Alcohol Acetyl Transferase Genes and Ester Formation in Brewer's Yeast

Chapter 18

Alcohol Acetyl Transferase Genes and Ester Formation in Brewer's Yeast Yukio Tamai

Downloaded by FUDAN UNIV on November 17, 2016 | http://pubs.acs.org Publication Date: August 13, 1996 | doi: 10.1021/bk-1996-0637.ch018

Central Laboratories for Key Technology, Kirin Brewery Co., Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236, Japan

Alcohol acetyl transferase (AATase), which catalyses the synthesis of acetate esters from alcohols and acetyl-CoA, was purified from Saccharomyces cerevisiae (sake yeast) and its partial amino acid sequence was determined. The AATase-encoding ATF1 genes were cloned from sake yeast and brewer's yeast (bottom fermenting yeast). The bottom fermenting yeast was found to have two types of ATF1 gene; ATF1 and a homologous gene Lg-ATF1, while sake yeast had one ATF1 gene. Strains carrying either the ATF1 genes carried on multiple copy plasmid or a single ATF1 gene with the native promoter replaced by the strong constitutive GPD (glyceraldehyde-3-phosphate dehydrogenase) promoter produced higher concentrations of acetate esters. On the other hand, disruption of the ATF1 gene resulted in a decrease in AATase activity and reduced acetate ester formation. Control of the ATF1 gene expression is a novel approach for the enrichment or reduction of acetate esters in yeast strains used in the brewing industry.

The volatile esters formed during fermentation are thought to be important flavor components in beer and other alcoholic beverages. Beer contains ethyl acetate (light fruity, solvent-like flavor) and isoamyl acetate (banana flavor) at concentrations near or just below threshold values and they are considered to determine the characteristics of a beer. Isoamyl acetate is also responsible for the excellent aroma of Japanese sake, especially of high quality sokes. Therefore, a number of studies have been carried out to determine the mechanism of formation of acetate esters in the various brewing processes. Nordstrom carried out extensive studies of acetate ester formation in yeast and proposed that the acetate esters were synthesized by the enzymatic condensation of acetyl Co A and the alcohols formed during fermentation (1-3). The higher alcohols, which are the substrates for AATase, are formed by both a catabolic route (Ehrlich pathway) and a anabolic route (biosynthetic pathway of amino acids). Yoshioka and Hashimoto were successful in partially purifying alcohol acetyl transferase (AATase), the enzyme responsible, and proposed a mechanism for acetate ester formation (4). As the AATase has a wide range of higher alcohol substrates, low or medium molecular weight esters are thought to be synthesized by AATase. Ester formation during fermentation is reduced by aeration of the medium or the addition of

0097-6156/96/0637-0196$15.00/0 © 1996 American Chemical Society

Takeoka et al.; Biotechnology for Improved Foods and Flavors ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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Alcohol Acetyl Transferase Genes in Brewer's Yeast

197

unsaturated fatty acids. As unsaturated fatty acids are synthesized from oxygen and saturated fatty acid by A9-desaturase in S. cerevisiae (5), aerobic conditions increase the concentration of unsaturated fatty acids in the cell membrane. As activity of the partially purified enzyme was inhibited by unsaturated fatty acids, Yoshioka and Hashimoto proposed that the unsaturated fatty acids synthesized in aerobic conditions reduce the ester formation by the inhibition of AATase activity (6-7). AATase was previously reported to be a membrane bound enzyme which was labile during purification. However, recently Minetoki et al. have purified AATase from S. cerevisiae and the partial amino acid sequence has been determined (#). The ATF1 genes encoding AATase was cloned from the sake yeast S. cerevisiae and the bottom fermenting yeast S. pastorianus (9). ATF1 is thought to be a useful tool for the study of ATF1 gene expression and for the regulation of ester formation in yeast. In this report, the structure of genes cloned from the bottom fermenting yeast S. pastorianus is summarized and the gene expression and the regulation of ester formation during brewing is described. Cloning of ATF1 Genes Encoding AATase Cloning of the K1-ATF1 Gene from Sake Yeast. Two mixed oligonucleotide probes were designed and synthesized on the basis of the amino acid sequences obtained from the purified AATase (8). Clones carrying sequences homologous to the probes were isolated from a genomic gene library constructed from the sake yeast Kyokai No.7 by the plaque hybridization method. Positive clones which hybridized to both probes were chosen for further analysis. Figure 1 shows a restriction map of the DNA fragment carrying K1-ATF1, which encoded AATase. The two synthetic oligonucleotides hybridized to a 1.0 kb EcoKl-BamHl fragment. Nucleotide Sequence of the K7-ATF1 Gene. An approximately 2.0-kb nucleotide sequence containing a EcoRl-BamHI fragment was determined. This fragment contained an open reading frame encoding a protein consisting of 525 amino acids. All amino acid sequences obtained from the purified AATase were found within this open reading frame. The molecular weight of the protein deduced from the nucleotide sequence was calculated as being 61,059 which was consistent with the value estimated by SDS-PAGE of the purified AATase. This gene was designated K1-ATF1: ATF1 derived from Kyokai No.7. Although AATase was reported to be a membrane bound enzyme (4,8), the hydrophobicity profile predicted by computer analysis showed that AATase has no obvious hydrophobic region which could act as a membrane anchor (8). Disruption of the ATF1 gene resulted in a drastic reduction in isoamylacetate synthesis. Thus, it was concluded that ATF1 encoded AATase in S. cerevisiae (Fujii, T., submitted). The ATF1 gene was mapped to the right arm of the XV chromosome of S. cerevisiae (Yoshimoto, H., in preparation). Cloning of the ATF1 and Lg-ATFl Genes from the Bottom Fermenting Yeast S.pastorianus. To analyze the ATF1 gene in the bottom fermenting yeast, Southern blotting was carried out using K7-ATF1 as a probe. The results showed that the bottom fermenting yeast contained two types of gene homologous with K1-ATF1 based upon the intensity of hybridization signal with the probe. One gene was highly homologous with YJ-ATF1, but the other showed less homology with K1-ATF1. The two homologous ATF1 genes were cloned from a gene library constructed from the bottom fermenting yeast KBY004 using a 0.4-kb Clal-EcoRl fragment of K1-ATF1 as a probe. Figure 1 shows the restriction maps of the strongly hybridizing gene and the weakly hybridizing gene. As the restriction map of the strongly hybridizing DNA


198

BIOTECHNOLOGY FOR IMPROVED FOODS AND FLAVORS Xb H H Hp H C

E

HB K HC

H

Xb

HB

H

Xb

K7-ATF1 Xb H

HpE H C

E

Pt HC


ATF1 Bg

Sea

XC

VBsm

Pt

K Sm C Sph

Bg

lkb Lq-ATF1

Figure 1. Restriction maps of the ATF1 genes. The restriction enzymes used were Xbal (Xb), ffindlH (H), Hpal (Hp), Clal (C), EcoKI (E), BamHl (B), Kpnl (K), Pstl (P), £g/II(Bg), Seal (Sea), Sail (S), Xhol (X), EcoRV (V), Bsml (Bsm), Smal (Sm), Sphl (Sph). The position and direction of the open reading frame are indicated by arrows.

Lg-ATFl Open Reading Frame 417

833

1249

1665

2080

395-'

e C

789

cd

C

1183-

O 1577-

1974

Figure 2. Harr plot comparison of nucleotide sequences of ATF1 and Lg-ATFl gene. Arrows indicate coding regions of the two genes.



18.

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Alcohol Acetyl Transferase Genes in Brewer's Yeast 199

fragment was quite similar to that of K1-ATF1, it is considered that the cloned DNA fragment encodes the ATF1 gene from the bottom fermenting yeast. The weakly hybridizing DNA differs in structure from Kl-ATFL Each of these two homologous genes was cloned into a multicopy yeast-E.coli shuttle vector and introduced into a laboratory yeast strain. As transformants carrying these genes homologous with ATF1 produce higher levels of AATase activity than the parental strain, it was clear that these cloned DNA fragments encoded AATase. The weakly hybridizing gene was thought to be specific to the bottom fermenting, brewery Lager yeast, and this was designated the Lg-ATFl gene. Comparison of ATF1 and K1-ATF1 showed that these two genes share an almost identical nucleotide sequence in the region which was sequenced. Thirteen nucleotide changes were observed in the open reading frame and three amino acid changes were found in these genes. However, Lg-ATF1 had less homology with ATF1. Figure 2 shows a Harr plot comparing the Lg-ATFl and ATF1 genes from the bottom fermenting yeast, indicating a relatively high homology (76%) for the open reading frames. However, the N-terminal region of the open reading frame and its 5' upstream region showed reduced homology. The amino acid sequence encoded by ATF1 shared 80% homology with that of Lg-ATFl. The AATase encoded by ATF1 contained fourteen cysteines and the AATase encoded by Lg-ATFl had eleven cysteines. Ten of these cysteine residues were found in the same position within their amino acid sequences. The hydrophobicity of the AATases encoded by ATF1 and Lg-ATFl also showed a similar profile. These results suggested that the protein structures of both AATases were analogous. ATF1 was mapped to a region close to the right arm telomere of XV chromosome in S. cerevisiae using an S. cerevisiae prime clone grid filter (ATCC 77284). The labelled ATF1 hybridized strongly to the 1050-kb chromosomes of S. pastorianus, which has the similar molecular weight as the XV chromosome of S. cerevisiae, and the 1000-kb chromosome of S. pastorianus. The labelled Lg-ATFl probe hybridized strongly with the 850-kb chromosome of both S. pastorianus and S. bayanus. The bottom fermenting yeast S. pastorianus is thought to be a hybrid between S. cerevisiae and S. bayanus (10-11). Therefore, ATF1 might be derived from S. cerevisiae and Lg-ATFl might be derived from S. bayanus (Yoshimoto, H., in preparation). Gene Dosage Effect of ATF1 and Lg-ATFl in Brewer's Yeast Construction of Vectors Carrying the ATF1 Genes for Brewer's Yeasts. Laboratory strains are usually heterothallic haploid and have several genetic markers which can be used to isolate trasformants for the study of yeast molecular genetics. However, brewer's yeasts, which include bottom fermenting yeast (Lager Yeast) and top fermenting yeast (ale yeast), are diploid or polyploid. Therefore, nutritional requirement mutations used for the yeast transformation as selectable markers are very difficult to isolate. A dominant marker gene is essential to the transformation of the brewer's yeast. As shown in Figure 3, two types of vector, pYT71 and pYT77, were constructed in this study: a single copy YCp vector and a multicopy YEp vector respectively, both carrying the G-418 resistance gene as a dominant selectable marker. To insert the genes to these vectors, the 5' upstream Hpal site and the 3' downstream Kpnl site of the ATFI gene were changed to Sal 1 by blunting and linker ligation. This 2.8-kb Sail DNA fragment was ligated to both vectors. Sail linker was ligated to the blunted Pstl site in 3' downstream region of Lg-ATFl. The resulting 3.0kb of Sail fragment was then ligated to both vectors.


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BIOTECHNOLOGY FOR IMPROVED FOODS AND FLAVORS


CEN3 P

ARS1

G-418 Resistance gene

2}im DNA reprication origin

G-418 Resistance gene

Figure 3. Structure of pYT71 and pYT77, vectors for brewer's yeast trans formation. pBR322 sequence is presented by the thin line.

_Bg_

Dominant marker

PGKy-BSR-PGKt

or PGKp-NEO-PGKt

x

ATF1

i

x Recombination

1)

Disrupted atfl on chromosome

ATF1

Figure 4. Construction of DNA fragments for atfl gene disruption and one step gene disruption. The restriction enzymes used were /fmdIII (H), Clal (C), EcoRl (E), BamUl (B), Pstl (P), #g/II(Bg), Sail (S), Sphl (Sph). The position and direction of the open readingframeare indicated by an arrow.



18.

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Laboratory Scale Fermentation Using Ale-Yeast Carrying ATF1 and Lg-ATFl Genes. Transformants carrying the ATF1 genes were pre-cultured in brewery wort, to which 50 pg/mL of G-418 had been added. Pre-culture was carried out anaerobically with agitation at 20°C for 3 days and the transformant cells were collected by centrifugation. The wort was pitched with 3.0g-wet weight cells/L without the addition of antibiotics and transferred to a 500 mL laboratory fermentation vessel (120-cm long, 2.5-cm diameter). Fermentation was carried out at 18°C for 4 days. There was no substantial difference in the fermentation performance of the transformants and the parental strain (data not shown). As shown in Table I, young beer produced by the transformants carrying the ATF1 gene on a multicopy vector had a 13 fold greater concentration of isoamyl acetate and a 9 fold greater concentration of ethyl acetate than the parental strain. However, transformants carrying ATF1 on single copy vector produced almost same concentration of both esters as parental strain. Transformants carrying Lg-ATFl on the multicopy vector produced less acetate esters than transformants carrying ATF1 on the multicopy vector. The enzyme activity of the transformants were compared on the third day of fermentation. All transformants carrying the ATF1 or Lg-ATFl on the multicopy vector exhibited high levels of AATase activity, but the AATase activity produced by ATF1 strains was higher than that of Lg-ATFl strains. Although the copy number of ATF1 and Lg-ATFl on the multicopy vector was determined by Southern blot analysis using a URA3 DNA fragment as a probe, no difference was observed (data not shown). Therefore, the expression level of the gene or the specific activity of the AATase encoded by the LgATF1 might be lower than that of the AATase encoded by ATFL Gene Disruption of Ale-Yeast ATFL To reduce ester formation, the ATF1 genes were disrupted in the top fermenting yeast (Ale-yeast). As shown in Figure 4, the EcoRl site within the open reading frame of ATF1 was filled using Klenow enzyme and a Xhol linker was ligated. DNA fragments for the ATF1 gene disruption were constructed by ligating the Sail DNA fragment containing the G-418 resistance gene (NEO) or blasticidine S resistance gene (BSR) to the Xhol site, The BglH fragment was used to transform the top fermenting yeast KTY001. The homologous ends pairs with the ATF1 locus on the chromosome, and recombination results in a chromosomal gene disruption, as shown in Figure 4. DNA was isolated from the transformants and digested with Clal for Southern blot analysis. Transformants in which at least one ATF1 gene was disrupted were used for re-transformation with the blasticidine resistance marker to isolate clones in which two ATF1 genes were disrupted. Southern blot analysis showed that transformants carrying the two disrupted ATF1 genes still maintained at least one ATF1 gene (data not shown). These results suggested that Aleyeast used in this study maintained at least three ATF1 genes. A yeast strain yYTl 13 contains one disrupted atf gene designated atflv.NEO, and yYTl27 contains two disrupted atf genes designated atflv.NEO and atflv.BSR. Laboratory Scale Fermentation Using ATF1 Disruptants. Transformants carrying the disrupted atfl genes were pre-cultured in YM10 medium (Yeast extract 1.25%, Malt Extract 1.25%, Glucose 10%), to which 50 pg/mL of G-418 had been added. Preculture was carried out anaerobically with agitation at 20°C for 3 days, and cells were collected by centrifugation. Brewery wort was pitched with 3.0g-wet weight cells/L without the addition of antibiotics and transferred to a 500 mL laboratory fermentation vessel (120-cm long, 2.5-cm diameter). Fermentation was carried out at 18°C for 4 days. There was no substantial difference in the fermentation performance of the transformants and the parental strain (data not shown). As shown in Table II, young


202



Table I. Gene dosage effects of ATF1 on the volatile esters and alcohols concentrations in the laboratory scale fermentation Isoamyl acetate (ppm)

Ethyl acetate (ppm)

n-propyl alcohol (ppm)

isobutyl alcohol (ppm)

Parental strain

0.67

7.4

14.4

21.9

77.8

ATF1 on pYT71

0.78

8.9

16.9

18.5

69.4

ATF1 on pYT77

10.10

62.7

17.8

22.0

74.1

Lg-ATFl on pYT71

0.69

7.8

17.1

20.3

76.8

Lg-ATFl on pYT77

2.20

16.6

16.7

22.1

79.3

Plasmids

Isoamyl alcohol (ppm)

Table II. Effects of ATF1 gene disruption on the volatile esters and alcohols concentrations in the laboratory scale fermentation Isoamyl acetate (ppm)

Ethyl acetate (ppm)



isoamyl alcohol (ppm)

Parental strain

0.83

9.6

14.9

13.9

73.8

yYT113 (atfl::NEO)

0.72

9.1

16.9

18.5

69.4

0.53 yYT127 (atfl::NEO,atfl::BSR)

8.3

17.8

22.0

74.1

Strains



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beer produced by the transformant carrying a single disrupted atfl gene contained approximately 15% less isoamyl acetate than the parental strain and there was a slight decrease in ethyl acetate production. Young beer fermented by the transformant carrying two disrupted atfl genes contained about 35% less isoamyl acetate than the parental strain and there is a small but significant decrease in ethyl acetate production. Although the haploid laboratory strain TIM has one ATF1 gene, disruption of ATF1 did not affect cell growth or fermentation ability (Fujii, T., submitted). This result indicated that ATF1 was not an essential gene in S. cerevisiae. Compared to the parental strain, laboratory strain carrying a disrupted atfl gene, TD4- Aatfl, only produced 30% isoamyl acetate in static culture. However, 70% production of ethyl acetate was maintained compared to the parental strain. These results showed that the AATase encoded by A TF1 mainly participates in isoamyl acetate formation, but play a lesser role in ethyl acetate formation. These results suggested that AATases encoded by genes other than ATF1 might be involved in the remaining ester formation in the atfl disruption strain. Minetoki et al showed that two separate AATase activities could be eluted from an ion exchange column (8). The major peak of AATase activity was purified and its amino acid sequences were used to clone the ATF1 gene. It will be interesting to elucidate enzyme characteristics of the minor peak of AATase activity and the gene expression. ATF1 Gene Regulation and Promoter Replacement Transcription Regulation of ATF1 Gene. The yeast cell adopts to circumstances by regulating protein function and gene expression. Many genes are controlled by transcription initiation. AATase enzyme activity is thought to be reduced by aeration or the addition of unsaturated fatty acids to the medium. To elucidate whether the A TF1 gene was regulated at the transcriptional level, RNA was extracted from cells cultured in repressed or derepressed conditions. The amount of mRNA transcribed from ATF1 was analyzed by Northern blotting. Transcription of ATF1 was reduced to basal levels in cultures containing unsaturated fatty acids or by aeration (Kobayashi, O., in preparation). It was also confirmed that expression of ATF1 and Lg-ATFl were co-regulated in the bottom fermenting yeast. A 1-kb DNA fragment containing the ATF1 or Lg-ATFl promoter region was obtained by PCR (polymerase chain reaction). Amplified ATF1 promoter or Lg-ATFl promoter was ligated to the E.coli 6-galactosidase gene lacZ on the plasmid pHY428 which was derived from pYT77. The resultant plasmid, pHY429 carrying the ATFlv.lacL fusion gene or pHY430 carrying the Lg-ATFlv.lacL, was introduced into the bottom fermenting yeast. The expression of ATF1 or Lg-ATFl was monitored by determining the level of 6-galactosidase activity under a variety of culture conditions. It was found that unsaturated fatty acids reduce lacZ expression to basal level. This result clearly indicated that ATF1 and Lg-ATFl were co-regulated at the transcriptional level in S. pastorianus (Yoshimoto, H., in preparation). Replacement of the ATF1 Promoter with a Constitutive GDP Promotor. AATase activity is known to be reduced by the addition of unsaturated fatty acids to the medium or shaking the culture for aeration (7). To enhance the expression of the ATF1 gene, a constitutive strong promoter was placed in front of the ATF1 coding region. As genes encoding glycolysis enzymes are reported to be strongly expressed in S. cerevisiae, the promoter of alcohol dehydrogenase (ADH), phospho-glycerol kinase (PGK) or glyceraldehyde-3-phosphate dehydrogenase (GPD) are usually chosen for heterologous gene expression. In this study, the GPD promoter was used for ATF1


204


gene expression. The 5' untranslated region of the ATF1 gene was deleted by ExolU endonuclease, blunted and the Pstl site in 3' untranslated region was also blunted by T4 DNA polymerase. This DNA fragment carrying the ATF1 open reading frame was ligated to the blunted HindlU site of the GPD promoter vector. The Sail fragment containing GPDp-ATFl was inserted into Sail site of pYT71. Laboratory scale fermentation experiments were carried out by the procedure described above. As shown in Table HI, the transformant carrying GAPp-ATFl on a single copy plasmid produced much greater levels of ethyl acetate and isoamyl acetate than ATF1 on the multicopy plasmid.


Conclusion and Discussion The S. cerevisiae AATase was purified and its internal peptide sequences were determined. ATF1 genes encoding the AATase were cloned from the bottom fermenting yeast S. pastorianus. The two genes isolated have different structures. One is very similar to the K1-ATF1 gene cloned from S. cerevisiae and the other, LgATF1, has less homology with ATFL Transformants carrying the ATF1 or the LgATF1 on a multicopy plasmid produced greater levels of acetate esters than the parental strain in laboratory scale fermentations. ATF1 and Lg-ATFl are present on two chromosomes of different molecular weight. ATF1 strongly hybridized to the X V chromosome of S. cerevisiae and two chromosomes in S. pastorianus, but not to S. bayanus. Lg-ATFl strongly hybridized to the 850-kb chromosome of S. bayanus and S. pastorianus, but not to S. cerevisiae. The bottom fermenting yeast S. pastorianus is thought to be a natural hybrid between S. cerevisiae and S. bayanus (10-11). Therefore, our results suggest that ATF1 might be derived from S. cerevisiae and Lg-ATFl might be derived from S. bayanus. Transformants carrying the disrupted atfl gene were constructed by a one step gene disruption method. As brewer's yeasts are not haploid, dominant marker genes are essential to select transformants. In this study, the G-418 resistance gene and blasticidine S resistance gene were used for gene disruption. G-418 (geneticine) is a standard reagent in yeast genetics. However, there are no reports describing the use of Blasticidine S in yeast transformation. The blasticidine resistance gene was placed downstream of the phospho-glycerol kinase promoter (PGKp) and this resistance gene allowed yeast cell growth on the medium containing 100 ug/mL of blasticidine S (Tamai, Y., in preparation). Double disruption of atfl in brewing yeast could be obtained by the use of these two resistance genes. ATF1 gene expression was monitored by measuring the B-galactosidase activity produced from the ATFlr.lacZ fusion gene. Interestingly, ATF1 was repressed by aeration or the addition of unsaturated fatty acids to the medium. ATF1 expression became constitutive by the replacement of the native promoter with the constitutive GPD promoter. These results suggested that ATF1 was regulated at the transcriptional level. Unsaturated fatty acids has been reported as repressing the OLE1 gene encoding A9-desaturase at the transcriptional level (12-13). However, the mechanism of this regulation has not been elucidated. Oxygen regulates the expression of many genes (14). It induces genes involved in respiration and represses genes involved in anaerobic fermentation. It will be interesting to know whether ATF1 is the oxygen regulated genes or whether it is regulated by the higher concentration of unsaturated fatty acids synthesized in aerobic culture. High temperature fermentation or high gravity fermentation produce greater levels of esters and high pressure in tall cylinder conical tanks reduces the ester formation in beer fermentation. Control of the ATF1 gene expression will provide a means of maintaining beer flavor characteristics in the different brewing processes. Takeoka et al.; Biotechnology for Improved Foods and Flavors ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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Alcohol Acetyl Transferase Genes in Brewer's Yeast

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Table III. Volatile esters and alcohols concentrations produced by the yeast carrying GPDp-ATFl in the laboratory scale fermentation Culture condition


Plasmids

Isoamyl acetate (ppm)

Ethyl acetate (ppm)



isoamyl alcohol (ppm)

pYT71

w/o Oleic acid

0.67

7.4

14.4

21.9

77.8

pYT71

+01eic acid

0.18

5.0

17.8

15.1

77.8

GPDp-ATFl on pYT71

w/o Oleic acid

9.24

68.4

15.3

12.8

69.4

GPDp-ATFl on pYT71

+01eic acid

2.16

14.5

10.7

10.0

47.6

Acknowledgement Research and cloning of ATF1 was carried out in collaboration with Ozeki Co., Ltd. I wish to express sincere thanks to Mr. Masaaki Hamachi and his colleagues at Ozeki Research Laboratories for their cooperation in this research and I would like to thank Dr. Reisuke Takahashi of KIRIN Brewery Co., Ltd. for permission to publish this work. I would also like to express my appreciation to Dr. Hiroyuki Yoshimoto for valuable discussion, and Miss Keiko Kanai and Mrs. Ritsuko Katoh for technical assistance. Literature Cited 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12. 13. 14.

Nodstrom, K. J. Inst. Brewing 1961, 67, 173-181. Nodstrom, K. J. Inst. Brewing 1962, 68, 398-407. Nodstrom, K. J. Inst. Brewing 1963, 69, 142-153. Yoshioka, K.; Hashimoto, N. Agric. Biol. Chem. 1981, 45, 2183-2190. Stukey, J. E . ; McDonough, V . M . ; Martin, C. E . J. Biol. Chem. 1989, 264, 16537-16544. Yoshioka, K.; Hashimoto, N. Agric. Biol. Chem. 1983, 47, 2287-2294. Inoue, T.; Tanaka, J.; Mitsui, S. Recent Advances in Japanese Brewing Technology; Japanese Technology Reviews; Gordon and Breach Science Publisher: Reading, U.K., 1992; vol. 2, 38-39. Minetoki, T.; Bogaki, T.; Iwamatsu, A . ; Fujii, T.; Hamachi, M . Biosci. Biotech. Biochem. 1993, 57, 2094-2098. Fujii, T.; Nagasawa, N.; Iwamatsu, A.; Bogaki, T.; Tamai, Y.; Hamachi, M . Appl. Envir. Microbiol. 1994, 60, 2786-2792. Vaughan-Martini, A.; Martini, A. Antonie van Leeuwenhoek 1987, 53, 77-84. Vaughan-Martini, A. Syntem. Appl. Microbiol. 1989, 12, 179-182. Bossie, M . A.; Martin, C. E . J. Bact. 1989, 171, 6409-6413. McDonough, V . M . ; Stukey, J. E.; Martin, C. E . J. Biol. Chem. 1992, 267, 5931-5936. Zitomer, R. S.; Lowry, C. V . Microbiol. Rev. 1992, 56, 1-11.


Alcohol Acetyl Transferase Genes and Ester Formation in Brewer's Yeast

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