Cloning and sequence determination of the gene encoding sorbitol dehydrogenase from Saccharomyces cerevisiae

Gene, 140 (1994) 121-126 0 1994 Elsevier Science B.V. All rights reserved.

GENE

121

0378-l 119/94/$07.00

QIIOI

Cloning and sequence determination dehydrogenase from Saccharomyces (Antibody;

Aparna

recombinant

DNA; sheep liver sorbitol

V. Sarthy, Cynthia

Molrculur Biology, Ahhott Laboratories, Received by G.P. Livi: 6 February

Schopp*

of the gene encoding sorbitol cerevisiae

dehydrogenase;

and Kenneth

hgt 11 yeast genomic

library)

B. Idler

Abbott Park. IL 60064. USA

1993; Revised/Accepted:

25 August/l0

September

1993; Received at publishers:

15 November

1993

SUMMARY

The identification of a sorbitol-induced sorbitol dehydrogenase (SDH) activity from Saccharomyces cereuisiae is described. The SDHl structural gene was isolated from a hgtll yeast genomic library using an antibody to a 40-kDa protein induced in yeast cells growing in medium containing sorbitol. The gene encodes a 3_57-amino-acid (aa) protein deduced from the nucleotide sequence. Comparison of the aa sequence of the yeast SDHl with that of sheep liver SDH reveals a 63% overall similarity. Yeast transformants containing the cloned gene carried on a multicopy plasmid express high levels of SDHl only when grown on sorbitol, suggesting that the cloned gene contains both regulatory and coding sequences.

to1 to the

INTRODUCTION

Various pathways for the utilisation of sorbitol have been described. Sorbitol can be metabolized via the phosphorylated intermediate sorbitol-6-PO, by the enzyme sorbitol-6-PO, dehydrogenase (Lengeler and Lin, 1972). An NAD-dependent enzyme, sorbitol dehydrogenase (SDH), has also been identified in a wide variety of species. This enzyme mediates the conversion of D-sorbiCorrespondence to: Dr. A.V. Sarthy, Abbott Laboratories, D-93D, One Abbott Park Road, Abbott Park, IL 60064, USA. Tel. (I-708) 937-1571; Fax (t-708) 938-6046. *Present address: 5413 62nd Street, N.W., Gig Harbor, Tel. (l-206) 858-8188.

WA 98335, USA.

Abbreviations: aa, amino acid(s); Ab, antibody(ies); ADHl, alcohol dehydrogenase 1; Ap, ampicillin; BME, B-mercaptoethanol; bp, base pair(s): kb, kilobases or 1000 bp; mu, milliunit(s); NAD, nicotinamide-adenine dinucleotide; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PAGE, polyacrylamide-gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; PYK, pyruvate kinase; R, resistance/resistant; S., Sacchuromyces; SC-Trp (medium), synthetic complete medium minus tryptophan; SDH, sorbitol dehydrogenase; SDHZ. gene encoding SDH; SDS, sodium dodecyl sulfate; u, unit(s): YP, 1% yeast extract/2% Difco Bactopeptone.

SSDI

0378-l

119(93)E0703-G

oxidized product fructose, which is then through the phosphorylated sugar metabolized fructose-l-phosphate (Jeffery and Jornvall, 1988). Preliminary experiments conducted in our laboratory suggested that Saccharomyces cerevisiae can utilize D-sorbitol as a sole carbon source for growth. Most strains, however, exhibit an unusually long lag period for growth which ranges between 2-4 weeks. We are interested in understanding the mechanism for the unusual lag and the regulation of expression of SDH(s) or other sorbitolinduced genes. In order to initiate these studies we have cloned the S. cerevisiae SDHl gene from a hgtll yeast genomic library. The gene is expressed only in cells grown in the presence of sorbitol.

EXPERIMENTAL

AND DISCUSSION

(a) Identification of yeast sorbitol dehydrogenase S. cerevisiae produces a sorbitol induced NAD-dependent SDH, as determined by the SDH activity staining assay of extracts electrophoresed on a non-denaturing gel (Fig. 1, panel A, lane 2). No detectable SDH enzyme

is a homotetrameric molecule made of 3539 kDa subunits (Jornvall et al., 1987; Maret and Auld, 1987). (b) molecular

cloning of the S. ce~ev~siae .SDHl

The yeast SDHl gene was identified

22

ing, using a polyclonal ol-induced primarily

611

antibody

yeast protein(s). with the 40-kDa

grown in sorbitol-containing

by immunoscreen-

raised against

the sorbit-

The polyclonal Ab reacted protein produced by cells medium

only, as determined

by Western blot analysis (data not shown). Furthermore, extracts of sorbitol-induced cultures separated in native

43

gels also showed a similar pattern of immunoreactivity and SDH activity as determined by both Western blot

23

analysis and by assaying SDH activity directly in the gel (data not shown). These results suggest that the Ab specifically reacts with a yeast SDH. A hgtll yeast genomic expression library was then screened with the Ab to isolate potential clones containing the yeast SDHl gene (Snyder et al., 1987). Eight positive clones were isolated from approx. 6 x IO5 phage plaques and EcoRI-generated inserts from the phage

18 14 Fig. 1. Analysis of SDH activity in S. ceret+siae. (A) SDH activity of X000-8B LAG. (B) SDS-PAGE analysis of sorbitol-induced proteins of 8000~8B LAG. Methods: Cell extracts from cultures grown in SC liquid medium (Sherman et al., 1983) containing 2% glucose, sorbitol or mannitol were loaded in lanes 1,2 and 3, respectively. Strain SOOO-8B (itfdTa udrdl::LEUZ uru3 trpf ku2) was obtained from Dr. ET. Young. A spontaneous mutant (8000~8B LAG) was isolated by its ability to grow with no lag in SC medium

supplemented

with 2% sorbitol

as a carbon

source. Saturated cultures of 8000-8B LAG grown at 30 C in 10 ml SC medium were pelleted, washed with water and resuspended in I ml extraction buffer (50 mM K.phosphate. pH 7.4/25 mM f3ME:l mM PMSF). The suspension was vortexed w-ith acid washed glass beads for 2 min. supernatants were analysed for SDH activity on 5% polyacrylamide gels (Fowler et a1.,1972: Williamson et al.. 1980). Sorbitol was substituted for ethanol. Supernatants were also boiled for 3 min in sample buffer and subjected to 0.1 ?&SDS-12.5% PAGE which was then stained with Coomassie blue. AFFOW points to the SDH band

activity was observed from both glucose and mannitolgrown cultures (lanes 1 and 3, respectively). The yeast SDH enzyme also oxidizes other secondary alcohols such as xylitol. However, mannitol and primary alcohols, e.g., ethanol, do not serve as substrates for the enzyme (data not shown). The activity-stained band in the native gel containing the yeast SDH enzyme, when re-electrophoresed on a SDS-polyacrylamide gel, contained a major species that migrated as a 48kDa poiypeptide. This protein also co-migrated with the predominant 40-kDa protein that is only produced by cells grown in sorbitol (unpublished results). Fig. 1 (panel B, lane 2) shows the synthesis of the sorbitol induced protein that migrates as a 40-kDa protein on SDS-PAGE. It is interesting to note that the SDH enzyme isolated from both sheep and human liver

genomes were subcloned into pUC18. In order to identify ?L clones containing coding sequences for the putative yeast SDHl protein, total RNA extracted from yeast cultures were blot hybridized against the genomic inserts. Two plasmids pS30 and pS32, hybridized specifically to yeast mRNA made in sorbitol containing medium. A Northern blot indicating hybridization of the pS32 insert DNA with total RNA extracted from cells grown in sorbitol-containing medium is shown in Fig. 2B. The size of the mRNA transcript is approx. 1.4 kb (Fig. 2B, lane 2) which is sufficient to encode a 40-kDa protein. There was no detectable hybridization to pS32 with RNA made from either glucose or mannitolgrown cells ( Fig. 2B. lanes 1 and 3). These results indicate that the pattern of transcription of the SL)lil gene from clones pS30 and pS32 corresponds to the induction of an SDH or other sorbitol-induced proteins. (c) Sequence analysis of the S, ceuevisiae gene SDHI and gene product (SDW ) Both regulatory and coding sequences for the yeast SDHl gene are present in the 1.7-kb XhoI-lrJindII1 fragment common to hS30 and hS32. The EcoRI fragment from pS32 subcloned into the multicopy yeast vector pMW5 (McKnight and McConaughy, 1987) results in an increase in levels of SDH activity when introduced into cells of S. cerevisiue.This increase in activity is only seen in cells growing in medium containing sorbitol and is not observed in cells carrying pMW.5 without insert (Table I). More direct evidence that pS30 and pS32 code for an SDH was obtained from the complete sequence of an

123 1 rb

A kb

12

3 H

9.5 -

J

7.5 -

4

--_) c--

4.4 -

st:P

M,“’ XB I

XH I

Fig. 3. Strategy

3 Kb I

2”

ND I

HNCNC H II I I

----_) (----+ C-

c--

for sequence

determination

H I w

I

of the S. cereaisiue

SDHI

gene. Initially, 5’ end-labeled fragments were sequenced according to Maxam and Gilbert (1980) to generate detailed information about genomic clone pS32. Determining the location of the Hind111 sites permitted subcloning of those fragments into M13mpll. The position of the

2.4 -

Hind111 site at approx. 2 kb was verified by end-labeling the EcoRI site of an overlapping genomic clone (pS30) and sequencing across the Hind111 site. Single-stranded (ss) DNAs, prepared from the Ml3 cloning and reverse orientations of the Hind111 fragments were screened for as described by Sambrook ments was accomplished

0.3-

et al., (1989). Sequencing using the dideoxy chain

of the Hind111 fragtermination method

(Sanger et al., 1977). Primers used for annealing to the ssDNA templates were either the -40 primer of Ml3 or synthetic custom oligos designed for ‘gene walking’ techniques. Bold arrows indicate regions sequenced by the Sanger method (1977), whereas dashed arrows indicate regions

Fig. 2. Northern blot analysis of total yeast RNA from strain 8000-8B LAG. The constitutively expressed PYK transcript (A) and the sorbitolinduced transcript (B) are shown by the arrowheads. Total RNA extracted according to Denis et al. (1986) from 8000-8B LAG cells grown in SC liquid medium containing 2% glucose, sorbitol or mannitol was electrophoresed in lanes 1, 2 and 3, respectively. resed on a 1.2% agarose/2.2 M formaldehyde

RNA was electrophogel (Sambrook et al.,

1989), blotted onto Hybond N filters (Amersham) and probed with nick-translated pPYK which contains the yeast pyruvate kinase gene (Burke et al., 1983) (panel A) or pS32 (panel B). An RNA ladder (Bethesda Research Laboratories) was used as size markers. TABLE

I

Sorbitol LAG

dehydrogenase

enzyme

activity

in transformants

Unitsb

pMW5 (2% sorbitol) pMW5S32 (2% sorbitol) pMAC561 (2% glucose)

291 690

alcohol

pADH 1SDH (2% glucose) were grown

under

selection

dehydrogenase

aa residues

NC,

based on the conservation

in the protein.

of 22

The yeast SDH has 19 of these

aa conserved. The aa residues Proz8, G1u32 and Cyslo3, which are apparently conserved in the other dehydrogenases, are not conserved in the yeast SDHl protein (Fig. 5). The predicted size (40 kDa) for the yeast SDH 1 for

is also similar

SDH

Jornvall,

from

to the known

sheep

1988; Maret

Comparison dehydrogenase

and

human

size of 3539 liver

(Jeffery

kDa and

and Auld, 1987).

of the deduced aa sequence enzyme of the xylose-utilizing

stipiris with that of SDHl

254 857

(1980). H, HindIII;

tween the two proteins. Jeffery and Jornvall (1988) have suggested an evolutionary relationship between SDH and

protein

8000-8B LAG transformed with”

“Transformants

of 8000~8B

sequenced by the Maxam and Gilbert method NcoI; ND, NdeI; XB, XhaI; XH, XhoI.

of S. cerevisiae,

of the xylitol yeast Pichia shows a 57%

sequence similarity (Kotter et al., 1990). Xylitol, an intermediate of the xylose catabolic pathway, is also oxidized in SC-trp

liquid medium

containing 2% sorbitol or 2% glucose. Fig. 1 legend describes aration of cell extracts and strain SOOO-8B LAG. “Units are expressed as umol/min per mg protein,

the prep-

approx. 3-kb Hind111 fragment generated from subfragments of pS30 and pS32 clones (Fig. 3). Analysis of the nt sequence (Fig. 4) revealed an ORF that predicts a 357-aa protein. Comparison of the deduced aa sequence of the yeast SDHl protein and that of the sheep liver SDH enzyme (Jeffery et al., 1987), as shown in Fig. 5, revealed a 63% overall similarity and 42% identity be-

by the S. cerevisiae

SDHl

enzyme

(data

Therefore, it is possible that the yeast SDHl functions in the degradation of xylose.

not

shown).

enzyme also

Potential promoter elements containing TATA-like sequences were also found upstream from the putative ORF (Fig. 4). Several (AATAAA) (Proudfoot within

the 3’-UTR.

putative polyadenylation signals and Brownlee, 1976) are located

The size of the predicted agrees

message

derived

from the nt sequence

with the observed

approx. analysis

1.4-kb mRNA, as seen in the Northern (Fig. 2B, lane 2).

blot

124

TGAAGCCTCTGTATCACCTTGCTAACCGCATTTCTTCCATCTAAAGTATGTTCATTGCCATAAGTTGCTTACTCTCTCTTTAATATATA;;ATC GACA~GGCTCAATGTCTTACCGTTCATCTTTATGAAGAGATATAGTATAGTGG

-119

AAAAAAGAAACATCAAACAATCAACAAGAAAAAATACTAA

-19

+1 AAAAAAAAATTGAAAAATATGTCTCAAAATAGTAGT~CCCTGCAGTAGTTCTAGAG~GTCGGCGATATTGCCATCGAGC~GACC~TCCCTACCATTA 82 MSQNSNPAVVLEKVGDIAIEQRP I P T I R 28 AGGACCCCCATTATGTCAAGTTAGCTATTAAAGCCACTGGTATCTGCGGCTCTGATATTCATTATTATAGAAGCGGTGGTATTGGTAAGTACATATTGAA 182 DPHYVKLAI K A T G I CGSDIHY YRSGGIGKYILK 61 GGCGCCAATGGTTTTAGGTCATGAATCAAGCGGACAGGTTGTGGAAGTTGGTGATGCCGTCACAAGGGTCAAAGTTGGTGACCGTGTTGCTATTGAACCT 202 APMVLGHES SGQVVEVG DAVTRVKVGDRVAIEP 94 GGTGTTCCTAGCCGTTACTCTGATGAGACCAAAGAAGGGATTGATGGTACTCTTG 382 GVPSRYSDETKEGRYNLCPHMAFAATPPIDGTLV 128 TGAAGTACTATTTATCTCCAGAAGATTTTCCTTGTG~TTGCCAGAAGGCGTCAGTTATG~GAGGGCGCTTGTGTCG~CCCTTATCAGTCGGTGTACA 482 KYYLSPEDFLVKLPEGVS YEEGACVEPLSVGVH 161 CTCTAATAAATTGGCTGGGGTCCGCTTTGGTACCAAAGTTCTGGCGCAGTCGCCCGCGCTTTTGGT 582 SNKLAGVRFGTKVVVFGAGPVGLLTGAVARAFG 194 GCCACCGACGTCATTTTCGTCGATGTATTCGACAACAAGCTTCTTCCCAGTTTTCCACCG 682 ATDVIFVDVFDNKLQRAKD FGATNTFNSSQFSTD 228 ATAAAGCCCAAGACTTGGCCGATGGGGTCCAAAAGCTTTTTGGGCGG~TCACGCAGATGTGGTGTTTGAGTGTTCAGGTGCTGATGTTTGCATTGATGC 782 KAQDLADGVQKLLGGNHA DVVFEC SGADVCIDA 261 CGCTGTCAAAACAACTAAGGTTGGAGGTACCATGGTGCAATG 882 AVKTTKVGGTMVQVGMGKNYTNFPIAEVSGKEM 294 AAATTGATTGGATGTTTCCGTTATTCATTCGGTGATTATCGTGACGCTGTG~CTTGGTTGCCACAGG~GTC~TGTCAAGCCATTGAT~CCCACA 982 KLIGCFRYSFGDYRDAVNLVATGKVNVKPLITHK 328 AATTTAAATTTGAAGATGCAGCCAAGGCTTACGACTTACGACTAC~CATTGCCCATGGTGGAGAGGTAGTCAAGACTATTATCTTTGGTCCTG~TG~AGTGAA 1082 FKFEDAAKAYDYNIAHGGEVVKTI IFGPE* 357 TACTTTTCGGCACTGGTTCATGTCCATATATATAGACCAATTCAAAAGCAGTAATACTTGAAAATAACACCGAAAAATAAATAGTAGACACG 1182 TTTAATGACTTAAAAACTAACTTTTTCATATCTAATATTGTCAGAGCGCTGACACATA 1282 TAGAGAGCTATATGATATGAGTGAGAGCAACTCTCCCGTATATGCTAAGAATATTGTCGCTTATTAGGATTGAAAGATAGATCRATGAGGAGG~T~TGT 1382 TACCCTTTTTTCTTAAAAATGTAAGAGGAAATTATGAAATATATACTCTGATTTGTTTATTATTGATTAAGAACAATATAATAACCGCTCTGGTAGCTACTG 1482 TACATATAATTTGACGGCATATATTGCTCATATATAAAACCGCATTACTTCCAGTTGATAGATTTTTACTCAGTTTCAGTACTGCCAGAACTGCTC 1582 ATTCGAGATTTTTTCTATTTTTAGAATAGGTAAAATTGCACTTAAATGTATAAGGGATGTACGAAGTGAGTGCCCAGACTGTTACTATGACAATTAAACT 1682 AATGTCGATGACCATTTGTTTCGACAACTCCATCTTCATTTTCTTCACGCGCCATACTCGGATGAGAAAGAATCTTTTCTCTAACTATACATTTCCAAAC 1782 Fig.4. The ntsequence of the yeast SDHI gene and deducedaa sequence. Position fl corresponds totheputative start codon. Negative numbers areascribed to DNA sequences representing theprobable 5'UTR. Potential TATA boxesand polyadenylation signals areindicated by solid lines. The accession No. of the ntsequence intheGenBank Nucleotide Sequence DatabaseisL11039.

(d) Over-production of SDHl in yeast Additional evidence to show that the cloned codes an SDH was obtained The

constitutive

pMAC561,

yeast

by expression ADHI

promoter

was fused to the coding

the resulting LAG strain

plasmid

was introduced

gene en-

in S. cerevisiue. carried

on

region of SDHl,

and

into the SOOO-8B

of S. cerevisiae (Fig. 6).

Previously

we demonstrated

that the yeast SDH (appa-

rently SDHl ) enzyme is made only in sorbitol-grown cells. No enzyme activity is detected in cells grown on glucose. with 240

/,/:_ ::,.:,,.I.:.:/:,:: .I. I,,:, I,:,.: I..,:.....:,:.: LI,~GNH~DWFECSGAD”CIDAAVKTTK”GGTMVQ”GMGKNYTNFPIAE”SGKEMKLI*C

300

,,,: ..:. ,:.::,. .,,,,,,:,,:I.:,. .I:: I .:,.:, FRYSFGDYRDAVNLVATGKVNVXPLITHKFKFEDARWLYDYNIAHGGEVVKTIIFGPE

I.

ADHI-SDHl

enzyme 357

Fig.5.Alignmentofsheep(S)liverSDH andyeast(Y)SDH(see Fig.4) utilizingtheGCG Gapanalysis program(GCG Package, 1991). Highly conserved residues within these two SDH sequences and otheralcohol dehydrogenases aredesignated by lowercaseboldtype.Upper-case bold-type aa indicate thoseconserved betweensheepliver SDH and otherdetermined alcoholdehydrogenases(ADH)which arenot present inyeast SDH (Jeffery and Jornvall, 1988).

However,

plasmid when

cells of 8000-8B

pADHlSDH fusion were grown

under

LAG

transformed

containing the shown to express non-inducing

(Fig. 7). We have also demonstrated

hybrid SDHl

conditions

that replacement

of

the transcriptional sequences of the putative yeast SDHl gene with transcriptional sequences of the constitutive ADHl

gene results in several-fold increase in the synthesis of SDH in cells grown in the presence of glucose (Table I). The precise nt sequence responsible for the regulated pression of SDHl is presently being investigated.

ex-

125

kDa

C

B

A 123

123

12

3

42-

1614Fig. 7. Synthesis of SDHl protein in cells (pADHlSDH). Detection of the SDHl protein

of S. crreaisiar as determined by

Coomassie blue staining (panel A); Western blot analysis (panel B); and native gel electrophoresis (panel C). Lane 1 contains soluble extract from cells of 8000-8B LAG (pMAC561), extracts of transformants of pADH 1SDH were loaded in lanes 2 and 3. Transformation of 8000-8B LAG was done according to Beggs (1978). Transformants were grown at 30 ‘C for 24 h in SC-Trp liquid medium containing 2% glucose. Cell lysates were prepared as described in Fig. 1. Preparation of Ab to sorbitol-induced protein(s): Cell pellet from a saturated culture of 8000-88 LAG grown at 30°C in 250 ml SC liquid medium containing 2% sorbitol was resuspended in 40 ml extraction buffer. Cells were disrupted using the Bead Beater (Biospec Products). Extracts were mixed with Fig. 6. Construction of pADHlSDH. A DNA segment corresponding to the 5’ UTR plus coding region for eleven N-terminal aa of SDH (based on the nt sequence shown in Fig. 4) was first excised from pS32 by digestion with SphI + XhaI. This sequence was replaced by an oligo adapter that contained a 5’SphI site, nt sequences encoding the deleted eleven N-terminal aa and a 3’XhaI site. The adapter DNA had the following sequence: S’-GCATGCGAATTCAAAAATATGTCTCAAAATAGTAACCCTGCAGTAGTTCTAGA. The EcoRI site at the 5’

SDS-PAGE 2 x sample buffer, boiled for 3 min and subjected to 0.1% SDS-7.5% PAGE. The gel was stained for 5 min in 25% isopropanol/lO% acetic acid/0.05% Coomassie blue; destained for 20 min in 5% methanol/7% acetic acid. A portion of the gel containing protein of approx. 40 kDa was excised, solubilised in 8% NaCl/0.2% KCl/O.l% SDS and used to immunise rabbits (New Zealand White). Polyclonal antisera was prepared as described by White and Wilcox (1984).

end of the adapter DNA was inserted to facilitate subsequent plasmid constructions. Oligo synthesis was performed on an Applied Biosystems 380A synthesizer using 5’-dimethoxytrityl nucleoside p-cyanoethyl phosphoramidites (Mandecki et al., 1990). For the construction of pADHlSDH, an EcoRI fragment encoding the SDHl ORF was isolated from pS32-0 (Fig. 6) and ligated into the EcoRI site of pMAC56l(McKnight and McConaughy, 1987) with T4 DNA ligase. The yeast vector pMAC561 (kind gift from Dr. B.D. Hall, University of Washington) contains the yeast ADHl promoter and CYCf terminator sequence. Darkened box represents the SDHl gene. E, EcoRI; S, SphI; XB, Xhal.

(e) Conclusions Our studies demonstrate that S. cereuisiae produces a sorbitol-induced SDH. A gene encoding an SDH was isolated from a hgtl 1 yeast genomic expression library and the nt sequence was found to predict a protein of 357 aa with a 63% overall similarity to the known aa sequence of sheep liver SDH. The cloned gene, when introduced into yeast on a multi-copy yeast vector, results in elevated levels of expression of SDHl enzyme only in cells grown in sorbitol medium.

ACKNOWLEDGEMENTS

We thank Dr. Leonard Katz and Dr. Gregory Okasinski for valuable discussions and continual support of this work. We acknowledge gifts of plasmids and yeast strains from Dr. Benjamin D. Hall and Dr. Elton T. Young and synthetic oligos from Thomas Kavanaugh. The manuscript was typed by Gail Swopes and Pat Plutz.

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