Enzyme and Microbial Technology 33 (2003) 163–172

Screening of two complementary collections ofSaccharomyces cerevisiaeto identify enzymes involved in stereo-selective reductions of specific

carbonyl compounds: an alternative to protein purification

Michael Katz, Bärbel Hahn-Hägerdal, Marie F. Gorwa-Grauslund∗Department of Applied Microbiology, Lund University, P.O. Box 124, 221 00 Lund, Sweden

Received 23 September 2002; received in revised form 21 March 2003; accepted 28 March 2003

Abstract

Two collections ofSaccharomyces cerevisiaestrains, having open reading frames (ORFs) either overexpressed or deleted, were screenedto identify cytosolic NADPH-dependent reductases involved in stereo-selective reductions of specific carbonyl compounds. As modelcompounds diacetyl (diketone) and ethyl acetoacetate (3-oxo ester) were used. The reductases encoded by YBR149w, YDR368w, YMR226cand YOR120w were found to reduce diacetyl and YOL151w, YHR104w, YGL157w, YOR120w and YDR368w reduced ethyl acetoacetate.

We cloned YBR149w, YDR368w and YMR226c using a vector with the strong constitutive GPD promotor and investigated theencoded reductases with respect to reduction of diacetyl. YBR149w, YDR368w and YMR226c were shown to encode (S)-specific diacetylreductases capable of further reducing racemic acetoin to butanediol. Furthermore, we demonstrated that the purified (S)-diacetyl reductase(EC 1.2.1.56) most likely is thed-arabinose dehydrogenase encoded by YBR149w (Ara1p).© 2003 Elsevier Science Inc. All rights reserved.

Keywords:Screening; Reductase; Yeast; NADPH; Deletion library; Overexpression library; Open reading frame; Diacetyl; Ethyl acetoacetate

1. Introduction

Ketoalcohols and hydroxy esters, that are importantchiral building blocks in the synthesis of pharmaceuti-cals and fine chemicals, can be prepared by whole cellreduction of diketones and ketoalcohols using the baker’syeastSaccharomyces cerevisiaeas biocatalyst[1]. Screen-ing of other yeast families revealed that different productstereo-selectivities can be obtained in the bioreduction ofa given compound[2–4]. For bioreduction it is of greatinterest to identify reductases, many of which are cytoso-lic and NADPH-dependent reductases, responsible for theconversion of a particular compound, since the reductaselevel can be genetically manipulated to enhance the yieldand productivity of the products[5].

Traditionally reductases were identified by protein purifi-cation from crude cell extracts using a combination of dif-ferent chromatographic purification steps such as NAD(P)Haffinity chromatography and size separation, ending with anN-terminal sequencing of the pure protein[6–12]. Howeverthis approach, based on the activity towards a specific com-

∗ Corresponding author. Tel.:+46-46-222-0619; fax:+46-46-222-4203.E-mail address:Marie-Francoise.Gorwa@tmb.lth.se

(M.F. Gorwa-Grauslund).

pound, is time-consuming and laborious since reductases arepurified towards a non-physiological substrate, which leadsto low activities and requires concentrating enzyme fractionsafter each purification step. Moreover, similar reductases aredifficult to separate due to their similar molecular weightand pI. Thus a “pure” enzyme fraction can in fact be a mix-ture of several similar reductases. Finally different researchlaboratories have purified the same enzymes towards differ-ent substrates[13,14] and therefore there is the possibilitythat the same reductase has been assigned different namesand EC numbers, notably since their physiological functionis often unknown.

The complete sequencing of theS. cerevisiaegenomewith 6144 open reading frames (ORFs)[15] now enableseach peptide sequence to be traced back to an ORF in thegenome. Recently an investigation based on sequence simi-larity showed that at least 49 potential NAD(P)H-dependentketone reductases (or 49 ORFs encoding putative reductases)are present inS. cerevisiae[14]. Many of these reductasespresent in all living organisms[16–18]have unknown phys-iological function. They have been shown to reduce a broadarray of ketones and ketoesters and to have overlapping ac-tivities [13]. Today two collections ofS. cerevisiaestrainshave been constructed: (1) a complete collection of dele-tion mutants[19] from the EuropeanS. cerevisiaearchive

0141-0229/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved.doi:10.1016/S0141-0229(03)00086-3

164 M. Katz et al. / Enzyme and Microbial Technology 33 (2003) 163–172

Fig. 1. The chemical structures and associated reactions for the two model compounds, diacetyl and ethyl acetoacetate.

for functional analysis (Euroscarf); and (2) a complete col-lection of overexpression strains, Yeast ORF ExpressionClones (ExClones) under the inducible Pcup1 promoter[20].

The aim of the present study was to establish a methodto identify the NADPH-dependent reductase(s) involved inthe reduction of specific carbonyl compounds, using an invitro activity screening assay of crude cell extracts preparedfrom the two collections (deletion clones and overexpres-sion clones) without the need for protein purification. Asmodel compounds, two well studied carbonyl compounds,the diketone diacetyl (2,3-butanedione) and the 3-oxo esterethyl acetoacetate (EAA) were used (Fig. 1).

2. Materials and methods

2.1. Chemicals

Ethyl acetoacetate, diacetyl, acetoin (racemic 3R/3S-acetoin or 3-hydroxy-2-butanone) and 2,3-butanediol (mix-ture of meso-2,3-butanediol, (2R,3R)-2,3-butanediol and(2S,3S)-2,3-butanediol) were purchased from Sigma-Aldrich(St. Louis, MO), Merck (Schuchardt, Germany), Sigma-Aldrich and Acros Organics (Princeton, NJ) respectively.(S)-Diacetyl reductase[6] (EC 1.2.1.56, baker’s yeast),glucose-6-phosphate dehydrogenase (EC 1.1.1.49, torulayeast), glucose-6-phosphate, NADPH and NADP+ werepurchased from Sigma-Aldrich.

2.2. Strains

Saccharomyces cerevisiaeCEN.PK 113-7A [MAT a,MAL2-8c; SUC; his3-∆1] and CEN.PK 111-32D [MAT a,MAL2-8c; SUC2; leu2-3,112] were obtained from Dr. P.Kötter, Institute of Microbiology, Frankfurt, Germany. Thedeletion mutants and their reference strain BY4741 [Mata his3∆1 leu2∆0 met15∆0 ura3∆0] were bought fromEUROSCARF (Frankfurt am Main, Germany). The Yeast

ExClone collection was bought from Resgen, InvitrogenCorporation (UK). The reference strain is EJ758 [Mat a;his3-200 leu2-3,112 ura3-52 pep4::HIS3] containing theslightly modified pYEX 4T-1 plasmid (Clonetech, CA)with the inducible Pcup1 promoter[20]. Escherichia coliDH5� (Life Technologies, Rockville, MD) was used forsubcloning.

2.3. Nucleic acid manipulation

Plasmid DNA was prepared with the Biorad miniprepkit (Hercules, CA). Restriction and modified enzymes wereobtained from Fermentas (St. Leon-Rot, Germany) or fromLife Technologies (Rockville, MD). DNA extractions fromagarose gels and purification of PCR products were madeby using the QIAquick extraction kit (Qiagen, Hilder,Germany).

2.4. Transformation

Competent cells ofE. coli DH5� were prepared andtransformed by the method of Inoue et al.[21]. Yeast trans-formations were made as described previously[22]. Es-cherichia colitransformants were selected on Luria–Bertani(LB) medium [23] plates with 100�g/ml ampicillin(IBI Shelton Scientific Inc., Shelton, CT).Escherichiacoli transformants were grown overnight in LB with100�g/ml ampicillin. S. cerevisiaetransformants were se-lected on yeast nitrogen base without amino acids (Difco,Detroit, MI).

2.5. Construction of TMB4091, TMB4092, TMB4093and TMB4094

The YMR226c, YBR149w and YDR368w genes wereamplified from chromosomalS. cerevisiaeDNA usingprimers containing specific restriction sites (Table 1).The cloning vectors, p423(HIS3) and p425(LEU2) [24]were digested using corresponding restriction enzymes

M. Katz et al. / Enzyme and Microbial Technology 33 (2003) 163–172 165

Table 1Primers and restriction sites for the construction of strains overexpressing the reductases encoded by YDR368w, YMR226c and YBR149w

Primer for amplification of genes Open readingframe

Restrictionsite primer

Restrictionsite vector

5′-GAAGATCTACCATGCCTGCTACG-3′ YDR368w BglII BamHI5′-AACTGCAGCTCATTGGAAAATTGGGAAAGG-3′ YDR368w Pst1 Pst15′-GCGGATCCTGATAATGTCCCAAGGTAGAAAAG-3′ YMR226c BamHI BamHI5′-CCATCGATCTAGGTTTATCCACGGAAGATATG-3′ YMR226c Cla1 Cla15′-ATTCTAGATATCTCCGCCTTCAATTGATAAAAGCGTCTT-3′ YBR149w Xba1 Spe15′-AAGGATCCAAGAAGCGAACTAAATAAAGTGAAAATAAAGTCGTTGTC-3 ′ YBR149w BamHI BamHI

(Table 1) and ligations were performed overnight at16◦C. The resulting plasmids p423-YMR226c(HIS3) andp423-YBR149w(HIS3) were used to transformS. cerevisiaeCEN.PK 113-7A, generating TMB4091 and TMB4092, re-spectively. The plasmid p425-YDR368w(LEU2) was usedto transformS. cerevisiaeCEN.PK 111-32D, generatingTMB4093. A controlS. cerevisiaestrain, TMB4094 con-taining the empty plasmid p423 (HIS3) was also generatedafter CEN.PK 113-7A transformation.

2.6. Cell growth

The collection strains were kept on rich medium agarplates (20 g/l glucose, 10 g/l yeast extract, 20 g/l tryptone,20 g/l agar). Colonies from the deletion mutants were takenfrom the agar plates with a sterile cotton plug and used forinoculation of 100 ml rich medium (40 g/l glucose, 40 g/ltryptone, 20 g/l yeast extract) in 250 ml shake-flask overnightat 30◦C and 200 rpm. Colonies from the ExClones weretaken from agar plates with a sterile cotton plug and used for24 h inoculation of 250 ml shake-flasks containing 100 mlSD-Ura omission medium with 40 g/l glucose as describedby the supplier (Resgen, Invitrogen Corporation, UK) withthe following modifications: Cu2+ ions, 80�M, were addedto medium for gene induction directly after inoculation and100 mM phosphate buffer (pH 6.8) was used.

Strains TMB4091, TMB4092, TMB4093 and TMB4094were kept on SD agar plates (20 g/l glucose, 6.7 g/l yeastnitrogen base, 20 g/l agar). Colonies were picked from theplates with a sterile cotton plug and used to inoculate 100 mlSD-medium (40 g/l glucose, 6.7 g/l yeast nitrogen base,100 mM phosphate buffer pH 6.8) in 250-ml shake-flaskovernight at 30◦C and 200 rpm.

All cells were harvested by centrifugation at 4000×g for5 min and washed once with 50 ml sterile milli-Q water.

2.7. Crude cell extract preparation

Cell extracts were prepared from yeast cells grown to sta-tionary phase or late exponential phase as described above.Wet cells, 0.6 g, were dissolved into 1.5 ml Y-Per yeast ex-traction protein reagent (Pierce, Rockford, IL) in a 2-mltube and incubated for 50 min at room temperature on arocking table. Cell debris was spun down for 30 s (Hermle

Labortechnik Z160M table centrifuge, Wehingen, Germany)at 14,000 rpm. The cell free crude extract was kept on ice.Protein concentration was determined by Coomassie ProteinAssay Reagent (Pierce) using bovine serum albumin (BSA)as a standard.

2.8. Activity measurements

Activity measurements were performed by followingthe change in absorbance at 340 nm from the oxidation ofNADPH using a Hitachi U2000 spectrophotometer (Tokyo,Japan). The assay was performed in potassium phosphatebuffer (100 mM, pH 7.0) at 30◦C. The initial change inabsorbance was followed during the first 40 s after additionof NADPH. One unit (U) of activity corresponds to 1�molNAD(P)H consumed per minute at 30◦C. Experimentswere done at least in duplicates.

For the screening of the deletion clones, the reduction ofethyl acetoacetate was performed using the following con-ditions: 186�l crude cell extract, 107�l ethyl acetoacetate(140 mM) dissolved in phosphate buffer (100 mM, pH 7.0)and 7�l NADPH (7.2 mM). For the reduction of diacetyl,the following conditions were used: 200�l crude cell ex-tract, 80�l diacetyl (80 mM), 20�l NADPH (7.2 mM) and0.7 ml phosphate buffer (100 mM, pH 7.0).

For the screening of the ExClone collection, the sameconditions were used. However, when high activity was ob-tained, i.e. more than two- to threefold increase compared tothe reference strain, 20–50�l crude cell extracts were usedinstead of 186 or 200�l.

The activities of diacetyl and (R/S)-acetoin reductionand oxidation ofd-arabinose were also compared betweenstrains TMB4091, TMB4092 and TMB4093 and the com-mercially available (S)-diacetyl reductase. Reductase activi-ties were measured as described above using 80�l 140 mMsubstrate (diacetyl or racemic acetoin), 20�l NADPH(3 mg/ml), 10�l crude cell extract or 80�l (S)-diacetylreductase (stock solution at 1.2 U/ml) in phosphate buffer(100 mM, pH 7.0).

The oxidation ofd-arabinose was measured by followingthe formation of NADPH using 100�l d-arabinose (1.0 M),20�l NADP+ (3 mg/ml), 10�l crude cell extract or 80�l(S)-diacetyl reductase (stock solution at 1.2 U/ml) in a phos-phate buffer (100 mM, pH 9.0).

166 M. Katz et al. / Enzyme and Microbial Technology 33 (2003) 163–172

2.9. Whole cell reduction of diacetyl

The whole cell reduction of 4 g/l diacetyl was performedin magnetically stirred 20-ml sealed serum vials using 5 g/lyeast cells (grown as described previously) in buffered water(50 mM citrate buffer, pH 5.5) containing 20 g/l glucose.

2.10. Analysis of substrates and products

Diacetyl, (R/S)-acetoin and 2,3-butanediol levels werefollowed using thin layer chromatography (TLC). The threecompounds could be visualized on the same TLC plate(silica 60 F254) using heptane/ethyl acetate (1:5) as mo-bile phase and developing with Seebach’s solution: 25 gphospho molybdic acid (ISN Biochemicals, Eschwege,Germany), 10 g ceric ammonium sulphate (ISN Biochemi-cals), 60 ml sulfuric acid (concentrated) and 920 ml H2O.A Gilson CLC, column liquid chromatography system(Gilson, Villiers-le-bel, France) was used for the quantita-tive analysis of diacetyl, (R/S)-acetoin and 2,3-butanediol.An Aminex HPX-87H column (BioRad, Richmond, CA)with a RID-10A refractive index detector (Shimadzu, Ky-oto, Japan) was used at 30◦C with 5 mM H2SO4 as mobilephase and a flow rate of 0.6 ml/min.

Enantiomeric excess of acetoin was determined using chi-ral gas chromatography in a Perkin-Elmer AutoSystem XLgas chromatograph (Stockholm, Sweden) equipped with aSupelco�-DEX 120 column (30 m length, 0.25 mm i.d.; Su-pelco, Bellefonte, PA) at 65◦C (isothermal) using helium asthe carrier gas (1.5 ml/min). The flame ionization detectorwas set to 275◦C. Cell free supernatant, 1.0 ml, from thewhole cell reduction or a standard mixture were extractedwith 1.0 ml dichloro-methane and 1�l of the organic phasewas injected for GC analysis.

3. Results

3.1. Identification of potential NADPH-dependentreductases in Saccharomyces cerevisiae

Saccharomyces cerevisiaeORFs encoding potentialNADPH-dependent reductases were identified and selectedfor screening, using several complementary strategies: (1)database search in MIPS (http://mips.gsf.de/proj/yeast/CYGD/db/index.html) for NADPH-dependent reductaseswith known physiological function inS. cerevisiae; (2) litera-ture search forS. cerevisiaereductases purified and/or repor-ted previously to reduce carbonyl compounds (3-oxo estersand diketones) but whose physiological function is stillunknown; (3) search forS. cerevisiaehomologous to thereductases found in strategy 2; and (4) search for heterol-ogous reductases involved in the reductions of diketonesor ketoesters using PubMed (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi) and BLAST these protein se-quences in the database against the completeS. cerevisiae

genome to identify the corresponding putative reductasesin S. cerevisiae (http://genome-www2.stanford.edu/cgi-bin/SGD/nph-blast2sgd) as described previously[14,25].

From MIPS database search (strategy 1), more than40 ORFs encoding characterized NADPH-dependent re-ductases/dehydrogenases were selected. The search wastherefore refined to exclude (i) mitochondrial reductases;(ii) reductases with only weak similarity to known re-ductases from other organisms; and (iii) reductases withFAD-binding motifs. NADPH-cytochrome P450 reduc-tase encoded by YHR042w was also excluded since themechanism of this reductase is not a reduction of a car-bonyl group but a hydroxylation of carbon atoms. Someof the selected reductases (Table 2) are directly involvedin the central carbon metabolism and amino acid synthe-sis, respectively and have been reported to be involvedin reductive biotransformation of carbonyl compounds[26].

According to Sybesma et al.[13], the followingS. cere-visiaeORFs have been shown to encode reductases involvedin NADPH-dependent reduction of ketoesters (oxo esters)and diketones: YDR368w (Ypr1p)[5,8,14], YOL151w(Gre2p) [5,14], YOR120w (Gcy1p) [27], YHR104w(Gre3p) [9], YBR149w (Ara1p) [11]. Alcohol dehydro-genase (ADH1 or YOL086c) and the� and � fatty acidsynthetase subunits (FAS1andFAS2encoded by YKL182wand YPL231w, respectively) have also been reported to beinvolved in the reduction of 3-oxo esters[13]. However,only FAS2 is an NADPH-dependent reductase and wasincluded in the present study.

Additional by searching for homologous to previouslyfound ORFs (strategy 3), putative NADPH-dependent reduc-tases were identified: YJR096w and YDL124w belong to thealdo-keto reductases; YGL157w, YGL039w and YDR541are homologous to YOL151w (Gre2p) and were also iden-tified during the purification of a novel ketoreductase fromZygosaccharomyces cerevisiae[12]. These four ORFs showhigh degree of homology[12] and show similarity to plantdihydro flavonol 4-reductase.

Homology searches at the protein level with the reduc-tases (PubMed) from other organisms (strategy 4) identifiedORFs in three families of reductases, namely the aldo-ketoreductase family (aldose reductases), the short chain dehy-drogenases/reductase family and the zinc-containing alcoholdehydrogenase family (medium chain dehydrogenase fam-ily). The identifiedS. cerevisiaealdo-keto reductases are ho-mologues to reductases already known to be involved in thereduction of oxo esters (YDR368w, YOR120w, YHR104w,YBR149w, YJR096w and YDL124w). The identified shortchain dehydrogenases ORFs are YNL202w, YMR226c,YDL114w, YIL124w, YBR159w, YIR035c, YKR009c,YKL055c, YIR036c and YOR246c. YBR159w was recentlysuggested to be a membrane-bound enzyme encoding a com-ponent of the microsomal fatty acid elongation[25] whereasYIR036c has been shown to be a cytosolic reductase in-volved in the reduction of the aromatic diketone, benzil[28].

M. Katz et al. / Enzyme and Microbial Technology 33 (2003) 163–172 167

Table 2Saccharomyces cerevisiaeORFs and their relative diacetyl reductase ac-tivities in the corresponding deletion mutants

Open reading frame Relative activity

Characterized reductases in MIPSYNL241c 111YHR183w 100YLR174w 93YHR179w 84YPL171c 110YPL091w 113YGR204w 103YFR030w 91YDR127w 97YPL231w a

YOR236w n.m.a

YBR166c n.m.YGL154c n.m.YNR050c n.m.YAL062w n.m.YOR375c n.m.YPL061w n.m.YPL061w n.m.YNL009w n.m.

Aldo-keto reductasesYOR120w (Gcy1) 107YDR368w (Ypr1) 73YBR149w (Ara1) 70YHR104w (Gre3) 102YJR096w 100YDL124w 100

Short chain alcohol dehydrogenasesYIL124w 111YIR035c 92YIR036c 90YDL114w 116YMR226c 67YBR159w 97YNL202w 118YKR009c 99YKL055c 85YOR246c 76

(Zn)-containing alcohol dehydrogenasesYMR318c 113YCR105w 101

Dihydro flavonol 4-reductasesYOL151w (Gre2) 103YDR541c 107YGL157w 103YGL039w 92

100% activity corresponds to the activity of the control strain BY4741(35 mU/mg total protein). n.m.: activity not measured because not availableat the time of screening.

a Non-viable deletion strain.

Although most short chain dehydrogenases with recentlyreported physiological function are membrane-bound, theywere included in the study since they may also appear inother subcellular locations. For example, YIL124w encod-ing aryl-glycerone phosphate reductase is located in theendoplasmic reticulum, in lipid particles and in the cytosol

(MIPS database). From the zinc-containing alcohol dehydro-genase family, we chose to study only YMR318c and its ho-mologous YCR105w, since YMR318c was recently shownto be an NADPH-specific alcohol dehydrogenase encodedby ADH6 [29]. On the contrary, YAL060W (2,3-butanedioldehydrogenase) and YLR070c (xylitol dehydrogenase) havebeen reported to be NADH-dependent[30,31]. Similarly,YJR159w (SOR1, EC 1.1.1.14), YOL086c (alcohol dehy-drogenase 1) and YDL168w are NADH-dependent (MIPSdatabase). YAL061w and YDL246c whose co-factor de-pendency remains unknown were not included in the study.

Reductases selected after the complementary searches aresummarized inTable 2.

3.2. Screening of the deletion clones

The deletion clones were screened by comparing theNADPH-activity of a deletion strain for a given substrateto the activity of the reference strain under the same condi-tions. The reference activity was set to 100% and the relativeactivities were rescaled accordingly. Assay conditions werechosen to obtain high activity of the reference strain (i.e.35 mU/mg). In some cases, the activity of a deletion clonewas a bit higher than 100%, most likely because of errorsin protein determination. Since the activities were neverhigher than 115%, any strain having an activity below 80%or more was considered to be significantly involved in thereduction of the studied substrate.

With diacetyl four strains giving a relative activity be-low 80% were identified: strains with deleted YBR149w(Ara1p), YDR368w (Ypr1p), YMR226c and YOR246c(Table 2). Four strains with activities less than 80% towardsethyl acetoacetate were also identified: strains with deletedYDR368w, YOL151w, YGL157w and YDR541c (resultsnot shown). Only few ORFs could not be tested becausetheir deletion was lethal to the cell (Table 2).

3.3. Screening of the ExClone collection

The ExClone collection was screened by comparing theNADPH activity of the reference strain for a given sub-strate to the activity of strains each having a reductasegene induced and overexpressed in the presence of copper.Any strain giving at least twofold increase in activity wasconsidered to be involved in the reduction of the studiedsubstrate. The following ORF overexpression clones werechosen: (1) identified ORFs in the screening of deletionclones (see above); (2) all ORFs from the aldo-keto reduc-tase family since most of the identified carbonyl reductaseshave been found in this family; and (3) ORF coding for thefatty acid synthetase�-subunit (FAS2or YPL231w) sincethis strain was not available as a deletion clone (non-viable).The results for diacetyl and ethyl acetoacetate are summa-rized inFigs. 2 and 3, respectively. When using diacetyl asa substrate, strains overexpressing reductases encoded byYBR149w, YDR368w and YMR226c indeed showed higher

168 M. Katz et al. / Enzyme and Microbial Technology 33 (2003) 163–172

Fig. 2. Relative NADPH-dependent diacetyl reductase activities of the tested overexpression clones. 100% activity for the reference strain (control)corresponds to a specific activity of 90 mU/mg protein.

activities: 5.2-, 3.8- and 4.1-fold increase respectively com-pared to the reference strain (Fig. 2). YOR246c showedonly 1.4-fold increase in activity and was therefore regardedas a false positive from the deletion screening. Althoughnot identified in the screening of the deletion clones, thereductase encoded by YOR120w (related to YBR149w andYDR368w) showed 3.4-fold increase in activity (Fig. 2). Wetherefore concluded that YBR149w, YDR368w, YOR120wand YMR226c encode NADPH-dependent reductases thatcan use diacetyl as a substrate.

When using ethyl acetoacetate as a substrate, higher activ-ities were also demonstrated for the reductases identified inthe screening of the deletion clones. YDR368w, YOL151wand YGL157w showed 7.3-, 4.7- and 4.3-fold increase inactivity, respectively, compared to the reference strain. On

Fig. 3. Relative NADPH-dependent ethyl acetoacetate activities of the tested overexpression clones. 100% activity for the reference strain (control)corresponds to a specific activity in the range of 8–13 mU/mg protein.

the contrary, YDR541c did not show increased activity.Here again, YOR120w showed high activity, 8.1-fold in-crease towards ethyl acetoacetate (Fig. 3) and YHR104walso showed 3.3-fold increase. We therefore concludedthat YOL151w, YGL157w, YOR120w, YHR104w andYDR368w are NADPH-dependent reductases acceptingethyl acetoacetate as a substrate.

3.4. Whole cell reduction of diacetyl using strainsoverexpressing YBR149w, YDR368w and YMR226c

To demonstrate the efficiency of the screening method,whole cell reductions of diacetyl were performed usingstrains overexpressing some of the identified reductases.The reductases encoded by YMR226c, YBR149w and

M. Katz et al. / Enzyme and Microbial Technology 33 (2003) 163–172 169

Table 3Diacetyl and (R/S)-acetoin reductase activities in crude cell extracts ofstrains overexpressing YMR226c (TMB4091), YBR149w (TMB4092) andYDR368w (TMB4093) compared to the control strain TMB4094

Sample Diacetyl reductaseactivity(mU/mg protein)

R/S-Acetoinreductase activity(mU/mg protein)

TMB4094 140 22TMB4091 1140 165TMB4092 815 250TMB4093 940 220

YDR368w were expressed under the strong constitutiveGPD promoter[24] to reach high activity and to avoid thecontrol by copper induction. Crude cell extracts were pre-pared from strains TMB4091 (overexpressing YMR226c),TMB4092 (overexpressing YBR149w) and TMB4093(overexpressing YDR368w) and NADPH-dependent di-acetyl reductase activities were measured and comparedwith the control strain TMB4094 (empty vector) (Table 3).In cell extract from TMB4091, TMB4092 and TMB4093activities of 1140, 815 and 940 mU/mg, respectivelywere found, while cell extract from the control strain,TMB4094, showed considerably less activity, 140 mU/mg(Table 3).

The reduction of diacetyl by whole cells was also in-vestigated with the three overexpressing strains and thecontrol strain TMB4094. The whole cell bioreduction wasinterrupted after 6 h and diacetyl and acetoin concentrationswere measured (Table 4). At the beginning of bioreductiondiacetyl was already lost, most likely due to absorption inand on the cells. After 6 h, more than 1.5 g/l acetoin hadbeen formed in all the bioconversions and trace amountsof 2,3-butanediol (less than 0.3 g/l) were detected withTMB4091 (overexpressing YMR226c). The enantiomericexcess ofS-acetoin at 6 h was 80% for the control strainTMB4094 and 87, 87 and 91% for TMB4091, TMB4092and TMB4093, respectively (Table 4), which indicatesthat the diacetyl reductases identified in the screening are(S)-specific.

Since trace amount of 2,3-butanediol were found forTMB4091 having the highest reductase activity, we tested

Table 4Diacetyl, (R/S)-acetoin concentrations and enantiomeric excess of ace-toin in the whole cell reduction of 4 g/l diacetyl with strains TMB4091,TMB4092 and TMB4093, overexpressing YMR226c, YBR149w andYDR368w, respectively, and with the control strain TMB4094

Strain Diacetyl (g/l) (R/S)-Acetoin(g/l)

e.e. (S)-acetoin(%)

T = 0 h T = 6 h T = 6 h T = 6 h

TMB4094 3.58 1.65 1.50 80TMB4091 3.48 0.62 2.51 87TMB4092 3.43 1.61 1.65 91TMB4093 3.44 0.96 2.13 87

the activity of the reductases towards racemic acetoin(R/S)-acetoin (Table 3). Indeed the strains overexpressingthe reductases showed an increased acetoin reductase ac-tivity (165, 250 and 220 mU/mg protein for YMR226c,YBR149w and YDR368w, respectively) whereas the controlstrain only showed low activity of 22 mU/mg protein. Com-mercially (S)-diacetyl reductase also showed activity in thepresence of racemic acetoin, which has not been reportedpreviously[6]. Using a co-factor recycling system[32], wecould demonstrate on TLC the formation of 2,3-butanediolfrom diacetyl at pH 7.0 and 30◦C by converting 5 g/l di-acetyl overnight with (S)-diacetyl reductase (2 U/ml) usingcatalytic amounts of NADPH and regenerating the co-factorwith glucose-6-phosphate dehydrogenase (6 U/ml) and26 g/l glucose-6-phosphate. NADPH-dependent diacetyl re-ductases are therefore most likely also NADPH-dependentacetoin reductases.

3.5. (S)-Diacetyl reductase hasd-arabinosedehydrogenase activity

(S)-Diacetyl reductase consists of at least two subunitsand the native protein has a size of 75 kDa[6]. Similarly,d-arabinose dehydrogenase consists of two subunits of40 and 39 kDa, which is unusual for an aldo-keto reduc-tases, and has a native size of 74 kDa[11]. The heavysubunit was sequenced N-terminally and this sequence cor-responded to YBR149w, whereas the small subunit couldnot be sequenced due to N-terminal blocking[11]. Basedon these similarities, (S)-diacetyl reductase was proposedto also be thed-arabinose dehydrogenase enzyme[13].We therefore comparedd-arabinose dehydrogenase activityin the three overexpression strains and in the (S)-diacetylreductase to elucidate whether (S)-diacetyl reductase hasd-arabinose dehydrogenase activity and was encodedby YBR149w. The results are summarized inTable 5.TMB4092 (YBR149w) showed ad-arabinose dehydro-genase activity of 33 mU whereas TMB4094 (referencestrain), TMB4091 (YMR226c) and TMB4093 (YDR368w)

Table 5Diacetyl reductase (DR) andd-arabinose dehydrogenase (AD) activity instrains TMB4091, TMB4092 and TMB4093, overexpressing YMR226c,YBR149w and YDR368w respectively, compared to the control strainTMB4094 and to commercial (S)-diacetyl reductase

Sample Diacetylreductase(mU)

d-Arabinosedehydrogenase(mU)

RatioDR/AD

TMB4094 (control) 19 3 6.3TMB4091 (YMR226c) 167 3 56.0TMB4092 (YBR149w) 127 33 3.8TMB4093 (YDR368w) 139 2 69.0(S)-Diacetyl reductase 96 28 3.4

Activities refer to total activities in 1.0 ml assays. The activities weremeasured using 10�l crude cell extract and 80�l (S)-diacetyl reductaseas described inSection 2.

170 M. Katz et al. / Enzyme and Microbial Technology 33 (2003) 163–172

gave background activities of about 3 mU in the assay. Theratios of diacetyl reductase activity tod-arabinose dehydro-genase activity were as follows: 6.3 for the control strainTMB4094, 3.8 for TMB4092 (YBR149w), 56 for TMB4091(YMR226c), 69 for TMB4093 (YDR368w) and 3.4 for thecommercial (S)-diacetyl reductase. These data together withthe purification results reported previously[6,11] stronglysuggests that commercial NADPH-dependent (S)-diacetylreductase (EC 1.2.1.56) is indeed thed-arabinose dehydro-genase (Ara1p) encoded by YBR149w.

4. Discussion

In this investigation we demonstrate that the screeningof two complementary libraries ofS. cerevisiaestrains, i.e.the copper-induced ExClones and the Euroscarf deletionstrain collection, is a tool to identify reductases involved inbioreduction of specific compounds. The study focused onNADPH-dependent reductases in the reductive direction.The same method can be used to identify NADH-dependentreductases and NAD(P)+-dependent dehydrogenases. Asimilar spectrophotometric assay has recently been usedto show that YBR149w, YHR104w and YOR120w wereinvolved in the reduction of carbonyl compounds[33]. Thedeletion clones have also been used to show that YBR159wencoded a microsomal�-keto-reductase having polyunsatu-rated fatty acid elongase activity[25]. However, to the bestof our knowledge, the present study is the first in which anentire library of candidates is screened for a specific activ-ity. Although crude cell extract screening limits the methodto cytosolic reductases, membrane-bound reductases canalso be targeted by using whole cell bioreduction and fol-low product formation rate and product formation pattern(enantiomeric excess and diastereomeric excess).

The screening method was tested using the well-studied3-oxo ester, ethyl acetoacetate, as a substrate. Proteinsencoded by YOL151w (Gre2p), YHR104w (Gre3p),YGL157w, YOR120w (Gcy1p) and YDR368w (Ypr1p)were identified as ethyl acetoacetate reductases, whereasthe role of YDR541c remains to be further investigated.YOL151w and YDR368w encode reductases involvedin 3-oxo ester reduction[8]. Furthermore, YOR120w,YHR104w and YBR149w were involved in the reduc-tion of ethyl pyruvate (2-oxo ester) and 2,3-hexanedione(diketone)[33]. Together with our results this shows thatscreening crude cell extracts from the twoS. cerevisiaecollections identifies the ORFs known from previous in-vestigations. Difficult and laborious protein purificationare thereby avoided. Difficulties in protein purification andN-terminal sequencing was most likely the reason whyGre2p was wrongly reported to be encoded by the ORFYJR105w[8]. This was later corrected[14,33] and Gre2pwas shown to be encoded by YOL151w. The screeningmethod also identified YGL157w and YOR120w to be in-volved in the reduction of ethyl acetoacetate, whereas the

role of YDR541c remains unclear since this ORF was onlyidentified from the deletion clone screening. The proteinsencoded by YDR541c and YGL157w have not been puri-fied but have more than 50% identity to YOL151w[12].YOR120w is closely related to YDR368w and encodesGcy1p that has been purified and crystallized[27]. Gcy1phas an unusual flat binding pocket and can therefore accepta broad array of substrates including unusually large sub-strates. This explains why Gcy1p was identified both as adiacetyl and an ethyl acetoacetate reductase.

Diacetyl reductase activities were demonstrated forproteins encoded by YBR149w, YOR120w, YMR226cand YDR368w. Furthermore YBR149w, YMR226c andYDR368w were shown to encode (S)-specific NADPH-dependent diacetyl reductases. Since these proteins alsoaccept racemic acetoin, diacetyl can be completely re-duced to 2,3-butanediol although at a lower rate for thesecond step. It has previously been suggested that severalNADPH-dependent diacetyl reductases should be presentin S. cerevisiae[30], however, the four reductases foundin the present study had previously not been identified. Itis also the first time that a substrate has been assigned forthe reductase encoded by YMR226c. Recently a diacetylreductase belonging to the short chain dehydrogenase fam-ily was cloned and characterized from several mammaliantissues, including hamster liver and human kidney[34,35].This reductase, that also hadl-xylulose reductase activity,accepted acetoin as a substrate although to a lower extentthan diacetyl. When we compared this reductase toS. cere-visiae short chain alcohol dehydrogenases, the YMR226csequence had the highest identity with 32% at the proteinlevel, which further confirms the precision of our screeningstrategy.

When comparing the screening of the overexpression andthe deletion clones it seemed as if some reductase activitieswere masked in the deletion clones by concomitant upregu-lation of other reductases. Deleting YOR120w did not resultin loss of activity even though high diacetyl and ethyl ace-toacetate reductase activities were found in the overexpres-sion clone. The upregulation of reductases/dehydrogenasesto replace a missing activity has previously been shown inS. cerevisiaefor the genesGPD1 and GPD2 involved inglycerol formation[36]. The upregulation of a reductasewas also recently suggested to explain non-expected e.e.values when using whole cell reduction of two oxo esters(3-oxo methyl hexanoate and 3-oxo ethyl heptanoate) ina S. cerevisiaestrain lacking Gre2p[33]. Therefore, theExClone library may be more efficient and accurate. How-ever, inserts are not fully guaranteed in the ExClone library,which might be the reason why YDR541c overexpressiondid not give significantly higher ethyl acetoacetate reduc-tase activity, as was expected from its strong similarity withother reductases[12]. Therefore, the two libraries shall beused in complementary way.

The strategy of screening crude cell extract based onthe two libraries was successfully implemented to identify

M. Katz et al. / Enzyme and Microbial Technology 33 (2003) 163–172 171

reductases and should enable the identification of activitiestowards other substrates without prior protein purification.Moreover, each activity can now be correlated to an ORF,which avoids inconveniences such as having one reductasewith different names, based on substrates used in proteinpurification.

5. Note added in proof

While this paper was reviewed, YMR226c was clonedfrom S. cerevisiaeinto E. coli and reported as a NADP+-dependentl-allo-threonine dehydrogenase also able to oxi-dize d- andl-serine by Fujisawa et al.[37]. The reductaseencoded by YDR368w (Ypr1p) was cloned, purified andcharacterized towards various aldehydes and ketones and de-scribed as a 2-methylbutyraldehyde reductase by Ford andEllis [38].

Acknowledgments

This work was financially supported by grants from theSwedish Research Council for Engineering Sciences (TFR)and Carl Trygger’s Foundation. MFGG was supportedby a Marie Curie fellowship QLK3-CT-1999-51355 fromthe European Community. We thank Nermina Naganjacfor helping with the screening of the two collections andcloning of YBR149w. We would also like to thank Kung-liga Fysiografiska Sällskapet i Lund for financial support topurchase the twoSaccharomyces cerevisiaecollections.

References

[1] Csuk R, Glänzer BI. Baker’s yeast mediated transformations inorganic chemistry. Chem Rev 1991;91:49–97.

[2] Katz L, King S, Greasham R, Chartrain M. Asymmetric bioreductionof a ketosulfone to the correspondingtrans-hydroxysulfone bythe yeastRhodotorula rubraMY 2169. Enzyme Microb Technol1996;19:250–5.

[3] Zmijewski MJ, Vicenzi J, Landen BE, Muth W, Marler P, AndersonB. Enantioselective reduction of 3,4-methylene-dioxyphenylacetoneusing Candida famata and Zygosaccharomyces rouxii. ApplMicrobiol Biotechnol 1997;47:162–6.

[4] Botes AL, Harvig D, van Dyk MS, Sarvary I, Frejd T, KatzM, et al. Screening of yeast species for the stereo-selectivereduction of bicyclo[2.2.2]octane-2,6-dione. J Chem Soc Perkin Trans2002;1:1111–4.

[5] Rodriguez S, Kayser M, Stewart JD. Improving the stereoselectivityof baker’s yeast reductions by genetic engineering. Org Lett1999;1:1153–5.

[6] Heidlas J, Tressl R. Purification and properties of two oxidoreductasescatalyzing the enantioselective reduction of diacetyl and otherdiketones from baker’s yeast. Eur J Biochem 1990;188:165–74.

[7] Heidlas J, Engel KH, Tressl R. Purification and characterizationof two oxidoreductases involved in the enantioselective reductionof 3-oxo, 4-oxo and 5-oxo esters in baker’s yeast. Eur J Biochem1988;172:633–9.

[8] Nakajima N, Ishihara K, Kondo S, Tsuboi S, Utaka M, Nakamura K.Differences in protein structure and similarities in catalytic functionof two l-stereoselective carbonyl reductases from baker’s yeast.Biosci Biotechnol Biochem 1994;58:2080–1.

[9] Kuhn A, van Zyl C, van Tonder A, Prior BA. Purification andpartial characterization of an aldo-keto reductase fromSaccharomycescerevisiae. Appl Environ Microbiol 1995;61:1580–5.

[10] Kim S, Huh W, Kim J, Hwang S, Kang S.d-Arabinose dehydrogenaseand biosynthesis of erythroascorbic acid inCandida albicans.Biochim Biophys Acta 1996;1297:1–8.

[11] Kim S, Huh W, Lee B, Kang S.d-Arabinose dehydrogenase fromSaccharomyces cerevisiae. Biochim Biophys Acta 1998;1429:29–39.

[12] Costello CA, Payson RA, Menke MA, Larson JL, Brown KA, TannerJE, et al. Purification, characterization, cDNA cloning and expressionof a novel ketoreductase fromZygosaccharomyces rouxii. Eur JBiochem 2000;267:5493–501.

[13] Sybesma WFH, Straathof AJJ, Jongejan JA, Pronk JT, Heijnen JJ.Reduction of 3-oxo esters by baker’s yeast: current status. BiocatalBiotrans 1998;16:95–134.

[14] Stewart JD, Rodriguez S. Cloning, structure, and activity of ketonereductases from baker’s yeast. New York: Marcel Dekker, Inc.; 2001.p. 175–208.

[15] Goffeau A, Aert R, Agostini-Carbone ML, Ahmed A, AigleM, Alberghina L, et al. The yeast genome directory. Nature1997;387:6632.

[16] Jörnwall H, Höög JO, Persson B. SDR and MDR: complete genomesequences show these protein families to be large, of old origin, andof complex nature. FEBS Lett 1999;445:261–4.

[17] Jez JM, Penning TM. The aldo-keto reductase (AKR) superfamily:an update. Chem Biol Interact 2001;130–132:499–525.

[18] Forrest GL, Gonzalez B. Carbonyl reductase. Chem Biol Interact2000;129:21–40.

[19] Giaever G, Chu AM, Ni L, Connely C, Riles L, Veronneau S, etal. Functional profiling of theSaccharomyces cerevisiaegenome.Nature 2002;418:387–91.

[20] Martzen MR, McCraith SM, Spinelli SL, Torres FM, Fields S,Grayhack EJ, et al. A biochemical genomics approach for identifyinggenes by the activity of their products. Science 1999;286:1153.

[21] Inoue H, Nojima H, Okayama H. High efficiency transformation ofEscherichia coliwith plasmids. Gene 1990;96:23–8.

[22] Gietz RD, Sugion A. New yeast—Escherichia colishuttle vectorsconstructed with in vitro mutagenized yeast genes lacking six-basepair restriction sites. Gene 1988;74:527–34.

[23] Ausubel FM, Kingston BR, Moore DD, Seidman JG, Smith JA,Struhl K. Current protocols in molecular biology. New York: Wiley;1995.

[24] Mumberg D, Muller R, Funk M. Yeast vectors for the controlledexpression of heterologous proteins in different genetic backgrounds.Gene 1995;156:419–22.

[25] Beaudoin F, Gable K, Sayanova O, Dunn T, Napier JA. ASaccharomyces cerevisiaegene required for heterologous fatty acidelongase activity encodes a microsomal�-keto-reductase. J BiolChem 2002;277:11481–8.

[26] Ward OP, Young CS. Reductive biotransformations of organiccompounds by cells or enzymes of yeast. Enzyme Microb Technol1990;12:482–93.

[27] Hur E, Wilson DK. The crystal structure of the GCY1 proteinfrom S. cerevisiaesuggests a divergent aldo-keto reductase catalyticmechanism. Chem Biol Interact 2001;130–132:527–36.

[28] Maruyama R, Nishizawa M, Itoi Y, Ito S, Inoue M. The enzymeswith benzil reductase activity conserved from bacteria to mammals.J Biotechnol 2002;94:157–69.

[29] Larroy C, Fernandez MR, Gonzales E, Pares X, Biosca JA.Characterization of theSaccharomyces cerevisiaeYMR318C (ADH6)gene product as a broad specificity NADPH-dependent alcoholdehydrogenase: relevance in aldehyde reduction. Biochem J2002;361:163–72.

172 M. Katz et al. / Enzyme and Microbial Technology 33 (2003) 163–172

[30] Gonzales E, Fernandez MR, Larroy C, Sola L, Pericas MA, ParesX, et al. Characterization of a (2R,3R)-2,3-butanediol dehydrogenaseas theSaccharomyces cerevisiaeYAL060w gene product: disruptionand induction of the gene. J Biol Chem 2000;275:35876–85.

[31] Richard P, Toivari MH, Penttilä M. Evidence that the gene YLR070cof Saccharomyces cerevisiaeencodes a xylitol dehydrogenase. FEBSLett 1999;457:135–8.

[32] Eguchi T, Kuge Y, Inoue K, Yoshikawa N, Mochida K, Uwajima T.NADPH regeneration by glucose dehydrogenase fromGluconobacterscleroides for l-leucovorin synthesis. Biosci Biotechnol Biochem1992;5:701–3.

[33] Rodriguez S. Baker’s yeast�-keto ester reductions: whole cellbiocatalysts with improved stereoselectivity by recombinant DNAtechniques. PhD thesis, University of Florida; 2000.

[34] Ishikura S, Isaji T, Usami N, Kitahara K, Nakagawa J, Hara A.Molecular cloning, expression and tissue distribution of hamster

diacetyl reductase. Identity withl-xylulose reductase. Chem BiolInteract 2001;130–132:879–89.

[35] Nakagawa J, Ishikura S, Asami J, Isaji T, Usami N, Hara A, et al.Molecular characterization of mammalian dicarbonyl/l-xylulose red-uctase and its location in kidney. J Biol Chem 2002;277:17883–91.

[36] Ansell R, Granath K, Hohmann S, Thevelein JM, Adler L. Thetwo isoenzymes for yeast NAD+-dependent glycerol 3-phosphatedehydrogenase encoded byGPD1 and GPD2 have distinct roles inosmoadaptation and redox regulation. EMBO J 1997;16:2179–87.

[37] Fujisawa H, Nagata S, Mosono H. Characterization of short-chaindehydrogenase/reductase homologues ofEscherichia coli (YdfG)and Saccharomyces cerevisiae(YMR226C). Biochim Biophys Acta2003;1645:89–94.

[38] Ford G, Ellis ME. Characterization of Ypr1p fromSaccharo-myces cerevisiaeas a 2-methylbutyraldehyde reductase. Yeast2002;19:1087–96.