AU707957B2 - DNA sequence coding for a protein of A. thaliana having a delta-5,7sterol,delta-7 reductase activity, delta7-red protein, production process, strains of transformed yeasts, uses - Google Patents

Description

SDNA sequence coding for a protein of A. thaliana havin a delta-5,7 sterol,delta-7 reductase activity, delta7-Red protein production process, strains of transformed yeasts, uses.

DNA sequence coding for a protein of A. thaliana having a delta-5,7 sterol,delta-7 reductase activity, delta7-Red protein, production process, strains of transformed yeasts, uses.

The present invention relates to a DNA sequence coding for a protein of A. thaliana having a delta-5,7 sterol,delta-7 reductase activity, delta7-Red protein, theirproduction process, strains of transformed yeasts, and their uses.

Delta-5,7 sterol,delta-7 reductase (E.C.1.3.1.21) is a microsomal enzyme whose presence has been revealed by its activity in homogenates of rat's liver E. Dempsey et al., Methods Enzymol., 15, 501-514, 1969) as well as in plant preparations of Zea mays Taton et al., Biochem. Biophys.

20 Res. Commun., 181, 465-473, 1991). This reductase is dependent on NADPH and reduces 7 -dehydrocholesterol to cholesterol in vitro.

Sterols are the main constituents of the membranes in eucaryotes but show structural differences depending on the 25 species. In the cells of eucaryotes such as yeasts, the principal sterol is ergosterol which contains a double unsaturation in position C-5 and C-7, a branched side chain in position C-24 and an unsaturation in position C-22 while in mammals cholesterol is characterized by an unsaturation in 30 position C-5 and a saturated side chain. Sitosterol, stigmasterol and campesterol, which represent the most common sterols in plants, have a branched but saturated side chain and, as with the sterols of vertebrates, do not have an unsaturation in position C-7. The enzyme responsible for this difference in structure of the sterol nucleus is delta-5,7 sterol,delta-7 reductase.

Delta-5,7 sterol,delta-7 reductase has never been purified to homogeneity and only a partial purification has been described E. Dempsey et al.; M. Taton et al., already quoted). The protein has not been characterized by its physical-chemical properties. No information on the sequence of the protein and no antibody directed against it have been described. Furthermore, an apparent deficiency in 7 -dehydrocholesterol reductase has been described in man associated with RSH/Smith-Lemli-Opitz (SLO) syndrome

M.

Opitz et al., Am. J. Med. Genet., 50, 326-338, 1994).

Cloning of a cDNA coding for delta-5,7 sterol,delta-7 reductase, which may enable the sequence of the corresponding protein to be identified as well as the characterization of the human gene or a congenital deficiency of it to be revealed, is not therefore achievable with known methods which make use for example of hybridization and/or immunological detection techniques. The obtaining of inventive screening methods allowing cloning to be carried out, in particular in the absence of information about the protein, is therefore particularly sought-after.

Ergosterol, the principal sterol of the membranes of fungi, contains a pair of conjugated double bonds in position C-5,7 which confers an antimycotic activity on compounds of the family of polyenes such as nystatin Bittman et al., J. Biol. Chem., 260, 2884-2889, 1985). The strong dependency of the action of nystatin on the membrane concentration of unsaturated sterols in position C-5,7 has allowed the selection of mutant strains vis-a-vis accumulated sterols in S. cerevisiae W. Molzahn et al., J. Gen. Microbiol., 72, 339-348, 1972). Thus the mutants erg2 and erg3 accumulate sterols which do not have conjugated double bonds in position C-5,7 because of a deficiency in sterol delta-8,7 isomerase Ashman et al., Lipids, 26, 628, 1991) and in sterol dehydrogenase Arthinton et al., Gene, 102, 39, 1991) respectively. Such mutants are viable because ergosterol, the principal natural sterol of yeast, can be replaced under certain conditions by different substitution sterols including cholesterol.

The advantage of increasing the resistance to nystatin of yeast strains enriched with sterols not having the double unsaturation in position C-5,7 was perceived by the inventors for cloning a cDNA coding for a heterologous protein having a delta-5,7 sterol,delta-7 reductase activity with a screening method using a metabolic interference in S. cerevisiae. The success of this approach which offers the advantage of being independent of knowing the DNA sequences or the protein as well as a detection solely based on the enzymatic activity, is not however foreseeable due to the many technical difficulties to be overcome.

A first limitation comes from the fact that the way nystatin works is not entirely elucidated W. Parks et al., CRC Critical Reviews in Microbiology, 301-304, 1978).

For example, the low specificity of the selection by nystatin is foreseeable due to the indirect nature of it which leads in yeast to the selection of spontaneous genomic mutations such as the erg mutants W. Molzahn et al. already quoted) or genomic mutations involving resistances independent of the S.sterol pathway. Similarly, cells transformed by a gene library can express heterologous genes conferring a S: 20 resistance to nystatin unrelated to the sterol pathway.

Another example of the expected limitation relates to the fact that the heterologous protein could be weakly active in the cell, leading to the absence of or to a low resistance to nystatin for example for one of the following reasons: 1) the gene coding for delta-5,7 sterol,delta-7 reductase is weakly expressed; 2) the protein is weakly or not active because of poor folding or as a result of an incorrect subcellular orientation; 3) the plant protein does not recognise the yeast's own sterols as substrates or 4) the 30 sterols which can be substrates are in the esterified form or stored in vesicles and are not in contact with the enzyme.

It can therefore be anticipated that the sterols accumulated in this way escape the action of delta-5,7 sterol,delta-7 reductase which, in eucaryotes, is reputed to be localized in the microsomes.

The present invention relates to the cloning of a cDNA of A. thaliana coding for a protein having a delta-5,7 sterol,delta-7 reductase activity, designated delta-7Red, 4 according to a cloning process carried out in a yeast by metabolic interference based on the resistance to nystatin.

The delta-7Red protein allows the sterols having an unsaturation in position C-7 to be reduced by a biological reduction process, which in addition provides an advantageous solution to the problem of the selective reduction in position C-7 of sterols or steroids having a double unsaturation in position C-5,7 which the reduction methods using chemical routes do not allow.

The invention also relates to transformed yeast cells expressing the delta-7Red which, in a surprising manner, accumulate products saturated in position C-7 and optionally saturated in position C-22 which, contrary to ergosterol, are substrates of the restriction enzyme of the side chain of cholesterol, i.e. cytochrome

P

450

SCC.

These unexpected properties allow the use of the transformed yeasts of the invention in a preparation process for sterols or steroids having an industrial and/or pharmacological use, in particular the preparation of pregnenolone by biological oxidation of endogenic yeast S: sterols, reduced in position C-7, by cytochrome

P

450

SCC

(P

450 SCC) in the presence of adrenodoxin reductase (ADR) and adrenodoxin (ADX. The transformed yeastsef- th invenLioncan be also used in a preparation process for intermediate steroids of the metabolization route of cholesterol into hydrocortisone in mammals and other vertebrates. This use has the advantage of allowing the preparation of hydrocortisone or its intermediates in a biological oxidation process which does not require the use of an exogenic sterol 30 such as cholesterol as the initial substrate and thus bypassing the problem of the penetration of a sterol into the yeast whose impermeability to exogenic sterols under aerobiosis conditions has been described T. Lorentz et al., J. Bacteriology, 981-985, 1986).

The invention also relates to the use of nucleic sequences obtained using the cloning process of the invention. An RNA or DNA sequence coding for delta-5,7 sterol,delta-7 reductase can be used for the diagnosis or the treatment of affections implicating the product of the gene of delta-5,7 sterol, delta-7 reductase. For example, a deficiency in delta-5,7 sterol,delta-7 reductase which converts 7-dehydro cholesterol into cholesterol is presumed to be implicated in RSH/SLO syndrome. Thus a human DNA sequence can be used as a probe for diagnosing a deficiency in delta-5,7 sterol,delta-7 reductase and can also be used in gene therapy to correct such a deficiency.

Therefore a subject of the present invention is a nucleic acid sequence which codes for a protein of A. thaliana having a delta-5,7 sterol,delta-7 reductase activity and having the nucleotide sequence of the sequence SEQ ID No. 1: GTGTGAGTAA TTTAGGTCAA CACAGATCAG AATCTGAGGC TTTGGCCGAG ACGAAGAGAA AAGCAGAAGA AGAAA ATG GCG GAG ACT GTA CAT TCT CCG ATC GTT ACT TAC 111 Met Ala Glu Thr Val His Ser Pro Ile Val Thr Tyr 1 5 GCA TCG ATG TTA TCG CTT CTC GCC TTC TGT CCA CCT TTC GTC ATT CTC 159 Ala Ser Met Leu Ser Leu Leu Ala Phe Cys Pro Pro Phe Val Ile Leu 20 CTA TGG TAC ACA ATG GTT CAT CAG GAT GGT TCT GTT ACT CAG ACC TTT 207 Leu Trp Tyr Thr Met Val His Gin Asp Gly Ser Val Thr Gin Thr Phe 35 GGC TTC TTT TGG GAG AAT GGA GTT CAA GGA CTT ATC AAC ATA TGG CCA 255 Gly Phe Phe Trp Glu Asn Gly Val Gin Gly Leu Ile Asn Ile Trp Pro 45 so 55 28/4/99CF8556.SPE,5

TI-'

v.VrC) AGA CCC ACT TTG ATT GCT TGG AAA ATT ATA TTT TGC TAT GGA GCA TTT Arg Pro Thr Leu Ile Ala Trp Lys Ile Ile Phe Cys Tyr Gly Ala Phe 70 303 GAA GCT ATT CTT CAG CTG CTT CTG CCT GGT AAA AGA GTT GAG GGT CCA GJlu Ala Ile Leu Gin Leu Leu Leu Pro Giy Lys Arg Vai Glu Gly Pro 85 ATA TCT CCA GCC GGA AAC Ile Ser Pro Ala Gly Asn CGA CCA GTT TAC AAG Arg Pro 100 Val Tyr Lys GCC AAT Ala Asn 105 GGT CTG GCT Gly Leu Ala TGG TTT GGA Trp Phe Gly GCT TAC Ala Tyr 110 TTT GTG ACA CTA Phe Val Thr Leu

GCA

Ala 115 ACC CAT CTT GGT Thr His Leu Gly CTT TGG Leu Trp 120 447

ATC

Ile 125 TTC AAC CCT GCA Phe Asn Pro Ala

ATT

Ile 130 GTC TAT GAT CAC Val Tyr Asp His

TTG

Leu 135 GGT GAA ATA TTT Gly Giu Ile Phe

TCG

Ser 140 495 a a a a a. a a.

C

C.

a a GCA CTA ATA TTC Ala Leu Ile Phe

GGA

Gly 145 AGC TTC ATA TTT Ser Phe Ile Phe

TGT

Cys 150 GTT TTG TTG TAC Val Leu Leu Tyr

ATA

Ile 155

AAA

Lys 2 5 GGC Gly CAT GTT His Val

GCA

Ala 160 CCT TCA TCA AGT Pro Ser Ser Ser

GAC

Asp 165 TCT GGT TCA TGT Ser Gly Ser Cys GGT AAC CTA Gly Asn Leu 170 ATT GGT AAG Ile Gly Lys 591 ATA ATT Ile Ile

GAC

Asp 175 TTC TAT TGG GGC Phe Tyr Trp Giy

ATG

Met 180 GAG TTG TAC CCT Giu Leu Tyr Pro

CGA

Arg 185 639 AGC TTT Ser Phe 35 190 GAC ATC AAG GTG Asp Ile Lys Val

TTT

Phe 195 ACT AAT TGC AGA Thr Asn Cys Arg

TTC

Phe 200 GGA ATG ATG TCT Gly Met Met Ser TGG GCA GTT CTT GCA GTC ACG Trp Ala Val Leu Ala Vai Thr 205 210 TAC TGC Tyr Cys ATA AAA CAG TAT GAA ATA AAT Ile Lys Gin Tyr Giu Ile Asn 215 220 735

GGC

Gly AAA GTA TCT Lys Val Ser GAT TCA Asp Ser 225 ATG CTG GTG AAC Met Leu Val Asn 230 ACC ATC CTG ATG CTG GTG Thr Ile Leu Met Leu Val 235 783 TAT GTC ACA AAA TTC TTC TGG TGG GAA GCT GGT TAT Tyr Val Thr Lys 240 Phe Phe Trp Trp Glu 245 Ala Gly Tyr TGG AAC Trp Asn 250 ACC ATG Thr Met 831

GAC

Asp ATT GCA Ile Ala 255 CAT GAC CGA GCT His Asp Arg Ala

GGA

Gly 260 TTC TAT ATA TGC Phe Tyr Ile Cys TGG GGT TGT CTA Trp Gly Cys Leu 265 TAC CTT GTG AAC Tyr Leu Val Asn 879 GTG TGG Val Trp 270 GTG CC? TCT GTC Val Pro Ser Val

TAC

Tyr 275 ACT TCT CCA GGC Thr Ser Pro Gly

ATG

Met 280

CAC

His 285

GGA

Gly CCC GTC GAA CTC Pro Val Glu Leu

GGA

Gly 290 ACT CAG TTG GCA Thr Gin Leu Ala

ATA

Ile 295 TAC AT? CTC GTT Tyr Ile Leu Val

GCA

Ala 300 9a a a a a a a a

S

a a

C

a ATT CTG TGC Ile Leu Cys

AT?

Ile 305 TAC ATA AAG TAT Tyr Ile Lys Tyr

GAC

Asp 310 TGT GAT AGA CAA AGG CAA Cys Asp Arg Gin Arg Gin 315 975 1023 1071 25 GAG TTC AGG Glu Phe Arg

AGG

Arg 320 ACA AAC GGG Thr Asn Gly AAA TGT Lye Cys 325 TTG GTT TGG GGA AGA GCC CCG Leu Val Trp Gly Arg Ala Pro 330 TCA AAG 30 Ser Lys AGT CTT Ser Leu 35 350

ATT

Ile 335 GTG GCG TCG TAT Val Ala Ser Tyr

ACT

Thr 340 ACA ACA TCT GGT Thr Thr Ser Gly

GAA

Glu 345 ACT AAA ACT Thr Lye Thr CTC TTA ACG TCT Leu Leu Thr Ser

GGA

Gly 355 TGG TGG GGA TTG GCT Trp Trp Gly Leu Ala 360 CGT CAT TTC CAT Arg His Phe His 1119 1167 1215

TAT

Tyr 365 GTT CC? GAG ATC TTA AG? GCT TTC TTC Val Pro Glu Ile Leu Ser Ala Phe Phe 370

TGG

Trp 375 ACC GTA CCG GCT Thr Val Pro Ala

CTC

Leu TTC GAT AAC TTC TTG GCA TAC TTC TAC GTC CTC ACC CT? CTT CTC TTT Phe Asp Aen Phe Leu Ala Tyr Phe Tyr Val Leu Thr Leu Leu Leu Phe 385 390 395 1263 -8- GAT CGA GCC AAG AGA GAC GAT GAC CGA TGC CGA TCA AAG TAT GGG AAA 1311 Asp Arg Ala Lye Arg Asp Asp Asp Arg Cys Arg Ser Lys Tyr Gly Lys 400 405 410 TAT TGG AAG CTG TAT TGT GAG AAA GTC AAA TAC AGG ATC ATT CCG GGA 1359 Tyr Trp Lys Leu Tyr Cys Glu Lys Val Lys Tyr Arg Ile Ile Pro Gly 415 420 425 ATT TAT TGATTGTAAC GAAGTCTGTT GTTCTCATTT TCTACTTATT ACGTTAATTC 1415 Ile Tyr 430 GAACGTTGGA ATCATCAAAA GACCGAGCCA AAACAAAAAT GCAAATTGAT GCGATAGACA 1475 TTCTTTTGCT GAAAAAAAAA A 1496 as well as the allelic variants of this sequence.

The delta-5,7 sterol,delta-7 reductase activity can be revealed for example using an enzymatic test in vitro described further on in the experimental part.

The above cDNA sequence which codes for a protein having 430 amino acids corresponding to the sequence shown in Figure 3 (SEQ ID No. 2) is a cDNA sequence which can be obtained for example by cloning a yeast, starting from an A. thaliana expression library, by using a screening method based on the appearance of a resistance of the yeast to nystatin, according to operating conditions a detailed description of which is given further on.

Knowledge of the nucleotide sequence SEQ ID No. 1 above allows the present invention to be reproduced by methods known to .a man skilled in the art, for example by chemical synthesis or by screening a gene library or a cDNA library using synthetic oligonucleotide probes by hybridization techniques or by PCR amplification.

Also a subject of the invention is a DNA sequence coding for a protein having a delta-5,7 sterol,delta-7 reductase activity and which hybridizes with the nucleotide sequence SEQ ID No. 1 under conditions of average or high stringency or which have a sequence identity of approximately 60% and more with this sequence.

:The sequences which hybridize in a detectable manner with sequence SEQ ID No. 1 hybridize under standard conditions of average stringency, for example a hybridization 28/4/99CF8556.SPE,8 at 42 0 C, for 12 hours in a 50% solution of formamide, SSCX6 followed by washings or under less stringent conditions, for example a hybridization at 42 0 C for 24 hours in a solution of formamide, SSCX6 followed by washings under known standard conditions Maniatis et al., Molecular cloning, Cold Spring Harbor Laboratory Press, 1989).

The percentage of nucleotide sequence identity can be determined by using for example the BLAST program "basic local alignment search tool" F. Altschul et al., J. Mol.

Biol., 215, 403-410, 1990) on the NCBI server.

The invention also relates to a DNA sequence coding for a protein having a delta-5,7 sterol,delta-7 reductase activity and amplifiable by the PCR technique using as primers the oligonucleotides coding for a consensus sequence having the amino acid sequence SEQ ID No. 3: Leu Leu Xaa Xaa Gly Trp Xaa Gly Xaa Xaa Arg Xaa Xaa Xaa Tyr 1 5 10 20 in which Xaa in position 7 is Trp or Tyr and Xaa in position 12 is His or Lys.

The above sequence SEQ ID No. 3 corresponds to a new consensus sequence which has been defined by alignment of the identity of amino acid sequences between the new sequence

SEQ

25 ID No. 2, deduced from the nucleotide sequence SEQ ID No. 1, and known sequences either of other sterol reductases having a specificity of action at a position other than position

C-

7, or of lamin B receptors, as is detailed further on in the experimental part. From the information given by the amino S 30 acid sequence SEQ ID No. 3, a primer constituted by at most 45 nucleotides can be defined and synthesized which, combined with a second primer oligodT (17 nucleotides) as rebound sequence, will allow using a commercially-available PCR assay kit (for example Stratagene) the amplification of a DNA coding for a protein having delta-5,7 sterol,delta-7 reductase activity.

The invention also relates to a protein of A. thaliana having a delta-5,7 sterol,delta-7 reductase activity and having the amino acid sequence SEQ ID No. 2: Met Ala Giu Thr Val His Ser Pro Ile Val Thr Tyr Ala Ser Met Leu 1 5 10 Ser Leu Leu Ala Phe Cys Pro Pro Phe Val Ile Leu Leu Trp Tyr Thr Met Val His Gin Asp Gly Ser Val 40 Thr Gin Thr Phe Gly Phe Phe Trp Giu Asn Gly Vai Gin Giy Leu 55 Ile Asn Ile Trp, Pro Arg Pro Thr Lau IleAia Trp Lys Ile Phe Cys Tyr Giy Aia Phe Giu Ala Ile Leu Gin Leu Leu Leu Gly Lys Arg Vai Glu Gly Pro Ile Ser Pro Ala 0S

S

S.

SW S 55 S S 555 5 V SW S S 555 5

S

*SSS

555( Gly Asn Arg Thr Leu Ala 115 Pro 100 Val Tyr Lys Ala Asn 105 Gly Leu Ala Ala Tyr Phe Vai 110 Phe Asn Pro Thr His Leu Gly Lau 120 Trp Trp Phe Gly Ile 125 Ala Ile Val Tyr ASP His Lau GIv nlii T1- nl 130 .Lw ClIft 140 3 0 Gly 145 Ser Phe Ile Phe Vai Leu Leu Tyr Lys Giy His Val S S 55 95 .55.

S

555.

S.

5

S

S

Ala 160 Pro Ser Ser Ser Asp 165 Ser Gly Ser Cys Asn Lu Ile Ile Asp Phe 175 Tyr Trp Gly Lys Val Phe 195 Met 180 Giu Leu Tyr Pro Arg 185 Ile Gly Lys Ser Phe Asp Ile 190 Thr Asn Cys Arg Phe Gly Met Met Ser Trp Ala Val Leu 200 205 Ala Val Thr Tyr Cys Ile Lys Gin Tyr Giu Ile Asn Gly Lys Val Ser 210 215 220 Met Asp Ser Met Leu Val Asri Thr Ile Leu 225 230 Leu Val Tyr Val Thr Lys 235 240 Phe Phe Trp Trp Glu Ala 245 Gly Tyr Trp Asn 250 Gly Thr Met Asp Ile Ala His 255 Asp Arg Ala Gly 260 Thr Phe Tyr Ile Cys Trp 265 Tyr Cys Leu Val Trp Val Pro 270 Pro Val Glu Ser Val Tyr 275 Thr Ser Pro Gly Met 280 Tyr Leu Val Aen His 285 Gly Leu Gly 290 Ile Tyr Gin Leu Ala Ile 295 Cys Ile Leu Val Ala 300 Gin Ile Leu Cys Ile Lye Tyr 305 Thr Asp 310 Leu Asp Arg Gin Arg 315 Ala Giu Phe Arg Arg 320 Asn Gly Lys Cys 325 Thr Val Trp Giy Arg 330 Thr Pro Ser Lys Ile Val 335 Ala Ser Tyr Thr 340 Trp Thr Ser Gly Giu 345 Arg Lye Thr Ser Leu Leu Leu 350 Val Pro Giu Thr Ser Gly 355 Trp Gly Leu Ala 360 His Phe His Tyr 365 iie Leu 370 Leu Ala 385 Ser Ala Phe Phe Trp 375 Leu Thr Val Pro Ala Thr Leu Leu Leu Leu 380 Phe Phe Asp Aen Phe Asp Arg Ala Lys Tyr Phe Tyr Val 390 Arg Asp Asp Asp Arg Cys Arg Ser Lye 405 Tyr Cys Giu Lye Val Lye Tyr Arg Ile 420 425 Tyr Gly Lye Tyr Trp Lye Leu 410 415 Ile Pro Gly Ile Tyr 430 as well as the allelic variants and analogues of this sequence.

By alleles and analogues, are included sequences modified by substitution, deletion or addition of one or more amino acids as long as these products retain the same function. The modified sequences can be for example prepared by using the site directed mutagenesis technique known to a man skilled in the art.

A particular subject of the invention is a protein of A.

thaliana having a delta-5,7 sterol,delta-7 reductase activity and having the amino acid sequence SEQ ID No. 2 above and designated delta-7Red.

The invention also relates to a protein having a delta- 5,7 sterol,delta-7 reductase activity having an amino acid sequence having an identity of sequence of approximately and more with the sequence SEQ ID No. 2.

The percentage of identity can be determined for exampleby using the BLAST program indicated above.

The invention also relates to a protein having a delta- 5,7 sterol,delta-7 reductase activity and having a crossed immunological reactivity with the protein of A. thaliana *o delta-7Red defined above.

The protein can be detected for example by immunoprecipitation using an anti-serum directed against the delta- 20 7Red protein, prepared according to known methods.

One of the aspects of the invention relates to a protein having a delta 5,-7 sterol,delta-7 reductase activity such as that obtained by expression in a host cell containing a DNA sequence defined previously and in particular relates to a S 25 protein of A. thaliana such as that obtained by expression in a host cell containing a DNA sequence coding for the amino acid sequence SEQ ID No. 2 above.

When the protein of the invention is obtained by expression in a host cell, this is carried out according to genetic engineering and cell culture methods known to a man skilled in the art.

Expression can be carried out in a procaryotic host cell, for example E. coli or in a eucaryotic host cell, for example a mammalian cell, an insect cell or a yeast containing the sequence coding for delta-7 Red of the invention preceded by a suitable promoter.

The recombinant protein obtained can be glycosylated or non-glycosylated.

In particular the invention relates to a protein according to the invention such as that obtained by expression in a yeast.

The invention also relates to an antibody directed against a protein having a delta-5,7 sterol,delta-7 reductase activity defined previously. The antibody can be a polyclonal antibody or a monoclonal antibody prepared according to methods known to a man skilled in the art.

The invention also relates to an expression vector containing a DNA sequence defined previously as well as to a host cell transformed by this vector.

The expression vectors are known vectors allowing the expression of the protein under the control of a suitable promoter. For the procaryotic cells, the promoter can be for example the lac promoter, the trp promoter, the tac promoter, the B-lactamase promoter or the PL promoter. For mammalian cells, the promoter can be the SV40 promoter or the promoters of the adeno-virus. Vectors of Baculovirus type can also be used for expression in insect cells. For yeast cells, the S 20 promoter can be for example the PGK promoter, the ADH promoter, the CYCl promoter or the GAL10/CYC1 promoter.

The host cells can be procaryotic cells or eucaryotic cells. Procaryotic cplls are for example E. coli, Bacillus or Streptomyces. Eucaryotic host cells include yeasts and 25 filamentous fungi as well as cells of higher organisms, for example cells of mammals or cells of insects. The mammalian cells can be hamster CHO cells or monkey Cos cells. The insect cells are for example SF9 cells. The yeast cells can be for example Saccharomyces cerevisiae, Schizosaccharomyces pombe or Kluyveromyces lactis.

Also a subject of the invention is a cloning process for a nucleic acid coding for a protein having a delta-5,7 sterol,delta-7 reductase activity in a microorganism comprising a screening method chosen from the resistance of the microorganism to nystatin or to an analogous compound whose toxicity depends on the presence of sterols carrying an unsaturation in position C-7, the hybridization of the nucleic acid with the nucleotide sequence of the sequence SEQ ID No. 1 above, the identification of the nucleic acid by using data processing techniques from DNA sequences isolated at random, the direct expression of the protein followed by immunodetection using antibodies directed against the protein having the amino acid sequence SEQ ID No. 2 above.

By microorganism is meant a yeast such as for example

S.

cerevisiae, S. pombe or K. lactis.

The compounds analogous to nystatin include for example amphotericin B or filipin.

By hybridization is meant a hybridization under average or high stringency conditions according to known standard conditions Maniatis et al., already quoted).

Identification using data-processing techniques can be carried out for example according to the BLAST program indicated above.

An example of the above cloning process in which the screening method uses the resistance to nystatin in a yeast is described further on in the experimental part.

20 A subject of the invention is also a DNA or RNA nucleic acid sequence, as obtained by the above cloning process. The nucleic acid sequence can be of procaryotic or eucaryotic origin according to the material from which the cloning is carried out, for example of human origin.

25 A subject of the invention is also a host cell transformed by a vector containing a DNA sequence as obtained by the above cloning process. The host cell can be a procaryotic cell or a eucaryotic cell. Examples of host cells and vectors have been indicated previously.

A particular subject of the invention is a host cell, chosen from a yeast or a filamentous fungus, transformed by a vector containing a DNA sequence according to the invention defined previously or a DNA sequence such as that obtained by the above cloning process.

One of the subjects of the invention also relates to a preparation process for a protein having a delta-5,7 sterol, delta-7 reductase activity in which a transformed host cell according to the invention is cultivated and the expressed protein is isolated and particularly relates to a process in which the host cell is a transformed yeast in which the coding DNA sequence is placed under the control of a yeast promoter.

One of the subjects of the invention also relates to a reduction process in vitro of a sterol unsaturated in position C-7 in which the sterol to be reduced is incubated with the protein obtained by the above process and the reduced sterol obtained is optionally isolated.

One of the subjects of the invention also relates to a reduction process in vivo of an exogenic sterol unsaturated in position C-7 in which the sterol is incubated with a transformed host cell according to the invention and the reduced sterol obtained is optionally isolated. The host cell can be a procaryotic cell or a eucaryotic cell, in particular a yeast or a filamentous fungus.

One of the subjects of the invention also relates to a reduction process in vivo of an endogenic sterol unsaturated in position C-7 in which a transformed host strain according 20 to the invention chosen from a yeast or a filamentous fungus is cultivated and the accumulated reduced sterol is optionally isolated.

In particular the invention relates to a reduction process in vitro or in vivo defined above in which the 25 reduced sterol obtained is a substrate of the restriction S* enzyme of the side chain of cholesterol (P 450 SCC) and quite particularly relates to a reduction process in vivo in which the endogenic sterol to be reduced is ergosta 5,7 diene 3-ol, ergosta 5,7,24(28) triene 3-ol or ergosta 5,7,22 triene 3-ol or a mixture of these.

A subject of the invention is also a production process for pregnenolone in which a transformed host cell according to the invention chosen from a yeast or a filamentous fungus is cultured, the accumulated endogenic sterol or sterols reduced in position C-7 are optionally isolated, the reduced sterols are incubated in the presence of P 450 SCC, and optionally in the presence of adrenodoxin reductase (ADR) and adrenodoxin (ADX), and the pregnenolone obtained is optionally isolated.

A particular subject of the invention is a production process for pregnenolone defined above in which the host cell is a yeast.

The invention also relates to a production process for pregnenolone in which a yeast transformed by one or more vectors allowing the coexpression of a protein having the activity of delta-5,7 sterol,delta-7 reductase and P 450

SCC

and optionally of ADR and ADX is cultured and the free or esterified pregnenolone is optionally isolated.

The invention particularly relates to the above process in which a transformed yeast coexpressing a protein having the activity of delta-5,7 sterol delta-7 reductase, P450SCC, ADR and ADX is cultured, quite particularly the above process in which the protein having the delta-5,7 sterol delta-7 reductase activity is the protein of A. thaliana delta-7Red and especially the above process in which the yeast strain is the strain EC01/pCD63.

Examples of the production of pregnenolone according to S 20 the invention are given further on in the experimental part.

The transformed yeast used for carrying out the production process for pregnenolone above can be for example a yeast co-transformed by an expression vector of the invention containing a DNA sequence coding for a protein 25 having a delta-5,7 sterol,delta-7 reductase activity and an expression vector of the cytochrome

P

450 SCC and optionally of ADX and ADR. The expression vectors of the cytochrome

P

450 SCC and/or ADX are known and preparations are described for example in the European Patent Application EP 0340878 as well as further on in the experimental part.

The transformed yeast used can also be a yeast in which the DNA sequence coding for the protein having delta-5,7 sterol,delta-7 reductase activity is integrated in a particular locus of the genome, for example at locus ADE2 and in which the ergosterol is no longer the majority sterol under the expression conditions of delta-7 reductase. The "integrated" yeast obtained can then be transformed by integrative expression cassettes or by expression vectors containing a DNA sequence coding for the cytochrome

P

450

SCC

and optionally for ADX and ADR.

An example of the construction of yeast strains producing pregnenolone or its acetic ester in vivo is given further on in the experimental part.

Therefore a subject of the invention is also a transformed yeast strain coexpressing a protein having the activity of delta-5,7 sterol delta-7 reductase, P 450 SCC, ADR and ADX and accumulating free or esterified pregnenolone, particularly an above yeast strain in which the protein having the activity of delta-5,7 sterol delta-7 reductase is the protein of A. thaliana delta-7Red and especially a yeast strain called EC01/pCD63 the precise construction of which is given further on in the experimental part.

The invention also extends to a human DNA sequence as obtained according to the cloning process defined above, used as a probe to diagnose a congenital deficiency in delta-5,7 sterol,delta-7 reductase. The deficiency in delta-5,7 sterol,delta-7 reductase includes for example the deficiency 20 in 7-dehydrocholesterol reductase responsible for the abnormally low levels of cholesterol in the plasma.

The invention also relates to a detection method for the deficiency in delta-5,7 sterol,delta-7 reductase which includes the incubation of a sample containing human genomic S. 25 DNA with the probe defined above under standard hybridization conditions and the revealing of the fixation or the absence of fixation of the probe to the genomic DNA, the absence of fixation or the reduction of the latter indicating a congenital deficiency in delta-5,7 sterol,delta-7 reductase.

The method of the invention can allow for example the detection of a congenital deficiency in prenatal delta-5,7 sterol, delta-7 reductase or in new-born babies as well as in patients suffering from various illnesses and in particular in patients having clinical manifestations of RSH/SLO syndrome.

The attached figures illustrate certain aspects of the invention: Figure 1 represents the profile in total sterols extracted from the clone F22 resistant to nystatin obtained by screening of the A. thaliana library in the yeast FY 1679.

Analysis is carried out by RP-HPLC at 205 nm or at 285 nm (Figure 1A) or by GC compared with the non-transformed yeast FY1679 (Figure lB).

Figure 2 represents a restriction map of the Not I fragment of the plasmid pF22 and the sequencing strategy.

Figure 3 represents the nucleotide sequence of the cDNA delta-7Red (SEQ ID No. 1) and the corresponding amino acid sequence (SEQ ID No. 2).

Figure 4 illustrates the measurement in vitro of the delta-5,7 sterol,delta-7 reductase activity of a microsomal fraction of FY1679 transformed with the inducible expression vector "delta-7/V8". Analysis is carried out with GC and shows the transformation of the substrate 7dehydrocholesterol (RT=16.528) into cholesterol (RT=15.887) in the presence of the endogenic sterols 5,22 diene 3-ol (RT=16.682); ergosterol (RT 17.249); ergosta 5-ene3-ol (RT=17.664).

20 Figure 5 illustrates the production of pregnenolone in vitro by restriction of ergosta 5-ene 3-ol (Fig. 5A); ergosta 5,24(28) diene 3-ol (5B) or ergosta 5,22 diene 3-ol purified from a transformed yeast expressing delta-7Red, then incubated with P 450 SCC, ADX and ADR. Analysis is carried out 25 by GC compared with a control restriction of cholesterol (full line).

Figure 6 illustrates the construction of the integrative plasmid pADdelta-7 allowing the integration of the cDNA coding for the delta-7Red (sterol delta-7 reductase) at locus ADE2.

Figure 7 represents the analysis by GC of the total sterols extracted by alkaline saponification of the strain FY1679 and the integrated strain ELR01 cultured in the presence of galactose.

Figure 8 schematizes the construction of the shuttle vector E. coli-S. cerevisiae V13 containing a single SalI (Sa) site in the multiple cloning site.

Figure 9 represents the stages of construction of the integrative plasmids pCD62-1 and pCD62-2 which allow the ADR expression cassette to be inserted in the intergenic region of genes leu2 and splldelta.

MCS1 and MCS2: multiple cloning sites.

Figure 10 represents the construction strategy of the strain CDR01.

Figure 11 represents stages of construction of the expression plasmid pCD63 containing the two expression cassettes ADX and P 450

SCC.

Figure 12 represents the analysis by GC of the sterols extracted from cells (cellular lysate) or culture medium (medium) isolated from the strain EC01/pCD63 after an induction by galactose for 9 hours (Figure 12a) or 24 hours (Figure 12b).

Figure 13 represents the structure of plasmid pTG 7457.

Figure 14 represents the structure of plasmid pTG 7453.

Figure 15 represents the structure of plasmid pTG 10014.

Figure 16 represents the structure of plasmid pTG 10004.

Figure 17 represents the structure of plasmid pTG 10031.

20 Figure 18 represents the structure of plasmid pTG 10033.

The following examples illustrate the invention without limiting it.

S* EXAMPLE 1: Cloning of the cDNA coding for delta-5,7 sterol,delta-7 reductase (delta-7Red) of A. thaliana 25 A Screening of the expression library of A. thaliana in yeast The starting cDNA expression library is the library S: described by M. Minet et al. (Plant 2, 417-422, 1992) o* which was prepared from mRNA of A. thaliana at the two-leaf germination stage and whose cDNA's bordered by the Not I site have been inserted at the Bst XI site of the expression cassette of the shuttle vector E. coli/S. cerevisiae pFL61.

This cassette contains the promoter and terminator sequences of the phosphoglycerate kinase gene (PGK). The origin of replication of the yeast derived from the 2u sequence and a selection indicator URA3 ensure the propagation of the vector in the yeast. The propagation of the vector in E. coli is derived from the plasmid pUC19.

The yeast strain FY 1679 (Mata), which is an isogenic strain of the strain S288C described by A. Thierry et al.

(Yeast, 6, 521-534, 1990), was transformed by the cDNA library using the lithium acetate method described by D.

Gietz et al. (Nucleic Acids Res., 20, 1425, 1992).

The cells were plated on a synthetic medium SGI containing 7 g/l of "yeast nitrogen base" (Difco), 1 g/l of bactocasaminoacids (Difco), 20 g/l of glucose, 20 mg/l of tryptophan and which is free from uracil. 105 prototrophic transformants for uracil were obtained then grouped and again plated on the same synthetic medium free from uracil and containing 2 or 5 gg/ml of nystatin at the rate of 5x10 4 cells per dish. 106 cells for each nystatin concentration were screened in this way. After incubation for 3 days at 280C, approximately 100 clones, which have grown at a concentration of 2 Ag/ml of nystatin, were collected by constituting groups of 5 clones whose sterol composition was analyzed by reversed-phase high performance liquid .:chromatography (called RP-HPLC in what follows) while a 20 single resistant clone, called F22, was obtained at a concentration of 5 Ag/ml of nystatin.

B Analysis of the sterols accumulated in the clone F22 The total sterols of the yeast are prepared according to the alkaline saponification method described by L. Parks S 25 et al. (Methods in Enzymol., 111, 333-346, 1985), then analyzed by RP-HPLC and/or by gas chromatography (called GC).

The residue of sterols obtained is dissolved in an ethanol-tetrahydrofuran-water mixture (65:10:25 v/v) then e* analyzed by RP-HPLC on an Applied Biosystems C18 bonded silica column (100 x 2.1 mm) at a flow rate of 1 ml/min and at 55 0 C using a linear gradient of methanol in water (50% to 100% over 18 min) and a photometric detection at 205 nm and 285 nm compared with the ergosterol, campesterol and cholesterol standards.

The composition of sterols is also analyzed by GC on an Alltech SE-30 capillary column (30 m x 0.32 mm) with helium as carrier gas, a temperature of 280 0 C and 3100C for the injector and the detector respectively, with an initial increase in the temperature of 110 0 C to 245 0 C at a speed of then of 3 0 C/min in order to reach 280 0

C.

The analysis by RP-HPLC (Figure 1A) and the analysis by GC (Figure 1B) show the profile of the accumulated sterols in the clone F22 obtained above characterized by the virtually complete disappearance of ergosterol, majority sterol of the non-transformed strain FY 1679, and its replacement by two majors sterols and in similar quantity which do not absorb at 285 nm and thus no longer have a conjugated double unsaturation according to analysis by RP-HPLC.

In Figure 1A, the campesterol (Sigma) (24-R-ergosta ene 3-ol) contains approximately 35% of dihydrobrassicasterol (24-S-ergosta 5-ene 3-ol).

C Cloning of the delta-7Red cDNA The plasmids originating from the clone F22 were amplified in E. coli according to the method described by J.

N. Strathern et al. (Methods in Enzymol., 194, 319-329, 1991), then digested by Not I. A fragment of about 600 base pairs (bp) and a fragment of 1.6 kbp were obtained. The 20 strain FY1679 was transformed with each of the above fragments respectively. The composition in sterols of each clone of the transformed yeast was analyzed as indicated above and allowed the plasmid carrying the gene responsible for the modified profile in sterols to be distinguished. The 25 plasmid identified in this way was named pF22.

D Determination of the CDNA sequence of delta-7Red The cDNA insert of pF22 was sub-cloned at the Not I site S of the vector pUC9N derived from pUC9 (Pharmacia) in which o*oo. the Eco RI site of the multiple cloning site is replaced by the insertion of a Not I restriction site while retainingthe reading frame of the LacZ gene. A restriction map was then determined (Figure 2).

Restriction fragments having the Not I external site and the EcoRI, PvuII or HindIII internal sites respectively were sub-cloned in the pBluescript plasmid (Stratagene). The nucleotide sequence was determined by the Sanger method with DNA polymerase Sequenase (Stratagene kit) on the two strands by using direct and inverse primers of pBluescript pUC9, T3 and T7 or deduced specific primers of the cDNA nucleotide sequence.

The compilation of all of the sequences obtained gives the delta-7Red cDNA nucleotide sequence of A. thaliana

(SEQ

ID No. 1) represented in Figure 3. It comprises 1496 nucleotides ending with a polyadenylation sequence. It has an open reading frame starting with a methionine initiator at nucleotide 76 and ending with a termination codon at nucleotide 1366. This results in an open reading frame of 1290 nucleotides coding for a protein of 430 amino acids.

The coding region of the delta-7Red cDNA codes for the protein delta-7Red the deduced amino acid sequence of which (SEQ ID No. 2) is shown in Figure 3. The sequence of the protein includes 430 amino acids amines having a calculated molecular mass of 49.286 kDa.

A sample of the E. coli DH5-1 strain containing delta- 7Red cDNA in the vector pUC9N (designated delta7red/pUC9N S: DNA) was deposited at the CNCM on 10th February 1995 under the number 1-1535.

20 E Determination of the consensus sequence SEQ ID No. 3 Using computerized searching of sequence databases (Genbank and EMBL), it has been shown that the sequence of the delta-7Red protein of A. thaliana has some sequence similarities with other sterol reductases, in particular sterol C-14 reductase and sterol C-24(28) reductase of S.

cerevisiae described by R. T. Lorentz et al. (DNA Cell Biol., 11, 685-692, 1992) and W. Chen et al. (Yeast, 7, 305-308, 1991) respectively as well as the product of the sts 1+ gene of S. pombe described by M. Shimanuki et al. (Mol. Biol.

Cell, 3, 263-273, 1992) and sterol C-14 reductase of Neurospora crassa (No. X77955 in the EMBL database). In addition, the delta-7Red protein shows a similarity with the 400 amino acids of the C-terminal end of the chicken lamine B receptor and with that of the corresponding human receptor described by H. J. Worman et al. Cell Biol., 111, 1535- 1542, 1990) and E. Schuler et al. Biol. Chem., 269, 11312-11317, 1994).

Sequence identity alignments were established between the amino acid sequence SEQ ID No. 2 deduced from the delta- 7Red cDNA obtained above and those of the three yeast sterol reductases and the two lamine B receptors, then a new consensus sequence having the amino acid sequence (SEQ ID No.

3): Leu Leu Xaa Xaa Gly Trp Xaa Gly Xaa Xaa Arg Xaa Xaa Xaa Tyr 1 5 10 in which Xaa in position 7 is Trp or Tyr and Xaa in position 12 is His or Lys, was defined in order to prepare oligonucleotides which can be used as primers to amplify, by PCR, new genomic DNA or cDNA sequences coding for a protein having a delta-5,7 sterol, delta-7 reductase activity.

F Expression of the delta-7Red protein in yeast.

a) construction of the expression vector delta-7/V8 inducible in yeast The deletion of the non-coding regions of cDNA of pF22 20 was carried out by the PCR amplification using the following specific oligonucleotides: TGGCGGAGAC TGTACATTC 3' (SEQ ID No. 4) and 25 5'CAGGGTACCT CAATAAATTC CCGGAATG 3' (SEQ ID No. which were defined in order to introduce a BamH I restriction site immediately upstream of the initiation codon and a Kpn I site immediately downstream of the stop codon.

The cDNA was amplified starting from 1 ng of "delta7red/pUC9N cDNA" plasmid in the presence of 2 units of Pfu DNA polymerase and 0.2 uM of each of the above primers by using the following amplification conditions: 94 0 C, 52°C, 50s; 74oC, 90s; 33 cycles with the Stratagene PCR kit.

A fragment of approximately 1300 bp was obtained, then digested by the restriction enzymes BamH I and Kpn I and inserted into the BamH I and Kpn I sites of the E. coli/S.

cerevisiae pYeDP1/8-2 shuttle vector (called V8) described by

I

C. Cullin et al. (Gene, 65, 203-217, 1988). V8 carries the selection indicator URA3 and contains an expression cassette in the yeast containing the promoter GAL10/CYC1 and the terminator sequence of the PGK gene. The vector thus obtained, called delta-7/V8, allows the inducible expression by galactose of the delta-7Red protein.

b) Production of the delta-7Red protein The yeast strain FY 1679 (Mata) was transformed with the delta-7/V8 plasmid obtained above by using the lithium acetate method described by D. Gietz et al. (already quoted).

The transformed yeast was cultured at 27 0 C, in the SGI selective medium described above but in which the glucose concentration is 5 g/l, until a cell density saturation (OD600nm=12) is obtained. The culture is then diluted by the addition of a volume of complete medium YP (10 g/1 in yeast extract (Difco) and 10 g/l in bactopeptone (Difco)), then the addition of ethanol v/v) as a source of carbon. When the growth has been allowed to reach a cellular concentration .:of at least 7x10 7 cells/ml, the expression of delta-7Red was 20 induced by the addition of galactose at a concentration of g/l.

The induction was also carried out in an SLI selective minimum medium, which corresponds to the SGI medium in which the glucose is replaced by galactose (20 until a 25 cellular concentration of 2 x 107 cells/ml is obtained.

c) Enzymatic test in vitro of the delta-5,7 sterol,delta-7 reductase activity Expression of the delta-7Red protein was revealed by using an enzymatic test described by M. Taton et al.

(Biochem. Bioph. Res. Commun., 181, 465-473, 1991) but without an NADPH regeneration system, with microsomal or cytosolic cell preparations of the above induced yeast.

The cellular fractions were obtained by mechanical rupture of the induced cells and isolation of the fractions by ultracentrifugation according to the method described by P. Urban et al. (Eur. J. Biochem., 222, 843-850, 1994). The cells are collected then washed twice with TE-KCl buffer mM Tris-HCl, pH 7.4; 1mM EDTA, 0.1 M KCl) and resuspended in 0.6 M TE-sorbitol lysis buffer. Glass beads with a diameter of 0.45-0.5 mm (Braun) are added until they show through the surface of the cellular suspension which is then agitated for min at 4 0 C. The cellular lysate on the surface is collected and the glass beads are washed three times with the lysis buffer. The lysate and the washings are united then centrifuged at 20,000 g for 13 min at 4 0 C so as to eliminate the mitochondrial fractions. The collected supernatant is centrifuged for 1 hour at 100,000 g and at 40C. The pellet which contains the microsomal fraction and the supernatant which represents the cytosolic fraction are collected separately.

The microsomal fraction or the cytosolic fraction obtained are incubated respectively for 90 min at 37 0 C in 100 mM Tris/HCl buffer at pH 7.3 containing as substrate 150 um of 7-dehydrocholesterol emulsified with Tween 80 (1.5 g/l) and in the presence of 2 mM of NADPH. The sterols are extracted by the addition of 3 volumes of a methanoldichloromethane mixture (50:50, v/v) then analyzed by GC in 20 comparision with standard products.

The formation of cholesterol (RT 15.887 min) from 7dehydrocholesterol (RT 16.528 min) is shown in Figure 4 with a microsomal fraction (3.5 mg/ml of protein) obtained above from the yeast FY 1679 transformed with the delta-7/V8 25 vector, induced for 3 hours and the endogenic sterols of which have higher retention times (RT 16.682 min for ergosta 5-22 diene 3-ol; RT 17.249 min for ergosterol and S: RT 17.664 min for ergosta 5-ene 3-ol).

e *These results show that the delta-7Red protein, on the one hand is expressed in the transformed yeast, and on the other hand has a delta-5,7 sterol,delta-7 reductase activity.

EXAMPLE 2: Reduction in vivo of endogenic sterols of yeast unsaturated in position C-7 Yeast strains whose majority sterols have a double bond in position C-5,7 were transformed with the delta-7/V8 vector obtained in Example 1, then cultured and induced as indicated in Example 1. The endogenic sterols, whose profile is analyzed by GC, were extracted and purified by RP-HPLC as indicated in Example 1 by using a preparative C18 column (100 X 4.6 mm), then identified by IR, UV, MS and NMR. The following three strains were used respectively: Strain FY1679 described in Example 1; Mutant strain erg5, called PLC 1051, characterized by a deficiency in sterol C-22 desaturase, which was constructed by crossing between the strain FY1679 and the original strain described by S. W. Molzahn et al. Gen. Microbiol., 72, 339-348, 1972) and which accumulates the ergosta 5,7diene 3-ol.

The mutant double strain erg4,5, called PLC 1451, characterized by a deficiency in sterol C-22 desaturase and sterol C-24(28) reductase (erg4), was obtained by crossing between the strain FY1679 and a strain described by S. W. Molzahn et al. (already quoted) having acquired a spontaneous resistance to nystatin during a systematic screening of yeasts searching for mutants of sterols and which accumulates ergosta 5,7,24(28) triene 3-ol.

20 The resultant haploid strain, which carries the double mutation erg4, erg5, grows in the presence of galactose and a non-fermentable substrate and is auxotrophic for uracil, tryptophan and histidine. The strains PLC 1051 and PLC 1451 were deposited at the CNCM on 10th February 1995 under the numbers 1-1536 and 1-1537 respectively.

The major reduced sterols identified respectively from the above three transformed strains are indicated in the following table: Initial Initial major Major endogenic strain endogenic sterol sterol reduced in position C-7 FY 1679 ergosta 5,7,22 triene ergosta 5-ene 3-ol 3-ol (ergosterol) (dihydrobrassicasterol) ergosta 5,22 diene 3-ol (brassicasterol) ergosta 5,7 diene 3-ol ergosta 5-ene-3-ol PLC 1051 erg4,5 ergosta 5,7,24(28) ergosta 5,24(28) PLC 1451 triene-3-ol diene 3-ol (ostreasterol) EXAMPLE 3: Production of pregnenolone in vitro by 20 restriction of yeast endogenic sterols reduced in position

C-

7 Production of pregnenolone is carried out by using the enzymatic restriction test of the side chain of cholesterol in vitro described by Wada et al. (Arch. Biochem. Biophys., 25 290, 376-380, 1991) in which 260 uM of a sterol reduced in position C-7 obtained in Example 2 is incubated in 150 ul of mM phosphate buffer, pH 7.4, 100 mM NaC1, 0.3% Tween 20 in the presence of 140 nM of adrenodoxin reductase, 1.16 uM of adrenodoxin and 0.68 uM of cytochrome

P

45 0 SCC of bovine origin obtained from the suprarenal glands for example according to D. W. Seybert et al., J. Biol. Chem., 254, 12088-12098, 1979.

The reaction is triggered by the addition of 150 uM of NADPH. After incubation for 80 min at 37 0 C, the reaction is stopped by the addition of a volume of a methanoldichloromethane mixture (50:50 The sterols are extracted and analyzed by GC as indicated in Example 1.

Figure 5 shows respectively that ergosta 5-ene 3-ol (Fig. 5A), ergosta 5,24(28) diene 3-ol (Fig. 5B) or ergosta 5,22 diene 3-ol (Fig. 5C) is the substrate of cytochrome

P

450 SCC and leads to a product having an identical RT to that of pregnenolone obtained under the same conditions by restriction of cholesterol.

The results obtained show that a transformed yeast expressing delta-7Red accumulates usable sterols directly to prepare pregnenolone by biological oxidation in vitro.

EXAMPLE 4: Construction of yeast strains producing pregnenolone or its acetic ester in vivo A Construction of the strain ELRO1 containing an expression cassette for the delta-7Red of A. thaliana integrated in the locus ADE2 of the haploid strain FY1679 mating type a (FY1679 Mata) a) Construction of the integrative plasmid pADdelta-7 (pADA7): Construction of the plasmid pAD 7 was carried out as Sdescribed in Figure 6. The BglII fragment (2244pb) S. containing the ADE2 gene of S. cerevisiae was isolated from 20 the plasmid pASZ 11 Stotz et al., Gene, 95, 91, 1990) and inserted in the pBluescriptII KS+ vector (Stratagene) at the BamHI site of the multiple cloning site.

The plasmid obtained, called pBS-ADE2, was then linearized at its single Stu I site and dephosphorylated.

25 A fragment of approximately 2.44 kb containing the promoter GAL10/CYC1, the coding phase of delta-7Red (sterol 7 reductase) and the terminator PGK (tPGK) was obtained from :the plasmid delta-7/V8, obtained in Example IF by the PCR technique using the following oligonucleotides as primers: GATTACGCCA AGCTTTTCGA AAC 3' (SEQ ID No. 6) and AGATCTTGAG AAGATGCGGC CAGCAAAAC 3' (SEQ ID N 0. 7) which have been defined to pair with the 3' end of tPGK and the 3' end of the URA3 gene respectively. The following were used: the plasmid delta-7/V8 as a template (80 ng), the above oligonucleotides (0.5 gM each), native Pfu DNA polymerase (1 29 unit in the buffer recommended by Stratagene) and the following amplification conditions: 35 cycles; 1 min at 95 0

C;

sec. at 95oC; 30 sec. at 56 0 C; 4 min 30 sec. at 70 0 The amplification fragment obtained was then cloned at the blunt ends in the above linearized plasmid pBS-ADE2 to give the plasmid pADA7 in which the NotI-PstI fragment of approximately 4720 pb carries the ADE2 gene interrupted by the expression cassette of delta-7Red.

b) Chromosomic integration in the yeast strain FY1679 Mata: The NotI-PstI fragment (4720 bp), isolated from the plasmid pADA7 digested with the restriction enzymes NotI and PstI, was introduced into the yeast FY1679 Mata by transformation using the lithium acetate method described by D. Gietz et al. (already quoted).

The transformants having integrated this fragment by e. homologous recombination were selected by their resistance to nystatin, this phenotype being due to the expression of delta-7Red which converts the delta-5,7 sterols of the yeast into delta-5 sterols.

20 The transformed cells were incubated at 28 0 C for 4 hours in the complete medium YP described in Example IF containing glucose (20 g/l) and supplemented with adenine (20 mg/l).

They were then concentrated then plated on an SLI-agar minimum medium (1g/l of bactocasaminoacids; 7 g/l of "yeast 25 nitrogen base"; 20 g/l of galactose; 20 mg/l of adenine; g/l of agar) and incubated overnight at 28°C to induce the expression of the delta-7Red gene. The absence of uracil complementation allows the growth of cells to be limited.

The clones were collected, grouped then plated on dishes on a complete medium YP containing galactose (20 g/l), supplemented with adenine (20 mg/l) and in the presence of increasing concentrations of nystatin (0 Ag/ml, 1 ug/ml, 2 gg/ml, 5 pg/ml, 20 Ag/ml respectively). On the 4th day, about twenty clones having grown at a concentration of /g/ml of nystatin were obtained. Twelve of these clones were sub-cultured on dishes in minimum medium WO (7g/l of "yeast nitrogen base" without amino acids, 20 g/l of glucose) enriched with uracil, leucine, tryptophan, histidine and adenine (20 mg/l each).

The auxotrophy of the adenine due to the disruption of the ADE2 gene was then confirmed by observing the absence of growth on the minimum medium WO described above enriched with uracil, leucine, tryptophan, histidine but free of adenine.

The presence of the expression cassette in the genome of the yeast was controlled by PCR amplification from the genomic DNA of the clones obtained and by using the primers having the sequence SEQ ID No. 6 and SEQ ID No. 7 above.

The functionality of the integrated delta-7Red gene was confirmed by GC analysis of the composition in sterols accumulated in the yeast and extracted by alkaline saponification according to the operating method described in Example 1B with a five meter SE 30 capillary column (Alltech). Analysis shows a modified profile containing sterols saturated in position C-7 when the clones are cultivated in the presence of galactose. The strain obtained, called ELR01, accumulates ergosta 5 ene 3-ol and ergosta 5,22 diene 3-ol instead of ergosta 5, 7, 22 triene 3- 20 ol (ergosterol), the majority sterol of the initial strain FY1679 as is shown in Figure 7.

The strain ELR01 constructed in this way expresses the delta-7Red gene when it is cultured in the presence of galactose due to the fact of transcriptional control by the 25 promoter GAL10/CYC1. Although the expression unit for delta- 7Red has the same transcription direction as that of the ADE2 gene, no expression of the delta-7Red is detectable by analysis of the composition of sterols by GC when the culture is carried out in the presence of glucose, as a result of the repression of the promoter GAL10/CYC1.

B Construction of the strain CDRO1 containing an expression cassette for the mature form of bovine adrenodoxin reductase (ADRm) integrated between the loci LEU2 and SPL1 of the haploid strain FY1679 mating type alpha (mat. alpha).

a) Construction of the shuttle vector E. coli-S. cerevisiae V13 The V13 vector corresponds to the V8 vector Cullin et al., already quoted) which carries the selection indicator URA3 and an expression cassette in yeast containing the GAL10/CYC1 promoter (pG/C) and in which an additional SalI site (Sa) has been introduced into the multiple cloning site, according to the construction diagram given in Figure 8.

The V8 vector was digested by the restriction enzymes HindIII and BamHI and the BamHI-HindIII fragment obtained (1722 pb) containing the URA3 gene and the GAL10/CYC1 promoter (called further on ura-gal"), was sub-cloned between the corresponding sites of the pUC18 vector (Pharmacia) digested by the restriction enzymes HindIII and BamHI.

The "ura-gal" fragment was then amplified by PCR from ng of the pUC18/"ura-gal" plasmid obtained denatured for sec. at 95 0 C using the following conditions: 30 cycles; sec. at 86 0 C; 10 sec. at 95 0 C; 40 sec. at 38 0 C; 5 sec. at 55 0 C and 2 min at 74 0 2 units of Taq DNA polymerase (Boehringer) in the manufacturer's buffer and 1AM of each of the primers having the following nucleotide sequences: 5' GGGGATCCGT GGTCGACGTA ATTTAGTGTG TGTATTTGTG TTTGCG 3' 20 (SEQ ID No. 8) and GTAAAACGAC GGCCAGT 3' (SEQ ID No. 9) The primer SEQ ID No. 8 contains a BamHI GGATCC site identical to that of the "ura-gal" fragment, 3 consecutive S. nucleotides at the BamHI site not hybridizing with the template, an Sail GTCGAC site and a sequence homologous to that of the GAL10/CYC1 promoter. The primer SEQ ID No. 9 matches the sequence preceding the Hind III site of the 30 multiple cloning site pUC18. The HindIII-BamHI fragment of 1722 pb obtained after amplification was digested by XhoI and Bam HI releasing a fragment of 254 bp containing the GAL10/CYC1 promoter (pG/C) which was then sub-cloned in the V8 vector digested by the restriction enzymes BamHI and XhoI.

The resultant V13 vector contains restriction sites allowing easy sub-cloning of the cDNA's coding for mature bovine adrenodoxin reductase (ADRm), mature bovine adrenodoxin (ADXm) and the bovine cytochrome

P

450

SCC.

32 Starting from the V13 vector, the V13-ADR vector, the V13-ADX vector and the V13-SCC10 vector were prepared respectively in the following manner: a) Construction of the V13-ADR vector A SalI-KpnI fragment of 1478 pb carrying the cDNA coding for ADRm was isolated from the plasmid pGBADR-2 described in Example 25 of the European Patent Application EP 340878 and sub-cloned in the corresponding sites of the V13 vector to give the V13-ADR vector.

b) Construction of the V13-ADX vector A Sail BamHI fragment of 390 pb carrying the cDNA coding for ADXm was isolated from the plasmid pGBADX-1 described in Example 23 of the European Patent Application EP 340878 and sub-cloned in the corresponding sites of the V13 vector to give the V13-ADX vector.

c) Construction of the V13-SCC10 vector A SalI-EcoRI fragment of 1554 pb carrying the cDNA coding for P 450 SCC was isolated from the plasmid described in Example 6 of the European Patent Application

EP

20 340878 and sub-cloned in the corresponding sites of the V13 :i vector to give the V13-SCC10 vector.

b) Construction of the integrative plasmids pCD62-1 and pCD62-2: The construction of the plasmids pCD62-1 and pCD62-2 was carried out as described in Figure 9.

a) Construction of the plasmid pFL26CD A NotI site was introduced into the plasmid pFL26 (N.

Bonneaud et al., Yeast, 7, 609-615, 1991), in the intergenic region separating the leu2 gene from the 5' end of the spll gene (called spll Kolman et al., J. Bacteriol., 175, 1433, 1993) according to the following operating method: Two DNA fragments of 704 bp and 347 bp carryingthe end of leu2 and the 3' end of Spll respectively were synthesized by PCR using primers having the following nucleotide sequences: TTGAAGGTTC AACATCAATT GATTG 3' (SEQ ID No. 10) and GTGTGGCGGC CGCCTCCTTG TCAATATTAA TGTTAAAG 3' (SEQ ID No. 11) for the amplification of the 704 bp fragment and the nucleotide sequences 5' CAAGGAGGCG GCCGCCACAC AAAAAGTTAG GTGT 3' (SEQ ID No. 12) and TCTGCTTCCC TAGAACCTTC TTATG 3' (SEQ ID No. 13) for the amplification of the 347 bp fragment.

The primers SEQ ID No. 11 and SEQ ID No. 12 have a sequence GGCGGCCG which corresponds to a NotI site and in which 3 bases do not match with the template. The primer SEQ ID No. 10 matches with a sequence situated 536 bp upstream S 20 from the stop codon of leu2 and the primer SEQ ID No. 13 with a sequence situated 194 bp upstream that of spllA.

The 704 bp and 347 bp fragments are first amplified by PCR by using the plasmid pFL26 as the template and Pfu DNA polymerase as the enzyme under standard conditions described by the supplier (Stratagene).

The two amplified fragments obtained matched over 20 bp at the level of the ends generated by the primer SEQ ID No.

11 (704 bp fragment) and the primer SEQ ID No. 12 (347 bp fragment) starting from the 5' end; these 20 bp correspond to 30 the first 20 nucleotides of each of these primers respectively.

The product resulting from the pairing of the DNA fragments of 704 bp (1 ng) and 347 bp (2 ng) was amplified using primer SEQ ID No. 10 and SEQ ID No. 13 and the following conditions: 30 cycles of 10 sec. at 95 0 C, 5 sec. at 0 C, 1 min at 45 0 C, 5 sec. at 650C and 2 min at 72 0

C

followed by a cycle of 7 min at 72 0 C; 50 pmol of each primer and 1 unit of Pfu DNA polymerase in 50 Al of reaction buffer (Stratagene). An amplified fragment of 1031 bp containing a NotI restriction site was obtained. This fragment was then digested by the BstXI and NsiI enzymes and the fragment of 920 bp obtained, containing the NotI site, was inserted in place of the initial BstXI-NsiI fragment of pFL26 to give the plasmid pFL26CD, whose map is represented in Figure 9a.

b) Construction of the plasmid Preparation of the plasmid pDP10036: A SalI-BamHI fragment of 390 bp carrying the cDNA coding for mature bovine adrenodoxin (ADXm) was isolated from the plasmid V13-ADX then sub-cloned in the SalI-BglII sites of the multiple cloning site of the plasmid pTG10033 which is flanked by the inducible promoter GAL10/CYC1 and the terminator ter PGK. The plasmid pTG10033, whose map is represented in Figure 18 and which corresponds to the expression vector pTG10031 (Figure 17), containing the promoter CYC1 and terPGK, in which the promoter CYC1 has been replaced by the promoter GAL10/CYC1 was prepared according to the operating method described further on.

20 The plasmid thus obtained, called pDP10034, carries the ADX expression cassette, i.e. the gene coding for ADXm under the transcriptional control of GAL10/CYC1 and terPGK.

Subsequently, the term "expression cassette" will be used for any gene under the transcriptional dependence of GAL10/CYC1 and terPGK.

A HindIII fragment of 3593 bp carrying the selection indicator URA3 and the ADR expression cassette was isolated from the plasmid V13-ADR digested by the restriction enzyme HindIII then inserted in the corresponding site of plasmid 30 pDP10034 digested by the restriction enzyme HindIII. The plasmid obtained, called pDPl0036, contains the ADX and ADR expression cassettes separated from each other by the marker URA3 (Figure 9b).

Preparation of the plasmid The AflIII-AccI fragment of 2770 bp carrying the ADR cassette was isolated by partial digestion of the plasmid pDP10036 with the restriction enzymes AflIII and AccI, bluntends were created by treatment with the klenow fragment of DNA polymerase I and sub-cloned after ligation at the blunt ends in the SmaI site of the plasmid pBlue-Script

KS+

(Stratagene). In the plasmid obtained, called pCD60, the ADR expression cassette is framed on either side by a NotI site, one situated at 209 bp upstream of the AflIII/SmaI ligation site and originating from the sub-cloned fragment and the other originating from the multiple cloning site (MCS1) of pBlue-Script KS+ (Figure 9b).

c) Construction of plasmids pCD62-1 and pCD62-2: The NotI fragment of 2558 bp, isolated from the plasmid digested by the restriction enzyme NotI, was then subcloned in the single NotI site of the plasmid pFL26CD.

Depending on the direction of the insertion of the fragment, two plasmids were obtained, called pCD62-1 and pCD62-2 (Figure 9c).

In the plasmid pCD62-1, the ADR expression cassette is oriented in the direction of the transcription of the leu2 :.gene while this orientation is reversed in the plasmid pCD62- 2.

20 The plasmid pCD62-1 was retained for subsequent constructions.

c) Chromosomic integration in the yeast strain FY1679 (Matalpha): The plasmid pCD62-1 contains regions homologous to a chromosomic locus of the strain FY1679. These regions correspond respectively to fragments BglII-ClaI of 1060 bp EcoRI-NotI of 707 bp and NotI-BglII of 602 bp (C) indicated in Figure The region corresponding to the ClaI-EcoRI fragment of 30 486 bp of the plasmid pCD62-1 was deleted in the strain FY1679, implying an auxotrophy of this strain vis-a-vis leucine (strain LEU2-) S. Sikorski et al., Genetics, 122, 19, 1989).

The plasmid pCD62-1 was linearized by digestion with the restriction enzyme XbaI whose restriction site is situated outside the homologous regions, then was introduced by transformation in the strain FY1679 (Matalpha) using the lithium acetate method Gietz et al., already quoted).

The cellular repair capacity of the DNA by the yeast ("gap repair") and the selection of recombinants having the phenotype LEU2 have allowed in the first instance two types of recombinants to be selected: the 1st type is obtained after homologous recombinations at the level of fragments

A

and B, the 2nd type is obtained after homologous recombinations at the level of fragments A and C. Only the latter type of recombination allowed the integration of the ADR expression cassette in addition to the restoration of phenotype LEU2.

To select this 2nd type of clone, a screening by PCR was carried out using the primer SEQ ID No. 10 above and the primer having the following nucleotide sequence: 5' TACATTAGGT CCTTTGTAGC 3' (SEQ ID No. 14) so as to confirm both the presence and the correct localization of the ADR expression cassette in the genome of the strain FY1679 (Matalpha).

S 20 In this screening, the primer SEQ ID No. 14 exclusively matches the sequence coding for ADRm and the primer SEQ ID No. 10 matches a chromosomic sequence (Figure The amplification reaction was carried out using as :template the genomic DNA (20 ng) isolated from the strain FY1679 (Matalpha) Hoffman et al., Gene, 57, 267, 1987), Taq DNA polymerase (lU, Boehringer), 50 pmol of each primerand 30 PCR cycles (10 sec. at 95 0 C, 1 min at 550C, 3 min at 72oC) under the standard conditions described by the supplier.

30 The amplification led to the isolation of a corresponding fragment of 2.9 kb in the case where the expression cassette has been integrated. In the opposite case, no amplification product was detected.

The strain FY1679 (Matalpha) selected in this way containing the ADR integrated expression cassette was called CDRO1.

The expression of ADR by this strain was revealed from cytosolic cellular fractions prepared according to the 37 protocol described in Example IF, by immunodetection of the protein recognised by the anti-ADR antibodies.

The functionality of the ADR expressed by the strain CDR01 was confirmed in the enzymatic restriction test of the side chain of cholesterol in vitro described in Example 3 and in which the purified ADR (0.28 pmol) was replaced by a cytosolic cellular fraction of the strain CDR01 containing 100 ug of total proteins. A bioconversion rate of cholesterol into pregnenolone of approximately 25% was observed, comparable to that obtained with purified

ADR.

C Construction of the diploid strain EC01 coexpressing delta-7Red of A. Thaliana and ADRm.

The diploid strain ECO1 was obtained by crossing the haploid strains CDRO1 and ELR01 obtained above according to the protocol described by G. Sprague et al. (Methods in Enzymology, 194, 77, 1991): A first selection on minimum medium WO described previously, enriched with uracil, tryptophan and histidine (20 mg/1 each) but free of leucine, allowed the diploid 20 clones LEU2+ (prototrophic character which shows the presence of the ADR expression cassette) to be isolated. These clones were then tested for their resistance to nystatin at 5 Ag/ml (resistance character which shows the presence of the expression cassette for delta-7Red) on the solid synthetic SLI-agar medium described previously.

A strain, called ECO1, was in this way arbitrarily isolated from the clones resistant to nystatin.

D Construction of the strain ECO1/pCD63 producing pregnenolone and pregnenolone 3-acetate.

30 a) Construction of the expression plasmid pCD63 The construction of the plasmid pCD63 was carried out as described in Figure 11.

The NotI fragment of 4961 bp containing the ADX expression cassette, the selection indicator URA3 and the ADR expression cassette, were isolated from the plasmid pDP10036 prepared above and digested with the restriction enzyme NotI, then cloned in the NotI site of the multiple cloning site of plasmid pFL45L Bonneaud et al., Yeast, 7, 609, 1991).

The vector thus obtained, called pDP10037, is represented in Figure 11.

On the one hand, the plasmid pDP10037 was linearized by digestion with the enzyme TthlllI whose restriction site is situated in the gene coding for ADRm.

On the other hand, a PvuII-EcoRV fragment of 3476 bp containing the expression cassette for P 450 SCC and the 5' end of the URA3 marker were purified from the plasmid V13-SCC10 obtained previously and digested with the restriction enzymes PvuII and EcoRV.

The two linear DNA's respectively obtained have homologous regions which correspond on the one hand to the end of the gene URA3 and to GAL10/CYC1, on the other hand to ter PGK as represented in Figure la.

These two fragments were then introduced into the yeast strain FY1679 (Mata) by co-transformation using the lithium acetate method Gietz et al., already quoted).

The following selection of prototrophic recombinants for uracil and tryptophan (URA3 RTP1+), allowed clones to be 20 isolated in which the double strand break generated by digestion by the restriction enzyme TthlllI has been repaired as a result of the integration of the expression cassette P450SCC by homologous recombination.

Selection of the recombinants (URA3 RTP1 was carried out on minimum medium WO enriched with leucine, histidine and leucine (20 mg/l each) but free from uracil and tryptophan.

Starting from 50 collected clones, the total DNA was extracted by the method described by C. Hoffman et al., (Gene, 57, 267, 1987), then introduced by electroporation 30 into the strain of E. coli XL1-Blue (Stratagene). The clones transformed by the plasmid generated by "gap repair" were selected on the rich medium LB (tryptone yeast extract NaCl containing 50 mg/l of ampicillin. Starting from one of the selected clones, the plasmid, called pCD63, was extracted according to the method described by J.

Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989. The plasmid pCD63 obtained contains expression cassettes ADX and P 45 0SCC separated by the selection indicator URA3, as represented in Figure lib.

b) Transformation of the strain EC01 by the plasmid pCD63 The plasmid pCD63 was introduced into the strain EC01 obtained above by transformation by the lithium acetate method Gietz et al., already described). The transformed yeast was then cultured on the minimum medium WO described previously and free from uracil, tryptophan, adenine and leucine but supplemented with histidine mg/1).

The strain, called EC01/pCD63, was isolated in this way.

A sample of the strain, under the reference EC01/pCD63, was deposited at the CNCM on 10th February 1995 under the number 1-1538.

c) Production in vivo of pregnenolone and pregnenolone 3acetate.

The strain EC01/pCD63 was cultured at 280C under aerobic conditions (130 r/min) in a 3-litre Erlenmeyer until the stationary growth phase is reached (D0600 12 to 13) in the selective medium SGI described in Example 1A and in which the 20 glucose concentration is 5 g/l. The culture is then diluted by the addition of a volume of the complete medium YP described above, then the addition of ethanol v/v) as a source of carbon. When a new stationary growth phase has been reached (DO600 12 to 13), galactose (20 g/l) is added to the culture, in the form of a concentrated solution (500 so as to simultaneously induce the expression of genes coding for ADXm, ADRm, P 450 SCC and delta-7 Red which are respectively under the control of the promoter GAL10/CYC1.

The co-expression of the four genes was revealed by 30 analysis of the sterols accumulated in the cells and in the supernatant of the culture respectively according to the following operating method: ml samples of the culture are harvested after 9 hours and 24 hours of induction. Each sample is centrifuged (4000 g, 10 min, 4 0 C) so as to separate the cells from the culture medium.

On the one hand, the cells are lyzed by mechanical rupture in the presence of glass beads according to the method indicated in Example 1F. Starting from the lysate thus obtained, the intracellular sterols are then extracted by the addition of a volume of hexane.

On the other hand, the sterols present in the culture medium are extracted directly by the addition of a volume of hexane.

The composition of sterols extracted is analyzed by GC as indicated in Example 1, compared with standard products.

The results obtained after 9 hours or after 24 hours of induction are shown in Figure 12a and in Figure 12b respectively.

After 9 hours of induction, Figure 12a shows: in the cellular lysate, the presence of a majority compound having an identical retention time to that of standard pregnenolone acetate (RT 11.8 min) while only a very weak peak is present at the retention time of the pregnenolone

(RT

9.9 min). Low quantities of endogenic sterols of the yeast reduced in position C-7 (ergosta 5-ene 3-ol and ergosta 5,22 diene 3-ol) are identified respectively at RT 18 min and RT 17 min. The alkaline saponification of the cellular lysate before analysis leads to the presence of a majority compound co-migrating with the pregnenolone. This allows confirmation that the accumulated compound having an RT of 11.8 min corresponds to pregnenolone acetate.

in the culture medium, the significant absence of pregnenolone or its acetate.

After 24 hours of induction, Figure 12b shows: -in the cellular lysate, the presence of low quantities of pregnenolone (RT 10.2 min) and of pregnenolone acetate

(RT

30 12 min) as well as of reduced endogenic sterols of the .yeast (RT 17 min and RT 18 min). The cholesterol (RT 16.2 min) is an internal standard added before the extraction.

in the culture medium, the majority presence of pregnenolone acetate and a minor quantity of pregnenolone.

The experiments carried out in parallel with the strain EC01 transformed by a control plasmid such as pFL45L already quoted, have shown no peak corresponding to free pregnenolone 41 or pregnenolone in the acetate form.

Identification of the sterols carried out in this way shows that the yeast strain EC01/pCD63 has accumulated the pregnenolone and the pregnenolone acetate in the absence of any source of exogenic sterols, after induction in the presence of galactose with a major production after an induction of 9 hours.

These results demonstrate on the one hand the effective mobilization of the endogenic sterols reduced in position C-7 of the strain ECO1, on the other hand the extreme effectiveness of the coupling of the restriction reaction of the side chain of the endogenic sterols.

o* o *oo Strains CDRO1 (Mat a) ELRO1 (Mata) 1 1 I Integration locus intergenic LEU2-SPLl Integrated gene ADRm Trans formant plasmid Plasmid marker Gene on the plasmid Selection indicators I

I

I t- 4 ECOl (diploid) ADE2 intergenic LEU2-SPL1 ADE2 intergenic LEU2-SPLI ADE2 delta-7Red ADRm delta-7Red ADRm delta-7RED H153, URA3, ADE2, URA3, LEtJ2, H153, TRP1 H153, URA3, TRPi HIS3 pCD63 ECO1/ pCD63 URA3 TRPl ADXm P450SCC L

I.

43 Preparation of Example 4: construction of the plasmid pTG10033 1. Derivative of pUC19 having a new multiple cloning site: The cloning vector M13mpl9 Yanish-Peron et al., Gene, 33, 103, 1985) was mutagenized using the following oligonucleotide: GCGCTCAGCG GCCGCTTTCC AGTCG 3' SEQ ID No. to introduce a NotI site into the sequence of the truncated lacI gene and to give the plasmid M13TG724.

A "polylinker" containing the EcoRI, SnaBI and NotI sites was then introduced into the EcoRI site of the plasmid M13TG724 by using the following oligonucleotides: AATTGCGGCC GCGTACGTAT G 3' SEQ ID No, 16 and 5' AATTCATACG TACGCGGCCG C 3' SEQ ID No. 17 to give the plasmid M13TG7244 in which a modification of the 0* insert was observed during the amplification stage. The Sinsert of the plasmid M13TG7244 has the following nucleotide S 25 sequence: 0..

GAATTCATACGTACGCGGCCGCAATTGCGGCCGGTACGTATAATTCACTGGCCGT

in which the EcoRI, SnaBI and SstI sites are underlined and 30 the lacZ sequence of pUC19 is in italics.

After digestion of the plasmid M13TG7244 by the restriction enzymes EcoRI and SstI, a "polylinker" containing the MluI *and AvrII sites was introduced using the following oligonucleotides: CAACGCGTCC TAGG 3' SEQ ID No. 18 and AATTCCTAGG ACGCGTTGAG CT 3' SEQ ID No. 19.

44 After digestion with the enzyme PvuII, the PvuII fragment obtained was sub-cloned in pUCl9 Yanish-Porch et al. already quoted) to give the plasmid pTG7457 (Figure 13).

2. Sub-cloning of the PGK terminator: pUC19 was digested with the restriction enzymes BamHI and EcoRI and a new "polylinker" BamHI SstI was introduced using the following oligonucleotides: GATCCGCAGA TATCATCTAG ATCCCGGGTA GAT 3' SEQ ID No. AGAGCTCAAG ATCTACCCGG GATCTAGATG ATATCTGCG 3' SEQ ID No. 21, CTTGAGCTCT ACGCAGCTGG TCGACACCTA GGAG 3' SEQ ID No. 22 and AATTCTCCTA GGTGTCGACC AGCTGCGT 3' SEQ ID No. 23.

The plasmid pTG7453 was obtained in this way (Figure 14) then was digested by the restriction enzymes BamHI and SstI.

The sites of the "polylinker" between BamHI and SstI were introduced into a derivative of plasmid pTG7457 obtained above and digested by the restriction enzymes BamHI and SstI.

The new plasmid obtained contains the PvuII, HindIII, BamHI, EcoRI, XbaI, SmaI, BglII, SstI (=SacI), Mlul, AvrII, EcoRI, SnaBI, NotI, SnaBI, PvuI sites.

This new plasmid was digested by the restriction enzymes BglII and HindIII and a BglII-HindIII fragment containing the PGK promoter A. Hitzeman et al., Nucleic Acids Res., 7791, 1982; G. Loison et al., Yeast, 5, 497, 1989) was 30 inserted in the former to give the plasmid pTG10014 (Figure 3. Sub-cloning of promoters: a) the CYC1 promoter The sites of the "polylinker" between BamHI and SstI of the plasmid pTG7453 were introduced into a derivative of the plasmid pTG7457 as indicated above. The new plasmid obtained was restricted by the restriction enzyme SnaBI, then the RsaI-DraI fragment of 456bp of the plasmid pEMBL8 Dente et al., Nucleic Acid Res., 11, 1645, 1983) containing the origin of replication of the phage fl was introduced to give the plasmid pTG7503.

The BamHI HindIII fragment of 0.78 kb of the plasmid pGBSCC-9, prepared in Example 6 of the European Patent Application EP 0340378 and containing the CYC1 promoter of S.

cerevisiae, a "polylinker" and the lactase terminator of K.lactis, was sub-cloned in the plasmid pTG7503 digested with the restriction enzymes HindIII and BamHI to give the plasmid pTG10004 (Figure 16).

The XhoI and MluI sites of the CYC1 promoter were then eliminated by site directed mutagenesis of the double strand DNA of the plasmid pTG10004 using the following oligonucleotide: GCGGATCTGC TCGAAGATTG CCTGCGCGTT GGGCTTGATC 3' SEQ ID No. 24.

The plasmid pTG10005 thus obtained was then digested by the restriction enzymes SalI and XhoI then a MluI site was introduced by using the following oligonucleotides: 5' TCGACGGACG CGTGG 3' SEQ ID No. 25 and TCGACCACGC GTCC 3' SEQ ID No. 26 S" to give the plasmid pTG10006.

b) the GAL10/CYC1 promoter The plasmid pYeDP1/8-2 Cullin et al., Gene, 203, 1988) was opened with the restriction enzyme XhoI. The cohesive ends created were filled using the Klenow fragment of DNA polymerase then the plasmid was religated. The plasmid pTG10010 thus obtained in which the GAL10/CYC1 promoter no longer contains the XhoI site is used as a template for a PCR amplification.

4. Construction of the expression vector pTG10031 The remaining part of the sequence coding for lacZ was eliminated in the plasmid pTG7503 by site directed mutagenesis using the following oligonucleotide: TGGCCGTCGT TTTACTCCTG CGCCTGATGC GGTAT 3' SEQ ID No. 27 to give the plasmid pTG7549.

The LacZ promoter present in the plasmid pTG7549 wasthen deleted using the following oligonucleotides: GGCCGCAAAA CCAAA 3' SEQ ID No. 28 and AGCTTTTGGT TTTGC 3' SEQ ID No. 29 which are inserted in the plasmid digested beforehand by the restriction enzymes NotI and HindIII and which restore the two sites to give the plasmid pTG7553.

A BamHI MluI fragment containing the CYC1 promoter was obtained from the plasmid pTG10006 digested by the restriction enzymes BamHI and MluI and a Mlul HindIII fragment containing the PGK promoter was isolated from the plasmid pTG10015 digested by the restriction enzymes Mlul and HindIII. These two fragments were ligated and the ligation 25 product obtained was inserted in the plasmid pTG7553 digested beforehand by the restriction enzymes MluI and HindIII.

The following oligonucleotide: S* 5' GATCTATCGA TGCGGCCGCG 3' SEQ ID No. hybridized with the following oligonucleotide: CGCGCGCGGC CGCATCGATA 3' SEQ ID No. 31 S 35 which constitutes a BamHI MluI "linker" containing the Clal and NotI sites, was added and ligated to give the expression vector pTG 10031 (Figure 17).

47 The fragment amplified by PCR obtained above from the plasmid pYeDP1/8-2 was digested with the restriction enzymes ClaI and Sal then was introduced into the plasmid pTG10031 digested beforehand with the same restriction enzymes to give the plasmid pTGlOO33 (Figure 18).

48 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: ROUSSEL

UCLAF

STREET: 102, Route de Noisy CITY: ROMAINVILLE COUNTRY:

FRANCE

POSTAL CODE (ZIP): 93230 TELEPHONE: 49.91.49.91 TELEFAX: 49.91.46.10 (ii) TITLE OF INVENTION: DNA sequence coding for a protein of A.

thaliana having a delta-5,7 sterol,delta-7 reductase activity, delta7-Red protein, production process, strains of transformed yeasts, uses.

(iii) NUMBER OF SEQUENCES: 31 (iv) COMPUTER READABLE

FORM:

MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM:

PC-DOS/MS-DOS

SOFTWARE: PatentIn Release Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: FR 9501723 30 FILING DATE: 15-FEB-1995 (vi) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: FR 9506517 FILING DATE: 01-JUN-1995 S INFORMATION FOR SEQ ID NO: 1: SEQUENCE

CHARACTERISTICS:

LENGTH: 1496 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Arabidopsis thaliana (ix) FEATURE: NAME/KEY: CDS LOCATION:76. .1365 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GTGTGAGTAA TTTAGGTCAA CACAGATCAG AATCTGAGGC TTTGGCCGAG

ACGAAGAGAA

AAGCAGAAGA AGAAA ATG GCG GAG ACT GTA CAT TCT CCG ATC GTT ACT TAC Met Ala Giu Thr Val His Ser Pro Ile Val Thr Tyr 1 5 GCA TCG ATG TTA TCG CTT CTC GCC TTC TGT CCA CCT TTC GTC ATT CTC ill 159 Ala Ser Met Leu Ser Leu Leu Ala Phe Cys Pro Pro Phe Val Ile Leu CTA TGG TAC Leu Trp Tyr ACA ATG GTT Thr Met Val

CAT

His 35 CAG GAT GGT Gin Asp Gly TCT GTT Ser Val ACT CAG ACC TTT Thr Gin Thr Phe 207 S S *5 S *S S S S

S

*5

S

S

S

GGC

Gly 45 TTC TTT TGG GAG Phe Phe Trp Giu GGA GTT CAA GGA Gly Val Gin Gly

CTT

Leu 55 ATC AAC ATA TGG Ile Asn Ile Trp

CCA

Pro 255 3 0 AGA CCC ACT TTG Arg Pro Thr Leu GCT TGG AAA ATT Ala Trp Lys Ile

ATA

Ile 70 TTT TGC TAT GGA Phe Cys Tyr Gly GCA TTT Ala Phe GAA GCT ATT CTT CAG CTG CTT CTG Giu Ala Ile Leu Gin Leu Leu Leu

CCT

Pro ATA TCT CCA Ile Ser Pro 95 GCC GGA AAC CGA Ala Gly Asn Arg 85 CCA GTT Pro Val 100 GGT AAA AGA GTT GAG GGT CCA Gly Lys Arg Val Giu Gly Pro TAC AAG GCC AAT GGT CTG GCT Tyr Lys Ala Asn Gly Leu Ala 303 351 399 GCT TAC Ala Tyr 110 TTT GTG ACA CTA Phe Val Thr Leu GCA ACC CAT CTT GGT CTT TGG TGG TTT GGA Ala Thr His Leu Gly Leu Trp Trp Phe Gly ATC TTC AAC CCT GCA ATT GTC TAT GAT Ile Phe Asn Pro Ala Ile Val Tyr Asp 125 130 CAC TTG His Leu 135 GGT GAA ATA Gly Glu Ile TTT TCG Phe Ser 140 ATA AAA Ile Lys 155 495 GCA CTA ATA TTC Ala Leu Ile Phe

GGA

Gly 145 AGC TTC ATA TTT Ser Phe Ile Ph.

TGT GTT TTG Cys Val Leu 150 TTG TAC Leu Tyr GGC CAT GTT GCA CCT TCA TCA AGT GAC TCT GGT TCA TGT Giy His Vai Ala 160 Pro Ser Ser Ser Asp 165 Ser Gly Ser Cys GGT AAC CTA Gly Asn Leu 170 ATT GGT AAG, Ile Gly Lys ATA ATT GAC Ile Ile Asp 175 AGC TTT GAC Ser Ph. Asp 190 TTC TAT TGG GGC Ph. Tyr Trp Gly

ATG

Met 180 GAG TTG TAC CCT Giu Leu Tyr Pro

CGA

Arg 185 591 639 687 ATC AAG GTG Ile Lys Val

TTT

Ph.

195 ACT AAT TGC AGA Thr Asn Cys Arg

TTC

Ph.

200 GGA ATG ATG TCT Gly Met Met Ser

TGG

Trp, 205 GCA GTT CTT GCA Ala Val Leu Ala

GTC

Val 210 ACG TAC TGC ATA Thr Tyr Cys Ile

AAA

Lys 215 CAG TAT GAA ATA Gin Tyr Glu Ile

AAT

Asn 220

S.

S S

S

55 S S

S

S

*5 S S 55 5 55 55 5* 5 5 5555*5

S

GGC AAA Gly Lys GTA TCT GAT Val Ser Asp 225 TCA ATG CTG GTG Ser Met Leu Vai TTC TGG TGG GAA Phe Trp Trp Giu 245

AAC

Asn 230 ACC ATC CTG ATG Thr Ile Leu Met CTG GTG Leu Val 235 ACC ATG Thr Met 3 0 TAT GTC ACA AAA TTC Tyr Val Thr Lye Ph.

240 GAC ATT GCA CAT GAC Asp Ile Ala His Asp 255 GCT GGT TAT TGG Ala Giy Tyr Trp,

AAC

Asn 250 CGA GCT GGA Arg Ala Gly 260 TTC TAT ATA TGC Phe Tyr Ile Cys TGG GGT TGT CTA Trp Gly Cys Leu 265 TAC CTT GTG AAC Tyr Leu Val Asn 783 831 879 927 975 GTG TGG Val Trp 40 270 GTG CCT TCT GTC Val Pro Ser Val

TAC

Tyr 275 ACT TCT CCA GGC Thr Ser Pro Gly

ATG

Met 280 CAC CCC GTC GAA CTC His Pro Val Giu Leu 285 GGA ACT CAG TTG GCA ATA Gly Thr Gin Leu Ala Ile 290 295 TAC ATT CTC GTT Tyr Ile Leu Val

GCA

Ala 300 GGA ATT CTG TGC Gly Ile Leu Cys

ATT

Ile 305 TAC ATA AAG, TAT Tyr Ile Lys Tyr

GAC

Asp 310 TGT GAT AGA CAA AGG CAA Cys Asp Arg Gin Arg Gin 315 1023 GAG TTC AGG AGG ACA AAC GGG AAA TGT TTG GTT TGG Giu Phe Arg Arg 320 Thr Asn Gly Lys TCA AAG Ser Lys AGT CTT Ser Leu 350

ATT

Ile 335 GTG GCG TCG TAT Vai Aia Ser Tyr

ACT

Thr 340 Cys Leu Vai Trp 325 ACA ACA TCT GGT Thr Thr Ser Giy TGG GGA TTG GCT Trp Gly Leu Aia 360 GGA AGA GCC CCG Gly Arg Aia Pro 330

GAA

Giu 345 ACT AAA ACT Thr Lys Thr 1071 1119 1167 CTC TTA ACG TCT Leu Leu Thr Ser GGA TGG Gly Trp 355 CGT CAT TTC CAT Arg His Phe His

TAT

Tyr 365 GTT CCT GAG ATC Val Pro Giu Ile

TTA

Leu 370 AGT GCT TTC TTC Ser Aia Phe Phe

TGG

Trp 375 ACC GTA CCG GCT Thr Val Pro Ala

CTC

Leu 380 1215 1263 TTC GAT AAC TTC Phe Asp Asn Phe

TTG

Leu 385 GCA TAC TTC TAC Aia Tyr Phe Tyr

GTC

Val 390 CTC ACC CTT CTT Leu Thr Leu Leu CTC TTT Leu Phe 395 555

O

0** 0 &5 2 5 GAT CGA GCC AAG Asp Arg Ala Lys 400 TAT TGG AAG CTG 3 0 Tyr Trp Lys Leu 415 AGA GAC GAT GAC CGA TGC CGA TCA AAG Arg Asp Asp Asp Arg Cys Arg Ser Lys TAT GGG AAA Tyr Gly Lys 410 1311 1359 TAT TGT GAG Tyr Cys Giu

AAA

Lys 420 GTC AAA TAC AGG Val Lys Tyr Arg ATC ATT Ile Ile 425 CCG GGA Pro Gly ATT TAT TGATTGTAAC GAAGTCTGTT GTTCTCATTT TCTACTTATT

ACGTTAATTC

Ile Tyr 430 GAACGTTGGA ATCATCAAA GACCGAGCCA AAACAAAAAT GCAAATTGAT

GCGATAGACA

TTCTTTTGCT GAAAAAAAA

A

INFORMATION FOR SEQ ID NO: 2: SEQUENCE

CHARACTERISTICS:

LENGTH: 430 amino acids TYPE: amino acid TOPOLOGY: linear 1415 1475 1496 52 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Ala Giu Thr Vai His Ser Pro Ile Val Thr Tyr Ala Ser Met Leu Ser Leu Leu Ala Phe Cys Pro Pro Phe 25 Val Ile Leu Leu Trp Tyr Thr Phe Phe Trp Met Val His Gin Asp Gly Ser Val 40 Thr Gin Thr Phe Gly Glu Asn so Gly Val Gin Gly Leu 55 Ile Asn Ile Trp Pro Arg Pro Thr Leu Ile Ala Trp Lys Ile Phe Cys Tyr Gly Aia 75 Phe Giu Ala Ile Leu Gin Leu Leu Leu Gly Lys Arg Val Glu Gly Pro Ile Ser Pro Ala Gly Asn Arg Thr Leu Ala 115 Pro 100 Val Tyr Lys Ala Gly Leu Ala Ala Tyr Phe Vai 110 Phe Asn Pro Thr His Leu Giy Leu 120 Trp Trp Phe Gly Ile 125 Ala Ile 130 Vai Tyr Asp His Gly Giu Ile Phe Ser 140 Ala Leu Ile Phe Gly 145 Ser Phe Ile Phe Val Leu Leu Tyr Ile 155 Lys Gly His Vai Ala 160 35 Pro Ser Ser Ser Asp 165 Ser Gly Ser Cys Giy 170 Asn Leu Ile Ile Asp Phe 175 Tyr Trp Gly too Met 180 Giu Leu Tyr Pro Arg 185 Ile Gly Lys Lys Val Phe Thr 195 Asn Cys Arg Phe 200 Giy Met Met Ser Tyr Giu Ile Asn 220 Ser Phe Asp Ile 190 Trp Ala Val Leu 205 Gly Lys Val Ser Ala Val Thr Tyr Cys Ile Lys Gin 210 215 Asp Ser Met Leu Val Asn Thr Ile Leu Met Leu 235 Asn Thr 250 Vai Tyr Val Thr Lys 240 Phe Phe Trp, Trp Glu 245 Ala Gly Tyr Trp Met Asp Ile Ala His 255 Asp Arg Ala Gly 260 Phe Tyr Ile Cys Trp 265 Gly Cys Leu Val Trp, Val Pro 270 Pro Val Giu Ser Val Tyr 275 Thr Ser Pro Giy Met 280 Tyr Leu Val Aen His 285 Leu Gly 290 Thr Gin Leu Ala Ile 295 Tyr Ile Leu Val Aia 300 Giy Ile Leu Cys Ile 305 Tyr Ile Lye Tyr Asp 310 Cys Asp Arg Gin Arg 315 Gin Glu Phe Arg Arg 320 Thr Asn Gly Lys Cys 325 Leu Vai Trp Gly Arg 330 Aia Pro Ser Lye Ile Val 335 Aia Ser Tyr Thr 340 Thr Thr Ser Gly Thr Lys Thr Ser Leu Leu Leu 350 Val Pro Giu 9

I

Thr Ser Ile Leu 370 Gly 355 Trp Trp Gly Leu Ala 360 Arg His Phe His Tyr 365 Ser Ala Phe Phe Trp 375 Thr Val Pro Ala Phe Asp Asn Phe Leu 385 Ala Tyr Phe Tyr Leu Thr Leu Leu Leu 395 Phe Asp Arg Ala Lys 400 Arg Asp Asp Asp Arg 405 Cys Arg Ser Lys Tyr 410 Gly Lye Tyr Trp Lye Leu 415 Tyr Cys Glu Lys Vai 420 Lye Tyr Arg Ile 425 Ile Pro Gly Ile INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Peptide LOCATION:7 OTHER INFORMATION:/note= "residue 7 Trp or Tyr" (ix) FEATURE: NAME/KEY: Peptide LOCATION:12 OTHER INFORMATION:/note= "residue 12 His or Lys" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Leu Leu Xaa Xaa Gly Trp Xaa Gly Xaa Xaa Arg Xaa Xaa Xaa Tyr 1 5 10 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGONUCLEOTIDE" (ix) FEATURE: NAME/KEY: misc feature LOCATION:10..29 OTHER INFORMATION:/note= "SEQUENCE ID No 1 FROM 76 TO S- a.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CGCGGATCCA TGGCGGAGAC TGTACATTC 29 40 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGONUCLEOTIDE" (ix) FEATURE: NAME/KEY: misc_feature LOCATION:complement (10..28) OTHER INFORMATION:/note= "COMPLEMENTARY SEQUENCE

ID

No 1 FROM 1350 TO 1368" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CAGGGTACCT CAATAAATTC

CCGGAATG

28 INFORMATION FOR SEQ ID NO: 6: SEQUENCE

CHARACTERISTICS:

LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid 25 DESCRIPTION: /desc "OLIGONUCLEOTIDE" (ix) FEATURE: NAME/KEY: miscfeature 30 LOCATION:complement (1..23) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GATTACGCCA AGCTTTTCGA AAC 23 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: 40 LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc

"OLIGONUCLEOTIDE"

56 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: AGATCTTGAG AAGATGCGGC CAGCAAAAC 29 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 46 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGONUCLEOTIDE" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GGGGATCCGT GGTCGACGTA ATTTAGTGTG TGTATTTGTG TTTGCG 46 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear S(ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc

"OLIGONUCLEOTIDE"

(ix) FEATURE: NAME/KEY: misc_feature LOCATION:complement (1..17) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GTAAAACGAC GGCCAGT 17 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 57 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc

"OLIGONUCLEOTIDE"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: TTGAAGGTTC AACATCAATT GATTG INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc

"OLIGONUCLEOTIDE"

(ix) FEATURE: NAME/KEY: misc feature LOCATION:complement (1..38) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GTGTGGCGGC CGCCTCCTTG TCAATATTAA TGTTAAAG 38 38 INFORMATION FOR SEQ ID NO: 12: SEQUENCE

CHARACTERISTICS:

LENGTH: 34 base pairs 35 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc

"OLIGONUCLEOTIDE"

SEQUENCE DESCRIPTION: SEQ ID NO: 12: CAAGGAGGCG GCCGCCACAC AAAAAGTTAG

GTGT

INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGONUCLEOTIDE" (ix) FEATURE: NAME/KEY: misc_feature LOCATION:complement (1..25) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TCTGCTTCCC TAGAACCTTC TTATG INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGONUCLEOTIDE" (ix) FEATURE: NAME/KEY: miscfeature LOCATION:complement (1..20) 35 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: TACATTAGGT CCTTTGTAGC 52 0 INFORMATION FOR SEQ ID NO: S- SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear

I

(ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGONUCLEOTIDE" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GCGCTCAGCG GCCGCTTTCC AGTCG INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGONUCLEOTIDE" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: AATTGCGGCC GCGTACGTAT G 21 INFORMATION FOR SEQ ID NO: 17: e..

SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs 30 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid 35 DESCRIPTION: /desc "OLIGONUCLEOTIDE" (ix) FEATURE: NAME/KEY: misc_feature LOCATION:complement (1..21) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: AATTCATACG TACGCGGCCG C 21 INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc

"OLIGONUCLEOTIDE"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: CAACGCGTCC

TAGG

INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc

"OLIGONUCLEOTIDE"

(ix) FEATURE: NAME/KEY: misc_feature 30 LOCATION:complement (1..22) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: AATTCCTAGG ACGCGTTGAG

CT

4 4 INFORMATION FOR SEQ ID NO: e SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs i: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGONUCLEOTIDE" 61 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GATCCGCAGA TATCATCTAG ATCCCGGGTA GAT 33 INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGONUCLEOTIDE" (ix) FEATURE: NAME/KEY: miscfeature LOCATION:complement (1..39) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: AGAGCTCAAG ATCTACCCGG GATCTAGATG ATATCTGCG 39 INFORMATION FOR SEQ ID NO: 22: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid 30 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGONUCLEOTIDE" a (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: S. CTTGAGCTCT ACGCAGCTGG TCGACACCTA GGAG 34 INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 62 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGONUCLEOTIDE" (ix) FEATURE: NAME/KEY: misc feature LOCATION:complement (1..28) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: AATTCTCCTA GGTGTCGACC AGCTGCGT 28 INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGONUCLEOTIDE" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: GCGGATCTGC TCGAAGATTG CCTGCGCGTT GGGCTTGATC 0* S(2) INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 15 base pairs TYPE: nucleic acid STRANDEDNESS: single S 35 TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGONUCLEOTIDE" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TCGACGGACG CGTGG o INFORMATION FOR SEQ ID NO: 26: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGONUCLEOTIDE" (ix) FEATURE: NAME/KEY: misc feature LOCATION:complement (1..14) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: TCGACCACGC

GTCC

14 INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc

"OLIGONUCLEOTIDE"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: TGGCCGTCGT TTTACTCCTG CGCCTGATGC GGTAT 35 335 INFORMATION FOR SEQ ID NO: 28: SEQUENCE

CHARACTERISTICS:

LENGTH: 15 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc

"OLIGONUCLEOTIDE"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: GGCCGCAAAA CCAAA INFORMATION FOR SEQ ID NO: 29: SEQUENCE CHARACTERISTICS: LENGTH: 15 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGONUCLEOTIDE" (ix) FEATURE: NAME/KEY: misc_feature LOCATION:complement (1..15) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: AGCTTTTGGT

TTTGC

INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs So TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGONUCLEOTIDE" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 GATCTATCGA

TGCGGCCGCG

INFORMATION FOR SEQ ID NO: 31: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc

OLIGONUCLEOTIDE"

(ix) FEATURE: NAME/KEY: misc feature LOCATION:complement (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: CGCGCGCGGC CGCATCGATA Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof.

e* *0 *0 0 0.00 00%0.

28/1/98VSAP8556.SPE,65a i 66 The claims defining the invention are as follows: 1 Nucleic acid sequence containing a sequence coding for a protein having a delta-5,7 sterol,delta-7 reductase activity, said nucleic acid being a DNA or an RNA and having the nucleotide sequence of SEQ ID No. 1: GTGTGAGTAA TTTAGGTCAA CACAGATCAG AATCTGAGGC TTTGGCCGAG

ACGAAGAGAA

AAGCAGAAGA AGAAA ATG GCG GAG ACT GTA CAT TCT CCG ATC GTT ACT TAC Met Ala Giu Thr Val His Ser Pro Ile Val Thr Tyr 1 5 GCA TCG ATG TTA TCG CTT CTC GCC TTC TGT CCA CCT TTC GTC ATT CTC ill 159 Ala Ser Met Leu Ser Leu Leu Ala Phe cys 20 CTA TGG TAC ACA ATG OTT CAT CAG GAT GGT Pro Pro Phe Val Ile Leu TCT OTT ACT CAG ACC TTT Ser Val Thr Gin Thr Phe Leu Trp Tyr Thr Met Val His 35 Gin Asp Giy 207

GCC

Cly TTC TTT TGG, GAG Phe Phe Trp Giu

AAT

Aen 50 GGA GTT CAA GGA Gly Vai Gin Giy

CTT

Leu 55 ATC AAC ATA TOG, Ile Asn Ile Trp

CCA

Pro 255 AGA CCC ACT TTG Arg Pro Thr Leu a.

a.

a a a.

a a

ATT

Ile 65 OCT TGG AAA ATT Ala Trp Lys Ile

ATA

Ile 70 TTT TGC Phe Cys CAA GCT ATT Giu Aia Ile ATA TCT CCA Ile Ser Pro 95 CTT CAG CTG Leu Gin Leu 80 CTT CTG Leu Leu CCT GGT AAA AGA Pro Gly Lys Arg 85 TAT GGA GCA TTT Tyr Giy Ala Phe GTT GAG GGT CCA Val Ciu Cly Pro AAT GOT CTG GCT Asn Gly Leu Ala 351 399 GCC GGA AAC CGA Ala Gly Asn Arg CCA OTT TAC AAG GCC Pro Val Tyr Lys Ala 100 105 a.

a a a a a

VT

28/4/99CF8556.SPE,66

Claims (27)

1. 2. DNA sequence coding for a protein having a delta-5,7 sterol,delta-7 reductase activity and amplifiable by the PCR technique using as primers oligonucleotides coding for a consensus sequence having the amino acid sequence SEQ ID No. 3: Leu Leu Xaa Xaa Gly Trp Xaa Gly Xaa Xaa Arg Xaa Xaa Xaa Tyr 1 5 10 in which Xaa in position 7 is Trp or Tyr and Xaa in position 12 is His or Lys.
2. 3. A. thaliana protein having a delta-5,7 sterol,delta-7 reductase activity and having the amino acid sequence SEQ ID No. 2: Met Ala Glu Thr Val His Ser Pro Ile Val Thr Tyr Ala Ser Met Leu Ser Leu Leu Met Val His 35 Ala Phe Cys Pro Gin Asp Gly Ser Pro Phe 25 Val Ile Leu Leu Trp Tyr Thr Phe Phe Trp Val 40 Thr Gin Thr Phe Gly a a a Glu Asn 50 Gly Val Gin Gly Leu 55 Ile Asn Ile Trp Pro Arg Pro Thr Leu Ile 65 Ala Trp Lye Ile Ile 70 Phe Cys Tyr Gly Ala 75 Phe Glu Ala Ile Leu Gin Leu Leu Leu Pro Gly Lys Arg Val Glu 90 Gly Pro Ile Gly Asn Arg Pro Val Tyr Lys Ala Asn Gly Leu Ala Ala Ser Pro Ala Tyr Phe Val 110 28/4/99CF8556.SPE,69 a a a. 9a~ 3I a. 14 1 Thr Leu Ala Thr His Leu Gly 115 Leu Trp Trp Phe Gly Ile 120 1 Phe Asn Pro Ala Ile 130 Gly Ser Val Tyr Asp His Leu 135 Val Gly Giu Ile Phe Ser Ala Leu Ile Phe 140 Phe Ile Phe 145 Pro Cys 150 Ser Leu Leu Tyr Ile Lys 155 Asri Leu Gly His Val Ala 160 Ser Ser Ser Asp 165 Giu Giy Ser Cys Giy 170 Ile Ile Ile Asp Phe 175 Tyr Trp Giy Lys Val Phe 195 Ala Val Thr Met 180 Thr Leu Tyr Pro Arg 185 Gly Gly Lys Ser Asn Cys Arg Phe 200 Gin Met Met Ser Trp 205 Gly Phe Asp Ile 190 Ala Val Leu Lys Val Ser Tyr Cys Ile 210 Lys 215 Thr Tyr Glu Ile Asn 220 Val Asp 225 Phe Asp 30 Ser Ser Met Leu Val Phe Trp Trp Glu 245 Phe Ser Asn 230 Ala Tyr Pro Gly Tyr Trp Arg Val Ile Leu Met Ala Tyr 275 Thr Asn 250 Gly Leu Leu 235 Thr Cys Val Tyr Vai Thr Met Asp Ile Ala 255 Val Val Lys 240 His Pro G lu Gly 260 Thr Ile Gly Ile 295 Cys Cys Met 280 Tyr Trp 265 Tyr Leu Asn Ala 300 Gin Vai His 285 Gly Trp 270 Pro Leu Gly 290 Ile Tyr Gin Leu Ala Ile Leu Val Ile Leu Cys Ile Lys Tyr 305 Thr Asp 310 Leu Asp Arg Gin Arg 315 Ala Giu Phe Arg Arg 320 Aen Gly Lys Cys 325 Thr Val Trp Gly Arg 330 Thr Pro Ser Lys Ile Val 335 Leu Leu Ala Ser Tyr Thr 340 Thr Ser Gly Giu Lye Thr Ser Leu 350 Thr Ser Giy Trp Trp 355 Gly Leu Ala Arg His Phe His Tyr Val Pro Glu 360 365 Ile Leu Ser Ala Phe Phe Trp Thr Val Pro Ala Leu Phe Asp Asn Phe 370 375 380 Leu Ala Tyr Phe Tyr Val Leu Thr Leu Leu Leu Phe Asp Arg Ala Lye 385 390 395 400 Arg Asp Asp Asp Arg Cys Arg Ser Lys Tyr Gly Lys Tyr Trp Lys Leu 405 410 415 Tyr Cys Glu Lys Val Lys Tyr Arg Ile Ile Pro Gly Ile Tyr 420 425 430
3. 4. A. thaliana protein having a delta-5,7 sterol,delta-7 reductase activity and having the amino acid sequence SEQ ID No. 2 and designated delta-7Red. Protein having a delta-5,7 sterol,delta-7 reductase activity having an amino acid sequence having a sequence identity of about 60% and more with the sequence SEQ ID No.2 defined in claim 4.
4. 6. Protein having a delta-5,7 sterol,delta-7 reductase activity and having a crossed immunological reactivity with the A. thaliana delta-7Red protein defined in claim 4.
5. 7. Protein having a delta-5,7 sterol,delta-7 reductase activity as obtained by expression in a host cell containing a DNA sequence according to claim 1 orclaim2.
6. 8. A. thaliana protein having a delta-5,7 sterol,delta-7 30 reductase activity as obtained by expression in a host cell containing a DNA sequence coding for the amino acid sequence SEQ ID No. 2.
7. 9. Protein according to claim 7 or claim 8 in which the host cell is a yeast. 35 10. Antibody directed against a protein having a delta-5,7 sterol,delta- 7 reductase activity according to any one of claims 3 to 9.
8. 11. Expression vector containing a DNA sequence according to claim 1 or claim 2. 40 12. Host cell transformed by a vector according to claim 11. J^- -72-
9. 13. Cloning process for a nucleic acid coding for a protein having a delta- 5,7 sterol,delta-7 reductase activity and which hybridizes with SEQ ID No. 1 under average or high stringency conditions or which has a sequence identity of approximately 60% or more with SEQ ID No. 1 in a microorganism comprising a screening method chosen from the resistance of the microorganism to nystatin or to an analogous compound whose toxicity depends on the presence of sterols carrying an unsaturation in position C-7, the hybridization of the nucleic acid with the nucleotide sequence of the sequence SEQ ID No. 1, the identification of the nucleic acid by using data processing techniques from DNA sequences isolated at random, the direct expression of the protein followed by immuodetection using antibodies directed against the protein having the amino acid sequence SEQ ID No. 2.
10. 14. DNA or RNA nucleic acid sequence as obtained by the process defined in claim 13. Host cell transformed by a vector containing a DNA sequence according to claim 14. 20 16. Host cell transformed according to claim 15 or claim 18 in which the host is a yeast or a filamentous fungus.
11. 17. Preparation process for a protein having a delta-5,7 sterol,delta-7 reductase activity in which a host cell transformed according to any one of S claims 12, 15 or 16 is cultured and the expressed protein is isolated.
12. 18. Process according to claim 17 in which the host cell is a transformed yeast in which the coding DNA sequence is placed under the control of a yeast promoter.
13. 19. Reduction process in vitro for a sterol unsaturated in position C-7 in which the sterol to be reduced is incubated with the protein obtained according :.3Ct to claim 17 or claim 18 and the reduced sterol obtained is optionally isolated. S28/499CF86SPE72 28/4/99CP8556.SPE,72 -73- Reduction process in vivo for an exogenic sterol unsaturated in position C-7 in which the sterol is incubated with a host cell transformed according to any one of claims 12, 15 or 17 and the reduced sterol obtained is optionally isolated.
14. 21. Reduction process in vivo for an endogenic sterol unsaturated in position C-7 in which a host strain transformed according to claim 16 is cultured and the reduced sterol accumulated is optionally isolated.
15. 22. Reduction process in vitro or in vivo according to any one of claims 19 to 21 in which the reduced sterol obtained is a substrate of the restriction enzyme of the side chain of cholesterol
16. 23. Reduction process in vivo according to claim 22 in which the endogenic sterol to be reduced is ergosta 5,7 diene 3-ol, ergosta 5,7,24(28) triene 3-ol or ergosta 5,7,22 triene 3-ol or a mixture of these.
17. 24. Production process for pregnenolone in which a host cell transformed according to claim 16 is cultured, the accumulated endogenic sterol or sterols reduced in position C-7 is/are optionally isolated, the reduced sterols are incubated in the presence of P46oSCC, and optionally in the presence of adrenodoxin reductase (ADR) and of adrenodoxin (ADX), and the pregnenolone obtained is optionally isolated.
18. 25. Process according to claim 24 in which the host cell is a yeast.
19. 26. Production process for pregnenolone in which a yeast transformed by 0 one or more vectors coding for a protein having the amino acid sequence of SEQ ID No: 2 allowing the coexpression of the protein having the activity of delta-5,7 sterol,delta-7 reductase and of P 45 oSCC, and optionally of ADR and 0 ADX, is cultured and the free or esterified pregnenolone is optionally isolated.
20. 27. Production process for pregnenolone according to claim 26 in which a transformed yeast coexpressing a protein having delta-5,7 sterol,delta-7 reductase, P460SCC, ADR and ADX activity, is cultured.
21. 28. Process according to claim 27 in which the protein having the delta-5,7 sterol,delta-7 reductase, activity is the A. thaliana delta-7Red protein. 14/5/99CF8556.73 -74-
22. 29. Process according to claim 28 in which the yeast strain is the strain EC01/pCD63. Transformed yeast strain coexpressing a protein having delta-5,7 sterol,delta-7 reductase, P450SCC, ADR and ADX activity and accumulating free or esterified pregnenolone wherein the protein has the amino acid sequence of SEQ ID No: 2.
23. 31. Yeast stain according to claim 30 in which the protein having the delta- 5,7 sterol,delta-7 reductase activity is the A. thaliana delta-7Red protein.
24. 32. Yeast strain according to claim 31 named EC01/pCD63.
25. 33. Human DNA sequence according to claim 14 used as a probe for diagnosing a congenital deficiency in delta-5,7 sterol,delta-7 reductase.
26. 34. Method for detecting the deficiency in delta-5,7 sterol,delta-7 reductase which comprises the incubation of a sample containing human genomic DNA with the probe according to claim 33, under standard hybridization conditions and revelation of the fixation or absence of fixation of the probe to the genomic DNA, the absence of fixation or the reduction of the latter indicating a i* congenital deficiency in delta-5,7 sterol,delta-7 reductase.
27. 35. Nucleic acid sequence according to any one of claims 1 to 3 or a protein produced therefrom, the transformed yeast strain according to claim or the method according to claim 34 substantially as hereinbefore described with reference to any one of the Examples. S DATED this 14 th day of May, 1999. ROUSSEL UCLAF By their Patent Attorneys: CALLINAN LAWRIE /T "e 14/5/99CF8556,74 l7- OJ Strains Integration Integrated Transformant Plasmid Gene(s) on Selection locus gene plasmid marker the plasmid indicators CDRO1 intergenic ADRm HIS3, URA3, (Mata) LEU2-SPL1 TRP1 ELRO1 ADE2 delta-7Red ADE2, UPA3, (Mata) LEU2, HIS3, TRP 1 intergenic ADRm -HIS3, URA3, EC0l LEU2-SPL1 delta-7Red TRP1 (diploid) intergenic ADRm pCD63 URA3 ADXm HIS3 ECOl! LEU2-SPLI delta-7RED TRP1 P450SCC pCD63 S S S S S S S S S S *SS SS 55 55 *S 5 5 5 5 55 55 5 5 55* *55 S S ABSTRACT A cDNA sequence coding for an A. thaliana protein having a delta-5,7 sterol,delta-7 reductase activity (SEQ ID No. 1). Cloning process. Reduction process for a sterol unsaturated in position C-7. Production process for pregnenolone. S**