- Email: [email protected]
Cloning and expression of the gene encoding α-acetolactate decarboxylase from Acetobacter aceti ssp. xylinum in brewer's yeast
journal of blotechnology ELSEVIER
Journal of Biotechnology32 (1994) 165-171
Cloning and expression of the gene encoding a-acetolactate decarboxylase from Acetobacter aceti ssp. xylinum in brewer's yeast Shigeyuki Yamano *'a, Junichi Tanaka a, Takashi Inoue b " Central Laboratory of Key Technology, lO'rinBrewery Co., Ltd., 1-13-5, Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa 236, Japan b Beer Division, Kirin Brewery Co., Ltd., Yokohama, Japan
(Received 18 January 1993; revision accepted 17 April 1993)
Abstract Acetobacter aceti ssp. xylinum genomic library was constructed using cosmid pJB8 in Escherichia coli. The gene encoding a-acetolactate decarboxylase (ALDC) was isolated from the library by direct measurement of ALDC activity. The ALDC gene was expressed by its own promoter in E. coli. The nucleotide sequence was determined, and an open reading frame which may encode a protein composed of 304 amino acids with a molecular weight of 33,747 was found. A brewer's yeast was transformed with the YEp-type plasmid containing the ALDC gene placed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The laboratory-scale growth test confirmed that the total diacetyl concentration was considerably reduced by the transformant. The analysis of the wort indicates that the Acetobacter ALDC reduces the concentration of diacetyl more effectively than that of 2,3-pentanedione. Key words: a-Acetolactate decarboxylase; Acetobacter aceti ssp. xylinum; Cloning; Brewer's yeast
I. Introduction
'Diacetyl-flavor' is one of the most common off-flavors in beer. During main fermentation, brewer's yeast produces a-acetohydroxyacids ( a acetolactate and a - a c e t o h y d r o x y b u t y r a t e ) w h i c h are intermediates of the isoleucine-valine synthetic pathway, a-Acetohydroxyacids are leaked into the fermented wort and converted to diacetyl
* Corresponding author.
(DA) and 2,3-pentanedione by slow nonenzymatic oxidative decarboxylation (Lewis and Weinhouse, 1958). Subsequently, D A and 2,3-pentanedione are converted to acetoin and 3-hydroxy2-pentanone by diacetyl reductase of yeast cells during maturation period of several weeks. Since maturation is the most time-consuming step, acceleration of this step is economically important to the brewing industry. For this purpose, A L D C is an enzyme that converts a-acetolactate directly to acetoin and a-acetohydroxybutyrate to 3-hydroxy-2-pentanone which have no effect on the
0168-1656/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0168-1656(93)E0027-U
166
s. Yamanoet al./Journal of Biotechnology 32 (1994)165-171
flavor of beer. This enzyme is found in many bacteria but not in yeasts (Godtfredsen et al., 1983). Recently, Sone et al. (1987) cloned the ALDC gene from Enterobacter aerogenes and expressed this gene in yeast to reduce the DA content of fermented wort (Sone et al., 1988). Growth tests showed that DA production of the transformants was considerably lower than that of the parent yeast. Gene engineering is an effective technique for improving brewer's yeast, but not fully accepted in public opinion. Therefore, microorganisms used for food making may be desirable as the donors of genes. In this report, we describe the cloning of the ALDC gene from Acetobacter aceti subsp, xylinum, formerly used for vinegar making, and the expression of that gene in a brewer's yeast.
for the selection of cells having /3-galactosidase activity.
Plasmids E. coli cosmid vector pJB8 (Ish-Horowicz and Burke, 1981) was used for construction of cosmid library. E. coli plasmid vectors pUC12, pUC18 and pUC19 (Yanisch-Perron et al., 1985) were used for plasmid construction. Yeast-E. coli shuttle vector YEpl3K (Sone et al., 1988) was used to construct the expression plasmid of the ALDC gene in a brewer's yeast.
2. Materials and methods
DNA manipulations The procedures for cleavage of DNA by restriction endonuclease, ligation, transformation of E. coli, transduction, plasmid preparation and gel electrophoresis were described by Maniatis et al. (1982). In vitro packaging was carried out as suggested by the supplier (Amersham-Japan, Tokyo, Japan).
Microorganisms and media Acetobacter strains were obtained from the Institute for Fermentation (IFO), Osaka (Japan). Escherichia coli DH1 (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1) was used for construction of the cosmid library. E. coli JM109 [recA1 enclA1 gyrA96 thi-1 hsdR17 supE44 reIA1 A(lac-proAB) / F' (traD36 proAB laclqZAM15)] was used for plasmid construction and sequencing. Acetobacter strains were grown in YPD medium (1% yeast extract, 2% polypeptone and 2% glucose) overnight at 30°C with shaking. E. coli strains were grown in LB medium (0.5% yeast extract, 1% tryptone and 1% NaCI) overnight at 37°C with shaking. Ampicillin (50/zg m1-1) was added to the medium for selection of transformants. Saccharomyces cerevisiae $288C (a SUC2 real gal2 CUP1; ATCC 26108) was used as a donor for genomic library. Brewer's yeast K1084 was from our stock collection and used as a parent strain. These strains were grown in YPD medium (1% yeast extract, 2% polypeptone and 2% glucose) overnight at 30°C with shaking. M63 medium (Guarente, 1983) containing X-gal (5-bromo-4chloro-3-indolyl-/3-D-galactopyranoside) was used
Isolation of chromosomal DNA and construction of the cosmid library A. aceti ssp. xylinum IFO 3288 was grown in YPD medium overnight at 30°C. After harvesting, the cells were lysed by incubation in 50 mM Tris-HCl (pH 8.0), 50 mM EDTA, 1 mg m1-1 lysozyme at 37°C for 20 min followed by the addition of 0.5% SDS, 50/zg m1-1 proteinase K and incubation at 65°C for 30 min. The lysate was extracted twice with phenol and once with phen o l / chloroform. After precipitation with an equal volume of ethanol, DNA was dissolved in TE buffer (10 mM Tris-HC1 (pH 8.0), 1 mM EDTA). After addition of 100 /xg m1-1 RNase A and incubation at 37°C for 60 min, the DNA solution was extracted once with phenol and once with phenol/chloroform. After precipitation with 2 vols. of ethanol, DNA was dissolved in TE buffer and dialyzed against buffer. Chromosomal DNA was partially digested with HindIII. DNA fragments of approx. 40 kb were purified by sucrose density gradient centrifugation and ligated to pJB8 digested with HindIII. The ligated DNA was packaged in vitro and transduced into E. coli DH1. Ampicillin-resistant transductants were ob-
S. Yamano et al. /Journal of Biotechnology 32 (1994) 165-171
tained at a frequency of 5.0 × 103 per /zg of chromosomal DNA.
Assay of A L D C Acetobacter strains were grown in YPD medium overnight at 30°C with shaking. The cells were harvested from 1 ml of culture by centrifugation and resuspended in 500 /zl of 30 mM potassium phosphate buffer (pH 6.2). Ten /~1 of toluene was added and cells were permealized by vortex mixing at high speed for 30 s. ALDC activity was measured as described by l_Oken and Stcmer (1970). E. coli was grown in LB medium supplemented with 50 /xg m1-1 ampicillin overnight at 37°C with shaking. The ceils were harvested from 5 ml of culture by centrifugation and resuspended in 200/xl of 30 mM potassium phosphate buffer (pH 6.2). Permealization of the cells and assay of ALDC activity were carried out in the same way as Acetobacter strains. ALDC assay in yeast was carried out as described previously (Sone et al., 1988). Isolation of yeast promoters and terminator Yeast genes were cloned from the EMBL3 phage library of $288C using oligonucleotide probes synthesized on the basis of the published DNA sequence. The glyceraldehyde-3-phosphate dehydrogenase gene (GPD; Holland and Holland, 1979) was obtained as the 2.1 kb HindlII fragment and subcloned into HindlII cleaved pUC18. The region spanning coding sequence of the GPD gene and the 5' noncoding sequence up to - 1 5 bp of initiation codon was eliminated by serial digestion with Exonuclease III to isolate the GPD promoter. After ligating HindlII linkers to the 3' end of the GPD promoter, the 1.0-kb HindlII fragment containing the GPD promoter was cloned into HindlII-cleaved pUC18 to construct pST18. The multi-cloning sites were located at the 5' end of the GPD promoter in pST18. The HindlII site at the 5' end of the GPD promoter was filled with Klenow fragment to construct pST18H. The phosphoglycerate kinase gene (PGK; Hitzeman et al., 1982) was obtained as the 2.9 kb HindIII fragment and subcloned into HindIIIcleaved pUC18 to construct pST2. The PGK ter-
167
minator was isolated as the 0.38 kb BgllI-HindlII fragment from pST2, and subcloned into BamHI +HindlII-cleaved pUC12. The HindlII site of this plasmid was converted to Sail site by treatment with Klenow fragment followed by ligating Sail linkers. The resultant plasmid containing the PGK terminator as the 0.38 kb SacI-SalI fragment was designated pSY104S.
Selection markers for brewer's yeast and transformation The E. coli lacZ gene, which lacks its promoter and the first eight N-terminal amino acids, was isolated as the 3.0 kb BamHI fragment from pMC1871 (Pharmacia) and inserted into the BgllI site of YEpl3K. The 1.5 kb BamHI-HindlII fragment containing the ADC1 promoter was inserted into this plasmid cleaved with BamHI + HindlII to adjust the reading frame. The resultant ADCI-lacZ fusion gene was isolated as the 4.5 kb B a m H I - X h o I fragment. The G418 (Geneticin)-resistant gene was isolated as the 1.7 kb SalI-BamHI fragment by partial Sail digestion and BamHI digestion of pUC4K (Pharmacia). Transformation of brewer's yeast was carried out by the method of Sone et al. (1988) Laboratory-scale growth test Laboratory-scale growth tests were carried out as described previously (Sone et al., 1988).
3. Results
Cloning of the A L D C gene ALDC activity of several Acetobacter strains isolated from vinegar was measured (Table 1). A. aceti ssp. xylinum IF03288 possessed the highest ALDC activity and was selected as a source of the A L D C gene. The cosmid library was constructed from the chromosomal DNA of A. aceti ssp. xylinum IF03288. Transductants were examined for the presence of the ALDC gene by directly measuring enzyme activity of permeabilized cell suspension. Among 600 transductants, two ALDC-positive clones were obtained. Restriction enzyme analysis
168
S. Yamano et al. /Journal of Biotechnology 32 (1994) 165-171
Table 1 ALDC activity of Acetobacter strains Strains
Acetobacter aceti ssp. xylinum IFO IFO IFO Acetobacter aceti ssp. aceti IFO IFO Acetobacter rancens IFO
ALDC activity (U m l - 1) 3288 13693 13773 3283 3284 3298
0.45 0.19 0.16 0.01 0.01 0.05
showed that two plasmids isolated from these clones contained several common fragments. These plasmids were designated pAX1 and pAX2. pAX1 was used in further study. pAX1 was digested partially with Sau3AI. Resultant fragments of about 3 kb were subcloned into pUC12 digested with BamHI. Several clones having ALDC activity were obtained. One of the plasmids isolated from these clones was designated pAXSll, pAXSll contained an insert fragment of about 3.6 kb. To identify the coding region of the ALDC on this fragment, subcloning experiments were carried out as shown in Fig. 1. Transformants with pAC2, pAX5, pAX6, pAX6R or pAX8 had ALDC activity, indicating that the ALDC gene locates in the 1.2 kb KpnI-EcoRI fragment and that the ALDC gene was expressed in E. coli by its own promoter. Southern blot analysis of the chromosomal DNA from A. aceti ssp. xylinum was performed (data not shown). The 1.2 kb KpnI-SacI fragment from pAX6 was used as a probe. There was a single hybridization fragment for each digestion with BamHI, BstEII, BamHI + BstEII and SphI as expected. It was confirmed that the ALDC gene derived from A. aceti ssp. xylinum IF03288 and that the copy number of the ALDC gene is one.
DNA sequencing and analysis As described above, it was confirmed that the ALDC gene was located in the 1.2 kb KpnIEcoRI fragment. The nucleotide sequence of this fragment was determined. A large open reading frame (ORF) of 304 amino acids was found (Fig. 2). Several possible initiation codons were found in the ORF. Plasmid pAX31 was constructed by
inserting the 1011-bp RsaI-SmaI fragment of pAX6 into the Sinai site of pUC19 with the same transcriptional direction of lac promoter, pAX31 retained higher ALDC activity than that of pAX6 in E. coli. It is not yet clear which is the initiation codon of the ALDC gene.
Construction of the expression plasmid for brewer's yeast and transformation The 0.38 kb SacI-SalI fragment containing the PGK terminator from pSY104S was cloned into YEp13K cleaved with SacI + SalI to construct YEp13K-PGKt. The ALDC gene whose translation-start point was considered to be at bp 70 in Fig. 2 was isolated as the RsaI-SacI fragment from pAX31. After treating the HindIII site of pST18H with Exonuclease III, the GPD promoter was isolated as the 1.0 kb B a m H I (HindIII) fragment. The GPD promoter and the
. , ~ I kb
Plasmid (E,Sa,Sm) i
(P) i
(E) = (sin) i
(sa)i
i
ALDC activity
pAXSt 1
+
pAC2
+
pAX8
+
pAX5
+
pAX6,6R
+
pAX4 (Sm)i
pAXt0
Fig. 1. Restriction map and deletion analysis of pAXSll. p A X S l l was digested with Ncol and SrnaI followed by treatment with Klenow fragment to create two fragments. The 0.8 kb fragment was ligated with pUC12 digested with SmaI to construct the plasmid pAX10, pAX4 was constructed by selfligation of the other 5.5 kb fragment, pAX5 was obtained by ligating p A X S l l digested with PstI. pAX6 was obtained by insertion of the 1.2 kb KpnI-SacI fragment into pUC19 digested with K.pnI and SacI. pAX6R contained the same insert as pAX6 in pUC18. The fragments in pAX6 and pAX6R were differently oriented with respect to the lac promoter present on the vector, pAX8 was obtained by insertion of the 1.7 kb SphI-EcoRI fragment into pUC19 digested with SphI and EcoRI. pAC2 was obtained by insertion of the 2.2 kb BamHI-EcoRI fragment into pUC12. Restriction site: B, BamHI; Bs, BstEII; E, EcoRI; H, HindlII; K, KpnI; N, NcoI; P, PstI; Sa, SacI; Sin, Sinai; Sp, SphI. Restriction sites in parentheses are derived from vectors.
S. Yamano et al. /Journal of Biotechnology 32 (1994) 165-171
169
GGTACCGGGACCATAGGGGGGCTTGGGGTCG l II I B a m HI
¢TTTCGGCATGGGCGCGCTGCTGCCCGCGATCAGGCCGCCCATGCAGAAGGCGGCGAGCG TCATGTAAAGTGGCTTCATATCCGGTCATCCTG~TTTCAATGCGCGTTCACAATGGCAG
Indlll)/Rsal
TCCTGAACGGCCAAGGGCAAGGCCAGGGCCTGCACGGCGGCATTTCCATATATTTTATAT
Jail.
ATGGAAATAGGCTTTAATATATATTGGACGTACGAACCTGCCTGCATCACCATTAGTCTG H £ I G F l I Y W T Y E P A C I T I S L
il
¢lll/Xho I
CAATCACAAATGACCGGGTTGAnAeGATGCCATGTGCCGCATTGTCCCCCGATGCAULG 120 O S Q M T G L R R C H V P H ¢
P P M OE
ACTGAGGTCGTGAAGCTTAAATGCTACTCGGTAGGGGATGTTGATACCCGGTCCAGCGCT T E V V i L [ C Y S Y G 0 V 0 T R S S A
Z40
GCTGATTCGACTGGCGTGCGTCCGCGCATGAACCGCCTGTACCAGACATCGACCATGGCC 300 k
0
S T G V i
P R M I
R L Y O T S T M k
GCGCTGCTTGATGCGGTCTATGATGGCGAGACCACGCTTGATGAACTGCTGATGCACGG¢ 360 A L L O k V Y 0 G E T T L D E L L H H G AATTTCGGGCTGGGCACGTTCAACGGCCTTGATGGCGAGATGATCGTCAATGACAGCGTA 420 H F G L G T
F H 6
L O G E M I
V H 0
S V
ATCCACCAGTTCCGTGCAGACGGGCAGGCCGGTCGTGTGCCGGGCGACCTCAGGACTCC6 410 I
H 0
F N k
D G O k
G R V
P G D L R T P
TTCGCCTGCGTTACCTTCTTCAACCCGGAGAAGGAATACATGATC~CACCGCGCAGGAT 540 F A C V
T F
F H P E K E Y M I
D T A O O
S
AAGGAAGGCTTCGAGGCGATCGTGGATCACCTCGTCAACAATCCCAACCTGTTCGCCGCC |00 K E G F
E k
V 0
H L V N N P N L F k
GTGCGCITTACCGGCATGTTCGAGCGGGTCGAGACCCGCACCGTGTTCTGCCAGTGCCAG i|0 V R F
T 6 M
E R V
E T R T V
F ¢
a
~
\BamHI
k
O C 0
CCCTACCCACCCATGCTGGAAGTGGTGGC¢CG¢¢AGCCCA¢CATGCA6CTTGGTG¢CTCC 720 P Y P P M L V V k R 0 P T M O L G A S
Fig. 3. Construction of the expression plasmid pAX43GL for brewer's yeast, pBR322 sequence is represented by the thin line. GPDp, GPD promoter; PGKt, PGK terminator; G418 r, G418-resistance gene.
ACCGGCACCATGCTTGGTTTCCGCACGCCGGGCTACAIGCAGGGCGTGAACGTGGCGGGT 710 T G T M L G R T P G Y M 0 G V H V A G TATCACCTGCACTTCCTGACTGAGGACGGACGCCGTGGCGGCCATGTGACCGATTACGGC 140 Y H L H F L E O G R R G G H V T D Y G GTGCTGCGCGGTCGGCTTGAGGTGGGCGTGATTTCCGATGTGGAAATCCAGCTGCCCCG¢ $0| V L R G R L V G V f S O V E I 0 L P R ACCGAACAGTTCGCGCGCGCCAACCTGTCCCCCGAAAACATTCACGAGGCCATTCGCGTG 160 T E O F A R A H L $ P E i I H [ A I | V GCCGAGGGCGGCTGAGGGTTTCCCCTCCCGCCTGAGCAACTGTCCGCTCCGCCCCGGCTG1120 AEGGt CGGTGCCAACCGTTCAGGATACCTGAAATCATGACTGACAAGACCAAATCGGCCGCGCCG ~aal
GAGTGCGGGGCGGACATGATCCCCGGGCGAGCTC6AATTC
Fig. 2. Nucleotide sequence and deduced amino acid sequence of the ALDC gene of A. ace~ ssp. ~linum. The putatwe ribosome-binding sites are underlined. * Shows s ~ p codon.
ALDC gene were cloned into YEp13K-PGKt cleaved with BgllI + SacI. The resultant plasmid was designated pAX43B. The 1.7 kb SalI-BamHI fragment containing the G418-resistance gene and the 4.5 kb BamHI-XhoI fragment containing the ADCI-lacZ fusion gene were inserted into the SalI site of pAX43B to construct pAX43GL (Fig. 3). Brewer's yeast KI084 was transformed with pAX43GL. Cells containing pAX43GL made blue colonies on M63 plate containing X-gal. The rate of the clones containing plasmid among G418-re-
Table 2 Analysis of wort Strain
KI084 KI084 (pAX43GL)
Diacetyl compounds a (mg 1- 1)
Apparent extract
Apparent attenuation b
DA
PD
TDA
(Plato)
(%)
0.67 0.12
0.39 0.16
1.00 0.26
2.85 2.84
75.3 75.4
a DA, Diacetyi; PD, 2,3-pentanedione; TDA, total diacetyl (diacetyl + 2,3-pentanedione × 0.86). b [(Extract content of wort-apparent extract of beer)/extract content of wort] × 100.
Ethanol (%, v V- 1)
4.61 4.61
170
S. Yamano et al. /Journal of Biotechnology 32 (1994) 165-171
sistant clones was not constant (50 ~ 80%). ALDC activity of the transformants was 4 to 5 U mg-1 protein.
Laboratory-scale growth test Analysis of fermented wort is shown in Table 2. The total DA in the wort with the transformant was reduced to one-fourth of that with the parent yeast. There was no substantial difference in process performances except total DA formation of both strains. Expression of the ALDC gene did not affect the growth of brewer's yeast. The content of diacetyl was reduced more effectively than that of 2,3-pentanedione in the fermented wort. This means that the ALDC from A. aceti ssp. xylinurn decreased a-acetolactate more effic.~ently than a-acetohydroxybutyrate in yeast, pAX43GL in brewer's yeast KI084 was maintained stably under unselective conditions. 96% of cells contained pAX43GL at the end of the growth test. 4. Discussion
The ALDC genes from Enterobacter aerogenes (Sone et al., 1988) and Bacillus brevis (Diderichsen et al., 1990) were already cloned. The deduced amino acid sequence of the ALDC gene from A. aceti ssp. xylinum was compared with those of other ALDCs. There was a 45.7% match of amino acids in 230 overlap residues with E. aerogenes ALDC, and a 35.6% match in 247 overlap residues with B. brevis ALDC. Many homologous stretches of amino acid were distributed over the sequences except their Ntermini. The highest homologous region was Glyl°a-Ile 115, whose homology was 85% with the three ALDCs. It was therefore reasonable to assume that this region is very important for ALDC activity. E. aerogenes ALDC of 260 amino acids was the same size as B. brevis ALDC. It seems that only A. aceti ssp. xylinum ALDC has an extra long N-terminal sequence of approx. 40 amino acids. If the GTG codon at bp 130 is the real initiation codon, A. aceti ssp. xylinum ALDC has almost the same size as other ALDCs. Consensus sequences for promoters of Acetobacter strains have been unknown. A typical ribosome-binding
site was not found close to a putative ATG initiation codon at bp 1. On the other hand, the typical ribosome-binding site (GAGG) was located 13 bp upstream from the GTG codon at bp 94. And, there was the ribosome-binding site (AGGA) 14 bp upstream from the GTG codon at bp 130. As described in Results, the truncated ALDC gene in pAX31 retained a higher ALDC activity than that of pAX6 in E. coli. It is likely that the translation-start point of the ALDC gene is located downstream from bp 70 in Fig. 2. Since brewer's yeast cells are polyploid prototrophs, a dominant marker such as a drug-resistant gene is necessary for transformation of brewer's yeasts. Although the G418-resistant gene is frequently used as a dominant marker, it makes brewer's yeast ceils weakly resistant. In the selection medium with low G418 concentration (10 ~g ml-1), resistant clones do not always contain a plasmid. In our experiment, the G418-resistant gene and ADCI-lacZ fusion gene were used for efficient selection of transformants. In the future, the G418-resistant gene should be expressed at higher levels by a stronger promoter to increase the transformation efficiency of brewer's yeast cells. Sone et al. (1988) reported that the 2-/zm DNA-based plasmid was stable in the brewer's yeast KI084. We observed that the expression plasmid for the Acetobacter ALDC gene was also maintained stably in KI084 cells. It is possible that the endogenous 2-/~m DNA in the brewer's yeast KI084 increases the stability of the heterologous 2-/zm DNA-based plasmid that contains only a portion of the 2-~m DNA genome. The substrate specificity of bacterial ALDCs has been little investigated. Since the ALDC from A. aceti ssp. xylinum had not yet been studied, its substrate specificity was unknown. We observed that this ALDC decreased a-acetolactate more efficiently than a-acetohydroxybutyrate in yeast. This substrate specificity is similar to that of the ALDC from Klebsiella terrigena (Suihko et al., 1990) Use of the ALDC from A. aceti ssp. xylinum for brewing has not yet been reported. Acceptance in public opinion is important for the use of genetically improved microorganisms
S. Yamano et al. /Journal of Biotechnology 32 (1994) 165-171
in the food industry. From this point of view, cloning of genes from microorganisms for food making may increase in the future. We cloned the ALDC gene from A. aceti ssp. xylinum and confirmed that this ALDC gene was useful in reducing total DA concentration in wort.
5. Acknowledgements We are grateful to Toshihiko Ohno, Fumio Shimizu and Hidetaka Sone for their valuable suggestions, and to Naomi Odagawa for her technical assistance.
6. References Diderichsen, B., Wedsted, U., Hedegaard, L., Jensen, B.R. and Sj~holm, C. (1990) Cloning of aldB, which encodes a-acetolactate decarboxylase, an exoenzyme from Bacillus brevis. J. Bacteriol. 172, 4315-4321. Guarente, L. (1983) Yeast promoters and LacZ fusions designated to study expression of cloned genes in yeast. Methods Enzymol. 101, 181-191. Godtfredsen, S.E., Lorck, H. and Sigsgaard, P. (1983) On the occurrence of ct-acetolactate decarboxylases among microorganisms. Carlsberg Res. Commun. 48, 239-247.
171
Hitzeman, R.A., Hagie, F.E., Hayflick, J.S., Chen, C.Y., Seeburg, P.H. and Derynck, R. (1982) The primary structure of the Saccharomyces cerevisiae gene for 3-phosphoglycerate kinase. Nucleic Acids Res. 10, 7791-7808. Holland, J.P. and Holland, M.J. (1979) The primary structure of a glyceraldehyde-3-phosphate dehydrogenase gene from Saccharomyces cerevisiae. J. Biol. Chem. 254, 9839-9845. Ish-Horowicz, D. and Burke, J.F. (1981) Rapid and efficient cosmid vector cloning. Nucleic Acids Res. 9, 2989-2998. Lewis, K.F. and Weinhouse, S. (1958) Studies in valine biosynthesis. II. a-Acetolactate formation in microorganisms. J. Am. Chem. Soc. 80, 4913-4915. LOken, J.P. and St~rmer, F.C. (1970) Acetolactate decarboxylases from Aerobacter aerogenes. Eur. J. Biochem. 14, 133-137. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Sone, H., Fujii, T., Kondo, K. and Tanaka, J. (1987) Molecular cloning of the gene encoding a-acetolactate decarboxylase from Enterobacter aerogenes. J. Biotechnol. 5, 87-91. Sone, H., Fujii, T., Kondo, K., Shimizu, F., Tanaka, J. and Inoue, T. (1988) Nucleotide sequence and expression of the Enterobacter aerogenes o~-acetolactate decarboxylase gene in brewer's yeast. Appl. Environ. Microbiol. 54, 3842. Suihko, M.L., Blomqvist, K., Penttilii, M., Gisler, R. and Knowles, J (1990) Recombinant brewer's yeast strains suitable for accelerated brewing. J. Biotechnol. 14, 285-300. Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33, 103-119.
Journal of Biotechnology32 (1994) 165-171
Cloning and expression of the gene encoding a-acetolactate decarboxylase from Acetobacter aceti ssp. xylinum in brewer's yeast Shigeyuki Yamano *'a, Junichi Tanaka a, Takashi Inoue b " Central Laboratory of Key Technology, lO'rinBrewery Co., Ltd., 1-13-5, Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa 236, Japan b Beer Division, Kirin Brewery Co., Ltd., Yokohama, Japan
(Received 18 January 1993; revision accepted 17 April 1993)
Abstract Acetobacter aceti ssp. xylinum genomic library was constructed using cosmid pJB8 in Escherichia coli. The gene encoding a-acetolactate decarboxylase (ALDC) was isolated from the library by direct measurement of ALDC activity. The ALDC gene was expressed by its own promoter in E. coli. The nucleotide sequence was determined, and an open reading frame which may encode a protein composed of 304 amino acids with a molecular weight of 33,747 was found. A brewer's yeast was transformed with the YEp-type plasmid containing the ALDC gene placed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The laboratory-scale growth test confirmed that the total diacetyl concentration was considerably reduced by the transformant. The analysis of the wort indicates that the Acetobacter ALDC reduces the concentration of diacetyl more effectively than that of 2,3-pentanedione. Key words: a-Acetolactate decarboxylase; Acetobacter aceti ssp. xylinum; Cloning; Brewer's yeast
I. Introduction
'Diacetyl-flavor' is one of the most common off-flavors in beer. During main fermentation, brewer's yeast produces a-acetohydroxyacids ( a acetolactate and a - a c e t o h y d r o x y b u t y r a t e ) w h i c h are intermediates of the isoleucine-valine synthetic pathway, a-Acetohydroxyacids are leaked into the fermented wort and converted to diacetyl
* Corresponding author.
(DA) and 2,3-pentanedione by slow nonenzymatic oxidative decarboxylation (Lewis and Weinhouse, 1958). Subsequently, D A and 2,3-pentanedione are converted to acetoin and 3-hydroxy2-pentanone by diacetyl reductase of yeast cells during maturation period of several weeks. Since maturation is the most time-consuming step, acceleration of this step is economically important to the brewing industry. For this purpose, A L D C is an enzyme that converts a-acetolactate directly to acetoin and a-acetohydroxybutyrate to 3-hydroxy-2-pentanone which have no effect on the
0168-1656/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0168-1656(93)E0027-U
166
s. Yamanoet al./Journal of Biotechnology 32 (1994)165-171
flavor of beer. This enzyme is found in many bacteria but not in yeasts (Godtfredsen et al., 1983). Recently, Sone et al. (1987) cloned the ALDC gene from Enterobacter aerogenes and expressed this gene in yeast to reduce the DA content of fermented wort (Sone et al., 1988). Growth tests showed that DA production of the transformants was considerably lower than that of the parent yeast. Gene engineering is an effective technique for improving brewer's yeast, but not fully accepted in public opinion. Therefore, microorganisms used for food making may be desirable as the donors of genes. In this report, we describe the cloning of the ALDC gene from Acetobacter aceti subsp, xylinum, formerly used for vinegar making, and the expression of that gene in a brewer's yeast.
for the selection of cells having /3-galactosidase activity.
Plasmids E. coli cosmid vector pJB8 (Ish-Horowicz and Burke, 1981) was used for construction of cosmid library. E. coli plasmid vectors pUC12, pUC18 and pUC19 (Yanisch-Perron et al., 1985) were used for plasmid construction. Yeast-E. coli shuttle vector YEpl3K (Sone et al., 1988) was used to construct the expression plasmid of the ALDC gene in a brewer's yeast.
2. Materials and methods
DNA manipulations The procedures for cleavage of DNA by restriction endonuclease, ligation, transformation of E. coli, transduction, plasmid preparation and gel electrophoresis were described by Maniatis et al. (1982). In vitro packaging was carried out as suggested by the supplier (Amersham-Japan, Tokyo, Japan).
Microorganisms and media Acetobacter strains were obtained from the Institute for Fermentation (IFO), Osaka (Japan). Escherichia coli DH1 (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1) was used for construction of the cosmid library. E. coli JM109 [recA1 enclA1 gyrA96 thi-1 hsdR17 supE44 reIA1 A(lac-proAB) / F' (traD36 proAB laclqZAM15)] was used for plasmid construction and sequencing. Acetobacter strains were grown in YPD medium (1% yeast extract, 2% polypeptone and 2% glucose) overnight at 30°C with shaking. E. coli strains were grown in LB medium (0.5% yeast extract, 1% tryptone and 1% NaCI) overnight at 37°C with shaking. Ampicillin (50/zg m1-1) was added to the medium for selection of transformants. Saccharomyces cerevisiae $288C (a SUC2 real gal2 CUP1; ATCC 26108) was used as a donor for genomic library. Brewer's yeast K1084 was from our stock collection and used as a parent strain. These strains were grown in YPD medium (1% yeast extract, 2% polypeptone and 2% glucose) overnight at 30°C with shaking. M63 medium (Guarente, 1983) containing X-gal (5-bromo-4chloro-3-indolyl-/3-D-galactopyranoside) was used
Isolation of chromosomal DNA and construction of the cosmid library A. aceti ssp. xylinum IFO 3288 was grown in YPD medium overnight at 30°C. After harvesting, the cells were lysed by incubation in 50 mM Tris-HCl (pH 8.0), 50 mM EDTA, 1 mg m1-1 lysozyme at 37°C for 20 min followed by the addition of 0.5% SDS, 50/zg m1-1 proteinase K and incubation at 65°C for 30 min. The lysate was extracted twice with phenol and once with phen o l / chloroform. After precipitation with an equal volume of ethanol, DNA was dissolved in TE buffer (10 mM Tris-HC1 (pH 8.0), 1 mM EDTA). After addition of 100 /xg m1-1 RNase A and incubation at 37°C for 60 min, the DNA solution was extracted once with phenol and once with phenol/chloroform. After precipitation with 2 vols. of ethanol, DNA was dissolved in TE buffer and dialyzed against buffer. Chromosomal DNA was partially digested with HindIII. DNA fragments of approx. 40 kb were purified by sucrose density gradient centrifugation and ligated to pJB8 digested with HindIII. The ligated DNA was packaged in vitro and transduced into E. coli DH1. Ampicillin-resistant transductants were ob-
S. Yamano et al. /Journal of Biotechnology 32 (1994) 165-171
tained at a frequency of 5.0 × 103 per /zg of chromosomal DNA.
Assay of A L D C Acetobacter strains were grown in YPD medium overnight at 30°C with shaking. The cells were harvested from 1 ml of culture by centrifugation and resuspended in 500 /zl of 30 mM potassium phosphate buffer (pH 6.2). Ten /~1 of toluene was added and cells were permealized by vortex mixing at high speed for 30 s. ALDC activity was measured as described by l_Oken and Stcmer (1970). E. coli was grown in LB medium supplemented with 50 /xg m1-1 ampicillin overnight at 37°C with shaking. The ceils were harvested from 5 ml of culture by centrifugation and resuspended in 200/xl of 30 mM potassium phosphate buffer (pH 6.2). Permealization of the cells and assay of ALDC activity were carried out in the same way as Acetobacter strains. ALDC assay in yeast was carried out as described previously (Sone et al., 1988). Isolation of yeast promoters and terminator Yeast genes were cloned from the EMBL3 phage library of $288C using oligonucleotide probes synthesized on the basis of the published DNA sequence. The glyceraldehyde-3-phosphate dehydrogenase gene (GPD; Holland and Holland, 1979) was obtained as the 2.1 kb HindlII fragment and subcloned into HindlII cleaved pUC18. The region spanning coding sequence of the GPD gene and the 5' noncoding sequence up to - 1 5 bp of initiation codon was eliminated by serial digestion with Exonuclease III to isolate the GPD promoter. After ligating HindlII linkers to the 3' end of the GPD promoter, the 1.0-kb HindlII fragment containing the GPD promoter was cloned into HindlII-cleaved pUC18 to construct pST18. The multi-cloning sites were located at the 5' end of the GPD promoter in pST18. The HindlII site at the 5' end of the GPD promoter was filled with Klenow fragment to construct pST18H. The phosphoglycerate kinase gene (PGK; Hitzeman et al., 1982) was obtained as the 2.9 kb HindIII fragment and subcloned into HindIIIcleaved pUC18 to construct pST2. The PGK ter-
167
minator was isolated as the 0.38 kb BgllI-HindlII fragment from pST2, and subcloned into BamHI +HindlII-cleaved pUC12. The HindlII site of this plasmid was converted to Sail site by treatment with Klenow fragment followed by ligating Sail linkers. The resultant plasmid containing the PGK terminator as the 0.38 kb SacI-SalI fragment was designated pSY104S.
Selection markers for brewer's yeast and transformation The E. coli lacZ gene, which lacks its promoter and the first eight N-terminal amino acids, was isolated as the 3.0 kb BamHI fragment from pMC1871 (Pharmacia) and inserted into the BgllI site of YEpl3K. The 1.5 kb BamHI-HindlII fragment containing the ADC1 promoter was inserted into this plasmid cleaved with BamHI + HindlII to adjust the reading frame. The resultant ADCI-lacZ fusion gene was isolated as the 4.5 kb B a m H I - X h o I fragment. The G418 (Geneticin)-resistant gene was isolated as the 1.7 kb SalI-BamHI fragment by partial Sail digestion and BamHI digestion of pUC4K (Pharmacia). Transformation of brewer's yeast was carried out by the method of Sone et al. (1988) Laboratory-scale growth test Laboratory-scale growth tests were carried out as described previously (Sone et al., 1988).
3. Results
Cloning of the A L D C gene ALDC activity of several Acetobacter strains isolated from vinegar was measured (Table 1). A. aceti ssp. xylinum IF03288 possessed the highest ALDC activity and was selected as a source of the A L D C gene. The cosmid library was constructed from the chromosomal DNA of A. aceti ssp. xylinum IF03288. Transductants were examined for the presence of the ALDC gene by directly measuring enzyme activity of permeabilized cell suspension. Among 600 transductants, two ALDC-positive clones were obtained. Restriction enzyme analysis
168
S. Yamano et al. /Journal of Biotechnology 32 (1994) 165-171
Table 1 ALDC activity of Acetobacter strains Strains
Acetobacter aceti ssp. xylinum IFO IFO IFO Acetobacter aceti ssp. aceti IFO IFO Acetobacter rancens IFO
ALDC activity (U m l - 1) 3288 13693 13773 3283 3284 3298
0.45 0.19 0.16 0.01 0.01 0.05
showed that two plasmids isolated from these clones contained several common fragments. These plasmids were designated pAX1 and pAX2. pAX1 was used in further study. pAX1 was digested partially with Sau3AI. Resultant fragments of about 3 kb were subcloned into pUC12 digested with BamHI. Several clones having ALDC activity were obtained. One of the plasmids isolated from these clones was designated pAXSll, pAXSll contained an insert fragment of about 3.6 kb. To identify the coding region of the ALDC on this fragment, subcloning experiments were carried out as shown in Fig. 1. Transformants with pAC2, pAX5, pAX6, pAX6R or pAX8 had ALDC activity, indicating that the ALDC gene locates in the 1.2 kb KpnI-EcoRI fragment and that the ALDC gene was expressed in E. coli by its own promoter. Southern blot analysis of the chromosomal DNA from A. aceti ssp. xylinum was performed (data not shown). The 1.2 kb KpnI-SacI fragment from pAX6 was used as a probe. There was a single hybridization fragment for each digestion with BamHI, BstEII, BamHI + BstEII and SphI as expected. It was confirmed that the ALDC gene derived from A. aceti ssp. xylinum IF03288 and that the copy number of the ALDC gene is one.
DNA sequencing and analysis As described above, it was confirmed that the ALDC gene was located in the 1.2 kb KpnIEcoRI fragment. The nucleotide sequence of this fragment was determined. A large open reading frame (ORF) of 304 amino acids was found (Fig. 2). Several possible initiation codons were found in the ORF. Plasmid pAX31 was constructed by
inserting the 1011-bp RsaI-SmaI fragment of pAX6 into the Sinai site of pUC19 with the same transcriptional direction of lac promoter, pAX31 retained higher ALDC activity than that of pAX6 in E. coli. It is not yet clear which is the initiation codon of the ALDC gene.
Construction of the expression plasmid for brewer's yeast and transformation The 0.38 kb SacI-SalI fragment containing the PGK terminator from pSY104S was cloned into YEp13K cleaved with SacI + SalI to construct YEp13K-PGKt. The ALDC gene whose translation-start point was considered to be at bp 70 in Fig. 2 was isolated as the RsaI-SacI fragment from pAX31. After treating the HindIII site of pST18H with Exonuclease III, the GPD promoter was isolated as the 1.0 kb B a m H I (HindIII) fragment. The GPD promoter and the
. , ~ I kb
Plasmid (E,Sa,Sm) i
(P) i
(E) = (sin) i
(sa)i
i
ALDC activity
pAXSt 1
+
pAC2
+
pAX8
+
pAX5
+
pAX6,6R
+
pAX4 (Sm)i
pAXt0
Fig. 1. Restriction map and deletion analysis of pAXSll. p A X S l l was digested with Ncol and SrnaI followed by treatment with Klenow fragment to create two fragments. The 0.8 kb fragment was ligated with pUC12 digested with SmaI to construct the plasmid pAX10, pAX4 was constructed by selfligation of the other 5.5 kb fragment, pAX5 was obtained by ligating p A X S l l digested with PstI. pAX6 was obtained by insertion of the 1.2 kb KpnI-SacI fragment into pUC19 digested with K.pnI and SacI. pAX6R contained the same insert as pAX6 in pUC18. The fragments in pAX6 and pAX6R were differently oriented with respect to the lac promoter present on the vector, pAX8 was obtained by insertion of the 1.7 kb SphI-EcoRI fragment into pUC19 digested with SphI and EcoRI. pAC2 was obtained by insertion of the 2.2 kb BamHI-EcoRI fragment into pUC12. Restriction site: B, BamHI; Bs, BstEII; E, EcoRI; H, HindlII; K, KpnI; N, NcoI; P, PstI; Sa, SacI; Sin, Sinai; Sp, SphI. Restriction sites in parentheses are derived from vectors.
S. Yamano et al. /Journal of Biotechnology 32 (1994) 165-171
169
GGTACCGGGACCATAGGGGGGCTTGGGGTCG l II I B a m HI
¢TTTCGGCATGGGCGCGCTGCTGCCCGCGATCAGGCCGCCCATGCAGAAGGCGGCGAGCG TCATGTAAAGTGGCTTCATATCCGGTCATCCTG~TTTCAATGCGCGTTCACAATGGCAG
Indlll)/Rsal
TCCTGAACGGCCAAGGGCAAGGCCAGGGCCTGCACGGCGGCATTTCCATATATTTTATAT
Jail.
ATGGAAATAGGCTTTAATATATATTGGACGTACGAACCTGCCTGCATCACCATTAGTCTG H £ I G F l I Y W T Y E P A C I T I S L
il
¢lll/Xho I
CAATCACAAATGACCGGGTTGAnAeGATGCCATGTGCCGCATTGTCCCCCGATGCAULG 120 O S Q M T G L R R C H V P H ¢
P P M OE
ACTGAGGTCGTGAAGCTTAAATGCTACTCGGTAGGGGATGTTGATACCCGGTCCAGCGCT T E V V i L [ C Y S Y G 0 V 0 T R S S A
Z40
GCTGATTCGACTGGCGTGCGTCCGCGCATGAACCGCCTGTACCAGACATCGACCATGGCC 300 k
0
S T G V i
P R M I
R L Y O T S T M k
GCGCTGCTTGATGCGGTCTATGATGGCGAGACCACGCTTGATGAACTGCTGATGCACGG¢ 360 A L L O k V Y 0 G E T T L D E L L H H G AATTTCGGGCTGGGCACGTTCAACGGCCTTGATGGCGAGATGATCGTCAATGACAGCGTA 420 H F G L G T
F H 6
L O G E M I
V H 0
S V
ATCCACCAGTTCCGTGCAGACGGGCAGGCCGGTCGTGTGCCGGGCGACCTCAGGACTCC6 410 I
H 0
F N k
D G O k
G R V
P G D L R T P
TTCGCCTGCGTTACCTTCTTCAACCCGGAGAAGGAATACATGATC~CACCGCGCAGGAT 540 F A C V
T F
F H P E K E Y M I
D T A O O
S
AAGGAAGGCTTCGAGGCGATCGTGGATCACCTCGTCAACAATCCCAACCTGTTCGCCGCC |00 K E G F
E k
V 0
H L V N N P N L F k
GTGCGCITTACCGGCATGTTCGAGCGGGTCGAGACCCGCACCGTGTTCTGCCAGTGCCAG i|0 V R F
T 6 M
E R V
E T R T V
F ¢
a
~
\BamHI
k
O C 0
CCCTACCCACCCATGCTGGAAGTGGTGGC¢CG¢¢AGCCCA¢CATGCA6CTTGGTG¢CTCC 720 P Y P P M L V V k R 0 P T M O L G A S
Fig. 3. Construction of the expression plasmid pAX43GL for brewer's yeast, pBR322 sequence is represented by the thin line. GPDp, GPD promoter; PGKt, PGK terminator; G418 r, G418-resistance gene.
ACCGGCACCATGCTTGGTTTCCGCACGCCGGGCTACAIGCAGGGCGTGAACGTGGCGGGT 710 T G T M L G R T P G Y M 0 G V H V A G TATCACCTGCACTTCCTGACTGAGGACGGACGCCGTGGCGGCCATGTGACCGATTACGGC 140 Y H L H F L E O G R R G G H V T D Y G GTGCTGCGCGGTCGGCTTGAGGTGGGCGTGATTTCCGATGTGGAAATCCAGCTGCCCCG¢ $0| V L R G R L V G V f S O V E I 0 L P R ACCGAACAGTTCGCGCGCGCCAACCTGTCCCCCGAAAACATTCACGAGGCCATTCGCGTG 160 T E O F A R A H L $ P E i I H [ A I | V GCCGAGGGCGGCTGAGGGTTTCCCCTCCCGCCTGAGCAACTGTCCGCTCCGCCCCGGCTG1120 AEGGt CGGTGCCAACCGTTCAGGATACCTGAAATCATGACTGACAAGACCAAATCGGCCGCGCCG ~aal
GAGTGCGGGGCGGACATGATCCCCGGGCGAGCTC6AATTC
Fig. 2. Nucleotide sequence and deduced amino acid sequence of the ALDC gene of A. ace~ ssp. ~linum. The putatwe ribosome-binding sites are underlined. * Shows s ~ p codon.
ALDC gene were cloned into YEp13K-PGKt cleaved with BgllI + SacI. The resultant plasmid was designated pAX43B. The 1.7 kb SalI-BamHI fragment containing the G418-resistance gene and the 4.5 kb BamHI-XhoI fragment containing the ADCI-lacZ fusion gene were inserted into the SalI site of pAX43B to construct pAX43GL (Fig. 3). Brewer's yeast KI084 was transformed with pAX43GL. Cells containing pAX43GL made blue colonies on M63 plate containing X-gal. The rate of the clones containing plasmid among G418-re-
Table 2 Analysis of wort Strain
KI084 KI084 (pAX43GL)
Diacetyl compounds a (mg 1- 1)
Apparent extract
Apparent attenuation b
DA
PD
TDA
(Plato)
(%)
0.67 0.12
0.39 0.16
1.00 0.26
2.85 2.84
75.3 75.4
a DA, Diacetyi; PD, 2,3-pentanedione; TDA, total diacetyl (diacetyl + 2,3-pentanedione × 0.86). b [(Extract content of wort-apparent extract of beer)/extract content of wort] × 100.
Ethanol (%, v V- 1)
4.61 4.61
170
S. Yamano et al. /Journal of Biotechnology 32 (1994) 165-171
sistant clones was not constant (50 ~ 80%). ALDC activity of the transformants was 4 to 5 U mg-1 protein.
Laboratory-scale growth test Analysis of fermented wort is shown in Table 2. The total DA in the wort with the transformant was reduced to one-fourth of that with the parent yeast. There was no substantial difference in process performances except total DA formation of both strains. Expression of the ALDC gene did not affect the growth of brewer's yeast. The content of diacetyl was reduced more effectively than that of 2,3-pentanedione in the fermented wort. This means that the ALDC from A. aceti ssp. xylinurn decreased a-acetolactate more effic.~ently than a-acetohydroxybutyrate in yeast, pAX43GL in brewer's yeast KI084 was maintained stably under unselective conditions. 96% of cells contained pAX43GL at the end of the growth test. 4. Discussion
The ALDC genes from Enterobacter aerogenes (Sone et al., 1988) and Bacillus brevis (Diderichsen et al., 1990) were already cloned. The deduced amino acid sequence of the ALDC gene from A. aceti ssp. xylinum was compared with those of other ALDCs. There was a 45.7% match of amino acids in 230 overlap residues with E. aerogenes ALDC, and a 35.6% match in 247 overlap residues with B. brevis ALDC. Many homologous stretches of amino acid were distributed over the sequences except their Ntermini. The highest homologous region was Glyl°a-Ile 115, whose homology was 85% with the three ALDCs. It was therefore reasonable to assume that this region is very important for ALDC activity. E. aerogenes ALDC of 260 amino acids was the same size as B. brevis ALDC. It seems that only A. aceti ssp. xylinum ALDC has an extra long N-terminal sequence of approx. 40 amino acids. If the GTG codon at bp 130 is the real initiation codon, A. aceti ssp. xylinum ALDC has almost the same size as other ALDCs. Consensus sequences for promoters of Acetobacter strains have been unknown. A typical ribosome-binding
site was not found close to a putative ATG initiation codon at bp 1. On the other hand, the typical ribosome-binding site (GAGG) was located 13 bp upstream from the GTG codon at bp 94. And, there was the ribosome-binding site (AGGA) 14 bp upstream from the GTG codon at bp 130. As described in Results, the truncated ALDC gene in pAX31 retained a higher ALDC activity than that of pAX6 in E. coli. It is likely that the translation-start point of the ALDC gene is located downstream from bp 70 in Fig. 2. Since brewer's yeast cells are polyploid prototrophs, a dominant marker such as a drug-resistant gene is necessary for transformation of brewer's yeasts. Although the G418-resistant gene is frequently used as a dominant marker, it makes brewer's yeast ceils weakly resistant. In the selection medium with low G418 concentration (10 ~g ml-1), resistant clones do not always contain a plasmid. In our experiment, the G418-resistant gene and ADCI-lacZ fusion gene were used for efficient selection of transformants. In the future, the G418-resistant gene should be expressed at higher levels by a stronger promoter to increase the transformation efficiency of brewer's yeast cells. Sone et al. (1988) reported that the 2-/zm DNA-based plasmid was stable in the brewer's yeast KI084. We observed that the expression plasmid for the Acetobacter ALDC gene was also maintained stably in KI084 cells. It is possible that the endogenous 2-/~m DNA in the brewer's yeast KI084 increases the stability of the heterologous 2-/zm DNA-based plasmid that contains only a portion of the 2-~m DNA genome. The substrate specificity of bacterial ALDCs has been little investigated. Since the ALDC from A. aceti ssp. xylinum had not yet been studied, its substrate specificity was unknown. We observed that this ALDC decreased a-acetolactate more efficiently than a-acetohydroxybutyrate in yeast. This substrate specificity is similar to that of the ALDC from Klebsiella terrigena (Suihko et al., 1990) Use of the ALDC from A. aceti ssp. xylinum for brewing has not yet been reported. Acceptance in public opinion is important for the use of genetically improved microorganisms
S. Yamano et al. /Journal of Biotechnology 32 (1994) 165-171
in the food industry. From this point of view, cloning of genes from microorganisms for food making may increase in the future. We cloned the ALDC gene from A. aceti ssp. xylinum and confirmed that this ALDC gene was useful in reducing total DA concentration in wort.
5. Acknowledgements We are grateful to Toshihiko Ohno, Fumio Shimizu and Hidetaka Sone for their valuable suggestions, and to Naomi Odagawa for her technical assistance.
6. References Diderichsen, B., Wedsted, U., Hedegaard, L., Jensen, B.R. and Sj~holm, C. (1990) Cloning of aldB, which encodes a-acetolactate decarboxylase, an exoenzyme from Bacillus brevis. J. Bacteriol. 172, 4315-4321. Guarente, L. (1983) Yeast promoters and LacZ fusions designated to study expression of cloned genes in yeast. Methods Enzymol. 101, 181-191. Godtfredsen, S.E., Lorck, H. and Sigsgaard, P. (1983) On the occurrence of ct-acetolactate decarboxylases among microorganisms. Carlsberg Res. Commun. 48, 239-247.
171
Hitzeman, R.A., Hagie, F.E., Hayflick, J.S., Chen, C.Y., Seeburg, P.H. and Derynck, R. (1982) The primary structure of the Saccharomyces cerevisiae gene for 3-phosphoglycerate kinase. Nucleic Acids Res. 10, 7791-7808. Holland, J.P. and Holland, M.J. (1979) The primary structure of a glyceraldehyde-3-phosphate dehydrogenase gene from Saccharomyces cerevisiae. J. Biol. Chem. 254, 9839-9845. Ish-Horowicz, D. and Burke, J.F. (1981) Rapid and efficient cosmid vector cloning. Nucleic Acids Res. 9, 2989-2998. Lewis, K.F. and Weinhouse, S. (1958) Studies in valine biosynthesis. II. a-Acetolactate formation in microorganisms. J. Am. Chem. Soc. 80, 4913-4915. LOken, J.P. and St~rmer, F.C. (1970) Acetolactate decarboxylases from Aerobacter aerogenes. Eur. J. Biochem. 14, 133-137. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Sone, H., Fujii, T., Kondo, K. and Tanaka, J. (1987) Molecular cloning of the gene encoding a-acetolactate decarboxylase from Enterobacter aerogenes. J. Biotechnol. 5, 87-91. Sone, H., Fujii, T., Kondo, K., Shimizu, F., Tanaka, J. and Inoue, T. (1988) Nucleotide sequence and expression of the Enterobacter aerogenes o~-acetolactate decarboxylase gene in brewer's yeast. Appl. Environ. Microbiol. 54, 3842. Suihko, M.L., Blomqvist, K., Penttilii, M., Gisler, R. and Knowles, J (1990) Recombinant brewer's yeast strains suitable for accelerated brewing. J. Biotechnol. 14, 285-300. Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33, 103-119.