the level of mxr1 gene expression in brewing yeast during beer fermentation is a major determinant...

The level of MXR1 gene expression in brewing yeast duringbeer fermentation is a major determinant for the concentration

of dimethyl sul¢de in beer

J�rgen Hansen �, Susanne V. Bruun, Lene M. Bech, Claes GjermansenCarlsberg Research Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen-Valby, Denmark

Received 17 December 2001; received in revised form 15 March 2002; accepted 16 March 2002

First published online 12 April 2002

Abstract

DMS (dimethyl sulfide) is an important beer flavor compound which is derived either from the beer wort production process or via thebrewing yeast metabolism. We investigated the contribution of yeast MXR1 gene activity to the final beer DMS content. The MXR1-CAgene from Saccharomyces carlsbergensis (synonym of Saccharomyces pastorianus) lager brewing yeast was isolated and sequenced, andfound to be 88% identical with Saccharomyces cerevisiae MXR1. Inactive deletion alleles of both genes were substituted for theirfunctional counterparts in S. carlsbergensis. Such yeasts fermented well and did not form DMS from dimethyl sulfoxide. Overexpressionin brewing yeast of MXR1 from non-native promoters with various strengths and transcription profiles resulted in an enhanced andcorrelated DMS production. The promoters of MXR1 and MXR1-CA contain conserved Met31p/Met32p binding sites, and in accordancewith this were found to be co-regulated with the genes of the sulfur assimilation pathway. In addition, conserved YRE-like DNAsequences are present in these promoters, indicating that Yap1p may also take part in the control of these genes. @ 2002 Federation ofEuropean Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Brewing; Yeast; Dimethyl sul¢de; MXR1

1. Introduction

Dimethyl sul¢de, DMS, is a thioether of marked impor-tance for the aroma and £avor of beer as well as otherfermented or distilled beverages, e.g. whiskies. In lagerbeers, the DMS content regularly exceeds the taste thresh-old level of approximately 30 Wg l31 [1], which o¡ers anexplanation for the focus that has been on this sulfurcompound. Between the taste threshold level and approx-imately 100 Wg l31, DMS contributes to the distinctivetaste of some lager beers, whereas above 100 Wg l31,DMS may impart a £avor described as ‘cooked sweet-corn’, a sensation usually unwanted. DMS found in beermay be derived through thermal degradation of S-methyl-

methionine during kilning and wort preparation, and itwas originally believed that this is the pathway of largestsigni¢cance for the ¢nal DMS content in beer [2,3]. Sub-stantial evidence suggests, however, that enzymatic con-version of DMSO (dimethyl sulfoxide) to DMS by thebrewing yeast is of importance and perhaps even the ma-jor source of the ¢nal DMS content in beer [4]. See Annessand Bamforth [5] for a review on DMS formation in beerproduction.Saccharomyces yeasts contain an enzymatic activity that

reduces DMSO to DMS in an NADPH-dependent man-ner [6^8], and a so-called MetSO (methionine sulfoxide)reductase isolated from yeast [9,10] was suggested to beidentical to the DMSO reductase [7,8,11^13]. MetSO re-ductase has a much higher a⁄nity for MetSO than forDMSO [12,13] and MetSO inhibits DMSO reduction[7,8,13], indicating that the native function of the enzymeis reduction of oxidized methionines, but also that thedegree of MetSO formation during kilning of the maltwill probably a¡ect DMSO reduction.

Peptide MetSO reductases in di¡erent organisms repairoxidized methionine species in proteins, thereby restoring

1567-1356 / 02 / $22.00 @ 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 0 8 4 - 3

* Corresponding author. Tel. : +45 3327 5376; Fax: +45 3327 4764.E-mail address: [email protected] (J. Hansen).

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www.fems-microbiology.org

their biological activity. Recently, the MXR1 gene wasdisrupted in a strain of Saccharomyces cerevisiae, andthe disruptant was shown not to be able to reduce pep-tide-bound MetSO and to retain only 33% of the parentalreduction activity against free MetSO [14]. In a previousstudy we observed that disruption of the MXR1 gene in S.cerevisiae also abolished formation of DMS from DMSO,and that an MXR1 deletant did not seem to be impaired inits growth properties under a variety of conditions [15].We set out to construct an Saccharomyces carlsbergensis(synonym of Saccharomyces pastorianus) brewing yeastmutant without MXR1 activity, and to study the e¡ectsthat the mutation would have on formation of DMS fromwort DMSO and on the fermentation properties of themutant yeast.

2. Materials and methods

2.1. Strains of bacteria and yeast and microbiologicalmethods

Yeast strains employed and constructed during thisstudy are described in Table 1. Escherichia coli DH5K(Gibco BRL Life Technologies, Paisley, UK) was usedfor selection and propagation of plasmid DNA. SC (syn-thetic complete) and SD (synthetic dextrose) media wereprepared as described [16]. YPD medium contained 1%Bacto yeast extract (Difco, Detroit, MI, USA), 2% Bactopeptone (Difco) and 2% glucose. Brewer’s wort for com-mercial production of lager beer had a gravity of 14.5‡P(Plato) and was autoclaved before use. Yeast was grown at

Table 1Yeast strains used in this study

Strain Genotype Reference

S. cerevisiae S288C MATK SUC2 mal mel gal2 CUP1 [40]S. cerevisiae X2180-1A MATa SUC2 mal mel gal2 CUP1 [40]S. cerevisiae JH418 MATa SUC2 mal mel gal2 CUP1 str1v [32]S. carlsbergensis F252 NM Carlsberg strain collectionS. carlsbergensis JH345 NM [23]S. carlsbergensis C80-CG65 MATa [28]S. carlsbergensis C80-CG110 MATK [28]S. carlsbergensis LG159 MATa ura3-ca (chromosome V monosomic) L. Ho¡mann, personal communicationS. carlsbergensis JH268 MATK met2-ce met2-ca [23]S. carlsbergensis JH334 MATa met2-ce met2-ca [23]S. monacensis CBS1503 Centraalbureau voor Schimmelcultures,

The NetherlandsS. carlsbergensis JH360 NM MET2-CE met2-ce met2-ca MET2-CA [23]S. carlsbergensis JH767 MATa mxr1-ce MXR1-CA met2-ce met2-ca This studyS. carlsbergensis JH770 MATK mxr1-ce MXR1-CA met2-ce met2-ca This studyS. carlsbergensis JH869 MATa mxr1-ce mxr1-ca met2-ce met2-ca This studyS. carlsbergensis JH875 MATK mxr1-ce mxr1-ca met2-ce met2-ca This studyS. carlsbergensis JH884 MATK met2-ce met2-ca mxr1-ce mxr1-ca: :MET2-CA This studyS. carlsbergensis JH892 MATK mxr1-ce MXR1-CA met2-ce MET2-CA This studyS. carlsbergensis JH894 MATK mxr1-ce mxr1-ca met2-ce MET2-CA This studyS. carlsbergensis JH899 MATa mxr1-ce mxr1-ca met2-ce met2-ca : :pYC020 (MET2-CA) This studyS. carlsbergensis JH902 MATa mxr1-ce mxr1-ca MET2-CE met2-ca This studyS. carlsbergensis JH917(JH892UJH899)

NM mxr1-ce mxr1-ce mxr1-ca MXR1-CA met2-ce met2-ce MET2-CA met2-ca : :pYC020 (MET2-CA)

This study

S. carlsbergensis JH921(JH894UJH899)

NM mxr1-ce mxr1-ce mxr1-ca mxr1-ca met2-ce met2-ce MET2-CA met2-ca : :pYC020 (MET2-CA)

This study


NM mxr1-ce mxr1-ce mxr1-ca mxr1-ca: :MET2-CA met2-ce met2-ce met2-camet2-ca : :pYC020 (MET2-CA)

This study


NM mxr1-ce mxr1-ce mxr1-ca MXR1-CA MET2-CE met2-ce MET2-CAmet2-ca

This study


NM mxr1-ce mxr1-ce mxr1-ca mxr1-ca MET2-CE met2-ce MET2-CA met2-ca This study


NM mxr1-ce mxr1-ce mxr1-ca mxr1-ca MET2-CE met2-ce MET2-CA met2-ca This study


NM mxr1-ce mxr1-ce mxr1-ca mxr1-ca: :MET2-CA MET2-CE met2-ce met2-camet2-ca

This study


NM mxr1-ce mxr1-ce mxr1-ca mxr1-ca: :MET2-CA met2-ce met2-ce met2-camet2-ca : :pYC020 (MET2-CA)

This study


NM mxr1-ce mxr1-ce mxr1-ca mxr1-ca: :MET2-CA MET2-CE met2-ce met2-camet2-ca

This study

S. carlsbergensis JH911 NM G418-R [PADE6-MXR1] This studyS. carlsbergensis JH913 NM G418-R [PYML131-MXR1] This studyS. carlsbergensis JH941 NM G418-R [PTPI1-MXR1] This study

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30‡C (S. cerevisiae) or 20‡C (S. carlsbergensis), and trans-formed using the lithium acetate method [17].

2.2. DNA manipulations and sequencing

Plasmid DNA was prepared from E. coli as described[18], or using Promega Wizard Plus Midiprep DNA Puri-¢cation System (Promega Inc., Madison, WI, USA). DNAmanipulations were performed according to the manufac-turers of enzymes (Roche Molecular Biochemicals, Hvido-vre, Denmark; Promega, Madison, WI, USA; or NewEngland Biolabs Ltd., Beverly, MA, USA). Polymerasechain reaction (PCR) was performed using the ExpandHigh Fidelity PCR System (Roche Molecular SystemsInc., Branchburg, NJ, USA) and according to the manu-facturer. Nucleotide sequencing was performed according

to the manufacturer using PRISM AmpliTaq FS Dye Ter-minator Cycle Sequencing kit (Applied Biosystems Inc.).Sequencing reactions were processed in Applied Biosys-tems 373A or 310 sequencers, according to the properuser’s manuals. All oligonucleotide primers used for nu-cleotide sequencing and PCR ampli¢cation are describedin Table 2.

2.3. Isolation, cloning and sequencing of the MXR1-CAgene

The MXR1-CA gene from S. carlsbergensis was isolatedand cloned in the following way (illustrated in Fig. 1): a402-bp fragment of the MXR1-CA open reading frame(ORF) was ampli¢ed from S. monacensis genomic DNA,employing the primers MXR1-CA-1 and MXR1-CA-4

Table 2Oligonucleotide primers used in this study for nucleotide sequencing and PCR ampli¢cation

Name Nucleotide sequence

MXR1-CA-1 5P-CGTAGCGGCCGCATTAAATACGATCCAGCTAAAGATAAG-3PMXR1-CA-2 5P-CGTAGCGGCCGCCCATTTAGGTTGCCATTCTCCTTTAATCTT-3PMXR1-CA-3 5P-CGTAGCGGCCGCATTAAGTATGATCCAGCCAAGGATAAA-3PMXR1-CA-4 5P-CGTAGCGGCCGCCCATTTTGGTTGCCATTCTTCCTTTATTTT-3PMXR1-CA-SEQ1 5P-GATTGTACTGAGTGCACCAT-3PMXR1-CA-SEQ2 5P-CAATACGCAAACCGCCTCTC-3PMXR1-CA-UP 5P-CGGCAAGCTTACACCCACATGCTAATGTGATTAG-3PMXR1-CA-DOWN 5P-CGGCGAATTCCATTCAGATGCTGATTTGAAAGAGC-3PYER041w-1 5P-CGGCGAATTCCTTGGATGAAGCTGTCGCTAAGTTC-3PYER041w-2 5P-CGGCGAATTCGTAAAGTCCAATCTGAATACTTGG-3PYER041w-3 5P-CGGCGAATTCCCATTCAAGGCCGACCACTACAGA-3PSAH1-1 5P-CGGCAAGCTTCACGCTCAAGCATCACAATCAATGC-3PSAH1-2 5P-CGGCAAGCTTGAACACTCCTCTGTCCTTGACCAAC-3PSAH1-3 5P-CGGCAAGCTTCTGTGACAGTGATAGCAGTAGCAC-3PMXR1-CA-SEQ-F1 5P-TAGCGTAGAATAAGTCGA-3PMXR1-CA-SEQ-F2 5P-CTTAAATGATCGCATAGT-3PMXR1-CA-SEQ-F3 5P-CCAAAATGGGGTAATAAG-3PMXR1-CA-SEQ-F4 5P-TGACAGGATCGTTAGATG-3PMXR1-CA-SEQ-R1 5P-TAACTTGGGTGTCAAATT-3PMXR1-CA-SEQ-R2 5P-ATTCTTCAACAGTAAATG-3PMXR1-CA-SEQ-R3 5P-GTTCCTTGATCAGGTCCT-3PMXR1-CA-SEQ-R4 5P-ACTTAATTGTTTTAGAGA-3PMXR1-CA-D1-1 5P-ATTAGGCCGGCCTAGCGTAGAATAAGTCGACGCG-3PMXR1-CA-D1-2 5P-CGGCGAGCTCCGTGGTGGGGTCATGAATTCTG-3PMXR1-CA-D2-1 5P-CGGCGAGCTCGGCGCGCCTGAAAGAGCTAACTCAAATCAAA-3PMXR1-CA-D2-2 5P-ATTAGGCGCCGGATCCCTCGAGTAACTTGGGTGTCAAATTGACC-3PMSR1 5P-GGTAAAGCTTGGCGAGTCGAGAAAGGAAATC-3PMSR2 5P-GGTATCTAGAATCGATGGTTTTTGAAATAAGCGACGAC-3PMSR3 5P-GGTAGGATCCCATTATCTGAGAGAAATGTAG-3PMSR4 5P-GGTAGAATTCGTCGCCTGGTTAAAGGCTAAC-3PMET2-CE-F 5P-CCACAATTGCAAAACTCCCAAGCCC-3PMET2-CE-R 5P-CCACATACAAATCCAAAACATGAAG-3PMXR1-ORF-F 5P-CGGCTCTAGAATGTCGTCGCTTATTTCAAAA-3PMXR1-ORF-R 5P-CGGCGGATCCCTACATTTCTCTCAGATAATG-3PTPI 3P-end-F 5P-CTGAGGATCCGATTAATATAATTATATAAAA-3PTPI 3P-end-R 5P-CTGAGTCGACTTTCATAGACACAGTACTTAC-3PADE6pr-F 5P-ATTACGGCCGCCAACGATGAGTGAAGCTGAC-3PADE6pr-R 5P-CGGCTCTAGATGAACTTGACTTCTTTTGTTATGG-3Pyml131pr-F 5P-ATTACCGCGGTAGTTTGTCAGCTGCGAATAAC-3Pyml131pr-R 5P-ATTATCTAGAATCGTTATGCGATCTTTTCTAG-3P

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(annealing at 38‡C, 1.25 mM MgCl2). The primers con-tained NotI sites at their 5P-ends, and the fragment wasinserted NotI in the pYC000 vector [19], creating plasmidpSB4. The insert of pSB4 was sequenced by using theprimers MXR1-CA-SEQ1 and MXR1-CA-SEQ1, whichare homologous to pYC000 sequences adjoining the NotIsite. The obtained nucleotide sequence was used to devisethe sequence-speci¢c primers MXR1-CA-UP (containing aHindIII site at its 5P-end) and MXR1-CA-DOWN (con-taining an EcoRI site at its 5P-end). A new series of PCRreactions, using S. monacensis genomic DNA as template,were carried out (annealing at 44‡C, 1.5 mM MgCl2), us-ing combinations of MXR1-CA-UP with the primersYER041w-1, -2, and -3 (HindIII sites included) or combi-nations of MXR1-CA-DOWN with the primers SAH1-1,-2, and -3 (EcoRI sites included). Fragments of the ex-pected size obtained with MXR1-CA-UP/YER041w-3and MXR1-CA-DOWN/SAH1-1 were inserted into plas-mid pUC18 [20], HindIII or EcoRI as proper, resulting inthe plasmids pSB13 and pSB8, respectively. Preliminarynucleotide sequences of the subcloned MXR1-CA ‘up-stream’ and ‘downstream’ regions were obtained, usingstandard pUC18 sequencing primers. The primersMXR1-CA-D1-1 (contains an FseI site at the 5P-end)and MXR1-CA-D2-2 (contains a NarI site at the 5P-end)were used to PCR-amplify the complete MXR1-CA re-gion, using S. carlsbergensis LG159 genomic DNA as tem-plate (annealing at 55‡C, 1.5 mM MgCl2). The obtainedDNA fragment was inserted FseI^NarI into plasmidpYC021 [19], creating plasmid pJH223. The complete nu-cleotide sequence of the 1162-bp MXR1-CA fragment wasobtained by sequencing the insert in plasmid pJH223 or bydirect sequencing of the PCR fragment, using the primersMXR1-CA-SEQ-F1, -F2, -F3, -F4, -R1, -R2, -R3 and-R4.

2.4. Construction of MXR1 deletion alleles andMXR1 overexpression cassettes

An inactive deletion allele of MXR1 to be used for two-step deletional inactivation of MXR1-CE in brewing yeastwas constructed as follows: a 727-bp DNA fragment, cov-ering nucleotide positions 3703 to +24 relative to the startcodon of MXR1, was generated by PCR using the oligo-nucleotide primers MSR1 and MSR2, and S. cerevisiaeS288C genomic DNA as template for the reaction. A719-bp fragment covering nucleotide positions +535 to+1253 was likewise generated, employing the oligonucleo-tide primers MSR3 and MSR4. The 727-bp DNA frag-ment was inserted into the HindIII and XbaI sites ofpUC18. The 719 bp, truncated to 483 bp due to an over-looked internal EcoRI restriction site, was inserted intothe BamHI and EcoRI sites of pUC18, creating plasmidpJH119. In the HindIII site of this plasmid was inserted a3.7-kb MET2-CA-containing DNA fragment from p19-6[21], thus creating pSB7 (Fig. 2). An inactive deletion allele

of MXR1-CA to be used for two-step deletional inactiva-tion of MXR1-CA in brewing yeast was constructed in thefollowing way: a 514-bp DNA fragment, covering nucle-otide positions 3203 to +311 relative to the start codon ofthe gene, was generated by PCR, using oligonucleotideprimers MXR1-CA-D1-1 and MXR1-CA-D1-2, andS. carlsbergensis LG159 genomic DNA as template forthe reaction. A 577-bp DNA fragment, covering nucleo-tide positions +586 to +1162 was generated likewise (usingthe primers MXR1-CA-D2-1 and MXR1-CA-D2-2). The514-bp fragment was inserted FseI^SacI and the 577-bpfragment SacI^NarI in plasmid pYC020 [19], thus creatingplasmid pJH227 (Fig. 2).

For one-step inactivation of MXR1-CA in brewingyeast, the plasmid pJH232 was constructed in the follow-ing way: An XhoI^SalI fragment containing the inactiveMXR1-CA gene from pJH227 was inserted in the SalIsite of pUC19 [20], thus creating plasmid pJH228. In theAscI site (incorporated within the SacI site of primerMXR1-CA-D2-1) of pJH228 was inserted the MET2-CA-containing AscI fragment of pJH227, creating pJH232(Fig. 2).

For overexpression of MXR1, plasmids pJH235 (PTPI1-MXR1), pJH238 (PADE6-MXR1) and pJH247 (PYML131-MXR1) were created as follows: a PCR-ampli¢ed MXR1ORF-containing DNA fragment (using the primersMXR1-ORF-F and MXR1-ORF-R) was inserted XbaI^BamHI in the same restriction sites of plasmid pPF9[22], which contains the TPI1 promoter, thus creatingplasmid pJH167. A 472-bp TPI1 3P-end fragment was am-pli¢ed from S. cerevisiae S288C DNA, using the primersTPI 3P-end-F and TPI 3P-end-R, and inserted BamHI^SalIin pJH167, creating pJH176. A 372-bp ADE6 promoterregion was PCR-ampli¢ed using the primers ADE6pr-Fand ADE6pr-R and substituted for the TPI1 promoterfragment (EagI^XbaI) of plasmid pJH176, creatingpJH220. Next, the SacII^SalI fragments of pJH176 andpJH220, containing the entire MXR1 expression cassettes,were transferred to plasmid pYC030 [19], creating plas-mids pJH235 and pJH238, respectively. Finally theSacII^XbaI ADE6 promoter-containing fragment ofpJH238 was substituted with a 1781-bp PCR-fabricatedYML131r promoter fragment (using primers YML131pr-F and YML131pr-R), thus creating plasmid pJH247.

2.5. Crossing of brewing yeast and selection forallotetraploid mating products

Allodiploid yeasts of opposite mating type were matedon the surface of YPD plates at 20‡C and allotetraploidmating products were enriched and selected for in richgalactose medium as described earlier [23].

2.6. Flask and pilot plant fermentation experiments

For £ask fermentation experiments, yeast was inoculat-

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ed from freshly grown plate cultures into 200 ml growthmedium (either SD medium or autoclaved brewer’s wort)in 500-ml polypropylene Erlenmeyer £asks. Growth pro-ceeded through a 10-day period at 20‡C (50 rpm shaking)with CO2 locks. Samples were taken through the side ofthe £asks with syringes into evacuated blood samplingtubes [24]. For manual EBC tall tube fermentations, yeastwas propagated in brewer’s wort at 14‡C, and 14 g of wetyeast was added to 2 l of aerated brewer’s wort in an EBCtube [25]. The temperature during the ¢rst 6 days ofgrowth was 14‡C, after which it was lowered to 7‡C andkept there for the last 24 h to allow the yeast to settle tothe bottom of the tubes. Samples were taken anaerobicallyeach day through silicone membranes using evacuatedblood sampling tubes [24]. For pilot plant fermentationtrials at 50-l scale, yeast was propagated in brewer’swort and then inoculated into fresh brewer’s wort (aeratedto 8^10 ppm O2) at 1.5U107 cells ml31 in 50-l cylindro-conical fermentation vessels. The main fermentation pro-ceeded at 14‡C until the decrease in apparent extract wasless than 0.1‡P a day. Then the temperature was decreasedto 8‡C for maturation, and the yeast was harvested. Theharvested yeast was used for the subsequent brewing gen-eration.

2.7. Northern analysis

Total yeast RNA was prepared from frozen cell pelletsusing the Bio101 FastRNA Kit-Red (Bio101 Inc.). FifteenWg RNA from each preparation was electrophoresed onformaldehyde gels, the separated RNA blotted onto Hy-bond-N nylon membrane (Amersham Pharmacia BiotechInc.), and the RNA cross-linked to the membrane byUV irradiation. [32P]dCTP-labelled DNA probes wereproduced by random primed labelling (Roche MolecularBiochemicals) of ACT1 (as an internal control) orMXR1 DNA (coding regions), and Northern hybridi-zations were performed at high stringency, as described[18].

2.8. Analysis of content of DMS aroma compounds

DMS was measured using static headspace gas chroma-tography with sulfur-speci¢c detection (Sievers 350B sulfurchemiluminescence detector). Headspace sampling wasperformed using an automated headspace sampler (PerkinElmer HS-40) with a 0.03-min injection time. The limit ofdetection for this method is 32 nM and the standard de-viation is always below 10%. Higher alcohols, esters, ace-taldehyde, diacetyl and pentanedione were measured bygas chromatography.

3. Results

3.1. The S. carlsbergensis brewing yeast MXR1-CA gene is88% identical to S. cerevisiae MXR1

S. carlsbergensis is usually found to contain two diver-gent types of each gene, of which one is identical to thecorresponding S. cerevisiae gene (the ‘-CE’ type, forS. cerevisiae-like) and one is di¡erent (the ‘-CA’ type, forS. carlsbergensis-speci¢c) (see, e.g., Kielland-Brandt et al.,1995, [26]). While the S. cerevisiae MXR1 gene was al-ready characterized [15], and thus the sequence of theS. carlsbergensis MXR1-CE known, the MXR1-CA genewas not, and hence work was initiated to isolate and char-acterize MXR1-CA. No screenable phenotype is associatedwith an mxr1 mutation, meaning that complementationcloning with gene libraries could not be used to obtainthe MXR1-CA gene. In our earlier study [15] it wasshown, by Southern analysis, that while S. carlsbergensiscontains two divergent types of MXR1, the MXR1-CEgene is the only type found in S. cerevisiae, while theMXR1-CA gene is the only type to be found in S. mona-censis strain CBS1503. To avoid any contaminatingMXR1-CE background, we therefore used genomicDNA from S. monacensis for PCR ampli¢cation of theMXR1-CA gene. The strategy was to ¢rst amplify a part

Fig. 1. Strategy for the isolation and sequencing of the MXR1-CA gene.

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of the S. monacensis MXR1-CA ORF, sequence this frag-ment, and then amplify fragments spanning the non-cod-ing ‘gap’ from the nearest upstream ORF to the sequencedpart of the MXR1-CA gene, and from the nearest down-stream ORF to the sequenced part of the MXR1-CA gene(Fig. 1). We thus hypothesized that the gene order is con-served between S. cerevisiae and S. monacensis, and alsothat ORF sequences are in general so conserved that PCRwith S. cerevisiae-derived oligonucleotide primers wouldbe feasible. To ¢rst amplify a part of the MXR1-CAORF, the primers MXR1-CA-1, -2, -3 and -4 were used.MXR1-CA-1 and -2 are homologous to the region of S.cerevisiae MXR1 just downstream of the start codon,while MXR1-CA-3 and -4 are homologous to regionssomewhat upstream of the stop codon. While MXR1-CA-2 and -4 are identical to the corresponding S. cerevi-siae nucleotide sequence, MXR1-CA-1 and -3 were de-signed to ¢t the codon bias of genes in S. cerevisiae forthe correspondingly encoded amino acids. All combina-tions of the four primers were tried out in preparativePCR, and at varying annealing temperatures and MgCl2concentrations, but only with the combination of MXR1-CA-1 and MXR1-CA-4 was the proper 402-bp MXR1-CAfragment obtained. Sequencing of this fragment in plasmidpSB4 allowed the de¢nition of the MXR1-CA sequence-speci¢c primers MXR1-CA-UP and -DOWN. MXR1-CA-UP reads ‘upwards’ from within the MXR1-CA ORF, inthe direction of the ORF YER041w 3P-end, while MXR1-CA-DOWN reads ‘downwards’ from within MXR1-CA, inthe direction of the SAH1 gene 3P-end. PCR reactionswere now performed, in which MXR1-CA-UP was com-bined with each of the primers YER041w-1, -2 and -3, andMXR1-CA-DOWN with SAH1-1, -2 and -3. MXR1-CA-UP combined with YER041w-3 resulted in a DNA frag-ment of around 450 bp, representing the MXR1-CA ORFupstream region, and MXR1-CA-DOWN combined withSAH1-1 and SAH1-3 resulted in fragments of approxi-mately 600 and 500 bp, respectively, representing MXR1-CA ORF downstream regions. Nucleotide sequencing ofthese three DNA fragments subcloned in plasmids pSB13,pSB8 and pSB10 resulted in a preliminary sequence of thewhole MXR1-CA region. This sequence, however, was ob-tained from S. monacensis, which could have small se-quence variations with the MXR1-CA allele of S. carlsber-gensis brewing yeast. Therefore the obtained sequenceinformation from S. monacensis MXR1-CA was exploitedto design sequence-speci¢c primers that could be used toPCR-amplify the MXR1-CA region from an S. carlsber-gensis strain. Strain LG159 is an allodiploid S. carlsber-gensis mater which has accidentally lost the one of itschromosome Vs that contain MXR1-CE sequences(L. Ho¡mann, personal communication and J. Hansen,unpublished results). Genomic DNA from LG159 wasused for PCR ampli¢cation of an 1162-bp MXR1-CAfragment with the primers MXR1-CA-D1-1 and MXR1-CA-D2-2. Direct sequencing of this PCR fragment or of a

cloned derivative in plasmid pJH223 con¢rmed the pre-liminary MXR1-CA sequence; no di¡erences were foundbetween S. monacensis and S. carlsbergensis MXR1-CA.The MXR1-CA coding region is 88% identical to S. cere-visiae MXR1, while the deduced amino acid sequencesfrom these two genes vary with 10%. The MXR1-CA se-quence has been submitted to GenBank with the accessionnumber AF367437.

3.2. A viable brewing yeast inactivated in the MXR1 genescan be constructed

To evaluate the importance of the brewing yeast MXR1genes for the DMS content in beer, we decided to inacti-vate all MXR1 genes in this yeast, or, alternatively, reducethe MXR1 gene dosage by three quarters. As described inSection 2, plasmid pSB7 was created for two-step inacti-

Fig. 2. Maps of plasmids containing MXR1 and MXR1-CA deletion al-leles (pSB7, pJH227 and pJH232). The pDRAW32 program was usedfor drawing the plasmids [41].

FEMSYR 1469 5-6-02


vation [27] of MXR1-CE, and plasmid pJH227 for two-step inactivation of MXR1-CA (Fig. 2). Both of theseplasmids are integration plasmids that contain theMET2-CA gene marker outside of the MXR1 gene dele-tion cassettes. The MET2-CA marker can be used forselection of integration in the allodiploid brewing yeastmaters JH268 and JH334 [23], which are met2v derivativesof the meiotic lager brewing yeast segregants C80-CG110and C80-CG65 [28] (see Table 1), that will form a func-tional allotetraploid brewing yeast upon mating with eachother. The plasmids pSB7 and pJH227 were linearized byrestriction digestion in their unique HpaI and BstZ17I re-striction sites, respectively, present within the MXR1 in-activation cassettes. This was performed in order to in-crease the integration frequency and to direct theintegration event to the MXR1-CE and -CA loci [29].The yeast strains JH268 and JH334 were thus transformedwith HpaI-linearized pSB7, and several hundred integrantswere selected as methionine prototrophs. Integration wascon¢rmed by PCR analysis showing the presence of anmxr1-ce deletion allele, and one integrant was subjectedto growth for several generations in rich medium(YPD+5 mM methionine) to allow for ‘loop-out’ of plas-mid DNA due to a secondary recombination eventthrough homologous MXR1 sequences. The culture wasdiluted and plated at a few thousand colonies per plateon YPD. The colonies were replica-plated to SC mediumwithout methionine, and methionine auxotrophs werepicked and pure-cultivated. Southern and PCR analysiswere applied to screen clones that had lost the wild-typeMXR1-CE gene from those that had lost the deletion al-lele together with the plasmid DNA. In this way the yeaststrains JH767 (MATa mxr1-ce MXR1-CA met2-ce met2-ca) and JH770 (MATK mxr1-ce MXR1-CA met2-ce met2-ca) were created (Table 1). Now these two strains weretransformed with BstZ17I-linearized pJH227, for a newround of gene inactivation, this time with MXR1-CA.Eventually a few strains were obtained, in which also theMXR1-CA gene had been substituted with the inactivemxr1-ca allele. These were strains JH869 (MATa mxr1-ce mxr1-ca met2-ce met2-ca) and JH875 (MATK mxr1-cemxr1-ca met2-ce met2-ca). We now wanted to re-introducefunctional MET2 genes in both of these strains, as well asstrain JH770, as it appears that two functional MET2 genecopies are necessary in a tetraploid brewing yeast for nor-mal sulfur assimilation to take place [23]. For strain JH875integration of a functional MET2 gene was obtained bytransformation of the yeast with a 3.7-kb HindIII MET2-CA-containing DNA fragment from p19-6 [21], followedby selection of prototrophic transformants on mediumwithout methionine. Proper integration of the wild-typeMET2-CA gene at the MET2-CA locus was con¢rmedby PCR. One such MET2-CA-containing yeast strainwas named JH894 (MATK mxr1-ce mxr1-ca met2-ceMET2-CA). Likewise, strain JH892 (MATK mxr1-ceMXR1-CA met2-ce MET2-CA) was created on the basis

of strain JH770. For unknown reasons it appeared di⁄cultto convert strain JH869 to methionine prototrophy in thismanner. Therefore, two alternative strategies were triedout: (i) insertion of a complete plasmid pYC020, contain-ing MET2-CA [19], at the MET2-CA locus (integrationwas directed to the MET2-CA locus by plasmid lineariza-tion in the MET2-CA gene at the PsiI site), or (ii) bytransformation with a MET2-CE fragment, prepared byPCR from S. cerevisiae S288C genomic DNA (using theprimers MET2-CE-F and MET2-CE-R). Both strategieswere successful, resulting in the strains JH899 (MATamxr1-ce mxr1-ca met2-ce met2-ca : :pYC020 [MET2-CA])and JH902 (MATa mxr1-ce mxr1-ca MET2-CE met2-ca).Yet another approach was tried out to disrupt the func-tional MXR1-CA gene in strain JH770 and at the sametime converting the strain to a methionine prototroph:JH770 was transformed with the SalI^PstI fragmentfrom pJH232 (Fig. 2), which contains the MXR1-CA se-quences of the plasmid used for MXR1-CA two-step dele-tion, embracing a functional MET2-CA gene. Thus, themiddle part of MXR1-CA in strain JH770 was substitutedwith MET2-CA. One of the resulting strains, the genotypeof which was con¢rmed by PCR, was named JH884(MATK mxr1-ce mxr1-ca : :MET2-CA met2-ca met2-ce).The genotypes of all these strains are described in Table 1.

Now the K-maters JH884, JH892 and JH894 werecrossed with the a-maters JH899 and JH902 to form allo-tetraploid hybrid yeasts that could be used in fermentationtrials, as described in Section 2. Of the resulting strains,JH917 and JH929 are yeasts in which only one out of theoriginal four MXR1 gene copies is still functional, and thestrains JH921, JH925, JH933, JH935, JH938, JH952 andJH953 are all yeasts completely without functional MXR1genes. From preliminary experiments, there were no indi-cations that any of these yeast strains would grow slowerthan their wild-type progenitors, whether on plates or inliquid growth medium.

3.3. A S. carlsbergensis mxr1v mutant yeast is unable toconvert DMSO to DMS

The ability of the S. carlsbergensis yeast strains JH917and JH929 (one active MXR1-CA gene) and JH921,JH925, JH933, JH935, JH938, JH952 and JH953 (no ac-tive MXR1 genes) to form DMS from DMSO was initiallytested by growth in SD medium containing 0.1 mMDMSO, as described in Section 2. After 10 days of semi-anaerobic growth, it was found that the reference strainJH345 was able to convert at least 7.3% of the DMSOpresent (some of the formed DMS may have evaporated),the strains with one active MXR1-CA gene could convertat least 2^3% of the DMSO, while less than 0.14% of theDMSO was converted by the strains without active MXR1genes (Table 3). Obviously, S. carlsbergensis yeast strainswithout MXR1 gene activity are basically unable to reduceDMSO to DMS.

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3.4. Up to 80% of the ¢nal beer DMS may be derived frombrewing yeast DMSO reduction

The strains JH917, JH929, JH921, JH933, JH938 andJH953 were tested for DMS formation in brewer’s wort inmanual 2-l EBC tall tube fermentations, with strain JH360as reference strain. As presented in Fig. 3, the concentra-tion of DMS is reduced by 80% after fermentation withthe mxr1 strains as compared to the reference strainJH360 (with 147.9 Wg l31). As 71.2 Wg l31 DMS waspresent in the wort from the beginning, and an averageof 32.2 Wg l31 was present in beer after fermentation withthe mxr1 strains, 39 Wg l31 or 55% of the original wortDMS has disappeared during fermentation with thesestrains, most probably due to CO2 purging. It is also evi-dent that the activity of one active MXR1-CA gene in theyeast strains JH917 and JH929 increases the ¢nal beerDMS content by 40%. However, this 13 Wg l31 only rep-resents 11% of the DMS formed when four active MXR1genes are present (reference strain JH360).

3.5. MXR1 overexpression enhances DMS productionduring beer fermentation

To further substantiate the hypothesis that MXR1 geneexpression is highly decisive for the amount of DMS pro-

duced, we proceeded to make hybrid DNA construct al-lowing MXR1 transcription at various strengths. The pro-moters of TPI1 (encoding triose phosphate isomerase),ADE6 (encoding phosphoribosylformyl glycinamidine syn-thetase), and YML131r (hypothetical ORF) were chosenfor their di¡ering dynamics and strength of expression oftheir native genes (K. Olesen and J. Hansen, CarlsbergResearch Laboratory, unpublished). Plasmids pJH235(PTPI1-MXR1) and pJH238 (PADE6-MXR1) were both lin-earized by restriction digestion with MunI at the uniqueMunI site in the TPI1 3P-end sequence, whereas pJH247(PYML131-MXR1) was linearized by restriction digestionwith BglII, cutting in the YML131r promoter sequence.The Carlsberg lager production strain F252 was trans-formed with the linearized plasmids and G418-resistanttransformant clones were isolated. The resulting yeaststrains JH911 (PADE6-MXR1), JH913 (PYML131-MXR1)and JH941 (PTPI1-MXR1) were tested in a 2-l EBC fer-mentation, and DMS content as well as MXR1 gene ex-pression were measured each day in the 10-day fermenta-tion (Fig. 4). Evidently, DMS production duringfermentation and the ¢nal DMS content are very wellcorrelated to the relative MXR1 transcription level. Itcan also be seen, however, that maximal DMS content isreached well after MXR1 transcription has peaked.

3.6. Inactivation of the MXR1 genes does not impede thefermentation performance of brewing yeast

It was imaginable that the lack of enzymatic activitiesfor reduction of oxidized protein-contained methionineresidues could impede fermentation performance. Wetherefore proceeded to evaluate the strains JH929 (oneactive MXR1-CA gene) and JH938 (no active MXR1-CAgenes) in a 50-l pilot plant fermentation. The Carlsbergbrewing strain F252 was chosen as the reference strain,so as to be able to compare the fermentation performanceand the £avor generation of the mxr1 mutants with a

Fig. 4. MXR1 overexpression and its e¡ect on DMS production duringbeer fermentation. Filled symbols denote relative MXR1 transcription,open symbols DMS content. Triangles, the MXR1 gene was transcribedfrom the YML131r promoter; squares, the MXR1 gene was transcribedfrom the ADE6 promoter; circles, the MXR1 gene was transcribed fromthe TPI1 promoter; diamonds, the MXR1 gene was transcribed from itsnative promoter. A.U.: arbitrary units.

Table 3DMS production from yeast strains with diminished or neutralizedMXR1 gene activity, grown under semi-anaerobic conditions in syntheticmedium (SD) containing 0.1 mM DMSO

Strain % of added DMSO present as DMS

JH345 7.3JH917 2.7JH929 2.6JH921 0.13JH925 0.10JH935 0.10JH933 0.11JH952 0JH938 0JH953 0

Fig. 3. DMS production from yeast strains with diminished or neutral-ized MXR1 gene activity (2-l EBC fermentations).

FEMSYR 1469 5-6-02


known pro¢le. The yeast strains were tested in three con-secutive fermentation generations, and as seen from Fig. 5,the fermentation performance of the mutant strains was atleast as good as that of the reference strain. The beersresulting from strains JH929 and JH938 were evaluatedby a trained taste panel. No signi¢cant di¡erences werefound between these beers and the one fermented by strainF252, whether the beer was fresh or force-aged by heating.Analysis of aroma compounds such as acetaldehyde, high-er alcohols and esters did not reveal any signi¢cant di¡er-ences between the reference beer and the beers producedusing the mutant yeasts (Table 4).

Table 5 shows the beer DMS content as an average ofthese three experiments. Evidently, production of DMS bythe reference strain was smaller in these experiments thanin the EBC tube fermentation experiments. However, thereference yeast in these experiments (F252) is not com-pletely isogenic with the mutant strains, and in consecutivefermentation experiments it was found that DMS produc-tion of JH360 is approximately twice as large as that ofF252 (data not shown). Anyhow, in this experiment therewas an average decrease in DMS content from F252- toJH938-fermented beer of 48%. The beer DMS contentafter fermentation with the mxr1 strain JH938 was notvery much lower than the original wort DMS content,indicating that only a small fraction of a start concentra-

tion of 30 Wg l31 DMS could be purged from the ferment-ing beer under these conditions.

3.7. At wort DMS concentrations normally encountered,61^72% of wort DMS appears to be purged by CO2

during fermentation

The construction of a brewing yeast without capabilityfor DMS production made possible studies of DMS purg-ing by CO2. The fermentations were performed in 2-l EBCtubes, and 10, 50, 100 or 200 Wg l31 DMS was added to 2 lof autoclaved brewer’s wort (autoclaving was performedto ensure that no inherent DMS was present) inoculatedwith yeast strain JH938 (mxr1). As a control experimentone tube containing autoclaved wort without DMS addi-tion was inoculated with the wild-type strain JH360. Ascan be seen from Fig. 6a, the majority of the DMS waspurged during the fermentation, represented in Fig. 6b asthe percentage decrease at the end of fermentation as afunction of the DMS start concentration. Under theseconditions, from 61 to 72% of wort DMS is purged atDMS concentrations most often found in brewer’s wort.That the ¢nal DMS level is reduced to a lower level inthese experiments than in the 50-l pilot fermentation ex-periments (Table 5) may be due to the di¡erence in dimen-sions between EBC tall tube fermentors and 50-l cylindro-conical fermentors, possibly leading to di¡erences in theability of carbon dioxide to purge DMS.

Fig. 5. Fermentation performance of yeast strains with diminished orneutralized MXR1 gene activity (50-l pilot plant fermentations). Circles,reference yeast strain F252; squares, mutant yeast strain JH929 (one ac-tive MXR1 gene); triangles, mutant yeast strain JH938 (no MXR1 activ-ity).

Table 4Production of aroma compounds from yeast strains with diminished or neutralized MXR1 gene activity in 50-l pilot fermentations (average of three fer-mentation generations)

F252 content (mg l31) S.D. JH929 content (mg l31) S.D. JH938 content (mg l31) S.D.

Acetaldehyde 1.7 0.61 2.3 0.57 2.4 0.511-Propanol 12 1.1 15 0.39 14 0.611-Butanol 0.41 0.12 0.3 0.045 0.48 0.122-Methyl-1-propanol 11 0.94 15 0.94 15 3.13-Methyl-1-butanol 54 1.9 61 2.6 56 5.7Ethylacetate 15 2.8 12 0.71 13 1.9Isobutylacetate 0.03 0.022 0.042 0.0098 0.041 0.0073Isoamylacetate 1.3 0.23 1 0.14 1.1 0.14Ethylhexanoate 0.1 0.012 0.09 0 0.083 0.0094Ethyloctanoate 0.13 0.025 0.12 0.024 0.11 0.037

S.D.: standard deviation.

Table 5DMS production from yeast strains with diminished or neutralizedMXR1 gene activity in 50-l pilot fermentations (average of three fermen-tation generations)

Strain DMS (Wg l31) S.D.

Wort 34.3 6.9F252 54.2 3.9JH929 35.1 4.3JH938 28.1 4.9

S.D.: standard deviation.

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3.8. The MXR1 gene is repressed by organic sulfur

The S. cerevisiae MXR1 gene is co-regulated in a cellcycle-speci¢c manner with the genes of the methioninebiosynthetic pathway (the MET genes) [30]. It is thereforequite possible that this gene is repressed by organic sulfuras are the MET genes [31,32], and that the content of, e.g.,methionine in the wort will therefore in£uence DMSOreduction. This hypothesis is strongly supported by thefact that the AAAAATGTG DNA motif, known to takepart in MET gene regulation through binding of theMet31p and Met32p transcription factors [31,33], ispresent in both MXR1 gene promoters, at a conservedlocation, 380 to 388 in MXR1-CA and 379 to 387 inMXR1. We studied MXR1 transcription in the S. cerevi-siae strain X2180-1A and in an str1 mutant of this yeast,JH418. The inability of methionine to repress the sulfurassimilation genes in an str1 strain indicates a cysteinebiosynthesis dependence of this repression mechanism[32]. Fig. 7 shows that MXR1 transcription can normallybe repressed by 5 mM L-methionine, but not in an str1mutant, consistent with a view that MXR1 transcription isregulated co-ordinately with the sulfur assimilation genes.However, it is also evident that MXR1 transcription is

induced in an str1 strain, and that the transcription de-creases with the addition of methionine to this strain.

4. Discussion

To be able to evaluate the contribution of MetSO re-ductase enzyme activity to the lager beer DMS content, weisolated and characterized the S. carlsbergensis-speci¢cMXR1 gene, MXR1-CA. The MXR1-CA ORF was foundto be 88% identical to the S. cerevisiae MXR1 ORF andthe deduced amino acid sequences 90% identical, quitecomparable to values from other sets of genes character-ized (e.g. [21,34,35]). Inactive deletion alleles of S. cerevi-siae MXR1 and S. carlsbergensis MXR1-CA were nowused to construct allodiploid S. carlsbergensis deletion mu-tants with partial or full inactivation of MetSO reductase,and after crossing of such mutants, reconstituted allotetra-ploid S. carlsbergensis brewing yeasts with no or one quar-ter of the normal MXR1 gene content were obtained.

Preliminary growth experiments indicated no impair-ment of the growth ability due to the gene deletions,and therefore we proceeded to test the impact of theMXR1 gene inactivations on production of DMS fromDMSO in synthetic medium. The most important ¢ndingfrom this experiment is that basically no DMS was formedfrom DMSO, if no active MXR1 genes were present, whilesome was formed if one single active MXR1-CA was left.Thus, approximately 7.3% of the DMSO added could befound as DMS after fermentation with the wild-type refer-ence strain (containing two active MXR1-CE and two ac-

Fig. 6. a,b: CO2 purging of DMS during fermentation. a: DMS contentin fermenting wort. Solid lines/open symbols represent fermentation ex-periments with mutant yeast strain JH938 (no MXR1 activity). Variousconcentrations of DMS were added to DMS-free wort. Dashed lines/¢lled symbols represent fermentation experiments with reference yeaststrain JH360, in which wort without DMS addition was used. b: De-crease in DMS concentration as a percentage of the start DMS concen-tration.

Fig. 7. Transcriptional regulation of the MXR1 gene in S. cerevisiae.Wt, wild-type reference S. cerevisiae strain X2180-1A; str1v, S. cerevisi-ae str1 mutant strain JH418. Yeast was grown to the exponential phasein SD medium. Then 5 mM L-methionine was added to half of eachculture, and after 4 h further incubation, the yeast cells were harvested.

FEMSYR 1469 5-6-02


tive MXR1-CA genes), while this fraction was decreased toapproximately 2.7% with the yeast mutants containingonly one active MXR1-CA gene. These conversion ratiosare somewhat lower than conversion ratios reported byothers [5,8]. However, those fermentation experimentswere usually performed at quite low temperatures (8‡C),and it was reported that between 8‡C and 20‡C, an up to10-fold decrease in DMS formation could be seen [8].

After testing of two of the strains with reduced, andfour with neutralized Mxr1p activity in 2-l EBC tubewort fermentations, several conclusions could be drawn.It appeared that as much as 80% of the ¢nal beer DMScontent originated from brewing yeast DMSO reduction inthis experiment (and hence only 20% came directly fromthe wort). This result is in very good accordance withearlier studies on brewing yeast DMS contribution [4].Furthermore, 55% of the original beer DMS content wasfound to disappear during fermentation, most probablydue to CO2 purging. Finally, one remaining MXR1-CAgene results in a DMS production that is only 11% ofthat in the strain with all four MXR1 genes active. Apossible explanation for this somewhat low value, wellbelow 25%, could be that MXR1-CA genes are less activethan MXR1-CE genes under the conditions chosen forfermentation, a situation that has been observed beforefor sets of homeologous genes in S. carlsbergensis brewingyeast [36].

We also tried overexpression of the S. cerevisiae MXR1gene in S. carlsbergensis brewing yeast, in a 2-l pilot fer-mentation. Overexpression was performed with three dif-ferent promoters of di¡erent strength (TPI1, ADE6 andYML131). It appeared that DMS production during fer-mentation increased in a manner proportional to the pro-moter strength. However, maximal DMS concentrationwas in all cases reached well after the MXR1 expressionhad peaked. This may either be due to the decrease in CO2

production and thus DMS purging during the course ofthe fermentation, or to competitive inhibition of Mxr1p byMetSO in the early period. In any event, the experimentshows very clearly that MXR1 expression is of utmostimportance for beer DMS content.

As the yeast MXR1 genes encode enzymes believed tosalvage oxidized methionine residues in cellular proteins[14], one could imagine that complete elimination of thesegenes would render the yeast cell quite susceptible to oxy-gen stress, and thus initial slow growth. We thereforetested strains JH929 (one remaining active MXR1-CAgene) and strain JH938 (MXR1 function abolished) in a50-l pilot fermentation trial. The Carlsberg productionstrain F252 was used as a reference strain in this experi-ment, as we wanted to judge the £avor character of thetest beers relative to an accepted £avor pro¢le. It appearedthat the mutant yeast strains attenuated the wort at leastas well as the reference yeast, and that the £avor pro¢lewas acceptable and very close to that of the reference beer.Thus, lack of MXR1 expression does not have any signi¢-

cant impact on fermentation ability or £avor pro¢le of theresulting beer. The results seem to suggest that oxidativedamage of protein methionine residues is not taking placein yeast cells during a beer fermentation, at least not to adamaging extent, quite in accordance with the mostly an-aerobic environment during the process. Alternatively,brewing yeast may contain an alternative enzymatic activ-ity that can reduce oxidized methionines.

In this experiment, the DMS content of the JH938 beerwas only reduced by 48% compared to the reference yeastbeer. This is probably due to the fact that F252 and JH938are di¡erent S. carlsbergensis isolates : in a wort fermenta-tion control experiment strain F252 produced approxi-mately half the amount of DMS of strain JH360, whichis isogenic to the mutant yeast strains. The fact that wortattenuation was actually slightly better with the mutantsthan with the reference strain is most likely also explainedby the strain di¡erence.

As strain JH938 does not produce DMS itself, we usedthis strain to evaluate to which extent CO2 purging ofDMS depends on the wort DMS concentration. It ap-peared that the degree of DMS stripping does dependon the DMS concentration in the wort; at typical wortDMS concentrations (10^100 Wg l31), from 61 to 72% waspurged during primary fermentation, at least under thechosen conditions (in 2-l EBC tall tubes). This is in verygood accordance with the results of earlier studies [4], butsomewhat in discrepancy with our results from the 50-lfermentation experiments (Table 5). The possible in£uenceof fermentor dimensions on DMS purging is a matter forfurther research.

When scrutinizing the MXR1 and MXR1-CA promoterregions, both were found to contain, at similar locations,the consensus sequence for the MET gene DNA elementresponsible for binding of the transcription factors Met31pand Met32p [31,33]. When studying MXR1 transcriptionwith or without methionine in the growth medium, wefound that MXR1 is indeed repressed by methionine. Fur-thermore, this repression seems to be cysteine biosynthe-sis-dependent, as complete repression by methionine wasnot possible in an str1 strain [32], strongly indicating thatMXR1 is regulated along with the genes of the sulfurassimilation and methionine biosynthesis pathway. How-ever, we also saw that MXR1 expression is induced in thestr1 mutant strain, without methionine addition, and thatthe addition of methionine lowers the expression level tothe wild-type level without methionine addition. It wasrecently shown that str1 mutants show signs of oxidativestress due to their lower content of cysteine and gluta-thione [38], and this led us to propose the following hy-pothesis: in an str1 mutant the more oxidative intracellu-lar environment results in a high MetSO/methionine ratio.This is somehow monitored and results in an induction ofMXR1 transcription. When methionine is added, the Met-SO/methionine ratio decreases and induction ceases.

At positions 3129 to 3123 in the MXR1 promoter and

FEMSYR 1469 5-6-02


at positions 3119 to 3113 in the MXR1-CA promoter, aconserved TGAGTAA sequence is found, very similar tothe so-called YRE sequence, which is a prerequisite toYap1p-mediated gene induction due to oxidative stress[39]. As it is known that the presence of MXR1 to someextent protects yeast cells against H2O2 oxidation damage[14,37], and that Yap1p transcriptional induction is en-hanced by H2O2 [39], Yap1p may be involved in inductionof MXR1 transcription in response to increases in theMetSO/methionine ratio. In any event, the methioninesensitivity of MXR1 implicates that the state of the intra-cellular sulfur metabolism as well as the wort content ofcompounds such as methionine will in£uence DMS pro-duction during fermentation.

To conclude, we were able to show that MXR1 expres-sion is a major determinant for the beer DMS content,and to construct a well-behaving lager brewing yeast with-out the ability to produce DMS. In addition, we showedthat the ¢nal DMS content in beer is dependent on manyfactors, amongst which are the wort DMS concentrationand probably also wort methionine content.

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the level of mxr1 gene expression in brewing yeast during beer fermentation is a major determinant...

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