| Title of Invention | GENETICALLY MODIFIED YEAST STRAIN AUTONOMOUSLY PRODUCING STEROIDS |
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| Abstract | The invention concerns genetically modified yeast strains autonomously producing steroids from a simple carbon source. The invention also concerns a method for producing steroids from said yeast strains. |
| Full Text | YEAST STRAINS AUTONOMOUSLY PRODUCING STEROIDS The present invention relates to the production of steroids in microorganisms, in particular yeast strains. Steroids, in particular cholesterol-derived steroids, are involved in many physiological processes, among which mention may be made of regulation of carbohydrates and cholesterol levels in the bloodstream, maintenance and development of muscle mass, and development of the central nervous system. Among the drawbacks observed in the event of an imbalance of circulating steroid levels, mention may be made of the possible triggering of autoimmune diseases, such as lupus, of certain cancers, for example breast cancer, of cardiovascular diseases, for example atherosclerosis. Problems with steroid regulation are also suspected in the case of the triggering of certain neurological diseases, such as Parkinson's disease or Alzheimer's disease. Steroids, in particular hydrocortisone, can be used as therapeutic agents as such or as supplements in other treatments. Thus, synthetic derivatives of glucocorticoids are used for their anti-inflammatory and, at high doses, immunosuppressive actions. The production of steroids is partly associated with expensive methods of extraction or synthesis. John Warcup Cornforth was the first to carry out the complete synthesis of a steroid, cholesterol, using a enzymatic method. However, it is important to have a method for obtaining steroids of interest, in particular cholesterol derivatives, at an affordable price. A certain number of proteins involved in steroid biosynthesis have been expressed in yeast. Thus, patent EP 360 361 demonstrates the activity of the proteins P450 17a and P450c21 in the yeast Kluyveromyces lactis. Similarly, the possibility of in vivo conversion of 11- deoxycortisol to hydrocortisone in a genetically modified yeast expressing P450cll have been described (Dumas et al. f 1996) , as has the production of 17a- hydroxyprogesterone from pregnenolone in yeast (Degryse et al., 1999) . In addition, Duport et ai,, (Duport et al,, 1998) describe the synthesis of pregnenolone and progesterone in a genetically modified yeast. It has also been described, in patent application WO 99/40203, that inactivation of the ATF2 gene in a yeast strain makes it possible to avoid acetylation of the steroids produced by this strain. The present invention makes it possible to carry out steroid synthesis, by fermentation of genetically modified yeast strains, in the presence of a simple carbon source, The method provided by the present invention therefore makes it possible to obtain a large amount of steroids of interest, at low cost, since the method uses fermentation of yeast and the addition of a simple carbon source which is readily commercially available. Definitions: According to the present invention, the expression "simple carbon source" is intended to mean carbon sources which can be used by those skilled in the art for the normal growth of a yeast. It is in particular intended to denote the various assimilable sugars, such as glucose, galactose or sucrose, or molasses, or the byproducts of these sugars. A most particularly preferred simple carbon source is ethanol and glycerol. According to the present invention, the term "steroid derivative" is intended to denote a compound which can be obtained, in particular with one or two enzymatic or chemical reactions, from said steroids• It is in particular intended to denote acetylated or hydroxylated steroids or steroids bearing a substituent such as a halogenated {fluorine, iodine) derivative, or a methyl group. There are various types of problems to be solved in order to be able to produce steroids in a microorganism: it is advisable to eliminate the parasitic reactions which may be observed due to the presence of endogenous enzymes in the microorganism chosen, it is advisable to introduce the genes for modifying the. synthesis intermediates such that the levels of expression obtained are as close as possible to the levels observed in mammals. Thus, recreating the correct balances is an important conditions for the success of such a project, it is advisable to obtain a level of expression of the various genes which makes it possible to preferentially direct the biosynthesis toward the chosen steroid. Steroid synthesis is a series of extremely complex reactions involving several enzymes in order to obtain Cortisol from cholesterol. 20,22-Dihydroxycholesterol should first be produced, which is then transformed into pregnenolone, itself hydroxylated to 17a-hydroxypregnenolone. This is then transformed into 17a-hydroxyprogesterone, which gives deoxycortisol, leading to Cortisol (hydrocortisone). An alternative pathway consists of the production of pregnenolone, and then of progesterone, transformed to 17a-hydroxyprogesterone. It has been demonstrated that pregnenolone can be produced in yeast, from a simple carbon source (Duport et ai., 1998, the content of which is incorporated into the present application by way of reference) . To do this, it was necessary to delete an endogenous pathway (by disruption of the A22-sterol desaturase {ERG5) gene of the endogenous ergosterol biosynthesis pathway)f in order to obtain an accumulation of C-22'-saturated products. Specifically, the ergosterol normally produced by yeast differs from cholesterol by an unsaturation at C-7 (8) of the B ring, an unsaturation at C-22 and an additional methyl group at C-24, Thus, the products saturated at C-22 and at C-7 in the B ring can be used as substrates by the enzymes in the Cortisol production chain. The present invention makes it possible to synthesize steroids, and in particular the steroids located further downstream than pregnenolone, simply, by fermentation of genetically modified yeast strains, in the presence of a simple carbon source. In a particular case of the invention, the steroids synthesized are excreted into the culture medium, which simplifies the purification thereof. The method provided by the present invention therefore makes it possible to obtain a large amount of steroids of interest, at low cost, since the method uses fermentation of yeasts and the addition of a simple carbon source which is readily commercially available. Preferably, the steroids which can be produced by the yeast strain according to the invention are steroids included in the Cortisol synthetic pathway, as stated above. It is also possible to produce other types of steroids, from 17a-hydroxypregnenolone, in particular dihydroepiandrosterone (DHEA), by the action of the enzyme 17a"hydroxylase and lyase, and the derived steroids (androstenediones, testosterone, etc.). These steroids can be produced by introducing the appropriate enzymes into the yeast strain, in the same way as for the strain exemplified in the present invention. In order to carry out the method according to the invention, the invention also relates to a genetically modified yeast strain producing a steroid or a steroid derivative, characterized in that it allows autonomous production from a simple carbon source. The fact that the production is carried out autonomously means that there is no need to add substrates in order to obtain the steroid of interest, so that the yeast can produce it only from the starting simple carbon source. It is also clear that the strain can produce a steroid of the metabolic pathway, using a substrate located upstream in the metabolic pathway, insofar as the yeast strain according to the present invention contains all the genes required to complete the metabolic pathway for steroid production, Preferably, the yeast strain according to the invention produces a steroid or steroid derivative which is a derivative of cholesterol metabolism, i,e, which is part of the cholesterol metabolic chain. Cholesterol metabolism is well known to those skilled in the art, and is explained in biochemistry and endocrinology publications• Thus, preferably, said steroid or steroid derivative is in particular included in the group consisting of 17α-hydroxypregnenolone, Cortisol, cortisone, cortexolone, 17α-hydroxyprogesterone, and derivatives of these steroids. It is also possible to produce pregnenolone and progesterone with a yeast strain according to the present invention. Thus, a subject of the present invention is in particular a genetically modified yeast strain autonomously producing, from a simple carbon source, a steroid or a steroid derivative, derived from cholesterol metabolism, characterized in that said steroid or steroid derivative is included in the group consisting of 17α-hydroxypregnenolone, hydrocortisone, cortexolone, 17α-hydroxyprogesterone, and derivatives of these steroids, As will be seen later, the yeast strain according to the invention has at least one genetic modification chosen from a group consisting of disruption or inactivation of an endogenous gene, modification of the promoter of an endogenous gene, duplication of an endogenous gene, and introduction of at least one heterologous gene, in one or more copies, episomally or chromosomally. It is, moreover, advantageous for the yeast strain of the invention to have a combination of said gene modifications. As explained later, in a first embodiment, the yeast strain has- at least one disruption of an endogenous gene chosen from the group consisting of ERGS, ATF2f GCYlf YPRl, AREl, ARE2, ATFl and ADE2. In a preferred embodiment, the yeast strain according to the invention has a disruption of the endogenous genes ERGS, ATF2, GCYl and YPRl. As described later, these genes encode proteins which induce parasitic reactions in the yeast. With regard to the ADE2 gene, it may also optionally be disrupted, in particular in order to integrate a heterologous gene into the yeast strain. In one embodiment, the yeast strain according to the invention has at least one heterologous gene integrated into the chromosome, at least one locus chosen from ADE2, HIS3, TRPl, LEU2, GCYl, ATF2 and YPRl, the integration being carried out intragenically or intergenically in the immediate vicinity of one of the loci. In one embodiment of the invention, said yeast strain has at least one heterologous gene located on a multicopy plasmid or a low copy plasmid, said multicopy plasmid being chosen from yeast 2-micron replicon-based plasmids which replicate in Saccharomyces cerevisiae and said low copy plasmid being chosen from plasmids based on a chromosomal ARS origin of replication with a yeast centromere. To implement an embodiment of the invention, and as developed below, the yeast strain according to the invention has at least one heterologous gene or cDNA chosen from the group consisting of the gene of sterol A7-reducta5e and of the cDNAs of cytochrome P450 SCC, of adrenodoxin, of adrenodoxin reductase, of cytochrome b5, of 3P-hydrosteroid dehydrogenase isomerase, of cytochrome P450 reductase, of cytochrome P450 C17, of cytochrome P450 C21 and of cytochrome P450 Cll, and of the sequences encoding these proteins, These heterologous genes or cDNAs are under the control of a promoter sequence chosen from the group consisting of the yeast endogenous promoter sequences TDH3, TEFl, PGKl, CYCl, GALlO, ATF2, TIRl, ARHl and ADE2r and the hybrid promoter GALlO-CYCl, It is necessary to use a terminator sequence for any heterologous gene or cDNA introduced, preferably chosen from the terminator sequences of the endogenous genes PGfCl, CYClr ATF2, ADE2 and NCPl. Expression cassettes or blocks are then obtained, which consist of a promoter, the heterologous gene (or cDNA, optionally encoding the mature protein preceded by the ATG codon encoding methionine (Met-mat) or encoding a fusion protein optionally having signals for addressing to cellular compartments), and a terminator sequence, In one embodiment, the yeast strain according to the invention has the sterol A7-reductase heterologous expression block integrated into the chromosome at the ADE2 locus. In a particular embodiment of the invention, the yeast strain comprises at least one cassette for expression of the genes encoding P4 50scc ^^id adrenodoxin cofactor of P450scc, located on a high copy plasmid, and it comprises a cassette for expression of adrenodoxin reductase cofactor of P450scc, located on a single copy plasmid or a low copy plasmid or integrated into the chromosome. Preferably, these expression cassettes contain the mature protein preceded by a methionine, and the protein is located in the cytosol. In one embodiment, the yeast strain comprises at least one expression cassette chosen from the cassettes for expression of 3P-hydrosteroid dehydrogenase isomerase, of cytochrome P450cl7 or cytochrome P450c21, located on a high copy plasmid or a low copy plasmid or integrated into the chromosome. In a particular embodiment, the yeast strain according to the invention comprises at least one expression cassette for P4501iP, located on a multicopy plasmid, the protein produced having a signal for addressing to mitochondria, and/or at least one expression cassette for adrenodoxin cofactor of P45011p, located on a multicopy plasmid, with a weak promoter (i.e. the strength of which is related to that of the CYCl promoter) , the protein produced having a signal for addressing to mitochondria. Preferably, the proteins are produced in the form of a precursor, - with a homologous or heterologous signal for addressing to mitochondria, the proteins taking their mature form in this cellular compartment. It is therefore interesting to note that, m a particularly preferred embodiment of the invention, two copies of the gene encoding adrenodoxin are introduced into the yeast strain, one of them being intended to express the protein in the cytosol of the cell, the other being produced such that the mature protein is in the mitochondria• In a particular embodiment, the yeast strain also comprises at least one expression cassette (expression promoter as mentioned above with the coding portion of the NCPlf ATRl and/or ATR2 gene, with its own terminator or terminator as defined above) located on a multicopy plasmid or low copy plasmid or integrated into the chromosome. The expression cassettes for NCPl, ATRl and ATR2 may in particular be integrated at the NCPl locus of S. cerevisiae. In a particular embodiment, the yeast strain according to the invention also expresses the ARHlp protein, a protein homologous to mammalian adrenodoxin reductase in yeast, at a level higher than the physiological expression level. Overexpression of this protein can be obtained using techniques well known to those skilled in the art, for example by introducing a new expression cassette (expression promoter, coding portion of the ARHl gene with, its own terminator or a terminator as defined above), in addition to the endogenous gene, into the yeast. Surprisingly, it has indeed been shown that expression of the ARHl gene at a level higher than the physiological expression level significantly increases the amount of steroids produced. However, this expression should not be too great to obtain the desired effect. Thus, if an expression of the ARHl protein at a level higher than a physiological level is desirable, care should be taken not to overexpress this protein too strongly, otherwise there is a risk of losing this increase in production of steroids. The yeast strain according to the present invention may be polyploid, diploid, haploid or aneuploid in nature, without this being harmful to the implementation of the invention. It is preferably a strain of Saccharomyces cerevisiaSf in particular derived from one of the strains FY 1679-28C and FY 1679-18b which are spores of the strain FY 167 9 deposited with the American Type Culture Collection under the number 96604, A subject of the invention is also a yeast strain, characterized in that it is the strain CDR07 Mat-α or TGY260, deposited with the CNCM on January 24, 2001, under the respective accession numbers 1-2616 and 1-2615. The invention also relates to a strain obtained after crossing of CDR07 Mat-a and TGY260, and optionally sporulation and transformation with a. plasmid from yeasty in particular the strains UCY2 and UCY4 and the strains UCY3 and UCY26 described in the present invention. A subject of the invention is also yeast strains obtained after crossing of UCY2 and TGY245, and optionally sporulation and transformation with at least one plasmid from yeast, in particular the strains UCY5, UCY6, UCY16, UCY19, UCY2 0, UCY24, UCY25 and UCY25, also described in the present invention. It is useful for the yeast strain according to the invention to have the elements required for excreting the steroid produced into the culture medium, in order to simplify purification of the final product. The invention also relates to a method for producing a steroid, characterized in that it comprises the steps of fermenting a yeast strain according to the invention in the presence of a simple carbon source, and of recovering the steroid produced. Finally, the subject of the invention is also a pharmaceutical preparation comprising a yeast strain according to the invention, optionally with a pharmaceutically acceptable excipient, such an excipient being well known to those skilled in the art. Although the yeast strain according to the invention produces a steroid autonomously from a simple carbon source, it is also possible to provide it with cholesterol or a related structure, or a substrate already present as a cholesterol derivative, in order to obtain the products located downstream- The possibility of being able to enter at any stage, in particular at the pregnenolone level or later in the metabolic pathway of the desired steroid therefore makes it possible in particular to be able to provide the yeast with unnatural substrates, which lead to the synthesis of unnatural and substituted steroids, in particular fluorinated steroids, In a first embodiment, the yeast strain according to the present invention in particular makes it possible to produce the desired steroid (in particular Cortisol) in an amount greater than 10 mg/1, preferably greater than 50 mg/1, more preferably 80 mg/1, more preferably 100 mg/1, and most preferably 200 mg/1- In another embodiment, the steroid of interest (preferably hydrocortisone) is present in a proportion greater than 20%, preferably 25%, more preferably 30%, more preferably 35%, more preferably 4 0%, more preferably 50%, and most preferably 65%, of the total steroids produced by the strain according to the invention (in particular the synthesis intermediates) . In order for it to be possible for the yeast strain according to the present invention to produce the steroids of interest, it is necessary for it to have genetic modifications. Thus, the yeast strain according to the invention has at least one genetic modification chosen from the group consisting of disruption or inactivation of an endogenous gene, modification of the promoter of an endogenous gene, duplication of an endogenous gene, and introduction of at least one heterologous gene (in particular an expression block with homologous promoter and/or terminator and a heterologous coding portion), in one or more copies, episomally or chromosomally. Preferably, the yeast strain according to the invention has several (at least four) genetic modifications as stated above. Thus, some endogenous genes of the yeast are favorably inactivated or disrupted. The genes can be inactivated or disrupted by introducing, into the coding sequence, an exogenous gene (in particular an expression block with homologous promoter and/or terminator and a heterologous coding portion) as described below, and/or a selectable marker. It is also possible to modify the promoters of these genes in order to decrease the level of expression. The yeast gene ATF2 (Cauet et ai., 1999) encodes an acetyl transferase which uses pregnenolone as substrate, and disruption thereof makes it possible to eliminate this parasitic acetylation reaction, the product of which cannot then be used, and thus to increase the yield of steroid of interest. Thus, the yields can be multiplied by values of between 3 and 7 after inactivation of the ATF2 gene. The GCYl and YPRl genes encode aldo-keto reductases, These genes are advantageously inactivated or disrupted. These two genes are part of a family of 6 more or less homologous genes, all six of which are supposed to encode aldo-keto reductases. However, the products of these genes are the most active on the substrates envisioned herein, in particular GCYl, and inactivation thereof is therefore extremely advantageous for obtaining hydrocortisone. As specified above, it is advantageous to inactivate the ERGS gene in order to accumulate a substrate which has a structure as close as possible to the structure of cholesterol. However, it has been shown that the yeast according to the invention can, nevertheless, produce the steroids of interest despite the activity of this gene. However, in order to optimize yields, it may be useful to inactivate it by mutation, deletion and/or insertion. It is also possible, without this being really essential for the overall success of the steroid production with the yeast according to the present invention, to inactivate other genes, such as AREl, ARE2, ADE2 or ATFl. These genes all encode proteins the absence of which may improve the overall yield of synthesis of the steroid of interest. As described in the article by Duport et al,, cited above (Duport et al., 1998) , it is indicated that the presence of an expression block with homologous promoter and/or - terminator and a sequence encoding Al-reductase is useful in that it makes it possible to desaturate the 7-8 double bond of ergosterol and of its derivatives, and thus to obtain one or more precursors with a structure closer to the structure of cholesterol, the starting substrate for the production of pregnenolone. Thus, it is advantageous for the yeast strain according to the present invention to contain this expression block. In a particular case, said block for expression of A7-reductase is integrated into the genome of the yeast, preferably at the locus of the ADE2 gene, by the same token leading to the disruption of the ADE2 gene* The promoter used for transcription is an inducible promoter, such as GALIO-CYCI, or a constitutive promoter, such as the GALIO-GALIO-CYCI promoter which is disrupted in the strain CAlO described in Duport et ai., 1998, The protein used is preferentially derived from Arabidopsis thaliana (but may also be derived from a mammalian species), the cDNA being cloned in the native form (complementary DNA just after a translation initiation methionine), and under the control of a transcription terminator which is conventional in yeast, such as PGKl, It should be noted that activity of the A7-reductase gene in yeast has been described by Lecain et al., 1996, the technical content of which (in particular the sequences of the A7-reductase gene and the constructs and procedures) is incorporated into the present application by way of. reference. The first step is the production of pregnenolone, obtained after introducing into the yeast the enzymes for normally transforming cholesterol into pregnenolone. In the present case, this is the enzyme for cleaving the side chain (P450scc for side chain cleavage), with two coenzymes (adrenodoxin, ADX, and adrenodoxin reductase, ADR) . The transformation of the yeast with these respective expression blocks is described in Duport et al., 1998/ cited above. A complementary DNA encoding the mature proteins, with a methionine added to the N-terminal end to allow translation, is preferably used. Promoters such as the GALlO-CYCl hybrid promoter or the TEFl promoter are used to ensure transcription of the cDNAs. Conventional terminators, in particular the FGKl terminator, are used. The various cDNAs encoding the P450scc/ ADR or ADX proteins may be of vertebrate origin, for example human or bovine origin, but also rat or fish. The genes encoding these proteins are preferably placed on plasmids; for P450scc and ADX, a multicopy low copy or high copy plasmid, in particular derived from a 2 micron yeast plasmid, is preferred, whereas a single copy plasmid or a low copy plasmid is rather used for the expression of ADR. The expression block for ADR may also be integrated into the chromosome of the yeast. This makes it possible to control expression of the ADR, since is appears that too much expression harms-the desired sec activity. The proteins are preferably expressed so as to be able to exert their activity in the cytosol* The following step is the conversion of pregnenolone to 17α-hydroxyprogesterone, by the combined action of ITa-hydroxylase (P450cl7) and 3P-hydroxysteroid dehydrogenase OP-HSD). To express these two proteins, strong promoters, such as TEFlr TDH3 or GALlO-CYClr are preferably used. However, a weaker promoter, such as CYCl, may also be suitable. The terminators used are conventional, and in particular come from the PGKl or NCPl genes. The complementary DNAs encoding the complete proteins are expressed. The species of origin of these proteins does not appear to modify the results obtained, and it is thus possible to use proteins of human origin (in particular one or other of the two iso types of SP-HSD), of bovine origin or originating from other organisms (in particular from fish) . For these two proteins, it is a question of obtaining the best possible expression, and they can therefore be expressed on single copy or multicopy, low copy or high copy plasmids, or by having integrated the expression blocks into at least one chromosome of the yeast, Conversion of the 17α-hydroxyprogesterone to deoxycortisol is then sought, via P450c21, which allows hydroxylation at position 21, It is a question of expressing the protein.from its cDNA in the most effective way possible, To do this, a strong promoter (TEFl, TDH3, GALIO-CYCI, etc.), or even the CYCl promoter, and a conventional terminator (in particular PGKl) are used to design the transcriptional unit. This unit is placed on a single copy or multicopy, low copy or high copy plasmid, or else integrated into the genome of the yeast. It should be noted that the species of origin appears to be important here, and that it is preferable to use P450c21 of human origin. The deoxycortisol is then converted to Cortisol, under the action of the P450cll system, which contains P450cll, allowing hydroxylation at the 11-p position, and an adrenodoxin and an adrenodoxin reductase as cofactors. The inventors of the present application have shown that the results obtained are better when this last system is expressed in the inner membrane of yeast mitochondria. Thus, it is advantageous to produce fusion proteins which carry, as precursor, the mitochondrial addressing sequence of the yeast Cox6p protein precursor. The cDNA encoding the mature proteins is also preferably used. The P450cll protein is thus the protein of human or bovine origin or a human-bovine hybrid protein * The latter construct is the preferred form for implementing the invention. The gene used in the transcriptional unit is preferably under the control of the CYCl promoter. It is advantageous to place a rabbit β-globin intron - and a terminator of the human growth hormone gene in the position 3' of the coding sequence. The transcriptional unit is preferably placed on a multicopy plasmid. The adrenodoxin is used in its mature form, placed with a sequence for addressing to mitochondria, for example chosen from those of the percursors of the Cox6p, Cox4p, fumarase, ARHlp and F9 ATPase proteins, and under the control of a promoter in particular chosen from TDH3, TEFl and CYCl. A protein of bovine or human origin is preferred- A terminator, which may be PGKl, should be placed in the transcriptional unit. The expression block is preferably expressed from a multicopy plasmid. The proteins acting as an adrenodoxin reductase is the ARHlp protein, an endogenous yeast protein, which is normally expressed in the host's mitochondria. Preferably, the yeast is, however, transformed in such a way that the host strain contains a natural copy of the ARHl gene and a second copy of the ARHl gene under the control of the CYCl promoter. The activity of this protein is essential for obtaining the desired effect, and it has even been observed that inactivation of the gene is lethal for yeast (Lacour et al,, 1998) , It should be noted that overexpression of this gene appears to be toxic for the organism, and that the promoter chosen should therefore allow a level of expression which leads to the desired 11-β hydroxylase activity without being deleterious for yeast. Surprisingly, it has in fact been shown that expression of the ARHl gene at a level higher than the physiological expression level significantly increases the amount of steroids produced. However, as mentioned above, this expression should not be too great to obtain the desired effect. Thus, if expression of the ARHl protein at a level greater than a physiological level is desirable, care should be taken not to overexpress this protein too strongly, otherwise there is a risk of losing this increase in production of steroids. By way of example, integration of the expression cassette for ARHl, comprising as promoter the CYCl promoter, at the LEU2 locus of S. cerevisiae gives satisfactory levels of expression and results, On the other hand, integration, at the same locus, of a cassette comprising the TEFl promoter, acknowledged to be much stronger than the CYCl promoter, gives less advantageous results, Thus, two copies of a transcriptional unit encoding the ADX co-enzyme protein are preferably introduced, one of them having activity outside the mitochondria, and in particular in the cytosol, the other having activity in the mitochondria of the host cell. It is also possible to introduce other genes, in particular the genes encoding proteins having NADPH P450 reductase activity, such as NCPl (yeast reductase also called CPRl) , ATRl or ATR2 (plant reductases) , or human reductase. These proteins improve P450cl7 and P450c21 activities. Either the-endogenous promoter [NCPl) is used, or the DNAs encoding the proteins are placed under the control of promoters such as GALlO-CYCl, CYCl, TEFl, etc. It is also possible to introduce the TGLl gene, which encodes a protein having deesterification activity, under the control of a strong promoter such as GALlO-CYCl, on a multicopy or single copy plasmid. This makes it possible to reduce the effect of parasitic reactions of sterol esterification which may remain even after inactivation of ATF2f and in particular those produced by the product of the AREl and ARE2 genes. It is also possible to add a plasmid expressing cytochrome b5 from yeast or another species, which is a cofactor in several of the reactions defined above. When it is desired to produce DHEA, it is also possible to introduce a low or high copy plasmid encoding desmolase (P450 17a), under the control of a promoter such as GALlO-CYClr CYCl or TEFlr with a PGKl terminator. It is also possible to introduce a cDNA encoding cytochrome P450cl7 having lyase activity, for example that of human origin, in the presence of an excess of NADPH P-450 reductase, for instance mammalian reductases, NCPl, ATRl or ATR2, These transcriptional units preferably use a strong promoter. Finally, it is possible to restore the activity of the endogenous ERG6 and ERG2 genes, inhibited by pregnenolone, by placing them under the control of a strong constitutive promoter. It is also possible to introduce other heterologous genesy- in particular encoding a protein with 24,25 sterol reductase activity, or the HMGl gene, present in the synthetic path with cholesterol in humans. It is also advantageous to introduce the human MDRl gene, which encodes a pump which is not inhibited by accumulation of the abnormal sterols which appear due to the inhibition of the ERGS gene by pregnenolone. This makes it possible to expel these products, too great an accumulation of which might prove to be toxic for the host cell. It is also possible to overexpress the yeast PDR12 gene, which will have a detoxification effect for the steroids which may inhibit the yeast growth. It is understood that, when reference is made to "gene" above, this is intended to mean not only the DNA fragment encoding the protein having the desired activity (and especially the cDNA fragment representing in particular the mature forms), but also the promoters (in particular TEFl, GALIO-CYCI, CYCl, TDH3) and the terminators (in particular the PGKl) . These transcriptional units are preferentially introduced on low or high copy plasmids, or integrated into the chromosome of the yeast. It is also understood that, depending on the desired aim, and in particular the steroid that is intended to be produced, it is possible to introduce only some of the genes encoding the various proteins of each step, and to provide the yeast with an intermediate in order to obtain a steroid which is downstream in the metabolic chain. It is also easy not to introduce the genes for obtaining the products located downstream, and therefore to be able to stop relatively high in the metabolic chain. The yeast used to implement the present invention is preferably: - the FY1679'-28c strain, described in Duport et al., 1998, which has the genotype [MATa, rho", ura3-52r trplA63, leu2Al, his3A200f GAL2, fenl). This strain has also been described by Thierry et al., 1990; - the FY1679-18b strain, having the genotype {MATa, rho", ura3-52, trplA63, leu2Al, his3A200, GAL2, fenl) . These two strains therefore have an identical genotype and an opposite sign. DESCRIPTION OF THE FIGURES Figure 1: Diagrammatic representation of a biosynthetic pathway for hydrocortisone as can be obtained according to the invention, Figure 2: Diagrammatic representation of construction of yeast strains exemplified according to the invention. Figure 3: Map of the plasmid pCV29. PGK term: PGK terminator. GALIO/CYCI prom: GALlO/CYCl promoter. HGH term: terminator of the human growth hormone gene. Intron β-globin: intron-of the rabbit β-globin gene. CYCl prom: CYCl promoter. PGK term: PGK terminator. S. cerevisiae 2-micron: 2-micron origin of replication of 5. cerevisiae, Cox6pre: presequence of cytochrome oxidase subunit 6. Bovine/human P450 lip: bovine/human fused cDNA of P45011p. mat-ADX: mature form of ADX with an NH2"terminal methionine. E.coli replicon: E, coli Replicon, Figure 4: Map of the plasmid pCC12. CYCl prom; CYCl promoter. PGK term: PGK" terminator. ARS CEN: S. cerevisiae chromosomal origin of replication. E.coli replicon: E.coli Replicon. TDH3 prom: TDH3 promoter* SP-HSDH: 3p-hydroxysteroid dehydrogenase cDNA. matADR: mature form of ADR preceded by a methionine. Figure 5: Map of plasmid pFMlO. CYClp: CYCl promoter, P45011p: bovine/human fused cDNA of P4501ip. ADE2: yeast ADE2 gene. TDH3p: TDH3 promoter, 3β-HSD: 3P-hydroxysteroid dehydrogenase cDNA, Rl: base for recombination, the sequence of which is given in SEQ ID No. 39, GALlO/CYClp: GALIO/CYCI promoter. matADX: cDNA encoding the mature form of ADX, URA3: yeast URA3 gene. P450scc; cDNA encoding the mature form of P450scc (CYPllAl). 2-micron origin: 2-micron origin of replication of S. cerevisiae. R2; base for recombination, the sequence of which is given in SEQ ID No. 40. EXAMPLES The examples below describe an embodiment of the present invention and should not be considered as limiting the invention. For the constructions, the FY1679-28c and FY1679-18b yeast strains described above are used as starting materials. Example 1: Disruption of the YPRl gene The construct comprising interruption of the YPRl gene (YDR368w) by the URA3 gene in the plasmid pPOLYIII was obtained by 4 successive PCRs. First, three independent PCRs were carried out to obtain the 5' portion of the YPRl gene (PCR 5) , the functional URA3 gene bordered by YPRl sequences (PCR 6), and the 3' portion of the YPRl gene (PCR 7). The PCRS DNA was obtained by amplification on a genomic DNA matrix with the oligonucleotides OTG11314 (SEQ ID No. 1) and OTG11315 (SEQ ID No. 2) and, similarly, the PCR7 DNA is obtained by amplification using the oligonucleotides OTG11316 (SEQ ID No. 3) and OTG11317 (SEQ ID No, 4) on the same matrix. The URA3 gene flanked by 5' and 3' YPRl region is amplified using the oligonucleotides OTG11463 (SEQ ID No, 5) and OTG11464 (SEQ ID No. 6) on a matrix pTG10054 described in Degryse et al,, 1995, incorporated herein by way of reference as regards the description of this plasmid. The products of PCR5, PCR6 and PCR7 were mixed in an equimolecular- fashion and then amplified by PCR using the oligonucleotides OTG11314 (SEQ ID No. 1) and OTG11317 (SEQ ID No. 4), so as to obtain a product of PCR8. This PCR8 product was digested with the Xhol enzyme and then subcloned into the plasmid pPOLYIII (described by Lathe et ai., 1987, and incorporated herein by way of reference as regards the description of this plasmid) digested with Xhol, to give the plasmid pTGl2011. The orientation of the insertion into the plasmid pPOLYIII was determined by digestion with the Ncol and EcoRI enzymes. The plasmid pTG12 011, which allows disruption of the parasitic YPRl gene with the URA3 gene, is digested with the Xhol enzyme. The digestion product is used to transform the FY1679-18b strain using the lithium chloride method well known to those skilled in the art. The transformants are selected on a uracil-free medium. The transformants are analyzed by PCR amplification using the oligonucleotides which were used to construct the plasmid pTG12011. The clones which are positive in this test are then screened by the 170H^progesterone bioconversion method described below, in the presence of glucose as carbon source. The capacity for bioconversion is analyzed by HPLC as described by Dumas et ai., 1996 and Degryse et al., 1999, the contents of which are incorporated into the present application by way of reference, in particular the explanations of the bioconversion studies, or as described in Kuronen at al., 1999. A clone, TGY195#4, is selected for further characterizations. The TGyi95#4 strain is transformed using both the plasmid YRp7 (Parent et al,, 1985, which is incorporated by way of reference as regards the description of this plasmid) (1 µg) and 5 µg of plasmid pTG12045 (described below) digested with Notl. The transformed strains are selected on a tryptophan-free medium. Colonies (678 colonies) are subcultured on a medium containing trytophan (so as to eliminate the plasmid YRp7) and on a medium containing tryptophan and 5-fluoroorotate (5F0) in order to select the colonies which have lost the URA3 gene interrupting the YPRl gene. Example 2: Construction of various plasmids For overexpression of the P450c21 protein in yeast two types of promoter were used, TEFl (transcription elongation factor 1) and TDH3 (glyceraldehyde-3-phosphate dehydrogenase 3). In all cases, the transcription terminator is the PGK terminator. In these plasmids, the Sail, Miul fragment carries the human P450c21 cDNA, a) Construction of the plasmids pTG10470 and pTG10469 The plasmid pTG10289 was obtained by modification of pMAc21 (Wu et al., 1991) by digestion with Kpnl and Mlul and introduction of the oligonucleotide OTG5868 (SEQ ID No. 27). The cDNA of this plasmid comes from the American Type Culture Collection (ATCC, Rockville, Maryland, USA) under the name pc21/3c. It is the 1.6 Kb EcoRl-BamHl fragment which was used as a base to construct the various plasmids. The modifications introduced are described in the article above and in the article by Hu et al,, 1990, In this procedure, the noncoding portion of P450c21 of the plasmid piyiAc21 which contains the expression cassette for p450c21 was removed, as was the Kpnl site located therein, The plasmid pTG102 92 was obtained by transferring the human c21 cDNA (Sail, MIuI fragment) of the plasmid pTG10289 into the plasmid pTG10031 (described in Degryse et al., 1995, which is incorporated into the application by way of reference) using the Sail and Mlul sites. The plasmid pTG10475 was obtained by PCR and recombination. Specifically, using the plasmid pTG10292, a fragment of the human P450c21 cDNA representing approximately 250 nucleotides was amplified using the oligonucleotides OTG7410 (SEQ ID No. 7) and OTG5927 (SEQ ID No. 8). This fragment represents the coding sequence of human P450c21, of a Sail site and of the sequence AAAA. This fragment was digested with Sail and then ligated onto the linear fragment of pTG10292 digested with Sail, and the recombination experiment was then carried out in the BJ5183 strain described by Degryse et al.r 1995. The plasmid obtained, pTG10475, carries a P450c21 cDNA with a coding sequence identical to that of the natural cDNA, unlike the plasmid pMAc21, on a fragment compatible with the vectors generally used by the inventors, i.e. a fragment bordered by the Sail and Mull restriction sites. This fragment has the following environment around the ATG codon for translation initiation: GTCGACAAAAATGCTGCTCCTGGGCCTGCTGC (SEQ ID No, 9). Using this plasmid, the Sail, Mull fragment carrying the human p450c21 cDNA was transferred into the plasmid pTG10158 (Degryse et al., 1995) by conventional cloning, to give the plasmid pTG10472. This same Sail, mluI fragment of the plasmid pTGl047 2 was then transferred by conventional cloning into the plasmid pTG10085 (Degryse et ai., 1995), to give the plasmid pTG10469. This plasmid therefore has the human P450c21 cDNA under the control of the TEFl promoter, with the PGKl terminator• This same fragment carrying the P4 50c21 cDNA on a Sail and Mlul restriction fragment is transferred into the plasmid pTG10092 by recombination in the BJ5183 strain, to give the plasmid pTG10470 (Degryse et al., 1996). The plasmid pTG104 7 0 therefore carries the human P450c21 cDNA under the control of the TEFl promoter and of a PGKl terminator, with a URA3-d selection marker with the environment of the ATG-initiator codon described above. b) Construction of the plasmid pTG12036 The plasmid pTG12036 was constructed in 4 steps from pTG10802, The plasmid pTGlOSOl (which is the origin of the plasmid pTG10802) is a plasmid of the pUC type into which a series of restriction sites has been inserted between the Xhol and Xhol sites, This series of sites includes the Hindlll, Snajbl, Clal and Spel sites. Between the Hindlll and Clal sites, the Hindlll, Clal cassette of pTG1047 0, comprising the TEFl promoter, the human P4 50c21 cDNA and the PGKl terminator, was inserted between the Hindlll and Cial sites of pTGlOSOl, to give pTG10802. This plasmid was then digested with Xhol and the cassette introduced is therefore deleted in order to introduce a PCR fragment bordered by Xhol sites. This 2.5 Kb fragment comes from amplification with the pair of oligonucleotides OTG1184'4 (SEQ ID No. 10) and OTG118 4 5 (SEQ ID No. 11) on the plasmid pTG12010#40 (cf. below) so as to obtain a fragment bordered by Xhol sites, containing the GCYl gene interrupted by the URA3 gene bordered in the 5' position by a CJal restriction site- This fragment was cloned between the Xhol sites of the plasmid pTG10802, so as to obtain the plasmid pTG12035. The plamid pTG12010#36 was used with the aim of introducing the missing HindIII site. This plasmid is essentially identical - to pTG12010#40, but has a HindIII site positioned 3' of the URA3 gene at the limit with the GCYl gene, but does not have a Clal site positioned 5' of the URA3 gene at the junction with the GCYl gene (cf, below) , By recombination in vivo in E. coli, between the 2.2 Kb Ncol, BaiuHl fragment which carries from 5' to 3' a fragment of the URA3 gene, the 3' fragment of the GCYl gene and, finally, a portion of the plasmid pTG12035, i.e. the large 4.45 Kb Stul, Aflll fragment. The plasmid pTG12036 is obtained. The plasmid obtained, pTG12036, has the GCYl gene interrupted by the URA3 gene bordered by Cial and HindlU sites in 5' and 3' position, respectively. This fragment is then replaced with the expression cassette for P4 50c21 carried by the 2.33 Kb Cial, Hindlll fragment of the plasmid pTG10469 (see above), so as to obtain the plasmid pTG12086. c) Construction of the plasmid pTG12045 The unique Sphl site of the plasmid pPOLYIII is destroyed by insertion of the pair of complementary oligonucleotides OTG1197 5 (SEQ ID No. 12) and OTG1197 6 (SEQ ID No. 13) . The Sphl site of pPOLYIII is destroyed and replaced with a Cial site, to give the plasmid pTGl2040, A Cial, EcoRl genomic DNA fragment corresponding to the 0.7 Kb 3' portion of the YPRl gene obtained by amplification with the oligonucleotides OTG11981 (SEQ ID No. 14) and OTG11982 (SEQ ID No. 15) is introduced into the plasmid pTGl204 0, between the unique Clal and EcoRl sites, to give the plasmid pTG12041. In this 2.84 Kb plasmid pTG12041, the 5' portion of the YPRl gene (0.66 Kb) , amplified by the oligonucleotides OTG11314 (SEQ ID No. 1) and OTG11980 (SEQ ID No. 16) from wild-type yeast genomic DNA^ is cloned in the form of an Xhol, Hindi 11 fragment, between the Sail and Hindlll sites of the plasmid pTG12041, The 3.5 Kb plasmid pTG12042 is obtained. This plasmid carries the YPRl gene interrupted by the Clal and HindiII sites. The cytochrome P450c21 cassette is cloned between these sites, in the form of a 2.33 Kb Cial, Hindlll fragment originating from the plasmid pTG10469. The plasmid pTG12045 is thus obtained. d) Construction of the plasmids pTG12010#36 and #40 The plasmid pTG 12010 was constructed based on the plasmid pUC19 (Yanisch-Perron et al., 1985), whereas the plasmid pTG12011 was constructed based on the plasmid pPOLYIII (Lathe et al., 1987). The construct comprising the disruption of the GCYl gene by the URA3 gene in the plasmid pUC19 was obtained by four successive PCR amplifications. First, three independent PCRs were carried out so as to obtain the 5' portion of the GCYl gene (PCRl), the functional URA3 gene bordered by. GCYl sequences (PCR2), and the 3' portion of the GCYl gene (PCR3). The 5' and 3' portions of the GCYl gene [lacuna] using the pairs OTG11285, OTG11286 and OTG11287, OTG11289 (respectively SEQ ID No. 17 to SEQ ID No. 20) on a matrix of genomic DNA of the FY1679-28c strain. The URA3 gene flanked by GCYl sequences (in such a way as to obtain a deletion of part of the coding sequence of the GCYl gene) is amplified using the oligonucleotides OTG11305 (SEQ ID No, 21) and OTG11306 (SEQ ID No. 22) from a linearized plasmid pTG10054 matrix (Degryse et al.f 1995). The buffer conditions and matrix and primer concentration conditions for the amplification are described by the producer or manufacturer of the Tag DNA polymerase enzyme, and in particular for the elongase enzyme developed by Life Technologies. The temperature cycles are as follows: a first cycle of 6'30 to denature primer and matrix, and then 30 cycles of 30s at 93°C, 2 min at 54°C and 3 min at 68°C, and the final cycle is 5 min at 72°C. The PCRl, PCR2 and PCR3 products are mixed in an equimolecular fashion and amplified again using the oligonucleotides OTG 11285 (SEQ ID No. 17) and OTG11289 (SEQ ID No. 20). The final product, PCR4, which is 2.9 Kb in size, is then subcloned between the Kpnl and BamHl restriction sites of the plasmid pUC19, so as to obtain the plasmid pTGl2010. - The structure of the plasmid was verified by restriction profile and nucleotide sequencing of the ends. Cloning pTG12010 in fact made it possible to obtain two versions of this plasmid, version pTGl2010#40 {pTG1204 0 clone 40) and pTG12010#3 6. The initial desire was to obtain the GCYl gene interrupted by the URA3 gene bordered by the Cial and HindiII sites respectively positioned 5' and 3' of the gene. In fact, two different plasmids were obtained, which differ only by the presence or absence of Cial and Hlndlll sites at the ends of the URA3 gene. The plasmid pTG12010#4 0 has a HindiII restriction site at the 3' end of the URA3 gene, but no Cial site positioned 5' . The plasmid pTGl2010#36 has no Hindi11 site at the 3' end, but a Clal site at the 5' end of the gene. This property is used to obtain the plasmid which has the URA3 gene bordered by the HindiII and Clal sites, interrupting the coding sequence of GCYl. e) Construction of the plasmid pTG12086 This plasmid is used to integrate an expression cassette for P4 50c21 and also to disrupt the GCYl gene at the same time. This plasmid was constructed from the plasmid pTG12036 and from the plasmid pTG10614. The construction of the plasmid pTG10614 was carried out as follows. This plasmid was constructed from the PTG10212 (Degryse et al., 1995), which is a yeast expression plasmid based on a TDH3 promoter, a PGKl terminator and a URA3-d selection marker. The selection marker is replaced with the selection marker of the plasmid pTG10054 (Degryse et al., 1995) by homologous recombination in E. coll; to do this, the 2,1 Kb Mlul, Fspl fragment of pTG10054 containing the URA3 marker flanked by recombination sequences is recombined with the large Hindlll fragment of pTG10212, to give the plasmid pTG10610 which has the same characteristics as pTG10212 (Degryse et ai., 1995) with a URA3 marker in the same orientation as pTG10054, The Sail, Mlul fragment carrying the human cytochrome P450c21 cDNA of the plasmid pTG10472 (cf. above) is transferred into the plasmid pTG0610, to give the plasmid pTG10614. The Cial, Hindlll fragment of this plasmid containing, from 5' to 3' , the TDH3 promoter, the human P450c21 cDNA bordered by Sail and Miul sites, and then the PGKl terminator is transferred into the plasmid pTGl2036, to give the plasmid pTGl2086 which therefore contains the sequence of the GCYl gene interrupted by the TDH3 expression cassette for human cytochrome P4 5 0c21. f) Construction of the plasmid pTGl2048 The plasmid pTGl2048 was constructed from the plasmids pFL26CD, pTG10925 and pTG10953, pTG10953 is identical to the plasmid described in Lacour et al., .1998,- but the TEFl promoter which is carried by a Clal, Sail fragment is replaced with a CYCl promoter (Degryse et al., 1995), The expression cassette is carried by a Notl-Notl DNA fragment. The plasmid pTGl0925 was constructed from pFL26CD described by Duport et al, 1998. The plasmid contains a genomic fragment of yeast which comprises the LEU2 and NFSl genes in 5' to 3' and 3' and 5' orientation respectively. An expression cassette for ADR bordered by Notl sites was introduced into the intergenic region to give the plasmid pTG10925, This cassette {TEFl - mature bovine ADR - PGKl terminator) was replaced with a new cassette comprising the ARHl gene under the control of the CYCl promoter and the PGKl terminator, to give the plasmid pTG12048, On this plasmid the LEU2 gene and the expression cassette are in the same transcriptional orientation. Example 3. Construction of the TGY212#1 strain A colony is selected in the screen described in example 1, It is named TGY212#1. This colony is subjected to a bioconversion experiment as described previously with 100 fxg/ml of 170H-progesterone substrate, and the strain is allowed to grow in a minimum medium supplemented with required amino acids and uracil. This strain is capable of converting 170H-progesterone to 11-deoxycortisol with an efficiency of the A clone, TGY243#1, was selected in order to introduce in place of the URA3 gene an expression cassette for human P450c21. Example 5: Construction of the TGY245 strain This TGY2 4 3#1 strain is transformed with the plasmid YRp7 (1µg) described by Parent et al. , 1985 and with the plasmid pTG12086 linearized with the Xhol enzyme (5 µg) . The pTG12086 transforming fragment contains the coding sequence of GCYl interrupted by an expression cassette for human P450c21 {TDH3:: human P450c21 cDNA:PGKi terminator) . The colonies which grow in the absence of tryptophan are selected. These 381 colonies are then transferred onto a medium containing tryptophan and 5-fluoroorotate, About ten colonies are then tested in a rich medium of the YPG type supplemented with tryptophan, histidine, leucine and uracil at a concentration of 50 µg/ml. The strains are allowed to convert 170H-progesterone at a concentration of 100 µg/ml, starting with an OD 600 nm of 0.1, for 16 hours, Among these 10 clones, one clone, TGY24 5#2D, is chosen based on two criteria: its capacity to convert 170H-progesterone to 11-deoxycortisol and, secondly, the absence of formation of 4-pregnene-17a, 2 0ot-diol-3-one, indicating the disruption of GCYl. Example 6: Construction of the TGY260 strain The strain was constructed by transformation of TGY2 4 5 with the plasmid pTG12048 which allows overexpression of the ARHl gene at the LEU2 locus. The TGY260 strain was constructed from the TGY245 strain which was transformed with the linearized plasmid PTG12048. This plasmid pTG1204 8 is an intergenie integration plasmid carrying the LEU2 selection marker. On this plasmid/ an expression cassette for the ARHl gene (Lacour et al., op, cit.) is integrated in the region between the LEU2 gene and the NFSl gene. This cassette comprises the CYCl promoter followed by the ARHl gene and the PGKl terminator bordered by Notl restriction sites. This plasmid pTG12048 was linearized with the Xhol and Sail restriction enzymes. This fragment is used to transform the TGY245 strain using a conventional method of transformation with lithium chloride. The transformant colonies are then selected on a medium containing no leucine. A strain, TGY2 60, was selected. The TGY2 60 strain was deposited with the CNCM [National Collection of Culture in Microorganisms] on January 24, 2001, under the number 1-2615. Example 7; Construction of the CDR07matα strain reductase is functioning correctly. A CDR07 MATa spore is selected based on the above criteria. and PGKl terminator was introduced into the unique blunt-ended Pmel site - of this plasmid after filling the Notl ends with DNA polymerase Klenow fragment. One of the plasmids obtained, pCV29, contains three expression cassettes for ADX, P450scc (both in mature form) and also a form of human P4 5011(3 targeted to mitochondria. In addition, this plasmid carries, as a marker, the URA3 gene framed by two expression cassettes. b) Construction of the plasmid pCC12 The plasmid pCC12 (cf. Figure 4) is a replicative yeast plasmid based on the chromosomal origin of replication such as ARSl and a CEN centromere (there are 16 of them in yeast), both from S, cerevislae. This plasmid replicates in the form of one or two copies in yeast. It was constructed from pFL38C2, which is derived from pFL38 (Bonneaud et al,, 1991), the content of which is incorporated into the present application by way of reference, in particular the descriptions of the plasmids) * An oligonucleotide which contains the recognition sequence of the AJotI, Pad and Ascl sites (OLIP FL SEQ ID No, 24) was introduced into this plasmid at the Hindlll site. The orientation of the polylinker was verified by sequencing. This plasmid carries a URA3 selection marker bordered by Bglll sites. The URA3 gene was replaced with a 2.7 Kb ADE2 gene obtained by PCR amplification on the genome of FYl679-28c using the oligonucleotides 5'ADE2090 (SEQ ID No. 37, CGATTCGGATCCACTAGTAACGCCGTATCGTGATTAACG) and 3'ADE2 0 8 9 (SEQ ID No. 38, CCTCAAGGATCCTCCTGACGTAGCGCTATCCTCGGTTCTG). To do this, the plasmid pFL38C2 was digested with the Bglll enzyme, and the URA3 Bglll fragment was replaced with the 2.7 Kb fragment containing the ADE2 gene. The plasmid obtained, pAMl, serves as a basis for constructing the plasmid pCC12, Construction of the plasmid from the plasmid pAMl: The plasmid pCC12 contains two expression cassettes, one for 3p-HSDH of bovine origin, the other for adrenodoxin reductase, also of bovine origin. The bovine 3β-HSDH cDNA was recloned by PCR from a bovine adrenal gland cDNA library. The coding sequence with the cDNA published by Zhao et al., 1989, was not modified. The two oligonucleotides add a Sail site and 4 adenines in front of the ATG, to give the sequence GTCGACAAAAATG (SEQ ID No. 25) . The Mlul site is located right next to the termination codon of the bovine 3|3-HSDH cDNA, to give the sequence: end of the cDNA TGACCTGGAGTGACAATGACGCGT (SEQ ID No. 2 6). The sequence recognized by the Mlul enzyme is ACGCGT. The cDNA is transferred, in the form of a Sail, Mlul fragment, into the plasmid pTG10212, to give the plasmid pCC4. This plasmid therefore has a NotI fragment containing the 3p-HSDH cDNA bordered by both a TDH3 promoter and a PGKl terminator, positioned 5' and 3' respectively. This expression cassette is then transferred into the plasmid pTG12018, and the cassette is thus now bordered in the 5' position by a NotI site and in the 3' position by an Ascl site. This fragment is then cloned into the yeast expression plasmid pAMl in the Notl and Ascl sites, to give the plasmid pCCll. Into the AJotI site of this plasmid are inserted the WotI fragment carrying the TEFl promoter, the cDNA of the mature form of adrenodoxin reductase of bovine origin, and the PGKl yeast terminator, respectively in this order, originating from the plasmid pTG10361, This plasmid carries the same cDNA which is described in Dumas et al., 1996 (the content of which is incorporated into the present application by way of reference, in particular the descriptions of the plasmids), except that the addressing sequence of the cytochrome oxidase precursor has been replaced with a methionine codon. The cDNA therefore encodes a mature form of the adrenodoxin reductase cDNA. Example 10: Construction of the CDR07 strain The CDR07 strain was obtained by crossing between the FY167 9-18b MAT a strain and the CAIO MAT a strain described by Duport et al., 1998. These two strains were first of all isolated on a rich medium containing glycerol and then on a medium in a medium containing potassium acetate as described in "Yeast Protocols Methods in Cell and Molecular Biology Edited by Ivor H Evans in 1996, Humana Press Totowa New Jersey". After sporulation, the tetrads were digested with zymolyase 100T for 30 minutes at 37°C. Several cycles of selection were applied in order to obtain a clone which produces campesterol as the major sterol, having lost the other characteristics of CAIO. The spores are first of all selected on a minimum medium containing nystatin with supplements (adenine, leucine, tryptophan, uracil, histidine) . The clones resistant to nystatin and auxotrophic for adenine and leucine are then subcultured on rich medium containing adenine and hygromycin • (200µg/ml) in order to detect deletion of the ERGS gene by the hygromycin resistance gene. Clones auxotrophic for adenine and leucine (and uracil and tryptophan) and also hygromycin-resistant and nystatin-resistant are analyzed in terms of their sterol profile. Two clones of opposite mating-type signs, producing campesterol as the major sterol, are selected. They are named CDR07 MAT a and CDR07 MAT a. Example 11: Construction of the integration plasmids PTG12093 and pLIP5 respectively for the HIS3 and TRPl loci a) Construction of pLIP5 pLIP5 is a plasmid which can be used for genomic integrations at the TRPl locus or as a multicopy plasmid in the yeast S. cerevlsiae. The basic plasmid which was used to construct this plasmid is the plasmid pFL45S described in Bonneaud et al.f 1991 (the content of which is incorporated into the present application by way of reference, in particular the descriptions of the plasmids). A genomic DNA fragment corresponding to the 5' portion upstream of the TRPl promoter was first cloned using the oligonucleotides 0LIP21 and OL1P22 (respectively SEQ ID No, 30 and 31) to carry out a PCR amplification. The PCR product was first subcloned into pCR-Script AmpSK(+) (stratagene, La Jolla, California, USA), The 0LIP21 and OLIP22 ends in fact have a Narl site in the 5' position and a Hindlll site in the 3' position. This Narl, Hindlll fragment replaced the multiple cloning sites of the plasmid pFL45S described above. The plasmid obtained, pLIPB, is further modified using the oligonucleotide OLIP20 (SEQ ID No. 32), which is used to replace the unique Hindlll site with the unique Notl site. A fragment containing., from 5' to 3', the TEFl promoter, the cytochrome P-4 50cl7alpha cDNA and then the PGKl terminator,-as described in Degryse et ai,, 1999 (the content of which is incorporated into the present application by way of reference, in particular the descriptions of the plasmids), is introduced into this Notl site. The final plasmid pLIP5 can both be used as a multicopy plasmid based on the TRPl marker, when the 2-micron portion is removed, and it can be used as a plasmid for integration at the TRPl locus. The cassette is thus integrated 5' of the TRPl gene. b) Construction of the plasmid pTG12093 This plasmid was a plasmid for intergenic integration at the HI S3 locus. This plasmid is constructed from the plasmid pUC-HIS3 described by Duport et al,, 1998. The unique Xhol restriction site of this plasmid was transformed into a unique Notl restriction site using a suitable linker while at the same time destroying the Xhol site, to give the plasmid pUC19-HJS3, Into the unique WotI site of this plasmid, [lacuna] a Notl fragment originating from pTGl0792, This fragment contains the TDH3 promoter and the cDNA encoding mature bovine ADX fused to the addressing sequence of the precursor of the COX6 cytochrome (of the yeast cytochrome oxidase subunit 6 described in Dumas et al,, 1996. The 5ail, Mlul restriction fragment of plasmid pTG10350 containing the cDNA described previously was transferred into the plasmid PTG10211, so as to form the plasmid pTG10792- This plasmid therefore has a 1,6 kilobase fragment containing the TDH3 promoter, the cDNA fused between mature bovine ADX and the yeast COXe presequence, and the PGKl terminator, from 5' to 3' . This Notl-Notl fragment is inserted into the plasmid pTG12093. HIS3 and the expression cassette are transcribed in the same orientation, Example 12: Crossing of CDR07 MATa with TGY260 MATa The SB14 strain (CDR07MATa X TGy260 MATa) is allowed to sporulate in a very depleted medium containing potassium acetate. The various asci are then allowed to grow in a minimum medium containing the following products: uracil, histidine, tryptophan and adenine. The spores are then selected on a correctly supplemented minimum medium containing from 8 to 12 jxg/ml of nystatin. The positive spores are then selected based on the presence of the human P450-c21 cDNA. To do this, the clones are screened in Kappeli medium (M Arreguin de Lorencez, 0 J Kappeli, 1987) with 2% ethanol (and 0,1% glucose) as carbon source in the presence of 300 mg/ml of 170H~progesterone. The bioconversion is incubated for up to 72 hours. The capacity for bioconversion is analyzed by HPLC as described by Dumas et al., 1996 and Degryse et al-, 1999 (the contents of which are incorporated into the present application by -way .of reference, in particular the explanations of the bioconversion studies). These clones are also analyzed in terms of their sterol profile by gas chromatography as described by Duport et al., 1998 (the content of which is incorporated into the present application by way of reference, in particular the explanations of the bioconversion studies), for the purpose of detecting campesterol and ergosta-5,22-dienol. Three spores, YCC3, YCC4 and YCC5, are selected on the basis that they produce ergosta-5,22-dienol and campesterol and that they convert 170H-progesterone to 11-deoxycortisol with a production efficiency of 25, 120 and 42 ^tg/ml in 72 hours, respectively. YCC4 and YCC5 are then selected for two further successive transformations with the plasmids pLIPS and pTG12093 linearized, respectively, with the ApaLl and EcoRl enzymes. The plasmids pLIP5 and pTGl2093 are intergenic expression plasmids for bovine P450cl7 and mitochondrial bovine ADX respectively. pTG12093 is a plasmid for integration into the intergenic region in the position 3' of the HIS3 locus, whereas pLIPS is a plasmid for integration into the intergenic region in the position 5' of the TRPl locus. In addition, these two plasmids carry a unique NotI site which allows integration of an expression cassette containing, in this order; TEFl promoter, bovine P450cl7 cDNA, PGKl terminator for pLIP5, and in this order; TDH3 promoter, COXVIpre: :mat bovine ADX cDNA, PGKl terminator for pTG12093, The YCC4 strain is transformed successively with the plasmids pLIP5 and pTG12093 which are linearized (with the ApaLI and EcoRl restriction enzymes). In the first transformation, the transformant clones are first of all selected on a medium which does not contain tryptophan. The clones which grow in the absence of tryptophan are then selected by PCR with the oligonucleotides C17-3 and C17-5 (respectively SEQ ID No. 33 and SEQ ID No. 34). A clone, YCC8, is selected for a further transformation with the linearized plasmid pTG12093. In the same way, the clones which grow in the absence of histidine are selected and then the presence of the ADX cDNA is verified by PCR using the oligonucleotides ADX-3 and ADX-5 (respectively SEQ ID No. 35 and SEQ ID No. 36). A clone, UCY2, is selected; its mating-type sign is MATa. Example 13: Construction of the UCY4 strain The UCY2 strain has a functional ATF2 gene, that is to say most of the pregnenolone produced is transformed to pregnenolone acetate by the ATF2p protein which is a pregnenolone acetyl transferase, the natural function of which in yeast is unknown (Cauet et al., 1999). In addition, this reaction is irreversible and therefore, once it has been produced, the acetyl pregnenolone can no longer be transformed into hydrocortisone, It therefore appears to be imiportant to destroy this activity so as to allow more efficient production of hydrocortisone. The ATF2 gene was therefore disrupted using a G418 resistance gene. To do this, a disruption plasmid, pAM3kanaC, was constructed, allowing introduction of a G418 resistance marker into the ATF2 gene. This plasmid was constructed from the plasmid pAMl (the construction of which was described above), and from the plasmid pTG12002, which is an expression plasmid for the ATF2 gene. pTG12002 is an expression plasmid for ATF2 based on the plasmid pTGl0260 (Degryse et al., 1995) in which the Xjbal restriction site of the 2-micron origin of replication has been inactivated- This plasmid therefore comprises an expression cassette for the ATF2 gene (Cauet et ai., 1999) comprising, the CYCl promoter, the ATF2 gene (framed by the Sail and Mlul restriction sites) and the PGKl terminator. This cassette was modified by PCR so as to contain the complete ATF2 gene comprising the promoter of the ATF2 gene, the coding sequence of the ATF2 gene, and the terminator of the ATF2 gene, on a Kpnl, Notl restriction fragment. This Kpnl-Notl fragment is introduced at the Kpnl-Notl sites of the plasmid pAMl, to give the plasmid-pAM3, In the plasmid pAM3, the expression cassette for G418 resistance is introduced into the ATF2 gene, causing the inactivation thereof. To do this, the plasmid pAM3 is digested with the Accl restriction enzyme, and then partially with the Sad restriction enzyme. A band of approximately 7 500 bp is purified on a gel. The plasmid pFA6a kanMX4 (Wach et al,. Methods in Microbiology Volume 2 6 Yeast Gene Analysis Chapter 5 PCR-based Gene Targeting in Saccharomyces cerevisiae) is digested with Sad and Accl, and the band at 1500 bp is purified on a gel. The two fragments are ligated and then transformed, A plasmid is obtained, pAM3kanaC, pAM3kanaC is digested with Pvull and AJotI, and the band at 2215 bp is purified on a gel and then transformed into UCy2, which is plated out on dishes of rich medium containing 130 µg/ml of G418. Approximately 600 clones are subcultured on this medium containing G418, and two clones are resistant to G418, A clone which does not contain the plasmid pAM3 is conserved. This method of gene activation is well known to those skilled in the art. The single clone thus obtained is allowed to carry out bioconversion with 100 µg/ml of pregnenolone in Kappeli medium. After 24 hours, the absence of pregnenolone acetate is verified by extraction and gas chromatography as described in Cauet et al., 1999. This phenomenon indicates that the ATF2 gene responsible for acetylation of pregnenolone has clearly been disrupted and is no longer functional. The strain is named UCY4. Example 14; Construction of the UCY3 and UCY26 strains Screening of the spores obtained by crossing the CDR07 and TGY260 strains gave several strains, including the YCC4 and YCC5 strains. As described above, the YCC4 strain was transformed with a series of 2 plasmids: pLIP5 and pTG12 0 93. A spore, UCY2, was then selected (cf. example 12) . In the same way, the YCC5 strain was also transformed with the plasmids pLIP5 and pTG12093 and an additional spore, named UCY3, was obtained. Like UCY2, UCY3 is characterized by the presence and expression of A. thaliana A7 reductase measured by nystatin resistance and the presence of brassicasterol and campesterol as major sterol of these recombinant yeasts, The presence of the ADX cDNA is verified by PCR using the oligonucleotides ADX-3 and ADX-S (respectively SEQ ID No. 35 and SEQ ID No. 36), and then by Western blot as described in Dumas et al. f 1996, incorporated herein by way of reference as regards the description of this technique. The activity of progesterone hydroxylation at position 17 and at position 21 is verified by bioconversion of progesterone to 170H-progesterone and 11-deoxycortisol. To do this, the strain is incubated in the presence of 200 mg/1 of progesterone as described in Dumas et al., 1996 and Degryse et al., 1999. UCY3 is capable of producing 11-deoxycortisol from progesterone, indicating the presence of the P450cl7 and P450c21 activities. The presence of 4-pregnene-17a,20a-diol-3-one is also detected, indicating that at least one of the two activities encoded by GCYl or YPRl is present in the strain. To avoid accumulation of pregnenolone acetate, the pregnenolone acetylation activity encoded by the ATF2 gene was eliminated. With this aim, the plasmid pAMSkanaC (cf, example 13) was used to transform the UCY3 strain. pAM3kanaC is first of all digested with PvuII and AfotI, the band at 2215 bp is purified on a gel and is then transformed into UCY3, which is plated on dishes of rich medium containing 130 |Ag/ml of G418. Colonies resistant to the G418 antibiotic, and which are no longer capable of transforming pregnenolone into pregnenolone acetate, are isolated; a colony, UCY26, is more particularly selected for further transformations and to test its production of hydrocortisone. Example 15: Construction of the UCY5 strain With the aim of .having better genetic variability, the UYC2 strain (cf. example 12) was crossed with the TGY245 strain (cf. example 5) , and a diploid strain, YSA2-2n/ was thus selected. This strain was placed under conditions for the production of asci and 85 asci were dissected (as described in 'Yeast Protocols in Molecular Biology Volume 53 p59-67, 1996"). The isolated spores are screened on the basis of their auxotrophic property. Thus, the clones capable of growing in the absence of tryptophan and histidine and requiring adenine are more particularly selected* Expression of A7 reductase is verified by making sure that the strain is resistant to nystatin and that campesterol and brassicasterol are present in the membranes of these strains. The latter analysis is carried out by gas chromatography of the total sterols of these strains as described in Duport et al., 1998. In addition, the presence of the cDNAs encoding P4 50cl7 and ADX is verified by PCR using the oligonucleotides C17-3 and C17-5 (respectively SEQ ID No, 33 and SEQ ID No, 34) and the oligonucleotides ADX-3 and ADX-5 (respectively SEQ ID No. 35 and SEQ ID No. 36), respectively. Finally, the functionality of the GCYl and YPRl genes in these positive spores is verified in two ways: either by PCR or by the absence of 2 0-alpha-reductase activity on 170H-progesterone. Disruption of the YPRl gene by the expression cassette for human P450c21 in 5.cerevisiae, containing the TEFl promoter, the human P450c21 cDNA and the PGK terminator, is detected by PCR using.the pair of oligonucleotides XlTEFl and X2C21 (respectively SEQ ID No. 47 and SEQ ID No. 48), Disruption of the GCYl gene by the expression cassette for human P450c21 in S,cerevisiae, containing the TDH3 promoter, the human P450c21 cDNA and the PGK terminator, is also detected by PCR using the pair of oligonucleotides X3TDH3 and X2C21 (respectively SEQ ID No. 49 and SEQ ID No, 48). The disappearance of the two GCYl and YPRl activities and the presence of the activity corresponding to the P450cl7 and P450c21 activity are finally verified by bioconversion of progesterone to 11-deoxycortisoodifiel under the conditions described by Dumas et al., 1996, md by Degryse et ai., 1999. The recombinant yeasts are incubated at 30°C in the presence of 200 mg/1 of progesterone with a cell density of 5 in a culture medium of the Kappeli type containing 2% of galactose or 2% of glucose as carbon source, in a volume of 10 ml. After incubation for 48 hours, the culture medium of these yeasts is extracted as described previously and the amount of 170H"progesterone and 11-deoxycortisol produced in particular are measured by HPLC. The spore selected no longer produces a detectable amount of 4-pregnene-17a,20a-diol-3-one, but produces 170H-progesterone and ll^deoxycortisol using the two carbon sources. The clone chosen produces the largest amount of 11-deoxycortisol. A strain which satisfies positively all these criteria is more particularly selected for further transformations; this strain is named UCY5. Example 16: Construction of the plasmids pFMlO and pTG10897 a) Construction of the plasmid pFMlO The plasmid pFMlO is a vector which allows the simultaneous expression of 4 proteins in yeast. It contains no origin of replication for E. coll nor an ampicillin resistance gene; this plasmid cannot replicate in E.coli, It is obtained by recombination in yeast of two plasmids: pFM7 and pCBl2, These plasmids, unlike the plasmid pFMlO, both replicate in E.coli since they possess the E.coli replicon. The plasmid pFM7 also replicates in S.cerevislae, since it also comprises the 2-micron origin of replication of the yeast S. cerevisiae. These two plasmids also have the Rl and R2 sequences (respectively SEQ ID No. 39 and SEQ ID No. 40) bordering the expression cassettes and also the selection markers. The Rl and R2 sequences come from the A. thaliana photochlorophyllide oxidoreductase gene and are each approximately 300 base pairs in size. The two sequences Rl and R2 are cloned in reverse orientation into the plasmids pFM7 and pCB12. Thus, between the Rl and R2 sequences, the pFM7 plasmid contains, in the following order: the 2-micron origin of replication contained in the 2-micron fragment (fragment bordered by the EcoRI restriction sites, as described by Urban et al., 1994), a fragment bordered by Not I sites originating from the plasmid pCD63 described in Duport et al., 1998 containing two expression cassettes for the mature form of P450scc ^nd the mature form of ADX separated by the functional yeast URA3 gene, and then the R2 sequence. Similarly, the plasmid pCB12 contains the Rl and R2 sequences, but they are cloned in the direction opposite to that of the plasmid pFM7. Thus, between the R2 and Rl sequences, there is the expression cassette for R450iip, the ADE2 selection marker and the expression cassette for Bp-HSD of bovine origin. The expression cassette for P45O113 comes from the plasmid pCV29 and contains the CYCl promoter, a hybrid cDNA encoding P450iip and a PGK terminator. The ADE2 gene comes from the plasmid pAMl; it is the Bglll-Bglll fragment, Similarly, the expression cassette for bovine 3p"HSD, i • e. the TDH3 promoter, the bovine 3(i-HSD cDNA and the PGK terminator, comes from the plasmid pCC12; it is the Cldl-AscI fragment. b) Construction of the plasmid pTG10897 The coding sequence of the ATF2 gene is amplified by PCR using two 20mer oligonucleotides corresponding respectively to the 5' and 3' portion of the coding sequence of the ATF2 gene (and also comprising the cloning sites) and using as matrix genomic DNA from the FY 1679-28c strain. This PCR product is then digested with the Clal and Hindlll enzymes and is cloned between the Clal and HindiII sites of the plasmid pTGlOQSl [Degryse, 1995 #102], to give the plasmid pTG10885. This plasmid is used as a basis for the construction of a plasmid for disrupting the ATF2 gene. The sequence of the URA3 gene was amplified from genomic DNA of the TGY156 strain (described in Cauet et al., 1999 and incorporated herein by way of reference); this strain in fact has an ATF2 gene interrupted by the URA3 gene. The oligonucleotides OTG10842 (SEQ ID No. 41) and OTG10841 (SEQ ID No, 42) were therefore used for the amplification on a matrix of genomic DNA from the TGY15 6 strain^ and the amplification product was used as an initiator of recombination with the plasmid pTG10885 digested with the BstXI and StuI restriction enzymes. The plasmid pTG10897 containing the coding sequence of the ATF2 gene interrupted by the URA3 gene is thus obtained. Specifically, the functional URA3 gene is located between 509 nucleotides of the 5' portion of the coding sequence of ATF2 and 444 nucelotides of the 3' portion of this coding sequence. Example 17 : Construction of the UCY6, UCY16, UCY16-pFM10, UCY19, UCY20, UCY24; UCY25 and UCY27 strains A series of new strains was constructed, based on the UCY5 strain, with the aim of improving the production of steroid of interest. Specifically, the UCY5 strain does not express adrenodoxin reductase, which is an essential component of the reaction for side chain cleavage by P450scc. Furthermore, as was described previously, the AT:F2 gene encodes an acetyl transferase which uses pregnenolone as substrate, and disruption thereof makes it possible to eliminate this parasitic acetylation reaction and to thus increase the yield of steroid of interest. Finally, the exogenous biosynthetic pathway uses the endogenous ARHlp activity, the latter being essential to yeast survival. However, this mitochondrial activity, which replaces mammalian adrenodoxin reductase, might be limiting in the strains producing hydrocortisone. A strain having certain modifications making it possible to considerably increase the yield of production of steroid of interest was therefore constructed based on the UCY5 strain. Thus, this strain lacks ATF2 activity, overproduces the adrenodoxin reductase protein and also overproduces the ARHl protein. In order to allow ADR to be expressed in the UCY5 strain, the latter was transformed with the vector pTG10925 (cf. example 2 f), which, when it is transformed in yeast in linear form, allows integration at the LEU2 locus and expression of ADR under the control of the TEFl promoter. The expression of ADR was verified by Western blotting as described in Dumas et ai., 1996. A clone expressing ADR, and named UCY6, was more particularly used for the subsequent transformations. With the aim of eliminating the parasitic acetylation reaction transforming pregnenolone into pregnenolone acetate catalyzed by the ATF2 enzyme, the ATF2 gene encoding this enzyme was disrupted- Specifically, this gene was interrupted by the URA3 marker; to do this, the NotI fragment of the plasmid pTG10897, which contains the sequence of the ATF2 gene interrupted by the functional yeast VRA3 gene, was used. This fragment is used to transform the UCY6 strain. The colonies capable of growing in the absence of uracil are selected, and then the capacity of these clones to transform pregnenolone into pregnenolone acetate is measured as described by Cauet et al., 1999. A clone capable of growing in the absence of uracil and incapable of transforming pregnenolone into pregnenolone acetate is more particularly isolated. This clone is called UCyi6. Since this strain cannot be transformed with plasmids carrying only the URA3 marker, two new plasmids were constructed: the plasmids pCB12 and pFM7. These two plasmids, when they are recombined together, make it possible to obtain the plasmid pFMlO based on the VRA3 and/or ADE2 markers (cf. above and figure 5). Thus, to obtain the plasmid pFMlO, the plasmid pFM7 is linearized with the Aatll restriction enzyme and the corresponding DNA fragment is isolated on a gel. The plasmid pCB12 is, for its part, digested with BamHl and the 9300 base pair band is isolated according to conventional molecular biology techniques. Approximately 5µg of the pCB12 and pFM7 fragments are mixed and are used to transform the UCY16 strain. Some UCY16-pFM10 clones, capable of growing in minimum medium (without uracil and amino acids), are isolated and the corresponding strains are tested for their level of steroid production according to the protocol described below. Alternatively, and with the aim of being able to subsequently use the plasmids of the pCV29 type based on a URA3 marker, the disruption of the ATF2 gene in the UCY6 strain was also obtained by transforming this strain with the linearized plasmid pAM3kanaC (cf. example 13) . The G418-resistant colonies are then tested for the absence or presence of pregnenolone acetyl transferase activity as described by Cauet et al,, 1999, A G418-resistant colony no longer having pregnenolone acetyl transferase activity is more particularly selected. This strain is called UCY24. With the aim of increasing the ARHl activity in a strain containing an exogenous pathway for production of hydrocortisone, the UCY5 strain was transformed with the plasmids pTG12048 or pTG12050 linearized beforehand with Xhol and Sap I respectively. The plasmid pTG12 04 8 which was described above allows overexpression of the ARHl gene at the LEU2 locus under the control of the CYCl promoter. The plasmid pTG12050 differs from the plasmid pTG12048 only in that the CYCl promoter controlling expression of the ARHl gene has been replaced with the TEFl promoter as described in Degryse et al., 1995 and Dumas et ai,, 1996. UCY5 was transformed with linearized pTG12048 or pTG12050. In each of the two cases, the colonies capable of growing in the absence of leucine were selected. In addition, the presence of the expression cassettes for the ARHl gene was verified by PCR. Specifically, two pairs of oligonucleotides make it possible to verify the presence of an additional copy of the ARHl gene (under the control of the CYCl or TEFl promoter). First, the pair arhlD (SEQ ID No. 43) and nfslR (SEQ ID No. 44) makes it possible to verify the presence of the junction between the ARHl gene and the NFSl gene. Secondly, the pair leu2D (SEQ ID No. 45) and arhlR (SEQ ID No. 46) makes it possible to verify the junction between the LEU2 gene and the ARHl gene. Besides the presence of the expression cassettes for the ARHl gene encoding the ARHl activity, the expression of the ARHlp protein was also tested in the clones obtained. It is not, however, easy to detect overexpression of ARHlp. This is due to the natural presence of the ARHlp protein at a low level in S. cerevisiae strains. Western blotting experiments carried out as described in Dumas et al., 1996 make it possible, however, to confirm the increase in the amount of ARHlp. In addition to the Western blotting experiments which prove to be tricky, the presence of an increased amount of ARHlp can be verified with experiments consisting of cytochrome c reduction or of llbeta-hydroxylation of 11-dioxyCortisol, which experiments are reconstituted in vitro with purified mitochondria from recombinant yeasts as UCY20 = UCY5, LEU2::TEFl;:ARHl. UCY25 = UCyi9, atf2-A::GA18R (resistant to nystatin and to G418). UCY27 = UCY20, atf2-A: -.GAIQR (resistant to nystatin and to G418). UCY24 = UCY6, atf2-A::GA18R (resistant to nystatin and to G418). Bibliography: Andersson S, et al,, Cloning, structure, and expression of the mitochondrial cytochrome P-4 50 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J, Biol, Chem, 1989- 264 (14): p. 8222-8229. Arreguin de Lorencez M and Kappeli 0 J, Regulation of gluconeogenic enzymes during the cell cycle of Saccharomyces cerevislae growing in a chemostat. Gen Microbiol, 1987, 133 (Pt 9): p, 2517-22, Bonneaud N. et al., A family of Iow and high copy replicative, integrative and single-stranded S. cerevislae/E. coli shuttle vectors. Yeast, 1991 Aug-Sep; 7(6): p. 609-15 Burgers P.M. and Percival K.J., Transformation of yeast spheroplasts without cell fusion. Anal Biochem, 1987, 163(2): p. 391-7. Cauet G. et al,^ Pregnenolone esterification in Saccharomyces cerevisiae, A potential detoxification mechanism- Eur J Biochem, 1999. 261(1): p. 317-24. Chua SC, et al,. Cloning of cDNA encoding steroid 11 beta-hydroxylase (P450cll) . Proc Natl Acad Sci USA, 1987. Oct; Valvo, L-, et al., General high--performance liquid chromatographic procedures for the rapid screening of natural and synthetic corticosteroids, J Pharm Biomed Anal, 1994., 12(6): p, 805-10. Wu^ DA, et ai., Expression and functional study of wild-type and mutant human cytochrome P4 50c21 in Saccharomyces cerevisiae, DNA Cell Biol, 1991. 10(3): p. 201-9, Yanisch-Perron et al., Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene, 1985, 33(1): p. 103-19. Zhao HF. et al. , Molecular cloning, cDNA structure and predicted amino acid sequence of bovine 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase. FEBS Lett, 1989. 259(1): p. 153-7. Claims 1, A genetically modified yeast strain autonomously producing, from a simple carbon source, a steroid or steroid derivative, derived from cholesterol metabolism, characterized in that said steroid or steroid derivative is 6. The yeast strain as claimed in one of claims 1 to 5, characterized in that it has at least one expression block for a heterologous gene integrated into the chromosome, at least one locus chosen from ADE2, HIS3, TRPlf LEU2, GCYl, ATF2 and YPRl, the integration being carried out intragenically or intergenically in the immediate vicinity of said locus. 7. The yeast strain as claimed in one of claims 1 to 6, characterized in that it has at least one expression block for a heterologous gene located on a multicopy plasmid or a low copy plasmid. 8. The yeast strain as claimed in claim 1, characterized in that the multicopy plasmid is chosen from yeast 2-micron rep1icon-based plasmids which replicate in Saccharomyces cerevislae and in that the low copy plasmid is chosen from plasmids based on a chromosomal ARS origin of replication with a yeast centromere. 9. The yeast strain as claimed in one of claims 1 to 10, characterized in that it has at least one heterologous gene or cDNA in a expression block, said gene or cDNA being chosen from the group consisting of the gene of sterol A7-reductase;- of cytochrome P450 SCC, of adrenodoxin, of adrenodoxin reductase, of 3P-hydrosteroid dehydrogenase isomerase, of cytochrome b5, of cytochrome P4 50 reductase, of cytochrome P450 C17, of cytochrome P450 C21 and of cytochrome P450 Cll, and of the sequences encoding these proteins, 10. The yeast strain as claimed in claim 9, characterized in that at least one heterologous gene or cDNA is under the control of a promoter sequence chosen from the group consisting of the yeast endogenous promoter sequences TDH3r TEFlf PGKl, CYCl, GALIO, ATF2, TIRl, ARHl and ADE2, and the hybrid promoter GALlO-CYCl. 11. The yeast strain as claimed in either of claims 9 and 10, characterized in that the terminator sequence of at least one heterologous gene or cDNA in the expression block is chosen from the terminator sequences of the endogenous genes PGKl, CYCl, ATF2, ADE2 and NCPl, 12. The yeast strain as claimed in one of claims 1 to 11, characterized in that it has the sterol A7-reductase heterologous expression block integrated into the chromosome at the ADE2 locus, 13. The yeast strain as claimed in one of claims 1 to 12, characterized in that it comprises at least one cassette for expression of the genes encoding the mature forms of P450scc snd of adrenodoxin, located on a high copy plasmid. 14. The yeast strain as claimed in one of claims 1 to 13, characterized in that it comprises a cassette for expression of adrenodoxin reductase for P450scc/ located on a single copy plasmid or a low copy plasmid or integrated into one or more yeast chromosomes. 15. The yeast strain as claimed in claim 14, characterized in that said cassette for expression of adrenodoxin reductase has the elements which ensure that the protein is present in the cytosol of the host cell. 16. The yeast strain as claimed in one of claims 1 to 15/ characterized in that it comprises at least one expression cassette chosen from the cassettes for expression of 3P-hydrosteroid dehydrogenase isomerase, of cytochrome P450cl7 and of cytochrome p250c21, located on a high copy plasmid- 17. The yeast strain as claimed in one of claims 1 to 16, characterized in that it comprises at least one expression cassette for p4 5011(3^ located on a multicopy plasmid, the protein produced having a signal for addressing to mitochondria. 18- The yeast strain as claimed in one of claims 1 to 17, characterized in that it comprises at least one expression cassette for a precursor of adrendoxin for P45011p, located on a multicopy plasmid, with a weak promoter, the protein produced having a signal for addressing to mitochondria, 19. The yeast strain as claimed in one of claims 1 to 18, characterized in that it has at least two expression cassettes for adrenodoxin, such that one protein is active outside the mitochondria, the other being active in the mitochondria of the host cell. 20. The yeast strain as claimed in one of claims 1 to 19, characterized . in that it comprises at least one expression cassette for NCPl, ATRl and/or ATR2, located on a single copy plasmid or integrated into the chromosome, 21. The yeast strain as claimed in one of claims 1 to 20, characterized in that it expresses the ARHl protein at a level higher than a physiological level. 22. The yeast strain as claimed in claim 21, characterized in that it has, in addition to the endogenous gene, a second expression cassette for the ARHl protein, 23. The yeast strain as claimed in claim 22, characterized in that the expression of the ARHl protein is placed under the control of the CYCl promoter in said cassette. 24. The yeast strain as claimed in one of claims 1 to 23, characterized in that it is polyploid, diploid, haploid or aneuploid, 25. The yeast strain as claimed in one of claims 1 to 24, characterised in that it is a strain of Saccharomyces cerevisiae. 26. The yeast strain as claimed in claim 24, characterized in that it is derived from the FY 1679-28C strain or the FY 1679-18b strain. 27. A yeast strain, characterized in that it is the CDR07 Mat-a or TGY2 60 strain, deposited with the CNCM on January 24, 2001, under the respective accession numbers 1-2 616 and 1-2615, or a strain obtained after crossing of CDR07 Mat-α and TGY2 60., and optionally sporulation and transformation with a plasmid from yeast, in particular the UCY2 or UCY4 strain. 28. The yeast strain as claimed in one of claims 1 to 27, characterized in that it has the elements required for excreting the steroid produced in the culture medium. 29 . A method for producing a steroid, characterized in that it comprises the steps of fermenting a yeast strain as claimed in one of claims 1 to 28 in the presence of a simple carbon source, and of recovering the steroid produced. 30. A pharmaceutical preparation comprising a yeast strain as claimed in one of claims 1 to 28, 31. A genetically modified yeast strain substantially as herein described with reference to the accompanying drawings. 32. A pharmaceutical preparation substantially as herein described with reference to the accompanying drawings. |
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| Patent Number | 212206 | ||||||||
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| Indian Patent Application Number | 1151/CHENP/2003 | ||||||||
| PG Journal Number | 02/2008 | ||||||||
| Publication Date | 11-Jan-2008 | ||||||||
| Grant Date | 26-Nov-2007 | ||||||||
| Date of Filing | 24-Jul-2003 | ||||||||
| Name of Patentee | M/S. AVENTIS PHARMA S.A | ||||||||
| Applicant Address | 20 avenue Raymond Aron, F-92160 ANTONY | ||||||||
Inventors:
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| PCT International Classification Number | C12P 33/00 | ||||||||
| PCT International Application Number | PCT/FR2002/000348 | ||||||||
| PCT International Filing date | 2002-01-29 | ||||||||
PCT Conventions:
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