CA1295568C - Cloning and utilization of aminotransferase genes - Google Patents

Abstract

ABSTRACT
A composition of matter comprising plasmids containing genes coding for the synthesis of aminotransferases was constructed. These plasmids are used in a method for synthesizing increased concentrations of aminotransferase enzymes and for the improved synthesis of amino acids in cells having the transamination reaction rate-limiting.

Description

Background of the Invention Field of the Invention This invention involves the construction and use of genetically engineered plasmids. These plasmids are constructed using restriction endonucleases to contain the genes coding for the aminotransferase enzymes used during the synthesis of amino acids. These plasmids promote the synthesis of aminotransferases in bacterial cells. This synthesis allows increased yields of aminotransferase enzymes and the elimination of the aminotransferase reaction as a rate-limiting step in amino acid biosynthesis.

Description of the Prior Art The transmination of amino acid precursor molecules by aminotransferase enzymes is reviewed by Umbarger (Ann.
Rev. Biochemistry, 47:533-606,1978. Umbarger at p. 534 indicates he is reviewing principally the gram-negative bacteria Escherichia coli and Salmonella typhimurium, but that their pattern of amino acid synthesis and regulation is not always the universal pattern.
Aminotransferase and transaminase are used as equivalent terms in this document. Present aminotransferase nomenclature defining the transaminases A, B and C and the evolution of this nomenclature is discussed by Umbarger at p. 550-552 and p. 581-582. To avoid the potential confusion of the transaminase A, B, C designation we will ~k 129S~68 use the genetic terminology of aspC, tyrB and ilvE gene products as defined by Umbarger at p. 582. The aspC gene product will catalyze the t~ansamination of amino acid precursors to produce aspartate, glutamate, phenylalanine and tyrosine. The tyrB gene product will catalyze the transamination of amino acid precusors to produce phenylalanine, tyrosine, glutamate, aspartate and leucine. The vE gene product will catalyze the transamination of the amino acid precusors to produce isoleucine, valine, leucine, phenylalanine, glutamate and alanine.
The location of the aspC, ~y_B and ilvE genes on the genetic map of E. coli K12 strain is disclosed and discussed by Bachmann et al. (Microbological Reviews, 44:
1-56, March 1980). The aspC gene located at E. coli K12 map position 20 minutes codes for an enzyme here called aspartate aminotransferase and given the Enzyme Commission Number E.C. 2.6.1.1 (Bachmann et al, p. 4).
The ~y_B gene located at E. coli K12 map position 91 minutes codes for an enzyme called tyrosine or aromatic aminotransferase and given the Enzyme Commission Number E.C. 2.6.1.5 (Bachmann, et al. p. 21). The ilvE gene located at E. coli K12 map position 84 minutes codes for an enzyme here called branched-chain-amino-acid aminotransferase and given Enzyme Commission Number E.C.

2.6.1.42 (Bachmann et al. p. 10).

12955~i8 Amino acid transamination by aspC and tyrB gene products are disclosed by Gelfand et al., (J. Bacteri-ology, 130:429-440, April 1977) to be responsible for essentially all the aminotransferase activity required for the de novo biosynthesis of tyrosine, phenylala-nine, and aspartate in E coli K12. However, the pres-ence of the ilvE gene product alone can reverse a phenylalanine requirement indicating it has the ability to transaminate a precursor or phenylalanine.
Transducing phage lambda has been used to carry DNA
fragments for E coli in the aspC region (Christensen and Pedersen Mol. Gen Genet, 181: 548-551, 1981). The aspC region in bacteriophage lambda was used as a source of the gene for the ribosomal protein Sl. Their Sl gene was closed onto plasmids for research purposes. The aspC
gene, however, was not cloned into a plasmid for the production of transaminases. The aspC region was used as a marker to facilitate isolation of a transducing phage in a tyrosine auxotroph lacking the aspC and tyrB genes.
This transducing bacteriophage was not suitable for producing high levels of aminotransferases.

Summary of the Invention Applicants invention describes the isolation of the genes coding for the aminotransferases used during the bacterial synthesis of amino acids and the construction of plasmids containing these aminotransferase genes.

l~gS568 Applicant's plasmids contain one or more copies of the aspC, tyrB or ilvE genes. Applicant's invention des-cribes a use for these aminotransferase genes in the synthesis of increased enzyme concentrations of the aminotransferase in bacterial cells. Applicant's in-vention describes a method for the production of in-creased amounts of aminotransferases. Applicant's invention describes a method of improved amino acid synthesis where the aminotransferase catalyzed reaction is rate limiting. Applicants invention describes a method for the increased synthesis of L-phenylalanine wherein the aminotransferase catalyzed transamination of phenylpyruvic acid to phenylalanine is rate-limiting.
Applicant's invention describes the nucleotide sequence of the tyrB gene encoding the enzyme aromatic amino-transferase. Applicant's invention describes the amino acid sequence of the aromatic aminotransferase encoded by the gene tyrB.
The aminotransferase genes, aspC, tyrB, ilvE, code for the synthesis of the aminotransferases which catalyze the transamination of the carbonyl precursors of amino acids. AspC gene codes for the transaminase A (aspar-tate aminotransferase) (EC2.6.1.1) and is catalytically active during the synthesis of aspartate, glutamate, phenylalanine and tyrosine. The tyrB gene codes for tyrosine or aromatic aminotransferase (EC 2.6.1.5) and is catalytically active during the synthesis of phe-nylalanine, tyrosine, glutamate, aspartate and leu-cine. The ilvE gene codes for transaminase B and is _4_ 12955~8 catalytically active during the synthesis of iso]eucine, valine, leucine, phenylalanine and glutamate. When the transamination reaction in bacterial amino acid synthesis becomes rate-limlting, the presence of these plasmid-borne aminotransferase genes allows for the synthesis of additional aminotransferases. Aminotransferase synthesis sufficient to overcome the rate limitation results in an increased rate of amino acid biosynthesis.
These plasmids are used to increase the total quan-tity of aminotransferase present in a cell and it may beused as follows. The transamination reaction may be used within the cell, in a cell extract, or the extract can be utilized for further purification of the specific am-inotransferase and then used in a purified state.

Objects of the Invention It is an object of this invention to construct an extra-chromosomal element, such as a plasmid, that contains one or more genes capable of coding for the synthesis of an aminotransferase.
Another object of the invention is to construct a plasmid containing one or more of the following amino-transferase genes, aspC, tyrB or ilvE.
Yet another object of the invention is a method for the synthesis of aminotransferases.
Still another object of the invention is a method for the synthesis of an aminotransferase that is the product of the aspC, tyrB or ilvE gene.

~ -5-" 129~i568 Another object of the invention is a method of in-creasing the rate of transamination of receptive amino acid precursor molecules accomplished by the insertion of a plasmid containing the aspC, tyrB or ilvE gene into a microorganism, followed by the synthesis of a physio-logically effective concentration of aminotransferase resulting in an increased rate of amino acid synthesis.
Still another object of the invention is a method of increasing the transamination of phenylpyruvic acid by the aminotransferases coded for by plasmids containing the a_ C, tyrB or ilvE genes and expressed in a micro-organism.
Yet another object of the invention is a method of increasing the synthesis of an amino acid in a cell where the aminotransferase is a rate-limiting enzyme which is accomplished by the introduction and expression of a plasmid containing one or more aminotransferase genes.
Still another object of the invention is the in-creased synthesis of phenylalanine from phenylpyruvic acid by the introduction and expression of a plasmid containing an aminotransferase gene in a microorganism wherein the wild type transamination of phenylpyruvic acid is a rate-limiting step in the synthesis of phe-nylalanine.
Another object of the invention is the increased synthesis of tyrosine from p-hydroxyphenylpyruvate by the introduction and expression of a plasmid containing an aminotransferase gene in a microorganism wherein the wild lZ9SS~

type transaminase of p-hydroxyphenylpyruvic acid is a rate-limiting step in the synthesis of tyrosine.
Still yet another object of the invention is the introduction and expression of a plasmid containing an aminotransferase gene in an enteric microorganism, one such microorganism being Escherichia coli.

Description of the Drawings Figure 1 describes the biosynthetic pathway for the synthesis of the aromatic amino acids tyrosine, phenylalanine and tryptophan.
Figure 2 is a native protein gel electropherogram showing the presence or absence of the aminotransferases encoded by tyrB, aspC or ilvE genes in various bacterial strains.
Figure 3 describes the DNA nucleotide sequence of the EcoRI-BamHI fragment carrying the ~y_B gene.
Figure 4 describes the amino acid sequence of the aminotransferase encoded for by the tyrB gene.
Figure 5 illustrates an E. coli Flowchart showing intermediate strains and processes resulting in deposited strain HW159.
Figure 6 illustrates the modification of the ~y_B+
plasmid.
Figure 7 illustrates the restriction map of the plasmid carrying the aspC gene.

1295S~3 Detailed Description of the Specific Embodiments The aminotransferases or tranaminases are a family of enzymes that covalently transfer an amino group from a donor molecule, such as aspartate or glutamate, to an acceptor a-keto-acid precursor of an amino acid, such as oxaloacetic acid. The reaction is freely reversible.
Therefore, the aminotransferases can be utilized both in the synthesis and the degradation of amino acids.
Amino acid biosynthesis often requires transamination as the final step in biosynthesis. An example of such à
process is the transamination of phenylpyruvic acid to produce phenylalanine with the simultaneous conversion of glutamate to a-ketoglutarate. This is shown as step 10 in Figure 1. The biosynthesis of the aromatic amino acids phenylalanine and tyrosine both require the action of an aminotransferase as the final step. Phenylpyruvate is converted to phenylalanine by an aminotransferase that transfers an amino group from glutamate (figure 1, step lO). Similarly, an amino group from glutamate is transferred to 4-hydroxyphenylpyruvate to produce tyrosine (figure l, step 12). Each aminotransferase has preferred substrates for the transamination reaction. However, residual activity is present on other substrates permitting each aminotransferase to participate in more than one amino acid's biosynthesis. The E. coli transaminase encoded by the aspC gene is active on 12~5S68 aspartate, glutamate, phenylalanine and tyrosine. The E. col transaminase encoded by the tyrB gene is active on phenylalanine, tyrosine, glutamate, aspartate and leucine. The E coli transaminase encoded by the ilvE
gene is active on isoleucine, valine, leucine, phenyl-alanine and glutamic acid (Umbarger at p. 582).
It is recognized that other pathways exist for synthesizing tyrosine and phenylalanine in other microorganisms. One such pathway is found in cyanobacteria and P aeruginosa and involves the conversion of prephenate directly to pretyrosine.
Pretyrosine is then a substrate for conversion directly to tyrosine or to phenylalanine. It is recognized that analogous cloned transaminase genes for the appropriate transaminase are similarly useful in increasing the reaction synthesizing pretyrosine when there are excess levels of prephenate. This transaminase would result in increasing the rate of synthesis of tyrosine and phenyl-alanine.
The enzymatic steps in the synthesis of the aromatic amino acids are shown in figure 1, steps 1-20. Normally each enzymatic step is under regulation by controlling the synthesis of the enzyme and/or by allosterically regulating the rate of enzymatic catalysis. Wild type microorganisms normally do not over-produce any one amino acid due to these controls. Mutant strains in which the controls on the synthesis of a particular amino acid have been inactivated or by-passed may be isolated and in _g_ X

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general these strains tend to over-produce that particu-lar amino acid. In the case of L-phe, the regulation is of necessity complex, since part of its synthetic pathway is shared with the other aromatic amino acids L-tyrosine (L-tyr) and L-tryptophane (L-trp) (see Figure 1 of the accompanying drawings).
In E coli, as in many microorganisms, the first step in the pathway is catalyzed by the three DAHP synthetase isozymes each of which is sensitive to one of the three final products. In addition, the first steps in the pathway after the branch point at chorismate are also sensitive to feed-back inhibition. Anthranilate syn-thetase is inhibited by L-trp and the two chorismate mutase isozymes are inhibited by L-phe and L-tyr. The synthesis of a number of the enzymes concerned is also subject to regulation. The tyrosine repressor, the product of the tyrR gene, controls expression of DAHP
synthetase (L-tyr) and chorismate mutase (L-tyr). The genes for these last two enzymes together constitute the tyrosine operon. The tryptophan repressor, the product of the trpR gene, controls expression of all the enzymes of the trp operon which convert chorismate to L-trp.
This regulator molecule also controls the expression of DAHP synthetase (L-trp). In addition, expression of the pheA gene which codes for the chorismate mutase (L-phe) and the expression of the trp operon are rendered sens-itive to the level of L-phe. The trp operon is rendered sensitive to the level of L-trp by an attenuator/leader peptide control system.

lZ9SS6~

Mutant strains that over-produce amino acids are often isolated by selecting for resistance to appropriate amino acid analogues. In the case of L-phe production, one may isolate strains derepressed for DAHP (L-tyr) and DAHP (L-trp) expression by selecting 3-fluoro-tyrosine and 5-methyl-tryptophan resistant mutants which generally have inactive tyrR and trpR repressors. Mutants in which the chorismate mutase (~-phe) is resistant to L-phe may be isolated by selecting for 2-thienyl-alanine resist-ance. A DAHP synthetase (L-phe) that is resistant to feed-back inhibition may be isolated by first construct-ing a mutant strain lacking the other two DAHP synthetase isozymes. In such a strain, the synthesis of L-tyr and L-trp is sensitive to the amount of L-phe in the medium.
Mutants able to grow in medium containing L-phe, but lacking L-tyr or L-trp, usually contain feed-back resis-tant forms of DAHP synthetase (L-phe).
Now, a combination of these approaches will result in strains that over-produce L-phe. Since the synthetic pathway is largely shared with that of L-tyr and L-trp, attention must be given to the prevention of L-tyr and L-trp build-up if one is interested in the optimisation of L-phe synthesis. One simple expedient is to incorporate a tyrA mutation into an L-phe over-producing strain so as to prevent L-tyr accumulation and to avoid the unwanted diversion of precursors to L-phe. Similarly, a deletion of the entire trp operon would prevent accumulation of L-trp. Such mutants would, of course, have to be cultured 12~5S68 in media that included these two amino acids so as to permit growth.
The combination of approaches outlined above results in a strain de-regulated for the synthesis of L-phe which accumulates that amino acid in significant amounts. Now, the rate limiting steps in a biosynthetic pathway are usually those at which control is exerted. In a de-regulated strain, it is likely that a new step will be-come rate-limiting. Thus, to increase the production of L-phe still further it is necessary to identify the new rate-limiting step and to increase the activity of the relevant enzyme. This may be done in one of three ways:
(a) by isolating mutant strains in which the relevant enzyme is present in greater amounts;
(b) by isolating mutant strains in which the relevant enzyme has a higher specific activity;
(c) achieving the same end as in (a), but by cloning the gene for the enzyme of interest onto a suitable multi-copy plasmid and introducing this into the L-phe over-producing strain.
If such an enzyme is not normally subject to regulation, isolating mutants of the first two classes may be dif-ficult. The best way to achieve the increase in activ-ity is to clone the gene for the relevant enzyme.
This is achieved by ligating DNA from a suitable do-nor strain carrying the gene for the enzyme one wishes to clone to a suitably prepared DNA from an appropriate vector. One can then transform a mutant strain lacking 12~35568 the activity of the newly cloned gene. The problem experimentally is to isolate the gene. If the lack of this activity bestows a recognizable phenotype on this recipient strain, such as auxotrophy for a particular amino acid, then one can simply select for clones carrying the gene of interest by virtue of the ability of such clones to complement the lesion in the recipient strain.
As an alternative, the gene could be cloned first into a low copy number plasmid or into a suitable bacteriophage vector and subsequently sub-cloned into the desired multi-copy plasmid. Alternatively a strain carrying a suitable prophage near the gene of interest can be constructed and a specialized transducing phage carrying the desired gene isolated by inducing the lysogenic donor strain and using the resultant lysate to transduce the recipient to prototrophy. The gene of interest can then be sub-cloned from the DNA of this specialized transducing phage such as lambda.
Should the mutation of the gene of interest not confer any readily identifiable phenotype, such as auxotrophy, then the cloning of such a gene can be effected by cloning nearby genes for which a selection exists and then screening these clones for the presence of the gene of interest by assaying for the product of the desired gene after introducing the clones into a recipient deficient in that enzyme. Such an approach is facilitated by using methods that allow the cloning of large DNA fragments, such as those involving bacterio-phages or cosmids. If no 12955~

readily selectable marker is known to be in the vicinity of the desired gene, then it may be possible to isolate a strain carrving the insertion of a readily selectable transposon, such as Tn 10, which encodes tetracycline resistance, near the gene of interest. Large DNA
fragments containing this marker can then be cloned and the resultant phage or cosmids screened for the presence of the gene of in erest. The gene could then be sub-cloned into a suitable high copy number plasmid.

The following are the strain of E. coli used in the examples. Beside each strain designation is a sumrnary of the bacterial genotype and source. The relationship of intermediate strains of E. coli leading to the production of the auxotroph HWl59 which is both ~C and tyrB
is shown in Figure 5. The process which converted one strain to another is also summarized on the Flowchart in Figure 5.

Strain HWl59, the aspC and ~B mutant was deposited in The American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, U.S.A. and has the number designation ATCC 39260.

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Table 1 STRAIN LIST
Strain Genot~pe Source HW22 metE M. Edwards BHB2688 recA~imm 434 CI857 b2 red 3 Eaml5 Sam7 J. Burke BHB2690 recA/~imm 434 CI857 b2 red 3 Daml5 Sam7 J. Burke E107 thrl leuB6 thyA6 thil dnaB107 deoCl B. Bachmann lacYl tonA21 rpsL67 SupE44 DG44 hsdS thil lacYl galK2 xyl5 mtlI proA2 B. Bachmann argE3 hisG4 hppT29 aspC13 rpsL31 tsx33 supE44 recB21 recC22 sbcB15 MC1061 araD139(ara-leu)del7697 M. Casadaban (lacIPOZY)del74 galU galK hsdR rpsL
DG30 thil proA2 argE3 hisG4 lacYl galK2 B. Bachmann aral4 xyl5 mtll rpsL31 tsx323 supE44 recB27 recC22 sbcB15 hsdS hppT29 ilvE12 tyrB509 aspC13 ES430 thi malB29 relAl spoTl B. Bachmann HW157 araD139(ara-leu)del7697 This work (lacIPOZY)del74 galU galK
hsdR rpsL aspC13 HW159 araD139(ara-leu)del7697 This work (lacIPOZY)del74 galU galK (deposited) hsdR rpsL aspC13 tyrB507 HW225 araD139(ara-leu)del7697 This work (lacIPOZY)del74 galU galK
hsdR rpsL aspC13 tyrB507 recA srl::TnlO
HW519 Prototroph W3110 Commercially Available 12955~3 EXAMPLES
Construction and Use of Transaminase Plasmids For the purpose of exemplification, a novel strain of - ~' E coli that over-produces L-phe has been constructed.
This was achieved by a multi-step process that involved systematically de-regulating the L-phe synthetic pathway.
In such de-regulated strains, it has been found that the final step, namely the transamination of PPA to L-phe, becomes rate-limiting during fermentation and the PPA

accumulates with detrimental effect. As a further example, therefore, the gene for the tyrosine (aromatic) amino-transferase from E coli has been cloned onto a multi-copy plasmid. Introduction of this plasmid into the L-phe over-producing strain reduces this build-up of PPA
and increases the efficiency of the fermentation. As an additional example, the gene for the aspartate aminotransferase of E coli has been cloned onto a high copy number plasmid. This enzyme also has L-phe aminotransferase activity and incorporation of this plasmid in the L-phe over-producing strain also enhances the efficiency of the process.
Strains carrying plasmids with ~y~B or aspC have added utility in that they over-produce the relevant enzyme to a considerable extent. They are therefore a novel and useful source of these enzymes.
The ~y~B or aspC genes are also useful on a plasmid, in a suitable genetic background, by providing a selective lZ95~

pressure for maintenance of that plasmid. This selective pressure has advantages over conventional antibiotic selection methods.
Example 1 Preparation of Plasmid DNA
Plasmid DNA was prepared as follows (adapted from the method of J. Burke and D. Ish-Horowitz) (Nucleic Acids Research 9: 2989-2998 (1981)) The desired bacterial strain was grown to saturation in 5ml of L-Broth plus suitable antibiotics. The culture was transferred to a 15ml corex tube and the cells deposited by centrifugation at 10,000 RPM for 1 minute. The supernatant was decanted and the pellet resuspended in 500 ~1 of fresh solution I, (50 mM D-glucose, 25 MM Tris pH8.0, 10 mM EDTA and 2 mg Lysozyme ml-1) and then incubated at room temperature for 5 minutes. The cells were lysed by the addition of 1 ml fresh solution II (containing 0.2 M NaOH and 1% SDS).
This was mixed in gently and then incubated on ice for 5 minutes. 750 ~1 of cold solution III (to 29 ml of glacial acetic acid add water to about 60 ml adjust the pH
to 4.8 with 10 m ~OH and make up to 100 ml) was then added and mixed in thoroughly. After a further 5 minutes on ice, the precipitated chromosonal DNA was pelleted by centrifugation at 10,000 rmp for 10 minutes. The supernatant was decanted to a fresh 15 ml corex tube and 3.75 ml of Ethanol was added. The tube was kept on ice for 15 minutes. The DNA was pelleted by centrifugation at 10,000 rpm for 10 minutes. The supernatant was then 129556~3 poured off and the pellet thoroughly resuspended in lOmM
Tris (ph8.0), lmMEDTA, 20 ~l of 3 M sodium acetate pH7.0 was added and the DNA transferred to an Eppendorf micro-test tube. The DNA was extracted twice with 200 ~l of ultra-pure phenol that contained 0.1% 8-hydroxy quinoline and had been pre-equilibrated twice against 1 m Tris pH8.0 and once against 100 mM Tris 10 mM EDTA pH8Ø
The residual phenol was removed by four extractions with 1 ml of diethyl ether. The DNA was then precipitated by the addition of 500 ~l ethanol and incubation in a dry-ice/ethanol bath for 10 minutes. The DNA was pelleted by centrifugation at 10,000 rpm for 10 minutes. The supernatant was discarded and the pellet resuspended in 500 ~l of 70% ethanol in water (v/v). The DNA was re-pelleted by centrifugation at 10,000 rpm for 10 minutes, the supernatant was discarded and the pellet containing the DNA was dried in a vacuum dessicator for 30 minutes. The DNA was redissolved finally in 50 ~l of 10 mM Tris 1 mM EDTA and stored at -20C
2n This preparation generally gives about 5 ~g of plasmid DNA when the strains used are derived from MC1061. The preparations also contain considerable amounts of RNA and so all the restriction digests described in this patent also include 100 ~g ml-1 RNASE
A that has been pre-treated at 90 for 15 minutes to destroy DNAses. The preparation can also be scaled up to cope with plasmid isolation from 50 ml cultures.

12955~3 Example 2 (a) Cloning of the tyr B gene Isolation of cosmid clones carrying tyrB and DNA from the E coli strain HW 22 (metE) was prepared according to the method of Marmur (PNAS 46:453, 1960). Aliquots of this DNA were partially digested with tlle restriction endonuclease Sau3A as follows: The DNA (80 ~g) was made up to 400 ~l in Sau3A buffer containing 50 mM
NaCl, 6 mM Tris-HCl (tris (hydroxymethyl) aminomethane hydrochloride) pH 7.5, 5 mM MgCl2, 100 ~g/ml gelatin.
The DNA solution was`then-split into 8 x 50 ~l aliquots. Sau3A (New England Biolabs) was then serially diluted (2-fold steps) in Sau3A dilution buffer containing 50 mM KCl, 10 mM Tris-HCl pH 7.4, 0.1 mM EDTA
(ethylene diamine tetra acetic acid, disodium salt), 1 mM
dithiothreitol (DTT), 200 ~g/ml bovine serum albumin (BSA) and 50% v/v glycerol. 5 ~l of each of seven serial dilutions along with 5 ~l of undiluted enzyme were then added to the DNA solution. The amount of enzyme added ranged from 2.5 to 0.02 u. The digestion was continued for l hour after which time the reactions were stopped by a five minute incubation at 65C. The tubes were then cooled on ice.
Two ~l aliquots of each of the digestions were taken and were subjected to electrophoresis on a 0.7% w/v agarose gel in TBE (50 mM tris-HCl pH 8.3, 50 mM boric acid and l mM EDTA) along with suitable markers. After staining with ethidium bromide (1 ~g/ml) for 15 minutes, the gel was observed under 254 nm illumination to ~2$55~

visualise the DNA. In this way, the sample giving the greatest amount of material in the 45 kb si~e range could be estimated.
The DNA from this sample was then ligated to DNA from the cosmid vector pHC79 which had been prepared as described below:
Two 25 ~g aliquots of pHC 79 DNA were completely digested one with the restriction endonuclease Hind III
(EC 3.2.12.21) and the other with the restriction endonuclease SalI (EC 3.1.23.37). The Hind III
digestion was carried out in lO0 ~l of Hind III
digestion buffer containing 60 mM NaCl, 7 mM MgC12, 7 mM

Tris-HCl pH 7.4, 100 ~g~ml gelatin and 15 units of Hind III (New England Biolabs). Incubation was at 37C for one hour. The enzyme was then inactivated by heating to 65C for five minutes. The SalI digestion was carried out in lO0 ~l of SalI digestion buffer containing 150 mM NaCl, 6 mM Tris-HCl pH 7.9, 6 mM MgCl2, 6 mM

2-mercaptoethanol, 100 ~g/ml gelatin and 25 units of SalI (New England Biolabs). Incubation was at 37C for one hour. The enzyme was then inactivated by heating to 65C for five minutes.
The two aliquots were pooled and digested with 10 units calf intestinal phosphatase (PL Labs) at 37C for 2 hours to remove 5' phosphates. The pooled pHC 79 was then adjusted to 0.3 M sodium acetate pH 7.0 and phenol extracted twice, followed by four ether extractions. The DNA was precipitated with 2.5 volumes of ethanol, washed ~295568 with 70% v/v ethanol, dried and finally re-suspended in 10 mM Tris-HCl, 1 mM EDTA to a final concentration of 500 ~g/ml. The ligation reactions contained 1 ~g of prepared pHC 79 and 1 ~g of Sau3A digested E coli DNA
in 8 ul of ligation buffer (10 mM Tris-HCl pH 7.9, 10mM
MgCl2, 20 mM dithiothreitol, 25 ~g/ml gelatin and 1 mM

ATP). The reaction mixture was incubated at 37C for 5 minutes, cooled and the ligation initiated by the addition of T4 DNA ligase (EC 6.5..1.1).
0 These reactions were continued for 4 hours at 15C.
Four ~l aliquots of the ligation mixtures were then subjected to in vitro packaging into bacterio-phage ~
particles. Strains BHB 2688 and BHB 2690 were inoculated from NZY agar plates into 50 ml of NZY broth (containing 10 g NZamine (from Humko Sheffield), 5 g yeast extract and 2.5 g NaCl/litre) and incubated with aeration at 30C
until the E600 reached 0.3. The cultures were then raised to 45C by immersion in a 60C water bath and incubated at 45C for 20 minutes without aeration. The two cultures were then vigorously aerated at 37C for 3 hours. The two cultures were pooled, chilled to 4C and harvested by centrifugation at 7000 rpm for 2 minutes.
The pellet was washed once in 100 ml of cold M9 minimal medium (per liter: 6g Na2 HPO 4, 3g KH2PO4, 0.5g NaCl, 1.0 NH4Cl; adjusted to pH7 with 8 N NaOH, after autoclaving, sterile glucose added to 0.2% W/V, CaCl2 added to 0.1 mM and Mg SO4 to 1 mM). The pellet was washed in 5 ml cold complementation buffer (40mM Tris-HCl - 1~2~55~ 51 pH 8.0, 10 mM spermidine-HCl, 10 mM putrescine-HCl, 0.1%
V/V 2-mercaptoethanol and 7% V/V dimethyl sulfoxide) and pelleted at 5000 rpm for 30 seconds. The cells were finally resuspended in 0.25 ml cold complementation buffer and dispensed into 20 ~l aliquots in micro test tubes.
These were frosell immediately in liquid nitrogen and stored at -80C.
For the packaging reaction, one 20 ~l packaging mix was removed from the liquid nitrogen and to it was immediately added 1 ~l of 30 mM ATP. The mix was then placed on ice for 2 minutes. The 4 ~l aliquots of ligated DNA were then added and mixed thoroughly. This was then incubated at 37C for 30 minutes. After 30 minutes, 1 ~l of 1 mg/ml DNAase (Worthington) was added and mixed in until the sample had lost its viscosity. To this packaged DNA preparation was then added 200 ~l of the strain E 107 which had been grown to an E600 of about 1.0 in tryptone maltose broth. MgSO4 was also added to a final concentration of 10 mM. The packaged cosmids were absorbed for 30 minutes at 30C after which time 500 ~l of L-broth (Luria broth: 1% W/V
bacto-tryptone, 0.5% W/V bacto-yeast extract, 0.5% W/V
NaCl, 1.2% W/V glucose, pH7) was added and the incubation continued for a further 30 minutes at 30C. The cells were then plated on L-agar containing 100 ~gjml carbenicillin (-carboxy benzyl penicillin) and incubated at 30C for 24 hours.

Carbenicillin resistant colonies were then picked to L-agar carbenicillin plates and incubated overnight at 42C. Strain E107 (3) carries the mutation dnaB which renders it unable to grow at 42C. Thus, only those colonies which had acquired a cosmid clone carrying dnaB from the wild-type donor should be able to grow at this temperature. In this way, 8 cosmid clones carrying dnaB were isolated. Since dnaB maps close to tyrB, the gene for the aromatic transaminase, and since cosmid cloning results in plasmids containing large inserts of donor DNA, it was reasonable to assume that some of the cosmid clones would also carry ~y~B. That some of the clones did indeed carry ~y_B was shown in the following way.
Cosmid DNA from each of the dnaB clones was purified and packaged into bacteriophage ~ particles as described above. The packaged cosmids were then introduced into the transaminase deficient strain DG 44 which carries the mutations ~y_B and aspC. These two mutations together ~0 bestow a Tyr Asp phenotype on DG 44. Introduction of an intact tyrB gene would be expected to restore the ability of this strain to grow in the absence of tyrosine and aspartate. Direct screening of clones for tyrB is not easy in this strain, since it will not maintain plasmids derived from colE1, such as pHC 79. After introducing the cosmid clones into DG 44, therefore, 2 hours were allowed for recombination between the ~y~B

gene on the cosmid and the mutant allelle on the bacterial -~3-12~S5~8 chromosome. The cells were then washed and plated on minimal medium lacking tyrosine and aspartate to screen for tlle occurrence of t~rB recombinants. Four of the original dnaB clones gave tyrB recombinants in this screen and must therefore carry at least some of the tvrB
gene.
(b) Sub-cloning of restriction fragments from cosmid dnaB
clone The ~y_B cosmid clones were of limited utility owing to the large size thereof which resulted in a low copy number. Therefore, restriction fragments carrying the tyrB gene were sub-cloned into the multi-copy plasmid pAT153, (see for example, Twigg, A.J., and Shenatt, D., (1980), Nature, 283, 216-218) as follows: Two ~g aliquots of dnaB cosmid number 5 which carries ~y_B was digested in 20 ~l reaction with the following restriction endonucleases BamHI, HindIII, SalI, SphI, ClaI, EcoRI and BglII.
In each case, the incubation was at 37C for one hour and the enzyme was inactivated by heating to 65C for five minutes. For the BamHI digestion, the reaction medium contained 150 mM NaCl, 6 mM Tris-HCl pH 7.9, 6 mM MgCl2, 100 ~g/ml gelatin and 4 units of BamHI (New England Biolabs). Three (3) units of HindIII (New England Biolabs) were used in 60 mM NaC1, 7 mM MgCl2, 7 mM

Tris-HCl pH 7.4 and 100 ~g/ml gelatin. In the case of SalI, 5 units of enzyme obtained from New England Biolabs were used in 150 mM NaCl, 6 mM Tris-HCl pH 7.9, 6 mM

lZ~?S56~3 MgC12, 7 mM 2-mercaptoethanol and 100 ~g/ml yelatin.

For SphI, the reaction contailled 50 mM NaCl, 6 mM
Tris-HCl pH 7.4, 6 mM MgC12, 10 MM 2-mercaptoethanol, 100 ~g/ml gelatin and 1 unit of the SphI enzyme (New England Biolabs). The reaction mixture for ClaI
digestion corresponded to that for HindIII, digestion, except thzt the enzyme used was 3 units of ClaI obtained from Boehringer Biochemicals. In the case of EcoRI 4.5 units of EcoRI (New England Biolabs) were used in 100 mM
Tris-HCl pH 7.5, 50 mM NaCl, 5 MM MgC12 and 100 ~g/ml gelatin. Lastly, the reaction medium for ~II digestion contained 60 mM NaCl, 10 mM Tris-HCl pH 7.6, 10 mM
MgC12, 10 mM 2-mercaptoethanol and 100 ug/ml gelatin.

These digested DNA preparations were then subjected to electrophoresis on a horizontal 1% w/v low gelling temperature (LGT) agarose gel in TBE for 6 hours at 5 V/cm. The DNA fragments were visualized by staining the gel for 15 minutes in a 1 ~g/ml solution OI ethidium bromide, followed by observation under a 366 nm UV light source. Individual bands were excised and stored at ~C.
One (1) ~g aliquots of pAT153 DNA were digested with the same set of restriction endonucleases in 20 ~1 reactions, except for BglII (which does not cut pAT153).
Cosmid BglII fragments were sub-cloned into BamHI-cut pAT153 (BamHI and BglII give the same 5' overhangs).

After digestion, the linearized vector was digested with 1 unit of calf intestinal phosphatase (PL Labs) for 1 hour, to remove 5' phosphates and prevent recircularization, lZ9~

incubated at 70C for lO minutes to inactivate the enzyme and then also run on a 1~ w/v LGT agarose gel as described fo. the cosmid fragmen's. The band corresponding to plasmid linearized by each enzyme was then excised from the gel as described above.
The cosmid fragments were then ligated to the appropriately-cut vector as follows. The gel slices were melted by incubation at 65C for lO minutes and then cooled to 37C. The melted gel slices were all about lO0 ~l. Two ~l of vector fragment and 8 ~l of a particular cosmid fragment were then added to 40 ~l of pre-warmed 1.25 x ligation buffer (62.5 mM Tris-HCl pH
7.8, 12.5 mM MgCl2, 25 mM dithiothreitol, 1.25 mM ATP
and 62.5 mg/ml gelatin) and mixed thoroughly. Ligase was added and the reaction continued at 15C overnight. The ligated samples were then re-melted at 65C for 5 minutes, cooled to room temperature and added to 200 ~l of competent cells of the E coli strain HW 87 which had been prepared as follows. An overnight culture of HW 87 in L-broth was diluted 1:50 into 50 ml of fresh pre-warmed L-broth and incubated at 37C with good aeration until the E600 reached 0.6. The cells were then pelleted by centrifugation at 10,000 rpm for 5 seconàs and resuspended in 25 ml of cold 50 mM CaCl2. The cells were left on ice for 10 minutes after which they were re-pelleted as above and re-suspended in 2 ml of cold 50 mM CaC12.
After a further 10 minute incubation at 0C, the cells were competent for transformation.

~z9~568 After addition of the ligated DNA, the cells were incubated at 0C for a further 10 minutes, heat-shocked at 37C for 2 minutes and finally mixed with 750 ~l of pre-warmed L-broth. The cells were incubated for 30 minutes at 37C to allow phenotypic e~pression of the plasmid encoded ~-lactamase gene before plating suitable aliquots onto L-agar plates containing 200 ~g/ml carbenicillin. The plates were incubated at 37C
overnight. Colonies containing recombinant plasmids were identified by the sensitivity thereof to tetracycline in the case of fragments cloned at the HindIII, SalI, SphI, BamHI and ClaI sites of pAT 153.
Recombinants isolated using the restriction endonuclease EcoRI were identified by examining the size of the plasmids in individual colonies. This was performed as follows. Potential recombinant colonies were patched onto L-agar plates containing 200 ~g~ml carbenicillin. These were incubated overnight at 37C.

The bacteria from about 1 cm2 of each patch were re-suspended in lytic mix containing 10 mM Tris-HCl p~ 8, 1 mM EDTA, 1% w/v sodium dodecyl sulfate (SDS), 2% w/v Ficoll and 0.5% W/V bromphenol blue (Sigma chemicals) 100 ~g/ml RNAse. This was then incubated at 65C for 30 minutes. Each sample was then vortex-mixed vigorously for 30 seconds. 50 ~l aliquots of each preparation were then loaded onto a 1% w/v agarose gel and subjected to electrophoresis for 4 hours at lO V/cm. The plasmid bands were stained with ethidium bromide as described above and visualized under ~2~S~13 254 nm UV illumination. ~ecombinallt plasmids were identified by the reduced mobility thereof compared to non-recombinant contl-ols. Using these methods, a number of recombinant plasmids carrying various fragments from the original cosmid dnaB clone were isolated. To facilitate the screening of these plasmids for those that carried the intact ~y_B gene, a new strain carrying ~y_B and ~C mutations was constructed.
Example 3 Construction of transaminase deficient strain.
It was decided to move the already characterised mutations in DG 44 into a background known to allow maintainance of the plasmids (that of strain MC 1061).
Bacteriophage Pl was grown on a mixed culture OI strain HW
22 carrying random insertion of the tetracycline resistant transposon Tn 10 prepared as follows: A saturated culture of HW22 was grown overnight in lambda YM broth (lO g bacto tryptone, 2.5 g NaCl and 0.2% w/v maltose per litre).
This was diluted x ljlOO into 100 ml of fresh pre-warmed lambda YM broth and incubated at 37C until the OD600 reached 0.6. The cells were deposited by centrifugation at lO,OOO rmp for five seconds and resuspended in 5 ml of lambda YM broth. NK 55, a bacteriophage lambda derivative carrying the tetracycline transposon TnlO, was then added to a multiplicity of infection of 0.2. The bacteriophage was absorbed for 45 minutes at 37C. Then 200 ml aliquots were spread onto L-agar plates containing tetracycline (20 ~g/ml) and sodium pyrophosphate (2.5 mM). The plates $5S~

were incubated at 37C for 24 hours. Approximately 5,000 TetR colonies were pooled by washing the colonies off the plates using a total of 5 ml of L-broth. This was then diluted into 50 ml of L-broth containing 20 ~g~ml tetracycline and grown overnight at 37C. The TnlO pool was stored at -20C after adding sterile glycerol to bring the concentration to 20% w/v.
In o-~der to grow bacteriophage P1 on the TnlO pool, a 200 ml aliquot of this glycerol stock was diluted with 5 1~ ml of L-broth including 10 ~g/ml tetracycline and incubated overnight with shaking at 37C. To 0.2 ml of this overnight culture was added 2 x 10 plaque forming units (pfu) of phage P1 clear and 10 ~1 of 50 mM
CaC12. The cells were incubated at 37C for five minutes and then added to 5 ml of L-broth + 2.5 mM CaC12 pre-warmed to 37C. The cells were then incubated at 37C
with vigorous aeration for four hours. The cell debris was removed by centrifugation at 10,000 rpm for five minutes and the phage-containing supernate stored over chloroform at 4C.
This supernate was used to transduce DG 30 to permit growth on minimal medium lacking tyrosine and aspartate.
(The minimal medium used was that of Vogel and Bonner; 0.2 g/l MgS04.7H20, 2 g/l citric acid, 10 g/l K2H P02, 3.5 g/l NaH2P04 .4H20 and 10 mM FeC12. After autoclaving, glucose was added to a final concentration of 0.2% w/v along with necessary supplements of amino acids at 50 ~g/ml. The Tyr Asp transductants were then 12955~3 replica-plated to the same medium including 10 ~g/ml tetracycline to detect those transductants that had simultaneously become TetR. A number of these Tet Tyr Asp recombinants were purified and used to prepare new Pl lysates. Each of these eight Pl preparations was then used to transduce DG 30 to TetR.

From each experiment, 50 TetR transductants were picked to minimal medium lacking tyrosine and aspartate to test for linkage between the site of the Tn 10 insertion involved and aspC or tYrB. From each of these experiments one TetR colony which remained tyrB ~C
was purified and again used to prepare a Pl lysate. These lysates were used to transduce MC 1061 to Tet . Cell extracts from these transductants were then run on native polyacrylamide gels and stained for L-phe transaminase activity as described by Gelfand.
The cell extracts were prepared by growing the desired transductants overnight in 10 ml of L-broth at 37C. The cells were harvested by cetrifugation at lO,000 rpm for fifteen seconds and re-suspended in 0.5-1.0 ml of sonication buffer depending upon the size of the.pellet.
The sonication buffer used was 25 mM Tris-HC1, 25 mM
KH2P04 pH 6.9, 0.2 mM pyridoxal phosphate, 0.5 mM

dithiothreitol, 0.2 mM EDTA and 10% v/v glycerol. The cell suspensions were transferred to Eppendorf micro test tubes and kept on ice. Each suspension was sonicated with a Davies sonicator using four five-second bursts at setting 2. The sonicated suspensions were then cetrifuged lZ~SS~i8 at 15,000 rpm for five minutes at 4C to remove cell debris and the supernatants transferred to a fresh micro test tube. The cell extracts were then kept on ice until they could be loaded on a gel or stored at -20C.
The native gel electrophoresis was performed as follows. The gel consisted of 8.5 % w/v acrylamide, 0.227% w/v bis-acrylamide in 375 mM Tris-HCl pH 8.3. This acrylamide stock was de-gassed and polymerized by the addition of 0.05% w/v ammonium persulfate and 0.016% w/v TEMED. After polymerization, the gels were pre-run in 37.5 mM Tris-HCl pH 8.3 for at least one hour at 4C.
Prior to loading the samples, the buffer was changed to running buffer consisting of 76.7 mM glycine, 1 mM
Tris-HCl pH 8.3. About 50 ~g protein from each cell extract was then loaded and subjected to electrophoresis at lO v/cm for from four to six hours at 4C. The protein concentration in the cell extracts was determined using the Bio-Rad protein reagent method. The gels were stained for L-phe transaminase activity as follows. The gel was washed briefly in 100 mM phosphate buffer pH 7.5 and then immersed in 500 ml of fresh staining solution containing 12.5 mM ~-ketoglutarate, 0.2 mM pyridoxal phosphate, 0.6 mM nitro blue tetrazolium, 0.2 mM phenazine methosulphate, 30 mM L-phe and lO0 mM K2HP04 pH 7.5 that had been pre-warmed to 37C. The gel was shaken gently in the staining solution for one hour at 37C in the dark. The gel was then washed in distilled water and left in distilled water until it could be photographed. (See 1.2955~8 figure 2.) This illustrates the presence or absence of detectable aminotransferases in various E. coli strains.
MC 1061 extracts prepared in this way gave three staining bands characteristic of the ilvE, aspC and ~y_B activities. By comparing the transaminase pattern of extracts prepared from the transductants with that of MC1061 it was possible to detect MC 1061 recombinants lacking the characteristic aspC activity.
One mutant which was devoid Of ~E_ activity was purified and named HW109. This strain was then cured of the TnlO transposon by the method of Bochner et al (J.Bact 143:926). HW109 was grown overnight in L-broth.

Approximately 106 cells per plate were spread onto Bochner selective medium which was prepared as follows:
Solution A (Bacto-tryptone lOg, Bacto yeast extract 5 g, chlortetracycline HCl 50 mg, agar 15 g, water 500 ml) and solution B (NaC1 10 g, NaH2P04.H2o 10 g, glucose 2 g, water 500 ml) were autoclaved separately for 20 min at 15 psi. The solutions were mixed and cooled to pouring ~0 temperature. 5 ml of ZnC12 (20 mM) and 6 ml of 2 mg ml 1 fusaric acid were then added prior to pouring. The plates were incubated for 24 hr at 37 and the fusaric acid resistant colonies isolated and purified by re-streaking on the same medium. These isolates were then tested for sensitivity to tetracycline. One tetracycline sensitive derivative of HW109 was chosen and named HW157.
This approach did not, however, yield tyrB

derivatives of HW 22. To isolate a strain with Tn 10 lZ~355~i8 linked to ~y_B, the procedure was as îollows. Strain ES430 carries a mutation in malB that renders it unable to use maltose as a carbon source The malB is reasonably closely linked with ty_B. Therefore, ES430 was transduced with Pl grown on the random Tn 10 pool in HW 22 described above, selecting for Tet R on maltose Maconkey agar plates suplemented with 10 ~g/ml tetracycline.
Pl transductions were performed as follows. The recipient strain was grown in L-broth to an OD600 of about 1Ø CaC12`was added to a final concentration of 2.5 mM. Phage Pl clear grown on the desired donor was then added at a multiplicity of insertion of between 0 2 and 1.0 (usually 108 pfu Pl clear per ml of recipient).
The cells were incubated at 37C for fifteen minutes, centrifuged at 10,000 rpm for five seconds, washed in the original volume of 0.1 M citrate buffer pH 7.0 and finally re-suspended in citrate buffer. Suitable aliquots were then plated on the selective medium.
2~ Transductants which had simultaneously become Tet and Mal were picked and purified. Pl lysates were then prepared from these strains and used to transduce DG 44 to TetR. The TetR colonies derived from each of the eight Pl lysates were then patched onto minimal agar plates lacking aspartate and tyrosine. In one of these experiments, good linkage between Tn 10 and ty~B was obtained. Therefore, one TetR tvrB recombinant from this experiment was isolated, a Pl lysate prepared from this recombinant and used to transduce HW157 described above to Tet . Fifty of these transducants were then patched onto minimal agar supplemented with leucine, but lacking L-aspartate and L-phenylalanine. In this way, a ~y_B aspC derivative of MC 1061 was detected.
This strain was designated HW 158. Finally, HW159, a tetracycline sensitive derivative of HW158 was isolated as described above for the isolation of HW157. HW 159 will not grow on minimal medium supplemented with leucine since it requires aspartate and tyrosine for growth unlike the parental strain MC1061 which only requires leucine.
Example 4 Screening of sub-clones for ability to complement the tyr B lesion in HW 159 All the recombinant plasmids obtained by subcloning DNA fragments from the dnaB cosmid clone (described above) were introduced into strain HW 159 by transformation selecting on L-agar supplemented with 200 ~g/ml carbenicillin. These transîormants were then streaked onto minimal medium supplemented only with 2U leucine to test for the ability of the presence of genes carried by the plasmid to complement the ~y_B lesion in HW 159. One sub-clone carrying a ClaI fragment from cosmid dnaB clone number 5 was found to restore the ability of HW 159 to grow on minimal medium supplemented with leucine and thus to carry the tyrB gene.
Sequencing of the ~_B gene was performed by the method of Maxam and Gilbert. The sequence of the ~y~B

gelle is shown in figure 3. Based upon this DNA sequence the amino acid sequence for the aminotransferase encoded for by ~y_B could be determined using the genetic triplet codons. This amino acid sequence is shown in figure 4.
~xample 5 Cloning of the aspC gene:
As a starting point for the cloning of the aspC gene, it was decided to use the specialized transducing phage lambda aspC2 obtained from M. Ono. The phage was prepared 1~ as follows. A culture of HW76, which is a double lysogen carrying lambda aspC and lambda CI 857 Sam 7, was grown to an OD600 of 0.6 at 30C. The culture was then incubated at 45C for fifteen minutes to induce the prophage and then shaken vigorously at 37C for three hours. Cell lysis was completed by the addition of 0.5 ml of CHC13 and the cell debris removed by centrifugation at 10,000 rpm for ten minutes. The phage was precipitated by the addition of NaCl to 2.4% w/v and polyethylene glycol (average molecular weight 6,000) to 10% w/v. The phage was precipitated overnight at 4C and then pelleted by centrifugation at 5,000 rpm for ten minutes. The phage pellet was gently re-suspended in 10 ml of phage buffer consisting of 10 mM Tris-HCl pH 7.5, 10 mM MgS04. The phage was pelleted by centrifugation for one hour at 40,000 rpm and finally re-suspended in 1 ml of phage buffer.
The phage was further purified by running on a CsCl block gradient as follows. The lml of phage was applied 12955~

to a two-step block gradient in celiulose nitrate tubes.
The gradient contained 1 ml of 5M CsCl, lO mM MgS04, 10 mM Tris-HCl pH 8.0 and 0.1 mM EDTA overlayed with 3 ml of 3M CsCl, 10 mM Mg S04, lO mM Tris-HC1 pH 8.0 and 0.1 mM

EDTA. The gradient was centrifuged in a Beckmann SW 65 rotor for one hour at 30,000 rpm at 20C. The phage band was removed in 0.5 ml from the side of the tube using a l ml syringe with a 5/8 inch (1.6 cm) 25 guage needle. The 0.5 ml of phage was then mixed with 0.5 ml of saturated CsCl solution (25C) in 10 mM Mg S04, 10 mM Tris-HCl pH

8.0 and 0.1 mM EDTA and mixed well in a cellulose nitrate centrifuge tube. This was then overlayed with 3 ml of 5M
CsCl in 10 mM Mg S04, 10 mM Tris-HCl pH 8.0 and 0.1 mM
EDTA and then 1 ml of 3M CsC1 in 10 mM Mg S04, lO mM

Tris-HCl pH 8.0 and 0.1 mM EDTA. This was again centrifuged at 30,000 rpm for one hour at 20C. The phage was again removed from the side of the tube in 0.5 ml as before.
The DNA was extracted from the phage particles as ~0 follows. To the 0.5 ml of phage in a 15 ml corex centrifuge tube was added 50 ~l of 2M Tris-HCl pH 8.5, 0.2 M EDTA and 0.5 ml of formamide was added. After 30 minutes, 0.5 ml of water was added and the DNA
precipitated by the addition of 3 ml of ethanol. The DNA
was pelleted by centrifugation for five minutes at 10,000 rpm. The supernate was discarded and the pellet rinsed with 70% v/v ethanol. The DNA was dried in vacuo and finally dissolved in TE (10 mM Tris-HCl and l mM EDTA pH

-~6-lZ~55~3 8.0) to give a DNA concentration of 500 ~g/ml. The lambda aspC DNA was partially digested with the restriction endonuclease Sau3A as described above for E.
coli DNA. The DNA from all of the digests were pooled and run on a 1% w/v low gelling temperature agorose gel in TBE. Also prepared and run on the same gel was 1 ~g of pAT153 DNA completely digested with the restriction endonuclease BamHI and calf intestinal phosphatase as described above. The DNA was visualized under 366 nm U.V.
light as described above.
The bands corresponding to linearised pAT153 and the partially restricted lambda aspC DNA between 2.5 and 6kb were excised. The DNA was extracted from the gel as follows. The gel slices were melted at 65C for five minutes in 15 ml convex tubes, cooled to 37C and diluted with two volumes of water at 37C. The agarose was then removed by the addition of an equal volume of phenol at 37C and vortexing for three minutes. The phases were separated by cetrifugation at 10,000 rpm for five minutes 2U and the upper aqueous phases removed to clean tubes.
Three phenol extractions were performed. The final supernate was then extracted four times by vortexing with an equal amount of diethyl ether, allowing the phases to separate and then removing the upper ether layer. The volume of the remaining aqueous phase was determined and 3M sodium acetate added to bring the salt concentration to 0.3 M. Yeast transfer RNA was added to a final concentration of 5 ~g/ml as a carrier. The DNA was 129556~3 precipitated by the addition of 2.5 volumes of ethanol, overnight incubation at -20C and centrifugation at 10,000 rpm for 30 minutes. The pellet was washed in 70% v/v ethanol, re-centrifuged at 10,000 rpm for 30 minutes and dried in vacuo. The DNA was then dissolved in TE to give a concentration of about 100 ~g/ml. The partially restricted lambda aspC DNA was then ligated to BamHI cut pAT153 by adding 2 ~l of prepared lambda aspC DNA to 2 ~l of prepared pAT153 DNA in 4 ~l of 2x ligation buffer (described above). This mixture was incubated at 37C for five minutes, cooled to room temperature and the ligation started by the addition of 1 ~l of T4 DNA
ligase (New England Biolabs). The ligation was continued at 15C for four hours. The entire ligation reaction mixture was then cooled on ice and added to 200 ~l of competent cells prepared from strain HW 159 as described above. The transformation was performed as above and the cells plated on L-agar containing 200 ~gjml carbenicillin. The plates were incubated overnight at 2~ 37C. About 5,000 colonies were obtained. These were then pooled, washed twice in 10 ml of 0.85% w/v NaCl and plated onto minimal agar containing leucine and carbenicillin, but lacking tyrosine and aspartate. Only HW 159 cells which carried a recombinant plasmid containing aspC could grow on this medium.
After incubation at 37C for twenty-four hours, about 100 colonies were obtained. Some of these were purified by successive streakings on the same medium. That these 5561~

contained a recombinant plasmid was confirmed by running single colony agarose gels as described above. That the recombinant plasmids in these strains indeed conferred the Asp Tyr phenotype was confirmed by isolating the plasmids, re-transforming into HW 159 and showing that these transformants had become Asp Tyr . That the transformants had been altered bv the presence of cloned aspC as opposed to tYrB was shown by running native acrylamide gels on cell-free extracts and staining for transaminase activity as described above. These experiments confirmed that the plasmids indeed carried an intact ~pC gene.
The actual level of aminotransferase activity is measured in arbitrary units indicating relative activity.
Relative aminotransferase activity on various strains containing the ~y_B and aspC genes on plasmid pAT153 is shown in table 2. The assay was performed using a sigma R
aspartate transaminase assay kit. (Sigma Technical Bulletin No. 56-UV, revised August 1980) lZ~SS68 Table 2 Transaminase Levels Measured Using Aspartate As Substrate (Sigma Aspartate Transaminase Assay I~it) mg Protein Units/Min. Relative Activity HW157 25 0.6 HW158 pHC79 15 0.37 HW158 ptyrB 494 12.3 HW158 paspCl-1 1738 43.5 Exam~le 6 Further Analysis of the tyrB Clone The ClaI insert carrying ~y~_ is approximately 4.5 kb. A preliminary restriction map was constructed by the commonly used technique of double restriction endonuclease digestion followed by gel electrophoresis to analyse the restriction fragments obtained. In one orientation (see figure 6) the ClaI insert had EcoRI, BamHI and NruI sites in positions that facilitated the reduction in plasmid size by in vitro deletion. For example, the small EcoRI
fragment was removed as follows. One ~g of ptyrB DNA
was digested to completion with EcoRI in a final volume of 20 ~1. The enzyme was inactivated by incubation at 65 ~2~$55i'~i~

for 5 minutes. A 2 ~l aliquot of this digest was then diluted into 50 ~l of ligase buffer, one ~l of T4 DNA
ligase added and the DNA ligated for 16 hours at 15.
Ligation at this low DNA concentration leads predominantly to the re-circularisation of the plasmid without the small EcoRI fragment. The DNA was then transformed into HW87 selecting for carbenicillin resistance as described above. Individual transformants were picked, purified and analysed on single colony lysate gels as described above.
Most of the transformants contained plasmids that exhibited higher mobility than the parental plasmid ptyrB. Plasmid DNA from several of these clones was isolated and the loss of the small EcoRI fragment confirmed by EcoRI digestion and analysis of these digests by electrophoresis on a 1% agarose gel. All the deleted plasmids gave just one band corresponding to the large EcoRI fragment of ptyrB. The parental plasmid gave the expected two bands.
The EcoRI deleted ptyrB was then subjected to a similar series of manipulations to remove the BamHI
fragment and this EcoRI BamHI deleted plasmid was further subjected to deletion using the enzyme NruI in the following buffer. (lOO mM NaCl 6 mM Tris pH7.4 6 mM
MgCl2 5 mM ~-mercaptoethanol lOO ~g/ml gelatin) All three plasmids were purified and used to transform HW225 (~y~ aspC recA) to carbenicillin resistance. All the transformants simultaneously acquired the ability to grow on minimal medium in the absence of L-tyrosine and ~29~568 L-aspartate. This demonstrates that the tyrB gene is still intact on all three deleted plasmids.
The BamHI-EcoRI fragment from the double deleted ptyrB
was subjected to sequence analysis following the protocols described by Maxam and Gilbert (Maxam, A. M., and W. Gilbert, 1977, PNAS 51: 382-389; Maxam, A. M., and W. Gilbert, 1977, MIE 65: CH 57 499-559, Part I). One substantial open reading frame was discovered that could code for a protein of 380 or 396 amino acids in length. The precise start point for translation is not known. There is a typical ribosome binding site about 10 nucleotides upstream from an in phase GTG (valine codon). Alternatively there is an in phase ATG
(methionine) but this is not preceded by a typical ribosome binding site. The DNA sequence of the Eco~I-BamHI fragment and the amino-acid sequence deduced from it are presented in figures 3 and 4.
Map of paspC
The restriction map of the smallest aspC sub-clone (paspC
3-4) was elucidated by a combination of single and double restriction digests as described above see figure 7. The aspC sub-clones do not possess restr c~ion sites that allow the insert to be excised cleanly since they were made by ligating Sau3A fragments into the ~amHI site of pAT 153, a procedure which does not regnerate the BamHI site.
The paspC3-4 (pME98) was used to transform the E. coli prototroph HW519 and during fermentation produced levels of transaminase equivalent to or better than that of paspCl-l.
The E. coli HW519 carrying pME98 has the deposit number ATCC 39501.

Example 6 Increased Yield of Amino Acids In the reactions ~-equiring aminotransferase enzymes in figure 1, reactions ~lO and #13, presence of additional aminotransferase enzymes facilitates amino acid synthesis. This increased level of the aminotransferase enzymes synthesized by the ~ C, tyr B or ilvE gene enables a cell to overcome a rate-limited reaction at the transamination step.

A plasmid of the present invention, carrying the asp C, tyr B or v E gene is inserted into a bacterial cell by techniques well known in biochemistry. This inserted gene produces a messenger RNA which catalyzes the synthesis of increased levels of aminotransferases when the bacterial cell is grown in a suitable media. The presence of these increased levels of aminotransferases catalyzes increased levels of amino acids. The amino acids are then harvested from the cells and media by purification procedures well known in fermentation science.
The addition of the tyrB containing plasmid into E.Coli 13281 (U.S. Patent 2,973,304) resulted in an 11%
increase in L-phenylalanine production as shown in Table 3.

1~95568 Table 3 _henylalanine Production in E. Coli (Strain 132~31) No Plasmid Plus tyrB Plasmid L-Phe Produced 100o 111%

Culture grown at 32C for 48 hours.

Claims (5)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A plasmid comprising a replicating extra-chromosomal element containing a gene coding for the synthesis of an E. coli aminotransferase selected from the group consisting of aspC, tyrB and ilvE.

2. The plasmid of claim 1 wherein said E. coli tyrB
gene comprises the following nucleotide sequence:

3. The plasmid of claim 1 designated pME98 (ATCC
39501).

4. A method for the synthesis of transaminating enzymes comprising the expression of an E. coli aminotransferase gene on a plasmid within a cell resulting in the synthesis of active aminotransferase, wherein said gene is selected from the group consisting of aspC, tyrB and ilvE.

5. A composition of matter comprising the biologically pure Escherichia coli strain ATCC 39260.