Yukio Tamai

Co-existence of two types of chromosome in the bottom fermenting yeast, Saccharomyces pastorianus

The bottom fermenting yeasts in our collection were classified as Saccharomyces pastorianus on the basis of their DNA relatedness. The genomic organization of bottom fermenting yeast was analysed by Southern hybridization using eleven genes on chromosome IV, six genes on chromosome II and five genes on chromosome XV of S. cerevisiae as probes. Gene probes constructed from S. cerevisiae chromosomes II and IV hybridized strongly to the 820‐kb chromosome and the 1500‐kb chromosome of the bottom fermenting yeast, respectively. Five gene probes constructed from segments of chromosome XV hybridized strongly to the 1050‐kb and the 1000‐kb chromosomes. These chromosomes are thought to be S. cerevisiae‐type chromosomes. In addition, these probes also hybridized weakly to the 1100‐kb, 1350‐kb, 850‐kb and 700‐kb chromosome. Gene probes constructed from segments including the left arm to TRP1 of chromosome IV and the right arm of chromosome II hybridized to the 1100‐kb chromosome of S. pastorianus. Gene probes constructed using the right arm of chromosome IV and the left arm of chromosome II hybridized to the 1350‐kb chromosome of S. pastorianus. These results suggested that the 1100‐kb and 1350‐kb chromosomes were generated by reciprocal translocation between chromosome II and IV in S. pastorianus. Three gene probes constructed using the right arm of chromosome XV hybridized weakly to the 850‐kb chromosome, and two gene probes from the left arm hybridized weakly to the 700‐kb chromosome. These results suggested that chromosome XV of S. cerevisiae was rearranged into the 850‐kb and 700‐kb chromosomes in S. pastorianus. These weak hybridization patterns were identical to those obtained with S. bayanus. Therefore, two types of chromosome co‐exist independently in bottom fermenting yeast: one set which originated from S. bayanus and another set from S. cerevisiae. This result supports the hypothesis that S. pastorianus is a hybrid of S. cerevisiae and S. bayanus.

Acetate ester production by Saccharomyces cerevisiae lacking the ATF1 gene encoding the alcohol acetyltransferase

Abstract Alcohol acetyltransferase (AATase) was believed to be the only enzyme responsible for acetate ester production of Saccharomyces cerevisiae . However, recent studies have shown that S. cerevisiae has several types of AATase. In order to determine the precise role of each AATase in ester production, the ATF1 gene encoding one type of AATase, was disrupted and the ability of the atf1 null mutant to form isoamyl acetate and ethyl acetate was studied. The results of AATase assays using isoamyl alcohol as a substrate revealed that although the AATase activity of the null mutant was dramatically reduced, 20% activity was retained. However, when ethanol was used as a substrate, more than 80% activity was retained. The production of isoamyl acetate and ethyl acetate during fermentation was also compared and it was shown that, compared to the control, the null mutant produced less than 20% isoamyl acetate but produced more than 60% ethyl acetate. These results suggest that the yeast cell has several different types of AATase, and that the Atf1 protein plays a major role in isoamyl acetate production but has a relatively minor role in acetate ester production during fermentation.

Nucleotide sequences of alcohol acetyltransferase genes from lager brewing yeast, Saccharomyces carlsbergensis

The nucleotide sequences of alcohol acetyltransferase genes isolated from lager brewing yeast, Saccharomyces carlsbergensis have been determined. S. carlsbergensis has one ATF1 gene and another homologous gene, the Lg‐ATF1 gene. There was a high degree of homology between the amino acid sequences deduced for the ATF1 protein and the Lg‐ATF1 protein (75·7%), but the N‐terminal region has a relatively low degree of homology.

Transcriptional co-regulation ofSaccharomyces cerevisiae alcohol acetyltransferase gene,ATF1 and Δ-9 fatty acid desaturase gene,OLE1 by unsaturated fatty acids

The ATF1 gene encodes an alcohol acetyl transferase which catalyzes the synthesis of acetate esters from acetyl CoA and several kinds of alcohols. ATF1 expression is repressed by unsaturated fatty acids or oxygen. Analysis using ATF1‐lacZ fusion plasmid revealed that ATF1 gene expression is widely repressed by a variety of unsaturated fatty acids, and the degree of ATF1 transcriptional repression varies according to the structure of the unsaturated fatty acids. Interestingly, it was noted that the degree of ATF1 transcriptional repression was related to the melting point of unsaturated fatty acids added to the medium. The OLE1 gene, which encodes Δ‐9 fatty acid desaturase, has been reported to be repressed by unsaturated fatty acids. Transcription of OLE1 was also repressed by a wide variety of unsaturated fatty acids under anaerobic conditions. The degree of transcriptional repression of OLE1 was also related to the melting point of the added unsaturated fatty acids. Therefore, it is considered that ATF1 and OLE1 transcription are regulated in response to cell membrane fluidity. As has been reported for OLE1, the repression of ATF1 by unsaturated fatty acids was relieved in a disruptant carrying a faa1 and faa4 double mutation, two fatty acid activation genes. However, the ATF1 transcript in this double gene disruptant was repressed by oxygen. These results suggested that ATF1 transcription was co‐regulated by the same mechanism as the OLE1 gene and that unsaturated fatty acids and oxygen repressed the ATF1 transcript by a different regulation pathway.

Isolation and characterization of the ATF2 gene encoding alcohol acetyltransferase II in the bottom fermenting yeast Saccharomyces pastorianus.

The ATF2 gene encodes alcohol acetyltransferase II, which catalyses the synthesis of isoamyl acetate from acetyl coenzyme A and isoamyl alcohol. To characterize the ATF2 gene from the bottom fermenting yeast Saccharomyces pastorianus, the S. pastorianus ATF2 gene was cloned by colony hybridization using the S. cerevisiae ATF2 gene as a probe. When an atf1 null mutant strain was transformed with a multi‐copy plasmid carrying the S. pastorianus ATF2 gene, the AATase activity of this strain was increased by 2·5‐fold compared to the control. The S. pastorianus ATF2 gene has 99% nucleic acid homology in the coding region and 100% amino acid homology with the S. cerevisiae ATF2 gene. Southern blot analysis of chromosomes separated by pulse‐field gel electrophoresis indicated that the ATF2 gene probe hybridized to chromosome VII in S. cerevisiae and to the 1100 kb chromosome in S. pastorianus. As S. pastorianus is thought to be a hybrid of S. cerevisiae and S. bayanus, the S. bayanus‐type gene, which has a relatively low level of homology with the S. cerevisiae‐type gene, is also usually detected. Interestingly, an S. bayanus‐type ATF2 gene could not be detected. These results suggested that the cloned ATF2 gene was derived from S. cerevisiae. Analysis using an ATF2–lacZ fusion gene in S. pastorianus showed that expression of the ATF2 gene was relatively lower than that of the ATF1 gene and that it is repressed by aeration but activated by the addition of unsaturated fatty acids. The S. pastorianus ATF1, Lg‐ATF1 and ATF2 Accession Numbers in the DDBJ Nucleotide Sequence Database are D63449, D63450 and D86480, respectively. Copyright

Molecular mechanism of the multiple regulation of the Saccharomyces cerevisiae ATF1 gene encoding alcohol acetyltransferase.

The ATF1 gene encodes an alcohol acetyl transferase (AATase), that catalyses the synthesis of acetate esters from acetyl CoA and several kinds of alcohols. ATF1 transcription is negatively regulated by unsaturated fatty acids and oxygen. A series of analyses of the ATF1 promoter identified an 18 bp element essential for transcriptional activation. Ligation of the 18 bp element into a plasmid carrying the CYC1 promoter deleted UAS‐activated transcription and conferred transcriptional repression by unsaturated fatty acids. The 18 bp element contains a binding sequence for Rap1p, which is a transcriptional repressor and activator. In vitro binding studies showed that Rap1p binds to the 18 bp element essential for transcriptional activation. The results of internal deletion studies of the promoter region suggested that there was also a region responsible for ATF1 oxygen regulation. This region contained the consensus binding sequence for the hypoxic repressor Rox1p. In vitro binding studies showed that Rox1p binds to the region responsible for oxygen regulation. To investigate the effect of the hypoxic repressor complex on transcription, ATF1 expression was measured in rox1, tup1 and ssn6 disruptant strains. It was found that rox1, tup1 and ssn6 disruption caused elevated expression of ATF1 under aerobic conditions. Thus, the activation of ATF1 transcription is dependent on Rap1p, and the Rox1p–Tup1p–Ssn6p hypoxic repressor complex is responsible for repression by oxygen. Furthermore, a study of ATF1 expression in a sch9 null mutant suggested that the Sch9p protein kinase is involved in ATF1 trancriptional activation. Copyright

Characterization of the ATF1 and Lg-ATF1 genes encoding alcohol acetyltransferases in the bottom fermenting yeast Saccharomyces pastorianus

The bottom fermenting yeast Saccharomyces pastorianus (formerly Saccharomyces carlsbergensis) has one ATF1 gene and a homologous gene called Lg-ATF1 which encode alcohol acetyltransferases. Southern blot analysis of chromosomes separated by pulsed-field gel electrophoresis in a variety of bottom fermenting yeasts showed that in most of the yeasts analyzed, the ATF1 gene is located on the 1000- and 1050-kb chromosomes or only on the 1050-kb chromosome, while the Lg-ATF1 gene is located on the 850-kb chromosome. Acetate ester synthesis in a bottom fermenting yeast was found to be reduced by aeration or the addition of an unsaturated fatty acid. The enzyme activities involved in the synthesis of acetate esters decreased under these conditions. Experiments using ATF-lacZ fusion genes showed that the transcription of the ATF1 and Lg-ATF1 genes is co-regulated and is repressed by aeration or the addition of an unsaturated fatty acid. These findings suggest that the reduction in acetate ester synthesis observed in the bottom fermenting yeast was due to a reduction in enzyme synthesis resulting from transcriptional repression of the ATF1 and Lg-ATF1 genes.

Diversity of the HO gene encoding an endonuclease for mating-type conversion in the bottom fermenting yeast Saccharomyces pastorianus.

Two types of HO gene were cloned, sequenced and characterized from the bottom fermenting yeast Saccharomyces pastorianus. The HO gene present on the 1500 kb chromosome was designated Sc‐HO (S. cerevisiae‐type HO), because the nucleotide sequence of its promoter region and the open reading frame (ORF) was almost identical to that of the S. cerevisiae laboratory strain HO gene (Lab‐HO). The other HO gene, designated Lg‐HO (Lager‐fermenting‐yeast specific HO), showed 64% and 83% homology with the promoter and ORF of the Lab‐HO at the nucleotide sequence level, respectively, and was located on the 1100 kb chromosome. Analysis of the 4 kb DNA fragment amplified from S. bayanus type strain indicated that the nucleotide sequence of S. bayanus‐HO is almost identical to that of the Lg‐HO. The SSB1 gene located downstream of the HO gene in S. cerevisiae was also found in the 3′ distal region of the Sc‐HO, Lg‐HO and S. bayanus HO genes. These results showed that the genetic arrangement around the HO loci both of S. pastorianus and S. bayanus is identical to S. cerevisiae. Southern analysis has revealed that Saccharomyces sensu stricto contain four types of HO genes; S. paradoxus‐type HO, the Sc‐HO, the Lg‐HO and S. uvarum‐type HO genes. This HO gene diversity provides useful information for the classification of strains belonging to Saccharomyces sensu stricto. The S. pastorianus Sc‐HO, Lg‐HO and S. bayanus‐HO Accession Nos in the DDBJ Nucleotide Sequence Database are AB027449, AB027450 and AB027451, respectively. Copyright

A novel hemiacetal dehydrogenase activity involved in ethyl acetate synthesis in Candida utilis.

Acetate ester synthesis was studied in vitro with the ethyl acetate-producing yeast Candida utilis. The level of enzyme activity observed for the NAD+-dependent hemiacetal dehydrogenase acting on hemiacetal, which was produced non-enzymatically from an alcohol and an aldehyde, was much greater than that for the other enzyme involved in ester synthesis, alcohol acetyltransferase. The level of ethyl acetate synthesis in vivo approximately paralleled the hemiacetal dehydrogenase (HADH) activity. The results suggest that the main pathway for ethyl acetate synthesis in C. utilis involves a novel hemiacetal dehydrogenase activity.

Pyruvate decarboxylase encoded by the PDC1 gene contributes, at least partially, to the decarboxylation of α-ketoisocaproate for isoamyl alcohol formation in Saccharomyces cerevisiae

Isoamyl alcohol is an important flavor component of yeast-fermented alcoholic beverages. To identify the enzyme and gene involved in the decarboxylation of alpha-ketoisocaproate (alpha-KIC) for isoamyl alcohol formation, the enzyme was partially purified and analyzed by mass spectrometry. The pyruvate decarboxylase encoded by the PDC1 gene was considered a likely candidate enzyme. Genetic analysis showed that the activity of alpha-KIC decarboxylase and production of isoamyl alcohol partially decreased in a pdc1 null mutant and increased in a transformant with a multi-copy plasmid carrying the PDC1 gene. These results indicate that pyruvate decarboxylase encoded by the PDC1 gene contributes, at least partially, to the decarboxylation of alpha-KIC for isoamyl alcohol formation.