CZ20001165A3 - A method for producing ergosterol and its intermediates by recombinant yeast - Google Patents

Abstract

The solution relates to a method for producing ergosterol and its intermediates using recombinant yeast and plasmids for yeast transformation.

Description

Technical area

This invention relates to a process for the production of ergosterol and its intermediates using recombinant yeast and plasmids for yeast transformation.

State of the art

Ergosterol is the final product of sterol synthesis in yeasts and fungi. The economic importance of this substance lies in the production of vitamin D£ from ergosterol by UV radiation and in the production of steroid hormones by biotransformation, starting from ergosterol. Squalene is used as a basic synthetic material for the synthesis of terpenes. Its hydrogenated form squalane is used in dermatology and cosmetics together with various derivatives as a component of skin and hair care products. The intermediate products of ergosterol metabolism are also of economic importance.

The most important of these intermediates are farnesol, geraniol and squalene. Other sterols are also economically useful, such as zymosterol and lanosterol, with lanosterol being a key natural and synthetic substance used for the chemical synthesis of saponins and steroid hormones. Lanosterol penetrates the skin well and spreads well, and is therefore used as an emulsifier and as an active ingredient in skin creams.

Genes controlling ergosterol metabolism in yeast • · • · ··· · · · · · · · · · · · · · · « ·· ·

2· · · · · ········ ·· · are sufficiently known and cloned, such as

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

Acyl-CoA:sterol acyltransferase (SATI) (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 sterol acyltransferase have not yet been fully investigated. High expression of the gene of these enzymes has already been verified, but has not led to any significant increase in the amount of ergosterol obtained. In the case of HMG1-high expression, increased squalene production has been described, and mutations have been introduced in this reaction to interrupt the further reaction after squalene is obtained (EP-0 486 290). Increased production of geraniol and farnesol has also been described, but there was no high expression of ergosterol metabolism genes, but rather an interruption of the reaction sequence in the direction of geraniol and farnesol formation (EP-0313 465).

Specific inhibitors of ergosterol biosynthesis can also lead to the accumulation of certain intermediates, such as allylamines, which prevent the conversion of squalene to squalene epoxide, resulting in a substantial increase in squalene levels (up to 600 times the normal amount) (Jandrositz et al., 1991).

Although the use of inhibitors allows for a significant increase in the amount of, for example, squalene, the addition of these substances is not advantageous, since even small amounts of them have the same effects in the organism, and therefore the method of producing ergosterol biosynthesis products by increased production is more advantageous.

• ·

Λ · · ··· * · · · ···· · ··· · ·· » • · ··· ······· · · · • · · ··· ··«· «·«··· 9 9 9 9 9 9 9

The aim of this invention is to develop a microbiological process for the production of ergosterol and its intermediates, the microorganisms required for this, such as yeast strains, which synthesize increased amounts of ergosterol or the necessary intermediates, and the preparation of plasmids required for the transformation of yeast strains.

The essence of the invention

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

The subject of the present invention is a method, the essence of which lies in the fact that

a) first a plasmid is created into which several genes suitable for the metabolism of ergosterol are inserted in a modified form or

b) first a plasmid is created into which one of the genes suitable for the metabolism of ergosterol is inserted in a transformed form, • · « · • · • · « · • « * • * · ····· ·· · · · ·

c) microorganisms are transformed with the plasmid prepared in this way, whereby these microorganisms are transformed with one plasmid according to a) or several plasmids according to

b) simultaneously or sequentially,

d) fermentation into ergosterol is carried out with the microorganisms prepared in this way,

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

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

The subject of the present invention is in particular a method, the essence of which consists in that a-i) a plasmid is first created into which the following genes are inserted:

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

i) the HMG-CoA reductase gene (t-HMG), and ii) the squalene synthetase gene (ERG9), • · «· »· · · · · · ««· ··· · « · 0 ···· « ··· · ·« · • · · · · «··««/-· · · 9 or a-iii) first a plasmid is created into which the following genes are inserted:

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

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

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

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

ii) squalene synthetase gene (ERG9) a

iii) acyl-CoA:sterol acyltransferase (SATI) gene • · ··· » · · ··· ·

ii) squalene synthetase gene (ERG9) a

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

iii) the acyl-CoA:sterol acyltransferase (SATI) gene; iv) the squalene epoxidase (ERG1) gene; or

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

c) using the plasmids thus prepared, microorganisms are transformed, whereby these microorganisms are transformed with one plasmid listed under a-i) to a-vii) or several plasmids listed under b) simultaneously or sequentially,

d) the microorganisms thus obtained are fermented to ergosterol,

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

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

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

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

The plasmids used are preferably the YepH2 plasmid, which contains the middle (mittler) ADH promoter, t-HMG (transformed variant of HMG1) and TRP terminator (see Fig. 1), the YDpUHK3 plasmid, which contains the middle ADH promoter, t-HMG (transformed variant of HMG1) and TRP terminator, the kanamycin resistance gene and the ura3 gene (see Fig. 2) and the pADL-SAT1 plasmid, which contains the SÁTI gene and the LEU2 gene from Yepl3.

These plasmids and their use for the production of ergosterol and its intermediates, such as squalene, farnesol, geraniol, lanosterol, zymosterol, 4,4-dimethylzymosterol, 4-methyl• · • · ······ ·· ·· ·· ·· zymosterol, ergost-7-enol and ergosta-5,7-dienol, in particular sterols with a 5,7-plum structure, are also subject of the present invention.

The host for introducing plasmids according to the invention can be essentially any microorganism, but in particular yeast.

Preferably, the species S. cerevisiae can be used, in particular the strain S. cerevisiae AH22.

The present invention also provides a yeast strain

S.cerevisiae AH22, which contains the plasmid pADL-SAT1.

Combined transformation of microorganisms with plasmids pADL-SAT1 and YDpUHK3 is also preferred, especially transformation of yeasts such as S.cerevisiae AH22.

The metabolic sequence of ergosterol is generally influenced as follows:

The reaction sequence in the direction of ergosterol is maximized, while the activity of several narrow-profile (flaschenhals) enzymes is simultaneously increased. Various enzymes play a decisive role, with the combination of deregulation or high expression having a decisive influence on the increase in the yield of ergosterol. The enzymes or their genes HMG1 (Basson et al., 1988), ERG9 (Fegueur et al., 1991), acyl-CoA:sterol acyltransferase (SÁTI) (Yu et al., 1996) and/or squalene epoxidase (ERG1) (Jandrositz et al., 1991) inserted in a modified form into the yeast strain are used as combinations, whereby the gene insertion is carried out using one or more plasmids and this plasmid(s) contains DNA sequences either • · • · · · · · · · » · · I » · · 4

9 4 4 individually or in combination. Altered means in the case of the HMG1 gene that only its catalytic region without the membrane-bound domain is expressed from the respective gene. This conversion has already been described (EP-0486 290). The aim of the HMG1 conversion is to prevent feed-back regulation caused by intermediates of ergosterol biosynthesis. This means that neither HMG1 nor the other two genes mentioned are subject to transcriptional regulation. In addition, the gene promoter is replaced by the middle ADH1 promoter. This promoter fragment of the ADH1 promoter is characterized by approximately constitutive expression (Ruohonen et al., 1995), so that transcriptional regulation no longer takes place via intermediates of ergosterol biosynthesis.

Products produced during high expression can be used for biotransformations or other chemical and therapeutic purposes, e.g. for obtaining vitamin D2 from ergosterol by UV radiation and for obtaining steroid hormones from ergosterol using biotransformations.

The subject of the present invention are also microorganisms, in particular yeast strains, which, using high expression in the process with the genes listed under a-i), enable the production of increased amounts of ergosterol and ergosterol in combination with increased amounts of squalene.

A modified variant of the HMG1 gene in which only the catalytic region without the membrane-bound domain is expressed is preferred. This modification has been described (EP-0486 290).

The subject of this invention is also a process for the production of ergosterol and intermediate products resulting from this reaction, which is characterized in that the genes mentioned in the process under a), in particular

0 00 «00 ·0 • •0 ♦ · · 0 · · • · · · 0 0 0 0 · ·· 0 · ··· ····(* ·· · «00 0 0 « «0 0 0C 00 in the procedure under a-i) to a-vii) (double, triple, quadruple combination of genes) together with plasmids are first independently inserted into microorganisms of the same species, fermentation is carried out with them to ergosterol and the ergosterol thus obtained is extracted from the cells, analyzed, purified and isolated using column chromatography.

The present invention also provides expression cassettes comprising a central ADH promoter, a t-HMG gene, a TRP terminator and a SÁTI gene with a central ADH promoter and a TRP terminator, and expression cassettes comprising a central ADH promoter, a t-HMG gene, a TRP terminator, a SÁTI gene with a central ADH promoter and a TRP terminator, and an ERG9 gene with a central ADH promoter and a TRP terminator.

The subject of this invention is also a combination of expression cassettes, this combination consisting of

a) the first expression 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 SÁTI gene and the TRP terminator are located,

c) a third expression cassette on which the ADH promoter is located, the ERG9 gene with the TRP terminator.

A further object of the present invention is the use of these expression cassettes for the transformation of microorganisms used for fermentation into ergosterol, these microorganisms preferably being yeasts.

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 performed.

Examples of embodiments of the invention

1. Restrictions

Restriction of plasmids (1 to 30 µg) was carried out in batches of 30 µl. To the DNA contained in 24 µl of J^O, 3 µl of the appropriate buffer, 1 µl of RSA (bovine serum albumin) and 2 µl of enzyme were added. The enzyme concentration was 1 unit/µl or 5 units/µl depending on the amount of DNA. In some cases, 1 µl of RNase enzyme was added to degrade tRNA. This restriction reaction mixture was incubated for 2 hours at 37 °C. Restriction was checked using a Minigel.

2. Gel electrophoresis

Gel electrophoresis was performed in Minigel (Minigelapparatur) or Vide-Minigel apparatus. Minigels (ca. 20 ml, 8 wells) and Vide-Minigels (50 ml, 15 or 30 wells) consisted of 1% agarose in TAE. 1 x TAE was used as buffer.

3 μΐ of stopper solution was added to the samples (10 μΐ) and the samples were loaded. I-DNA with HindIII (bands: 23.1 kb; 9.4 kb; 6.6 kb; 4.4 kb; 2.3 kb;

• · · · • · · · · · ·

2.0 kb; 0.6 kb). The separation was performed at a voltage of 80 V for 45 to 60 minutes. The gel was then stained in ethidium bromide solution and evaluated under UV light using an INTAS video documentation system or photographed using an orange filter.

3. Gel elution

The desired fragments were isolated by gel elution. The restriction reaction mixture was applied to several wells of the Minigel and separated. Only lambda-//i«dlll and a trace of a substance were stained in the ethidium bromide solution, these substances were monitored under UV light and the desired fragment was labeled. This prevented damage to the DNA in the other wells by the ethidium bromide and UV radiation. The desired fragment could be excised from the unstained gel by the labeling after the stained and unstained parts of the gel were placed on top of each other. The agarose portion with the isolated fragment was placed in a dialysis tube, a little air-bubble-free TAE buffer was added and this portion was placed in the BioRad Minigel apparatus. 1 χ TAE was used as the washing buffer, the voltage was 100 V for 40 minutes. The polarity of the electric current was then changed for 2 minutes to release the DNA stuck to the dialysis tube. The buffer from the dialysis tube containing the DNA fragments was transferred to a reaction vessel and precipitated with ethanol. 1/10 volume of 3M sodium acetate, tRNA (1 μΐ per 50 μΐ of solution) and two and a half volumes 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 taken up in 10 to 50 μΐ of water (depending on the amount of DNA).

· · · · » * · ···· · ··· · * · ·

4. Klenow modification

The protruding ends of the DNA fragments were filled in using the Klenow procedure, resulting in blunt ends. The following components were pipetted onto 1 pg of DNA:

DNA pellet + 11 μΐ ø^O + 1.5 μΐ lOx Klenow buffer + 1 μΐ 0.1 M DTT + 1 μΐ nucleotide (dNTP 2 mM) + 1 μΐ Klenow polymerase (1 unit/μΐ)

DNA should be prepared by ethanol precipitation to avoid inhibition of Klenow polymerase by impurities. Incubation was performed for 30 minutes at 37°C, followed by heating to 70°C for 5 minutes to stop the reaction. DNA was recovered from this reaction mixture by ethanol precipitation and removed into 10 μΐ ř^O.

5. Ligation

The DNA fragments to be ligated were combined. The final volume of 13.1 μΐ contained approximately 0.5 μΐ DNA with a vector ratio of 1:5. This sample was incubated for 45 seconds at 70 °C, cooled to room temperature (approximately 3 minutes) and then cooled on ice for 10 minutes. Then ligation buffer was added: 2.6 μΐ 500 mM Tris.HCl pH 7.5 and 1.3 μΐ 100 mM MgC^ and this mixture was incubated on ice for another 10 minutes. After the addition of 1 μΐ 500 mM DTT and 1 μΐ 10 mM ATP and a further 10 minutes of incubation on ice, 1 μΐ ligase (1 unit/μΐ) was added. The entire reaction procedure was carried out as without shaking as possible so that the DNA ends lying next to each other would not move away again. Ligation was performed overnight at 14°C.

6. E.coli transformation

The relevant 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 performed simultaneously and as a zero control, a reaction without the addition of DNA was performed. For each transformation reaction, 100 μΐ of 8% PEG solution, 10 μΐ DNA and 200 μί competent cells (E.coli NM522) were pipetted into a benchtop centrifuge tube. The reaction mixtures were placed on ice for 30 minutes and shaken occasionally. Then a heat shock was performed: 1 minute at 42 °C. For regeneration, 1 ml of LB-media was added to the cells and the reaction mixture was incubated for 90 minutes at 37 °C on a shaker. 100 μί of the undiluted reaction mixture, the reaction mixtures diluted 1:10 and 1:100 were plated on LB + Ampicillin plates and culture was carried out overnight at 37 °C.

7. Plasmid isolation from E.coli (minipreparation)

E.coli colonies were placed overnight in 1.5 ml of LB+Ampicillin medium in a test tube in a benchtop centrifuge at 37 °C and 120 rpm. The next day, the cells were centrifuged for 5 minutes at 5000 rpm and 4 °C, the resulting pellet was removed into 50 μί TE-buffer. 100 μΐ 0.2 N NaOH, 1% SDS solution was added to each reaction mixture, the mixture was mixed and placed on ice for 5 minutes (cell lysis). Then 400 μΐ Na-acetate/NaCl solution (230 μΐ H2O, 130 μΐ 3 M Na-acetate, 40 μΐ 5 M NaCl) was added, the reaction mixture was mixed and placed on ice for another 15 minutes (protein precipitation). After 15 minutes of centrifugation at 11,000 rpm. The supernatant containing the plasmid DNA was transferred to an Eppendorf tube. If the supernatant was not completely clear, centrifugation was repeated. 360 μΐ 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 1/min., 4 °C), the supernatant was removed, the pellet was washed with 100 μΐ of ice-cold 96% ethanol, incubated for 15 minutes at -20 °C and centrifuged again (15 min.,

000 1/min., 4 °C). The obtained pellet was dried in a Speed Vac dryer and then removed into 100 μΐ 1^0. The plasmid DNA was characterized by restriction analysis. In each case, restriction was performed with 10 μΐ of the reaction mixture and separation was performed by electrophoresis on a Vide-Minigel gel (see earlier).

8. Processing of plasmids from E.coli (maxi preparation)

For isolation of larger amounts of plasmid DNA the maxipreparation method (Maxipráp) was used. Two flasks containing 100 ml of LB+Ampicillin medium were inoculated with a colony or 100 μΐ of frozen culture carrying the isolated plasmid and were cultured overnight at 37 °C and 120 1/min. The following day this culture (200 ml) was poured into a GSA-vessel and centrifuged at 4000 1/min. (2600 x g) for 10 minutes. The cell pellet was removed into 6 ml of 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 by treating with 12 ml of 0.2 N NaOH, 1% SDS solution and incubation was carried out for another 5 minutes at room temperature. Proteins were precipitated by adding 9 ml of chilled 3 M Na-acetate solution (pH 4.8) during fifteen-minute incubation on ice.

After centrifugation (GSA-13000 rpm (27500 x g), 20 min., 4 °C), the supernatant containing DNA was transferred to another GSA-tube.

0

0 • 0 • · • 0 0 0 0 0 0 * « and DNA was precipitated with 15 ml of ice-cold isopropanol during a 30-minute incubation at -20 °C. The DNA pellet was washed with 5 ml of ice-cold ethanol and air-dried (approx. 30 - 60 minutes). It was then taken up in 1 ml of H2O. Plasmid control was performed by restriction analysis. The concentration was determined by applying the diluted solution to a Minigel. To reduce the salt content, 30 to 60-minute microdialysis (pore size 0.025 pm) was performed.

9. Yeast transformation

A pre-culture (Voranzucht) of the Saccharomyces cerevisiae (S. cerevisiae) strain AH22 was used for yeast transformation. A flask with 20 ml of YE-media was inoculated with 100 μΐ of the frozen culture and cultured overnight at 28 °C and 12 1/min. The main cultivation was carried out under the same conditions in a flask with 100 ml of YE-media, inoculated with 10 μΐ, 20 μΐ or 50 μΐ of the pre-culture.

9.1 Preparation of competent cells

The next day, the contents of the flasks were evaluated using a Thom chamber (Thomakammer) and a flask containing 3 - 5x10' cells/ml was further processed. The cells were obtained by centrifugation (GSA: 5000 1/min. (4000 x g), 10 minutes). The cell pellet was removed into 10 ml TE buffer and divided into two benchtop centrifuge tubes (5 ml each). The cells were centrifuged for 3 minutes at 6000 1/min. and washed twice more with 5 ml TE buffer. Finally, the cell pellet was removed into 330 μΐ lithium acetate buffer per 10^ cells, transferred to a sterile 50 ml Erlenmeyer flask and shaken for one hour at 28 °C. In this way, competent cells for transformation were obtained.

• 0 ·· *0 * 00 00 »·* 0 · * 0 * > ♦

00« * ««· 0 «« « • » 0 « 0 <««···· «0 0

00 «0 · 0000 ♦ 0 0 0 0 0 0« * · · *·

9.2 Transformation

For each transformation reaction, 15 μΐ of herring sperm DNA (Heringssperma DNA) (10 mg/ml), 10 μΐ of transformed DNA (approx. 0.5 μg) and 330 μΐ of competent cells were pipetted into a benchtop centrifuge tube and this mixture was incubated for 30 minutes at 28 °C (without shaking!). Then 700 μΐ of 50% PEG 6000 was added and incubation was carried out for another hour at 28 °C without shaking. This was followed by a heat shock for 5 minutes at 42 °C.

100 μΐ of this suspension was plated on selection medium (YNB, Difco) to select for leucine prototrophy. For selection for G418-resistance, cell regeneration was performed after heat shock (see Regeneration phase, section 9.3).

9.3 Regeneration phase

Since the selection marker indicates resistance to G418, the cells needed time to express the resistance gene. 4 ml of YE-media were added to the transformation reaction mixture and this reaction mixture was cultured overnight at 28 °C on a shaker (120 1/min.). The following day, the cells were centrifuged (6000 1/min., 3 min.), removed into 1 ml of YE-media and 100 μΐ or 200 μΐ of this mixture were plated on YE-G418 plates. These plates were cultured for several days at 28 °C.

10. Reaction conditions for PCR

The reaction conditions for the polymerase chain reaction (PCR) had to be optimized • · • · · · for each experiment, so they are not universally valid for every reaction mixture. Among other things, the amount of DNA, salt concentration and melting temperature (Schmelztemperatur) can be changed. For our conditions, it was suitable to use an Eppendorf cap designed for the Thermocycler device, into which the following substances were added: To 2 μΐ (approx. 0.1 U) of Super Taq Polymerase, 5 μΐ of Super Buffer, 8 μΐ dNTP (0.625 μΜ each), 5’-primer, 3’-primer and 0.2 pg of template DNA dissolved in such an amount of water as to obtain a total volume of the PCR reaction mixture of 50 μΐ were added. This reaction mixture was centrifuged briefly and overlaid with a drop of oil. 37 to 40 cycles were used for amplification.

The following examples describe the preparation of plasmids according to the invention, the preparation of yeast strains and their use, but the invention is not limited to the examples given.

Example 1

Expression of tHMG in S.cerevisiae AH22

The DNA sequence for tHMG (Basson et al., 1988) was amplified by standard methods using 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 obtained DNA fragment was inserted into the cloning vector pUC19 (Yanish-Perron et al., 1985) after modification according to Klenow, thus giving the vector pUC19-tHMG. After isolation of the plasmid and restriction of pUC19-tHMG with the endonucleases EcoRI and EamHI, the obtained fragment was inserted into the yeast expression vector pPT2b (Lang and Looman, 1995), which was also modified with EcoRI and EamHI. The resulting plasmid pPT2b-tHMG contained the ADH1 promoter (Bennetzen and Halí, 1982) and the TRPΙ terminator (Tschumper and Carbon, 1980), between which a tHMG-DNA fragment was inserted. A DNA segment containing the so-called middle ADH1 promoter containing tHMG and the TRPΙ terminator was isolated from the pPT2b-tHMG vector using the endonucleases EcoRV and NruI. This DNA segment was inserted into the yeast vector YEpl3 (Fischhoff et al., 1984), modified with the endonuclease SphI and DNA polymerase. The thus formed vector YEPH2 (Fig. 1) was modified with the endonucleases EcoRV and NruI.

This created a DNA fragment with the following regions:

the transcriptionally active region from the tetracycline resistance gene (Sidhu and Bollon, 1990), from the middle ADH1 promoter, from tHMG and from the TRP terminator (expression cassette). This DNA fragment was inserted into the YdpU vector (Berben et al., 1991), modified with Stul. The vector YdpUH2/12 thus formed was modified with the endonuclease Smal and ligated to the DNA sequence encoding kanamycin resistance (Vebster and Dickson, 1983). This resulting construct (YDpUHK3, Fig. 2) was modified with EcoRV. The yeast strain Saccharomyces cerevisiae AH22 was transformed with this construct. This transformation of the yeast by the action of the linearized vector according to this example leads to the chromosomal integration of the entire vector at the URA3 gene locus. In order to eliminate regions from the integrated vector that do not belong to the expression cassette (originating from E. coli, the E. coli Ampicillin resistance gene, the TEF promoter and the kanamycin resistance gene), the transformed yeast were subjected to selection pressure (Selektionsdruck) by means of FOA selection (Boeke et al., 1987), which was positively influenced by uracil-auxotrophic yeast. The uracil-auxotrophic strain resulting from this selection is designated AH22/tH3ura8 and contains the tHMG1 expression cassette as a chromosomal integration in the URA2 gene.

• * • · · · • ·

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

Cultivation conditions:

The starting material for the pre-grown VMVIII culture was as follows:

A mixture of 20 ml of VMVIII + histidine (20 gg/ml) + uracil (20 gg/ml) was inoculated with 100 μΐ of frozen culture and this mixture was incubated for 2 days at 28 °C and 120 1/min (reciprocating motion). This pre-grown culture of 20 ml was inoculated with a mixture of 100 ml of VMVIII + histidine (20 gg/ml) + uracil (20 gg/ml). In the main cultivation, 50 ml of YE (in 250 ml culture flasks) were inoculated with 1 x 10^ cells. These flasks were incubated at 160 1/min on a rotary shaker at 28 °C for 48 hours. The HMG-CoA reductase activity was then determined (method of Quareshi et al., 1981) and the following values were obtained:

Table 1

specific HMG-CoA reductase activity * (units/mg protein) AH22 3.99 AH22/tH3ura8 11.12

* Unit is defined as the conversion of 1 nmol NADPH per minute in one milliliter of reaction mixture. Measurements were performed with whole protein isolates.

» · • · ► · · · • · » · · • · · · · · • · · · · · ···« ·· · í*

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

Table 2

Squalene (% wt.) Ergosterol (% wt.) AH22 0.01794 1,639 AH22/tH3ura8 0.8361 1.7024

Percentage data are based on dry yeast.

Example 2

Expression of SÁTI in S.cerevisiae AH22

The sequence for Acyl-CoA: steroltransferase (SATI, Yang et al., 1996) was obtained by the procedure described above using PCR from genomic DNA from Saccharomyces cerevisiae S288C. The primers used were DNA oligomers SAT1-5’ and SAT1-3’ (see SEQ ID NOS. 3 and 4). The obtained DNA fragment was cloned in the cloning vector pGEM-T (Mezei and Storts, 1994) to form the vector pGEM-SAT1. After modification of pGEM-SAT1 with EcoRI, the fragment was obtained and cloned in the yeast expression vector pADH1001, which was also modified with EcoRI. The thus formed vector pADH-SAT1 was modified with endonuclease NruI and ligated to a fragment from Yep13 containing the L£Z/2 gene.

Thus, the yeast expression vector pADL-SATl was created • · « · ·« ♦·· ·· • · t ♦ · ú * · · ···· · · · » ·· • · · · · ······· ·· «··*·· ·· 9 · · (Fig. 3), which was inserted into the yeast strain AH22. The thus prepared AH22/pADL-SATl strain was incubated for 7 days in VMVIII (Lang and Looman, 1995) on Minimalmedia. Cultivation conditions: (pre-grown culture see earlier). Main cultivation: a mixture of 50 ml VMVIII + histidine (20 pg/ml) + uracil (20 pg/ml) (in 250 ml culture flasks) was inoculated with 1x10^ cells. The flasks were incubated at 160 1/min on a circular shaker at 28 °C for 7 days. The resulting sterols were analyzed by gas chromatography (see Table 3).

Table 3

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

Percentage data are based on dry yeast.

Example 3

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

Example 3.1

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

Table 4

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

Percentage data are based on dry yeast.

Example 3.2

Yeast cultures were grown for 7 days in VMVIII, but different amounts of uracil were added to the cultures. The amount of uracil in the medium was set to 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 strain AH22/tH3ura/pADL-SAT1 were significantly dependent on the amount of uracil added to the VMVIII medium.

Example 3.3

Yeast cultures were grown for 7 days in VMVIII. Then, the total amount of sterols was determined as previously described. Free sterols were extracted with n-hexane from yeast disrupted with glass beads.

The results are shown in Table 5.

« · * • · · • · · ·

- 24 - .· :

These results indicate that the enzyme sterol acyltransferase (Sat1) causes highly efficient reesterification, especially for those sterols that do not have a 4,4-dimethyl group. This means that this procedure could be used to separate 4,4-dimethylsterols from their corresponding demethylated forms.

Table 5

Percentage of free sterols. Each sterol was determined as free sterol (without saponification) and this value was related to the total amount of that sterol. The respective absolute values of the total sterol content are given in brackets, based on area/g dry matter. Lanosterol and 4,4-dimethylzymosterol are sterols containing a 4,4-dimethyl group.

% free sterols Check AH22/tH3ura/pADL-SAT1 Lanosterol 54 (0.99) 59 (2.90) 4,4-dimethylzymosterol 58 (0.77) 84 (2.37) 4-methylzymosterol 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 images in drawings

In Fig. 1 the plasmid YEpH2 is shown with the relevant interface sites.

In Fig. 2, the plasmid YDpUHK3 with the relevant interface sites is shown.

t · • · • · • · · · • ·

Figure 3 shows plasmid pADL-SAT1 with the relevant junction sites.

Figure 4 shows the cultivation behavior and the ergosterol and squalene contents at different uracil dosages. In this figure, the following means:

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

SEQUENCE OVERVIEW

(1) GENERAL INFORMATION (i) APPLICANT: (A) NAME: Schering AG (B) STREET: Müllerstrasse 178 (C) CITY: Berlin (E) COUNTRY: Germany (F) Postal code: D-13342 (G) PHONE: (030)-4681 2085 (H) FAX: (030)-4681 2058

(ii) TITLE OF THE INVENTION:

• · • · · · « · · · · » ·

PROCESS FOR THE PRODUCTION OF ERGOSTEROL AND ITS INTERMEDIATES USING RECOMBINANT YEAST (iii) NUMBER OF SEQUENCES: 4 (iv) COMPUTER-PROCESSABLE FORM:

(A) MEDIA TYPE: floppy disk (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 NO:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 bases (B) TYPE: nucleic acid (C) STRANDINGNESS: single (D) TOPOLOGY: linear (ii) HYPOTHETICAL SEQUENCE: no (iii) SEQUENCE DESCRIPTION: SEQ ID NO:

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

(i) SEQUENCE CHARACTERISTICS:

• · • 0

- 27 - 0 · · 0 0 « · • · · · · · · · * » · · * 0 ······» 0 0·· · · · · ·

(A) LENGTH: 23 bases (B) TYPE: (C) CHAIN ARRANGEMENT: nucleic acid is simple (D) TOPOLOGY: linear (i i) HYPOTHETICAL SEQUENCE: no (iii) SEQUENCE DESCRIPTION: SEQ. ID No.2:

5’ - AGTCACATGGTGCTGTTGTGCTT (2) INFORMATION FOR SEQ ID NO.3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 bases (B) TYPE: (C) CHAIN ARRANGEMENT: nucleic acid simple (D) TOPOLOGY: linear (ii) HYPOTHETICAL SEQUENCE: no (iii) SEQUENCE DESCRIPTION: SEQ. ID No.3:

5’ - GAATTCAACCATGGACAAGAAGAAG (2) INFORMATION FOR SEQ ID NO.4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 bases (B) TYPE: (C) CHAIN ARRANGEMENT: nucleic acid is simple

• · • · (D) TOPOLOGY: linear (ii) HYPOTHETICAL SEQUENCE: no (iii) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

5' - AGAATTCCACAGAACAGTTGCAGG

Review of cited literature

Basson, M.E., Thorsness, Μ. , Finer-Moore, J., Stroud, R.M., Řine, J. (1988) Structural and functional conservation between yeast and human 3-hydroxy-3-methylgluataryl coenzyme A reductases, the rate-limiting enzyme of sterol biosynthesis. Moth. Cell. Biol. 8: 3793-3808.

Bennetzen, J.L., Halí, B.D. (1982) The primary structure of the Saccharomyces cerevisiae gene for alcohol dehydrogenase. J. Biol. Chem. 257: 3018-3025.

Berben, G., Dumont, J., Gilliquet, V., Bolle, P.A., Hilger, F. (1991) The YDp plasmids: a uniform set of vectors 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 as a selective agent in yeast molecular genetics. Methods in Enzymology 154: 164-175.

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

Fischhoff, D.A., Vaterston, R.H., Olson, Μ. V. (1984) The yeast cloning vector YEpl3 contains a tRNALeu3 gene that can mutate to an amber suppressor. Genes 27: 239-251.

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

Mezei, L.M., Storts, D.R. (1994) in: PCR technology: current innovations, Griffin, H.G. and Griffin, A.Μ. , eds CRC Press, Boča Raton, 21.

Mortimer, R.K., Johnston, J.R. (1986) Genealogy of principal strains of the yeast genetic stock center. Genetics 113: 35-43.

Lang C., Looman A.C. (1995) Efficient expression and secretion of Aspergillus niger RH5344 polygalacturonase in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 44: 147-156.

Parks LV, Bottema CDK, Rodriguez RJ, Lewis TA (1985) Yeast sterols: yeast mutants as tools for the study of sterol metabolism. Meth. Enzymol. 111: 333-346.

Qureshi, N., Nimmannit, S., Porter, J.V. (1981)

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

Ruohonen, L., Aalto, M.K., Keranen, S. (1995) Modifications to the ADH1 promoter of Saccharomyces cerevisiae for efficient production of heterologous proteins. Journal of Biotechnology 39: 193-203.

Siduh, R.S., Bollon, A.P. (1990) Bacterial plasmid pBR322 sequences serve as upstream activating sequences in Saccharomyces cerevisiae. Yeast 6: 221-229.

Tschumper, G., Carbon, J. (1980). Genes 10: 157-166.

Webster, T.D., Dickson, R.C. (1983) Direct selection of Saccharomyces cerevisiae resistant to the antibiotic G418 following transformation with a DNA vector carrying the kanamycin-resistance gene of Tn903. Genes 26: 243-252.

Yang, Η., Bard, M., Bruner, D.A., Gleeson, A., Deckelbaum,

R.J., Aljinovic, G., Pohl, Τ. Μ., Rothstein, R., Sturley,

S. L. (1996) Sterol esterification in yeast: a two-gene process. Science 272: 1353-1356.

Yanisch-Perron, C., Vieira, J., Messing, J. Gene 33 (1985) 103-119.

Yu, C., Rothblatt, J.A. Cloning and characterization of the Saccharomyces cerevisiae acyl-CoA:sterol acyltransferase (1996), The Journal of Bioiogical Chemistry, 271: 2415724163.

Claims (24)

PATENT CLAIMS Process for the production of ergosterol and its intermediates, characterized in that: (a) a plasmid is first constructed into which several genes suitable for the metabolism of ergosterol are inserted; or b) first, the construction of a plasmid into which one of the genes suitable for ergosterol metabolism is inserted, c) transforming the plasmid thus produced with microorganisms, wherein said microorganisms are transformed with a single plasmid according to a) or several plasmids according to (b) 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. The method according to claim 1, wherein the plasmid into which it is introduced - 33 characterized in that a-i) the structure of the following genes is first inserted: (i) the HMG-CoA reductase (t-HMG) gene; (ii) the squalene synthetase (ERG9) gene; (iii) the acyl-CoA: sterol acyltransferase (SATI) gene; and (iv) the squalene epoxidase (ERG1) gene; constructs a plasmid into which the following genes are inserted: (i) HMG-CoA reductase (t-HMG) gene; and (ii) the squalene synthetase (ERG9) gene; or (a-iii) first construct a plasmid into which the following genes are inserted: (i) HMG-CoA reductase (t-HMG) gene; and (iii) the acyl-CoA gene: sterol acyltransferase (SATA); or (iv) a plasmid construction is first carried out in which the following genes are inserted: (i) HMG-CoA reductase (t-HMG) gene; and (iv) the squalene epoxidase (ERG1) gene; or (a-v), first, the construction of plasmids into which the following genes are inserted: the squalene synthetase (ERG9) gene; and (iii) the acyl-CoA: sterol-acyltransferase (SATA) gene; or (a-vi) first, the construction of plasmids into which the following genes are inserted: the squalene synthetase (ERG9) gene; and (iv) squalene epoxidase (ERG1) gene; or (A-vii) a plasmid is constructed first, into which the following genes are inserted: (iii) acyl-CoA gene: sterol acyltransferase (SATI); and (iv) squalene epoxidase (ERG1) gene; or (b) the construction of the plasmids is carried out, in which one of the genes mentioned under a-i is inserted, 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-vii) or several plasmids mentioned 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 also extracted from the cells and analyzed and finally (f) the ergosterol thus obtained and its intermediates are purified and isolated by column chromatography. • · · · · · · · · · · · · · · · · ¹ Method according to claim 2, characterized in that the squalene epoxidase (ERG1) gene is additionally inserted into the plasmid under a-ii), a-iii) and av) and in addition the acyl-CoA gene is inserted in plasmid a-ii): sterol acyl transferases. 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 a-vii) and in claim 3 under a-ii), a-iii (a) and (v) are first independently introduced 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-pulp 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). Process according to Claims 1 to 4, characterized in that the yeast is present. that microorganisms are • · «· • ·« 37 that the strain used is that the strain used is The method according to claim 8, characterized in that they are S. cerevisiae. A method according to claim 9, characterized by 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 SATI gene and the LEU2 gene of YEp13. Use of the plasmids according to claim 12 for the manufacture of ergosterol. Use of the plasmids according to claim 12 for the production of intermediates in the manufacture of ergosterol which are squalene, farnesol, geraniol, lanosterol, zymosterol, 4,4-dimethylzymosterol, 4-methylzymosterol, ergost-7-enol, and ergosta-5,7-dienol. Use of plasmids according to claim 12 for the production of sterols with 5-7-diene structure. 16. Expression cassettes comprising the middle ADH promoter, the HM-HMG gene and the TRP terminator and the SAT1 gene with the middle ADH promoter and the TRP terminator. 17. 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 intermediate ADH promoter and TRP terminator. 18. Combination of expression cassettes, forming the combination a) the first cassette on which the ADH promoter, t-HMG gene and TRP terminator are located b) a second expression cassette on which the ADH promoter, SAT1 gene and TRP terminator is located 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 the transformation 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 18. 22. A microorganism according to claim 21, characterized in that it is a yeast. 23. Use of a microorganism according to claims 21 and 22 for fer39 meirtation to ergosterol. Use of a microorganism according to claims 21 and 22 for fermentation to ergosterol intermediates.