DD259637A1 - PROCESS FOR THE PREPARATION OF ISOZITRATLYASE IN RECOMBINANT MICROORGANISMS - Google Patents

Abstract

The invention relates to a process for the preparation of isocitrate lyase, recombinant microorganisms used being both bacterial and yeast strains. The invention aims to produce said enzyme with increased effectiveness. The object associated with this object is essentially solved in such a way that microorganism cells which have been transformed with one of the expression plasmids produced in the process, which in turn regulates one of its native expression unit in an insert coding for the amino acid sequence of isocitrate lyase DNA (ICL1 gene from the donor strain Yarrowia lipolytica H222-27), cultured in a liquid nutrient medium under aerobic conditions and then the gene product is isolated from the cell extract. As a result of the method, the shuttle expression plasmids pYLB40 and pYLB90 as well as various ICL1 recombinant E. coli and yeast strains, the latter from the species Yarrowia lipolytica, Saccharomyces cerevisiae and Pichia pinus, have become available as pBR322 derivatives in particular.

Description

Field of application of the invention

The invention relates to a microbiological production process for the enzyme isocitrate lyase (C 4.1.4.1) using genetically modified microorganism strains.

The enzyme isocitrate lyase (shorthand: ICL) - it converts isocitrate to glyoxylate and succinate - has been detected except in mammalian cells in a variety of pro- and eukaryotic microorganisms as well as plant and other animal cells. Isocitrate lyase has gained importance as a biochemical and a fine chemical, it is used in a variety of forms for the detection of isocitric acid and compounds.

The field of application of the invention is thus primarily in the production of biochemicals and fine chemicals. In addition, the inventive solution for genetic and biotechnological research of importance.

Characteristic of the known state of the art

The enzyme isocitrate lyase has hitherto been obtained from natural microbial production strains by the well-known methods of enzyme isolation and purification. However, these processes are very material and costly in the case of ICL extraction because the enzyme is synthesized in the cells of the native formers only in relatively small quantities. ICL overproducing microorganisms or cell cultures are not yet known (S.Fukui et al., Takaaka, in: A. Flechter [Ed.], Products from Alkanes, Cellulose and other Feedstocks, Akademie-Verlag, Berlin, 1981).

ICL production by recombinant, d. H. Genetically modified microorganisms have so far been described neither in the patent literature nor in scientific publications. Recently, only the isolation of a gene coding for an isocitrate lyase (name: ACUD gene) from the filamentous fungus Aspergillus nidulans (D. J. Ballance and G. Turner, Mol. Gen. Genet., 202, 1886, 271) has recently been disclosed. However, expression of this ACUD gene in heterologous microorganism species was not investigated. Nor has any attempt been made to achieve an increase in microbial ICL production based on a genetic engineering construction which results in an increased copy number per transformant cell for the ACUD gene.

Object of the invention

The invention aims to produce the enzyme isocitrate lyase in genetically engineered microorganism cells, optionally in both bacterial and yeast cells, with increased efficiency.

Explanation of the essence of the invention

The invention has for its object to describe a genetic engineering-microbiological process, with the selected microorganism strains, both bacterial and yeast strains are produced, which have the ability to synthesize the enzyme isocitrate lyase

 According to the invention, this object is achieved in its basic feature as follows:

With an expression plasmid of the pYLB family respectively prepared using standard genetic engineering operations of the DNA linkage, which carries a DNA coding for its amino acid sequence of the enzyme isocitrate lyase, regulated by its native expression unit (short description: ICL1 gene), microorganism cells are grown in transformed in a known manner. The transformed microorganism cells are cultured under aerobic conditions in a liquid nutrient medium containing assimilable sources of carbon and nitrogen as well as mineral salts, and the gene product synthesized according to the method, i. the isocitrate lyase is finally isolated in a manner known per se from the cell extract.

This basic feature of the method is initially further eliminated in its gene technology part in that the pYLB expression plasmid used is an ICLI-recombinant shuttle derivative of an E. coli cloning vector, preferably a derivative of the vector pBR 322. For the construction of such pYLB shuttle plasmids, an ICL1 gene-carrying fragment from a yeast Yarrowia lipolytica strain is used. Preferably, a citric acid-producing strain of this yeast is used as the gene donor

 In particular, the shuttle expression plasmids of the pYLB type are prepared by

Genomic DNA of the respective Y. lipolytica donor is initially fragmented with a small-cutting restrictase, for example with BamHI, EcoRI or BglII,

The resulting digestion mixture is then inserted into an E. coli cloning vector which has been linearized in the associated or in a compatible restriction-site cleavage site,

- transformed with the ligation mixture obtained in this way - either directly or, if necessary, after intermediate cloning in a restriction-negative E. coli host-ICL-negative E. coli recipient,

- Thereafter, the transformed cells are cultured for selection on ICL1 recombinant clones in a medium with acetate or ethanol as the sole carbon source, and

- From the replicable E. coli transformants, the plasmid DNA is finally isolated in a known manner. In this way, for example, as pBR322 derivatives pYLB shuttle-type expression plasmids are produced whose molecular size is mainly in the range of 7.0 kg to 9.0 kb.

Mjt DNA samples of an ICL1-recombinant expression plasmid of the pYLB family constructed according to the above partial method are transformed in the continuation of the overall production process now both ICL-negative bacterial production strains and ICL-negative yeast production strains. Bacterial strains of the species E. coli, on the one hand, and strains from the yeast genera Yarrowia, Accaromyces or Pichia, on the other hand, are preferably selected for this partial section.

In the biotechnological part of the basic feature of the method is further designed so far that in one variant each transformed with an ICL1 recombinant pYLB expression plasmid E. coli production strain, ie each "ICL1 recombinant E.coli strain obtained according to the method, in a Nursing medium containing acetate and / or ethanol as the sole carbon source is cultivated at temperatures of 28 ° C to 40 ⁰ C and acidities of pH 6.5 to pH 8.5 for a period of 4 hours to 48 hours.

In the other variant of the present method, each method according to the method prepared ICLI-recombinant yeast strain in a nutrient medium containing as carbon source alcohols, acetate, fatty acids and / or n-alkanes, at temperatures of 25 C to 40 C and Aciditäten of pH 4.5 to pH 7.0 for a period of 6 hours to 48 hours. In a preferred embodiment of the method, the ICL1 gene-carrying fragments from the donor strain Y.Iipolytica H 222-27, whose genomic DNA is digested for this purpose with the small-cutting restrictase EcoRI, recovered. The digestion mixture obtained in this way (see Fig. 1) is then ligated in this preferred form with EcoRI-cut DNA of the plasmid pBR322. To suppress self-ligation, this reaction uses dephosylated linearized DNA from pBR322. As a result of this ligation variant arise. Recombinant plasmids in which both antibiotic resistance markers of the parent vector are intact.

Cells of the known restriction-negative recipient E. coli SK1590 are transformed with the ligation mixture obtained. From the gene bank for genomic H 222-27 DNA obtained by this intermediate cloning, the entire mixture of the recombinant pBR322 derivatives is removed and converted to the ICL-negative E. coli strain K8-5m. The transformed cells are then cultured in a medium containing both acetate (as sole carbon source) and tetracycline and / or ampicillin for selection. This cultivation can be carried out, for example, on an acetate agar.

From the replicable transformants of this variant of the preferred embodiment of the method, the DNA of an ICL1 recombinant new shuttle expression plasmid, which was named pYLB40, can then finally be isolated in a manner known per se. This plasmid has an approximately 4.3kb ICLI gene-carrying insert from the H222-27 genome; its resulting molecular size is thus 8.6kb.

In another variant of the preferred embodiment of the method, the intermediate cloning is omitted in a restriction-negative E. coli recipient; rather, the ligation mixture obtained after the insertion of EcoRI-fragmented H222-27 DNA into EcoRI-linearized pBR322-OES is used directly to transform K8-5m cells. The transformant culture is introduced after a short incubation in complete medium (LB medium plus ampicillin and / or tetracycline) in liquid selective medium with acetate as the sole carbon source and also tetracycline and / or ampicillin addition and cultured, so that again only the recombinant clones are capable of replication.

In this variant of the preferred embodiment of the method, the DNA of a second novel ICLI recombinant shuttle expression plasmid can be isolated from the resulting transformants. For this plasmid, which was named pYLB90, the molecular size is 8.3 kb. Its approximately 4.0kb insert has a different restriction pattern compared to the pYLB40 insert (see Figures 2 and 3).

The preferred embodiment is further characterized in that when using bacterial production strains for the industrial production of isocitrate lyase the E. coli strains K8-5 m and SM1104 are used. These strains are optionally transformed with both pYLB40 and pYLB90 DNA. Transformant clones of said E. coli strains are cultured in the preferred embodiment in a fermentation vessel under the conditions mentioned above, but preferably at a temperature of 37 ° C.

On the other hand, if the ICL production process is carried out using yeast production strains, then in the pYLB40 as in the pYLB90 case, the technical recovery of the enzyme in the corresponding transformants of the ICL-negative parent strains

Yarrowia lipolytica B204-12C-212, Sacaromyces cerevisiae IICI 1-1A, and Pichiapinus N798

performed.

The Y. lipolytica strain mentioned here is a highly stable mutant of the known starting strain Y.üpolytica B 204-12 C. To increase the transformation rate, in this variant of the preferred embodiment, linearized, in particular EcoRI or BglII cut, is used for this purpose provided pYLB40 or pYLB90 DNS. In each of said recombinant yeast production strains is an integrative transformation of pYLB DNA.

Transformant clones of said yeast strains are then cultivated in this preferred method form in a fermentation vessel under the conditions mentioned above, in particular at a temperature of 28C.

For the ICL1-recombinant E. coli strains fermented in this way, ICL enzyme activities of 50 μΜΜΙ of reacted isocitrate / hr. reached χ mg protein; for the yeast strains fermented by this method, the enzyme activities of ICL ranged from 10μ.ΜοΙ to 120μ.ΜοΙ of reacted isocitrate / hr. χ mg protein (see table).

In connection with the invention described above are in the depository for microorganisms of the GDR, Central Institute of Microbiology and Experimental Therapy AdW, DDR-6900 Jena, Beutenbergstr. 11 following root deposits have been made:

Root registration number

Yarrowia lipolytica H 222-27 ZIMET43 728

Yarrowia lipolytica B 204-12C ZIMET43 834

E. coli HB101 (pYLB90) ZIMET11236.

The main advantages of the described invention are seen in that

The gene product isocitrate lyase can be produced in particular in the production strains of the species E. coli used with a significantly increased amount of enzyme compared to the prior art, and

In carrying out the method, DNA fragments with the native ICL 1 gene of the donor strain Y. lipolytica H 222-27 have become available for further genetic engineering research.

embodiments Example 1)

1.1) Isolation of chromosomal DNA from strain Y. lipolytica H 222:

10 ml liquid YEP-G medium (1% yeast extract, 2% peptone, 2% glucose, pH 6.5) are inoculated with a loop of the strain Y. lipolytica H222-27 and cultured for 24 hours at 28 ° C with shaking. With 4 ml of this yeast cell suspension, 80 ml liquid YEP-G medium are inoculated and shaken for 12 hours at 28 ° C. 1.51 mitThiamin minimal medium and 0.4% acetate as sole carbon source (BARTH & KUNKEL, Z.AIIg. Microbiol., 19.1979, 381-390) are inoculated with 75 ml of this culture broth and 12 hours at 28 ⁰ C with shaking incubated. These cells are harvested by centrifugation, resuspended with 50 ml of 0.9% NaCl solution, spun down and then resuspended in 50 ml wash buffer (40 mM Tris, 50 mM EDTA, pH 7.5). The cells are then recentrifuged and resuspended in 25 ml of TES buffer (0.2 M Tris, 0.02 M EDTA, 1 M NaCl, pH 8.0). 1.5mi of β-mercaptoethanol and 0.5 g of a yeast cell wall-dissolving enzyme preparation are added to this cell suspension. After incubation for 25 minutes at 37 ^° C., the resulting spheroplasts are centrifuged off and washed once with 50 ml of TES buffer. The cell mass is taken up in 30 ml lysis buffer (50 mM Tris, 15 mM NaCl, 10 mM EDTA, pH 8.0); then 10 mg Proteinase Kund is added to 2 ml of a 20% sodium dodecyl sulphate solution. The mixture is left for 1 hour at 37 ⁰ C with occasional gentle shaking. With 50 ml of cold, water-saturated phenol, the lysate is extracted twice, the aqueous phase is carefully collected after centrifuging jewe.ils. This aqueous supernatant containing the DNA * is shaken out twice with distilled ether. The residual ether is removed under vacuum. The aqueous DNS solution is mixed with 0.1 volume of 3 M sodium acetate (pH 8.0) and overlaid with 2.5 volumes of pure ethanol. With a rod, both phases are mixed, whereby the precipitating DNA is carefully wound around the rod.

The DNA collected by means of the bar is washed with 70% ethanol, dried in vacuo, and iiVIOmlTE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) is dissolved in the refrigerator overnight. To this solution is 100μΙ RNase solution (10 mg RNAse A / ml double-distilled water.) Was added and after 1 hour, incubated at 37 ⁰ C again. By twice shaking with cold, water-saturated phenol and subsequent treatment with ether, the RNAse is eliminated again. Subsequently, VioVolumenteile 3M sodium acetate; pH 8.0; and 0.54 parts by volume of isopropanol. The precipitated DNA is separated by centrifugation, washed with 70% ethanol three times and then dissolved in 3 ml of sterile TE buffer overnight at 4 ° C. The thus isolated DNA is measured at 260 nm and 280 nm to determine the DNA content and the protein content. Agarose gel electrophoresis (0.4%) is used to determine the average fragment size of DNA recovered

 In this way, about 2.6 mg of H222-27 DNA with a fragment size greater than 50 kb was recovered.

1.2) Cleavage of the DNA of Y. lipolytica H 222-27 with the restrictive endonuclease EcoRI

2 / ^ g of the obtained according to point 1.1) genomic DNA are in TM buffer (1OmM Tris, 1OmM digested MgCl ₂ 1 mM Dithiothreitol) and 10OmM NaCl restrictive with 10 units of endonuclease Eco RI at 37 ⁰ C for 1 hour, pH 7.8. 15μΙ of this approach are mixed with 5μΙ stoppers (50% glycerol, 7.45% EDTA, 0.25% bromophenol blue) and applied to a 0.7% agarose gel to control fragmentation of the DNA (Figure 1).

After digestion, the mixture is shaken twice with cold phenol and ether. Subsequently, the DNA is precipitated with ethanol, washed with 70% ethanol and dried in vacuo. The DNA is dissolved in double-distilled sterile 10μΙ Aqua and stored until further use at +4 ⁰ C.

1.3) Cleavage of the plasmid pBR322 with the endonuclease EcoRI and dephosphorylation of the DNA: ends

3 μg of the plasmid pBR322 are cleaved with 15 units of EcoRI under the conditions given above. The DNA linearized in this manner is treated with calf intestinal phosphatase according to PERBAL protocol (A Practical Guide to Molecular Cloning, John Wiley & Sons, 259, 1984) to prevent religation of the vector without yeast DNA insert. ·

1.4) Ligation of genomic DNA from Y. lipolytica H 222-27 with pBR 322, transformation to E. coli and isolation of a recombinant plasmid

2 μg of the EcoRI digested genomic DNA of strain H222-27 are digested with 3 μg of EcoRI-linearized, dephosphorylated pBR322 DNA in ligation buffer (25 mM Tris, pH 7.8, 10 mM MgCl ₂ , 1 mM DTT, 0.4 mM ATP ) (final concentration: 5 μg DNA / ml) and incubated for 1.8 hours at 8 ° C. after addition of 1 unit of T4 ligase. The ligation mixture is divided into portions of 200 / Ag DNA and transformed to 200 μΙ of competent cells of strain E. coli K8-5m. The transformation of this strain is carried out by the CaCl ₂ method (MANIATIS et al., Molecular Cloning, Cold Spring Harbor Laboratory, NY, 1982). The transformation mixture is incubated in liquid LB medium (10 g bacto-yeast extract, 5 g NaCl, pH 7.5) with ampicillin (50 / xg / ml) for 1 hour at 37 ° C.

The cells are then centrifuged off and washed with 10 ml minimal medium supplemented with sodium acetate (0.4%), methionine (25 / xg / ml), ampicillin (25 ^ g / ml) and tetracycline (10 ^ g / ml) ((MMA + A + T) medium). These cultures are shaken at 37 ° C. After 5 days of culture, the cells from growing cultures (MMA + A + T) ausgespatelt agar plates and re-incubated at 37 ⁰ C. Of the colonies grown on these plates, 20 each per batch are inoculated and tested for the presence of recombinant plasmids by the minipreparation method of HOLMS & QUIGLEY (Anal. Biochem., 114, 193-197, 1981).

In this way, the plasmid pYLB90 was isolated, which contains a chromosomal DNA sequence from H 222-27 of about 4.0 kb. This chromosomal DNA insert was digested with various restrictive endonucleases by standard methods to establish the appropriate sites. The resulting physical map of this DNA sequence is shown in Figure 3.

1.5) Preparation of Isozitratlyase in E.coli K8-5m - cells of the recombinant strain E. coli K8-5m (pYLB90) are cultured in 10 ml LBTetrazyklin and / or ampicillin for 20 hours at 37 ⁰ C. The cells are then centrifuged off, washed once with MMA and resuspended in 11 MMA + methionine (25 μg / ml). After 48 hours of cultivation at 37 ⁰ C, the E. coli cells are centrifuged off and then

unlocked by homogenization. The determination of the specific activity of Isozitratlyase and the isolation and purification of this enzyme is carried out in a conventional manner. The specific activity of ICL detected in the cell extract of E. coli K8-5m (pyLB90) is shown in Table 1.

1.6) Linearization of the plasmid pYLB90 and transformation of Y. lipolytica B204-12C-212

10 μl of isolated DNA from plasmid ρYLB90 are linearized in TM buffer with the aid of the enzyme BglII (20 units) in the presence of 10 mM NaCl. The linear DNA thus obtained is used for the transformation of Y. lipolytica strains, especially of strain B 204-12C-212, according to the modified lithium acetate method (ITO et al., J. Bacteriol., 153, 1083, 163-168). used. In this case, 50 ml of a YEP-G culture of strain B 204-12C-212 from the late logarithmic growth phase (5 × 10 ⁷ cells / ml) are centrifuged off, with 25 ml of sterile TE buffer; pH 7.5; washed, centrifuged again and taken up with 8 ml lithium acetate solution (0.1 M Li-acetate in TE buffer, pH 7.5). The cell suspension is shaken slowly at 28 ° C for one hour, then centrifuged, and the cells are resuspended in 1 ml lithium acetate solution. 300μΙ each of this suspension are placed in 2 sterile tubes to which each 50 / xg fractionated fish sperm DNA is added. To a tube is added 10 / xg of the linearized DNA of pYLB90 (in 50 μM TE buffer), while the second tube (serving as a control) is filled with 50 μM of sterile TE buffer. Both tubes are incubated at 28 ° C for 30 minutes. Thereafter, 1 ml of a 40% polyethylene glycol 4000 (PEG) solution (in 0.1 M Li-acetate solution) is added to each batch, mixed and incubated for 1 hour at 28T. Subsequently, both tubes are incubated for 8 minutes at 37 ° C and then cooled in an ice bath. The cells are centrifuged off, in sterile A. dist. resuspended and plated on MMT acetate + methionine agar (BARTH & KUNKEL, supra).

In this way it was possible to isolate 35 transformants per μg of DNA, which were designated Y. lipolytica B 264-12 C-121 (pYLB90).

1.7) Preparation of isocitrate lyase in strain Y. lipolytica B204-12C-212 (pYLB90)

From a YEP-G culture of strain B204-12C-212 (pYLB90) in the stationary growth phase 10 ml are removed, centrifuged, with sterile A. dist. washed and transferred to 11MMT acetate supplemented with methionine. The cell culture is shaken for 8 hours at 28 ° C. Thereafter, the cells are centrifuged off. The determination of the specific activity of isocitrate lyase is carried out according to BARTH (Curr Genet., 10, 1985, 194-124). The isolation and purification of the isocitrate lyase is carried out in a manner known per se

 The specific activity of isocitrate lyase detected in B204-12C-212 (pYLB90) is shown in Table 1.

Example 2)

2.1) Ligation of genomic DNA from Y. lipolytica H 222-27 with pBR322; Transformation to E. coli and isolation of a recombinant plasmid

As described in Example 1 under 1.4, 2 ug of the EcoRI-cut genomic DNA of strain H222-27 with 3 ug EcoRI-linearized, dephosphorylated pBR322 DNA are ligated. The ligation mixture is divided into portions of 200 .mu.-g of DNA and transformed per 200 .mu.Ι competent cells of the strain E. coli SK1590 in a conventional manner. The transformation mixtures are spiked onto LB agar plates with ampicillin + tetracycline and incubated for 24 hours at 37 ° C. Thereafter, washed all washed clones (about 20,000) with 10ml LB, mixed with ampicillin and tetracycline, and shaken for 16 hours at 37 ° C in a 50ml Steilbrustkölbchen. From the E. coli cells, the plasmid DNA is isolated by the method of HOLMS & QUIGLEY (a.a.

The strain E. coli SM1104 is subsequently transformed with this mixture of recombinant plasmids. The transformation mixtures are spiked onto (MMA + A + T) agar plates and incubated at 37 ° C. The colonies washed on these plates are inoculated and tested for the presence of recombinant plasmids by the minipreparation method of HOLMS & QUIGLEY (supra).

In this way, the plasmid pYLB40 was isolated, which contains a chromosomal DNA sequence from H 222-27 of about 4.3 kb. ,

2.2) Preparation of isocitrate lyase in E. coli SM 1104 (pYLB40) isocitrate lyase is prepared from the strain SM 11β4 (pYLB40) in the same manner as described in Example 1 under 1.5) for the case of the host organism K8-5m (pYLB90).

Example 3)

3.1) Transformation of strain Saccharomyces (Sacch.) Cerevisiae icl1-1 A with plasmid pYLB90

10 ml of YEP-G are injected with a vaccine loop of the strain Sacch. cerevisiae icl 1-1 A and shaken at 28 ° C for 24 hours. 2 ml of this culture solution are then transferred into 100 ml YEP-G and incubated again at 28 ° C for 10 hours with shaking. The cells of this suspension are centrifuged off, washed once with 10 ml of TE buffer and resuspended in 10 ml of lithium acetate solution (0.1 M in TE buffer, ρ 7.5), centrifuged again and then with 2 ml of 0.1 M lithium acetate Solution added. This cell suspension is allowed to stand for 1 hour at 28 ° C. 300μΙ of this culture are mixed ηηίίδΟμΙ pYLB90 solution (containing 10μg plasmid DNA) and 100μΙ of a genomic DNA from E. coli (50μg DNA) and then incubated at 28 ° C for 30 minutes. 2.1 ml of a 40% solution of polyethylene glycol 4000 in 0.1 M lithium acetate solution are added and then a heat shock treatment is carried out at 42 ° C for 5 minutes. The cells are centrifuged off, washed twice with TE buffer and taken up in 0.4 ml of TE buffer.

This suspension is spiked onto acetate agar plates (MMT acetate agar according to BARTH & KUNKEL, supra). After 7 days incubation of these agar plates at 28 ° C, the washed colonies are inoculated on Schrägargarröhrchen with MMT acetate agar, incubated for two days at 28 ⁰ C and then stored in the refrigerator.

3.2) Preparation of isocitrate lyase in strain Saccharomyces cerevisiae icl 1-1A (pYLB90)

The preparation of isocitrate lyase in Saccharomyces cerevisiae icl 1-1 A (pYLB90) is carried out as described in Example 1 under 1.7) for the case of the host organism Y. lipolytica B204-12C-212 (pYLB90).

Legends to the 3 pictures:

Fig. 1: Chromosomal DNA of Y. lipolytica digested with the restriction enzyme EcoRI Fig. 2: Cleavage of the plasmids pBR322, pYLB40 and pYLB90 with different restrictases Fig. 3: Restriction map of the DNA insert of Y. lipolytica H 222-27 in plasmid pYLB90

Table 1: Specific activities of the enzyme isocitrate lyase in E. coli and Y. lipolytica transformed with plasmid pYLB90 compared to the untransformed isocitrate lyase-negative mutants

strain designation

ICL1 gene Specific activities

derlCLInMol / mgxh)

E.coli K8-5m E. coli 8-5m (pYLB90) Y. lipolytica B 204-12 C-212 Y.iipolytica B204-12C-212 (pYLB90)

4 42

0 36

Claims (22)

claims: 1. A process for the preparation of Isozitratlyase in recombinant microorganisms using standard genetic engineering operations of DNA linkage, characterized in that - With an expression plasmid of the pYLB series, which carries a regulated by their native expression unit, for the amino acid sequence of the enzyme isocitrate lyase encoding DNA (ICL1 short gene), transformed microorganism cells in a liquid nutrient medium with assimilable carbon and nitrogen sources and mineral salts are cultured under aerobic conditions and - The gene product isolated from the cell extract. 2. The method according to claim 1, characterized in that is used as an expression plasmid ICL1 recombinant shuttle derivative of an E. coli cloning vector. 3. The method according to claim 2, characterized in that is used as an expression plasmid ICL1 recombinant shuttle derivative of the E. coli vector pBR322. 4. The method according to claim 1 to 3, characterized in that one provides the ICL1 gene-carrying insert for the shuttle expression plasmids from a strain of the yeast Yairrowia lipolytica. 5. The method according to claim 4, characterized in that one provides the ICL1 gene-carrying insert for the shuttle expression plasmids from a citric acid-producing Y.Iipolytica strain. 6. The method according to claim 5, characterized in that one provides the ICL1 gene-carrying insert for the shuttle expression plasmids from the Y. lipolytica strain H 222 or from a derivative of this strain. 7. The method according to claim 1, 2,4 and 5, characterized in that one produces an ICL1 recombinant expression plasmid of the pYLB family as a shuttle derivative of an E. coli cloning vector by Genomic DNA of the respective Y. lipolytica donor strain fragmented with a small-cutting restrictrase, The inserted digestion mixture is inserted into an E. coli cloning vector linearized in the associated or in a compatible cleavage site, If necessary, ICL-negative E.coli recipients are transformed with the ligation mixture obtained after intermediate cloning in a restriction-negative E. coli strain, - Cultured the transformed cells for selection in a medium with acetate or ethanol as the sole carbon source and - isolated from the replicable E. coli transformants, the plasmid DNAin a conventional manner. 8. The method according to claim 1, 3, 6 and 7, characterized in that the ICL1 recombinant expression plasmid pYLB40 (molecular size 8.6 kb) is prepared by Genomic DNA of the donor strain Y. lipolytica H 222-27 fragmented with the EcoRi restriction gene, The resulting digestion mixture is inserted into the EcoRI site of plasmid pBR322, - transformed with the ligation mixture obtained after Zwischenkolonierung in restriction-negative E. coli recipient SK1590 the ICL-negative E. coli strain K8-5m, - The transformed cells for the purpose of selection in a medium containing both acetate as the sole carbon source and tetracycline and / or ampicillin, cultured and - isolated from the replicable transformants, the pYLB40 DNA in a conventional manner. , 9. The method according to claim 1, 3, 6 and 7, characterized in that the ICL1-. recombinant expression plasmid pYLB90 (molecular size 8,3kb) is prepared by Genomic DNA of the donor strain Y. lipolytica H 222-27 fragmented with the EcoRi restriction gene, The resulting digestion mixture is inserted into the EcoRI site of plasmid pBR322, Transformed with the obtained ligation mixture the ICL-negative E. coli strain K8-5m, - The transformed cells for the purpose of selection in a medium containing both acetate as the sole carbon source and tetracycline and / or ampicillin, cultured and - isolated from the replicable transformants, the pYLB90 DNA in a conventional manner. 10. The method according to claim 1 and 7, characterized in that is transformed with DNA samples of an ICL1 recombinant expression plasmid of the pYLB family ICL-negative bacterial production strains. 11. The method according to claim 1,7 and 10, characterized in that transformed with DNA samples of an ICL1 recombinant expression plasmid of the pYLB family ICL-negative production strains of the species E. coli. 12. The method according to claim 1 and I _1, characterized in that is transformed with DNA samples of an ICL1 recombinant expression plasmid of the pYLB family ICL-negative yeast production strains from the genera Pichia-Saccharomyces and Yarrpwia. 13. The method according to claim 1,8 and 11, characterized in that one transforms the ICL-negative E. coli production strains K8-5m and SM1104 with DNA samples of the ICL1 recombinant plasmid ρYLB40. 14. The method according to claim 1, 9 and 11, characterized in that one transforms the ICL-negative E. coli production strains K8-5m and SM1104 with DNA samples of the ICL1 recombinant piasmide pYLB90. 15. The method according to claim 1,8 and 12, characterized in that one transforms the ICL-negative production strain Pichia pinus N 789 with samples of EcoRiged DNA of the ICL1 recombinant plasmid pYLB40. 16. The method according to claim 1, 9 and 12, characterized in that one transforms the ICL-negative production strain Pichia pinus N 789 with samples of BGIII-cut DNA of the ICL1 recombinant plasmid pYLB90. 17. The method according to claim 1,8 and 12, characterized in that one transforms the ICL-negative production strain S. cerevisiae icl-1-1 A with samples of EcoRI-cut DNA of the ICL1 recombinant plasmid pYLB40. 18. The method according to claim 1, 9 and 12, characterized in that "is transformed with samples of BglII-cut DNA of the ICL1 recombinant plasmid pYLB90 the ICL negative production strain S. cerevisiae icl 1-1A. 19. The method according to claim 1,8 and 12, characterized in that the ICL-negative production strain Y. lipolytica B204-12C-212, a highly stable mutant of strain Y. lipolytica B with samples of EcoRiged DNA of the ICLI recombinant plasmid pYLB40 204-12 C, transformed. 20. The method according to claim 1, 9 and 12, characterized in that the ICL-negative production strain Y.lipolytica B204-12C-212 is transformed with samples of BglII-cut DNA of the ICLI recombinant plasmid pYLB90. 21. The method according to claim 1,11,13und14, characterized in that the transformed with an ICL1 recombinant pYLB plasmid E. coli strains in a nutrient medium containing only acetate and / or ethanol as a carbon source, at temperatures from 28 ° C to 40 ° C, preferably 37 ⁰ C, and cultivating acidities of pH 6.5 to ρ 8.5 for a period of 4 hours to 48 hours. 22. The method according to claim 1,12 and 15 to 20, characterized in that the transformed with an ICLI recombinant pYLB plasmid yeast strains in a nutrient medium, which as a carbon source alcohols, acetate, fatty acids and / or n- Alkanes, cultivated at temperatures of 25C to 40C, preferably 28C, and acidities of pH 4.5 to pH 7.0 for a period of 6 hours to 48 hours. For this 3 pages drawings