Note: Descriptions are shown in the official language in which they were submitted.
1341352
Bac)~ground of the Invent10I1
Field of the Tm~e~Wion
This invention involves the construction and use of
genetically engineered plasmids. 'These plasmids are
constructed using restriction endonucleases to ~~ontain the
panes coding for the amipotransferase 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 ec;uivalent
terms in this document. (resent aminotransfera:>e
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
-1-
1 341 35 2
use the genetic terminology of as~C, tyrB and ilvE gene
products as defined by Umbarger at p. 582. The aspC gene
product v.il1 catalyse the t:ansamination of amino acid
precursors to produce aspartate, glutamate, phenylalanine
and tyrosine. The tyzB gene product will catalyze the
transamination of amino acid precusors to produce
pl:enylalanine, tyrosine, glutamate, aspartate and
leucine. The ilvE gene product will catalyze the
transamination of the amino acid precusors to produce
isoleucine, valine, leucine, phenylalanine, glutamate and
alanine.
The location of the as C, tyrB and ilvE genes on
the genetic map of E. col.i K12 strain is disclosed and
discussed by Bachmann et al. (Microbological Reviews, 44:
1-56, b3arch 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. a).
The tyrB gene located at E. coli 1'12 map poaition 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 a1. p. 21). The ilvE gene
located at E coli f~12 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).
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1 341 ~5 2
Amino acid transamination by a~C and t~rB gene
products are disclosed by Gelfand et al., (J. Bacteri-
ology, 130:429-440, April 1.977) 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 as~C region (Christensen
and Pedersen Mol. Gen Genet., 181: 548-551, 1981). The
as~C region in bacteriophage lambda was used as a source
of the gene for the ribosomal protein S1. Their ;31 gene
was closed onto plasmids for research purposes. 'Phe as~C
gene, however, was not cloned into a plasmid for i:he
production of transaminases. The as~.nC region was used as
a marker to facilitate isolation of a transduc.ing phage
in a tyrosine auxotroph lacking the as~C and ~B 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 gene_>s.
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~ 341 35 2
Applicant's plasmids contain one or more copies of the
as~C, t~rB 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 t~rB gene encoding the enzyme aromatic amino-
transferase. Applicant's invention describes the amino
acid sequence of the aromatic aminotransferase encoded by
the gene t r~rB.
The aminotransferase genes, as~C, t~rB, ilvE, code
for the synthesis of the aminotransferases which <:atalyze
the transamination of the carbonyl precursors of amino
acids. ABC gene codes for the transaminase A (aspar-
tate aminotransferase) (EC2.6.1.1) and is catal.yt_Lcally
active during the synthesis of aspartate, glutamate,
phenylalanine and tyrosine. The t~rB 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
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'1 341 35 2
catalytically active during the synthesis of isoleucine,
valine, leuc.ine, phenylalanine and glutamate. Then the
transamination reaction in bacterial amino acid synthesis
becomes rate-limiting, the presence of these plasmid-
borne aminotransferase genes allows for the synthesis of
additional aminotransferases. Aminotransferase synthesis
sufficient to overcome the rate limitation result, 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 be
used 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 t:he
synthesis of an aminotransferase.
Another object of the invention is to construct
a plasmid containing one or more of the following amino-
transferase genes. as~C, 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 as~C, t~rB or ilvE gene.
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1 341 35 2
Yet another object of the invention is; a method for
producing amino acids using microorganisms wherein the
amino acid transamination reaction becomes rate-limiting,
the improvement comprising the introduction into the
microorganism's chromosome additional genes coding for an
amino transferase capable of transaminating the keto acid
precursors of the amino acids.
", 5 a
~~+~ ~5 2
Another object of the invention is a method of in-
creasing the rate of transamination of receptive amino
acid precursor molecules accomplished by the inse>rtion of
a plasmid containing the aspC, tyrB or :i.lvE gene into a
microorganism, followed by the synthesis of a physio-
logically effective concentration of aminotransfe~rase
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 as~C, t~rB 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
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1 341 35 2
type transaminase of p-hydroxyphenylpyruvic acid is a
rate-limiting step in the synthesis of tyrosine.
Still yet another object of tile 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 aminotra;nsferases
encoded by tyrB, as~C or ilvE genes in various
bacterial strains.
Figure 3 describes the DNA nucleotide sequence of the
EcoRI-BamHI fragment carrying the t~rrB 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 they tyrB+
plasmid.
Figure 7 illustrates the restriction map of the
plasmid carrying the aspC gene.
1 341 35 2
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 c-keto-acid preci.irsor of an amino acid, such as
oxaloacetic acid. The reaction is freely rover:>ible.
Therefore, the aminotransferases can be ut.ilizecl both in
the synthesis and the degradation of amino acidw~.
Amino acid biosyntl-aesis often requires tran~;amination
as the final step in biosynthesis. An example e~f such a
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 ~igure 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
10). Similarly, an amino group from glutamate is
transferred to 4-hydroxyphenylpyruvate to produce tyrosine
(figure 1, 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
_g_
1 341 35 2
aspartate, glutamate, phenylalanine and tyrosine. The
E. coli transaminase encoded by the ~rB 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, ph~~nyl-
alanine and glutamic acid (Umbarger at p. 582).
It is recognized that other pathways exist fo:r
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 l, steps 1-20. Normally
each enzymatic step is under regulation by controlling
the synthesis of the enzyme and/or by allost.erical.ly
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_
1341352
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 t~rrR 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 tr~R gene, controls expression of all the enzymes
of the try 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 try operon are rendered sens-
hive to the level of L-phe. The trp operon .is rendered
sensitive to the level of L-trp by an attenuator/lcsader
peptide control system.
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1341352
Mutant strains that over-produce amino acids are
often isolated by selecting for resistance to appropriatf=
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 tr~.R repressors. Mutants in which
the chorismate mutase (L-phe) is resistant to L-pile may
be isolated by selecting for 2-thienyl-alanine re;sist-
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
t~rA 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 try operon would prevent accumulation of
L-trp. Such mutants would, of course, have to be cultured
-11-
'I 341 35 2
in media that included these two amino acids so ass 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 regu:Lation,
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 washes
to clone to a suitably prepared DNA from an appropriate
vector. One can then transform a mutant strain 1<~cking
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'1341352
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 fir~~t 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
l0 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
-.13-
1 341 35 2
readily selectable marker is known to be in the vicinity
of the desired gene, then it may be possible to isolate a
strain carrying the insertion of a readily selectable
transposon, such as Tn 10, which encodes tetracycline
resistance, near the gene of interest. Large DNA
fragments contalnlng this marker can then be cloned and
the resultant phaae or cesmids screened for the presence
'of the gene of interest. The gene could then be
sub-cloned into a suitable high copy number plasmid.
lp The following are the strain of E. coli used in the
examples. Beside each strain designation is a summary of
the bacterial genotype and source. The relationship of
intermediate strains of E. coli leading to the production
of the auxotroph HW159 which is both asnC and tr~rB
is shown in Figure 5. The process which converted one
strain to another is also summarized on the Flowchart in
Figure 5.
Strain HW159, the aspC' and tyrB mutant was
deposited in The American Type Culture Collection, 12301
20 Parklawn Drive, Rockville, MD 20852, U.S.A. and has the
number designation ATCC 39260.
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1 341 35 2
Table 1
STR.~1IN LIST
Strain Genotype Source
HW22 metE M. Edtrards
BH82bsag recA,'\.in~m 334 CIQ57 b2 red 3 EamlSJ. Burke
Sam7
BT3B2690 recA;'?,imm =34 CI857 b2 red 3 DamlS J. 3urke
Sam?
E107 thrl leuB6 thr.~,6 thil dnaB107 deoClB. Bachmann
lacYl tonA21 rpsL,67 SupE4a
DG44 hsdS thil lacYl galfi2 xyl5 mtll proA2B. Bachmann
argE3 hisG4 hppT29 aspCl3 rpsL31 tsx33
supE44 recB21 recC22 sbc815
MC1061 araD139(ara-leu)de17G97 M. Casadaban
(lacIPO~Y)de174 galU gall: hsdR rpsL
DG30 thil proA2 argE3 hisG4 lacYl JalF~2 B. Bachmann
aral4 xyl5 mtll rpsL31 tsx323 supE44
recB27 recC22 sbcBlS h sdS hppT29
ilvEl2 tyrB509 aspCl3
ES430 thi ma1B29 relAl spoil B. Bachmann
HW157 araD139(ara-1eu)de17b97 This work
(lacIPOZY)de174 galU galK
hsdR rpsL aspCl3
HW159 araD139(ara-leu)de17697 This work
(lacIPOZY)de174 galU galK (deposited)
hsdR rpsL aspCl3 tyrB507
HW225 araD139(ara-leu)de17697 This work
(lacIPOZY)de174 galU galK
hsdR rpsL aspCl3 tyrB507
recA srl::TnlO
HW519 Prototroph W3110 Commercially
j0 Available
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1 341 35 2
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 mufti-step process that involved
systematically de-regulating the L-phe synthetic pathway.
In such de-regulated strains, it leas 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
mufti-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 tyrB 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 tyrE or as C genes are also useful on a plasmid,
in a suitable genetic background, by providing a selective
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1 341 35 2
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 5m1 of L-Broth plus
suitable antibiotics. The culture was transferred to a
15m1 corex tube and the cells deposited by centrifugation
at 10,000 RPM for 1 minute. The supernatant wa.s decanted
and the pellet resuspended in 500 ul of fresh solution
I, (50 mP9 D-glucose, 25 MM Tris pH8.0, 1G 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 D9 NaOH and lj; SDS).
This was mixed in gently and then incubated on ice for 5
minutes. 750 ul 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 k: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 pells:ted by
centrifugation at 10,000 rmp for 10 minutes. '.Che
supernatant was decanted to a fresh 15 ml core;K tube and
3.75 ml of Ethanol was added. The tube was keI?t on i<:e
for 15 minutes. The DNA was pelleted by centrifugation at
10,000 rpm for 10 minutes. The supernatant was then
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1 341 35 2
poured off and the pellet thoroughly resuspended in lOmM
Tris (ph8.0), lmMEDTA, 20 ul of 3 M sodium acetate pH7.0
was added and tl:e DNA transferred to an Eppendorf
micro-test tube. The DNA was extracted twice with 200
ul of ultra-pare phenol that contained 0.1°0 8-hydroxy
quinol ine and lad been pr~:=-enuilibrated twice ac_fainst 1 m
Tris pH8.0 and once against 100 mf~7 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 ul ethanol and incubation in a _
dry-ice; ethanol bath for 10 minutes. The DNA wa,s pelleted
by centrifugation at 10,000 rpm for 10 minutes. The
supernatant was discarded and the pellet resuspe~nded in
500 ul of 70;o ethanol in water (v/v). The DNA was
re-pelleLed by centrifugation at 10,000 rpm for 10
minutes, the supernatant taas discarded and the ~>ellet
containing the DNA was dried in a vacuum dessicator for 30
minutes. The DNA was redissolved finally in 50 ul of 10
mM Tris 1 mM EDTA and stored at -20°C_
2n This preparation cJenerally gives about 5 ug of
plasmid DNA when the strains used are derived from
MC1061. The preparations also contain considerable
amounts of RNA and so <31i tine restriction digest=s
described in this patent also include 100 ug ml-~1 RNASE
A that has been pre-treated at 90° for 15 minute's to
destroy DNAses. The preparation can also be scaled up to
cope with plasmid isolation from 50 ml cultures.
-1R-
1 341 35 2
Example 2
(a) Cloning of the tyr B gene
Isolation of cosmid clones carrying t~rB anc: DNA from
the E coli strain HW 2'? (metE) was prepared according
to the method of Marmur (PNAS 46:453, 1960). A:Liquots of
this DNA were partially digested with the restriction
endonuclease Sau3A as follows: The DNA (80 ug) was
made up to 400 ul in Sau3A buffer containing 50 mM
NaCl, 6 mM Tris-HC1 (tris (hydroxymethyl) aminomethane
l~ hydrochloride) pH 7.5, 5 mM MgCl2, 100 ug/ml gelatin.
The DNA solution was then split into 8 x 50 ul
aliquots. Sau3A (New England Biolabs) was then serially
diluted (2-fold steps) in Sau3A dilution buffer
containing 50 mM I:C1, 10 mM Tris-HC1 pH 7.4, 0.1 mM EDTA
(ethylene diamine tetra acetic acid, disodium salt), 1 mM
dithiothreitol (DTT), 200 ug/ml bovine serum albumin
(BSA) and 50% v/v glycerol. 5 pl of each of seven
serial dilutions along with S yl of undiluted enzyme
' were then added to the DNA solution. The amount of enzyme
20 added ranged from 2.5 to 0.02 u. The digestion was
continued for 1 hour after which time the reactions were
stopped by a five minute incubation at 65°C. The tubes
were then cooled on ic:e.
Two yl aliquots of each of the digestions were taken
and were subjected to electrophoresis on a 0.7;o w/v
agarose gel in TBE (5U mM tris-HC1 pH 8.3, 50 mM boric
acid and 1 mM EDTA) along with suitable markers. After
staining with ethidium bromide (1 ug/ml) for 15 minutes,
the gel was observed under 254 nm illumination to
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1341352
visualise the DNA. In this way, the sample giv:Lng the
greatest amount of material in the 45 kb sire range could
be estimated.
The DNA from this sample was then ligated to DNA from
the cosmid vector pHC79 t:~hich had been prepared as
described below:
Two 25 ug aliquots of pHC 79 DNA were completely
digested one o;ith the res~ri,~.ti~n e=ndonuclease Hind III
(EC 3.2.12.21) and the other with the restriction
°ndonuclease SalI (EC 3.1.23.37). The Hind III
digestion was carried out in 100 ul of Hind III
digestion buffer containing 60 mM NaCl, 7 rnM MgCl2, 7 mM
Tris-HC1 pH 7.4, 100 ug;ml gelatin and 15 units of Hind
III (New England Biolabs). Incubation was at 37°C for
one hour. The enzyme was then inactivated by heating to
65°C for five minutes. The SalI digestion was carried
out in 100 ul of SalI digestion buffer containing 150
mM NaCl, 6 mM Tris-HC1 pF3 7.9, 6 mM MgCl2, 6 mM
2-mercaptoethanol, 100 yg/m1 gelatin and 25 units of
SalI (New England Biolabs). Incubation was at 37°C for
one hour. The enzyme was then inactivated by heating to
65°C for five minutes.
The two aliquots were pooled and digested with 10
units calf intestinal phosphatase (PL Labs) at 37°C for 2
hours to remove S' 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
-20-
1 341 35 2
with 70% v/v ethanol, dried and finally re-.,uspende~' in 10
mM Tris-HC1, 1 mM EDTA to a final concentration of 500
vg/ml. The ligation reactions contained 1 ug of
prepared pHC 79 and 1 ug of Sau3A digested E coli DDIA
in S ul of ligation buffer (10 mM Tris-HC1 pH 7.9, lOmM
L~lgCi2, 20 nnLQ dithiothreitol, 25 ug/rnl gelatin and 1 rnM
ATP). The reaction mixture was incubated at 37°C for 5
minutes, cooled and the ligat:ion initiated :~y the addition
of T4 DNA lipase (EC 6.5..1.1).
to These reactions were continued for 4 hours at 15°C.
Four u1 aliquots of the ligation mixtures were then
subjected to in vitro packaging into bacterio-phage a
particles. Strains BHB 2688 and B1-3B 2690 were inoculated
from NZY agar plates into 50 ml of NZY broth (containing
i0 g NZamire (from F?umko Sheffield), 5 g yeast extract and
2.5 g NaC1/litre) and incubated with aeration at 30°C
until the E600 reached 0.3. The cultures were then
raised to 45°C by immersion in a 60°C water bath and
incubated at 45°C for 20 minutes without aeration. The
20 two cultures were then vigorously aerated at 37°C for 3
hours. The two cultures were pooled, chilled to 4°C and
harvested by centrifugation at 7000 rpm for 2 minutes.
The pellet was washed ence in 100 ml of cold M9 minimal
medium (per liter: 6g Na2 HPO 4, 3g KH2P04, 0.5g
NaCl, 1.0 NH4C1; adjusted to pH7 with 8 N NaOH, after
autoclaving, sterile glucose added to 0.2% ta/V, CaCl2
added to 0.1 mM and Mg S04 to 1 mM). The pellet: was
washed in 5 ml cold complementation buffer (40mM Tris-HC1
-21-
'1 341 35 2
pH 8.0, 10 mM spermidine-HC1, 10 mM putrescine-HC1, 0.1%
V;'V 2-mercaptoethanol <~nd 7°o V/V dimethyl sulfoxide) and
pell~ted at 5000 rpm for 30 seconds. 'fhe cells were
finally resuspended in 0.5 ml cold complementa'~ion buffer
and dispe:lsed into 20 ill :i'_iq~_iots in micro test tubes.
These were tro~ei: immediately in liquid nitrogen and
stored at -SO°C.
For the packaging reaction, one 20 ul packaging mix
was removed from the liqmid nitrogen and to it was
immediately added 1 ul of 30 mM ATP. The mix was then
placed on ice for 2 mivnutes. The 4 ul_ aliquots of
ligated DNA were then added and mixed thoroughly. This
was then incubated at 37°C for 30 minutes. After 30
minutes, 1 ul of 1 mg,/ml DNAase (Worthington) w<3s added
and mia:ed in until the sample had lost its viscosity. To
this packaged DNA preparation was then added 20a ul of
the strain E 107 which ilad been grown to ail E600 °'
about 1.0 in tryptone maltose broth. MgS04 was also
added to a final concentration of 10 mM. The packaged
cosmids were absorbed for 30 minutes at 30°C after which
time 500 ul 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 30°C. Th.e cells
were then plated on L-agar containing 100 yg/ml
carbenicillin (a-carboxy benzyl penicillin) and
incubated at 30°C for 24 hours.
-22-
1 341 35 2
Carbenicillin resistant colonies were then picked to
L-agar carbenicillin plates and incubated overnight at
~42°C. Strain E107 (3) carries the mutation dnaB which
renders it unable to grow at 42°C. Thus, only those
colonies v:~hicl: had acquired a cosmid clone carrying
dnaB from the wild-type donor should be able to grow
at this te:rperature. In this way, 8 co.~mid 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 tyrB. That
some of the clones did indeed carry t~rB was shown in 'the
following way.
Cosmid DNA from each of the dnaB clones was purified
and packaged into bacteriophage a particles as described
above. The packaged cosmids were then introduced into the
transaminase deficient strain DG 44 which carries the
mutations tyrB and asDC. These two mutations together
10 bestocv 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 colEl, such as pHC 79. After
introducing the cosmid clones into DG 44, therefore, 2
hours were allowed for recombination between the ~rB
gene on the cosmid and the mutant allelle on the bacterial
-l3-
1341352
chromosome. The cells were then washed and played on
minimal medium lacking tyrosine and aspartate to screen
for the occurrence of t~,~_B+ ~wecombinants. Four of the
original dnaB clones gave tvrB recombinants in this
screen and mast therefore carry at least some o:E the ~~rB
gene.
(b) Sub-cloning of restriction fracaments from cosmid dnaB
clone
The ~rB cosmid clones were of limited utility owing
to the large size thereof which resulted in a low copy
number. Therefore, restriction fragments carrying the
ty~rB gene were sub-cloned into the multi-copy p:lasmid
pATl53, (see fer example, Twigg,
A.J., and Shenatt, D., (1980), Nature, 283, 216-218) as
follows: Two ug aliquots of dna8 cosmid number 5 which
carries tyrB was digested in 20 ul reaction with the
following restriction endonucleases BamHI, HindIII,
SalI, SphI, ClaI, EcoRI and B~lII.
In each case, the incubation was at 3'7°C for one hour
,.20. and .the.. enzyme w.as inactivated by heating to 65°C for five
minutes. For the BamHI digestion, the reaction medium
contained 150 mM NaCl, 6 mM Tris-HC1 pH 7.9, 6 ;mM MgCl2,
100 ug/ml gelatin and 4 units of BamHI (New England
Biolabs). Three (3) units of HindIII (New England
Biolabs) were used in 60 mM NaCl, 7 mM MgCl2, 7 mM
Tris-HC1 pH 7.4 and 100 ug/ml gelatin. In the case of
SalI, 5 units of enzyme obtained from New England Biolabs
were used in 150 mB9 NaCl, 6 mM Tris-HC1 pH 7.9, 6 mM
-24-
1 341 35 2
;~1gC12, 7 mM 2-mercapteethanoi and 100 ug;'ml c~~latin.
For SohI, the reaction contained 50 mM NaCl, 6 mM
Tris-HC1 pH 7.4, 6 mM I~igCl2, 10 mff 2-mercaptoet:hano:L,
100 ug;iml gelatin and 1 unit of the Sr~hI enzyme (New
England Biolabs) . The r~~~act~on rniat:ure fo:- i.laI
dic_restion corresponded to that for :?in.iIII, ciig~=stion,
eYCept that the enzyme u:.ed was 3 units of CIaI obtained
from Boehringer Biochenicals. In the case of EcoRI 4._'i
units of EcoRI (New Ennland Biolabs) were used in 100 mM
Tris-TIC1 pH 7.5, 50 mM 1'!aC.l, 5 MM MgCl3 and 100 ugjml
gelatin. Lastly, the reaction medium for B~1II digestion
contained 60 mM NaCl, 10 rnM Tris-HC1 pH 7.6, 10 mM
f~7gC12, 10 mM 2-mercaptoethanol and 100 ug/ml gelatin.
These digested DNA preparations were then subjected to
electrcphoresis on a horizontal l;o w/v low gelling
temperature (LGT) agarose gel in TEE for 6 hours at 5
V;~cm. The DNA fragments were visualised by staining the
gel for 15 minutes in a 1 ug;ml solution of eth:Ldium
bromide, followed by observation under a 366 nm UV light
source. Iuidividual bands were excised and stored at 4"C.
One (1) ug aliquots of pAT153 DNA were digested with
the same set of restriction endonucleases in 20 ul
reactions, except for )III (which does not cut pAT153).
Cosmid III fragments were sub-cloned into BamHI-cut
pATl53 (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,
-25-
1 341 35 2
incubated at 70°C for 10 minutes to inactivate the enzyme
and then also run on a 1°o w/v LGT agarose gel as described
for the cosmid fragments. The band cor:-esponding to
plasmid linearized by each enzyme was then excised from
the gel as described above.
The cosmid fragments were th~_n ligated to the
appropriately-cut vector as follows. The gel slices were
melted by incubation at 65°C for 10 minutes and. then
cooled to 37°C. The melted gel slices were all about 100
ul. Two u1 of vector fragment and 8 ul o.f a
particular cosmid frac~memt were then added to 9~0 ul of
pre-warmed 1.25 x ligation buffer (62.5 mM Trig;-HC1 pH
7.8, 12.5 mP9 MgCl2, 25 mM dithiothreitol, 1.25 mM ATP
and 62.5 mgjml gelatin) and mixed thoroughly. Ligase was
added and the reaction continued at 15°C overnight. The
ligazed samples were t=hen re-melted at 65°C for 5 minutes,
cooled to room temperature and added to 200 ul of
competent cells of the E coli strain HW 8? which had
been prepared as follows. An overnight culture. of HW 87
LO in L-broth was diluted 1:50 into 50 ml of fresh pre-warmed
L-broth and incubated at 37°C with good aeration until the
E600 reached 0.6. Th<~ cells were then pelleted by
centrifugation at 10,000 rpm for 5 seconds and resuspended
in 25 ml of cold 50 mM CaCl2. The cells were .Left on
ice for 10 minutes after which they were re-pe:Lleted as
above and re-suspended in 2 ml of cold 50 mM C;~C12.
After a further 10 minute incubation at 0°C, the cells
were competent for transformation.
-26-
1 341 35 2
After addition of the ligated DNA, the cells were
incubated at 0°C for a further 10 minutes, heat-shocked at
37°C for 2 minutes and finally mil:ed with 750 u:L of
pre-warmed L-broth. The cells were incubated for 30
minutes at 37°C to allow phenotypic e::~_~ession of the
plasmid encoded ~-lactamase gene before plating suitable
aliquots onto L-agar plates containing 200 ug,%m:L
carbenicillin. The plates were incubated at 37°C
overnight. Colonies containing recombinant plasmids were
identified by the sensitivity thereof to tetracvycline 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 si<:e
of the plasmids in individual colonies. 'This was
performed as follows. Potential recombinaait colonies were
patched onto L-agar plates containing 200 yg/ml
carbenicillin. These were incubated overnight at 37°C.
The bacteria from about 1 cm2 of each patch were
re-suspended in lytic mix COIltaining 10 mM Tris-HC1 pH 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
ug/ml RNAse. This was then incubated at 65°C for 30
minutes. Each sample was then vortex-mixed vigorously for
seconds. 50 ul aliquots of each preparation were
then loaded onto a 1% w/v agarose gel and subjected to
electrophoresis for 4 hours at 10 V/cm. The plasmid bands
were stained with ethidium bromide as described. above and
visualized under
_27_
1 341 35 2
254 nm UV illuminatlOIl. Recombinant plasmids were
identified by the reduced mobility thereof compared to
non-recorzbinant controls. Using these methods, a number
of recombinant plasmids carrying various fragments from
the oricJinal cosmid dnaB clone ~,~ez.-e isolated. '.Co
facilitate the screening of these plasmi<is for those that
carried tl:e intact t;,rrB gene, a new strain carrying
tvrB and as_pC 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 P1 was grown on a mixed culture of strain HW
22 carrying random insertion of the tetracycline resistant
transposon Tn 10 prepared as follows: A saturated culture
of HV22 was grown overnight in lambda x'M broth (10 g bacto
tryptone, 2.5 g NaCl and 0.2w/v maltose per litre).
This was diluted x 1j100 into 100 ml of fresh pre-warmed
2p lambda YM broth and incubated at 37°C until the OD600
reached 0.6. The cells were deposited by centrifugation
at 10,000 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 37°C. Then 200 ml aliquots
were spread onto L-agar plates containing tetracycline (20
ug/ml) and sodium pyrophosphate (2.5 mM). The plates
_2$_
1 341 35 2
were incubated at 37°C for 24 hours. Approximately 5,000
TetR ~:,lonies wire pooled by washing the colonies off
the plates using a total of 5 ml of L-broth. Tluis was
then diluted into 50 ml. of L-broth containing 20 ug;ml
tetracycline and grown overnight at 37°C. 'The T'n10 pool
was stored at -20°C after adding sterile glycerol to bring
the concentration to 20~o w/v.
In order to grow bact~riophage Pl on t:he Tnl.O pool, a
200 ml aliauot of this glycerol stock ',aas diluted with 5
lU ml of L-broth including 10 ugjml tetracycline an,d
incubated overnight with shaking at 37°C. To 0.2 ml of
this overnight culture was added 2 x 106 plaque forming
units (pfu) of phage P1 clear and 10 ul of 50 mlvl
CaCl2. The cells were incubated at 37°C for five
minu:.es and then added to 5 ml of L-broth + 2.5 mM CaCl2
pre-warmed to 37°C. The cells were then incubat=ed at 37°C
with vigorous aeration for four hours. The cell debris
was removed by centrifugation at 10,000 rpm for five
minutes and the phage-~~ontaining supernate stored over
2p . , . ~hlor~oform~at a~~C:' . . ,
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/1 MgS04.7H20, 2 g/1 citric acid, 10 g/1 1:2H P02,
3.5 g/1 NaH2P04 .4H20 and 10 mM feCl2. After
autoclaving, glucose was added to a final concentration of
0.2% w/v along with necessary supplements of amino acids
at 50 ug/ml. The Tyr+ Asp+ transductants were then
-29-
1 341 35 2
replica-plated to the same medium including 10 ugjml
tetracycline to detect those transductants that had
simultaneously become T'etP. A number of these TetR
+ +
Tyr Asp recombinants were purified and used to
prepare nev; P1 ly_::;t:es. F:ach of tl~~~se eight °1
preparations ;:as then usec_1 to transduce DG 30 to TetR.
From each experiment, 50 TetR tramm:uctants wire picked
to minimal medium lacking tyrosine and aspartate to test
for linkage between the site of the Tn 10 insertion
involved and as'oC or tyrB. From each of these
experiments one TetR colony which remained t~B asyDC
was purified and again used to prepare a P1 lysate. These
lysates were used to transduce I~7C 1061 to TetR. Cell
extracts from these transductants were then run on native
polyacrylamide gels and stained for L-phe transa.minase
activity as described by Gelfand.
The cell extracts were prepared by growing t:he desired
transductants overnight in 10 ml of L-broth at 3~7°C. The
cells were harvested by cetrifugation at ,10,000 rpm for
LO 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 mid Tris-HC1, <:5 mM
KH2P04 pH 6.9, 0.2 mM pyr:idoxal phosphate, 0.5 nrM
dithiothreitol, 0.2 mM ED'rA and 10% v/v glyceroT~. The
cell suspensions were transferred to Eppendorf rnicro test
tubes and kept on ice. Each suspension was sonicated with
a Davies sonicator using four five-second burst:a at
setting 2. The sonicated suspensions were then cetrifuged
-30-
1341352
at 15,000 rpm for five minutes at 4°C 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 =20°C.
The native gel electrophoresis was performed as
follows. The gel consisted of 8.5 °o w,/v acrylamide,
0.227;0 vtiv bis-acrylarnide in 375 mL4 Tris-HC1 pH 8.3. This
acrylamide stock was de-gassed and polymerized by the
addition of 0.05°a w;/v ammonium persulfate and 0.016% w/v
TEP7ED. After polymeri:;ation, the gels were pre-run in
37.5 mT~1 Tris-HC1 pH 8.3 for at least one hour at: 4°C.
Prior to loading the samples, the buffer was changed to
running buffer consisting of 76.7 mM glycine, 1 mM
Tris-HC1 pH 8.3. About 50 ug protein from each cell
extract was then loaded and subjected to electrophoresis
at 10 v/cm for from four to six hours at 4°C. 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..'> and then
. ~. ..3s~_
immersed in 500,m1~of fresh staining solution containing
12.5 mM a-ketoglutarate, 0.2 mM pyridoxal phosphate, 0.6
mM nitro blue tetrazol.ium, 0.2 mM phenazine metlzosulphate,
mM L-phe and 100 mM I~2HP04 pH 7.5 that had been
pre-warmed to 37°C. The gel was shaken gently in the
staining solution for one hour at 37°C in the dark. The
gel was then washed in distilled water and left in
distilled water until it could be photographed. (See
-31-
1 341 35 2
figure 2.) This illustrates the preseace or ab~:ence of
detectable aminotransferases in various E. coli strains.
MC 1061 extracts prepared in this way gave three
staining bands characteristic of the ilvE, as~C and
tvrB 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 v;hich was devoid of a~C activity was
purified and named HW1G9. 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-trypt:one lOg, Bacto yeast extract 5 g,
chlortetracycline HC1 50 mg, agar 15 g, water 500 ml) and
solution B (NaCl 10 g, NaH2P04.H20 10 g, glucose: 2
g, water 500 ml) were autoclaved separately for 20 min at
psi. The solutions were mixed and cooled to pouring
te~npe,rature.. .:5~,n1,l,,Qf "ZnC.l2._(zO,mM). and,b 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 f~usaric
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 tr~rB
derivatives of HW 22. To isolate a strain with Tn 10
-32-
. 1 341 35 2
lint:ed to tyrB, the procedure was as follows. S~tL-ain
ES430 carries a mutation i.n malB that renders it. unable
to use maltose as a carbon source. The malB is
reasonably closely linked with tyrB. Therefore, ES430
was transduced with Pl. gro~,:~n on the random 'rn 1f pool in
Hid 22 described abo~.:~e, selecting for Tet R on maltose
P~laconkey agar plates ~uplemented v:ith 10 yc~,'m1
tetracvcline-
F1 transductions were performed as follows. The
recir~i~~nt ~ train vas cy-o~:w in L-broth to an OD600 of
about 1Ø CaCl2 was added to a final concentration of
2.5 mM. Phage P1 clear grown on the desired donor was
then added at a multiplicity of insertion of between 0.2
and 1.0 (usually lOg pfu P1 clear per ml of recipient).
The cells were incubated at 3;°C For fifteen minutes,
centrifuged at 10,000 fpm for five seconds, washed in the
original volume of 0.1 1'i citrate buffer pH 7.0 a.nd finally
re-suspended in citrates buffer. Suitable aliquots were
then plated on the selective medium.
Transductants which had simultaneously become TetR
and Mal+ were picked and purified. P1 lysates were then
prepared from these strains and used to transduc:e DO a4 to
TetR. The TetR colonie>s derived from each of the
eight P1 lysates were then patched onto minimal agar
plates lacking aspartat:e and tyrosine. In one of these
experiments, good linkage between Tn 10 and tyrB was
obtained. Therefore, one TetR t_yrB recombinani~ from
this experiment was isolated, a P1 lysate prepared from
this recombinant and used to transduce HW157 described
-33-
11 341 35 2
above to TetR. Fifty of these transducants were then
patched onto minimal agar supplemented with leuc:ine, but
lacking L-aspartate and L-phenylalanine. In this way, a
tyrB aspC derivative of MC 1061 was detected.
This strain was designated HS9.158. Finally, HW159, a
tetracycline sensitive derivative of HW158 was isolated as
described above for the i~>olation of Hw157. HW 159 will
not grow on minimal medium supplemented with leucine since
it requires aspartate and tyrosine for growth unlike the
1p parental strain t~7C1061 which only requires leucine.
Example 4
Screening of sub-clones for abili~ to complement the t~
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 H49 159 by
transformation selecting on L-agar supplemented with 200
ugjml carbenicillin. These transformants were then
streaked onto minimal medium supplemented only with
2U. , leucine. to test ;for .the,.ability.,:of tOe,"p.reseyce .of .qe.nes
carried by the plasmid to complement the t~rB lesion in.
HW 159. One sub-clone carrying a ClaI fragment from
cosmid dnaB c'_one number 5 was found to restore the
ability of HW 159 to grow on minimal medium. supplemented
with leucine and thus to carry the ~rB gene.
Sequencing of the t~B gene was performed by the
method of Maxam and Gilbert. The sequence of the tyrB
-34-
1341352
gene is shown in figure 3. Based upon this DNA sequence
the amino acid sequence for the aminotransferase encoded
for by tyzB could be determined using the genetic triplet
codons. This amino acid sequence is shown in figure 4.
E~:an~, le S
.p __._
Cloning of the ast~~C-cJene_
As a starting point for the cloning of the asoC c3ene,
it was decided to use the specialized transducin.g phage
lambda aspC2 obtained from M. Ono. The phacJe was prepared
lU as follows. A culture of HW76, which is a double lysogen
carrying lambda as~C and lambda CI 857 Sam 7, wa.s grown
to an OD600 °f 0.6 at 30°C. The culture was then
incubated at 45°C for fifteen minutes to induce the
prophage and then shaken vigorously at 37°C for three
hours. Cell lysis was completed by the addition of 0.5 ml
of CHC1.. and the cell debris removed by centrifugation
J
at 10,000 rpm for ten minlates. The phage was precipitated
by the addition of NaCl to 2.4;o w/v and polyethylene
glycol (average molecular weight 6,000) to 10°,o w/v. The
20 , phac~e~,was precipitated overnight, at 4°C, and, then pelleted
by centrifugation at S,OOU rpm for ten minutes. The phage
pellet was gently re-suspended in 10 ml of phage buffer
consisting of 10 mM Tri.s-HC1 pH 7.5, 10 mM MgSO~~. The
phage cvas 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 waa applied
-35-
1 341 35 2
to a two-step block gradient in celimiose nitrate tubes.
The gradient contained 1 ml of 5L~9 CsCl, 10 mL~i MgS04, 10
mM Tris-HC1 pH 8.0 and 0.1 mM EDTA overlayed with 3 ml of
3M CsCl, 10 mM r9g SO~, 10 mM Tris-HC1 pH 8.0 and 0.1 mM
EDTA. The gradient: ',:as cent: ifud='d W : a )ackmann S1~ E5
rotor for one hour at 30,000 ~-ym at 2C°C. Tloe phage band
was remo-.-ed in 0.5 ml :_rc~m the side ~f the tube usitxg a 1
ml syringe with a 5,,8 inclu (1.6 cm) 25 c)uacre needle. The
0.5 ml of phaae ~.~as th=_-n mixed with 0.5 ml. of saturated
CsCl solution (25°C) in 10 mM t~lg SO;I, 10 n~I~1 Trigs-HCl pH
8.0 and 0.1 mM EDTA and mixed well in a cellulose nitrate
centrifuge tube. This was then overlayed with ~ ml of 5M
CsCl in 10 mi~7 Mg SOa, 10 mM Tris-HC1 pH 8.0 and 0.1 mM
EDTA and then 1 ml of 3M CsCl in 10 mhl L4g SO~, 1.0 mM
Tris-HC1 pH 8.0 and 0.1 mL~9 EDTA. This was ac3ain
centrifuged at 30,000 rpm,for OI'1e hour at 20°C. 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
ZO follows. To the 0.5 m1 of phage in~a l5 ml coreex
centrifuge tube was added 50 ul of 2M Tris-HC1 pH 8.5,
0.2 M EDTA and 0.5 ml of formamide Laas 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 pellets rinsed
with 70% v/v ethanol. The DNA was dried in vacuo and
finally dissolved in TE (10 mM Tris-HC1 and 1 mLn EDTA pa
-.s 6-
1 341 35 2
S.0) to give a DNA COIICentration of SOO ug/ml. The
lambda aspC DNA was partially digested with the
restriction endonuclease Sau3A as described above fo~_- E.
coli DNA. The DNA from all of the digests were pooled
and run on a 1°o w/v low c~c:lling temperature agorose gel. in
TBE. Also prepared and run on the same gel was 1 ug ef
p.~~.T153 DNA ccmpletel y digested with the re:;t~-ici~ion
lendonuclease BamHI and calf intestinal phosphatase as
described above. The DN.4 was visualized under 366 nm U.V.
licJht as described above.
The bat:ds corresponding to linearised pAT153 and the
partially restricted lambda aspC DNA between 2._'> and 6kb
were excised. The DNA was extracted from the gel as
follows. The gel slices were melted at 65°C fox five
minutes in 15 m1 convex tubes, cooled to 37°C and diluted
with two volumes of water a~ 37°C. The agarose was then
removed by the addition of an equal volume oz phenol at
37°C 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.~~ Tyre final
supernate was then extracted four times by vorte:xing 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 ug/ml as a carrier. The DNA was
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precipitated by the addition of 2.5 volumes of ethanol,
overnight incubation at. -20°C and centrifugation at 10,000
rpm for 30 minutes. Tlve pellet was washed in 70% v;v
ethanol, re-centrifuged at 10,000 rpm for 30 minutes and
dried in vacuo. 'flm Dr~A was then dissolved in T'E to
give a concentration of about 100 uc3;ml. The partially
rests icted lambJa a PLC DNA was then lirated to E~amE3I cut
pAT153 by adding 2 ul of i>repared lamb<ja asr~C DNA to 2
ul of prepared pATl53 DNA in 4 u1 of 2x ligation
bt;ffar (described above). This mixture was incubated at
37°C for five minutes, cooled to room temperature and the
ligation started by the addition of 1 ul of T4 DNA
ligase (New England Biolabs). Tine ligation was continued
at 15°C for four hours. The entire ligation reaction
mixture was then cooled on ice and added to 200 ul 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 ugjml
carbenicillin. The plates were incubated overnight at
2U 37°C. About 5,000 colonies were obtained. These were
then 'pooled, wasl-ied 'ti,;~ice in' 10 ml of 0.$5% wjv 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 37°C for twenty-four hours, about
100 colonies were obtained. Some of these were purified
by successive streakings on the same medium. That these
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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 H~~7 159 and showing that
+ +
these transformants had become Asp Tyr . That the
transformants had been altered by the presence of cloned
asoC as opposed to t~)w°as 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 aspC gene.
The actual level of aminotransferase activity is
measured in arbitrary units indicating relative activity.
Relative aminotransferase activity on various strains
containing the t~rB and a ~C genes on plasrnid 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)
.> . v: ~. ~..::. r ., . ; ; , ~ ~ : r:, . . ., ,_ : , . . ~.,.... . . ... . .
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1 341 35 2
Table 2
Transaminase Levels b7easured-Usilyg-Ast~artate_As Subs-trate
Sigma Aspaytate _T.ransaminase-Assa_y_ ISit
mg Frotein
Umits~t~IlI1- Rei ative i4ctivity
t~IC 1061 40 1
fisJ157 25 0.6
fitd 15 8 I~ID -
HW158 pHC79 15 0.37
HSa158 p tyrB X94 12 . 3
Ht°7158 paspCl-1 1738 -I3.5
E~arnnie 6
Further Analysis of the tvrB Clone
The ClaI insert carrying tyrB is approt:imately 4.5
kb. A preliminary restriction map was constructed by the
commonly used technique of double restriction endonucle<3se
digestion followed by gel electrophoresis ~tci'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 ug of ptyrB DNA
was digested to completion with EcoRI in a final volume of
20 ul. The enzyme was inactivated by incubation at 65°
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for 5 minutes. A 2 ul ;sliquot of this digest waa them
diluted into 50 ul of lipase buffer, one ul of T4 DNA
lipase added and the DNA ligated for 16 hours at 15°.
Ligaticn at this low DNA concentration leads pre~dominani~ly
to the re-circularisation of tlw:e plasn~id .,~it.hout the small
EcoRI fragment. Th~s Dr7A was then transformed into HW87
selecting for carbenicillin resista:~ce as described
above. Indi~~idual transfozmants were picked, purified and
analysed on single colony lysate gels as described above.
Most of the transformants contained r~lasmids that
exhibited higher mobility than the parental plas;mid
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 correspornding 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. (100 mM NaCl 6 mM Tris pH7.4 6 mM
MgCl2 5 mM S-mercaptoethanol 100 ug/ml gelatin) All
three plasmids were purified and used to transform HW225
(tyrB aspC recA) to carbenicillin resistance. A.11 the
transformants simultaneously acquired the ability to grow
on minimal medium in the absence of L-tyrosine and
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1341352
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 i~ia:w:m and GilbeLt (Mar: am, .~,. M. , and W. Gilbert,
1977, Ft~IAS 51: 3S2-389; Maxam, A. M., and W. Gilbert, 1977,
P~II~ 65: CH 57 a°°-'59, Pant 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
bindlIlg 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 Ecol;I-Bam.HI fragment
and the amino-acid sequence deduced from it are presented in
figures 3 and 4.
Mao of paspC
The restriction map of the smallest aspC sub-clone (paspC
3-~) was elucidated by a combination of single a.nd double
l0 restriction digests as described above see figure 7. The
'aspC sub-c'Iones do not pos~sess-restric~ion siteee that allow
the insert to be excised cleanly since they were made by
ligating Sau3A fragments into the BamHI 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 produce>d level.s of
transaminase equivaleni~ to or better than that of paspCl-1.
The E. toll HW519 carrying pME98 has the deposii~ number
ATCC 39501.
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1 :341 35 2
Exam le 6
Increased Field of amino Acids
In the reactions ~ecruiring aminotransferase enzymes in
figure 1, reactions #10 and #13, presence of additional
aminotransfe:.-ase en~vm~~:: f,~cilitate~- amino ac:d
synthesis. This increa<;ed levol of the aminotrar~sferase
enz.~mes synthesised by the ash C, tyr 6 or i lyE gene
enables a cell to overcome a rate-limited reaction at the
transamination step.
A plasmid of the preseat invention, carrying the asp
C, ~r B or ilv E gene is inserted into a bacterial cell
by techniques well known in biochemistry. This inserted
gone produces a messenger RNA which catalyzes they
synthesis of increased levels of aminotrar_sferase~s when
the bacterial cell is aro~.an in a suitable media. The
presence of these increased levels of aminotransi:arases
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.
2U The addition of the tyrB containing plasmid into
E.Coli 13281 (U:S. pateilt'2;°73,304resulted i~ri ari 11
increase in L-phenylalanine production as shown in Table 3.
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1 341 35 2
Table 3
Phenylalanine Production in E. Coli
(Strain 13281)
210_Flas:nid Pl.us_tyr8__Plasmid
L-Phe Produced 1C0,°~ 111°0
Culture grcw,i at 32°C for 48 hou_s.
... . , .. , , ~ . , . . . _: ~~:_:, , . . .
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