic acid; NMR, nuclear magnetic resonance; GC, gas chromatography; GC-MS, combined sequential gas chromatography and mass spectrometry. 1The abbreviations used are: TCA, tricarboxylic acid; NMR, nuclear magnetic resonance; GC, gas chromatography; GC-MS, combined sequential gas chromatography and mass spectrometry. cycle. It has been known for many years that yeasts do not operate the same metabolic routes because branched-chain amino acids can serve as the sole source of nitrogen but not carbon (2Cooper T.G. Strathern J.N. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces cerevisiae: Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 39-99Google Scholar, 3Large P.J. Yeast. 1986; 2: 1-34Crossref Scopus (108) Google Scholar). The predominant view, in a rather sparse literature, is that yeasts first use transamination but that decarboxylation of the keto acids proceeds via a 舠carboxylase舡 to an aldehyde that is then reduced in an NADH-linked reaction producing the appropriate 舠fusel舡 alcohol (2Cooper T.G. Strathern J.N. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces cerevisiae: Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 39-99Google Scholar, 3Large P.J. Yeast. 1986; 2: 1-34Crossref Scopus (108) Google Scholar, 4Derrick S. Large P.J. J. Gen. Microbiol. 1993; 139: 2783-2792Crossref PubMed Scopus (67) Google Scholar). This scheme is sometimes called the 舠Ehrlich pathway舡 to honor the originator of the ideas (5Ehrlich F. Ber. Dtsch. Chem. Ges. 1907; 40: 1027-1047Crossref Scopus (174) Google Scholar), which were slightly modified later (6Neubauer O. Fromherz K. Hoppe-Seyler's Z. Physiol. Chem. 1911; 70: 326-350Crossref Scopus (47) Google Scholar). Acceptance of the so-called Ehrlich pathway is problematical for at least four reasons. First, the supposed pathway has never been proven to exist. Simply showing that e.g. radioactively labeled leucine is converted into isoamyl alcohol does not prove that the individual steps are those envisaged in the scheme. Other routes are conceivable. The wrongly named carboxylase (which should at least be called a 舠decarboxylase舡) has never been isolated; a few authors assume that pyruvate decarboxylase is responsible (4Derrick S. Large P.J. J. Gen. Microbiol. 1993; 139: 2783-2792Crossref PubMed Scopus (67) Google Scholar), without proof. The second problem is that this scheme cannot explain all of the products that yeast makes; some fusel alcohols do not correspond to any known amino acid. Third, even if the pathway did exist as envisaged by Webb and Ingraham (7Webb A.D. Ingraham J.L. Adv. Appl. Microbiol. 1963; 5: 317-353Crossref Scopus (83) Google Scholar), the production of fusel alcohols in this manner would require a mixing of the synthetic and degradative reactions involving the branched-chain amino acids. This is something that cells normally avoid by coordinate regulation. Synthetic enzymes are usually repressed and/or inhibited if the end product is present. Conversely, catabolic enzymes are not induced if the compound is being synthesized at rates that are satisfying biosynthetic demand. Furthermore, if this unusual situation did exist, then operation of the metabolic pathways would require some extraordinary channeling of metabolites between different subcellular compartments. Fourth, the Ehrlich pathway does not explain all of the known facts as Ehrlich himself was fully aware. Two of the most serious concerns are that the kinetics of amino acid utilization do not match fusel alcohol formation in complex media, and in media containing low levels of amino acids, there is a poor correlation between the amino acid composition and the composition of the resulting fusel oil (7Webb A.D. Ingraham J.L. Adv. Appl. Microbiol. 1963; 5: 317-353Crossref Scopus (83) Google Scholar). Recent examination of the catabolism of the branched-chain amino acids in Saccharomyces cerevisiae confirms that the first step is transamination. There is an aminotransferase specific to branched-chain amino acids that is encoded by a single gene called BAA1(8Dickinson J.R. Norte V. FEBS Lett. 1993; 326: 29-32Crossref PubMed Scopus (47) Google Scholar). Branched-chain α-keto acid dehydrogenase has also been demonstrated in this yeast (9Dickinson J.R. Dawes I.W. J. Gen. Microbiol. 1992; 138: 2029-2033Crossref PubMed Scopus (43) Google Scholar). This enzyme is distinct from both α-ketoglutarate dehydrogenase and pyruvate dehydrogenase because it could not be detected in assay conditions that permitted the measurement of α-ketoglutarate dehydrogenase and vice versa. Also, a strain lacking α-ketoglutarate dehydrogenase due to a deletion and disruption of the KGD1 coding sequence and another strain lacking pyruvate dehydrogenase due to a disruption of the gene encoding the E1α sububunit of that latter enzyme both retained branched-chain α-keto acid dehydrogenase activity (9Dickinson J.R. Dawes I.W. J. Gen. Microbiol. 1992; 138: 2029-2033Crossref PubMed Scopus (43) Google Scholar). Branched-chain α-keto acid dehydrogenase has been purified fromS. cerevisiae, and a number of its properties have been characterized (10Sinclair D.A. Dawes I.W. Dickinson J.R. Biochem. Mol. Biol. Int. 1993; 31: 911-922PubMed Google Scholar). Our experience with 13C nuclear magnetic resonance (NMR) spectroscopy for metabolic analysis in wild-type and mutant yeast (11Dickinson J.R. Dawes I.W. Boyd A.S.F. Baxter R.L. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5847-5851Crossref PubMed Scopus (52) Google Scholar, 12Dickinson J.R. Hewlins M.J.E. J. Gen. Microbiol. 1988; 134: 333-337PubMed Google Scholar, 13Dickinson J.R. Hewlins M.J.E. J. Gen. Microbiol. 1991; 137: 1033-1037Crossref PubMed Scopus (9) Google Scholar, 14Dickinson J.R. Sobanski M.A. Hewlins M.J.E. Microbiology. 1995; 141: 385-391Crossref PubMed Scopus (10) Google Scholar) led us to use this technique to determine the metabolic pathways used in fusel alcohol formation from branched-chain amino acids. This paper reports the catabolism of leucine to isoamyl alcohol. The strains used are shown in Table I. Strains JRD815–1.2 and JRD815–6.1 were constructed by mating YSH5.127.-17C to FY1679-YDL080c(α) and sporulation of the resultant diploid. Standard genetic techniques were used in the strain construction (15Sherman F. Methods Cell Biol. 1975; 11: 189-199Crossref PubMed Scopus (13) Google Scholar, 16Mortimer R.K. Hawthorn D.C. Methods Cell Biol. 1975; 11: 221-233Crossref PubMed Scopus (89) Google Scholar). Progeny segregating all three pdc mutations were identified by a failure to grow on glucose-containing media and by enzyme assays; segregation of the disrupted YDL080c gene was followed by the ability to grow in the presence of the antibiotic G418 (Geneticin, at 200 ॖg/ml). Starter cultures were grown in a medium comprising 17 yeast extract, 27 Bacto-peptone, and 27 carbon source. For studies of leucine catabolism, cells were grown in minimal medium containing (per liter) 1.67 g of yeast nitrogen base (Difco), 20 g of leucine, and either 20 ml of ethanol or 20 g of glucose for the carbon source. Experiments involving 13C labeling used [2-13C]leucine (99.9 atom 7) from Cambridge Isotope Laboratories (Cambridge, MA). Auxotrophic requirements were supplied as required at 20 ॖg/ml. Liquid cultures were grown in conical flasks filled to 407 nominal capacity in a gyrorotatory shaker. Agar (27) was used to solidify media. All cell cultures were at 30 °C.Table IYeast strains usedStrainComplete genotypeSource or Ref. No.IWD72MATα (9Dickinson J.R. Dawes I.W. J. Gen. Microbiol. 1992; 138: 2029-2033Crossref PubMed Scopus (43) Google Scholar)MML22MATa leu2 ura3 his3 ade2 lys2 met8 gal80∷HIS3 lpd1∷URA3(19Lanterman M.M. Dickinson J.R. Danner D.J. Hum. Mol. Genet. 1996; 5: 1643-1648Crossref PubMed Scopus (18) Google Scholar)YSH5.127.-17CMATaleu2 ura3 trp1 pdc1Δ∷LEU2 pdc5Δ∷URA3 pdc6Δ∷TRP1 GALS. Hohmann1-aS. Hohmann, Department of General and Marine Microbiology, Göteborg University, Medicianregatan 9C, S-41390 Göteborg, Sweden.FY1679MATα leu2 ura3 his3 TRP1This workFY1679-YDL080c(a)MATa LEU2 ura3 HIS3 TRP1 ydl080c∷KanrThis workFY1679-YDL080c(α)MATα leu2 ura3 HIS3 trp1 ydl080c∷KanrThis workJRD815–1.2MATα leu2 ura3 trp1 pdc1Δ∷LEU2 pdc5Δ∷URA3 pdc6Δ:TRP1 ydl080c∷KanrThis workJRD815–6.1MATa leu2 ura3 trp1 pdc1Δ∷LEU2 pdc5Δ∷URA3 pdc6Δ∷TRP1 ydl080c∷KanrThis work1-a S. Hohmann, Department of General and Marine Microbiology, Göteborg University, Medicianregatan 9C, S-41390 Göteborg, Sweden. Open table in a new tab 13C NMR spectra were recorded at 20 °C in culture supernatants (adjusted to pH 6 and containing 1572H2O and 50 mm potassium phosphate buffer) as described previously (12Dickinson J.R. Hewlins M.J.E. J. Gen. Microbiol. 1988; 134: 333-337PubMed Google Scholar, 13Dickinson J.R. Hewlins M.J.E. J. Gen. Microbiol. 1991; 137: 1033-1037Crossref PubMed Scopus (9) Google Scholar, 14Dickinson J.R. Sobanski M.A. Hewlins M.J.E. Microbiology. 1995; 141: 385-391Crossref PubMed Scopus (10) Google Scholar) using a Bruker AMX360 NMR spectrometer operating at 90.5 MHz using 32,000 data points over 22,000 Hz with Waltz-16 1H decoupling and by the DEPT method to determine the number of protons attached to each carbon atom. Signals were identified by comparison with spectra of standard compounds recorded under identical conditions and confirmed by of of the compounds to Standard Bruker was used are to in of a at under the conditions used In isoamyl alcohol was in culture supernatants by gas chromatography using a mm of to a mm with a The of the and the was the was from 50 to °C over was used as Standard of isoamyl alcohol were in used gas on a mm with a of The was a the mass spectrometer was a mass spectrometer named The was and the were at was the alcohol and alcohol had identical but could be because the a of mass which the latter Standard of isoamyl alcohol a over the were with the were by on with of in mm potassium phosphate pH containing mm and mm and disrupted using a as described previously J.R. J. Gen. Microbiol. 1988; Scholar). Cell was used as a source of decarboxylase was as described by and J. PubMed Google Scholar). reductase was at °C in ml of containing mm The reaction was by the of was by the at in a The assay all were also to assay the reaction in the using either and or at cell were examined for the ability to convert individual α-keto acids into isoamyl alcohol in of was at 30 °C with of α-keto acid in ml of with or without or A from the of the were to prevent the of the were in there was a for the were for in a and the supernatants were and a were then at °C for no or by as described the 13C NMR of a culture of a wild-type strain that had been in a minimal medium in which ethanol was the carbon source and [2-13C]leucine was the sole nitrogen source. The was of leucine at A number of were due to leucine as a Hz due to the labeled leucine as a Hz due to the labeled leucine leucine leucine ethanol and ethanol The other that were were of α-ketoisocaproate of isovalerate of α-hydroxyisocaproate and of isoamyl alcohol the metabolites identified and the that were labeled with a number of routes between leucine and isoamyl alcohol All of these transamination to α-ketoisocaproate The first was via branched-chain α-keto acid dehydrogenase A in to which would then be converted to isovalerate by acyl-CoA The second possibility was via pyruvate decarboxylase pathway α-ketoisocaproate reductase to α-hydroxyisocaproate followed by decarboxylation to isoamyl alcohol. The possibility was that isovalerate was from envisaged a pyruvate decarboxylase-like pathway was as a could not a metabolic route in yeast between α-ketoisocaproate and isovalerate using known or potential enzymes the was were to between the four metabolic routes for the metabolism of leucine to isoamyl alcohol. The carbon in that were labeled with 13C in the wild-type that are known are as amino Large The mutant is in dehydrogenase because it a disruption in the that dehydrogenase M.M. Dickinson J.R. Danner D.J. Hum. Mol. Genet. 1996; 5: 1643-1648Crossref PubMed Scopus (18) Google Scholar). a of this the strain pyruvate α-ketoglutarate and branched-chain α-keto acid dehydrogenase J.R. D.J. Dawes I.W. Mol. Gen. Genet. 1986; PubMed Scopus Google Scholar, D.A. Dawes I.W. 1995; PubMed Google Scholar, M.M. Dickinson J.R. Danner D.J. Hum. Mol. Genet. 1996; 5: 1643-1648Crossref PubMed Scopus (18) Google Scholar). strain branched-chain α-keto acid it a of this activity is required for the formation of isoamyl alcohol from lpd1 do not grow on carbon M.M. Dickinson J.R. Danner D.J. Hum. Mol. Genet. 1996; 5: 1643-1648Crossref PubMed Scopus (18) Google Scholar, J.R. D.J. Dawes I.W. Mol. Gen. Genet. 1986; PubMed Scopus Google Scholar, A. Mol. Biol. 1991; 11: PubMed Google Scholar), strain was in minimal medium comprising glucose as carbon source and leucine as sole source of these conditions culture supernatants of isoamyl alcohol This that branched-chain α-keto acid dehydrogenase any of the other keto acid dehydrogenase is not required for the formation of isoamyl of isoamyl alcohol by strains in glucose minimal medium with leucine as sole source of 6 and JRD815–6.1 pyruvate decarboxylase and cannot these had ethanol as carbon pdc5 and JRD815–6.1 pyruvate decarboxylase and cannot these had ethanol as carbon pdc5 pdc6 and JRD815–6.1 pyruvate decarboxylase and cannot these had ethanol as carbon pdc5 pdc6 alcohol was in are the of between were Strains and JRD815–6.1 pyruvate decarboxylase and cannot these had ethanol as carbon source. Open table in a new tab alcohol was in are the of between were The 13C NMR of a culture of mutant due to all of the in leucine as All of the of glucose were due to and The and of ethanol were also due to In the lpd1 were due to the of glucose to in the were because ethanol was the carbon source. The due to of α-ketoisocaproate and of isoamyl alcohol were also There was no resonance due to This that the isovalerate by the was via isovaleryl-CoA due to the of branched-chain α-keto acid dehydrogenase and subsequent by acyl-CoA isovalerate had been from either α-ketoisocaproate or then one would to it by possible routes for the conversion of α-ketoisocaproate to isoamyl pyruvate of α-ketoisocaproate to α-hydroxyisocaproate followed by decarboxylation of α-hydroxyisocaproate to isoamyl or a pyruvate decarboxylase-like The mutant strain YSH5.127.-17C pyruvate decarboxylase activity due to in all three for pyruvate decarboxylase S. J. 1991; PubMed Google Scholar). This strain as well as wild-type strains on ethanol minimal medium with leucine as sole source of nitrogen and of isoamyl alcohol The of isoamyl alcohol in the medium from the for this strain to be grown with ethanol as carbon source. The wild-type strain ethanol was the carbon source rather glucose J.R. Microbiology. 1996; PubMed Scopus Google Scholar). It to from this that pyruvate decarboxylase is not required for the formation of isoamyl alcohol from The of α-ketoisocaproate to α-hydroxyisocaproate pathway with subsequent decarboxylation an The of α-ketoisocaproate and has been described in J. J. PubMed Scopus Google Scholar). S. cerevisiae three on on on identical at the amino acid to the each of which is of encoding an S. cerevisiae the ability to convert α-ketoisocaproate to α-hydroxyisocaproate using as activity at 6 mm α-ketoisocaproate and with a pH at pH It to assay the reaction in the using α-hydroxyisocaproate with any one of or The reductase activity appears to the not from cells that had been grown on ethanol minimal medium in which leucine was the sole source of nitrogen did not convert α-hydroxyisocaproate into isoamyl alcohol. the presence of α-ketoisocaproate reductase the presence of α-hydroxyisocaproate in spectra as but it does not to be to the formation of isoamyl alcohol. on this enzyme be reductase activity in S. strain was in ethanol minimal medium with leucine as nitrogen source. Cell homogenates were and as described under are the of between were Open table in a new tab strain was in ethanol minimal medium with leucine as nitrogen source. Cell homogenates were and as described under are the of between were The possibility to be a pyruvate decarboxylase-like activity in The conversion of α-ketoisocaproate to isoamyl alcohol in confirms this The activity at α-ketoisocaproate mm and was to the The rates in in from which had been in ethanol minimal medium with leucine as the sole nitrogen source for explain the of of isoamyl alcohol in cultures at this A of data base the presence of the gene YDL080c on the amino acid sequence of which has to the enzymes encoded by and no has been to YDL080c although a for YDL080c is which as a of K. A. J. 1992; PubMed Google Scholar). Strains and both of which a disruption in this and strain do not grow in ethanol minimal medium with leucine as nitrogen source. all three strains do not grow well even in complex medium supplied with carbon as or a reduced ability to carbon in the genetic of the and the YDL080c disruption were of in glucose minimal medium with leucine as nitrogen source with identical kinetics to the wild-type used this It is that the strains with in isoamyl alcohol the of isoamyl alcohol for the of this YDL080c were the gene encoding the ability to one would have the of isoamyl alcohol to be in and The JRD815–1.2 and which are in all three pyruvate decarboxylase and disrupted in the levels of isoamyl alcohol to those by strains disrupted in YDL080c This that the gene YDL080c the major α-ketoisocaproate decarboxylase but that there be at least one other decarboxylase that is in the decarboxylation of α-ketoisocaproate and the formation of isoamyl alcohol from in conversion of α-ketoisocaproate to isoamyl alcohol by cell converted to isoamyl isoamyl alcohol strain was for in ethanol minimal medium with leucine as sole source of Cell homogenates were with α-ketoisocaproate then isoamyl alcohol was as described under are the of between were Open table in a new tab of the amino acid of the three of pyruvate decarboxylase with the gene product of The was using the with a of and a of The was using the that identical in a and in a The the with and in which is in all enzymes A. FEBS Lett. PubMed Scopus Google Large of wild-type strains and disruption mutant in glucose minimal medium with leucine as sole source of and of the YDL080c disruption Large strain was for in ethanol minimal medium with leucine as sole source of Cell homogenates were with α-ketoisocaproate then isoamyl alcohol was as described under are the of between were of the metabolism of [2-13C]leucine by the wild-type strain four routes from α-ketoisocaproate to isoamyl alcohol The first was via branched-chain α-keto acid dehydrogenase to this would then be converted to isovalerate by acyl-CoA hydrolase gene which has a this J. Biochem. PubMed Scopus Google Scholar, J. Biol. Chem. PubMed Google Scholar). This pathway operates in the as as isovalerate the end product. The pathway is not used for the synthesis of isoamyl alcohol because abolition of branched-chain α-keto acid dehydrogenase in an mutant did not prevent the formation of isoamyl alcohol. The second possibility was via pyruvate decarboxylase as by and Large (4Derrick S. Large P.J. J. Gen. Microbiol. 1993; 139: 2783-2792Crossref PubMed Scopus (67) Google Scholar). the of pyruvate decarboxylase activity in a pdc1 pdc5 pdc6 triple mutant had no on the levels of isoamyl alcohol produced. The and most possibility was via α-ketoisocaproate a novel activity as as are has not been identified previously in S. The metabolic of this enzyme in yeast is not at this It is that it has no role in the formation of isoamyl alcohol from α-hydroxyisocaproate because cell homogenates could not convert α-hydroxyisocaproate to isoamyl alcohol. in that this enzyme is in the between the yeast and and the of S. cerevisiae and acid it is to that this activity was from acid at some in The final possibility to be a pyruvate decarboxylase-like enzyme encoded appears to be the major route of decarboxylation of α-ketoisocaproate to isoamyl alcohol although disruption of this gene reveals that another decarboxylase can substitute to a minor extent. The of this minor decarboxylase at this the of and enzyme is to be the same as a gene identified previously as a of K. A. J. 1992; PubMed Google Scholar). is known at of the in which is by the gene product. the pathway of to be in all α-ketoisocaproate decarboxylase is a has a A. FEBS Lett. PubMed Scopus Google and be a yeast cell is to the role of decarboxylation and of would to be a metabolic the of the enzyme by in that in activity would be low was which with the known low rates of formation of isoamyl alcohol in media (7Webb A.D. Ingraham J.L. Adv. Appl. Microbiol. 1963; 5: 317-353Crossref Scopus (83) Google Scholar). It to us that because its role as is its role in of a appropriate for the YDL080c would be cerevisiae, a number of enzymes have a role as metabolic in this are the gene which as an of and is also in carbon Mol. Microbiol. 1992; PubMed Scopus Google Jones E.W. J.R. Broach J.R. The Molecular and Biology of the Yeast Cold Spring Harbor Laboratory, Cold Spring Harbor, Scholar), and which the major of pyruvate decarboxylase for on glucose and a on the of S. J. Biochem. PubMed Scopus Google Scholar). It be to of different of the gene can of the in either or gene regulation. at and for the of isoamyl alcohol by