FR2588015A1 - STRAIN OF HOST MICROORGANISM, PLASMID RECOMBINANT TRANSFORMANT, AND PROCESS FOR PRODUCTION OF THERMALLY STABLE TRANSAMINASE ENZYME - Google Patents

Abstract

THE INVENTION RELATES TO THE USE OF MICROORGANISMS WITH CONSIDERABLY AMPLIFIED TRANSAMINASE ACTIVITY FOR THE PRODUCTION OF L-AMINOACIDS. ACCORDING TO THE INVENTION, A STRAIN OF A HOST MICROORGANISM IS TRANSFORMED BY A RECOMBINANT PLASMID CONTAINING A FRAGMENT OF DNA WHICH INCLUDES A SALMONELLA TYPHIMURIUM GENE CODING FOR A THERMALLY STABLE TRANSAMINASE ENZYME; THE ATTACHED DRAWING GIVES A SCHEMATICAL REPRESENTATION OF A PARTIAL RESTRICTION MAP OF PAH27. THE INVENTION APPLIES IN PARTICULAR TO THE PRODUCTION OF AMINOACIDS.

Description

The present invention relates to the use of microorganisms having

  significantly amplified transaminase activity in the

  production of L-amino acids (hereinafter "amino acids").

  More particularly, the gene encoding this transaminase activity is transferred from the original bacterial genome to the multiple copy plasmids. The microorganisms transformed by these recombinant plasmids exhibit a considerably increased activity of transaminase under appropriate conditions. Moreover, this enzymatic activity is thermally stable. As a result, in the amino acid production by transamination, the genetically engineered organisms described here significantly exceed the isolates.

natural.

  Transamination is normally defined as the reaction between an amino acid and a keto acid that results in the transfer of amino and keto groups between the two raw materials. It is known that precursor ketoacids can be converted enzymatically to corresponding amino acids. The enzymes involved in the actual transfer of these groups are alternatively referred to as "transaminases" or "aminotransferases". As an example of this type of reaction, US Pat. No. 3,183,170 (Kitai et al.) Discloses the transamination of precursor keto acids in the presence of a multienzyme system obtained from various sources, including harvested bacterial cells, dried cells, cell macerates or enzyme solutions. The multi-enzyme system of Kitai et al. Consists of dehydrogenase, hydrogenase, transaminase, hydrogen acceptors and

electron transport.

  As an example, the E. coli aminotransferase genes have been described in "Escherichia coli Mutants Deficient in the Aspartate and Aromatic Amino Acid Aminotransferases" by Gelfand et al, volumel30, pp. 42940 (1977) and have been designated by ilvE, tyrB and aspC. It is known that the presence of E. coli tyrB and aspC functional genes alleviates auxotrophy for tyrosine, phenylalanine or aspartic acid. European Patent Application No. 84100521.8 (published August 29, 1984 under No. 0 116 860) (GD Searle and Co.) discloses that the ilvE, tyrB and aspC aminotransferase genes can be isolated and used to construct microorganisms. recombinants capable of producing these aminotransferase product genes. The recombinant DNA methods are used to construct a new microbial strain comprising a thermally stable transaminase gene of Salmonella typhimurium in a higher copy number than that normally found on the bacterial genome. This gene, encoding an enzyme that has been designated as AT sty 2, is inserted into a multi-copy plasmid, and the recombinant plasmid is introduced into a suitable host. The resulting high levels of the expressed transaminase product gene in the transformed host cause an overproduction of L-amino acid by transamination of an exogenously produced amino donor and a ketoacid precursor. The thermally stable enzyme expressed is therefore less sensitive to inactivation at high temperature exposure. The bioconversion of the precursor by the expressed enzyme of this invention results in considerably higher commercial quantities of the amino acid produced compared to natural isolates. The main subject of the present invention is a strain of microorganisms comprising multiple copies of a transaminase gene whose thermally stable product is responsible for the stereospecific conversion of alpha-ketoacids to amino acids. Such a strain has a greatly improved rate of conversion to amino acid, requiring substantially reduced residence times in the reaction vessel. Its other objects are to produce recombinant microorganisms which can be immobilized by conventional methods and which express a transaminase which has better half-life characteristics, that is to say which does not degrade or is not

  easily inactivated, even after immobilization.

  Another object of the present invention is to place the desired transaminase gene in a

  plasmid construction which is both

  actually stable and stable in the number of copies in order to reduce processing problems associated with deletions or transpositions of the gene's DNA sequence, or loss of the plasmid. An additional objective is to place the transaminase gene on a high copy number plasmid for high levels of

the expressed enzyme.

  Another subject of the present invention is the use of the spectinomycin resistance gene.

supported by a plasmid.

  The invention will be better understood and other purposes, features, details and advantages thereof

  will become clearer during the description

  explanatory text which will follow with reference to the accompanying schematic drawings given solely by way of example illustrating several embodiments of the invention and in which: FIG. 1 is a schematic representation of a partial restriction map of pAH27; FIG. 2 is a schematic representation of a partial restriction map of pGA108; and FIG. 3 is a schematic representation of a partial restriction map of pGA115. A thermally stable Salmonella typhimurium transaminase gene is cloned and expressed in a host for use in the production of amino acids. The transaminase gene is first isolated and preferably bound in a multicopic selectable vector. A suitable host is transformed by the recombinant vector containing the transaminase gene. Selection of the desired transaminase gene is demonstrated by complementation of an auxotrophic host microorganism for the desired amino acid and by the thermal stability of the expressed enzyme. This enzyme has been designated as AT sty 2. An expression host transformed with the S. typhimurium transaminase gene of interest, or a portion or extract of the transformed host, is capable of producing and accumulation of the amino acid in the presence of a precursor alpha-keto acid and an amino donor. Catalysts with the described transaminase activity have very long half-lives when

  uses them for this bioconversion.

  In the description, references to the "gene of

  transaminase "are considered to incorporate DNA fragments comprising the gene for AT sty 2 itself, the additional gene plus DNA or a functional portion of the transaminase gene, the AT sty 2 transaminase gene indicated is the S gene. typhimurium whose expression product is a transaminase enzyme which is characterized by a thermal stability and the ability to rapidly convert a precursor keto acid into the corresponding amino acid The transaminase gene is isolated directly from the S. typhimurium genome or can be obtained from any convenient source of S. typhimurium genetic material.This microorganism is available from the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852 (the 1985 ATCC catalog lists 51 available strains Strains are also available from various laboratories

  undertaking research on S. typhimurium.

  The genome or genetic material is partially digested with the restriction endonuclease and fragments of a practical size, such as about 25 kilobases, are isolated. These fragments are then linked to appropriate vectors to allow the isolation of active transaminase clones that express the thermally stable gene product. When lambda cloning vectors such as those described below are selected, Sau 3A restriction endonuclease is used because lambda vectors accept Sau 3A generated fragments to form libraries.

relatively erratic.

  In the preferred embodiment, E. coli aminotransferase negative hosts are conveniently used to determine complementation. An E. coli strain is obtained which is aspC-tyrB-ilvE-, i.e. a strain lacking any transaminase activity. The DG30 strain of E. coli K-12 described by Gelfand et al., J. Bacteriology, "Escherichia coli Mutants Deficient in the Aspartate and Aromatic Amino Acid Aminotransferases,", Volume 130, pp. 429-40 (1976), may be used. . This strain is available at E. coli Genetic Stock Center, Yale University, New Haven, Connecticut. DG30 is auxotrophic for tyrosine, phenylalanine and aspartic acid. Alternatively, E. coli K-12 strain DG44, also available at E. coli Stock Center, can be used. DG44 is aspC-tyrB and is auxotrophic for aspartic acid, tyrosine and phenylalanine. Other strains

  auxotrophs can be obtained or created.

  The DG30 and DG44 strains of transaminase-negative E. coli are transformed at very low frequencies, which decreases the possibility of detecting clones with transaminase activity. Since lambda phage can transduce these hosts at a much higher frequency, the likelihood of isolating transaminase-active clones is greatly increased.

  with the use of lambda cloning vectors.

  It is quite preferable to use a phage construct that can exist as a plasmid at low temperatures and as a lytic phage at higher temperatures. This "phasmid" approach is preferred for the efficiency of transduction and for easy isolation of DNA containing a transaminase gene identified by conventional plasmid recovery technique, although this is not required for implementation. practice of the present invention. The phasmid attempt as well as the method of constructing the appropriate lambda cloning vectors are described in detail in S. Elledge et al., J. Bacteriology, "Phasmid Vectors for Identification of Genes by Complementation of Escherichia.

  coli mutants ", Volume 162, pages 777-83 (1985).

  Prior to using E. coli transaminase negative hosts to select the recombinant phage library, the host strain is prepared by transformation with a plasmid containing a gene which

  expresses the lambda repressor sensitive to temperature.

  In the presence of the temperature-sensitive repressor, the described lambda phage can exist in the form of a plasmid at about 30 ° C. and in the form of a lytic phage at about 37 ° C. A suitable plasmid by which auxotrophs of E. coli is the plasmid pSE103 containing the ci857 gene which expresses the lambda temperature-sensitive repressor. The construction of this plasmid is described in the above cited article by Elledge et al. Any plasmid containing the cI857 gene can be used. The only condition is the presence of

  lambda repressor sensitive to temperature itself.

  It is an essential condition of the present invention that the expressed transaminase catalyst is not subject to degradation or degradation.

  rapid inactivation at the use temperatures.

  This thermal stability will substantially increase the half-life of use of the biocatalyst at temperatures in the range of about 10 ° C to 42 ° C. In addition, the thermally stable enzyme will be less susceptible to inactivation at typically used temperatures. immobilisation, which can

reach around 45 to 50 C.

  A determination of amino acid production by heat inactivation is used to select the clones containing the recombinant transaminase. This determination allows differentiation between transaminase genes that are heat sensitive and those that are heat stable. Sonicated cells are contacted with alpha-keto acid and an amino donor (eg, aspartic acid), before and after thermal shock at temperatures of about 55 C for 20 to 30 minutes. The ability to enzymatically convert the precursor to the amino acid after this treatment is measured in

  determining the amino acid product, for example by HPLC.

  Clones that have this ability contain the

AT gene desired st 2.

  The alpha-keto acid selected for use with this method will depend on the amino acid that is the desired product. For example, phenylpyruvic acid

  is converted to L-phenylalanine, alpha-keto acid

  L-leucine caproic acid, L-valine alpha-ketoisovaleric acid, alpha-keto-D-methyl-valeric acid L-isoleucine, pyruvic acid L-alanine, acid

  oxaloacetic acid L-aspartic acid, the acid: -hydroxy-

  alpha-ketobutyric acid to L-threonine, p-hydroxy-

  phenylpyruvic L-tyrosine, indole pyruvic acid L-tryptophan, and others. As can be seen, conventional Lamino acid precursors are used in the process of the invention. However, it has been demonstrated that the AT sty 2 enzyme has a particularly high activity in the conversion of phenylpyruvic acid to L-phenylalanine. Of course, these precursors can be used both in the determination of the production of amino acid by heat inactivation.

  and in the production of the desired L-amino acid.

  After isolating the thermally stable transaminase-active clones, confirmation is made to ensure that the activity is the result of the presence of a recombinant phasmid containing the AT sty 2 gene rather than a chromosomal revertant. The clones are grown and phage production is induced at about 420C. The phage titres are measured to ensure that there are large quantities. Next, the phasmid vectors considered to comprise the AT sty 2 gene are isolated from the clones in plasmid form. Isolated plasmids are introduced into a transaminase negative E. coli strain using standard procedures, for example, the transformation process described by Maniatis et al in "Molecular Cloning: A Laboratory Manual", pages 250-51 (Cold Spring Harbor Laboratory 1982), or the transduction processes described by Davis et al. In Advanced Bacterial Genetics (Cold Spring Harbor 1980). Only the genetic material comprising a functional transaminase gene will complement the lesions of the host strain, releasing it from its auxotrophy. Indeed, only transformants comprising an expressed transaminase gene can grow on minimal medium plates without additional amino acids. It can be deduced that the plasmids in these clones comprise and express a transaminase gene, since the auxotrophy of the host is delivered. Having identified clones containing the hybrid phage with the desired thermally stable transaminase gene, phages of these clones are transduced into a host containing a chromosomal cI857 gene to prevent contamination by the original plasmid, ie ie, pSE103, upon isolation of recombinant phasmid DNA. The transduced cells are incubated at temperatures sufficient to induce phage production and lysis (i.e., 37 C). The phage containing the desired gene is thus isolated. This confirms the extrachromosomal state of the desired transaminase gene. The isolated phage can be used for the production of restriction maps, if desired, and for the

  subcloning into a multi-copy vector.

  Once the genetic material has been isolated which comprises the AT sty 2 gene, it is desirable to transfer the gene to a high copy number plasmid. Multiple copies of the plasmid in the transformed host will provide multiple copies of the transaminase gene. In this way, the transaminase activity of each cell can be increased multiple times. It may also be desirable at this time to reduce the size of the recombinant DNA fragment by cleavage with restriction endonucleases. The DNA fragment containing the transaminase gene is subcloned into a multi-copy vector suitable for transformation of the desired host strain. It is preferable to use a selectable plasmid, for example a plasmid with antibiotic resistance genes allowing for biological selection and maintenance of the plasmid. If E. coli is to be the host microorganism, a particularly suitable vector is plasmid pBR322, which carries genes for resistance to ampicillin (Apr) and tetracycline (Tcr) and which will typically be present in about 10 to 20 copies. When antibiotic resistance is used to maintain the plasmid, only microorganisms that maintain the plasmid (with its antibiotic resistance gene (s) and also with the transaminase gene) will grow in a medium to which the appropriate antibiotic has been used. been added. Other antibiotics or other methods for plasmid maintenance and stability may be

to be used, if desired.

  The above isolated DNA fragment which has been determined to contain the S. typhimurium AT sty 2 transaminase gene is bound to the selected plasmid by conventional methods. The fragment can be excised from the recombinant phasmid isolated above by digestion with a suitable restriction enzyme. The

  Excised fragment of DNA is then bound to the chosen plasmid.

  Partial restriction maps of prepared plasmids

  according to this description are shown in Figures 1 and 2.

  FIG. 1 represents the restriction enzyme analysis of the plasmid pAH27 prepared in Examples IV and V. FIGS. 2 and 3 represent the restriction enzyme analysis of plasmids pGA108 and pGA115,

  respectively, prepared in Examples VI and VII.

  With reference to the preferred embodiment, where lambda SE4 (see Example I) or a similar construct is used, the Spr gene is placed in the vicinity of the transaminase gene and care should be taken to select an appropriate restriction endonuclease which will excise a DNA fragment containing both genes. A plasmid which contains the Spr gene upstream of the transaminase AT sty 2 gene has been shown to give a

  remarkable and is the most preferred construction.

  Spectinomycin is well suited for maintaining or preserving the plasmid because of its relatively low price and durability. In addition, the expression of the transaminase gene can be improved by the promoter r of the Spr gene. In any case, this embodiment results in unexpectedly high levels of

  expression of transaminase and stability.

  In order to confirm that the recombinant plasmid contains the desired transaminase gene, the plasmid can be re-isolated and introduced into a transaminase negative strain, as described above. Only organisms that have received the plasmid with the transaminase gene will form colonies on a minimum medium. Further confirmation can be obtained by growth so as to select the plasmid itself. For example, if the plasmid contains genes encoding spectinomycin resistance, growth on plates containing this antibiotic

confirms the presence of the plasmid.

  In the most preferred embodiment of the present invention, the olasmid is prepared as described in Examples I-IV. Indeed, the DNA fragment will contain both the thermally stable AT sty 2 gene of S. typhimurium and the spectinomycin resistance gene. The preferred plasmid vector is pBR322

  and the preferred host microorganism strain is E. coli.

  This results in unexpectedly high and unexpected levels of transaminase activity. This is unusual because of the relatively large size of the DNA fragment, which would be expected to result in low copy number and hence low levels of expression. In addition, one would expect a plasmid of this size to be relatively unstable. However, attempts to decrease the size of the DNA fragment and the plasmid (Example VI) resulted in a decrease in transaminase activity (Example VIII)

and stability.

  Once the AT sty 2 gene is placed on the desired plasmid, the recombinant plasmid is introduced into the desired host. E. coli strains are particularly suitable hosts because of the profusion of information concerning their handling. Furthermore, as indicated above, the introduction of the S. typhimurium transaminase gene into E. coli has been shown to result in uniquely high and stable levels of expression and stability of expression. . A strain of Salmonella can itself

  be used for the host, if desired.

  A newly created microorganism strain of this invention comprises multiple copies placed on the plasmid of the thermally stable AT sty 2 gene of S. typhimurium and exhibits a multiple increase in transaminase activity compared to natural isolates. The recombinant microorganisms are prepared by growing them under conditions suitable for maximizing transaminase activity in the cells. In general, conventional conditions for healthy cell growth will be appropriate. Indeed, one can grow microorganisms at a pH

  from about 6.5 to about 8, preferably from about 7 to 8.

  The temperature should be about 20 to 42 C,

preferably about 370C.

  Conventional growth media can be used to prepare the recombinant cells of this invention. The media should contain a source of carbon, a source of nitrogen, inorganic salts and, if desired, other minor organic or inorganic foods that may be required for the growth of the selected organism. In general, the following guidelines are appropriate. As a carbon source, fermentable sugars can be used. As nitrogen sources, urea, protein hydrolysates, ammonium salts of organic acids (such as ammonium acetate or ammonium oxalate) and / or ammonium salts of inorganic acids (such as ammonium sulphate, ammonium nitrate or ammonium chloride) can be satisfactory. Inorganic elements (such as

  potassium phosphate or magnesium phosphate) -

  and / or vitamins (such as thiamine, biotin) can be added. The specific choice of growth medium components will depend on the host microorganism chosen. The cells are harvested after growth and either the cells or a portion or an extract thereof can be used in the bioconversion of a precursor keto acid and a corresponding amino acid amino donor. Any practical process or process can be used that brings the transaminase enzyme into contact with the precursor and the amino donor. For example, whole cells, sonicated cells or enzyme extracts (crude or purified) can be used. Alternatively, the bioconversion can be undertaken by fermentation of a living culture, growing actively with the precursor

  and the amino donor added to the growth medium.

  It may be preferable to immobilize the cells or the enzyme for use in a batch or continuous reactor. In this way, the use of cell or enzyme transaminase activity can be maximized, while simultaneously minimizing manipulation and eliminating

  the needs of growth media and conditions.

  The thermally stable transaminase of the present invention is ideal for immobilization by methods requiring high temperatures. Even after being subjected to the temperatures required for the immobilization step, a transaminase sensitive activity is retained. The immobilized cells or enzymes can be contacted directly with a solution of the precursor and the amino donor. The molar ratio of the amino donor to the precursor keto acid,

  that is, aspartic acid with phenyl acid

  pyruvic, should be from about 1: 1 to about 1.5: 1.

  The microorganism strains of this invention offer significant advantages in the

  conversion of alpha-ketoacids to amino acids.

  The transaminase activity is such that the rate of conversion to amino acids is very fast. This allows the reduction of residence times in the reactor vessel. Problems associated with precursor failure can be solved, in turn, by reducing the reaction time. The microorganism strains of this invention produce a transaminase enzyme characterized by a long half-life, greatly extending the useful life of the catalyst. The amount and stability of transaminase activity in cells is greatly enhanced by recombinant techniques

described here.

  The following examples are given to illustrate the invention and should in no way limit the scope. The following abbreviations have been

  used throughout the description of the invention:

  Apr - ampicillin resistant ATP - adenosine triphosphate C - degree (s) Celsius DNA - deoxyribonucleic acid DTT - dithiothreitol EDTA ethylenediamine tetraacetic acid g - gram (s) HPLC - high pressure liquid chromatography IAA - isoamyl alcohol kb - kilobase (s) ) M - mole (s) mM - millimole (s) pg - microgram (s)) .l - microlitre (s) mn minute (s) nm - nanometer (s) OD - optical density UDO - Optical density units P- 5-P - Pyridoxal-5-phosphate PEG - polyethylene glycol rpm rotations SDS - sodium dodecyl sulfate Spr - spectinomycin resistant TCA - trichloroacetic acid Tcr - tetracycline resistance Tcs - tetracycline sensitive TES - chloride Tris-EDTAsodium Tris-tris (hydroxymethyl) -aminomethane. Media, tamper or concentrated solutions

  were prepared for use in the examples.

  All were produced with deionized water.

  Minimum medium MgS04. 7H20 0.20 g / 1 Citrate monohydrate 2.00 g / 1 K2HP04 10.00 g / 1 NaNH4HP04.4H20 3.50 g / i Glucose 0.50% Succinate 0.25% Malate 0.25% Histidine 0.10 mg / ml Arginine 0.10 mg / ml Asparagine 0.10 mg / ml Glutamine 0.10 mg / ml Isoleucine 0.10 mg / ml Leucine 0.10 mg / ml Valine 0.10 mg / ml Glutamic acid 0.10 mg / ml Alpha-keto-glutarate 1.00 mg / ml Thiamine-HCl 0.01 mg / ml noble agar (Difco) 1.50% The first four ingredients were mixed and autoclaved at 121 ° C for 15 minutes. 0 minutes to sterilize them. The agar was autoclaved separately. The sterilized components were mixed and cooled and the ingredients added. Complex medium (Luria broth) Bactotryptone (registered trademark) (Difco) Yeast extract Bacto (deposited) (Difco) NaCl NaOH, 0 g / l, 0 g / l, 0 g / l 1.5 ml / l The medium was autoclaved at 121 ° C. for 15 minutes to sterilize it. It was used in liquid form or in the form of solid plates, which were prepared by solidification with 1.5% agar

noble (Difco).

  Lysozyme buffer Tris-HC1 (pH 8.0) Na2EDTA NaCl1 Lysozyme sucrose, 0 mM, 0 mM, 0 mM, 0% 1.0 mg / ml

  Lysozyme was added before use.

  SDS SDS buffer (10%) 20.0 ml Na2EDTA (500, OMM) (pH 8.0) 20.0 ml TES 120.0 ml TES buffer Tris-HCl (pH 7.5) NaCl Na2EDTA (500, 0 mM) (pH 8.0) ATP ligase buffer DTT Tris-HCl (pH 7.8) MgCl 2

0.020M

0.100M

0.005M

  1.0 mM, 0 mM, 0 mM, 0 mM Modified Medium of Lowry Bactotryptone (Trade Mark) (Difco) Yeast Extract Bacto (Trade Mark) (Difco) NaCl1

KH2PO4

K2HP04

  MgSO4.7H2O, 00 g / l, 00 g / l, 00 g / l 0.50 g / l 4.00 g / l

0.25 g / l.

  The medium was autoclaved at

   C for 20 minutes to sterilize it.

  Transaminase solution Phenylpyruvic acid Aspartic acid ZnSO4. 7H20 Pyridoxal-5-phosphate 31.00 g / l 28.90 g / l, 00 g / l 0.02 g / l The aqueous solution was prepared and its pH

adjusted to 7.8 with NaOH.

  Reagent (Example IV) Pyridoxal-5-phosphate 2.5 mg Alpha-keto-glutarate 105.0 mg L-phenylalanine 245.0 mg Phenazine methosulfate 3.0 mg Tetrazolium blue Nitro 24.0 mg Ingredients above, 50.0 ml of the reagent, 0.1 mol of KHPO4 (a mixture of-K2HPO4 and KH2PO4 in proportions sufficient to give the indicated pH) was added with stirring,

to bring the mixture to pH 7.5.

Example I.

  (Isolation of a transaminase gene) A genetic library of Salmonella t phimurium in the form of the hybrid phage lambda SE4 was obtained from G. Walker, Massachusetts Institute of Technology. Lambda SE4 was constructed by binding the replication plasmid and the spectinomycin resistance gene (Spr) of the low copy number plasmid pDPT427 in lambda 1059. The whole is described in "Phasmid Vectors for Identification of Genes by Complementation of Escherichia coli Mutants of S. Elledge et al., J. Bacteriology, Vol. 162, pp. 717-83 (1985). This lambda construct in the presence of the temperature-sensitive lambda repressor can exist in the form of a plasmid, that is to say in the lysogenic state, at low temperatures and in the lytic state.

at high temperatures.

  Salmonella typhimurium DNA was partially digested with restriction endonuclease

  Sau 3A and 12-17 kb fragments were isolated.

  Lambda SE4 was digested with Bam HI restriction endonuclease. The Salmonella DNA fragments were bound to the linear SE4 lamnbda. After in vitro packaging, recombinant phages containing the 12-17 kb DNA fragments were chosen. For these steps, the processes described in Elledge et al.'S article were followed. The selected recombinant phages were introduced into E. coli

  M5158 after passing through a P2 lysogen.

  SE4 lambda recombinant phages were isolated from E. coli M5158 using high temperature induction. The cells were grown at 30 ° C to the log mean phase (OD 550-0.4), moved to a 43 ° C water bath shaker for 30 minutes and then incubated at 37 ° C for about 2 hours. CHCl3 was added after incubation. Concentrations of 1.0 x 104 to 5.0 x 108 were recovered

phage / ml.

  Prior to selecting the lambda phage library, E. coli K-12 strains DG30 and DG44, both obtained from E. coli Genetic Stock Center, Yale University, New Haven, Connecticut, were transformed with plasmid pSE103 (obtained from G. Walker and described by Elledge et al) containing the cI857 gene which expresses the temperature-sensitive lambda repressor. In addition, pSE103 carries the kanamycin resistance gene (Kmr) for the conservation of the plasmid. When the transformed strains of E. coli (DG30 / pSE103 and DG44 / pSE103) were transduced with the recombinant lambda phage obtained above, lambda could be maintained in the lysogenic state at low temperatures and in the state. lytic at high temperatures. The DG30 and DG44 strains of E. coli K-12 are described in Escherichia coli Mutants Deficient in the Aspartate and Aromatic Amino Acid Aminotransferases, by Gelfand et al., J. Bacteriology, Volume 130, pp. 429-40 (1977). strain DG30 is lambda-sensitive and aminotransferase-negative (i.e., aspC-tyrB-ilvE) .The DG44 strain has the genotype of DG30 except that it is ilvE +; It is sensitive to lambda and aspC tyrB ilvE + The ilvE gene encodes the amino acid aminotransferase of the branched-chain amino acid The transduction and complementation of E. coli DG44 cells containing the temperature-sensitive repressor by The lambda library was performed as follows: DG44 / pSE103 was grown at 30 ° C in Luria broth containing 50.0 μg / ml kanamycin and 0.2% maltose to an average log growth phase. (approximately D0550 = 0.4) The culture was centrifuged and ise suspended in 1/10 volume of Luria broth. A 0.1 ml portion of the cell culture was transduced with the lambda SE4 recombinant phage library isolated above using an infection multiplicity of 0.5. After adding lambda phage, the cultures were incubated at 30 ° C. for 30 minutes without shaking, and then washed twice with 1.0 ml of a solution.

  containing 10.0 mM Tris and 10.0 mM MgSO4 (pH 7.5).

  The cells were centrifuged and resuspended in 0.1 ml of the same solution and plated on the Minimum Medium, i.e., a medium lacking phenylalanine, tyrosine and aspartic acid. The plates were incubated at 30 C until

  colonies appear on the plates, about 4 days.

  The only clones capable of growing on this Minimum Medium are those containing the recombinant lambda phage

transaminase.

Example II.

  (detection of transaminase activity

thermally stable).

  The clones of Example I were determined in order to select those containing a gene encoding a transaminase enzyme having a thermal stability, that is to say the AT sty 2 enzyme. Strains were examined for examination. in a broth of Luria at 30 C overnight. The cells were sonicated and each sample was divided into two portions. A portion (about 1.0 ml) was held on ice for 23 minutes as a control. The other (about 1.0 ml) was incubated in a water bath shaker at 55 ° C for 23 minutes. To 0.5 ml of each portion (i.e. the control and the heated portion for each clone), 4.5 ml of a solution of 90.0 ml of aspartic acid (0) was added. , 1M), 30.0 mg of P-5-P,

  and 1860.0 mg of phenylpyruvic acid. It was incubated at 30 C.

  Samples were taken initially at 60 minutes, and the reaction stopped by the addition of 40.0 μl of TCA (50% concentrated solution) per 0.8 ml sample. The samples were analyzed for phenylalanine by HPLC. Two clones designated DG44-I and DG44-IV clearly show thermally stable transaminase activity, with about 75% of the transaminase activity conserved after heating.

comparison to the control portion.

Example III.

  (Confirmation of hybrid phage containing a

aminotransferase activity).

  Clones DG44-I and DG44-IV of Example II were tested to confirm that transaminase expression was the result of the hybrid phage containing the AT sty 2 gene, rather than a chromosomal reversion agent. Clones were grown on

  complex media and induced phage production.

  From each clone, phage concentrations of approximately 2.0 x 10 3 phage / ml were detected. Isolated phages were transduced to DG44 / pSE103 and DG30 / pSE103 from E. coli, each containing the temperature-sensitive lambda repressor. The cells were plated on the minimum medium (i.e., a medium lacking phenylalanine, tyrosine and aspartic acid), and incubated at 30 C. After 5 days, replenished DG44pSE103 clones were apparent and incubated. extended

  also revealed clones DG30 / pSE103 recomplete.

  A determination of phenylalanine production by heat inactivation as described in Example II was accomplished and all clones showed activity.

  transaminase stable to heat.

  To obtain large amounts of DNA containing the AT sty 2 gene, large scale plasmid preparations were made by the following process. Complex medium with 50 μg / ml spectinomycin was inoculated with transduced cells and incubated overnight. The cells were centrifuged and the pellet resuspended in 4.0 ml of lysozyme buffer and placed in a Sorvall SS-34 centrifuge tube. The cell suspension in lysozyme was incubated at 37 ° C for 15 minutes and 16.0 ml SDS buffer was added with gentle stirring until clear. 5 ml of 5M NaCl was added and incubated at -20 ° C for one hour followed by centrifugation in an SS-34 rotor at 20,000 rpm for 30.0 minutes. 5 ml of 50% Type 8000 PEG were added to the supernatant which was incubated on ice for 1 hour, centrifuged at 10,000 rpm for 10.0 minutes and the supernatant was rejected. The pellet was gently dissolved in 5.0 ml of TES buffer and 0.33 ml of a 2.0 mg / ml solution of RNase was added. It was incubated at 65 ° C. for 20 to 30 minutes. To each tube, 9.3 g of cesium chloride was added and mixed. The final volume was adjusted to 10.0 ml per tube using TES buffer. Then 100.0 μl of ethidium bromide at 10.0 mg / ml was added to each tube and mixed. Each sample was loaded into a self-sealing ultracentrifuge tube and centrifuged for about 48.0 hours at 40,000 rpm at 4 ° C. The plasmid band was collected by syringe and bromide was collected. ethidium was removed by extraction with IAA. Samples were dialyzed overnight in a 10 mM Tris-HCl and 10.0 mM EDTA (pH 8.0) buffer solution with several

buffer changes.

  In addition, phasmids were isolated from clones DG44-I and DG44-IV using the following rapid isolation of plasmids. The cells were grown overnight in 5.0 ml of Luria broth with pg / ml kanamycin. The culture was centrifuged for 10 minutes at 10,000 rpm and the pellet was resuspended in 1.4 ml of lysozyme buffer, which was then subdivided into two 0.7 ml portions (A and B). B) and placed in Eppendorf microfuge tubes. The cells were incubated at 37 ° C for 20 minutes and then 40 μl of% SDS was added to each microfuge tube while inversion mixing. Then, 60 μl of 5M NaCl was added to each tube and mixed by inversion. The tubes were centrifuged for 15 minutes in a microfuge and the supernatant (containing the plasmid DNA) decanted into the fresh tubes, taking care not to remove any precipitates. To the supernatant was added 5.0 μl of a 2.0 mg / ml solution of RNase. It was incubated at 37 ° C. for 15 minutes and then extracted once with an equal volume of phenol: CHCl 3 (1: 1) and once with an equal volume of CHCl 3: IAA (24: 1). The tubes were filled with cold ethanol, mixed and incubated for 1 hour at -20 C. The samples were microfuged

  for 5 minutes and the supernatant liquid was spilled.

  The remaining precipitate in portions A and B for each clone after second microfugging was combined by adding 100 μl of water to each, dissolving the pellets and combining in a 200 μl tube, 20 μl was added. of 3 M sodium acetate (pH 5.5) and 0.5 ml of cold ethanol. The samples were microfuged for 5 minutes, the supernatant liquid discarded and the pellet resuspended in 150 μl of 10.0 mM Tris-HCl buffer solution and 10.0 mM EDTA (pH 8.0). The samples (20 μl each) were placed on agarose gels. DG44 / pSE103, also prepared by the process just described, was used as a control on agarose gels. This was the original strain whose transduction was originally carried out by the hybrid phage without containing the lambda phasmid. The control showed two bands, corresponding to chromosomal DNA and plasmid pSE103. For samples DG44-I and DG44-IV, a third band, more than 50 kb, appeared, corresponding to the large phasmid, as one would expect. The phages of DG44-I and DG44-IV have been designated by lambda SE4 AH-I and

lambda SE4 AH-IV, respectively.

Example IV

  (Subcloning the thermally stable transaminase gene into a multi-copy plasmid using Hind III endonuclease) The heat-stable AT sty 2 gene of S. typhimurium was subcloned from lambda phage DNA

  SE4 hybrid AH-IV using Hind III endonuclease.

  A complete hybrid phage digestion product was prepared by Hind III endonuclease. Restriction enzyme analysis of iambda SE4 AH-I DNA revealed that two Hind III sites, approximately 17 kb apart, overlapped with the transaminase gene and the spectinomycin resistance gene (Spr. ) (figure 1). Plasmid pBR322 was also digested with Hind III. The 17 kb DNA fragment of hybrid phage DNA was ligated to the unique Hind III site of pBR322, inactivating the tetracycline resistance gene, but leaving intact the ampicillin resistance gene (Apr) . Transformed microorganisms containing the hybrid plasmids were therefore expected to exhibit the SprAprTcs phenotype. Ligation was accomplished by incubation of a mixture of 1.0 μg of Hind III dephosphorylated pBR322 DNA, 1.0 μg of HindIII-cleaved SE4 AH-IV DNA of 25, 0 1l

  of ligase buffer and a T4 DNA ligase unit (E.C.

6.5.1.1) at 16 C overnight.

  After incubation, the ligation mixture was transformed into DH2 competent cells of E. coli. The

  strain DH2 was obtained from E. coli Genetic Stock Center.

  The DH2 strain was chosen because it is recA-, which means that the plasmid is relatively stable and will not combine with the genome. The transformation was carried out by the following process: DH2 was grown in Luria broth at 37 ° C with vigorous stirring to OD 50 = 0.5, then cooled on ice and centrifuged for 10 minutes. minutes to

  7000 rpm in a sterile 50 ml centrifuge tube.

  The pellet was resuspended in an equal volume of 0.1 M cold sterile MgCl 2 (4 C). The mixture was centrifuged as above and the pellet resuspended in a half volume of 0.1 M CaCl 2 (4 C) and chilled on ice for 20 minutes. This suspension was centrifuged as above and put back into

25880-15

  suspension in 1/20 volume of 0.1 M cold CaCl 2 (4 C). A volume of 0.2 ml of these competent cells was added to a sterile tube containing the mixture

  Ligation reaction described in the preceding paragraph.

  The DNA / cell suspension was incubated on ice for 30 minutes and then pulsed with heat at 42 ° C. for 2 minutes. The cells were diluted 10-fold with Luria broth and incubated at 37 ° C for 2 hours to allow expression of

plasmid.

  The cells were plated on Luria broth plates containing 50 μg / ml spectinomycin (0.1 ml cell suspension per plate) and incubated at 37 ° C for 16-24 hours. Spr colonies were placed on separate plates of Luria broth containing either 50 μg / ml ampicillin, 20 μg / ml tetracycline or 50 μg / ml spectinomycin. 22 Spr r r clones showed the Ap Sp phenotype. All except

of one of these was Tcs.

  Rapid isolation of the plasmid was performed on the AprSpr clones, following the procedures described in Ex. III. Agarose electrophoresis of a Hind III restriction endonuclease digest of these DNA preparations revealed that four clones contained the expected DNA fragment pattern: a fragment of about 17 kb (transamninase gene ) and a 4.3 kb fragment (pBR322). The four plasmid-containing clones were subjected to a qualitative determination of transaminase. The test cells were grown overnight in complex medium with 50.0 μg / ml spectinomycin. 25-45 μl of the cell suspension was added to a 24-well titration cup with 450 μl Reagent (Example IV). The cup was incubated for about 2 hours at 37 C. A blue color was

  indicated transaminase activity.

  The four clones were shown to have significantly enhanced transaminase activity (e.g., by color intensity) compared to the parent E. coli strain DH2, with the parent strain yielding approximately + 1 and clones giving relative activity values of about +5. The four clones were designated DH2 / pAH5, DH2 / pAH17, DH2 / pAH18 and DH2 / pAH27. One of the clones, DH2 / pAH27 was irrevocably deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, for making available to the public, and

  received accession number ATCC 53248.

Example V.

  (Restriction analysis of the plasmid

Example IV)

  Three of the plasmids of Example IV appeared to have the same insert size and orientation determined by double restriction enzyme digestion and tetracycline sensitivity. One of them, designated pAH27, was subjected to restriction analysis by complete digestion with each of the nine restriction endonucleases, the list of which is given in Table I. The resulting fragments were

  resolved by size on electrophoresis agarose gels.

  The first six endonucleases of Table I cut pAH27 to produce 3-8 fragments. These fragments, in each case, reached 21-22 kb for the hybrid plasmid corresponding roughly to the 17 kb insert and

pBR322 of 4.3 kb.

Table I.

  Endonuclease Fragments Pvu I Fragment size (kb) 7.2, 1.7, 4.4, 1.5 4, 0, 1.0, 2.5 1.0 Pvu II Cla I Sal I Bam HI Bgl II Bcl I Pst I Eco RI

8.0, 4.5, 4.3, 2.3, 2.0

7.4, 5.0, 4.7, 4.0

7.6, 7.0, 5.0, 1.3

9.5, 6.6, 5.0

, 1.0, 1.0

  The relationships and distances between the restriction sites were thus determined by endonuclease double digestion. The resulting map of

  restriction is shown in Figure 1.

Example VI.

  (Subcloning the thermally stable transaminase gene into a multiple copy plasmid using Sau 3A endonuclease) The heat stable transaminase gene was also subcloned from hybrid lambda SE4 AH-IV phage using restriction endonuclease Sau 3A. Lambda SE4 AH-IV refers to the phage obtained from clone DG 44-IV of Examples II and III. A product of partial digestion of the hybrid phage with Sau 3A endonuclease was prepared, monitoring to obtain DNA fragments in the size range of 4 to 12 kb. Plasmid pBR322 was opened at Bam HI site and Sau 3A fragments of hybrid phage were inserted using the procedures of Example IV. Insertion of the fragments at the Bam HI site of pBR322 inactivated the Tcr gene but left the Apr gene intact. These clones are not expected to be spectinomycin resistant because smaller fragments of DNA containing the transaminase gene have been inserted. After ligation, the mixture was transformed into competent E. coli DG (aminotransferase-negative) cells by the procedure of Example IV, except that the cells were grown to OD 550 = 0.25. The cells were plated on Luria broth plates containing 50.0 μg / ml ampicillin and then stained on similar plates containing ampicillin and tetracycline to test the AprTc phenotype and plated onto the minimum medium to test the complementation of auxotrophy. Rapid isolation of the plasmids was accomplished on the AprTcs clones following the procedures described in Example III. Agarose electrophoresis of a Hind III restriction endonuclease digest of plasmids of thirteen AprTcs clones determined that two clones contained hybrid plasmids (designated pGA115 and pGA108) with 8.3 kb insertions, respectively. 9.4 kb. These clones were subjected to the quantitative determination of Example IV and gave improved activity values of about + 2 to +3. To demonstrate that the transaminase activity was derived from plasmids + ybrids, pGA115 and pGA108 were isolated by the procedure of Example III and reintroduced into DG30 (aminotransferase-negative) hosts by the transformation process. of Example IV, growing the cells to OD 550 = 0.25. After transformation with isolated plasmids pGA108 or pGA115, the DG30 hosts were released from their auxotrophy. One of the clones, DH2 / pGA115 has been irrevocably deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, for the provision of

  public, and received accession number ATCC 53247.

Example VII.

  (restriction analysis of the plasmids of

Example VI).

  Plasmids pGA108 and pGA115 of Example IV were subjected to restriction analysis by complete digestion with restriction endonucleases listed in Table II. The resulting fragments were resolved by size or size on agarose electrophoresis gels. Fragments, in each case, were added up to the approximate size of the plasmid, roughly corresponding to an 8.3 kb insert (for pGA115) or a 9.4 kb insert

  (for pGA108) and the pBR322 of 4.3 kb.

TABLE II.

  Endonuclease Fragments Size of fragment (kb) pGA115 (12.6kb): Pst I Sal I Hind III Eco RI Cla I Bgl II Bam HI pGA108 (13.7kb): Mr ... T Sal Sal I Pst I Cla I Eco RI Hind III Bam HI Bcl I 6.5, 7.7, 6.9, 12.6 12.6 6.2, 6, 3, 8.2, 7.4, 13.7 13.7 13.7

4,5, 1,6

4.9, 6

3,4, 2,3, 1,8

, 3, 1,2, 0.9

3.8, 1.7

  6.3 Relationships and distances between restriction sites were determined by further fragmentation using dual digests with endonuclease. The resulting cards of restrictions

are shown in Figures 2 and 3.

Example IX.

  (Relative increases in transaminase activity). Quantitative determination was made to measure the relative increases in E. coli DH2 transaminase activity at E. coli DH2 / pAH27, and E. coli-DG 30 and DG27 at E. coli DG 30 / pGA108 and DG30 / pGA115. The ferric chloride colorimetric determination for phenylpyruvic acid (process described below) measures the ability of a cell-free extract to catalyze the following reaction: alpha-ketoglutarate + L-phenylalanine

glutamate + phenylpyruvate.

  Cell-free extracts of each strain were prepared by sonicating the cells and then centrifuging at 15,000 rpm for one hour in an SS-34 rotor. A sample of each of the cell-free extracts was combined with a solution of P-5-P, alpha-ketoglutarate and L-phenylalanine substrates and incubated in a 37 C water bath by diluting with 0.25 moles of phosphate. of potassium as needed. The samples were swirled every 10 minutes. After one hour, a 50.0 μl sample was taken, added to 50.0 ml of Development Solution and swirled. After 5.0 minutes, the optical density at 640 nm was read. This determination has determined the relative activities of

  each strain shown in Table III.

25. 8015

25880 15

TABLE III.

  (Comparative transaminase activities) DH2 strain Relative activity DH2 / pAH27 DG 30 DG 27 DG 30 / pGA108 DG 30 / pGA115 0o 11.6 13.7 Example X.

(Stability of pAH27).

  The stability of plasmid pAH27 in the presence and in the absence of spectinomycin as selective pressure was sought. To 500 ml Erlenmeyer flasked flask was added portions of 100 ml Lowry Modified Medium, with and without 50 ppm spectinomycin. The flasks were inoculated with 1.0 ml of a cell suspension prepared by washing two sloping plates of DH2 / pAH27 (Example IV), grown at 37 ° C. for 48 hours, with 16 ml of brine saline solution. 0.9%. The shake flasks were incubated at 37 ° C at 250 rpm. After 24 hours, fresh shake flasks were inoculated by serial transfer. This serial transfer was repeated every 24 hours. Whole cell transaminase activities were determined every 24 hours. After 5 days of serial transfer, the average activity of the reproduced flasks indicated that 93% of the original phenylalanine-specific 1C production rate was conserved in the presence of spectinomycin, as opposed to 42% in its absence. This demonstrates that plasmid pAH27 was stably maintained in E. coli DH2 using

  ppm of spectinomycin in the culture medium.

  Colorimetric determination of ferric chloride of phenylpyruvic acid, sodium salt. Principle: Phenylpyruvic acid (PPA) forms a blue complex with ferric ions. The intensity of the blue color is proportional to the amount of PPA present. Reagents:

  1. Development Solution (SD) (1000 ml).

  Mix the following ingredients and cool to room temperature in an ice bath. Add the contents to a 1 liter volumetric flask and reach the volume with deionized water. Ingredients: 0.5 g of FeCl3. 6:20; 20.0 ml of glacial acetic acid; 6GO0ml

  dimethyl sulfoxide; 200.0 ml of deionized water.

  2. Na-PPA standard solution (10 g / l) - add 500.0 mg of Na-PPA.H 2 O to 50.0 ml of a 0.1 M T-ris: HCl buffer (pH 8.0). C. Agitate until dissolved Place the solution into a 50.0 ml volumetric flask and make up to volume with Tris-HCl buffer Store under refrigeration For 0.1 mole Tris / HC1 buffer: add 900 0 ml of deionized water at 12.11 g of Tris, adjust the pH to 8.0 with HCl, pour into a volumetric flask, add deionized water to 100 ml.

Calibration curve.

  Add the standard solution of Na-PPA and 5.0yl of the Development Solution to tubes of

  glass spectrophotometer. Mix with swirls. Let stand for 5 minutes (starting at

  count the time at the addition of SD to the first tube).

  Read the optical density (OD) at 640 nm. Prepare a standard calibration curve of Na-PPA.H20 (the vertical axis is the absorbance, the horizontal axis represents

* mg Na-PPA.H 2 O / ml SD).

  Standard Na-PPA mg Na-PPA.HO20 per ml SD

1 0.01

  y1 0.02 pl 0.03 20 Fi 0.04 pl 0.05 pl 0.06

25880 15

  Calculation: Na-PPA.H20 = (mg / ml) OD x 100 x dilution versus standard curve or PPA (g / l) OD x 100 x dilution slope versus standard curve x 164.4 204.1

Claims (18)

  A strain of a host microorganism characterized in that it is transformed by a recombinant plasmid containing a DNA fragment which contains a Salmonella typhimurium gene coding for an enzyme of thermally stable transaminase.   2. Strain according to claim 1, characterized in that the expression product of said gene catalyzes the stereospecific transamination of an alpha-keto acid and an amino donor corresponding L-amino acid. 3. Strain according to claim 1, characterized in that said transaminase enzyme is AT sty 2.   4. Strain according to claim 1, characterized in that said DNA fragment can   reach up to about 25 kilobases.   5. A strain according to claim 1, characterized in that said recombinant plasmid is a multi-copy plasmid.   6. Strain according to claim 1, characterized in that said recombinant plasmid contains one or more genes which confer to the other,   resistance to one or more antibiotics.   7. Strain according to claim 1, characterized in that said recombinant plasmid has one of the restriction maps according to the Figures 1, 2 and 3.   8. Strain according to claim 1, characterized in that it is deposited under the number ATCC 53247 or 53248.   9. Recombinant plasmid characterized in that it comprises a Salmonella typhimurium transaminase gene whose gene product has the 2580O15   characteristic of thermal stability and the ability to detect an auxotrophic host microorganism for the   phenylalanine, tyrosine and aspartic acid.   10. Recombinant plasmid according to claim 9, characterized in that the gene produced is the AT enzyme. sty 2.   11. Plasmid having the characteristic of the presence of a gene coding for the Salmonella typhimurium transaninase enzyme characterized in that it is prepared by: (a) isolating fragments of the Salmonella typhimurium genome by digestion with the restriction endonuclease, (b) ligation of said fragments into lambda phage, (c) transformation of a negative strain to E. coli transaminase by a plasmid containing a gene which expresses the lambda repressor sensitive to temperature, (d) transduction of the transformed E. coli strain by the hybrid phage prepared in step (b), (e) identification of hybrid phage-containing clones with the thermally transaminase gene stable,   (f) isolating a DNA fragment comprising the transaminase gene, and (g) ligating said DNA fragments in a plasmid. 12. Plasmid according to claim 11, characterized in that the expression product of said gene is thermally stable.   13. Plasmid according to claim 11, characterized in that the expression product of said gene is the enzyme AT sty 2.   A process for producing a thermally stable transaminase enzyme characterized in that it comprises putting a strain of a transformed host microorganism containing one or more recombinant plasmids which comprise a DNA encoding the thermally stable transaminase enzyme of Salmonella typhimurium in culture.   15. The method of claim 14   characterized in that said enzyme is AT sty 2.   16. A process for producing L-amino acids characterized in that it consists in: (a) selecting a strain of a transformed host microorganism containing one or more plasmids comprising a thermally stable transaminase gene of Salmonella typhimurium, and (b) placing said host microorganisms or a portion or extract thereof in contact with an alpha-keto acid and an amino donor to form the L-amino acid corresponding.   17. Method according to claim 16, characterized in that the expression product of said gene is the enzyme AT sty 2.   18. The method of claim 16, characterized in that whole cells of the selected strain or a portion or an extract thereof are immobilized.