GB2188050A - pWS101 plasmid, method of obtaining same and use thereof - Google Patents

Abstract

The invention relates to the novel pWS101 plasmid, which is obtained from a gene of Saccharomyces cerevisiae yeast, as well as its use for the production of the enzyme L-aspartate : 2-oxoglutarate aminotransferase E.C.2.6.1.1. An object of the invention is to provide a means for high expression of the enzyme L-aspartate : 2- oxoglutarate aminotransferase, E.C.2.6.1.1., so that production is achieved with minimal expenditures of time and money, as well as easily and with a high degree of purity. This is achieved by the pWS101 plasmid, characterised by the features: 1) it is capable of being multiplied in microorganisms of the species Saccharomyces cerevisiae and of the species Escherichia coli; 2) it carries the ASP5 gene of Saccharomyces cerevisiae, which encodes the enzyme L-aspartate : 2- oxoglutarate aminotransferase, E.C.2.6.1.1., on 3) a "shuttle" vector containing a centromere region (CEN4) of Saccharomyces cerevisiae (YCp50). A novel pWS101 plasmid comprising a vector containing the CEN4 centromere region of Saccharomyces cerevisiae, and the ASP5 gene of Saccharomyces cerevisiae encoding the enzyme L- aspartate : 2-oxoglutarate aminotransferase (E.C.2.6.1.1), the plasmid being capable of multiplying in a microorganism selected from the group consisting of Saccharomyces cerevisiae and Escherichia coli. A process for cloning the plasmid and a simple method of producing highly pure 1-aspartate : 2-oxoglutarate aminotransferase (E.C.2.6.1.1) comprising high expression of the enzyme gene with low expenditure of time and money.

Description

SPECIFICATION pWS101 plasmid, method of obtaining same and use thereof BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to the novel pWS101 plasmid and to a method of obtaining the plasmid utilizing Saccharomyces cerevisiae yeast DNA. This invention also relates to the use of the novel plasmid for the production of the enzyme L-aspartate: 2-oxoglutarate aminotransferase (E.C.2.6.1.1).

Description of the Background The enzyme L-aspartate: 2-oxoglutarate aminotransferase (E.C.2.6.1.1) is used in clinical chemistry for the production of control sera. In the past, this enzyme was isolated from porcine hearts. The prior art procedure for isolating the enzyme only yields limited amounts of the enzyme as determined by the availability of porcine hearts as the starting material.

SUMMARY OF THE INVENTION It is an object of the invention to provide a means for high expression of the enzyme Laspartate: 2-oxoglutarate aminotransferase (E.C.2.6.1.1) with minimal expenditures of time and money. The present method is simpler than the method of purifying the enzyme from porcine hearts and attains higher yields.

This and other objects are attained according to the present invention by the pWS101 plasmid comprising the YCp5O vector containing the CEN4 centromere region of Saccharomyces cerevisiae, and the ASP5 gene of Saccharomyces cerevisiae encoding the enzyme L-aspartate: 2oxoglutarate aminotransferase (E.C.2.6.1.1), wherein the plasmid is capable of being multiplied in a microorganism selected from the group consisting of Saccharomyces cerevisiae and Escherichia coli.

This plasmid makes possible the microbial production of the above-mentioned enzyme.

The above and other objects are also attained by a process for obtaining the pWS101 plasmid, comprising (a) obtaining and amplifying the YCp50 vector in a 290 A Escherichia coli bacterium; (b) incubating the YCp5O vector with Bam H1 restriction endonuclease enzyme in a proportion thereof and under reaction conditions effective to attain complete restriction of the vector; (c) obtaining chromosomal yeast DNA and incubating the DNA with Sau 3A restriction endonuclease enzyme in a proportion thereof and under reaction conditions effective to attain partial restriction of the DNA and obtain DNA fragments of different sizes; (d) isolating DNA fragments of a size between about 3 kilobase pairs (kbp) and 9 kbp;; (e) cloning the isolated DNA fragments into the restricted vector obtained in step (6) in a proportion thereof and under conditions effective to obtain a plasmid carrying the isolated DNA fragments; (f) incubating a 290 A Escherichia coli bacterium with the plasmid carrying the isolated DNA fragments in a proportion thereof and under conditions effective to attain the transformation of the bacterium; (g) selecting and combining at least 10,000 ampicillin; (h) amplifying and purifying the combined transformants under conditions effective to obtain a gene bank containing at least one pWS101 plasmid; (i) incubating a ura3 and asp5 Saccharomyces cerevisiae yeast mutant with the gene bank obtained in step (h) in a proportion and under conditions effective to attain the transformation of the mutant;; (j) selecting uracil and aspartate prototrophic transformants from the transformants of the mutant obtained in step (i) containing the pWS101 plasmid; and (k) isolating the pWS101 plasmid.

The above and other objects are attained as well by a method of preparing the enzyme Laspartate: 2-oxoglutarate aminotransferase (E.C. 2.6.1.1) comprising incubating the pWS101 plasmid in Saccharomyces cerevisiae yeast for a period of time and under conditions effective to obtain the enzyme.

This and other objects are also attained by a process for the production of the amino acid asparagine comprising overproducing the enzyme, which in turn converts aspartate and oxoglutarate to asparagine, thereby obtaining the amino acid in the growth culture. The amino acid may then be separated from the enzyme and the rest of the growth culture.

A more complete appreciation of the invention and many of the attendant advantages thereof will be readiiy perceived as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWING The figure depicts a preliminary endonuclease restriction map of the novel plasmid pWS101.

Other objects, advantages and features of the present invention will become apparent to those skilled in the art from the following discussion.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The method of producing the desired enzyme utilizing the pWS101 plasmid as a starting material utilizes techniques which individually are standard in the art of molecular yeast genetics.

One significant feature in the production of the enzyme is the molecular cloning of the Saccharomyces cerevisiae yeast gene in a vector, thereby encoding the enzyme in a plasmid vector multipliable in the Saccharomyces cerevisiae yeast and in the Escherichia coli bacterium.

In addition, the pWS101 plasmid can also be used for the microbial production of asparagine.

This is attained by overproducing the enzyme, which in turn leads to the transformation of cellular substrates into the amino acid asparagine. The amino acid is in turn excreted by the microorganism into the growth medium from which it is purified by known methods. The procedures utilized herein are also individually known in the art.

The present method for generating a yeast genomic library described in detail below has nowbeen published (Siede, W., and Eckardt-Schupp, S., Curr. Genet. 11:205 (1986).

Chromosomal yeast DNA of any desired aspartate prototrophic yeast strain is isolated and partially purified by methods known in the art (e.g., Cryer, D. R., Eccleshall, R., and Marmur, J., Meth. Cell. Biol. 12:39 (1975)).

Experiments are conducted in a small scale to obtain optimal conditions for incomplete or partial digestion of the chromosomal yeast DNA witn restriction endonuclease Sau 3A in a manner known in the art. (Maniatis, T., Fritsch, E. F., and Sambrook, J., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Publ., New York (1982)).

A larger quantity of chromosomal yeast DNA in various different suitable concentrations is then cut with the restriction endonuclease enzyme Sau 3A. The thus obtained DNA fragments having different lengths are separated in agarose gel, e.g., a 0.5% agarose gel, and visualized with ultraviolet radiation after dying with ethidium bromide.

A fraction of the gel containing fragments of DNA of a size between about 3 kbp and 9 kbp is cut out from the agarose gel, the agarose pieces are transferred to a dialysis tube and the DNA fragments are eluted by methods known in the art (Mariatis et al, supra).

The thus obtained DNA is purified, e.g., by means of a single column system (Schleicher & BR< Schuell, Elutip), precipitated with ethanol and stored in, e.g., ligase buffer.

A yeast gene bank is then prepared with the chromosomal DNA obtained hereinabove.

The vector used to construct the yeast gene bank in the present case is the YCp50 plasmid.

This plasmid contains the replication starting points ORI and ARS, respectively, which are recognized by Escherichia coli and Saccharomyces cerevisiae microorganisms. The plasmid also contains the E. coli genes for tetracycline and ampicillin resistance, the yeast gene URA3 and the centromere region of chromosome 4 (CEN4) (Kuo, C. L., and Campbell, J. L., Proc. Natl.

Acad. Sci. (USA) 79:4243 (1982)).

However, the utilization of other vectors capable of being multiplied in S. cerevisiae and E. coli is also possible and contemplated within the confines of this invention (Botstein,D., and Davis, R. W., in "The Molecular Biology of the Yeast Saccharomyces-Metabolism and Gene Expression"; Strathern, J. N., Jones, E. W., and Broach, R. D., eds., Cold Spring Harbor Laboratory Publ., New York, 607 (1982)).

The exemplary vector used for this purpose is distinguished by having a low number of copies but a high mitotic stability. However, multicopy vectors are also contemplated within the invention. The YCp50 plasmid is amplified in E. coli by means of, e.g., the chloramphenicol technique according to Maniatis, T., Fritsch, E. F., and Sambrook, J., in Molecular Cloning-A Laboratory Manual, supra.

After alkaline lysis of the cells conducted using procedures known in the art, the plasmid is then purified by methods also known in the art (e.g., Birnboim, H. C., and Doly J., Nucl. Acids Res. 7:1513 (1979). Alternative methods can also be used such as those disclosed by Maniatis, T., Fritsch, E. F., and Sambrook, J., in Molecular Cloning-A Laboratory Manual, supra.

The thus obtained plasmid is then completely cut with the restriction endonuclease Bam H1 at its tetracycline resistance gene. The thus obtained vector, which is now tetracyclin-sensitive, is then treated with alkaline phosphatase to avoid religation of the cut fragments (Chaconas, G., and van de Sande, J. H., in Methods in Enzymol. 65:75 (1980)). After ethanol precipitation, the plasmid is stored in, e.g., a ligase buffer.

The partially digested yeast DNA obtained supra is ligated with the thus treated vector overnight at 7"C utilizing methods known in the art.

A recombination-defective E. coli strain (290A, deposited under the number DSM 3616 with the Deutschen Sammlung Fur Mikroorganismen (German Collection of Microorganisms, Grisebach strasse 8, Göttingen, Federal Republic of Germany as a plasmid containing strain) is transformed with the ligation mixture by, e.g., a calcium chloride technique to be made resistant to ampicillin technique under conditions known in the art (Dagert, M., and Ehrlich, S. D., Nature' 275:104 (1978)).

Optimal conditions leading to high transformant frequencies and to high insertion frequencies are tested in a small scale utilizing methods known in the art, and a high percentage of tetracycline-sensitive colonies are obtained among the transformants.

Transformation is implemented as is known in the art in a large scale with the thus checked quantity ratios, whereby at least 10,000 tetracycline-sensitive transformants are combined by washing off from the plates where they have been grown.

The plasmids are isolated from some of the preparations for examination, and the sizes of their insertion are then determined by known methods. On the average, the insertions should not have a size below about 4 kbp.

The thus obtained yeast gene bank is stored at -700C in the form of a transformant mixture in a medium and under conditions according to, e.g., Beggs, J. D., Nature 275:104 (1978).

For the preparation of the plasmids, samples thereof are used for inoculating the microorganisms and the plasmids are isolated from cells in the stationary growth phase utilizing procedures known in the art.

A uracil and asparatate auxotrophic yeast strain (WS8106-5B ura3-52 asp5) deposited with the Deutschen Sammlung Fur Mikroorganismen (German Collection of Microorganisms), Grisebach strasse 8, Goftingen, Federal Republic of Germany under the number DSM 3617, is transformed with this plasmid mixture utilizing, e.g., a lithium acetate technique which is known in the art (e.g., modified according to Ito, H., Fukuda, Y., Murata, K., and Kimura, A., J. Bacteriol.

153:163 (1983)).

Alternatively, a spheroblasting method according to Hinnen, A., Hicks, J. B., and Fink, G. R., Proc. Natl. Acad. Sci. (USA) 75:1929 (1978) can also be used for this purpose.

An extract is obtained from one of the resulting uracil and aspartate prototrophic transformants utilizing cells in the logarithmic growth phase and the plasmid DNA is enriched and purified therefrom utilizing known procedures (Wiede, W., and Eckardt-Schupp, supra).

The 290A E. coli strain is then transformed with this preparation by known procedures and the plasmid of an ampicillin-resistant transformant is purified therefrom by means of methods known in the art.

The characteristics of this plasmid (pWS101), with which its complements the asp5 mutation, are confirmed after renewed yeast transformation.The plasmid is also characterized by means of restriction enzymes under reaction conditions known in the art (see Figure).

The ASP5 gene encodes the enzyme glutamate oxalacetate transaminase (GOT, L-aspartate:2oxoglutarate aminotransferase (E.C.2.6.1.1). The activity of this enzyme is easily determined in yeast extracts which may be prepared according to Sigurdson, D. C., Gaarder, M. E., and Livingston, D. E., Mol. Gen. Genet. 183:59 (1981) by means of a commercially available test (Boehringer-Mannheim, West Germany).

Without the plasmid, the asp5 mutant employed has no detectable GOT activity. However, significant GOT activity can be ascertained after the mutant is transformed with the pWS101 plasmid.

In addition, the presence of this plasmid in a haploid strain which already has some GOT activity increases this activity by a factor of about 2.

The strains of E. coli (290A) and S. cerevisiae (WS8106-5B) transformed with the pWS101 plasmid have been deposited in the Deutsche Sammlung fur Mikroorganismen (German Collection of Microorganisms), Grisebachstrasse 8, Göttingen, Federal Republic of Germany, under the numbers DSM 3616 and DSM 3617, respectively.

Since the plasmid may be spontaneously lost with a certain probability, the starting strains mentioned here are thus also available as indicated hereinabove.

Without the plasmid, the 290A E. Coli strain is tetracycline-sensitive and the WS8106-5B S.

cerevisiae strain requires uracil and aspartate.

Having now generally described this invention, the same will be better understood by reference to a certain specific example, which is included herein for purposes of illustration only and is not intended to be limiting of the invention or any embodiment thereof, unless so specified.

EXAMPLE (I) Chromosomal DNA was prepared from 1 liter of yeast culture (Saccharomyces cerevisiae) in a logarithmic growth phase by methods known in the art.

(II) A broad range of conditions for partial digestion with restriction endonuclease Sau 3A (Boehringer-Mannheim) were tested utilizing standard techniques.

(III) Selected digestion conditions for the partial digestion of chromosomal yeast DNA are as follows.

Sample A 200 ijg DNA 5.6 units Sau 3A Sample B 200 jug DNA 2.8 units Sau 3A Each sample was placed in 2 ml Sau 3A buffer (20 mM TRIS HCI, 50 mM NaCI, 6 mM MgCl2, pH 7.5) and was incubated for 1 hour at 37"C.

The reaction was stopped by cooling on ice with the addition of EDTA to a final concentration of 2 mM EDTA. The preparation was then subjected to a chloroform extraction, an ethanol precipitation, and was then stored in TE buffer (100 mM TRIS HCI, 10 mM EDTA, pH 8.0) utilizing customary procedures.

(IV) The differently digested DNA samples were combined, and 200,ug thereof electropho resed in an 0.5% agarose gel (18 hr, 35 V, 4"C, with a standard TRIS borate/ethidium bromide buffer).

The DNA fractions having a size between about 3 kbp and 9 kbp (as measured against A/Hind III size standards, Boehringer-Mannheim) were cut out from the rest of the gel, transferred into a dialysis tube, and eluted from the gel by electrophoresis (3 hr, 100 V, and 3 minutes there after at reversed polarity) by means of standard procedures.

(V) The buffer was removed from the dialysis tube and the DNA was then purified and concentrated by means of a single column system (Schleicher & Schuell, Elutip). The thus obtained DNA is precipitated in ethanol and stored in ligase buffer (20 mM TRIS-HCI, 10 mM MgCl2, 10 mM dithio erythritol, 0.6 mM ATP, pH 7.6).

(VI) The vector plasmid YCp50 was amplified in the E. coli 290 A strain by means of a known chloramphenicol technique described supra, isolated from a 1 1 logarithmic culture after alkaline lysis and purified using cesium chloride density gradient centrifugation for 36 hr at 45,000 rpm which is standard in the art.

After dialysis and ethanol precipitation, the plasmid was stored in Bam H1 buffer (10 mM TRIS-HCI, 100 mM NaCI, 5 mM MgCI2, 1 mM mercaptoethanol, pH 8.0).

(VII) A complete digestion (restriction) of the plasmid with restriction endonuclease Bam H1 (Boehringer-Mannheim) was conducted overnight at 37"C under standard conditions, followed by extraction with chloroform and ethanol precipitation.

The thus obtained restricted vector was stored in 50 I of (50 mM TRIS HCI, 1 mM MgCl2, 0.1 mM ZnCl2, 1 mM spermidin) phosphatase buffer.

(VIII) Testing of the ligation conditions in a small scale resulted in the selection of the following conditions.

3 jug YCp50 plasmid DNA, 3 ijg partially digested yeast DNA, 5 units T4 DNA ligase (Renner), and 100 il ligase buffer.

The above enzyme reaction components were incubated for 16 hr at 7"C.

(IX) The 290 A E. coli strain was transformed with the starting mixture described in (VIII) supra (10 AI samples). About 70% of the ampicillin-resistant transformants were found to be tetracycline-sensitive.

(X) samples were prepared from the plasmids of 10 tetracycline-sensitive transformants and their innortion size was determined after digestion with Eco R1 restriction endonuclease enzyme (Boehringer-Mannheim) and the fragment sizes were determined by comparison with molecular weight markers (Boehringer-Mannheim) as is known in the art. The average insertion size was found to be 4.5 kbp.

(XI) 30,000 colonies were washed off from the plates and stored as a suspension at -700C in a medium as described by Beggs, J. D., Nature 275:104 (1978). Samples of the colonies were grown overnight on plates (about 30 LB plates containing ampicillin). The plasmid mixture was then isolated by alkaline lysis and purified by means of a cesium chloride gradient as is standard in the art.

(XII) Transformation of the WS8106-5B yeast strain (ade2- 1 ura3-52 asp5) according to the lithium acetate method.

200 ml of a WS8106-5B logarithmic growth culture were transformed with a total of 40 ,ug of DNA obtained in (XI) supra, then plated out on a uracil-free aspartate-free medium (Sherman, F., Fink, G. R., and Lawrence, C. W., Methods in Yeast Genetics--Laboratory Manual, Cold Spring Harbor Laboratory, New York (1971)) by a method known in the art and described supra.

(XIII) an extract was produced from the thus obtained transformants by digesting with the enzyme zymolyase (Sekagaku, Kogyo, via Bayer) under reaction conditions known in the art.

The plasmid DNA was enriched by means of denaturing/renaturing steps, and then precipitated with ethanol and used for the transformation of E. coli bacteria (290A) as described supra.

The plasmid was isolated from the transformant and characterized by restriction enzymes digestion utilizing procedures which are standard in the art (see Figure).

(XIV) the possibility of complementing an asp5 mutation of yeast with this plasmid was confirmed by renewed yeast transformation already described above.

GOT ACTIVITY The following activities of the glutamate oxalacetate aminotransferase enzyme encoded by the ASP5 gene were then measured in the yeast extract using the commercially available Boehringer Mannheim GOT monotest.

(A) GOT ACTIVITY PROVIDED BY THE PLASMID WS8106-5B 0 (without plasmid) (t2 units/g protein) WS8106-5B 53 units/g protein (pWS101) The determination of the protein content of the samples was conducted by the method of Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193:265 (1951) using albumin standards.

(B) increased got activity by addition of plasmid to a haploid strain with got activity In a haploid strain which already has GOT activity, the plasmid increases the enzyme activity.

Independently of the absolute starting value, the expected result is an increase of the enzyme activity by a factor of 2.

WS8069/i 15 61 units/g protein (ASP5, without plasmid) We8069/115 126 units/g protein.

(ASP5, pWS 101) Fragmentation lengths of the plasmid shown in the accompanying figure are true to scale. The gene localization, however, may not be totally accurate according to Kuo, C. L., and Campbell, J.

L., Proc. Natl. Acad. Sci. (USA) 79:4243 (1982).

The restriction enzymes Eco R1, Sal 1, Xho 1 utilized in the above experiments were all obtained from Boehringer-Mannheim.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein.

The present disclosure relates to the subject matter disclosed in German Application P 3605035.0 filed in the Federal Republic of Germany on February 18th, 1986, the entire specification of which is incorporated herein by reference.

Claims (16)

1. A plasmid comprising: a DNA vector capable of being multiplied in a microorganism selected from the group consisting of Saccharomyces cerevisiae and Escherichia coli; and the ASP5 gene of Saccharomyces cerevisiae encoding the enzyme L-aspartate:2-oxoglutarate aminotransferase (E.C.2.6.1.1).

2. The plasmid of claim 1, wherein the vector is a fragment of the YCp5O vector containing the CEN4 centromere region of Saccharomyces cerevisiae.

3. The plasmid of claim 1 being pWS101.

4. A 290A E. Coli microorganism containing the plasmid of claim 1.

5. A WS8106-5B Saccharomyces cerevisiae microorganism containing the plasmid of claim 1.

6. A process of obtaining the plasmid of claim 1, comprising: (a) obtaining and amplifying the vector in a 290 A Escherichia coli bacterium; (b) incubating the vector with Bam H1 restriction endonuclease enzyme in a proportion thereof and under reaction conditions effective to attain complete restriction of the vector; (c) obtaining chromosomal yeast DNA and incubating the DNA with Sau 3A restriction endonuclease enzyme in a proportion thereof and under reaction conditions effective to attain partial restriction of the DNA and obtain DNA fragments of different sizes; (d) isolating chromosomal yeast DNA fragments of a size between about 3 kilobase pairs (kbp) and 9 kbp;; (e) cloning the isolated DNA fragments into the restricted vector obtained in step (b) in a proportion thereof and under reaction conditions effective to obtain a plasmid family carrying the isolated DNA fragments; (f) incubating a 290 A Escherichia coli bacterium with the plasmid family carrying the isolated DNA fragments in a proportion thereof and under conditions effective to attain the transformation of the bacterium; (g) selecting and combining at least 10,000 ampicillin-resistent tetracyclin-sensitive transformants of the bacterium; (h) amplifying and purifying the combined transformants under conditions effective to obtain a gene bank containing at least one copy of the plasmid;; (i) incubating a ura3 and asp5 Saccharomyces cerevisiae yeast mutant with the gene bank obtained instep (h) in a proportion and under conditions effective to attain the transformation of the mutant; (I) selecting uracil and aspartate prototrophic yeast transformants from the transformants of the mutant obtained in step (i) containing the plasmid; and (k) isolating the plasmid.

7. The process of claim 6, further comprising: separating the plasmid from the transformants of the yeast mutant.

8. The process of claim 7, further comprising purifying the plasmid.

9. A method of producing the enzyme L-aspartate:2-oxoglutarate aminotransferase (E.C.2.6.1.1., GOT) comprising: incubating the plasmid of claim 1 in Saccharomyces cerevisiae yeast for a period of time and under conditions effective to obtain the enzyme.

10. The method of claim 9, further comprising: separating the enzyme from the Saccharomyces cerevisiae yeast.

11. The method of claim 10, further comprising purifying the enzyme.

12. The method of claim 9, further comprising the following steps prior to the step of incubating the plasmid in Saccharomyces cerevisiae yeast.

(a) obtaining and amplifying the vector in a 290 A Escherichia coli bacterium; (b) incubating the vector with Bam H1 restriction endonuclease enzyme in a proportion thereof and under reaction conditions effective to attain complete restriction of the vector; (c) obtaining chromosomal yeast DNA and incubating the DNA with Sau 3A restriction endonuclease enzyme in a proportion thereof and under reaction conditions effective to attain partial restriction of the DNA and obtain DNA fragments of different sizes; (d) isolating DNA fragments of a size between about 3 kilobase pairs (kbp) and 9 kbp; (e) cloning the isolated DNA fragments into the restricted vector obtained in step (b) in a proportion thereof and under reaction conditions effective to obtain a plasmid family carrying the isolated DNA fragments;; (f) incubating a 290 A Escherichia coli bacterium with the plasmid family carrying the isolated DNA fragments in a proportion thereof and under conditions effective to attain the transformation of the bacterium; (g) selecting and combining at least 10,000 ampicillin-resistant tetracyclin-sensitive transformants of the bacterium; (h) amplifying and purifying the combined transformants under conditions effective to obtain a gene bank containing at least one copy of the plasmid; (i) incubating a ura3 asp4 Saccharomyces cerevisiae yeast with the gene bank obtained instep (h) in a proportion and under conditions effective to attain the transformation of the mutant; (j) selecting uracil and aspartate prototrophic yeast transformants from the transformants of the mutant obtained in step (i) containing the plasmid; and (k) isolating the plasmid.

13. A method of producing asparagine, comprising: incubating the pWS101 plasmid of claim 1 in Saccharomyces cerevisiae yeast for a period of time and under conditions effective to overproduce the enzyme to obtain asparagine.

14. The method of claim 13, further comprising: separating the asparagine from the enzyme and the Saccharomyces cerevisiae yeast.

15. The method of claim 14, further comprising: purifying the asparagine.

16. A method of obtaining and use of pWS101 PLASMID substantially as hereinbefore described with reference to the accompanying drawing.