Henderson, R. C. A., B. S. Cox, and R. Tubb. 1985. The transformation of brewing yeasts witha plasmid containing the gene for copper resistance. Curr. Genet.
Vol. 57, No. 10
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1991, p. 2796-2803
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 phosphoglycerokinase (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.
usually disrupts the locus in the chromosome, genes have mainly been targeted to nonessential regions such as to the HO locus involved in mating (32) or to the rRNA locus (7), which is present in over 100 copies in the genome. Integration into other loci such as LEU2 (14, 21) and ILV2 (4) has also been carried out in polyploid yeast strains. In this paper, we describe the construction of four different types of (x-ALDC-active, bottom-fermenting brewer's yeast strains. The genes from the bacteria Klebsiella terrigena and Enterobacter aerogenes encoding o-ALDC were integrated into the PGKJ or ADHI locus of the yeast genome by cotransformation and gene replacement. In addition, we compare the brewing properties of seven a-ALDC-active integrant yeast strains in 50-liter, pilot-scale brewing trials as well as the qualities of beers produced with these strains.
During beer fermentation, yeast produces a-acetolactate and ao-aceto-a-hydroxybutyrate, which are intermediates in the synthesis of valine and isoleucine, respectively (5). However, minor amounts of these compounds leak out of the cells into the fermenting wort. These are spontaneously decarboxylated to the respective diketones, diacetyl and 2,3-pentanedione, but only slowly under brewing conditions (23). The taste and smell of diacetyl is detected at a very low level, 0.02 to 0.10 mg/liter depending on the type of beer and the method of analysis, and most people find it very unpleasent. Thus, for production of high-quality beer, a separate maturation period (lagering) of 2 to 6 weeks for green beer is needed, during which time diacetyl is taken up by the yeast cells and enzymatically reduced to acetoin. This lagering period is costly for breweries. 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 enzyme (a-ald or budA) is not found in yeasts but has been isolated from several bacteria (2, 8, 28). The a-ald gene cloned into autonomously replicating plasmids has been transformed into brewer's yeast (27, 29) and shown strongly to reduce formation of diacetyl during fermentation without affecting the quality of the final beer (29). However, plasmid strains contain extra foreign DNA and are usually unstable in long-term usage (29, 30). The gene has also been integrated as multiple copies into a yeast rRNA gene (7) without affecting the fermentation performance of the yeast or the quality of the final product (31). Integration of a gene into the yeast genome, resulting in stable strains which carry minimal amounts of foreign DNA, is preferred to plasmid-carrying strains. Different techniques are now available for the transfer and expression of foreign genes in industrial yeast strains (12, 20). However, little is known about the effect of integration on the process behavior of industrial polyploid yeast strains. Because integration
MATERIALS AND METHODS
Microorganisms, vectors, and media. The a-ald (budA) isolated from the bacteria K. terrigena VTT-E74023 and E. aerogenes VTT-E-87292 (2). The bacteria and the industrial bottom-fermenting brewer's yeast strain Saccharomyces cerevisiae VTT-A-63015 (hereafter called A15), which was used as the host strain, were from the VTT Collection of Industrial Microorganisms. The PGKI and ADH1 promoter and terminator sequences were taken from plasmids pMA91 (17) and pAAH5 (1), respectively. The ot-ald genes were taken from plasmids pKB101 (29) and pPL4 (29). Bluescribe M13+ (Stratagene, La Jolla, Calif.) was used as a vector for the integration cassettes. Plasmid pET13:1 (9) was used as a selection plasmid in cotransformation. YPD and NEPRA media were used in transformation and selection (21). Construction of plasmids and expression cassettes. Plasmid 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 this plasmid a 2.8-kb-long HindIII fragment containing the PGKJ/a-ald expression cassette was released and cloned into Bluescribe M13+ at the Hindlll site to give plasmid pKB107 (Fig. 1). To obtain a similar ADHJ/ao-ald expression cassette, the ADHI promoter and terminator were first genes were
* Corresponding author. t Present address: Institute of Microbiology, University of Umea, Umea, Sweden. t Present address: Glaxo Institute for Molecular Biology,
Geneva, Switzerland. 2796
VOL. 57, 1991
a-ALDC BREWER'S YEAST STRAINS
2797
(Hind 111)
(Hind III/Sall)
HindIll1
(Hindil/Hindill)
(Hind III/BgIll) 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 and then cloned at the BamHI site of a Bluescribe M13+ vector from which the HindlIl site had been removed by filling in and religation. Into this plasmid, pKB102, a bluntended EcoRI-HindIII fragment containing the a-ald gene of K. terrigena from plasmid pKB101 (29) was ligated at the blunt-ended Hindlll site between the ADHI promoter and terminator sequences, giving plasmid pKB103 (Fig. 1) carrying a 2.9-kb-long ADHI/a-ald expression cassette. The construction of plasmid pKB105 (Fig. 1) carrying the oa-ald gene of E. aerogenes as a 2.7-kb-long PGKJla-ald expression cassette has been described earlier (29). The oa-ald gene of E. aerogenes was also linked to the ADHI promoter in plasmid pKB102. The gene was first released from plasmid pPL4 (29) as a SalI-HindIll fragment. The fragment was blunt ended by Klenow polymerase and ligated to the blunt-ended HindlIl site of plasmid pKB102, giving plasmid pKB106 (Fig. 1) carrying a 2.8-kb-long ADHIaot-ald expression cassette. Cotransformation of brewer's yeast, screening, and aALDC measurement of transformants. The cotransformation
method (21), based on copper resistance for selection (9), was used. Five micrograms of a linear expression cassette blunt ended by Klenow polymerase, derived from either plasmid pKB103 or pKB106 with BamHI or from pKB105 or pKB107 with Hindlll digestion, was transformed into spheroplasts of the bottom-fermenting brewer's yeast strain A15 together with 5 ,ug of the selection plasmid pET13:1. Transformants carrying the oa-ald gene were identified by colony hybridization (25) with the oa-ald gene of either K. terrigena or E. aerogenes as a probe or by a-ALDC activity measurement from YPD-medium-grown yeast cell extracts (29), using hydrolyzed acetolactic acid ethyl ester acetate as the substrate (Oxford Organic Chemicals Ltd., Brackly Northamptonshire, United Kingdom). Acetoin formed was detected by the Voges-Proskauer test (29), and some of the final reaction mixtures were also analyzed by gas chromatography (29). The protein content of the extracts was determined with the Folin phenol reagent (15). Rapid small-scale preparation of total DNA from yeast cells. Yeast cells were grown to the stationary phase (16 to 20 h) in 5 ml of YPD medium at 30°C with shaking. Cells were
2798
BLOMQVIST ET AL.
harvested by centrifugation at 5,000 rpm for 5 min and resuspended in 380 ,ul of 1.2 M sorbitol-0.1 M EDTA, pH 7.5. Zymolyase lOOT (5 mg/ml in 50% glycerol; Seikagaku Kogyo, Tokyo, Japan), 7 ilI, was added, and the mixture was incubated at 30°C for 60 min. Cells were pelleted by centrifugation at 3,500 rpm for 5 min, resuspended in 690 ,ul of 0.1% sodium dodecyl sulfate-50 mM Tris-1 mM EDTA (pH 7.5), mixed vigorously, and centrifuged at 15,000 rpm for 2 min to rupture the cells. A 4-,ul portion of RNase A (5 mg/ml; Sigma) was added to the supernatant, and the mixture was incubated at 37°C for 30 min. The mixture was extracted once with phenol and once with chloroform-isoamyl alcohol (24:1). DNA was precipitated with ethanol and resuspended in 50 ,ul of 50 mM Tris-1 mM EDTA, pH 7.5. DNA hybridizations. Southern analysis was carried out by conventional methods (16). The K. terrigena gene was probed by a 0.95-kb-long EcoRI-HindIll fragment containing the oa-ald gene isolated from plasmid pKB101 (29), and the E. aerogenes gene was probed by a 0.89-kb-long Sall-HindIII fragment isolated from plasmid pPL4 (29). Probes specific for the PGKI and ADHI genes were derived from plasmid pMA91 (17) by HindIlI digestion and from pAAH5 (1) by BamHI digestion, respectively. The fragments were labelled with [ot-32P]dCTP (The Radiochemical Centre, Amersham, United Kingdom) by using the Random Primed DNA Labelling Kit (Boehringer Mannheim, Mannheim, Germany) in accordance with the manufacturer's instructions. Brewing trials and analysis. Industrial worts (10.5%, wt/ wt) were used in the brewing trials, which were carried out in the 50-liter pilot brewery at 10°C as described earlier (29). Unless otherwise stated, the brewing and the beer analysis were carried out as described in Analytica-EBC (3). The growth was monitored by determining the amount of yeast (dry weight) in fermenting wort. Diacetyl (29), flavor compounds (18), and amino acids (6) were determined by chromatography. The beers were evaluated by a tasting panel (12 to 15 persons), using international flavor terms and scores from 1 to 5.
RESULTS Construction of a-ALDC-active brewer's yeast strains. The o-ald genes of K. terrigena and E. aerogenes were integrated into the yeast genome so that the endogenous PGKI or
APPL. ENVIRON. MICROBIOL.
ADHI yeast locus was replaced with the a-ald genes. To achieve this, the a-ald genes were first coupled between the promoter and terminator regions of the PGKI and ADH1 genes (see Materials and Methods; Fig. 1) and then excised from the plasmids by cutting at the 5' side of the promoter and the 3' side of the terminator. The expression cassettes were cotransformed as a linear molecule into the bottomfermenting brewer's yeast strain A15 together with plasmid pET13:1 carrying the copper chelatin gene as a selection marker. The transformants were first screened for copper resistance. Positive clones also containing the a-ald gene were screened either by colony hybridization, using at-aldspecific probes, or by confirmation of a-ALDC activity in cell extracts, using a-acetolactate as the substrate. Plasmid pET13:1 was removed from the yeast transformants exhibiting a-ALDC activity by growing the cells in YPD. The a-ALDC yeast strains obtained are summarized in Table 1 along with their relevant characteristics. Southern analysis of strains. It was anticipated that in the cotransformation procedure the oa-ald genes would replace the endogenous PGKI or ADHI locus by homologous recombination between the promoter and terminator regions of the expression cassette and those of the corresponding chromosomal genes. The integration pattern of the strains was studied by Southern analysis by digesting total chromosomal DNA of the recombinant strains with EcoRI. There is no site for this enzyme within the expression cassettes or in the endogenous S. cerevisiae ADHI locus, and there is only one site in the endogenous coding region of PGKJ. If the expression cassette had replaced the endogenous ADHI locus, the expected size difference in the EcoRI digest would be only 100 bp compared with the endogenous locus and would not be easily distinguishable in the analysis. This is evident in Fig. 2A, in which a fragment of about 7.0 kb can be seen in the recombinant strains A90 and A91, probed with either ADHI- or a-ald-specific probes. This is a band of approximately the same size as seen in the control strain A15 with the ADHI probe. This result indicates that a copy of the a-ald gene had integrated into and replaced an endogenous chromosomal ADHI gene in these strains. Strain A86, however, appeared to carry the a-ald gene elsewhere in the genome, and the presence of a tandem copy of the expression 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
2799
ct-ALDC BREWER'S YEAST STRAINS
A
B
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control strain A15; PGKI integrants FIG. 3. Yeast growth and flocculation (A) and fermentation rate (B) in brewing trials. Symbols: 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 strains (data not shown). The Southern pattern of the PGKI integrants was more complex (Fig. 2B). All four recombinant strains carried bacterial vector sequences. Strains A85, A92, and A95 all gave the same pattern when compared with each other when hybridized with a-ald-, PGKI-, and Bluescribe M13+-specific probes, suggesting a common mode of integration. The bacterial vector appeared to have integrated together with
the a-ald gene into a single locus in the genome. Disappearance of the 4.0-kb PGKI promoter-specific band, seen in the control strain A15, indicates that integration occurred at the PGKI locus. Overall, the hybridization pattern supports the proposal that one copy of plasmid pKB105 (strain A92) or pKB107 (strains A85 and A95) integrated through single recombination events via the promoter sequences into the PGKI locus, generating an EcoRI fragment of about 7.1 kb 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 Yeast strain
Origin of gene
Promoter
Site of
integration mtegratlon Copy no.*
Control strain A15 PGKI integrant A85 A95 A89 A92
K. terrigena K. terrigena E. aerogenes E. aerogenes
PGKI PGKI
PGKI PGKI Unknown PGKI
ADHI integrant A86 A91 A90
K. terrigena K. terrigena E. aerogenes
ADHI ADHI ADHI
Unknown ADHI ADHI
PGKI PGKI
1 1 >1? 1 1-2? 1 1
a-ALDC activity2
Bacterial vector
~sequences present
A^4
ai-Acetolactate (%)
-
0.36
0
+ + + +
3.24 NDb 3.07 ND
39.3 ND 46.4 ND
-
1.13 ND 0.57
30.0 ND 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 decarboxylated to acetoin in yeast ceil extracts (protein content, about 1.6 mg/ml) after incubation for 30 min. b ND, not determined.
enzymatically
2800
APPL. ENVIRON. MICROBIOL.
BLOMQVIST ET AL.
vector sequence and fragments of 2.5 and 1.8 kb containing the endogenous PGKI gene with the promoter and terminator, 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 sequences and both of the terminator sequences remained 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 have been shown to carry two structurally different forms of many chromosomes (11), and this could also be the case with chromosome XV on which the PGK1 gene is located. In transformant A89, all endogenous PGKl-specific bands remained intact, indicating integration of the expression cassette with vector sequences elsewhere in the genome. The presence of bacterial sequences in the PGK1 integrant strains could be a result of, e.g., incomplete digestion of the plasmids upon release of Bluescribe vector sequences from the expression cassettes. A single recombination event of the complete plasmid at the PGK1 locus might have been favored over gene replacement with the linear expression cassette. Gene replacement of the PGK1 locus of strain A15 is, however, possible with this strategy, as we demonstrated when constructing glucanolytic brewer's yeast strains (30). Growth and fermentation. Growth of the integrant yeast strains during the 50-liter fermentations (Fig. 3A), as well as the fermentation rate with these strains (Fig. 3B), was the same or faster than with the control strain A15. Alcohol contents were approximately the same in all trial beers (Table 2). Formation of diketones and lagering. The a-ALDC activity of the PGK1 integrant strain A89, carrying the oa-ald gene of E. aerogenes, was so effective that the total diacetyl content (free diacetyl plus a-acetolactate) never reached the taste threshold value of 0.02 mg/liter during fermentation (Fig. 4). With the other PGK1 integrant strains A85, A92, and A95, some diacetyl was formed during fermentation, but by the end of fermentation it had already decreased at least to its taste threshold value. No lagering was required for the trial beers produced with these PGK1 integrant strains, and the production time of beer was shortened by 2 weeks. With all ADH1 integrant yeast strains, A86, A90, and A91, the formation of diacetyl was relatively high during fermentation, and lagering for 4 to 5 days was necessary to remove diacetyl from the trial beers produced with these strains. However, even with these strains the lagering time was shortened by as much as 10 days compared with that 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-pentanedione contents found in green beers do not affect beer flavor, becatise its taste threshold value is as high as 1.0 to 1.5 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 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 beers. The foam stability of beer was also unaffected. The formation of fusel alcohols (higher alcohols) is growth associated and linked to amino acid synthesis (5, 24). In transamination, yeast prefers the amino groups of the amino
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2801
,a-ALDC BREWER'S YEAST STRAINS VOL. 57, 1991
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FIG. 4. Formation and reduction of total diacetyl during fermentation and lagering. Symbols represents the taste threshold value of diacetyl.
acids relevant in this study, leucine, isoleucine, and valine. The hydroxy acids formed in deamination reactions are decarboxylated to the respective alcohols, resulting in 3-methyl butanol (i-amyl alcohol), 2-methyl butanol (optically active amyl alcohol), and i-butanol, respectively. Thus, due to the slightly better growth of the recombinant yeast strains, the contents of fusel alcohols were also slightly higher in beers produced with the oe-ALDC yeasts than in the control beer (Table 2). The formation of esters is considered to be competitive with growth (19). Ester formation results from esterification of ethanol or higher alcohols with fatty acids, resulting mainly in acetate esters. The ester contents were very similar in all trial beers (Table 2). Presumably due to the slightly lower amount of other flavor compounds, e.g., fusel alcohols and esters, diacetyl was tasted in beer produced
as
in the legend to Fig. 3. The broken line
with strain A90 but not in beer produced with strain A86, although in both beers the concentration of diacetyl was the limit value, 0.02 mg/liter. Strain stability. One strain of each type of the ao-ALDC yeasts was recycled in comparison with the control strain A15 in seven successive 50-liter fermentations. No significant alteration was observed in either the fermentation patterns or the flavor profiles of beers produced with these recycled yeasts. The formation of diacetyl in fermenting wort also remained typical for beers produced with each strain (Fig. 6). The fermentation results with strains A85, A86, A89, and A90 were very similar to those obtained earlier (31). Between the two sets of trials the strains were maintained under liquid nitrogen. Altogether during this study, 44 pilot-scale (50-liter) fermentations were carried out, and 19 bottled trial beers were produced with these
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8
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APPL. ENVIRON. MICROBIOL.
BLOMQVIST ET AL.
2802
TABLE 3. Amounts of free amino acids in wort and in green beers produced with a-ALDC-active yeast strainsa
Cycle A15
1 2 44
i
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i
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6
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I 7-71 ---I I
Amt of amino acid (mg/liter)
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71
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5 6 7~
Yeast strain
Amino acid A15
A85
A86
A89
A90
Group 1 Arginine Histidine Leucine Lysine
237
