Note: Descriptions are shown in the official language in which they were submitted.
1228~8
PROCESS FOR PRODUCING TYROSINE
-
Back~round of the Invention
For the production of tyrosine by direct
fermentation, the methods using mutant strains requiring
phenylalanine for its growth or being resistant to tyro-
sine of the bacteria belonging to the genus Coryne-
bacterium, Brevibacterium, and the like are known Cgr.
Chem. Soc., Japan 50 (1) R79 - R87 (1976 ~.
The present inventors have constructed plasmid
vectors autonomously replicable in a microorganism
belonging to the genus Corynebacterium or Brevibacterium
and having selectable markers and adequate cloning si~es
and have developed a highly efficient transformation
system (Japanese Published Unexamined Patent Application
15 Nos. 183799/82 published Nov. 12, 1982, 186492/82 and
186489/82 both published Nov. 16, 1982. Further, the
present inventors have found that the plasmid vectors
are useful for expressing a foreign gene in a host
microorganism and increasing the productivity of amino
acids by ligating a DNA fragment containing a foreign
gene involved in the biosynthesis of amino acids such as
glutamic acid, lysine, tryptophan, histidine and phenyl-
alanine to the plasmid vectors according to the pro-
cedures in recombinant DNA technology (U.S. Patent No.
25 4,237,224 and Methods in Enzymology 68, Recombinant DNA,
edited by Ray Wu, ~cademic Press 1979) and transforming
Corynebacterium glutamicum L-22 or its derivatives using
the transformation methods (Japanese Published Unexamin-
ed Patent Application No. 126789/83, and Canadian Patent
30 Applications S.N. 447,439, Feb. 15, 1984, S.N. 447,442,
Feb. 15, 1984, S.N. 455,139, May 25, 1984).
Furthermore, the present inventors have found
that a microorganism prepared by the same method has
acquired an increased productivity of tyrosine.
- 2 - 1228038
Summary of the Invention
This invention relates to a process for producing
tyrosine by recombinant DNA technology. More specifically, the
present invention is a process for producing tyrosine by
transforming a host microorganism belonging to the genus
CorYnebacterium or Brevibacterium with a recombinant DNA of a
DNA fragment containing a gene involved in the biosynthesis of
tyrosine and a vector DNA, culturing the transformant in a
nutrient medium, accumulating tyrosine in the culture medium and
recovering tyrosine therefrom.
Brief Description of the Drawinqs
Fig. 1 illustrates the cleavage pattern with
restriction endonucleases for plasmid pEaroF-l.
Fig. 2 illustrates the cleavage pattern with
restriction endonucleases for plasmid pKmlaroFl.
In Figs. 1 and 2, the horizontal arrows show
orientation of transcription of genes.
Description of the Invention
The present invention provides a process for producing
tyrosine by culturing in a medium a transformant which is
obtained by transforming a microorganism belonging to the genus
CorYnebacterium or Brevibacterium with a recombinant DNA of a
DNA fragment containing a gene involved in the biosynthesis of
tyrosine and a vector DNA.
As the DNA fragment containing the gene used in the
present invention, the DNA fragment containing a gene involved
in the biosynthesis of tyrosine derived from prokaryotes,
viruses, bacteriophages or plasmids is used.
Metabolic pathway and regulation systems of aromatic
amino acids in microorganisms have been studied in detail on
Escherichia coli, Bacillus subtilis, and glutamic acid-producing
microorganisms such as strains of the genus of CorYnebacterium
3S and Brevibacterium [Agr. Chem. Soc. Japan, 50 (1), R79 - R87
(1976) and Ann. Rev. Biochemistry 47, 533 (1978)]. The gene
involved in the biosynthesis cf tyrosine of the present
invention is a DNA carrying a genetic information of at least
122B038
-- 3 --
one of enzymes involved directly or indirectly in the
biosynthesis of these aromatic amino acids. The genes
encoding for enzymes subjected to regulation on the bio-
synthetic pathway, i.e. 3-deoxy-D-arabino-heptulosonate
7-phosphate (referred to as DAHP hereinafter) synthetase
(referred to as DAHPase hereinafter), chorismate mutase
(referred to as CMase hereinafter), prephenate dehydro-
genase (referred to as PDGase hereinafter) and pre-
tyrosine aminotransferase are preferably used. Further,
the genes of strains which are relieved from feed back
inhibition and repression _ prlori or a posteriori are
applicable.
As the desired gene of the present invention,
any gene is used so long as it is expressed in the
microorganisms belonging to the genus Corynebacterium or
Brevibacterium and strengthens at least one enzyme
activity on the biosynthesis of tyrosine. As the source
of the gene, microorganisms which are used for the pro-
duction of amino acids such as aromatic amino acids by
Eermentation and microorganisms wherein the mechanism of
biosynthesis and regulation of aromatic amino acids is
clarified are preferably used. The gene derived from a
bacterium belonging to the genus Escherichia, Coryne-
bacterium, Brevibacterium, Microbacterium, Bacillus,
-
Staphylococcus, Streptococcus or Serratia and involved
in the biosynthesis of tyrosine or the metabolism re-
lating to the biosynthesis is preferably used. In the
example of the present invention, Escherichia coli JA194
~ roc. Natl. Acad. Sci., 74, 487-491 (1977 ~ was used.
The vector used in the present invention
should autonomously replicate in cells of the host
microorganism. Plasmids isolated by the present in-
ventors from microorganisms belonging to the genus
Corynebacterium or derivatives thereof are available.
pCGl (Japanese Published Unexamined Patent Application
*
~;
.~
12X8038
-- 4 --
No. 134500/82 published Aug. 19, 1982), pCG2 (Japanese
Published Unexamined Patent Application No. 35197/83),
pCG4 (Japanese Published Unexamined Patent Application
No. 183799/82 published Nov. 12, 1982), pCE51, pCE52
(Japanese Published Unexamined Patent Application No.
126789/83 published June 24, 1983), pCE53 (Canadian
Patent Application S.N. 447,442, Feb. 15, 1984), pCE54,
pCGll and pCB101 are preferably used.
Microorganisms carrying the plasmids have been
deposited with the Fermentation Research Institute,
Agency of Industrial Science and Technology, Ibaraki,
Japan and the American Type Culture Collection, Rock-
ville, Maryland, U.S.A. under the following accession
numbers.
Plasmid FER~l P- ATCC
pCGl 5865 31808
pCG2 5954 31832
pCG4 5939 31830
pCE54 - 39019
pCGll - 39022
pCB101 - 39020
Of the foregoing plasmids, pCG51 is most preferred.
pCE51 is prepared as follows.
pCGl is isolated from the cultured cells of
Corynebacterium glutamicum 225-57 (FERM P-5865, ATCC
31808) by the method described in the specification of
Japanese Published Unexamined Patent Application No.
134500/82 published Aug. 19, 1982. pGA22 is isolated
from the cultured cells of Escherichia coli harboring
the plasmid by a conventional method C n, G. et al.,:
J. Bacteriol., 140, 400 (1979 ~. Plasmid pCGl is
linearized with restriction endonuclease BglII and a
fragment of pGA22 digested with BamHI and containing
kanamycin resistant (KmR) gene is ligated to the linear-
~ ~,
1228038
-- 5 --
ized pCGl using the same cohesive ends of both-pladmids.
Isolation of pCE51 from the ligated DNA mixture is
achieved by selecting the transformants belonging to the
genus Corynebacterium or Brevibacterium and containing
KmR derived from pGA22, and analyzing the plasmid in the
transformant.
Transformation with the ligated DNA mixture is
carried out using protoplasts of the genus Coryne-
bacterium or Brevibacterium according to the method de-
10 scribed in Canadian Patent Application S.N. 401,124,
April 16, 1982 and Japanese Published Unexamined Patent
Application Nos. 186492/82 and 186489/82 both publish~d
Nov. 16, 1982. Kanamycin is used for selection of the
transformants. Transformants are recovered as a colony
regenerated on a hypertonic agar medium containing 100 -
800 ~g/mQ kanamycin which does not allow the reversion
to normal cells of protoplast which are not treated with
the ligation mixture. Alternatively, transformants are
regenerated unselectively on a regeneration medium, and
the resultant cells are scraped and resuspended, follow-
ed by the isolation of those cells grown on an agar
medium containing a drug at a concentration wherein the
recipient normal cells cannot grow, that is, generally
2 - 25 ~g/mQ kanamycin.
Plasmid DNAs in the transformants can be iso-
lated from cultured cells of the transformants and puri-
fied according to the methods described in Canadian
Patent Application S.N. 395,976, February 10, 1982 and
Japanese Published Unexamined Patent Application Nos.
30 134500/82 published Aug. 19, 1982 and 186489/82 publish-
ed Nov. 16, 1982. The structures of the DNAs can be
determined by digesting them with various restriction
endonucleases and analyzing the DNA fragments by agarose
gel electrophoresis. Although several types of plasmids
different in physical struct~re are obtained, the
lZ2803~3
- 5a -
plasmid isolated from one of the transformants is named
pCE51.
pCE51 has a molecular weight of about 6 Xb and
cleavage sites for HincII, HindIII, SmaI, XhoI and EcoRI
and gives Km phenotype.
Plasmids from the strains are recovered accord-
ing to the methods described in Canadian Patent Appli-
cations S.N. 395,976, filed February 10, 1982, S.N.
401,241, filed April 19, 1982 and S.N. 410,113, filed
10 August 25, 1982 and Japanese Published Unexamined Patent
Application Nos. 134500/82 published Aug. 19, 1982,
183799/82 published Nov. 12, 1982 and 35197/83 published
March 1, 1983.
Preparation of a recombinant DNA of a vector
DNA with a DNA fragment containing a gene is carried out
by conventional in vitro recombinant DNA technology,
e.g. cleavage and ligation of a donor DNA containing a
desired gene to a vector DNA (refer to Japanese Publish-
ed Unexamined Patent Application No. 126789/83 published
20 June 24, 1983, USP 4,237,224).
The ligase reaction gives recombinants con-
taining genes other than the desired gene. The desired
recombinant DNA can be obtained by directly transforming
a microorganism of the genus Corynebacterium or Brevi-
bacterium with the DNA mixture, selecting the trans-
formants having the phenotype derived from the desired
gene and isolating the desired recombinant DNA from
-
'~
;~ '
- 6 - ~Z28038
the cultured cells of the transformants. Instead of cloning the
desired gene directly in a microorganism of the genus
Corynebacterium or BreYibacterium, the desired gene can be
cloned by using another host-vector system such as Escherichia
coli. Then, it is recloned ln vitro into a vector of the genus
Corynebacterium or Brevibacterium to transform these
microorganisms and transformants containing the desired
recombinant plasmid are selected as mentioned above.
The following references are helpful for the
construction of recombinant DNA:
S.N. Cohen, et al., U.S.P. No. 4,237,224;
Idenshi Sosa Jikkenho, edited by Yasuyuki Takagi~
printed by Kodansha Scientific (1980);
Methods in Enzymology 68, Recombinant DNA, edited by
Ray Wu, Academic Press 1979
Japanese Published Unexamined Patent Application
No. 126789/83
Microorganisms belonging to the genus Corynebacterium
or Brevibacterium and which are competent for incorporating DNAs
may be used as the host microorganisms in the present invention.
The following are examples of a suitable host microorganism.
Accession Number
FERM P- ATCC
CorYnebacterium qlutamicum L-15 5946 31834
CorYnebacterium qlutamicum R-38 7087
CorYnebacterium qlutamicum K-43 7162
CorYnebacterium herculis 13868
CorYnebacterium herculis L-103 5947 31866
Brevibacterium divaricatum L-204 5948 31867
Brevibacterium lactofermentum 13869
Brevibacterium lactofermentum L-312 5949 31868
Brevibacterium flavum 14067
Tyrosine-producing mutant strains derived from these
strains are preferably used as host microorganisms. The mutant
strains are obtained as amino acid-requiring mutants, amino acid
analog-resistant mutants or mutants having both the properties.
- 7 - 1228038
Transformation of the host microorganisms with
recombinant DNAs is carried out through the following steps:
l) Preparation of protoplasts of host cells;
2) Transformation of the protoplasts with a reco~binant
DNA;
3) Regeneration of the protoplasts to normal cells and
sPlection of a transformant;
Practical method are described in Japanese Published
Unexamined Patent Application Nos. 186492/82, 186489/82 and
lOS999/83.
The thus obtained transformant is cultured in a
conventional manner used in the production of tyrosine by
fermentation. That is, the transformant is cultured in a
conventional medium containing carbon sources, nitrogen sources,
lS inorganic materials, amino acids, vitamines, etc. under aerobic
conditions, with adjustment of temperature and pH. Thus,
tyrosine accumulated in the medium is recovered.
As the carbon source, various carbohydrates such as
glucose, fructose, sucrose, maltose, mannose, sorbitol and
2Q mannitol, sugar alcohol, glycerol, starch, starch hydrolyzate,
molasses, various organic acids such as pyruvic acid, lactic
acid, acetic acid, fumaric acid and gluconic acid, and lower
alcohols such as ethanol may be used.
As the nitrogen source, ammonia, various inorganic or
organic ammonium salts such as ammonium chloride, ammonium
sulfate, ammonium carbonate and ammonium acetate, urea, and
nitrogenous organic substances such as peptone, ~2-amine, meat
extract, yeast textract, corn steep liquor, casein hydrolyzate,
fish meal or its digested product, and chrysalis hydrolyzate are
appropriate.
As the inorganic materials, potassium
dihydrogenphosphate, dipotassium hydrogenphosphate, magnesium
sulfate, sodium chloride, ferrous sulfate, manganese sulfate and
calcium carbonate may be used. Vitamines and amino acids
required for the growth of microorganisms may not be added,
provided that they are supplied with other components mentioned
above.
- 8 ~ ~ ~28~3~
Culturing is carried out under aerobic conditions with
shaking or aeration-agitation. Culturing temperature is
preferably 20 to 40C. The p~ of the medium during culturing is
maintained around neutral. Culturing is continued until a
considerable amount of tyrosine is accumulated, generally for 2
to 5 days.
After completion of the culturing, cells are removed
and tyrosine is recovered from the culture liquo~ by
conventional manners such as treatment with active carbon or ion
exchange resin.
In spite of the high similarity in microbiological
characteristics, so called ~lutamic acid-producing
microorganisms which produce glutamic acid in large amounts are
classified into various species and even into different genera
such as Corynebacterium and Brevibacterlum, which is probably
because of their industrial importance. However, it has been
pointed out that these microorganisms should belong to one
species because of nearly the same composition of amino acids in
the cell wall and the base composition of DNAs. Recently, it
~0 has been reported that these microorganisms have at least 70 to
80% homology in DNA-DNA hybridization, indicating that these
microorganisms are closely related. See, e g., Romatsu, Y.:
Report of the Fermentation Research Institute, No. 55, 1 (1980),
and Suzuki, K., Kaneko, T., and Romagata, K.: Int. J. S~st.
Bacteriol., 31, 131 11981).
In the present specifi~ation, the usefulness of the
present invention is illustrated using derivatives of
Corynebacterium qlutamicum as host microorganisms. However, in
consideration of the facts mentioned above, it is readily
assumed that the usefulness of the present invention is
applicable to all the glutamic acid-producing microorganisms.
In order to stably maintain recombinant DNA molecules and
express the DNA in these species, slight differences of such
properties of the host microorganisms as homology in the ~A are
negligible and it is sufficient for host microorqanisms to allow
the autonomous replication of plasmids and expression of ~enes
on them. That these microorganisms have such abilities is
apparent from the fact that plasmid ?CG4 which was isolated ~rom
9 12X803~3
CorYnebacterium glutamicum 225-250 (Japanese Published
Unexamined Patent Application No. 183799/82) and having an
streptomycin and/or spectinomycin resistant gene could replicate
in microorganisms belonging to the genus Corynebacterium or
Brevibacterium and that the gene responsible for the resistance
could be expressed (Japanese Published Unexamined Patent
Application No. 186492/82). Further, as described in Japanese
Published Unexamined Patent Application No. 126789/83, the
plasmid involved in histidine production functioned in
Cor~nebacterium herculis, Brevibacterium flavum and
Brevibacterium lactofermentum. Therefore, the present invention
is applicable to all the glutamic acid-producing microorganisms
including those microorganisms belonging to the genus
CorYnebacterium or Brevibacterium as well as to CorYnebacterium
qlutamicum
.
Certain specific embodiments of the present invention
are illustrated by the following representative examples.
Example 1
(1) Preparation of the chromosomal DNA and plasmid DNA:
The chromosomal DNA of Escherichia coli (hereinafter
referred to as E. coli) JA194 strain [Proc. Natl. Acad. Sci.,
94, 487-491 (1977)] was prepared by the following method.
A seed culture was inoculated into 400 mQ of L-broth
(pH 7.0) consisting of 10 g/Q Bacto-tryptone, 5 g/Q Bacto-yeast
extract and 5 g/Q NaCl (hereinafter referred to as LB).
Culturing was carried out with shaking at 37C and continued to
a latter stage of the logarithmic growth phase. Cells were
harvested from the culture broth and high molecular chromosomal
DNAs were isolated from the cells by the method of Saito et al.,
Biochim. Biophys. ~cta, 72, 619 (1963).
Separately pBR322 used as a vector was prepared from
the cultured cells of E. coli JA194 strain harboring pB~322 by
the following method.
The strain was grown with shaking at 37C in 400 mQ of
LB containing 100 ~g/m~ ampicillin to a latter stage of the
logarithmic growth phase. Cells were harvested from the culture
broth and lysed by the method of Tanaka, et al., ~J. Bacteriol.
121, 35~-362 (197i)].
- 10 - 122803~3
The whole lysate was centrifuged at 4C at 23,000 rpm
for one hour. The supernatant fluid was recovered and one fifth
volume of 50~ (W/V) polyethyleneglycol (PEG) 6,000 aqueous
solution was added. The mixture was stirred slowly and allowed
to stand at 4C overnight.
Formed precipitates were collected by centrifugation
at 3,000 rpm at 4C for 5 minutes and dissolved in 5 mQ of TE
buffer solution (pH 7.5) consisting of 10 mM Tris HCl and 1 mM
EDTA-Na2. Then, one mQ of 1.5 mg/mQ ethidium bromide was added,
and TE buffer solution was added to the total volume of 7.5 mQ.
To the mixture was added 7.875g of CsCl, and dissolved
completely. The solution was centrifuged at 105,000 x 9 at 20C
for 40 hours. A plasmid band detected under W irradiation was
taken out with a injector. Ethidium bromide was extracted three
times with isopropanol containing 15% (V~V) TE buffer solution.
The residue was dialysed against TE buffer solution at 4C
overnight, and used as a plasmid DNA.
(2) Cloning of a DNA fragment containing the gene coding
for DAHPase, CMase and PDGase:
In this step, 5 units each of restriction enzymes
EcoRI and HindIII (product of Takara Shuzo Co.) were added to
100 ~Q of a HindIII reaction solution containing 3 ~g of pBR322
plasmid DNA prepared above, and 5 units each of EcoRI and
HindIII (product of Takara Shuzo Co.) was added to 100 ~Q of
restriction enzyme HindIII reaction solution containing 9 l~g of
the chromosomal DNA. Each of the mixtures was allowed to react
at 37C for 60 minutes and heated at 65C for 10 minutes to stop
the reaction. Both of the mixtures were admixed with each other.
Then, 40 ~Q of a T4 ligase buffer solution (pH 7.6) consisting
of 660 mM Tris, 66 mM MgC12 and 100 mM dithiothreitol, 40 ~ of
5 mM ATP, 0.4 uQ of T4 ligase (product of Takara Shuzo Co., 1
unit/~Q) and 120 ~Q of H2O were added. The mixture was allowed
to react at 12C for 16 hours.
The above ligation mixture was provided ~or the
following transform~tion. As the recipient for the
transformation, ~AHPase-deficient E. coli AB3248 strain [J.
Bact., 93, 237-244 (1967)] or tyrA gene (CMase)-deficient E.
coli AT2273 strain [J. Bact., 91, 1494 ~1966~ was used.
- ll - 12~8038
The seed culture of the strain was inoculated into LB
and competent cells were prepared according to the method of
M. Dagert, [Gene, 6, 23-28 (1979)].
Fifty microliter of the ligation mixture was added to
0.2 mQ of a solution containing 109/m~ competent cells and the
mixture was allowed to stand under ice cooling for 10 minutes.
After heating at 37C for 5 minutes, 2 m~ of LB was added,
followed by standing at 37C for 90 to 120 minutes. Cells were
subjected to washing with physiological saline solution and
centrifugation twice. The cells were spread on M9 plate medium
(pH 7.0) consisting of 1 g/Q NH4Cl, 6 g~ Na2HPO4, 3 g/Q KH2PO4,
5 g/Q NaCl, 0.1 g/Q MgSO4 7~20, 0.015 g/Q CaC12 2H2O, 3 g/~
glucose, 4 mg~ vitamine Bl and 15 g/Q agar, [J. ~act. 121, 354-
362 (1975)] and containing 50 ~g/mQ each histidine, proline,
arginine, isoleucine and valine. Colonies grown on M9 plate
medium were plated on LB plate media containing 100 ~g/m~
ampicillin and 20 ~g/mQ tetracycline, respectively. Colonies
which grew on the ampicillin-containing medium and did not grow
on the tetracycline-containing medium were selected.
Plasmid DNAs were isolated from the thus selected
transformants which grew on M9 plate medium containing histidine,
proline, arginine, isolucine and valine and which were resistant
to ampicillin and sensitive to tetracycline by the same method
as described above. Plasmid pEaroFl isolated from one of the
transformants was digested with various restriction endonucleases
and analysed by agarose gel electrophoresis. The analysis showed
that an EcoRI-HindIII cleaved DNA fragment of about 4.2 Kb was
inserted into the larger EcoRI-HindIII cleaved fragment of
pBR322.
E. coli. AB3248 and E. coli AT2273 were transformed
using pEaroFl obtained above by the same method as described
above. Both transformants grew on M9 medium containing
histidine, proline, arginine, isoleucine and valine and
simultaneously were resistant to ampicillin. The fact showed
that the transformants had the same plasmids as pEaroFl.
The results described above showed that the genes
encoding for DAHPase, CMase and PDGase exist on the DNA fragment
of about 4.2 Xb cloned in pEaroFl.
- 12 ~ 1Z~8~3~
Further, pEaroFl having cleavage sites with various
restriction endonucleases such as EcoRI, BamHI, HindIII and the
like as shown in Fig. 1 was compared with plasmid pKB45 reported
by G. Zurawski, Proc. Natl. Acad. Sci. USA 75, 4271 (1978) to
show that the DNA fragment of about 4.2 Kb in pEaroFl possesses
aroF, tyrA and pheA genes of E. coli.
(3) Subcloning of aroFtyrA gene:
A DNA fragment containing aroFtyrA qene was recovered
from plasmid pEaroFl DNA prepared above and ligated to pCE51
which is a shuttle vector for E. coli and CorYnebacterium
qlutamicum.
pCE51 is a recombinant plasmid wherein plasmid pCG1
(Japanese Published Unexamined Patent Application No. 134500/82)
of CorYnebacterium qlutamicum is ligated with plasmid pGA22 of
E. coli ~refer to An, G. et al: J. Bacteriol., 140, 400
(1979)]. The ligation is carried out using the same cohesive
ends of BglII cleaved pCGl and BamHI fragment of pGA22
containing the kanamycin-resistant gene.
pCE51 is practically prepared by the method described
in Japanese Published Unexamined Patent Application
Nos. 105g99/83 and 126789/~3.
Five units of HincII (product of Takara Shuzo Co.) was
added to 100 ~1 of HincII reaction solution containing 3 Yg of
plasmid pEaroFl DNA and reaction was carried out at 37C for 60
minutes. Three-tenth units of HincII was added to 100 yQ of
HincII reaction solution containing 3 ~9 of plasmid pCE51 DNA
prepared from pCE51-carrying E. coli JA194 strain by the same
method as mentioned above and reaction was carried out at 37C
for 60 minutes to cut pCE51 at one of two HincII cleavage sites.
Both of the reaction mixtures were mixed with each other. 40 yQ
of T4 ligase buffer solution (pH 7.6) consisting of 660 mM Tris,
66 mM MgC12 and 100 mM dithiothreitol, 40 ~Q of 5 mM ATP, 0.4 yQ
of T4 ligase (product of Takara Shuzo Co., 1 unit/~Q) and 120 ~Q
of water were added. The mixture was allowed to react at 12QC
for 16 hours and reaction was stopped by heating at 55C for 10
minutes.
- 13 ~ 12 Z ~03 8
The ligation mixture was provided for the following
transformation. As the recipient for the transformation, E.
coli AB3248 strain was used. Transformation was carried out by
the same method as described above to obtain colonies grown on
M9 plate medium containing histidine, proline, arginine,
isoleucine and valine. Colonies grown on LB plate medium
containing 20 ~g/mQ kanamycin were selected from the colonies
obtained above.
Plasmid DNAs were isolated from the transformants
which grew on M9 plate medium containing histidine, proline,
arginine, isoleucine and valine and were resistant to kanamycin
by the same method as described above.
Plasmid pKmlaroFl obtained from one of the
transformants were digested with various restriction
endonucleases and analysed by agarose gel electrophoresis. The
analysis showed that a HincII cleaved DNA fragment of about
3.8 Kb bearing aroFtyrA of pEaroFl was inserted in one of two
HincII cleavage sites of pCE51. Plasmid pKmlaroF1 obtained
above has the restriction pattern as illustrated in Fig. 2.
(4) Preparation of tyrosine- and tyrosine analog-resistant
plasmid pKmlaroFl-m-18 from pKmlaroFl-carrying strain:
pKmlaroFl-carrying AB3248 strain was grown in LB
medium containing 20 ~g/m~ kanamycin to a latter stage of the
logarithmic growth phase. Cells were harvested by centrifugation
and washed with 50 mM Tris-malate buffer solution IpH 6.0) twice
and incubated with 400 ~g/mQ N-metyl-N'-nitro-N-nitrosoguanidine
in 50 mM Tris-malate buffer solution (pH 6.0) at room temperature
for 30 minutes. The treated cells were harvested by
centrifugation and washed with 50 mM Tris-malate buffer solution
(pH 6.0) twice. The washed cells were cultured in LB medium
containing 20 ~g/mQ kanamycin at 30C for 16 hours and plasmid
DNAs were isolated by the same method as mentioned above.
_. coli AB3248 was transformed using the isolated
?lasmids. Selection of transformants was carried ou~ on M9
plate medium containing 0.25 mg/mQ tyrosine and 50 ~g/mQ each
histidine, ~roline, arginine, isoleucine and valine. Colonie~
grown on ~9 pl~te medium containing 0.25 mg/mQ tyrosine and
- 14 -
lZ28038
50 ~g/mQ each histidine, proline, arginine, isoleucine and
valine and on LB plate medium containing 20 ~g/mQ kanamycin were
selected from the developed colonies.
The thus obtained colonies were resistant to 3-
aminotyrosine (3-AT) (product of Shigma Co.) since they could
grow on M9 plate medium containing 0.2 mg/mQ 3-AT and 50 ~g/mQ
each histidine, proline, arginine, isoleucine and valine.
The plasmids obtained from the thus prepared
transformant can confer resistance to tyrosine or a tyrosine
analog on a microorganism. E. coli AB3248 strain carrying one
of such plasmids, pKmlaroF-m-18 was able to grow on M9 plate
medium containing 1 mg/mQ tyrosine or 0.5 mg/mQ 3 AT, and
50 ~g/mQ each histidine, p{oline, arginine, isoleucine and
valine.
(5) Transformation of CorYnebacterium qlutamicum K43 with
pKmlaroFl and pKmlaroFl-m-18:
A seed culture of Corynebacterium qlutamicum K43 (FEF~I
P-7162, FERM BP-457) was inoculated in Semi-synthetic medium SSM
(pH 7.2) consisting of 20 g/Q glucose, 10 g/Q (NH4)2SO4, 3 g/Q
urea, 1 g/Q yeast extract, 1 g/Q KH2PO4, 0.4 g/Q MgC12 6H2O,
10 mg/Q FeSO4 7H2O, 0.2 mg/Q MnSO4 (4-6)H2O, 0.9 mg/Q ZnSO4
7H2O, 0.4 mg/Q CuSO4-SH2O, 0.09 mg/Q Na2B47 10H20~ 0.04 mg/Q
(NH4)6Mo7O24 4H2O, 30 ~g/Q biotin and 1 mg/Q thiamine
hydrochloride and containing 50 ~g/mQ phenylalanine and
culturing was carried out with shaking at 30C. NB medium (pH
7.2) consisting of 20 g/Q powdered bouillon and 5 g/Q yeast
extract was used for seed culture. The optical density (OD3 at
660 nm was monitored with a Tokyo Koden colorimeter and, at OD
0.2, penicillin G was added to the broth to a final
concentration of 0.5 unit/mQ. Culturing was continued to an OD
value of about 0.6.
Cells were harvested at an OD value of 0.6. The cells
were suspended at about 109 cells/mQ in RCGP medium (pH 7.6)
consisting of 5 g/Q glucose, 5 g/Q casamino acid, 2.5 g/Q yeast
extract, 3.5 g/Q K2HPO4, 1.5 g/Q KH2PO4, 0.41 g/Q ~IgC12 6H2O,
10 mg/Q FeSO4 7H2O, 2 mgJl MnSO4 (4-5)H2O, 0.9 mg/Q ~nSO4 7H2O,
0.04 mg/Q (NH4)~Mo7O24-4H2O, 30 ~g/Q biotin, 2 mg/Q thiamine
- 15 - :12Z80~
hydrochloride, 135 g/Q sodium succinate and 30 g/Q polyvinyl
pyrrolidone with a molecular weight of 10,000 and containing
1 mg/mQ lysozyme~ The suspension was put in an L-tube and
stirred slowly at 30C for 5 hours to obtain protoplasts.
Then, 0.5 mQ of the protoplast suspension was put in a
small test tube and centrifuged at 2,500 x g for 5 minutes. The
protoplasts were resuspended in 1 mQ of TSMC buffer tpH 7.5)
consisting of 10 mM magnesium chloride, 30 mM calcium chloride,
50 mM Tris and 400 mM sucrose and again subjected to
centrifugation and washing. The washed protoplasts were
resuspended in 0.1 mQ of TSMC buffer solution. One hundred
microliter of a mixture (1 : 1 by volume) of a two-~old
concentrated TSMC buffer and the ligated DNA mixture described
above was added to the protoplast suspension. Then, 0.8 mQ of a
solution containing 20% PEG 6,000 in TSMC buffer solution was
added to the mixture. Plasmid DNAs, pRmlaroFl and pRmlaroFl-m-
18 were prepared from E. coli AB3248 carrying these plasmids as
described above. After 3 minutesr 2 mQ of RCGP medium (pH 7.2)
was added and the mixture was centrifuged at 2,500 x g for 5
minutes. The supernatant fluid was removed and the precipitated
protoplasts were suspended in 1 mQ of RCGP medium. Then, 0.2 mQ
of the suspension was spread on RCGP agar medium (pH 7.2)
containing 200 ~g/mQ kanamycin and 1.4% agar and incubated at
30C for 7 days. Colonies resistant to kanamycin were grown on
the selection plate.
t6) Production of tyrosine by the transformant:
The thus obtained transformants carrying pKmlaroFl and
pKmlaroFl-m-18 have been deposited with the Fermentation
Research Institute as CorYnebacterium qlutamicum K44 (FE~M P-
7163, FERM BP-458) and Corynebacterium qlutamicum K45 (FE~M P-
7164, FE~M BP-460), respectively.
L-tyrosine production by pKmlaroFl and pKmlaroFl-m-18-
carrying strains was carried out as follows.
The strain was cultured in NB aqueous medium at 30C
for 16 hours and 0.5 mQ of the culture broth was inoculated in
5 mQ of a production medium P4 (p~ 7.2) consisting of 100 g/Q
molasses, 20 g/Q (NH~)2SO4, 0.5 g/Q KH2PO4, 0.5 g/Q K2~PO4,
- 16 ~ ~Z 2 803 8
0.25 g/Q MgSO4 7H2O and 20 g/Q CaCO3 and containing 0.25~ NZ
amine. Culturing was carried out at 30C for 96 hours.
After culturing, 6N NaOH solution was added to the
broth to a concentration of 50 ~Q/mQ and the mixture was heated
at 65C for 5 minutes to dissolve precipitated tyrosine
completely. The culture filtrate was subjected to paper
chromatography and color reaction with ninhydrin, and the amount
of L-tyrosine formed was determined colorimetrically. As a
control, CorYnebacterium glutamicum K43 was similarly treated.
The results are shown in Table 1.
Table 1
Amount of L-tyrosine
strain (mq/m~)
Corynebacterium qlutamicum K43 4.8
Corynebacterium qlutamicum K44 5.3
CorYnebacterium qlutamicum K45 7.7
