OL-Acetolactate Decarboxylase Genes in Brewer's Yeast
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1991, p. 2796-2803 Vol. 57, No. 10
0099-2240/91/102796-08$02.00/0
Copyright © 1991, American Society for Microbiology
Chromosomal Integration and Expression of Two Bacterial
oL-Acetolactate Decarboxylase Genes in Brewer's Yeast
K. BLOMQVIST,t M.-L. SUIHKO,* J. KNOWLES,t AND M. PENTTILA
Biotechnical Laboratory, Technical Research Centre of Finland (VTT),
P.O. Box 202, SF-02151 Espoo, Finland
Received 29 April 1991/Accepted 16 July 1991
A bacterial gene encoding a-acetolactate decarboxylase, isolated from Klebsiella ternigena or Enterobacter
aerogenes, was expressed in brewer's yeast. The genes were expressed under either the yeast phosphoglycer-
Downloaded from http://aem.asm.org/ on May 25, 2021 by guest
okinase (PGKI) or the alcohol dehydrogenase (ADHI) promoter and were integrated by gene replacement by
using cotransformation into the PGKI or ADHI locus, respectively, of a brewer's yeast. The expression level
of the aL-acetolactate decarboxylase gene of the PGKI integrant strains was higher than that of the ADHI
integrants. Under pilot-scale brewing conditions, the a-acetolactate decarboxylase activity of the PGKI
integrant strains was sufficient to reduce the formation of diacetyl below the taste threshold value, and no
lagering was needed. The brewing properties of the recombinant yeast strains were otherwise unaltered, and
the quality (most importantly, the flavor) of the trial beers produced was as good as that of the control beer.
During beer fermentation, yeast produces a-acetolactate usually disrupts the locus in the chromosome, genes have
and ao-aceto-a-hydroxybutyrate, which are intermediates in mainly been targeted to nonessential regions such as to the
the synthesis of valine and isoleucine, respectively (5). HO locus involved in mating (32) or to the rRNA locus (7),
However, minor amounts of these compounds leak out of which is present in over 100 copies in the genome. Integra-
the cells into the fermenting wort. These are spontaneously tion into other loci such as LEU2 (14, 21) and ILV2 (4) has
decarboxylated to the respective diketones, diacetyl and also been carried out in polyploid yeast strains.
2,3-pentanedione, but only slowly under brewing conditions In this paper, we describe the construction of four dif-
(23). The taste and smell of diacetyl is detected at a very low ferent types of (x-ALDC-active, bottom-fermenting brewer's
level, 0.02 to 0.10 mg/liter depending on the type of beer and yeast strains. The genes from the bacteria Klebsiella terri-
the method of analysis, and most people find it very un- gena and Enterobacter aerogenes encoding o-ALDC were
pleasent. Thus, for production of high-quality beer, a sepa- integrated into the PGKJ or ADHI locus of the yeast genome
rate maturation period (lagering) of 2 to 6 weeks for green by cotransformation and gene replacement. In addition, we
beer is needed, during which time diacetyl is taken up by the compare the brewing properties of seven a-ALDC-active
yeast cells and enzymatically reduced to acetoin. This lager- integrant yeast strains in 50-liter, pilot-scale brewing trials as
ing period is costly for breweries. well as the qualities of beers produced with these strains.
The enzyme a-acetolactate decarboxylase (a-ALDC; EC
4.1.1.5) decarboxylates a-acetolactate directly to acetoin
without formation of diacetyl (29). The gene coding for this MATERIALS AND METHODS
enzyme (a-ald or budA) is not found in yeasts but has been
isolated from several bacteria (2, 8, 28). The a-ald gene Microorganisms, vectors, and media. The a-ald (budA)
genes were isolated from the bacteria K. terrigena VTT-E-
cloned into autonomously replicating plasmids has been 74023 and E. aerogenes VTT-E-87292 (2). The bacteria and
transformed into brewer's yeast (27, 29) and shown strongly the industrial bottom-fermenting brewer's yeast strain Sac-
to reduce formation of diacetyl during fermentation without
affecting the quality of the final beer (29). However, plasmid charomyces cerevisiae VTT-A-63015 (hereafter called A15),
strains contain extra foreign DNA and are usually unstable which was used as the host strain, were from the VTT
in long-term usage (29, 30). The gene has also been inte- Collection of Industrial Microorganisms. The PGKI and
grated as multiple copies into a yeast rRNA gene (7) without ADH1 promoter and terminator sequences were taken from
affecting the fermentation performance of the yeast or the plasmids pMA91 (17) and pAAH5 (1), respectively. The
quality of the final product (31). ot-ald genes were taken from plasmids pKB101 (29) and
Integration of a gene into the yeast genome, resulting in pPL4 (29). Bluescribe M13+ (Stratagene, La Jolla, Calif.)
was used as a vector for the integration cassettes. Plasmid
stable strains which carry minimal amounts of foreign DNA, pET13:1 (9) was used as a selection plasmid in cotransfor-
is preferred to plasmid-carrying strains. Different techniques mation. YPD and NEPRA media were used in transforma-
are now available for the transfer and expression of foreign
genes in industrial yeast strains (12, 20). However, little is
tion and selection (21).
known about the effect of integration on the process behav- Construction of plasmids and expression cassettes. Plasmid
ior of industrial polyploid yeast strains. Because integration pKBOO7 carries the coding region without the 5'-flanking
region of the a-ald gene of K. terrigena linked between the
promoter and terminator of the yeast PGKI gene (29). From
*
Corresponding author. this plasmid a 2.8-kb-long HindIII fragment containing the
t Present address: Institute of Microbiology, University of Umea, PGKJ/a-ald expression cassette was released and cloned
Umea, Sweden. into Bluescribe M13+ at the Hindlll site to give plasmid
t Present address: Glaxo Institute for Molecular Biology, pKB107 (Fig. 1). To obtain a similar ADHJ/ao-ald expression
Geneva, Switzerland. cassette, the ADHI promoter and terminator were first
2796VOL. 57, 1991 a-ALDC BREWER'S YEAST STRAINS 2797
(Hind 111)
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HindIll1 (Hind III/Sall)
(Hind III/BgIll) (Hindil/Hindill)
1 ~~~~~Hind IIIBanH
EcoRi
FIG. 1. Plasmids pKB107 and pKB103 carrying the a-ald gene of K. terrigena and plasmids pKB105 and pKB106 carrying the ax-ald gene
of E. aerogenes.
isolated from vector pAAH5 as a 1.95-kb BamHI fragment method (21), based on copper resistance for selection (9),
and then cloned at the BamHI site of a Bluescribe M13+ was used. Five micrograms of a linear expression cassette
vector from which the HindlIl site had been removed by blunt ended by Klenow polymerase, derived from either
filling in and religation. Into this plasmid, pKB102, a blunt- plasmid pKB103 or pKB106 with BamHI or from pKB105 or
ended EcoRI-HindIII fragment containing the a-ald gene of pKB107 with Hindlll digestion, was transformed into
K. terrigena from plasmid pKB101 (29) was ligated at the spheroplasts of the bottom-fermenting brewer's yeast strain
blunt-ended Hindlll site between the ADHI promoter and A15 together with 5 ,ug of the selection plasmid pET13:1.
terminator sequences, giving plasmid pKB103 (Fig. 1) car- Transformants carrying the oa-ald gene were identified by
rying a 2.9-kb-long ADHI/a-ald expression cassette. colony hybridization (25) with the oa-ald gene of either K.
The construction of plasmid pKB105 (Fig. 1) carrying the terrigena or E. aerogenes as a probe or by a-ALDC activity
oa-ald gene of E. aerogenes as a 2.7-kb-long PGKJla-ald measurement from YPD-medium-grown yeast cell extracts
expression cassette has been described earlier (29). The (29), using hydrolyzed acetolactic acid ethyl ester acetate as
oa-ald gene of E. aerogenes was also linked to the ADHI the substrate (Oxford Organic Chemicals Ltd., Brackly
promoter in plasmid pKB102. The gene was first released Northamptonshire, United Kingdom). Acetoin formed was
from plasmid pPL4 (29) as a SalI-HindIll fragment. The detected by the Voges-Proskauer test (29), and some of the
fragment was blunt ended by Klenow polymerase and ligated final reaction mixtures were also analyzed by gas chroma-
to the blunt-ended HindlIl site of plasmid pKB102, giving tography (29). The protein content of the extracts was
plasmid pKB106 (Fig. 1) carrying a 2.8-kb-long ADHIaot-ald determined with the Folin phenol reagent (15).
expression cassette. Rapid small-scale preparation of total DNA from yeast cells.
Cotransformation of brewer's yeast, screening, and a- Yeast cells were grown to the stationary phase (16 to 20 h) in
ALDC measurement of transformants. The cotransformation 5 ml of YPD medium at 30°C with shaking. Cells were2798 BLOMQVIST ET AL. APPL. ENVIRON. MICROBIOL.
harvested by centrifugation at 5,000 rpm for 5 min and ADHI yeast locus was replaced with the a-ald genes. To
resuspended in 380 ,ul of 1.2 M sorbitol-0.1 M EDTA, pH achieve this, the a-ald genes were first coupled between the
7.5. Zymolyase lOOT (5 mg/ml in 50% glycerol; Seikagaku promoter and terminator regions of the PGKI and ADH1
Kogyo, Tokyo, Japan), 7 ilI, was added, and the mixture was genes (see Materials and Methods; Fig. 1) and then excised
incubated at 30°C for 60 min. Cells were pelleted by centrif- from the plasmids by cutting at the 5' side of the promoter
ugation at 3,500 rpm for 5 min, resuspended in 690 ,ul of 0.1% and the 3' side of the terminator. The expression cassettes
sodium dodecyl sulfate-50 mM Tris-1 mM EDTA (pH 7.5), were cotransformed as a linear molecule into the bottom-
mixed vigorously, and centrifuged at 15,000 rpm for 2 min to fermenting brewer's yeast strain A15 together with plasmid
rupture the cells. A 4-,ul portion of RNase A (5 mg/ml; pET13:1 carrying the copper chelatin gene as a selection
Sigma) was added to the supernatant, and the mixture was marker. The transformants were first screened for copper
incubated at 37°C for 30 min. The mixture was extracted resistance. Positive clones also containing the a-ald gene
once with phenol and once with chloroform-isoamyl alcohol were screened either by colony hybridization, using at-ald-
(24:1). DNA was precipitated with ethanol and resuspended specific probes, or by confirmation of a-ALDC activity in
in 50 ,ul of 50 mM Tris-1 mM EDTA, pH 7.5. cell extracts, using a-acetolactate as the substrate. Plasmid
DNA hybridizations. Southern analysis was carried out by pET13:1 was removed from the yeast transformants exhib-
Downloaded from http://aem.asm.org/ on May 25, 2021 by guest
conventional methods (16). The K. terrigena gene was iting a-ALDC activity by growing the cells in YPD.
probed by a 0.95-kb-long EcoRI-HindIll fragment containing The a-ALDC yeast strains obtained are summarized in
the oa-ald gene isolated from plasmid pKB101 (29), and the E. Table 1 along with their relevant characteristics.
aerogenes gene was probed by a 0.89-kb-long Sall-HindIII Southern analysis of strains. It was anticipated that in the
fragment isolated from plasmid pPL4 (29). Probes specific cotransformation procedure the oa-ald genes would replace
for the PGKI and ADHI genes were derived from plasmid the endogenous PGKI or ADHI locus by homologous re-
pMA91 (17) by HindIlI digestion and from pAAH5 (1) by combination between the promoter and terminator regions of
BamHI digestion, respectively. The fragments were labelled
with [ot-32P]dCTP (The Radiochemical Centre, Amersham, the expression cassette and those of the corresponding
United Kingdom) by using the Random Primed DNA Label- chromosomal genes. The integration pattern of the strains
ling Kit (Boehringer Mannheim, Mannheim, Germany) in was studied by Southern analysis by digesting total chromo-
accordance with the manufacturer's instructions. somal DNA of the recombinant strains with EcoRI. There is
Brewing trials and analysis. Industrial worts (10.5%, wt/ no site for this enzyme within the expression cassettes or in
wt) were used in the brewing trials, which were carried out the endogenous S. cerevisiae ADHI locus, and there is only
in the 50-liter pilot brewery at 10°C as described earlier (29). one site in the endogenous coding region of PGKJ.
Unless otherwise stated, the brewing and the beer analysis If the expression cassette had replaced the endogenous
were carried out as described in Analytica-EBC (3). The ADHI locus, the expected size difference in the EcoRI digest
growth was monitored by determining the amount of yeast would be only 100 bp compared with the endogenous locus
(dry weight) in fermenting wort. Diacetyl (29), flavor com- and would not be easily distinguishable in the analysis. This
pounds (18), and amino acids (6) were determined by chro- is evident in Fig. 2A, in which a fragment of about 7.0 kb can
matography. The beers were evaluated by a tasting panel (12 be seen in the recombinant strains A90 and A91, probed with
to 15 persons), using international flavor terms and scores either ADHI- or a-ald-specific probes. This is a band of
from 1 to 5. approximately the same size as seen in the control strain A15
with the ADHI probe. This result indicates that a copy of the
RESULTS
a-ald gene had integrated into and replaced an endogenous
chromosomal ADHI gene in these strains. Strain A86,
Construction of a-ALDC-active brewer's yeast strains. The however, appeared to carry the a-ald gene elsewhere in the
o-ald genes of K. terrigena and E. aerogenes were integrated genome, and the presence of a tandem copy of the expres-
into the yeast genome so that the endogenous PGKI or sion cassette cannot be excluded. No vector sequences
FIG. 2. Southern analysis of EcoRI-digested total DNA of the ADH1 (A) and PGKI (B) integrants. The specificities of the probes used
are indicated at the top (see Materials and Methods for further details). The approximate sizes (kilobases) of the hybridizing bands are shown
on the right of each panel. A15 is the control strain.VOL. 57, 1991 ct-ALDC BREWER'S YEAST STRAINS 2799
A B
4
8
71
6
.0
e
4-0 5
u
m
1-4
4-b
x 4
cu
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C14 0
._ 2
a)
64 3
m
C14
04
2
M)a
^s
3 4 5 6 7 1 1
jw3 4 5 6 7
Fermentation time, d Fermentation time, d
FIG. 3. Yeast growth and flocculation (A) and fermentation rate (B) in brewing trials. Symbols: control strain A15; PGKI integrants
carrying the a-ald gene of K. terrigena-*, A85, and A, A95; ADH1 integrants carrying the a-ald gene of K. terrigena-0, A86, and A, A91;
PGKI integrants carrying the a-ald gene of E. aerogenes-4, A89, and Q, A92; O, an ADHI integrant carrying the a-ald gene of E.
aerogenes, A90.
(B3luescribe M13+) were present in these three recombinant the a-ald gene into a single locus in the genome. Disappear-
strains (data not shown). ance of the 4.0-kb PGKI promoter-specific band, seen in the
The Southern pattern of the PGKI integrants was more control strain A15, indicates that integration occurred at the
complex (Fig. 2B). All four recombinant strains carried PGKI locus. Overall, the hybridization pattern supports the
bacterial vector sequences. Strains A85, A92, and A95 all proposal that one copy of plasmid pKB105 (strain A92) or
gave the same pattern when compared with each other when pKB107 (strains A85 and A95) integrated through single
hybridized with a-ald-, PGKI-, and Bluescribe M13+-spe- recombination events via the promoter sequences into the
cific probes, suggesting a common mode of integration. The PGKI locus, generating an EcoRI fragment of about 7.1 kb
bacterial vector appeared to have integrated together with containing the a-ald expression cassette and the Bluescribe
TABLE 1. a-ALDC-active brewer's yeast strains constructed and tested in the pilot brewery
Site of Bacterial vector a-ALDC activity2
Yeast strain Origin of gene Promoter mtegratlon Copy no.*
integration ~sequences present A^4 ai-Acetolactate (%)
Control strain
A15 - 0.36 0
PGKI integrant
A85 K. terrigena PGKI PGKI 1 + 3.24 39.3
A95 K. terrigena PGKI PGKI 1 + NDb ND
A89 E. aerogenes PGKI Unknown >1? + 3.07 46.4
A92 E. aerogenes PGKI PGKI 1 + ND ND
ADHI integrant
A86 K. terrigena ADHI Unknown 1-2? - 1.13 30.0
A91 K. terrigena ADHI ADHI 1 - ND ND
A90 E. aerogenes ADHI ADHI 1 - 0.57 15.5
a
Determined by measuring the intensity of red color (A540) developed in the Voges-Proskauer test and the amount of added a-acetolactate enzymatically
decarboxylated to acetoin in yeast ceil extracts (protein content, about 1.6 mg/ml) after incubation for 30 min.
b ND, not determined.2800 BLOMQVIST ET AL. APPL. ENVIRON. MICROBIOL.
vector sequence and fragments of 2.5 and 1.8 kb containing .0
the endogenous PGKI gene with the promoter and termina- 0)'
tor, respectively.
Unexpectedly, two bands instead of one were seen in the
untransformed strain A15 with both the PGK1 promoter and
the terminator-specific probe, and one of the promoter Cl4
ClNN C" e ef
sequences and both of the terminator sequences remained ~-0) 6 6666666
intact in the recombinant strains. It is possible that two
different copies of the PGKJ gene exist in the brewer's yeast
host strain and that only one of these was hit by the oa-ald
gene in the integration event. Lager brewer's yeast strains 0000 0C "It " o0
have been shown to carry two structurally different forms of ;
-u
many chromosomes (11), and this could also be the case with '_
C- t- -4 -f -Cf -
f
0-4 "Cl 0- 000
chromosome XV on which the PGK1 gene is located. r-- r-
In transformant A89, all endogenous PGKl-specific bands
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remained intact, indicating integration of the expression MT llz 't t t m
cassette with vector sequences elsewhere in the genome. ._
The presence of bacterial sequences in the PGK1 integrant E ED er -F -Cl00
otn C606 ocl tn
strains could be a result of, e.g., incomplete digestion of the 0
U.
plasmids upon release of Bluescribe vector sequences from CC 1H
cl ^o
tr
the expression cassettes. A single recombination event of cis-4
the complete plasmid at the PGK1 locus might have been cas
favored over gene replacement with the linear expression 0
cassette. Gene replacement of the PGK1 locus of strain A15 aL) 00 00 oo N 0%C' 0 CN /tn
is, however, possible with this strategy, as we demonstrated --- ---4 ,-4
when constructing glucanolytic brewer's yeast strains (30). COE_.
Growth and fermentation. Growth of the integrant yeast 000% N~
0 u) 00o
-~ -i00N.
M M
00
cn
strains during the 50-liter fermentations (Fig. 3A), as well as C0
the fermentation rate with these strains (Fig. 3B), was the '~ o~ XC7> a) C) C? O
co
same or faster than with the control strain A15. Alcohol
contents were approximately the same in all trial beers U) 8 C) CD O
00000000H
C"C r-4 r-- "l~
(Table 2). 0) 66666666Q 0
Formation of diketones and lagering. The a-ALDC activity 0
O
. 0 C;
O;
O O.4
OOO CS
0 OO . ..
of the PGK1 integrant strain A89, carrying the oa-ald gene of ._
E. aerogenes, was so effective that the total diacetyl content u
(free diacetyl plus a-acetolactate) never reached the taste .C
H = V
threshold value of 0.02 mg/liter during fermentation (Fig. 4). a~00)
With the other PGK1 integrant strains A85, A92, and A95, (A
some diacetyl was formed during fermentation, but by the .200m0 o Q*
00)E
end of fermentation it had already decreased at least to its
taste threshold value. No lagering was required for the trial 0_
[n
beers produced with these PGK1 integrant strains, and the N- N- 04 en 00 W)
production time of beer was shortened by 2 weeks. With all ,-4 ,-4
0)C
r-4
r-r-4
,-4l -4
'--4 V-4
,--4 r-q r-
ADH1 integrant yeast strains, A86, A90, and A91, the F0 vF N oo
formation of diacetyl was relatively high during fermenta-
tion, and lagering for 4 to 5 days was necessary to remove 0. o
diacetyl from the trial beers produced with these strains. 0.0)
However, even with these strains the lagering time was
shortened by as much as 10 days compared with that cl; cl Cl Cl
required for the control beer.
The a-ALDC activity of the integrant yeast strains also
decreased the formation of 2,3-pentanedione (Fig. 5), but not
as strongly as that of diacetyl. However, the 2,3-pentanedi-
one contents found in green beers do not affect beer flavor, O E -4 00000 00
becatise its taste threshold value is as high as 1.0 to 1.5 U,
.0
mg/liter (23).
Bottled beer and its flavor. The beers produced with the
recombinant yeast strains were similar to the control beer
(Table 2). The differences recorded were within the limits of 0.0
between-batch variation. The omission of lagering (beers
produced with strains A85, A89, A92, and A95) did not affect
turbidity, chemical stability, or polyphenol contents of 6666oooo
beers. The foam stability of beer was also unaffected. r-4 -4 -4
The formation of fusel alcohols (higher alcohols) is growth
associated and linked to amino acid synthesis (5, 24). In o o "
transamination, yeast prefers the amino groups of the amino,a-ALDC BREWER'S YEAST STRAINS
VOL. 57, 1991 2801
0.4H
E 0.3-
a)
0
.w
0.2F
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0
0.1 -
_I__
__
__ _ __ I_
3
1
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Fermentation, d Lagering, d
FIG. 4. Formation and reduction of total diacetyl during fermentation and lagering. Symbols as in the legend to Fig. 3. The broken line
represents the taste threshold value of diacetyl.
acids relevant in this study, leucine, isoleucine, and valine. with strain A90 but not in beer produced with strain A86,
The hydroxy acids formed in deamination reactions are although in both beers the concentration of diacetyl was the
decarboxylated to the respective alcohols, resulting in limit value, 0.02 mg/liter.
3-methyl butanol (i-amyl alcohol), 2-methyl butanol (optical- Strain stability. One strain of each type of the ao-ALDC
ly active amyl alcohol), and i-butanol, respectively. Thus, yeasts was recycled in comparison with the control strain
due to the slightly better growth of the recombinant yeast A15 in seven successive 50-liter fermentations. No signifi-
strains, the contents of fusel alcohols were also slightly cant alteration was observed in either the fermentation
higher in beers produced with the oe-ALDC yeasts than in the patterns or the flavor profiles of beers produced with these
control beer (Table 2). recycled yeasts. The formation of diacetyl in fermenting
The formation of esters is considered to be competitive wort also remained typical for beers produced with each
with growth (19). Ester formation results from esterification strain (Fig. 6). The fermentation results with strains A85,
of ethanol or higher alcohols with fatty acids, resulting A86, A89, and A90 were very similar to those obtained
mainly in acetate esters. The ester contents were very earlier (31). Between the two sets of trials the strains were
similar in all trial beers (Table 2). Presumably due to the maintained under liquid nitrogen. Altogether during this
slightly lower amount of other flavor compounds, e.g., fusel study, 44 pilot-scale (50-liter) fermentations were carried
alcohols and esters, diacetyl was tasted in beer produced out, and 19 bottled trial beers were produced with these
E
t0.2d
F
0
C.
CI
0.
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Fermentation, d Lagering, d
FIG. 5. Formation and reduction of total 2,3-pentanedione during fermentation and lagering. Symbols as in the legend to Fig. 3.;>.B§
2802 BLOMQVIST ET AL. APPL. ENVIRON. MICROBIOL.
Cycle TABLE 3. Amounts of free amino acids in wort and in green
beers produced with a-ALDC-active yeast strainsa
A15 1 I Amt of amino acid (mg/liter)
2
i `
7-71
44 ---I Amino acid Yeast strain
Wort
5iri----- I A15 A85 A86 A89 A90
2
7 Group 1
Arginine 237 39 41 27 31 29
A85 i HistidineVOL. 57, ,a-ALDC BREWER'S YEAST STRAINS
1991 2803
oa-ald genes used in this study, is used for spontaneous European Brewery Convention, Proceedings of the 22nd Con-
production of Lambic beer and thus can be considered a gress, Zurich, 1989. IRL Press, Oxford University Press, Ox-
food microbe. Apart from the ot-ald gene of E. aerogenes, ford.
the ADHI integrant strain A90 contains no foreign DNA. 14. Liljestrom-Suominen, P. L., V. Joutsjoki, and M. Korhola. 1988.
Construction of a stable a-galactosidase-producing baker's
For these reasons, this strain should be suitable for industrial yeast strain. Appl. Environ. Microbiol. 54:245-249.
production of beer. By using this strain in batch production 15. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.
of beer, the lagering time is shortened dramatically, from 2 to 1951. Protein measurement with the Folin phenol reagent. J.
3 weeks to 4 to 5 days. The saving of time, space, and energy Biol. Chem. 193:265-275.
would be even more significant if the use of immobilization 16. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular
techniques (13) and a-ALDC-active yeasts were to be com- cloning: a laboratory manual. Cold Spring Harbor Laboratory,
bined in the main fermentation. Cold Spring Harbor, N.Y.
17. Mellor, J., M. J. Dobson, N. A. Roberts, M. F. Tuite, J. S.
Emtage, S. White, P. A. Lowe, T. Patel, A. J. Kingsman, and
ACKNOWLEDGMENTS S. M. Kingsman. 1983. Efficient synthesis of enzymatically
This research was financed by Oy Panimolaboratorio-Bryggeril- active calf chymosin in Saccharomyces cerevisiae. Gene 24:1-
aboratorium Ab, the Technology Development Centre (TEKES), 14.
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Stiftelsen Svensk Etanolutveckling, the Technical Research Centre 18. Pajunen, E., V. Makinen, and R. Gisler. 1987. Secondary
of Finland (VTT), and the Nordic Yeast Research Program (NYRP). fermentation with immobilized yeast, p. 441-448. In European
We are grateful to T.-M. Enari for inspiration and helpful discus- Brewery Convention, Proceedings of the 21st Congress,
sions during this work. We warmly thank all those involved in this Madrid, 1987. IRL Press, Information Printing Ltd., Oxford.
work at VTT Biotechnical Laboratory. 19. Peddie, H. A. B. 1990. Ester formation in brewery fermenta-
tions. J. Inst. Brew. (London) 96:327-331.
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