Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• the present invention relates to a method for identifying cholesterol modulator compounds by detecting enzymatic activities involved in the synthesis or catabolism of cholesterol.
• the present invention also relates to the use of the compounds detected in the treatment of cardiovascular pathologies or metabolism.
• Cholesterol is the most important animal sterol. It is a fundamental component of cell membranes, which it controls fluidity, and is present in all animal tissues and particularly in the brain. The dysfunctions in its synthesis and its catabolism are related to numerous pathologies: atherosclerosis, angina pectoris, cardiovascular accident, metabolic syndrome, diabetes, Smith-Lemli-Opitz syndrome ...
• Synthesis inhibitors or cholesterol catabolism activators are known, such as DHCR14 / sterol-A14 reductase inhibitor triparanol, DHCR24 / sterol-A24 reductase, DHCR7 / sterol-A7 reductase and ⁇ 8-7 isomerase, AY9944 DHCR7 inhibitor / sterol-A7 reductase (J. Sanchez-Wandelmer et al 2009), SR31747 sterol inhibitor ⁇ 8-7 isomerase (R. Paul et al 1998), Amorolfine inhibitor DHCR14 / sterol-A14 reductase and sterol ⁇ 8-7 isomerase (AJ Carrillo-Munoz et al, 2006).
• a cholesterol-synthesizing enzyme inhibitor or an activator of cholesterol-catabolising enzymes causes cell death.
• the disappearance of cholesterol is deleterious for the life of the cell, especially for the mammalian cells used until now. This does not allow the discovery of new modulators of metabolism or transport of cholesterol.
• the Applicant has surprisingly shown using yeasts that it is possible to detect cholesterol modulators by means of reverse selection media, where cholesterol results in less resistance to toxins. This allows a determination of inhibitors of cholesterol synthesis and / or cholesterol catabolism activators when toxin resistance is observed.
• the yeast cells producing the cholesterol used in this test are genetically modified yeasts. Indeed, in the natural state, the yeasts produce ergosterol.
• the Applicant uses genetically modified yeasts to divert production of ergosterol to produce cholesterol.
• the yeasts are modified as mentioned in patent EP 0727489B1 or by deletion of non-essential ERG family genes (ERG 6, ERG 5, ERG 4, ERG 2 and ERG 3).
• ERG 6, ERG 5, ERG 4, ERG 2 and ERG 3 One way to obtain this type of yeast is also described in the international patent application WO2005 / 121315 (Aventis Pharma).
• the enzymes of the end of the synthesis and catabolism of ergosterol or cholesterol described in the present application are not essential for wild yeast or modified yeast to produce cholesterol; their activation or inhibition is not related to the growth or death of these yeasts. This makes it possible to study cholesterol modulators without having any problem of survival of these cells: in fact, without cholesterol mammalian cells, used until then for this kind of tests, die. It is therefore impossible to observe the effect of compounds lowering cholesterol synthesis since the cells die as soon as the cholesterol level is insufficient.
• the present invention solves this problem through the use of genetically modified yeasts where the survival of the cell is not strictly dependent on its cholesterol production.
• the invention which is the subject of the present application thus provides a new alternative to the means for detecting modulators of cholesterol synthesis.
• yeasts consists of transforming yeasts genetically to produce both cholesterol and enzymes related to catabolism of cholesterol.
• the recombinant yeasts are contacted with a potentially modulating cholesterol compound on a medium initially toxic to the yeasts producing cholesterol.
• the observation of the yeast growth restoration or on the contrary of the yeast death makes it possible to determine whether the test compound inhibits or increases the production of cholesterol by the yeasts.
• the yeasts used may be Saccharomyces cerevisae, Schizosaccharomyces pombe, Kluyveromyces (lactis and others) or Pichia pastoris.
• the yeasts used are Saccharomyces cerevisae.
• Such enzymes may be: DHCR14, DHCR24, DHCR7 and A8-A7-isosterol isomerase for cholesterol synthesis.
• this method makes it possible to identify cholesterol-catabolising enzyme activators, for example cytochromes P450 (for example CYP27A1 CYP46A1, CYP1 1A1 and CYP7A1).
• the toxins according to the invention may be syringomycin E, phytosphingosine, telomycin, iturin A, vibrio cholera toxin and cholesterol-dependent toxins.
• the toxin of the medium according to the invention is syringomycin E.
• the yeast strains are transformed by introduction of a vector allowing the expression of genes coding for the production of enzymes related to catabolism of cholesterol.
• the applicant uses a recombinant protein expression vector optimized for the functional expression of cytochromes P450 and their electron-carrying cofactors.
• these recombinant vectors are plasmids.
• the medium used to culture yeast is advantageously a Kappeli medium consisting of 600 ml demineralized H 2 0, 50 ml of the concentrated salt solution 20 times (Table 1), 10 g casamino acids (Difco) or 8.9 g (NH 4 ) 2 S0 4 (PROLABO 21 333.296) (Nitrogen source), 2 ml of the 500-fold concentrated vitamin solution ((Mix2), 0.2 ml of concentrated CaCl 2 and FeCl 3 solution 5000 times ((Mix3) and 100 mg / L of amino acids and 50 mg / L of bases required (except adenine at 100 mg / l) for 1 L of medium in total.
• Kappeli medium consisting of 600 ml demineralized H 2 0, 50 ml of the concentrated salt solution 20 times (Table 1), 10 g casamino acids (Difco) or 8.9 g (NH 4 ) 2 S0 4 (PROLABO 21 333.296) (Nitrogen source), 2 ml of the
• the pH of this solution is between 2.6 -2.7.
• the solution is stored at + 4 ° C. and aliquoted in 50 ml per tube.
• the solution is stored at -20 ° C. and aliquoted in 2.2 ml per tube
• the solution is kept at + 4 ° C. and aliquoted in 500 ⁇ l per tube.
• the sporulation media used for yeasts are as follows:
• Sporulation medium SP1 yeast extract 2.5 g / L, potassium acetate 9.8 g / L, glucose 1 g / L, agar 20 g / L.
• ACK sporulation medium potassium acetate 10 g / L, agar 20 g / L.
• Example A Construction of plasmid vectors: Construction of expression vector for DHCR24 and cytochrome P450
• This plasmid was constructed in order to obtain on the same vector 3 expression cassettes for DHCR24 (dehydro-cholesterol-24-reductase), mature CYP27A1 and the mature adrenopyrin electron (ADX) transporter conferring in a modified yeast. cholesterol synthesis and its catabolism via the activities of DHCR24 and CYP27A1 respectively.
• the activity of electron transporters such as ADX (provided on the plasmid) and ADR (carried on the genome of the strain) is necessary for functional cytochrome P450 mitochondrial activity such as CYP1 1A1 (international patent application WO02 / 061 109 ) or CYP27A1.
• Plasmid pM580 is obtained by methods of molecular biology and recombination in yeast known to those skilled in the art. This expression vector is derived from plasmids pCD63 (Pompon et al., 1998 Nat Biotechnol.) And pYeDP60 (Pompon et al 1994 Eur J. Biochem), and contains a 2 ⁇ replication origin for S. cerevisiae and the URA3 selection marker. (Fig 7).
• the TRP1 selection marker of pCD63 is deleted by homologous recombination in yeast.
• the fragment of pCD63 open at the Bsul and SmaI restriction sites at the level of the selection marker TRP1 is brought into contact with the PCR fragment obtained with the primers of SEQ ID NO 1 and 2 on the pCD63 template, at the BglII sites hybridizing with on both sides of the TRP1 marker.
• This mixture of DNA fragments is then introduced into a W303 type yeast by LiAc / PEG transformation method described by Gietz et al; 1995 and 2002.
• the recombinant clones are selected for the presence of the selection marker URA3 and for the absence of the TRP1 marker by growth on medium lacking uracil and lack of growth on a medium lacking tryptophan.
• the DNA is extracted from the yeasts by zymolysis lysis (12.5 mg / ml, 1M Sorbitol, phosphate buffer 0.1 M ph 7.2 for 1 h at 37 ° C.) followed by alkaline lysis of the Qiagen kit ref 12106 then transferred into TG1-type bacteria (TSB method adapted from Chung et al. 1989) for analysis by standard molecular biology techniques.
• This new vector is named plM565.
• the DHCR24 expression cassette modified on the 2nd and 3rd codon under the control promoter TPI (Triose Phosphate Isomerase) from the Nael fragment plM330 (derived from pYX212 # MBV-028-10 R & D System) is introduced to the site PvuII of plM565 to form plasmid pM578.
• TPI Teriose Phosphate Isomerase
• the DHCR24 expression cassette changed for the 2 nd and 3 rd codon is described in international patent application WO2005 / 121315.
• the promoter chosen here is the TPI promoter, constitutive promoter in place of CYC1.
• the CYP1 1A1 expression cassette (from pCD63) is substituted by that of mature (i.e., mitochondrial targeting-free) CYP27A1 by homologous recombination in yeast between the open plM578 vector. at the Nael site and the PCR fragment derived from plM558 with the primers of SEQ ID NO 3 and 4 containing the mature CYP27A1 expression cassette under the control of the Gal10 / CYC1 chimeric promoter derived from pYeDP60 (D. Pompon et al., 1994 and 1996).
• the mature CYP27A1 cDNA is from the GenBank number clone NM_000784 whose N-terminal part lacking the first 33 amino acids was modified by PCR with the primers of SEQ ID NO 5 and 6.
• the plasmid thus obtained containing the three expression cassettes for mature DHCR24, ADX and CYP27A1 is named plM580.
• plM584 The equivalent plasmid containing the coding sequence of CYP46A1 is named plM584 is obtained in a similar manner.
• the cDNA of CYP46 from GenBank number clone NM_006668 is introduced into pYeDP60 and the expression cassette (promCYC1 / GAL10-CYP46-TermPGK) is transferred into pM578 by homologous recombination in yeast.
• the control plasmid lacking a coding sequence for cytochrome P450 is similarly obtained by homologous recombination between the pYeDP60 expression cassette and plM578 open at the Nael site.
• This control vector without P450 is named plM582.
• Plasmid P450 DHCR24 References pCD63 CYP1 1A1 mature absent Pompon ei a / 1998 Nat.Biotechnol. pYeDP60 none missing Pompon ei a / 1994 Eur.J.Biochem. plM565 CYP1 1A1 mature absent present patent application plM578 CYP1 1A1 mature present present plM580 patent application CYP27A1 mature present plM584 CYP46A1 patent present present patent application plM582 none present the present patent application Example B: Construction of Modified Yeast Strains
• yeast strains producing cholesterol have been constructed as follows:
• the YIM126 strain was constructed to prevent the functioning of genes involved in cholesterol modifications and in particular the two enzymes responsible for esterification with aliphatic chains ARE1 and ARE2 and the enzyme encoded by the ERG5 gene. responsible for desaturation in position 22-23 of the side chain of ergostas and cholestas. These characteristics were gathered in the same strain containing the elements necessary for the production of cholesterol.
• haploid strains containing interesting characteristics are brought into contact to give a diploid strain which is then sporulated by growth on a very rich medium and very poor (as described in the international patent application WO2005 / 121315).
• the mixture of diploid cells and asci is then brought into contact with an aqueous medium containing 30% of ether for 3 and 6 minutes to preferentially lyse the diploid strains.
• the surviving clones are then plated on a selective medium according to the desired characters to obtain a haploid strain combining the parental characters.
• the ylM126 strain was obtained by selection of haploid clone resulting from three successive crosses of different strains by methods known to those skilled in the art.
• strain Fy1 1679-28c (international patent application WO2002 / 061 109) is crossed with the strain ERT (strain WGIF01 of the international patent application WO2005 / 121315).
• a diploid clone resulting from this selected cross on the complementary autotrophies of the parent strains is made to sporulate and then treated as mentioned above to obtain haploid clones.
• the haploid strains capable of growing on a medium free of adenine and tryptophan, which signify the autotrophic adenine character of Fy1679-28c and the erg6: TRP1 disruption of ERT were selected.
• the haploidy of clones was checked by crossing with control strains W303 MATa or MATalpha.
• Clones thus obtained combining an intact ADE2 locus and disruption of the ERG6 gene by the TRP1 tryptophan selection marker were named ylM1 and ylM1 1 1. Then the ylM1 strain is crossed with the haploid strain CDR06.
• CDR06 is a strain of genetic origin FY1679 whose ade2 locus is non-functional by the integration of an expression cassette for the A7-sterol reductase plant Arabidopsis thaliana.
• CDR06 and CDR07 (described in WO2002 / 061,109 or Duport et al; 2003) were obtained from the same cross and differ in the presence of the ERG5 gene which is functional in CDR06.
• a diploid clone ylM1 10XCDR06 is isolated and put under the spore production conditions.
• the spores are prepared as described above and isolated on a rich medium and then tested on different media in the presence and absence of adenine and in the presence of nystatin to verify the resistance of the strain to this antifungal linked to the combination of functional absence. ERG6p activity and the presence of A7-sterol reductase activity.
• the sterol composition is verified by saponification followed by organic extraction of the total sterols. The identity of the sterols is analyzed in GC / FID and verified in GC / MS. The presence of a product having the same retention time as desmosterol is observed for clones 4 and 6 from a pellet of cultured cells and processed as previously described in the international patent application WO2005 / 121315.
• clones 4 and 6 are also resistant to nystatin when galactose is a carbon source. These haploid clones 4 and 6 are referred to as IM are respectively 1 15 and y IM 1 16. Finally, a 3rd crossing with the ylM1 16 and CA23 strains is carried out in order to introduce the interruption of three genes of interest c ' that is, ARE1, ARE2 and ERG5 in a strain capable of producing cholesterol and also the expression cassette of an electron transporter necessary for the functioning of cytochrome P450.
• the CA23 strain described in the international patent application WO2005 / 121315 contains a non-functional form of these 3 ARE1 genes, ARE2 er ERG5 and the expression cassette of the adrenodoxine reductase electron transporter (ADR) integrated at the LEU2 locus.
• a 16XCA23 ylM1 diploid was isolated on a minimal medium containing no tryptophan, histidine and leucine but containing adenine and uracil. In this way, only diploid cells from a cross between ylM1 16 and CA23 can grow when none of the partners of the cross can develop under these conditions.
• a diploid clone is set to sporulate under the conditions described above.
• spores are isolated after treatment with ether of a mixture of spores and the diploid strain as previously described. These clones are characterized for their sexual sign, their ability to grow on a medium devoid of adenine, leucine, histidine and tryptophan, and their ability to resist three antifungals, nystatin, hygromycin and geneticin. Finally clone # 4 thus selected was named ylM126. This clone can grow in the absence of leucine indicating the presence of a functional LEU2 gene and therefore the presence of an expression cassette for the mature form of ADR under the control of the GAL10 / CYC1 promoter.
• This clone can also grow in the absence of tryptophan and histidine indicating the potential disruption of the ERG6 and ARE2 genes. It is resistant to high levels of nystatin and geneticin, probably indicating the presence of the DHCR7 enzyme as well as the disruption of the ARE1 gene respectively.
• the free and esterified sterols are analyzed to confirm or deny the presence of ARE1 and ARE2 gene products as well as the sterol quality to reverse or confirm the interruption of the ERG6 gene and the presence of DHCR7.
• ade2 GAL10 / CYC1-A7stérol-Reductase
• ade2 prom GAL10 / CYC1 - ⁇ 7sterolReductase
• Example C Strain construction combining the production of cholesterol and its catabolism by functional enzymatic activities in yeast.
• Plasmids containing the DHCR24 expression cassettes with or without cytochrome P450 and the ADX electron transporter are introduced into the ylM126 yeast by LiAC / PEG transformation method (Gietz et al, 1995 and 2002).
• the clones are selected on a medium devoid of uracil. Two to four clones of each combination are cultured for analysis of total sterols in GC / FID and GC / MS according to the procedure described in International Patent Application WO2005 / 121315. The clones are cultured for 48 hours under shaking at 30 ° C. in YBN medium supplemented with 2% glucose and adenine at 100 ⁇ g ml. The optical density (OD600nm) reaches an average value of 7 units. The cell pellets of a volume V of culture are harvested and transferred into an identical volume V of Kappeli medium containing 20% casa-amino acid, 2% glucose, 100 ⁇ g ml of adenine.
• the sterol profiles of the yeast samples are compared with the retention times of GC / FID gas chromatographic reference solutions such as cholesterol (FIG. 1A), desmosterol (FIG. 1B), pregnenolone (FIG. 1C), and 240H-cholesterol. (Fig. 1D), 270H-cholesterol (Figl E) and the products are identified by retention time similarity.
• GC / FID gas chromatographic reference solutions such as cholesterol (FIG. 1A), desmosterol (FIG. 1B), pregnenolone (FIG. 1C), and 240H-cholesterol.
• Fig. 1D 270H-cholesterol
• the products are identified by retention time similarity.
• the analyzes and the measurement of the peak area show a cholesterol production for the ylM126 strain containing the plasmid p5355 without P450 with a cholesterol / desmosterol ratio greater than 3.
• the ylM126 strains containing the P450 expression constructs produce a derivative. specific cholesterol of each P450 that uses cholesterol as substrate: that is, production of pregnenolone for CYP1 1A1 (Fig 3), 270H-cholesterol and 270H-sterols for CYP27A1 (Fig 4) and 240H-sterols cholesterol for CYP46A1 (Fig 5).
• the cholesterol / desmosterol ratio decreases by a factor of 2 in the presence of CYP46 and by a factor of 5 to 6 in the presence of CYP1 1A1 or CYP27A1 indicating that cholesterol is probably metabolized.
• New 27-hydroxylated sterols such as 270H-desosterol appear specifically in the presence of CYP27A1 activity. (Fig 4)
• Example D In Cell Biological Method for Detecting Cholesterol Metabolism: a) Culture Medium:
• a culture medium is developed that allows a simple phenotypic growth test or growth defect to distinguish yeasts producing cholesterol from those that do not produce or catabolize it.
• This culture medium is a rich medium of the YPG agar type (Complete medium YPG: yeast extract (Difco) 10 g / L, bacto-peptone (Difco) 20 g / L, glucose (Merck) 20 g / L) supplemented with the syringomycin E toxin of Pseudomonas syringae B-301D (Sigma # S6946) of 150 to 250 ng / ml.
• YPG agar type Complete medium YPG: yeast extract (Difco) 10 g / L, bacto-peptone (Difco) 20 g / L, glucose (Merck) 20 g / L
• Agar (20 g / L) is added to obtain solid media. b) detection of cholesterol metabolism
• the ylM126 yeast strains containing an expression vector for DHCR24 and cytochrome P450 such as plM580 described above, are cultured for 48 hours with stirring at 30 ° C. in YNB (Yeast nitrogen base / amino acid (Difco) 6 medium. , 7 g / L, glucose (Merck) 20 g / L.) Supplemented with 2% glucose and adenine at 100 mg / ml.
• YNB yeast nitrogen base / amino acid (Difco) 6 medium. , 7 g / L, glucose (Merck) 20 g / L.) Supplemented with 2% glucose and adenine at 100 mg / ml.
• the culture reaches an optical density at 600 nm of the order of 7 units.
• the cell pellet is harvested by centrifugation and transferred to an identical volume of Kappeli medium containing 20% casa-amino acid, 2% glucose, 100 ⁇ g / ml adenine and stirred at 30 ° C. for 24 hours reaching optical density at 600 nm of the order of 40 units.
• the cultures are diluted to 1 unit of optical density at 600 nm and then at 1/50 and 25 at 25 to perform a drop test on YPG agar plates containing syringomycin E according to methods known to those skilled in the art. .
• the yeasts containing the cholesterol-producing expression vector plM582 (without P450, Fig 6 column 1) are not able to grow while the yeasts containing the expression vector plM580 (with CYP27A1, Fig. 6 column 2) producing cholesterol and converting it into derived products are able to grow on YPG agar containing syringomycin E at 170 ng / ml (Fig 6A and 6B).
• the toxicity of this YPG medium containing syringomycin E depends on the amount of cholesterol available in the yeast observed in GC / FID or GC / MS.
• This modified yeast growth test for cholesterol synthesis also makes it possible to identify products that interfere with either synthesis or cholesterol catabolism.
• the ylM126 yeasts containing the expression plasmids for DHCR24 with or without P450 are cultured in YNB medium, 2% glucose, adenine at 100 ⁇ g ml for 48 h at 30 ° C. and then in Kappeli medium for 24 h at 30 ° C. as described. upper.
• the yeast suspensions are diluted to an optical density at 600 nm of 0.005 units (1/200) in YNB medium to inoculate a YPG agar containing syringomycin E at a toxic dose for the strain, ie 160 ng / ml for ylM126 containing PlM582 plasmid, or 250 ng / ml for the ylM126 strain containing the plasmid plM580.
• the inoculation of the agar medium is carried out by flooding of its surface and immediate suction of the excess liquid. Products of interest are deposited on the agar surface thus seeded and then dried. After an incubation period of 48 h to 72 h at 30 ° C, yeast growth halos are observed around the deposits of products interfering with the synthesis or catabolism of cholesterol. The products that induce the decrease in the amount of cholesterol either by inhibition of its synthesis or by activation of its catabolism confer a growth restoration on this initially toxic medium.
• Fig 1A cholesterol retention time by GC / FID gas chromatography
• Fig 1 B retention time of desmosterol by gas chromatography GC / FID
• Fig 1C Retention time of pregnenolone by GC / FID gas chromatography
• Fig 1 D retention time of 240H-cholesterol by gas chromatography GC / FID
• Fig 1 E retention time of 270H-cholesterol by gas chromatography GC / FID
• FIG. 2 The sterol profile of the ylM126 strain in the absence of P450 activity, shows the predominant presence of cholesterol (TR 21, 98 min.) And desmosterol
• FIG. 3 In the presence of CYP1 1A1 activity, the sterol profile of the ylM126 strain is modified with a reduction in the ratio of cholesterol (TR 21, 97) to the benefit of Desosterol (TR 23, 13) with appearance of a sterol corresponding to Prégenolone (TR
• FIG. 4 In the presence of CYP27A1 activity, the sterol profile of the ylM126 strain is modified with a reduction in the ratio of cholesterol (TR 21, 96) to the benefit of Desosterol (TR 23.1 1) and appearance of 2 other sterols corresponding to the 270H-Cholesterol (TR 33.53 min) and 270H-Desmosterol (TR 33.93 min).
• FIG. 5 In the presence of CYP46A1 activity, the sterol profile of the ylM126 strain is modified with the appearance of a sterol corresponding to 240H-cholesterol
• Fig 6 Growth test of different strains producing and or catabolizing cholesterol on YPG agar containing syringomycin E (SRG): Yeast culture dilutions deposited on YPG agar without syringomycin E (6A) or with syringomycin E at 170 ng / ml (6B); Dilution OD at 600 nm from top to bottom: 0.02 / 0.0008 / 00003.
• SRG syringomycin E
• Fig. 7 Growth restoration test on YPG agar containing syringomycin E inoculated with a cholesterol producing strain.
• a YPG agar medium containing syringomycin E at 250 ng / ml is seeded with a slurry of ylM126-plM580-CYP27A1 yeast suspension. 2 ⁇ l of products at 1 mM or 0.1 ⁇ g / ml are deposited on the dried and seeded surface.
• Fig 7A from top to bottom Cholesterol, 270H-Cholesterol and Amorolfine 0.1 mg / ml.
• Fig 7B from top to bottom Triparanol, AY9944, SR31747 at 1 mM.
• Fig. 8 Plasmid map plM580.

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