SK4382000A3 - METHOD FOR PRODUCING ERGOSTEROL AND INTERMEDIATE PRODUCTS THEREOFì (54) BY MEANS OF RECOMBINANT YEASTS - Google Patents

Abstract

The invention relates to a method for producing ergosterol and intermediate products thereof by means of recombinant yeasts and plasmids for transforming yeasts.

Description

The present invention relates to a process for producing ergosterol and its intermediates using recombinant yeast and yeast transformation plasmids.

BACKGROUND OF THE INVENTION

Ergosterol is the end product of the synthesis of sterols in yeast and fungi. The economic importance of this substance lies both in obtaining vitamin D ₂ from ergosterol by UV-radiation and secondly in obtaining steroid hormones by biotransformation, starting from ergosterol. Squalene is used as a basic synthetic material for the synthesis of terpenes. Its hydrogenated form of squalane is used in dermatology and cosmetics along with various derivatives as part of skin and hair care products. The intermediates of ergosterol metabolism are also of economic importance. The most important of these intermediates are farnesol, geraniol and squalene. Other sterols, such as e.g. zymosterol and lanosterol, with lanosterol being a key natural and synthetic substance used for the chemical synthesis of saponins and steroid hormones. Lanosterol penetrates well into the skin and spreads well and is therefore used as an emulsifying agent and as an active ingredient in skin creams.

The genes controlling the metabolism of ergosterol in yeast are well known and cloned, such as e.g.

HMG-CoA reductase (HMG1) (Basson et al., 1988), squalene synthetase (ERG9) (Feguer et al., 1991),

Acyl-CoA: sterol acyltransferase (SAT1) (Yu et al., 1996) and squalene epoxidase (ERG1) (Jandrositz et al., 1991).

Squalene synthetase catalyzes the reaction of farnesyl pyrophosphate via presqualene pyrophosphate to squalene. The reaction mechanisms of sterolacyltransferase have not been fully investigated. High gene expression of these

The 31430 / H enzymes were already verified, but did not lead to any significant increase in the amount of ergosterol recovered. In the case of HMG1-high expression, an increase in squalene production has been described, in addition to this, mutations have been introduced to interrupt the further reaction upon obtaining squalene (EP-0 486 290). The increased production of geraniol and farnesol has also been described, but there has been no high expression of ergosterol metabolism genes but interruption of the reaction sequence in the direction of geraniol and farnesol formation (EP-0 313 465).

Specific inhibitors of ergosterol biosynthesis may also lead to the accumulation of larger amounts of certain intermediates, such as allylamines, that prevent the conversion of squalene to squalene epoxide. Thus, a substantial increase in the amount of squalene (up to 600 times the normal amount) can be achieved (Jandrositz et al., 1991).

Although the use of inhibitors makes it possible to achieve a substantial increase in the amount of e.g. squalene, however, the addition of these substances is not advantageous because the same effects are already present in the body in small amounts, and therefore the method of producing ergosterol biosynthesis products by the method of increased production is more advantageous.

It is an object of the present invention to provide a microbiological process for the production of ergosterol and its intermediates, the microorganisms necessary for this, such as yeast strains, which synthesize increased amounts of ergosterol and / or ergosterol. intermediate and preparation of plasmids necessary for transformation of yeast strains.

SUMMARY OF THE INVENTION

It has now been found that it is possible to increase the amount of ergosterol and its intermediates by inserting the HMG1 genes (Basson et al., 1988), ERG9 (Fegueur et al., 1991, Current Genetics 20: 365-372), SAT1 (Yu and et al., 1996) and ERG1 (Jandrositz et al., 1991) in converted form into microorganisms such as e.g. yeast, wherein these genes are inserted individually into a single plasmid or in combination into one or more plasmids and inserted into the host cells simultaneously or sequentially.

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It is an object of the present invention to provide a process comprising:

(a) first, a plasmid is formed into which several genes suitable for the metabolism of ergosterol are inserted in a converted form; or

b) first, a plasmid is formed into which one of the genes suitable for the metabolism of ergosterol is inserted in a transformed form,

c) transforming the microorganisms with the plasmid thus produced, wherein the microorganisms are transformed with one or more of the plasmids according to (a) simultaneously or sequentially.

d) fermentation to ergosterol is carried out with the microorganisms thus prepared,

(e) after fermentation, ergosterol and its intermediates are extracted and analyzed from the cells and finally

(f) the ergosterol thus obtained and its intermediates are purified and isolated by column chromatography.

In particular, it is an object of the present invention to provide a plasmid into which the following genes are inserted:

(i) the HMG-CoA reductase (t-HMG) gene; (ii) the squalene synthetase (ERG9) gene; (iii) the acyl-CoA: sterol acyltransferase (SAT1) gene; and (iv) the squalene epoxidase (ERG1) gene; first creates a plasmid into which the following genes are inserted:

(i) the HMG-CoA reductase (t-HMG) gene; and (ii) the squalene synthetase (ERG9) gene; or, (a-iii) a plasmid is first formed into which the following genes are inserted:

(i) HMG-CoA reductase (t-HMG) gene

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(iii) the acyl-CoA: sterol-acyltransferase (SAT1) gene; or (a-iv), a plasmid is first generated into which the following genes are inserted:

(i) HMG-CoA reductase (t-HMG) gene; and

(iv) the squalene epoxidase (EKG1) gene, or a-v), is first produced a plasmid into which the following genes are inserted:

the squalene synthetase (ERG9) gene; and

(iii) the acyl-CoA: sterol-acyltransferase (SAT1) or a-vi) gene is first generated into a plasmid into which the following genes are inserted:

the squalene synthetase (ERG9) gene; and

(iv) the squalene epoxidase (ERG1) gene, or a-vii), first generates a plasmid into which the following genes are inserted:

(iii) the acyl-CoA gene: sterol acyltransferase (SAT1); and

(iv) the squalene epoxidase (ERG1) gene; or

(b) a plasmid is constructed first into which one of the genes listed under a-i is inserted, and

(c) transforming microorganisms with the plasmids thus prepared, wherein said microorganisms are transformed with one or more of the plasmids mentioned under (a) (i) to (a) (v) simultaneously or sequentially;

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(d) fermentation to ergosterol is carried out with the micro-organisms thus obtained,

(e) after fermentation, ergosterol and its intermediates are extracted from the cells and analyzed and finally

(f) the ergosterol thus obtained and its intermediates are purified and isolated by column chromatography.

The squalene epoxidase (ERG1) gene can be additionally inserted into the plasmids listed in a-ii), a-iii) and a-v) and the acyl-CoA: sterol-acyltransferase (SAT1) gene can be additionally inserted into the plasmid mentioned in a-ii. These plasmids are also an object of the present invention.

Intermediates include squalene, farnesol, geraniol, lanosterol, zymosterol, 4,4-dimethylzymosterol, ergost-7-enol and ergosta-5,7-dienol, especially sterols with a 5,7-diene structure.

The plasmids used are preferably the plasmid YepH2, which contains the mittler ADH promoter, the t-HMG (variable variant HMG1) and the TRPterminator (see Fig. 1), the plasmid YDpUHK3, which contains the intermediate ADH promoter, the t-HMG (variable variant HMG1). and the TRP-terminator, the kanamycin resistance gene and the ura3 gene (see FIG. 2) and the plasmid pADL-SAT1, which contains the SAT1 gene and the LEU2 gene from Yep13.

These plasmids and their use for the production of ergosterol and its intermediates, such as squalene, farnesol, geraniol, lanosterol, zymosterol, 4,4-dimethylzymosterol, 4-methylzymosterol, ergost-7-enol and ergosta-4,7-dienol, in particular sterols with 5 The 7-diene structure is also an object of the present invention.

The host for introduction of the plasmids of the present invention can be essentially any microorganism, especially yeast.

Preferably, a S. cerevisiae species, in particular a strain, may be used

S.cerevisiae AH22.

The present invention also relates to the yeast strain S. cerevisiae AH22, which contains the plasmid pADL-SAT 1.

Also preferred is the combined transformation of microorganisms with the plasmids pADL-SAT1 and YDpUHK3, especially the transformation of yeasts such as e.g.

S.cerevisiae AH22.

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The sequence of metabolism of ergosterol is generally affected as follows:

The reaction sequence in the ergosterol direction is maximized, while the activity of several flaschenhals enzymes increases at the same time. Various enzymes play a decisive role in this, the decisive influence on the increase in ergosterol yield being due to the combination of deregulation and resp. high expression. Enzymes resp. their HMG1 genes (Basson et al., 1988), ERG9 (Fegueur et al., 1991), acyl-CoA: sterol acyltransferase (SAT1) (Yu et al., 1996) and / or squalene epoxidase (ERG1) (Jandrositz and et al., 1991) inserted in altered form into a yeast strain, wherein the insertion of the genes is carried out by means of one or more plasmids and the plasmid (s) contain the DNA sequence either singly or in combination. "Altered" in the case of the HMG1 gene means that only its catalytic region without the membrane-bound domain is expressed from the gene of interest. This transformation has already been described (EP-0486 290). The goal of HMG1 conversion is to prevent feed-back regulation caused by ergosterol biosynthesis intermediates. This means that both HMG1 and the other two genes will not have transcriptional regulation. In addition, the gene promoter is replaced by the "mittler" ADH1 promoter. This ADH1-promoter fragment is characterized by approximately constitutive expression (Ruohonen et al., 1995), so that transcriptional regulation is no longer via ergosterol biosynthesis intermediates.

High-expression products can be used for biotransformation or biotransformation. other chemical and therapeutic purposes, e.g. for the recovery of vitamin D ₂ from ergosterol by UV radiation and for the recovery of steroid hormones from ergosterol using biotransformations.

Microorganisms, particularly yeast strains, which, by using high expression in the process of the genes mentioned under a-i), allow the production of increased amounts of ergoserol and ergosterol in combination with increased amounts of squalene, are also objects of the present invention.

Preferred is the variable variant of the HMG1 gene in which it was

31430 / H expressed only the catalytic region without the membrane bound domain. This conversion has been described (EP-0486 290).

The present invention also relates to a process for the production of ergosterol and an intermediate resulting from this reaction, characterized in that the genes mentioned under process (a), in particular under process (ai) to (a-vii) (double, triple, quadruple combination of genes) are together with the plasmids, are first introduced independently of each other into microorganisms of the same species, with which the ergosterol is fermented and the ergosterol thus obtained is extracted, analyzed, purified and isolated by column chromatography.

The present invention also provides expression cassettes comprising the middle ADH promoter, the t-HMG gene, the TRP terminator and the SAT1 gene with the middle ADH promoter and the TRP terminator, and expression cassettes comprising the middle ADH promoter, the t-HMG gene, TRP- a terminator, the SAT1 gene with the middle ADH promoter and the TRP terminator, and the ERG9 gene with the middle ADH promoter and the TRP terminator.

It is also an object of the invention to provide a combination of expression cassettes

a) first expression cassette on which the ADH promoter, tHMG gene and TRP terminator are located

b) a second expression cassette on which the ADH promoter is located, a gene

SAT 1 and TRP terminator, a

c) a third expression cassette on which the ADH promoter is located, a gene

ERG9 with TRP terminator.

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It is a further object of the present invention to use these expression cassettes for the transformation of microorganisms used for fermentation to ergosterol, wherein the microorganisms are preferably yeast.

The present invention also relates to microorganisms, such as yeast, which contain these expression cassettes as well as their use in fermentation to ergosterol and its intermediates.

The following examples are intended to illustrate the procedures employed.

DETAILED DESCRIPTION OF THE INVENTION

1. Restriction

Plasmid restriction (1-30 µg) was performed in batches of 30 µl. 3 µl of the appropriate buffer, 1 µl RSA (bovine serial albumin) and 2 µl enzyme were added to the DNA contained in 24 µl H ₂ O. The enzyme concentration was 1 single / µl or 5 single / µl according to the amount of DNA. In some cases, 1 µl of RNase enzyme was added to break down the tRNA. This restriction reaction mixture was incubated at 37 ° C for 2 hours. Restriction was controlled using Minigel.

2. Gel electrophoresis

Gel electrophoresis was performed in Minigel (Minigelapparatur) or Wide-Minigel apparatuses. Minigels (ca. 20 ml, 8 wells) and Wide Minigels (50 ml, 15 or 30 wells) consisted of 1% agarose inTAE. 1 x TAE was used as a buffer. To the samples (10 µl) 3 µl of the stopper solution was added and the samples were loaded. A7 / nd111 1-DNA (bands: 23.1 kb; 9.4 kb; 6.6 kb; 4.4 kb; 2.3 kb; 2.0 kb; 0.6 kb) was used as standard. The separation was carried out at a voltage of 80 V for 45 to 60 minutes. The gel was then stained in a solution of ethidium bromide and evaluated in UV light using the INTAS video documentation system or photographed using an orange filter.

3. Gel elution

The desired fragments were isolated by eluting the gel. The restriction reaction mixture was applied to several wells of Minigel and split.

Only lambda-H / ndII and a trace of some were stained in the ethidium bromide solution

The 31430 / H compounds were monitored under UV light and the desired fragment was labeled. This prevented DNA damage in the other wells by exposure to ethidium bromide and UV radiation. The desired fragment was able to be excised from the unstained gel by placing the stained and unstained portions of the gel on top of each other. An aliquot of the isolated fragment was placed in a dialysis tube, a little TAE buffer without air bubbles was added, and this portion was placed in an RioRad Minigel apparatus. The wash buffer used was 1 x TAE, the voltage was 100 V for 40 minutes. The polarity of the electric current was then changed for 2 minutes to release the DNA adhered to the dialysis tube. The dialysis tube buffer containing the DNA fragments was transferred to the reaction vessel and precipitated with ethanol. 1/10 volume of 3M sodium acetate, tRNA (1 μΙ per 50 μΙ solution) and two and a half times the volume of ice cold 96% ethanol were added to the DNA solution. This reaction mixture was incubated for 30 minutes at -20 ° C and then centrifuged at 12,000 rpm for 30 minutes at 4 ° C. The DNA pellet was dried and removed into 10 to 50 μΙ of water (depending on the amount of DNA).

4. Klenow adjustment

Following the Klenova procedure, the protruding ends of the DNA fragments were filled, resulting in bluntends. The following components were pipetted per 1 µg of DNA:

DNA pellet + 11 μΙ H ₂ O + 1.5 μΙ 10x Klenow buffer + 1 μΙ 0.1 M DTT + 1 μΙ nucleotide (dNTP 2 mM) + 1 μΙ Klenow polymerase (1 unit / μΙ)

DNA should be prepared by ethanol precipitation to prevent inhibition

Klenow polymerases caused by impurities. Incubation was carried out for 30 minutes at 37 ° C, followed by heating for 5 minutes at 70 ° C to stop the reaction. DNA was recovered from this reaction mixture by ethanol precipitation and was removed to 10 μΙ H ₂ O.

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5. Ligation

The DNA fragments to be bound were pooled. In the final volume

The sample was incubated for 45 seconds at 70 ° C, cooled to room temperature (about 3 minutes) and then cooled on ice for 10 minutes. . Ligation buffer was then added: 2.6 μΙ 500 mM Tris. HCl pH 7.5 and 1.3 µ1100 mM MgCl 2 and this mixture was incubated on ice for a further 10 minutes. After addition of 1 μΙ 500 mM DTT and 1 μ110 mM ATP and an additional 10 min incubation on ice, 1 μΙ ligase (1 unit / μΙ) was added. The whole reaction procedure was carried out as far as possible, without shaking, so that the ends of the superimposed DNA did not move away again. The ligation was carried out overnight at 14 ° C.

6. Transformation of E.coli

Appropriate Escherichia coli (E.coli) NM522 cells were transformed with DNA from the ligation reaction. As a positive control, a reaction with 50 ng of plasmid pScL3 was simultaneously performed and as a zero control, a reaction without addition of DNA was performed. For each transformation reaction, 100 μΙ of 8% PEG solution, 10 μΙ DNA and 200 μΙ competent cells (E.coli NM522) were pipetted into a centrifuge tube. The reaction mixtures were placed on ice for 30 minutes and shaken occasionally. Heat shock was then performed: 1 minute at 42 ° C. For regeneration, 1 ml of LB medium was added to the cells and the reaction mixture was incubated for 90 minutes at 37 ° C on a shaker. The LB + Ampicillin plates were coated with 100 µL of undiluted reaction mixture, diluted 1: 10 and 1:10, and cultured overnight at 37 ° C.

7. Isolation of plasmid from E. coli (miniprep)

E. coli colonies were placed overnight in 1.5 ml LB + Ampicillin medium in a centrifuge tube at 37 ° C and 120 rpm. The following day, cells were centrifuged for 5 minutes at 5000 rpm. and 4 ° C, obtained

The 31430 / H pellet was removed into 50 μΙ TE buffer. 100 µl was added to each reaction mixture. 0.2 N NaOH, 1% SDS, the mixture was stirred and placed on ice for 5 minutes (cell lysis). Then, 400 μΙ Na-acetate / NaCl solution (230 μΙ H2O, 130 μΙ 3 M Na-acetate, 40 μΙ 5 M NaCl) was added, the reaction mixture was stirred and placed on ice for 15 minutes (protein precipitation). After centrifugation for 15 minutes at 11,000 rpm. the supernatant containing the plasmid DNA was transferred to an Eppendorf flask. If the supernatant was not completely clear, centrifugation was performed again. 360 μΙ of ice-cold isopropanol was added to the supernatant and the mixture was incubated for 30 minutes at -20 ° C (DNA precipitation). The DNA was centrifuged (15 min, 12000 rpm, 4 ° C), the supernatant was removed, the pellet was washed with 100 µL of ice-cold 96% ethanol, incubated for 15 min at -20 ° C and centrifuged again (15 min, 12 min). 000 rpm, 4 ° C). The obtained pellet was dried in a Speed Vac oven and then removed to 100 μΙ H2O. Plasmid DNA was characterized by restriction analysis. Restriction was always performed with 10 μΙ of the reaction mixture and separation was performed by Wide-Minigel electrophoresis (see above).

8. Processing of plasmid from E. coli (maxiprep)

Maxiprep (Maxipräp) was used to isolate larger amounts of plasmid DNA. Two flasks containing 100 ml of LB + Ampicillin medium each were inoculated with the colony, respectively. 100 μΙ frozen cultures carrying the isolated plasmid and were cultured overnight at 37 Ca Ca 120 l / min. This culture (200 ml) was poured into a GSA vial the next day and centrifuged at 4000 l / min. (2600 x g) for 10 minutes. The cell pellet was removed into 6 ml TE buffer. For digestion (Abdau) of the cell wall, 1.2 ml of lysozyme solution (20 mg / ml TE buffer) was added and the reaction mixture was incubated for 10 minutes at room temperature. Finally, the cells were lysed with 12 ml of 0.2 N NaOH, 1% SDS solution and incubated for an additional 5 minutes at room temperature. Proteins were precipitated by the addition of 9 ml of cooled 3 M Na-acetate solution (pH 4.8) in a 15 minute incubation on ice. After centrifugation (GSA: 13000 rpm (27500 x g), 20 min, 4 ° C), the supernatant was

31430 / H containing DNA transferred to another GSA-vial and DNA was precipitated with 15 ml of ice isopropanol by incubation for 30 minutes at -20 ° C. The DNA pellet was washed with 5 ml of ice-cold ethanol and air dried (about 30-60 minutes). It was then taken up in 1 ml H ₂ O. Plasmid control was performed by restriction analysis. The concentration was determined by applying the diluted solution to Minigel. A 30-60 minute microdialysis (pore size 0.025 µm) was performed to reduce the salt content.

9. Transformation of yeast

A pre-cultured culture (Voranzucht) of Saccharomyces cerevisiae (S. cerevisiae) AH22 was used to transform the yeast. A flask of 20 ml YE-medium was seeded with 100 μΙ frozen culture and cultured overnight at 28 ° C and 12 L / min. The main culture was performed under the same conditions in a flask with 100 ml YE-medium inoculated with 10 μΙ, 20 μΙ or 50 μΙ of the pre-cultured culture.

9.1. Preparation of competent cells

The next day, the contents of the flasks were evaluated by Thom's chamber (Thomakammer) and the flask was maintained at 35x10 ⁷ cells / ml. Cells were harvested by centrifugation (GSA: 5000 rpm (4000 x

g), 10 minutes). The cell pellet was taken up in 10 ml TE buffer and divided into two centrifuge tubes (5 ml each). Cells were centrifuged for 3 minutes at 6000 l / min. and washed twice more with 5 ml TE buffer. Finally, the cell pellet was taken up in 330 μL of lithium acetate buffer per 10 ⁹ cells, transferred to a sterile 50 ml Erlenmeyer flask and shaken at 28 ° C for one hour. In this way, competent cells for transformation were obtained.

2.9 transformation

For each transformation reaction, 15 μΙ herring sperm DNA (10 mg / ml) was pipetted into the centrifuge tube,

31430 / H μΙ of transformed DNA (approx. 0.5 µg) and 330 μΙ of competent cells and this mixture was incubated for 30 minutes at 28 ° C (without shaking!). Then, 700 μΙ of 50% PEG 6000 was added and incubated for an additional hour at 28 ° C without shaking. This was followed by a thermal shock for 5 minutes at 42 ° C.

100 μΙ of this suspension was applied to selection medium (YNB, Difco) to allow selection for leucine prototrophy. On selection for G418 resistance, cell regeneration was performed after heat shock (see Regeneration Phase, section 9.3).

9.3. Regeneration phase

Since the selectable marker represents resistance to G418, the cells needed time to express the resistance gene. 4 ml of YE-medium was added to the transformation reaction mixture and the reaction mixture was cultured overnight at 28 ° C on a shaker (120 L / min). The next day, the cells were centrifuged (6000 L / min, 3 min), taken up in 1 ml YE-medium and 100 µL and 100 µL were added from this mixture. 200 μΙ on YE-G418 boards. These plates were cultured for several days at 28 ° C.

10. Reaction conditions for PCR

Polymerase Chain Reaction (PCR) reaction conditions had to be optimized for each experiment, so they are not generally valid for each reaction mixture. Among other things, it is possible to vary the amount of DNA, salt concentration and dissolution temperature (Schmelztemperatur). For our conditions, it was appropriate to use an Ependorf cap designed for the Thermocycler to which the following substances were given: To 2 μΙ (approx. 0.1 U) of Super Taq Polymerase, 5 μΙ of Super Buffer, 8 μΙ of dNTP (0.625 μ po each) ), 5'-primer, 3'-primer, and 0.2 µg of DNA matrix dissolved in the amount of water to obtain a total PCR reaction volume of 50 μΙ. The reaction mixture was centrifuged briefly and overlaid with a drop of oil. 37 to 40 cycles were used for amplification.

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The following Examples illustrate the preparation of plasmids of the present invention, the preparation of yeast strains and their use, but the present invention is not limited thereto.

Example 1

Expression of tHMG in S. cerevisiae AH22

The DNA sequence on tHMG (Basson et al., 1988) was amplified by standard methods by PCR from genomic DNA from Saccharomyces cerevisiae S288C (Mortimer and Johnston, 1986). The primers used were the DNA oligomers tHMG-5 'and tHMG-3' (see SEQ ID NOs 1 and 2). The DNA fragment obtained, after Klenow treatment, was inserted into the pUC19 cloning vector (YanishPerron et al., 1985) to give the pUC19-tHMG vector. After plasmid isolation and restriction of pUC19-tHMG with EcoRI and BamHI endonucleases, the fragment was inserted into the yeast expression vector pPT2b (Lang and Looman, 1995), which was also engineered with EcoRI and BamHI. The resulting plasmid pPT2b-tHMG contained an ADH1 promoter (Bennetzen and Hali, 1982) and a TRP1terminator (Tschumper and Carbon, 1980), in which a tHMG-DNA fragment was inserted. From the vector pPT2b-tHMG, a DNA fragment containing the so-called DNA fragment was isolated using EcoRV and Nru I endonucleases. a middle ADH 1-promoter containing tHMG and a TRRf-terminator. This DNA strand was inserted into the yeast vector YEp13 (Fischhoff et al., 1984), treated with SphI endonuclease and DNA polymerase. The resulting vector YEpH2 (Fig. 1) was treated with EcoRV and Λ / rul endonuclease. This resulted in a DNA fragment with the following regions: the transcriptionally active region from the tetracycline-resistant gene (Sidhu and Bollon, 1990), the middle ADH1 promoter, the tHMG and the TRP terminator (expression cassette). This DNA fragment was inserted into the YdpU vector (Berben et al., 1991) modified by SuI. The resulting vector YdpUH2 / 12 was engineered with SmaI and ligated to a DNA sequence encoding kanamycin resistance (Webster and Dickson, 1983). This resulting construct (YDpUHK3, Fig. 2) was treated with EcoRV. This construct transformed the yeast strain Saccharomyces cerevisiae AH22. This transformation of yeasts by the action of the linearized vector of

31430 / H of this example results in chromosomal integration of the entire vector at the gene site (Genlocus) of URA3. In order to eliminate regions that do not belong to the expression cassette (derived from E. coli, E.co//- anmpicillin resistant gene, TEF promoter and kanamycin n resistant gene) from the integrated vector, transformed yeasts were subjected to selection pressure (Selektionsdruck) by FOA- a selection (Boeke et al., 1987) positively influenced by uracil-auxotrophic yeast. The uracil-auxotrophic strain produced by this selection is designated AH22 / tH3ura8 and contains the tHMG1-expression cassette as chromosomal integration in the URA2 gene.

The yeast strain AH22 / tH3ura8 and the starting strain AH22 were cultured for 48 hours in YE at 28 ° C at 160 l / min in culture flasks (Schikanekolben).

Conditions for cultivation:

The pre-cultured WMVIII culture was as follows: a mixture of 20 ml WMVIII + histidine (20 pg / ml) + uracil (20 pg / ml) was inoculated with 100 μΙ of frozen culture and incubated for 2 days at 28 ° C and 120 l / min. (reciprocating movement). This pre-cultured 20 ml culture was seeded with a mixture of 100 ml WMVIII + histidine (20 pg / ml) + uracil (20 pg / ml). In the main culture, 50 ml YE (in 250 ml culture flasks) were seeded with 1 x 10 ⁹ cells. These flasks were incubated at 160 L / min on a circular shaker at 28 ° C for 48 hours. HMG-CoA reductase activity was then determined (Quareshi et al., 1981) and the following values were obtained:

Table 1

specific HMG-CoA- reductase activity * (single / mg protein) AH22 3.99 AH22 / tH3ura8 11,12

A unit is defined as a conversion of 1 nmol NADPH per minute

31430 / H in one milliliter of reaction mixture. Measurements were performed with whole protein isolates.

Sterols were extracted (Parks et al., 1985) and analyzed by gas chromatography. The following values were obtained:

Table 2

Squalene (% by weight) Ergosterol (wt.%) AH22 0.01794 1,639 AH22 / tH3ura8 .8361 1.7024

The percentages are based on yeast dry matter.

Example 2

Expression of SAT1 in S. cerevisiae AH22

The sequence for Acyl-CoA: steroltransferase (SAT1; Yang et al., 1996) was obtained as described above by PCR from genomic DNA from Saccharomyces cerevisiae S288C. The primers used were DNA oligomers of SAT1-5 'and SAT1-3' (see SEQ ID Nos. 3 and 4). The obtained DNA fragment was cloned in the pGEM-T cloning vector (Mezei and Storts, 1994) to give the pGEM-SAT1 vector. After treatment with pGEM-SAT1 with EcoRI, the obtained fragment was cloned in the yeast expression vector pADH1001, which was also EcoRI-treated. The resulting pADH-SAT1 vector was treated with Nru I endonuclease and ligated to the zYep 13 fragment containing the LEC / 2 gene.

This resulted in the yeast expression vector pADL-SAT1 (Fig. 3), which was inserted into the yeast strain AH22. The AH2 / pADL-SAT1 strain thus prepared was incubated for 7 days in WMVIII (Lang and Looman, 1995) for Minimalmedia. Culture conditions: (pre-cultivated culture see above). Main culture: a mixture of 50 ml WMVIII + histidine (20 µg / ml) + uracil (20 µg / ml) (in 250 ml culture flasks) was seeded with 1 x 10 ⁹ cells. The flasks were incubated at 160 L / min on a circular shaker at 28 ° C for 7 days. The resulting sterols were analyzed by gas chromatography (see Table 3).

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Table 3

Squalene (% by weight) Ergosterol (wt.%) AH22 cannot be determined 1,254 AH22 / Paddles-SAT1 cannot be determined 1,831

The percentages are based on yeast dry matter.

Example 3

Combined expression of truncated 3-hydroxy-3-methylglutaryl-CoA-reductase (tHMG) and acyl-CoA: sterol-acyltransferase (SAT1)

Example 3.1

The AH22 / tH3ura8 yeast strain was transformed with the SA-expression vector pADL-SAT1 to give AH22 / tH3ura8 / pADL-SAT1. This combined strain was cultured for 7 days in WWVIII. Sterols were extracted (see above) and analyzed by gas chromatography. The following values were obtained (see Table 4).

Table 4

Squalene (% by weight) Ergosterol (wt.%) AH22 / tH3ura8 1,602 3,798 AH22 / tH3ura / Paddles-SAT1 1,049 5,540

The percentages are based on yeast dry matter.

Example 3.2

Yeast cultures were cultured for 7 days in WMVIII, but different amounts of uracil were added to the cultures. The amount of uracil in the medium was set at 10, 20, 40 and 100 pg / ml. The amount of ergosterol and squalene was maximal when supplemented with 20 pg / ml uracil. The results are shown in FIG. 4. The results show that the yields of ergosterol and squalene in the AH22 / tH3ura / pADL-SAT1 strain hurt significantly from the amount of uracil added to WMVIII medium.

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Example 3.3

Yeast cultures were cultured for 7 days in WMVIII. The total amount of sterols was then determined as described above. Free sterols were extracted with n-hexane from yeast disrupted by glass beads.

The results are shown in Tab. 5th

These results indicate that the enzyme sterol-acyl transferase (Sate) causes highly efficient re-esterification, especially for those sterols that do not have a 4,4-dimethyl group. Thus, this process could be used to separate 4,4-dimethylsterols from their respective demethylated forms.

Table 5

Percentage of free sterols. Each sterol was determined as free sterol (no saponification) and this value was based on the total amount of sterol. The brackets show the respective absolute values of the total sterol content per area / g dry matter. Lanosterol and 4,4-dimethylzymosterol are sterols containing 4,4-dimethyl.

% free sterols inspection AH22 / tH3ura / Paddles-SAT1 lanosterol 54 (0.99) 59 (2.90) 4,4-dimethylzymosterol 58 (0.77) 84 (2.37) 4-metylzymosterol 7 (2.43) 10 (7.62) zymosterol 10 (1.67) 11 (5.85) ergost-7-enol, ergosta-5,7-dienol 24 (4.55) 12 (9.00)

Explanation of the drawings

In FIG. 1 is a plasmid YEpH2 with respective interface sites.

In FIG. 2 shows plasmid YDpUHK3 with respective interface sites. In FIG. 3 shows plasmid pADL-SAT1 with respective interface sites.

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In FIG. 4 shows the culture behavior and the contents of ergosterol and squalene at different dosages of uracil. In this picture it means:

OD = optical density, cultivation time = yeast dry weight, yeast dry weight, uracil supplementation = uracil dosage.

SEQUENCE OVERVIEW

(1) GENERAL INFORMATION (I) APPLICANT: (A) TITLE: Schering AG (B) STREET: Mullerstrasse 178 (C) THE CITY: Berlin (E) COUNTRY: Germany (F) Zip: D-13342 (G) PHONE: (030) -4681 2085 (H) FAX: (030) -4681 2058 (Ii) NAME OF THE INVENTION:

PROCEDURE FOR THE PRODUCTION OF ERGOSTEROL AND ITS INTERMEDIATES BY RECOMBINANT YEAST (iii) NUMBER OF SEQUENCES: 4 (iv) COMPUTER-FORMATABLE FORM:

(A) MEDIA TYPE: diskette (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentin Release # 1.0,

Version # 1.25 (EPO) (2) INFORMATION FOR SEQ. ID # 1:

31430 / H (i) SEQUENCE CHARACTERISTICS:

(A) 25 bases long (B) TYPE: nucleic acid (C) STRUCTURE (strandedness): simple (D) TOPOLOGY: linear (ii) HYPOTHETICAL SEQUENCE: no (iii) SEQUENCE DESCRIPTION: SEQ. IDČ.1:

5'- ACTATGGACCAATTGGTGAAAACTG (2) INFORMATION FOR SEQ. ID 2:

(i) SEQUENCE CHARACTERISTICS:

(A) 23 base length (B) TYPE: nucleic acid (C) CHAIN ORGANIZATION: simple (D) TOPOLOGY: linear (ii) HYPOTHETICAL SEQUENCE: no (iii) SEQUENCE DESCRIPTION: SEQ. ID 2:

R'- AGTCACATGGTGCTGTTGTGCTT (2) INFORMATION FOR SEQ. ID. No.3:

(i) SEQUENCE CHARACTERISTICS:

(A) 25 base length (B) TYPE: nucleic acid (C) CHAIN ORGANIZATION: simple (D) TOPOLOGY: linear (ii) HYPOTHETICAL SEQUENCE: no (iii) SEQUENCE DESCRIPTION: SEQ. ID 3:

5'- GAATTCAACCATGGACAAGAAGAAG (2) INFORMATION FOR SEQ. ID. No.4:

31430 / H (i) SEQUENCE CHARACTERISTICS:

(A) 24 base length (B) TYPE: nucleic acid (C) CHAIN ORGANIZATION: simple (D) TOPOLOGY: linear (ii) HYPOTHETICAL SEQUENCE: no (iii) SEQUENCE DESCRIPTION: SEQ. ID # 4:

5'- AGAATTCCACAGAACAGTTGCAGG

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Berben, G., Dumont, J., Gilliquet, V., Bolle, P. A., Hilger, F. (1991) The YDp plasmids: a uniform set of vector bearing versatile gene disruption cassettes for Saccharomyces cerevisiae. Yeast 7: 475-477.

Boeke, J.D., Trueheart, J., Natsoulis, G., Fink, G. (1987) 5-Fluorootic acid and a selective agent in yeast molecular genetics. Methods in Enzymology 154: 164175.

Fegueur, M., Richard, L., Charles, A. D., Karst, F. (1991) isolation and primary structure of the ERG9 gene of Saccharomyces cerevisiae encoding squalene synthetase. Currrent Genetics 20: 365-372.

Fischhoff, D.A., Waterston, R.H., Olson, M.V. (1984) The yeast cloning vector

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YEp13 contains a tRNALeu3 gene that can be mutated to an amber suppressor. Gene 27: 239-251.

Jandrositz, A., Turnowsky, F., and Hôgenauer, G. (1991) The gene encoding squalene epoxide from Saccharomyces cerevisiae: cloning and characterization. Gene 107: 155-160.

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Mortimer, R.K., Johnston, J.R. (1986) Genealogy of Principal Strains of the Yeast Genetic Stock Center. Genetics 113: 35-43.

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3-Hydroxy-3-methylgluraryl-CoA reductase from Yeast, Meth. Enzymol. 71: 455-461.

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Siduh, R. S., Bollon, A. P. (1990) The bacterial plasmid pBR322 sequences serve as upstream activating sequences in Saccharomyces cerevisiae. Yeast 6: 221229.

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Tschumper, G., Carbon, J. (1980) Sequence of a DNA fragment containing a chromosomal replicator and the TRPI gene. Gene 10: 157-166.

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Claims (24)

Process for the production of ergosterol and its intermediates, characterized in that: (a) the construction of a plasmid into which several genes suitable for the metabolism of ergosterol is inserted, or (b) a plasmid is constructed first, in which one of the genes suitable for the metabolism of ergosterol is inserted, d) transforming the microorganisms with the plasmid thus produced, wherein the microorganisms are transformed with one or more plasmids according to a) or several plasmids simultaneously or sequentially. d) fermentation to ergosterol is carried out with the microorganisms thus prepared, (e) after fermentation, ergosterol and its intermediates are extracted and analyzed from the cells and finally (f) the ergosterol thus obtained and its intermediates are purified and isolated by column chromatography. Method according to claim 1, characterized in that a-i) is first carried out the construction of a plasmid into which the following genes are inserted: (i) the HMG-CoA reductase (t-HMG) gene; (ii) the squalene synthetase (ERG9) gene; (iii) the acyl-CoA: sterol acyltransferase (SAT1) gene; and (iv) the squalene epoxidase (ERG1) gene; first construct the plasmid into which the following genes are inserted: i) the HMG-CoA reductase (t-HMG) gene; and 31430 / H replacement page ii) the squalene synthetase (ERG9) gene, or a-iii) first construct the plasmid into which the following genes are inserted: (i) HMG-CoA reductase (t-HMG) gene; and (iii) the acyl-CoA: sterol-acyltransferase (SAT1) gene; or (a-iv), a plasmid construct is inserted first into which the following genes are inserted: (i) the HMG-CoA reductase (t-HMG) gene; and (iv) the squalene epoxidase (ERG1) gene; or (a-v), the construction of a plasmid into which the following genes are inserted is carried out: (ii) the squalene synthetase (ERG9) gene; and (iii) the acyl-CoA: sterol-acyltransferase (SAT1) gene; or (a-vi), a plasmid is constructed first, into which the following genes are inserted: (ii) the squalene synthetase (ERG9) gene; and (iv) the squalene epoxidase (ERG1) gene; or (a-vii), a plasmid construct is inserted into which the following genes are inserted: (iii) the acyl-CoA gene: sterol acyltransferase (SAT1); and (iv) the squalene epoxidase (ERG1) gene; 31430 / H replacement side or (b) a plasmid is constructed first, in which one of the genes listed under a-i is inserted, and (c) transforming microorganisms with the plasmids thus prepared, the microorganisms being transformed by one or more of the plasmids listed under (a) to (a) (v) or several plasmids listed under (b) simultaneously or sequentially; (d) fermentation to ergosterol is carried out with the micro-organisms thus obtained, (e) after fermentation, ergosterol and its intermediates are extracted from the cells and analyzed and finally (f) the ergosterol thus obtained and its intermediates are purified and isolated by column chromatography. Method according to claim 2, characterized in that the squalene epoxidase (ERG1) gene is additionally inserted in the plasmid under a-ii), a-iii) and av) and in addition the acyl-CoA gene: sterol- acyl-transferase. A process for the production of ergosterol and its intermediates, characterized in that the plasmid genes mentioned in claim 1 under a), in claim 2 under ai) to avii) and in claim 3 under a-ii), a-iii) and av ), are first introduced independently into microorganisms of the same species and fermented to ergosterol, and the ergosterol thus obtained is extracted, analyzed, purified and isolated by column chromatography. Process according to claims 1 to 4, characterized in that the intermediates are squalene, farnesol, geraniol, lanosterol, zymosterol, 4,4-dimethylzymosterol, 4-methylzymosterol, ergost-7-enol and ergosta-5,7-dienol. Process according to claims 1 to 4, characterized in that the intermediates are sterols with a 5,7-diene structure. Method according to claims 1 to 4, characterized in that the plasmids are YEpH2 (Fig. 1), YDpUHK3 (Fig. 2) and pADL-SAT1 (Fig. 3). 31430 / H replacement page Π Process according to claims 1 to 4, characterized in that the microorganisms are yeasts. Method according to claim 8, characterized in that the species used are S. cerevisiae. Method according to claim 9, characterized in that the strain used is S. cerevisiae AH22. A yeast strain of S. cerevisiae AH22, comprising one or more of the genes mentioned in method a-i). Plasmid pADL-SAT1 (FIG. 3), consisting of the SAT1 gene and the LEU2zYEp13 gene. Use of the plasmid according to claim 12 for the manufacture of ergosterol. Use of the plasmid according to claim 12 for the manufacture of intermediates in the manufacture of ergosterol which are squalene, farnesol, geraniol, lanosterol, zymosterol, 4,4-dimethylzymosterol, 4-methylzymosterol, ergost-7-enol, and ergosta5,7-dienol. Use of the plasmid according to claim 12 for the production of sterols with 5-7-diene structure. 16. Expression cassettes comprising the middle ADH promoter, the t-HMG gene and the TRP terminator and the SAT1 gene with the middle ADH promoter and the TRPterminator. Expression cassettes comprising the middle ADH promoter, the t-HMG gene, the TRP terminator, the SAT1 gene with the middle ADH promoter and the TRP terminator, and the ERG9 gene with the middle ADH promoter and the TRP terminator. 18. Combinations of expression cassettes forming the combination a) the first cassette on which the ADH promoter, the t-HMG gene and the TRP terminator are located b) a second expression cassette on which the ADH promoter, the SAT1 gene and the TRP terminator is located; and c) a third expression cassette on which the ADH promoter, the ERG9 gene with the TRP terminator, is located. Use of expression cassettes according to claims 16 to 18 for transformation 31430 / H replacement side of microorganisms used for fermentation to ergosterol. Use according to claim 19, characterized in that the microorganism is yeast. Microorganisms comprising the expression cassettes according to claims 16 to 21 18th A microorganism according to claim 21, characterized in that it is a yeast. Use of the microorganism according to claims 21 and 22 for fermentation to ergosterol. Use of a microorganism according to claims 21 and 22 for fermentation to ergosterol intermediates.