Recombinant brewer's yeast strains suitable for accelerated brewing

Journal of Biotechnology, 14 (1990) 285-300 Elsevier

285

BIOTEC 00497

Recombinant brewer's yeast strains suitable for accelerated brewing M.-L. Suihko, K. Blomqvist *, M. Penttil~i, R. Gisler and J. Knowles VTT, BiotechnicalLaboratory, Espoo, Finland

(Received 25 October 1989; accepted 26 November 1989)

Summary Four brewer's yeast strains carrying the a-aid gene of Klebsiella terrigena (ex. Aerobacter aerogenes) or of Enterobacter aerogenes on autonomously replicating plasmids were constructed. The a-aid genes were linked either to the A D C 1 promoter or to the PGK1 promoter of yeast Saccharornyces cerevisiae. In pilot scale brewing (50 1) with three of these recombinant yeasts the formation of diacetyl in beer was so low during fermentation that lagering was not required. All other brewing properties of the strains were unaffected and the quality of finished beers was as good as that of finished beer prepared with the control strain. The total process time of beer production could therefore be reduced to 2 weeks, in contrast to about 5 weeks required in the conventional process. a-Acetolactate decarboxylase; Brewer's yeast; Recombinant strains; Accelerated brewing

Introduction The conventional process for production of lager beer includes preparation of wort, fermentation (also called main or primary fermentation), lagering (also called secondary fermentation), stabilization, filtration, pasteurization and filling. During fermentation the fermentable sugars are converted to ethanol and carbon dioxide

Correspondence to: M. Suihko, VTT, Biotechnical Laboratory, SF-02150 Espoo, Finland. * Present address: Institute of Microbiology,University of Ume~, 90187 Ume~i, Sweden

0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

286 and the flavour compounds are formed. During lagering the flavour formed is matured. The whole process takes at least 4 to 5 weeks. The lagering is the most time consuming step, lasting 2 weeks or more and requiring a large refrigerated space. Acceleration of this step would therefore be of great economic importance to the brewing industry. During fermentation brewer's yeast produces the a-acetohydroxyacids, aacetolactic acid and a-acetohydroxybutyric acid. These are used as precursors in the synthesis of valine and isoleucine (Haukeli and Lie, 1972; Petersen, 1985; Nakatani et al., 1984), but a small amount leaks out of the yeast cells and is spontaneously decarboxylated to the corresponding diketones, diacetyl and 2,3-pentanedione. This decarboxylation is slow at the temperatures used in lager beer processes. Due to its strong buttery flavour diacetyl is considered to be very unpleasant in finished beer. It has been proposed that the main reaction needed for maturation of beer is the enzymatic reduction of diacetyl to acetoin by yeast cells during lagering (Dellweg, 1985; Masschelein, 1986). The flavour threshold value of diacetyl has been reported to be from 0.02 to 0.10 mg l-1 depending on the type of beer and the method of determination. The colorimetric methods give much higher values, because they measure, at least partially, both diketones and their precursors, whereas the gas chromatographic methods used most frequently today measure the different compounds individually (Morrison and Benediak, 1987). The taste threshold values of 2,3-pentanedione and acetoin are much higher than that of diacetyl, 1.00-1.50 mg 1-1 and 30-100 mg 1-l, respectively. The reaction continues from acetoin to 2,3-butanediol and other components. The threshold level of 2,3-butanediol is as high as 500 mg 1-1 (Scherrer, 1972). The maximal diacetyl content in the beer is usually between 0.3 and 2.5 mg 1-1, depending on the process conditions, composition of wort and yeast strain used. At the end of the fermentation the total diacetyl content varies between 0.1 and 0.5 mg 1-1, demanding different times for lagering. The enzyme a-acetolactate decarboxylase (a-ALDC), EC 4.1.1.5, decarboxylates a-acetolactate directly to acetoin and a-acetohydroxybutyrate to 3-hydroxy-2-pentanone without the formation of diacetyl or 2,3-pentanedione. This enzyme exists in some bacteria, but not in higher organisms such as yeasts (Godtfredsen et al., 1983a, 1984b). It has been purified and characterized from the bacteria Aerobacter aerogenes (Loken and Stormer, 1970), Lactobacillus casei (Rasmussen et al., 1985) and Bacillus brevis (Jensen et al., 1987). These enzymes have been used successfully for accelerating the maturation of green beers (Godtfredsen and Ottesen, 1982; Godtfredsen et al., 1983b, 1984a). However, only the Bacillus enzymes have been stable under the conditions of fermentation (Godtfredsen et al., 1983b, Jensen et al., 1987). The gene coding for the enzyme (a-ald) has been isolated from the bacteria Enterobacter aerogenes (Sone et al., 1987a, Blomqvist et al., 1989), Streptococcus lactis subsp, diacetylactis (Goelling and Stahl, 1988) and Klebsiella terrigena (Blomqvist et al., 1989). K. terrigena is a new name for Aerobacter aerogenes in the current bacterial taxonomy (Orskov, 1984). In this paper we describe the construction of brewer's yeast strains carrying two different a-ald genes on autonomously replicating plasmids and the results of brewing at pilot scale with these recombinant strains.

287 Materials and Methods

Vectors and strains The plasmids Bluescribe M13 + (Stratagene, USA), pMA91 (MeUor et al., 1983), pET13:I (Henderson et al., 1985) and pAAH5 (Ammener, 1983) were used in construction of the yeast plasmids. The bacteria Klebsiella terrigena VTT-E-74023 and Enterobacter aerogenes VTT-E-87292 and the lager brewer's yeast strains VTT-A-63015 and VTT-A-66024 were from the VTT Collection of Industrial Microorganisms (Suihko, 1989). Deletion mutagenesis Deletion mutagenesis was carried out as described by Blomqvist et al. (1989) using PstI digestion before transformation of E. coli JM109 to reduce the background of wild type plasmids. The deletion was confirmed by dideoxy sequencing of the relevant regions from denatured plasmids (Zagursky et al., 1986). Transformation of brewer's yeast Method and growth media used in transformation of brewer's yeast using resistance to copper as selection (Henderson et al., 1985) have been described in detail earlier (Penttil~i et al., 1987). Measurement of a-ALDC activity of yeast transformants The diester, a-acetolactic acid ethylester acetate, was supplied by Oxford Chemicals, Ltd. (Brackley Northamptonshire, U.K.). It was hydrolyzed to a-acetolactate immediately before the assay as described by Olsen and Aunstrup (1984). Copper resistant transformants from NEPRA plates were inoculated into 5 ml of YPD medium containing 0.6 mM CuSO4 and incubated at 30 °C for 17-20 h. The cells were harvested by centrifugation at 3000 rpm for 10 min, washed once with 5 ml deionized water and resuspended with 1 ml 0.1 M phosphate buffer, pH 7.0. Ten microlitres of Zymolyase 60 000 enzyme solution (5 mg ml- a) was added, mixed and incubated at 37°C for 1 h. The cell suspension was then mixed vigorously by vortexing and centrifuged to remove the cell debris. To this cell extract was added 80 t~l of freshly hydrolyzed a-acetolactate substrate, the mixture was incubated at 30 ° C for 15-60 rain and acetoin formed, was detected by the Voges-Proskauer (VP) test by adding 500 t~l of 0.3% creatine solution, 600 /~1 of freshly prepared 5% a-naphthol in absolute ethanol and 300/~1 of 40% KOH to the tube. The intensity of red colour formed in 30 min was measured at 540 nm. Some of the final reaction mixtures were also analyzed by gas chromatography. The protein content of the samples was determined by the method of Lowry et al. (1951). Production of trial beers Yeast cells were inoculated from wort agar into 25 ml of wort-sugar solution (11% wort, w/w, and 10% sucrose solution, w/v, 1 : 1 supplemented with 0.1% yeast extract) and were incubated by shaking at 200 rpm and 25 ° C overnight. Three litres

288 of wort-sugar solution was inoculated with this culture and incubated by shaking at 125 r p m and 25 ° C for 2 d, after which the yeast cells were allowed to sediment for 2 d, the clear solution was decanted and the sedimented yeast cells were centrifuged; 125 g of this yeast mass was used for inoculation of 50 1 wort. Wort was obtained from a brewery and diluted to an original extract of 10.5% (w/w). The wort was aerated in keg vessels (h 50 1) using filtered compressed air at 3 1 min -1 for 30 rain. Immediately after aeration the wort was transferred to steel tubes (h 50 1) using filtered compressed air and inoculated with yeast (2.5 g 1-1). During the fermentation the temperature was 10°C. The fermentation was monitored daily ( 3 - 7 d) by determining the amount of yeast in suspension (dry weight, 10 mg ml-1), apparent extract (specific gravity) and total diacetyl (free diacetyl and a-acetolactate). At the end of the fermentation the alcohol content and yeast yield (the amount of sedimented yeast) were also determined. The green beer was transferred to lagering at 10 o C, or directly to stabilization for 3-5 d at 0 ° C if the amount of total diacetyl was less than 0.025 mg 1-1. After stabilization the beer was filtered (sheet filter + membrane), bottled, pasteurized for 30 min at 60 ° C and analyzed. Unless otherwise stated, the brewing and beer analyses were carried out as described in detail in Analytica-EBC (1987). Flavour compounds were determined by gas chromatography (Pajunen et al., 1987). The beers were evaluated by a tasting panel (12-15 persons) using the international flavour terms and scores from 1 to 5.

Analysis of vicinal diketones (VDK compounds) and acetoin by gas chromatography A head-space method with 2,3-hexanedione as internal standard was used. All samples were filtered through 0.22 /~m m e m b r a n e filters before analysis. The samples were pretreated in three different ways for different determinations. In the first procedure free diacetyl and free 2,3-pentanedione were determined from a 6 ml sample after 60 min incubation at 31°C encapsulated in a 20 ml glass vial containing 700 mg NaC1 to stabilize the liquid-vapour equilibrium. In the second procedure a-acetolactate and a-acetohydroxybutyrate were determined after thermal conversion of a-acetolactate to diacetyl and aacetohydroxybutyrate to 2,3-pentanedione by incubation for 1.5 h at 60 ° C. The vials were cooled in an ice-water bath for 15 min and diacetyl and 2,3-pentanedione were determined as in procedure 1. Procedure 2 resulted in free diacetyl + aacetolactate ( = total diacetyl) and free 2,3-pentanedione + a-acetohydroxybutyrate ( = total 2,3-pentanedione). The content of a-acetolactate or a-acetohydroxybutyrate was expressed as the difference in diacetyl or 2,3-pentanedione content between procedures 2 and 1. Total V D K could be expressed as the amount of both diketones and their precursors. In the third procedure acetoin was determined after oxidation of acetoin to diacetyl by boiling the samples for 20 rain with FeC13 (50%, w / v ) , FeSO 4 (30%, w / v ) and H2SO 4 (approx. 15%). After cooling the sample in an ice-water bath for 15 min, diacetyl was determined as in procedure 1. Procedure 3 resulted in free diacetyl + a-acetolactate + acetoin. The content of acetoin was expressed as the difference in diacetyl content between procedures 3 and 2. Procedure 3 also

289 provided the 3-hydroxy-2-pentanone content of the sample from the results of 2,3-pentanedione, analogously to acetoin. Gas chromatograph HP 5790 with EC detector and integration system HP 3396A were used for determination of the diacetyl and 2,3-pentanedione contents of the samples. The column used was DB-WAX 30 N (25 m, film 0.5 /~m). Injector temperature was 90 °C and detector temperature 125 ° C. The temperature program was: 30°C, 5 rain, 6 ° C rain -1, final temperature 95°C. Helium (2.5 ml min -1) was used as carrier gas and nitrogen (30 ml min -1) as make-up-gas. The typical retention time for diacetyl was approx. 7.4 min, for 2,3-pentanedione 9.8 min and for 2,3-hexanedione 11.8 min. The detection limit of the method was 0.005 mg 1-1 and the standard deviation for diacetyl +_4% and for 2,3-pentanedione +_7%.

Results

Construction of yeast plasmids carrying the a-aid gene of Klebsiella terrigena The a-aid gene of K. terrigena has been expressed in active form in a laboratory strain of S. cerevisiae (Blomqvist et al., 1989). For that experiment a plasmid pKB101 was constructed, carrying the a-aid gene without its 5' flanking regions, with the bacterial initiator codon GTG changed to ATG and the - 3 position G preceding the initiator changed to A. From this plasmid the a-aid gene was isolated and inserted between the yeast PGK1 gene promoter and terminator regions in the plasmid pMA91 to obtain the plasmid pKB007 (Fig. 1A) (Blomqvist et al., 1989). The plasmid pKB007 lacks a dominant selection marker and so it cannot be used directly for transformation of brewer's yeast. Thus the PGK1/a-aid expression cassette was released from the plasmid pKB007 and ligated to the vector pET13 : 1 as described in Fig. 1A to obtain the final expression plasmid pKB002. Plasmid pET13 : 1 carries the yeast CUP1 gene, which enables selection of transformants on the basis of their resistance to copper. To express the a-aid gene of K. terrigena under the control of the ADC1 promoter of yeast, the plasmid pKB006 (Fig. 1B) was first constructed. The ADC1 promoter and terminator regions were obtained from the expression vector pAAH5 as a 1.95 kb BamHI fragment which was filled in with Klenow and ligated to the vector pET13: 1, digested with HindlII and blunt-ended with Klenow. The same a-aid fragment which had been ligated to the PGK1 promoter (Fig. 1A), was inserted between the ADC1 promoter and terminator regions of the vector pKB006 as described in Fig. lB. This final expression vector was designated pKB003. Construction of yeast plasmids carrying the a-aid gene of Enterobacter aerogenes The E. aerogenes a-aM gene had previously been localized on a 3.5 kb fragment in plasmid pPL2 (Blomqvist et al., 1989). Sequence determination had shown that the complete gene could be released from this plasmid as a 1.4 kb EcoRI-BamHI fragment. This fragment was ligated after S1 treatment to Bluescribe M13 + vector at the SphI site which had been made blunt-ended with $1 nuclease. The resulting

290

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Fig. 2. The oligonucleotide(A) used for deletion mutagenesis (B) of the plasmid pPL3 to remove the 5' flanking region of the E. aerogenesa-aM gene and to change the -1 position C to T. plasmid pPL3 is shown in Fig. 2B. This clone still carries approx. 400 bps of the 5' flanking region of the a-aM gene, which were removed by deletion mutagenesis (Fig. 2B) using a specific oligonucleotide (Fig. 2A) in the same manner as previously described for the a-all gene of K. terrigena (Blomqvist et al., 1989). In the deletion mutagenesis of the E. aerogenes gene the -1 position T in the original sequence was changed to C (Fig. 2A), because C at this position is found in highly expressed yeast genes (Cigan and Donahue, 1987). The E. aerogenes gene starts with three A T G codons (Fig. 2A), which were left unchanged in the deletion procedure. To couple the a-all gene of E. aerogenes to the PGK1 promoter, the promoter and terminator regions of the PGK1 gene were first released from the vector pMA91 as a HindlII fragment and ligated to the HindlII site of the vector Bluescribe M13 + to obtain the plasmid pKB104 (Fig. 3A). Thereafter the a-all gene of E. aerogenes was released from the plasmid pPL4 as a 0.9 kb S a l I - H i n d l I I fragment and cloned to the plasmid pKB104 at the BgllI site as shown in Fig. 3A to obtain the plasmid pKB105. The P G K 1 / a - a l d cassette was released from this plasmid with HindlII and ligated to the HindlII site of the plasmid pET13 : 1 to obtain the final expression vector pKB008 (Fig. 3A) for brewer's yeast. In order to transfer the a-all gene of E. aerogenes into brewer's yeast under the

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control of the A D C 1 promoter, the same 0.9 kb S a l I - H i n d I I I fragment of plasmid p P I A as described above was cloned to the vector p K B 0 0 6 (Fig. 1B) at the H i n d I I I site as shown in Fig. 3B. The expression plasmid obtained was designated pKB009.

293 TABLE 1 Amount of free diacetyl, a-acetolactate and acetoin in the reaction mixture of yeast cell extracts incubated with a-acetolactate for 30 min. The a-aid gene of K. terrigena was expressed from the PGK1 (VTI'-A-87083) or the ADC1 (VTT-A-87076)promoter in the recombinant strains Yeast strain

mg 1-1 Free diacetyl

a-Acetolactate

Acetoin

Total

5 2 2

134 39 40

4 87 62

143 128 104

Control

VTT-A-63015 VTT-A-87083 VTT-A-87076

Construction of brewer's yeast strains producing a - A L D C The four expression vectors constructed, carrying the a-aid gene of K. terrigena or E. aerogenes linked to either the PGK1 or ADC1 promoter of yeast, were transformed to a commercial brewing strain using sphaeroplast transformation (Penttil~i et al., 1987). Expression of the a-aM gene was tested by measuring the a - A L D C activity of cell extracts of the copper resistant transformants. N o significant variation in a - A L D C activity was found between different transformants harboring the same plasmid. The brewer's yeast strains VTT-A-87083 and VTT-A-87076, transformed with plasmids pKB002 (regulatory regions of the PGK1 gene) and pKB003 (regulatory regions of the ADC1 gene), and carrying the a-aid gene of K. terrigena were chosen for further studies, as well as the corresponding strains VTT-A-88087 and VTT-A88088 transformed with plasmids pKB008 and pKB009, respectively, carrying the a-aid gene of E. aerogenes. The reaction mixtures of the a - A L D C activity measurement of the recombinant strains VTT-A-87083 (protein content 1.0 mg 1-1) and VTT-A-87076 (protein content 1.3 mg 1-1) were analyzed for free diacetyl, a-acetolactate and acetoin by gas chromatography (Table 1). The added substrate, a-acetolactate, remained unaltered in the reaction mixture of the control strain. Small amounts of diacetyl and acetoin were detected due to hydrolysis of the diester during storage and instability of a-acetolactate in the conditions of activity measurement. As a result of a - A L D C activity of the recombinant strains most of the added a-acetolactate was decarboxylated to acetoin and no diacetyl was formed. The total amount of reaction compounds detected was lower in the reaction mixtures of the recombinant strains than in that of the control strain, because the reaction continues from acetoin to other components in the recombinant strains. Brewing trials The brewing properties and formation of total diacetyl during fermentation with the recombinant strains and the control strain are shown in Figs. 4A-D. The fermentation rate, growth and flocculation of the recombinant strains were unaltered compared with the control strain. The only detectable difference was in the

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formation of diacetyl. The formation of diacetyl with the recombinant strains VTT-A-87083 (K. terrigena gene, PGK1 promoter) (Fig. 4A) and VTT-A-88087 (E. aerogenes gene, PGK1 promoter) (Fig. 4B) remained below the taste threshold value (0.020 mg 1-1) and so no lagering was carded out. The maximal amount of diacetyl with strain VTT-A-87076 (K. terrigena gene, ADC1 promoter) reached the threshold value (Fig. 4C), but by the end of fermentation it was only 0.005 mg 1-1 and again no lagering was required. Compared with the other recombinant strains the formation of diacetyl with strain VTT-A-88088 (E. aerogenes gene, ADC1 promoter) (Fig. 4D) was relatively high (0.190 mg 1-1 after 3 d), but was still significantly lower than that of the control strain (0.395 mg 1-1 after 3 d). With the recombinant strain VTT-A-88088 a lagering of 4 d was required, whereas the control strain required 21 d. The presence of the enzyme a - A L D C can be seen to affect not only the content of diacetyl but also that of 2,3-pentanedione (Fig. 5). The analyses of the bottled beers are shown in Table 2. The quality of the beers produced using the recombinant strains was as good as that of the control beer.

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Stability of the strain VTT-A-87083 An immobilized fermentation column (Kronl~Sf et al., 1989) was used for testing the stability of the strain VTT-A-87083. The percentage of cells containing plasmids in the carrier material as well as in the output from the column decreased linearly to 33% and 46%, respectively, during the 54 d of experiment. However, the a-ALDC activity of the strain was so strong that the diacetyl content of beer remained on the same level until the end of experiment. The diacetyl content in the beer using a conventional strain (VTT-A-66024) was on average 0.95 mg 1- ] but only 0.25 mg 1-1 when using the recombinant strain.

Discussion Shortening of the conventional brewing process has been one of the most favoured alternatives for improving the economy and increasing the capacity of a brewery with minimal investment cost. The process can be shortened either by accelerating the fermentation or lagering, or by producing beer in a continuous process. However, higher process temperatures, large amounts of yeast and stirring

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Fig. 5. Formation of total 2,3-pentanedione during fermentation. The symbols for the recombinant strains are: VTT-A-87076 (e), VTT-A-87083 (A), VTT-A-88087 (In) and VTT-A-88088 (tl). The values of the control strain VTT-A-63015 in three different runs are illustrated using the open corresponding symbols ( o , zx, t3).

TABLE 2 Quality of the bottled beers Analyses

Original extract Alcohol content Apparent extract Apparent attenuation Total diacetyl Total 2,3-pentanedione Flavour compounds Ethyl acetate i-Amyl acetate Ethyl caproate n-Propanol i-Butanol 2-Methylbutanol 3-Methylbutanol Taste evaluation *

Yeast strain used Control

VTT-A87083

VTT-A87076

Control

VqT-A88087

VTT-A88088

% (w/w) % (w/w) % (w/w) % mg 1-1 mg 1- ~

10.6 3.65 1.85 82.5 0.020 0.015

10.4 3.55 1.80 82.5 0.005 0.005

10.5 3.60 1.75 83.5 0.005 0.015

10.6 3.75 1.55 85.5 0.030 0.025

10.4 3.60 1.60 84.5 0.015 0.020

10.5 3.70 1.60 85.0 0.025 0.020

mg mg mg mg mg mg mg

13.3 1.0 0.2 11.2 10.2 14.3 40.6 4

16.8 1.3 0.2 10.4 9.7 14.4 36.5 3

13.1 0.9 0.2 11.5 8.2 12.3 36.8 3

13.5 1.0 0.2 11.6 10.1 14.7 41.8 3

1-1 1-1 1-1 1-1 1-1 1-1 1-1

10.2 0.7 < 0.05 11.2 8.4 13.6 41.7 3

19.8 2.3 < 0.05 12.4 12.5 18.4 51.7 4

* Score: 5 = excellent, 4 = very good, 3 = good, 2 = satisfactory, 1 = unacceptable.

297 or aeration during fermentation used for accelerating the process strongly stimulate the formation of off-flavours, mainly vicinal diketones, which necessitate a prolonged lagering (Pajunen and Makinen, 1975; Ahvenainen and Makinen, 1981). The same problem is present in a continuous beer process (M~ikinen, 1971; Baker and Kirsop, 1973) and in immobilized systems (Linko, 1985; Onaka et al., 1985; Masschelein et al., 1985; Curin et al., 1985; Nakanishi et al., 1985; KronKSf et al., 1989). Furthermore, the amounts of other flavour compounds have been reported to change in these systems. In order to control the amount of a-acetolactate produced by brewer's yeast, the genes responsible for the synthesis of valine and isoleucine (ILVI-ILV5) have been studied intensively (Petersen et al., 1983; Petersen, 1985; Dillemans et al., 1987; Holmberg et al., 1987). Strain improvement by breeding has resulted in some low-diacetyl lager yeast strains (Ramos-Jeunehomme et al., 1985; Gjermansen and Sigsgaard, 1986; Galvan et al., 1987; Goossens et al., 1987). Another possibility, investigated in this work, is to construct recombinant yeasts producing a-ALDC, an approach also taken by Sone et al. (1987b, 1988; Shimizu et al., 1989). The results presented in this study showed that in pilot scale trials the total brewing process can be shortened from the conventional 5 weeks to 2 weeks when recombinant strains are used. Furthermore, the quality of the trial beers was at least as good as that of the beer produced using the conventional yeast strain and process. Thus it seems that lagering is needed mainly for the removal of diacetyl and that other flavour compounds of the beer are of less importance. Although a-acetolactate and a-acetohydroxybutyrate are central intermediates in the synthesis of some amino acids (valine, isoleucine) as well as of some higher alcohols (fusel oils) and acetate esters (Dellweg, 1968), the recombinant strains constructed appeared not to require the addition of valine or isoleucine, since the growth of yeasts and the flavour profiles of beers were unaffected. The expression level of the a-ALDC enzyme was optimal when the Klebsiella gene was linked to the ADC1 promoter, but was apparently unnecessarily high when the gene was linked to the PGK1 promoter. However, the amount of diacetyl formed depends not only on the strain but also on the growth of yeast, which in turn is dependent on the composition of wort used for fermentation. Thus, in some conventional conditions and e.g. in immobilized systems where high amounts of diacetyl are often formed, the PGK1 promoter could be more advantageous.

Acknowledgements This research was financed by the Technology Development Centre (TEKES), Oy Panimolaboratorio-Bryggerilaboratorium Ab and Stiftelsen Svensk Etanolutveckling. We wish to thank warmly all those involved in this work at VTT Biotechnical Laboratory.

298 References

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