Note: Descriptions are shown in the official language in which they were submitted.
TITLE OF THE INVENTION
PF(OCESS FOR PRODUCING L-ISOLEUCINE
Background of the Inv2ntion
For the direct production of L-isoleucine by
fermentation methods using glutamic acid-producing
microorganisms belonging to the genus Corynebacterium or
Brevibacterium, the method using L-isoleucine-producing mutant
strains derived from wild-type strains are known.
As the L isoleucine-producing mutant strains, those
requiring amino acids for their growth, those resistant to amino
acid analogs or those having both characteristics thereof are
described in Japanese Published Unexamined Patent Application
Nos. 38995/72, 6237/76 and 32070/79.
The present inventors have studied the production of
L-isoleucine using a microorganism belonging to the genus
Corynebacterium or Brevibacterium by recombinant DNA technology
different from the conventional mutational breeding t~chnology
for the purpose OL improYing the L-isoleucine productivity. As
the result, the present inventors have ~ound that a microorganism
harboring a recombinant DNA of a gene involved in the
biosynthesis of threonine which is a precursor of L-isoleucine
and a vector plasmid of the microorganism belonging to the genus
Corynebacterium or Brevibacterium is superior in production of
L-isoleucine to a microorganism which does not harbor such
recombinant~
The facts that the introduction of a recombinant DN~
containing a gene involved in the biosynthesis of threonine into
an L-isoleucine-unproducing microorganism belonging to the genus
Corynebacterium or revibacterium confers L-isoleucine
productivity on the microorganism and that the introduction of
such recombinant DNA into an L-isoleucine producing microorganism
belonging to the genus Corynebacterium or Brevibacterium improve
its L-isoleucine productivity have been found first by ~he
present in~entors.
- 2 - ~Z~8039
In the Japanese Published Unexamined Patent Application
No. 893/83, production of L-isoleucine by a microorganism
belonging to the genus CorYnebacterium or Brevibacterium wherein
a chromosomal gene region involved in the resistance to an
S isoleucine antagonist is introduced is described. However, the
gene is different from that of t~e present invention.
Summary of the Invention
This invention relates to a process for producing L-
isoleucine by a novel expression method of a gene. Morespecifically, the present invention is a process for producing
L-isoleucine bY transforming a host microorganism belonging to
the genus Corynebacterium or Brevibacterium with a recombinant
DNA of a DNA ~ragment containing a gene encoding for the enzyme
involved in the biosynthesis of threonine from aspartic acid and
a vector DNA, cult~ring the transformant in a nutrient medium,
accumulating L-isoleucine in the culture medium and recovering
L-isoleucine therefrom.
Brief Description of The Drawinq
Fig. 1 illustrates the process for construction of and
the cleavage map for restriction endonucleases of plasmid pEthrl
wherein "Bgl II~Bam HI" with a broken line indicates a
recombina~ion site at the same cohesive ends formed by cleavage
with both restriction endonucleases. The restriction
endonucleases used in the preparation of the cleavage map are
PstI, EcoRI and XhoI. Molecular weight of the plasmid is
indicated as Kilobase (Kb).
Description of the Invention
The present invention provides a process for producing
L-isoleucine by culturing in a medium a transormant which is
obtained by transforming a microorganism belonging to the genu~
Corvnebacterium or Brevibacterium with a recombinant DNA of a
DNA fragment containing a gene encoding for the enzyme involved
in the biosynthesis of threonine from aspartic acid and a vector
DNA.
~ 3 ~ 1228039
As the host microorganism belonging to the genus
CorYnebacterium or Brevibacterium, all of the microorganisms
known as so-called glutamic acid-producing microorganisms are
applicable. The following are examples of suitable host
microorganisms.
CorYnebacterium qlutamicum ATCC 13032
CorYnebacterium acetoacidoPhilum ATCC 13870
CorYnebacterium herculis ATCC 13868
CorYnebacterium lilium ATCC 15990
Brevibacterium divaricatum ATCC 14020
Brevibacterium flavum ATCC 14067
Brevibacterium immarioPhillum ATCC 14068
Brevibacterium lactofermentum ATCC 13869
Brevibacterium thioqenitalis ATCC 19240
As the host, either wild-type strains which do not
produce isoleucine or strains which already have an ability to
produce isoleucine can be employed. As the latter strains,
amino acid-requiring mutant strains and amino acid analog-
resistant mutant strains are used.
As the enzyme involved in the biosynthesis of
threonine from aspartic acid, aspartate kinase, aspartate-
semialdehyde kinase, homoserine dehydrogenase, homoserine kinase
and threonine synthase [Agr. Biol. Chem., 38, 993 (1974)] are
mentioned.
As the gene encoding for the enzymes involved in the
biosynthesis of threonine, the DNA carrying the genetic
information of at least one of these enzymes is used. Any DNA
may be used so long as it is derived from prokaryotes,
eukaryotes, bacteriophages, viruses or plasmids. The genes
involved in the biosynthesis of threonine derived from
prokaryotes, bacteria such as the microorganisms belonging to
the genus Escherichia, Corvnebacterium, Brevibacterium,
Microbacterium, Bacillus, StaPhylococcus, StrePtococcus or
Serratia are preferable and especially the genes derived from
threonine- or isoleucine-producing mutants belonging to such
bacteria are preferably used. The threonine operon of
Escherichia coli K12 is the most preferable example.
~ 4 ~ 12 2 ~03 9
As the vector used to incorporate the DNA, the
plasmids constructed by the present inventors, pCGl, pCG2, pCG4,
pCGll, pCE54 and pCB101 are preferably used. The methods of
producing these vectors are described in Japanese Published
Unexamined Patent Application Nos. 134500/82, 183799/82,
35197/83 and 105999/83.
The recombinant DNA of the donor DNA encoding for the
enzyme involved in the biosynthesis of threonine and the vector
DNA is obtained by the recombinant DNA technology which
comprises cleaving ln vitro both DNAs with restriction enzymes,
recombining the cleaved DNAs by DNA ligase, transforming a
mutant strain belonging to the genus CorYnebacterium or
Brevibacterium and defective in the gene encoding for the enzyme
involved in the biosynthesis of threonine with the ligation
mixture, and selecting the transformants wherein the defective
phenotype is restored. The method of recombinant DNA technology
is described in Japanese Published Unexamined Patent Application
Nos. 186492/82 and 186489/82.
Instead of cloning the recombinant DNA directly in a
microoganism belonging to the genus Corynebacterium or
Brevibacterium, the recombinant DNA can also be obtained by
using another well established host-vector system as exemplified
with Escherichia coli system. That is, recombinant DNAs can be
obtained by the method which comprises transforming an
Escherichia coli mutant which lacks the gene encoding for the
enzyme involved in the biosynthesis of threonine with the in
vitro ligation mixture of the donor DNA encoding for the enzyme
involved in the biosynthesis of threonine and the vector DNA,
and selecting transformants wherein the defective phenotype is
restored. Subsequently the cloned DNA isolated from the
transformants and a vector DNA of the microorganism belonging to
the genus Corynebacterium or Brevibacterium are cleaved with a
restriction enzyme and religated with T4 ligase. With the
ligation mixture, a mutant strain belonging to the genus
Corynebacterium or Brevibacterium and defective in the gene
encoding for the en2yme involved in the biosynthesis of
threonine is transformed, and the transformants, wherein the
defective phenotype is restored, are obtained. The desired
recombinant DNA is isolated from such transformants.
- 5 - ~28~9
The threonine operon of Escherichia coli K12 is used
as a preferable e~ample of the DNA containing the gene involved
in the biosynthesis of threonine.
The present invention is explained in more detail usin~
the recombinant plasmid harboring the DNA fragment containing
the threonine operon o Escherichia coli, pEthrl.
The DNA fragment containing the threonine operon oE
Escherichia coli can preliminarily be cloned by the cloning
system in Escherichia coli. The method of cloning a gene in a
host of Escherichia coli is described, for example, in ~ethods
in Enzymology, 68, Ray Wu (Ed), Academic Press, New York (1979).
Practical cloning is carried out as follows~
The chromosomal DNA extracted ~rom Escherichia coli
having wild-type threonine operon and vector plasmid pGA2~ of
Escherichia coli are digested with res~riction en7yme Hlnd III,
followed by T4 pha~e ligase treatment. Escherichia coli K12
variant strain GT-3, a mutant strain which is defective in three
kinds of aspartate kinase and which re~uires homoserine and
diaminopimelic acid is transformed by a conventional method with
the ligation mixture to select transformants growing on a
minimal medium containing diaminopimelic acid and kanamycin
which is a selection marker of pGA22.
The plasmid which the resulting transformant harbors
is isolated from the cultured cells of the transEormant by a
conventional method. The structure of the plasmid is determine~
by digesting the plasmid with various restriction enzymes and
analyzing the resulting DNA fragments by agarose gel
electrophoresis. One of the plasmids thus obtained is pGH2
having the structure illustrated in Fig. 1. The ~NA fragment
containing the threQnine operon of Escherichia coli has already
been cloned and the restriction sites thereo~ ha~-e been
determined [re~er to Cossart P et al, Molec. Gen. Genet., 175,
39 (1979)]. pGH2 contains the cloned DNA fragment exhibiting
the same restriction sites, an~ is thus confirmed to possess the
~hreonine operon.
The defec' of aspartate kinase Oc GT-3 strain is
restored by the aspartate kinase present on the threonine operon
of p~H~ and G~-3 strain becomes protoproph ~or homoserine and
diaminopimelic acid.
1228iO3~
pEthrl is obtained as a recombinant of pGH2
and pCGll which is a vector plasmid of the genus Coryne-
bacterium and srevibacterium.
Plasmid pCGll is a plasmid constructed by the
present inventors and described in Japanese Published
Unexamined Patent Application No. 134500/82 published
Aug. 19, 1982 and Canadian Application S.N. 395,976,
filed Feb. 10, 1982. Plasmid pCGll is prepared by
inserting the BamHI fragment containing a gene responsi-
ble for resistance to streptomycin and/or spectinomycinof plasmid pCG4 isolated from Corynebacterium glutamicum
225-250 (ATCC 31830, FERM P-5939) into the unique BglII
cleavage site of plasmid pCGl isolated from Coryne-
bacterium glutamicum 225-57 (ATCC 31808, FERM P-5865)
using the same cohesive ends of both fragments.
pCGll is concentrated and isolated from the
cultured cells of Corynebacterium glutamicum ATCC 39022
harboring pCGll by the method described in Japanese
Published Unexamined Patent Application No. 186492/82
20 published Nov. 16, 1982. By a convantional method, pGH2
is digested with BamHI and pCGll is digested with BglII
and the digests axe mixed and treated with T4 ligase.
Corynebacterium glutamicum L~201, a mutant strain which
is defective in homoserine dehydrogenase is transformed
with the DNA mixture. Corynebacterium glutamicum LA201
is a mutant which is derived by a conventional mutagene-
sis from lysozyme-sensitive mutant strain L-22 derived
from Corynebacterium glutamicum ATCC 31833 (~apanese
Published Unexamined Patent Application No. 186492/82
30 published Nov. 16, 1982 and which requires homoserine
(alternatively, threonine and methionine) and leucine
for its growth. The homoserine-requirement depends on
the defect of homoserine dehydrogenase gene which acts
upon the threonine biosynthesis pathway to metabolize
aspartate-semialdehyde into homoserine.
~Z2~039
- 6a -
Transformation is carried out by the trans-
formation method using protoplasts of the genus Coryne-
bacterium or Brevibacterium described in Japanese
Published Unexamined Patent Application Nos. 186492/82
and 186489/82 both published Nov. 16, 1982. The practi-
cal embodiment of the method is described in the example
of the present application. Corynebacterium glutamicum
LA201 is transformed by the method using protoplasts and
the protoplasts are unselectively regenerated to normal
cells on a regeneration
~ 7 ~ 12 Z ~03 9
medium. The regenerated cells are scraped, washed with sterile
physiological saline solution and spread on a minimal medium
supplemented by leucine to isolate growing colonies.
Some of the thus obtained homoserine non-requiring
transformants obtain simultaneously kanamycin resistance
phenotype derived from pGH2 and spectinomycin resistance
phenotype derived from pCGll. The plasmid DNAs in the
transformants are isolated and purified from cultured cells by
the same method as in the isolation of pCGll described above.
The structure of the plasmid DNA is determined by analysis of
agarose gel electrophoresis after digestion with various
restriction enzymes. pEthrl is a plasmid obtained from one of
the transformants. The restriction map and the process for
producing the plasmid are illustrated in Fig. 1. As is apparent
from Fig. 1, pEthrl is a plasmid wherein a fragment containing
the threonine operon of pGH2 cleaved with 8amHI is inserted in
pCGll.
From another transformant, a plasmid wherein the
orientation of the BamHI fragment containing the threonine
operon is opposite to that in pEthrl is obtained. When either
recombinant plasmid is reintroduced into CorYnebacterium
qlutamicum LA201 and transformants are selected with kanamycin
or spectinomycin, the transformants restore homoserine requiring
property and have the donor plasmids characterized by cleavage
pattern for various restriction enzymes.
The capability to restore homoserine requiring
property depends on the expression of the homoserine
dehydrogenase gene on the threonine operon of Escherichia coli
present in the recombinant plasmid. It is known that the
threonine operon of Escherichia coli has the genes encoding for
aspartate kinase, homoserine dehydrogenase, homoserine kinase
and threonine synthase and the genes are transcribed as a single
transcription unit l-efer to Theze, J. et al,: J. Bacteriol.,
115 990 (1979)] and it is elucidated that the function of the
3S expression of all genes is located on the region between Hind
III site at 5.4 Rb and BamHI site at 11.3 Rb on pGH2 illustrated
in Fig. 1 ~refer to Cossart, P. et al.: Molec. Gen. Genet., 175,
39 (1979)]. Therefore, it is manifest that pEthrl has the
- 8 ~ 1~ X 8 03 9
function of expression of all genes and not only homoserine
dehydrogenase gene but also aspartate kinase, homoserine kinase
and threonine synthase gene are expressed in CorYnebacterium
qlutamicum harboring pEthrl.
An L-isoleucine-producing strain belonging to the genus
CorYnebacterium or Brevibacterium and harboring pEthrl is
obtained by transforming protoplasts of the genus
Corynebacterium or Brevibacterium with pEthrl and selecting for
spectinomycin and/or streptomycin resistance by the same method
as described above.
The presence of pEthrl in the transformant is detected
by isolating the plasmid from the transformant, digesting the
plasmid with various restriction enzymes and analyzing DNA
fragments by agarose gel electrophoresis as described above. L-
isoleucine producing strains are exemplified by pEthrl-carrying
strains of CorYnebacterium qlutamicum R40 (FERM P-7160, FERM BP-
455) and Brevibacterium flavum ATCC 14067 (refer tG the example
below). CorYnebacterium qlutamicum K40 is an L-isoleucine
producing strain derived from CorYnebacterium qlutamicum ATCC
31833 as an S-(2-aminoethyl)-cysteine resistant strain.
CorYnebacterium qlutamicum K40 has been deposited with the
Fermentation Research Institute, Agency of Industrial Science
and Technology, Ibaraki, Japan together with R41 strain (FERM P-
7161, FE~M BP-456) prepared by introducing pEthrl into R40
strain, and Brevibacterium flavum K42 (FERM BP-355) prepared by
introducing pEthrl into Brevibacterium flavum ATCC 14067.
Production of L-isoleucine by the transformant
harboring pEthrl is carried out by a conventional ~ermentation
method used in the production of L-isoleucine.
That is, the transformant is cultured in a
conventional medium containing carbon sources, nitrogen sources,
inorganic materials, amino acids, vitamins, etc. under aerobic
conditions, with adjustment of temperature and pH. Isoleucine,
thus accumulated in the medium, is recovered.
As the carbon source, various carbohydrates sucn as
glucose, glycerol, fructose, sucrose, maltose, mannose, starch,
starch hydrolyzate and molasses, polyalcohols and various
organic acids such as pyruvic acid, fumaric acid, lactic acid
- 9 - 12Z13039
and acetic acid may be used. Hydrocarbon and alcohols are
employed in the strains which can assimilate them. Blackstrap
molasses is most preferably 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, NZ-amine, meat
extract, yeast extract, corn steep liquor, casein hydrolyzate,
fish meal or its digested product, defatted soybean or its
digested product and chrysalis hydrolyzate are available.
As the inorganic materials, potassium
dihydrogenphosphate, dipotassium hydrogenphosphate, ammonium
sulfate, ammonium chloride, magnesium sulfate, sodium chloride,
ferrous sulfate, manganese sulfate and calcium carbonate may be
used. Vitamins and amino acids required for the growth of
microorganisms may not be added, provided that they are supplied
with other components mentioned above.
Culturing is carried out under aerobic conditions with
shaking or aeration-agitation. Culturing temperature is
preferably 20 to 40C. The pH of the medium during culturing is
maintained around neutral. Culturing is continued until a
considerable amount of L-isoleucine is accumulated, generally
for 1 to 5 days.
After completion of the culturing, cells are removed
and L-isoleucine is recovered from the culture broth by
conventional manners such as treatment with active carbon or ion
exchange resin.
Higher amount of L-isoleucine is obtained using the
strains of the genus CorYnebacterium and Brevibacterium
harboring pEthrl compared with the strains which does not
contain pEthrl.
The usefulness of the present invention lies in the
fact that, in an expressible form, introduction of the
recombinant DNA constructed wlth a gene involved in the
biosynthesis of threonine and a vector DNA of the genus
Corvnebacterium or Brevibacterium into a microorganism belonging
to the genus Corvnebacterium or Brevibacterium can give or
improve L-isoleucine productivity. The example of using
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Escherichia coli threonine operon is provided in the present
specification, but the purpose of the present invention is
accomplished using a gene involved in the biosynthesis of
threonine derived from other organisms. Therefore, the gene
involved in the biosynthesis of threonine is not restricted to
the threonine operon of Escherichia coli described in the
present specification. Further, the vector plasmid merely
provides its autonomously replicating ability to stably maintain
the recombined gene involved in the biosynthesis of threonine.
Therefore, plasmids autonomously replicable in the genus
CorYnebacterium or Brevibacterium other than pCGll described in
the present specification are used in the present invention.
In spite of many common microbiological properties,
microorganisms with high glutamic acid productivity (so-called
glutamic acid-producing microorganisms) are classified to
various species and even genera such as Corynebacterium and
Brevibacterium probably because of their industrial importance.
However, it has been pointed out that these microorganisms
should be classified as one species because they have homology
in the amino acids in the cell walls and the CC content of DNA.
Recently, it has been reported that these microorganisms have
more than 70% homology in DNA-DNA hybridization, indicating that
the microorganisms are very closely related [refer to Komastsu,
Y.: Report of the Fermentative Research Institute, No. 55, 1
(1980), and Suzuki, K., Kaneko, T., and Komagata, K.: Int. 3.
Sy~t. Bacteriol., 31, 131 ~1981)].
In the present specification, a case where a gene
involved in the biosynthesis of threonine is introduced into
CorYnebacterium qlutamicum R40 and Brevibacterium flavum ATCC
14067 and where the improvement in L-isoleucin production
depends on the expression of the gene is given. Considering the
above-mentioned very close relationship of glutamic acid-
producing microorganisms, it is readily assumed that the present
invention is applicable to all of the glutamic acid-producing
microorganisms. The effect of the present invention depends on
whether the recombinant DNA autonomously replicates in the
glutamic acid-producing microorganism and whether the gene
involved n the biosynthesis of threonine is expressed, and 50
11 - 122~039
slight difference of such DNA homology between glutamic acid-
producing microorganisms are negligible. That the glutamic
acid-producing microorganims have the common function to allow
replication of plasmids and expression of genes is apparent from
the fact that plasmid pCG4 which is isolated from
CorYnebacterium qlutamicum 225-250 (Japanese Published
Unexamined Patent Application No. 183799/82) and which has a
spectinomycin and/or streptomycin resistance gene can be
generally replicated and expressed in glutamic acid-producing
microorganisms such as strains of the genera Corynebacterium and
Brevibacterium tJapanese Published Unexamined Patent Application
No. 186492/82). Therefore, all of the glutamic acid-producing
microorganisms including the genera CorYnebacterium and
Brevibacterium such as Corynebacterium qlutamicum K40 and
Brevibacterium flavum ATCC 14067 fall within the scope for
application of the present invention to the end that L-
isoleucine-producing microorganisms are prepared by introducing
a recombinant DNA containing the gene involved in the
biosynthesis of threonine.
Example 1
(1) Cloning of a DNA fragment containing Escherichia coli
threonine operon:
Cloning was carried out using a host-vector system of
Escherichia coli. Plasmid pGA22, used as a vector, was isolated
from cultured cells of a derivative of Escherichia coli K12
carrying the present plasmid according to the method of An [An,
G. et al.: J. Bacteriol., 140, 400 (1979)].
The chromosomal DNA used as a donor DNA was isolated
from cultured cells of Escherichia coli K12 Hfr (ATCC 23740) by
the phenol-extraction method of Smith, M.G.: Methods in
Enzymology, 12, part A, 545 (1967). Then, 0.4 unit of HindIII
(product of Takara Shuzo Co., 16 units/~) was added to 60 ~Q of
a HindIII reaction solution (pH 7.5) consisting of 10 mM Tris-
HCl, 7 mM MgC12 and 60 mM NaCl and containing 4 ~g of pGA22
plasmid DNA. The mixture was allowed to react at 37C for 30
minutes and heated at 65C for 10 minutes to stop the reaction.
pGA22 plasmid DNA was digested with HindIII under the same
- 12 ~ 1 Z 2 8 03 9
conditions and subjected to agarose gel electrophoresis. It was
confirmed that one of the two HindIII cleavage sites present in
pGA22 was cleaved.
Separately, 4 units of HindIII was added to 140 ~Q of
the HindIII reaction solution containing 8 ~g of the chromosomal
DNA. The mixture was allowed to react at 37C for 60 minutes
and heated at 65C for 10 minutes to stop the reaction.
Then, 40 ~Q of the T4 ligase buffer solution (pH 7.6)
consisting of 660 mM Tris (hydroxymethyl) aminomethane (referred
to as "Tris" hereinafter), 66 mM MgC12 and 100 mM
dithiothreitol, 40 ~Q of ATP (5 mM, 0.3 ~Q of T4 ligase (product
of Takara Shuzo Co., 1 unit~Q) and 120 ~Q of H2O were added to
a mixture of the above digests and reaction was carried out at
12C for 16 hours. The reaction mixture was extracted twice
with 400 ~Q of phenol saturated with TES buffer solution (pH
8.0) consisting of 0.03M Tris, 0.005M EDTA and 0.05M NaCl and
subjected to dialysis against TES buffer solution to remove
phenol.
The ligase reaction mixture was used to transform
Escherichia coli GT-3 (J. Bacteriol. 117, 133-143, 1974) which
is a derivative of Escherichia coli K12 and requires homoserine
and diaminopimelic acid. Competent cells of the GT-3 strain
were prepared according to the method of Dagert, M. et al.,
Gene, 6, 23 (1979). That is, the strain was inoculated in 50 mQ
of L-medium (pH 7.2) consisting of 10 g/Q Bacto-tryptone, 5 g/Q
yeast extract, 1 g/Q glucose and 5 g/Q sodium chloride and
containing 100 ~g/mQ diaminopimelic acid and cultured at 37C to
an optical density ~OD) value at 660 nm of 0.5. The culture was
cooled with ice water for 10 minutes and cells were recovered by
centrifugation. The cells were suspended in 20 mQ of cooled
O.LM calcium chloride. The suspension was allowed to stand at
0C foc 20 minutes and then centrifuged to recover the cells.
The cells were suspended in 0.S mQ of 0.lM calcium chloride and
allowed to stand at 0C for 18 hours.
Then 200 ~Q of the ligase reaction mixture mentioned
above was added to 400 ~Q of the cell suspension treated with
calcium chloride. The mixture was allowed to stand at 0C for
10 minutes and then heated at 37C for 5 minutes. Thereafter,
- 13 - ~X28039
9 mQ of the L-medium was added and the mixture was incubated
with shaking at 37C for 2 hours. Cells were recovered by
centrifugation and washed with a physiological saline solution
twice. The cells were spread on Mg minimal agar medium (pH 7.2)
consisting of 2 g/Q glucose, 1 g/Q NH4Cl, 6 g/Q Na2HPO4, 3 g/Q
KH2PO4, 0-1 g/Q MgSO4 7H20, 15 mg/Q CaC12-2H2O, 4 mg/Q thiamine
hydrochloride and 15 g/Q agar and containing 12.5 ~g/mQ
kanamycin. Culturing was carried out at 37C for 3 days. Only
one colony was formed and the cells from this colony could also
grow on an L-agar medium containing 25 ~g/mQ ampicillin,
25 ~g/mQ chloramphenicol or 25 ~g/mQ ~anamycin, which is a
selection marker of pGA22.
A plasmid DNA was isolated from cultured cells of the
transformant by the same method as in the isolation of pGA22.
The plasmid DNA was digested with restriction endonucleases and
analyzed by agarose gel electrophoresis. The plasmid DNA had
the structure illustrated as pGH2 in Fig. 1. Since the DNA
fragment inserted in pGA22 had the same cleavage sites for
restriction endonucleases as the cloned DNA fragment containing
Escherichia coli operon (Cossart, P. et al.: Molec. Gen.
Genet., 175, 39, 1979), it is confirmed that pGH2 had the
threonine operon.
(2) In vitro recombination of pCGll and pGH2
CorYnebacterium qlutamicum LA103/pCGll harboring pCGll
~ATCC 39022) were cultured to an OD value of 0.8 in a 400 mA of
NB medium (pH 7.2) consisting of 20 g/Q powdered bouillon and
5 g/Q yeast extract. Cells were harvested from the culture
broth, and washed with TES buffer solution. The cells were
suspended in 10 mQ of a lysozyme solution (pH 8.0) consisting of
25% sucrose, 0.~ NaCl, 0.05M Tris and 0.8 mg/mQ lysozyme, and
incubated at 37C for 4 hours. Then, 2.4 mQ of 5M NaCl, 0.6 mQ
of 0.5M EDTA (pH 8.5) and 4.4 mQ of a solution consisting of 4
sodium lauryl sulfate and 0.7M NaCl were added successively.
The mixture was stirred slowly and allowed to stand on an ice
water bath for 15 hours. The whole lysate was centrifuged at
4C under 69,400 x g for 60 minutes. The supernatant fluid was
recovered and 10~ (by weight) polyethyleneglycol (PEG) 6,000
- 14 - 1228039
(product of Nakarai Kagaku Yakuhin Co.) was added. The mixture
was stirred slowly to dissolve completely and then kept on an
ice water bath. After 10 hours, the mixture was subjected to
centrifugation at 1,500 x g for 10 minutes to recover a pellet.
The pellet was redissolved gently in 5 mQ of TES buffer and
2.0 mQ of 1.5 mg/mQ ethidium bromide was added.
Then, cesium chloride was added to adjust the density
of the mixture to 1.580. The solution was centrifuged at 18C
at 105,000 x g for 48 hours. After the density gradient
centrifugation, a convalently closed circular DNA was detected
under W irradiation as a high density band located in the lower
part of the centrifugation tube. The band was taken out from
the side of the tube with an injector to obtain a fraction
containing pCGll DNA. To remove ethidium bromide, the fraction
was treated five times with an equal amount of cesium chloride
saturated isopropyl alcohol solution consisting of 90% by volume
isopropyl alcohol and 10~ TES buffer solution. The residue was
dialysed against TES buffer solution. Thus, a plasmid pCG11,
was obtained.
The plasmid pCGll was digested with restriction
endonucleases and analysed by agarose gel electrophoresis to
determine the molecular weight and clea~age sites for
restriction endonucleases. Fig. 1 illustrates the cleavage map
for various restriction endonucleases of plasmid pCGll.
2 units of BglII (product of Takara Shuzo Co., 6
units/~Q) was added to 100 yQ of the BglII reaction buffer
solution (pH 7.5) consisting of 10 mM Tris-HCl, 7 mM MgC12,
60 mM NaCl and 7 mM 2-mercaptoethanol and containing 2 ~g of
pCGll plasmid DNA. The mixture was allowed to react at 37C for
60 minutes. Separately, 2 units of BamHI (product of Takara
Shuzo Co., 6 units/yQ) was added to 100 ~Q of BamHI reaction
buffer solution (pH 8.0) consisting of 10 mM Tris-MCl, 7 mM
MgC12, 100 mM NaCl, 2 mM mercaptoethanol and 0.01% bovine serum
albumin and containing 2 ~g of pGH2 plasmid DNA. The mixture
was allowed to react at 37C for 60 minutes. Both digests were
heated at 65C for 10 minutes, and mixed. Then, 40 ~Q of T4
ligase buffer solution, 40 yQ of ATP (5 mM), 0.2 yQ of T4 ligase
and 120 ~Q of H2O were added to the whole mixture of both the
1;~28039
digests. Reaction was carried out at 12C for 16 hours. The
reaction mixture was extracted twice with 400 ~Q of phenol
saturated with TES buffer solution and subjected to dialysis
against TES buffer solution to remove phenol.
(3) Recovery of plasmid pEthrl
Protoplasts of CorYnebacterium qlutamicum LA201 which
requires homoserine and leucine were used for transformation. A
seed culture of Corynebacterium qlutamicum LA201 was inoculated
in the NB medium and cultured with shaking at 30C. Cultured
cells were collected at an OD value of 0.6 and suspended in an
RCGP medium (pH 7.6) containing 1 mg/mQ lysozyme at a
concentration of about 109 cells/mQ. The RCGP medium consists
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 MgC12 6H2O, 10 mg/Q
FeSO4 7H2O, 2 mg/Q MnSO4 (4-6) H2O, 0.9 mg/Q ZnSO4 7H2O,
0.04 mg/Q (NH4)6Mo7O24 4H2O, 30 ~g/Q biotin, 2 mg/Q thiamine
hydrochloride, 135 g/Q sodium succinate and 30 g/Q
polyvinylpyrrolidone of a molecular weight of 10,000. The
suspension was put into an L-tube and allowed to react with
gentle shaking at 30C for 5 hours to make protoplasts.
Then, 0.5 mQ of the protoplast suspension was
transferred into a small tube and centrifuged at 2,500 x g for 5
minutes to obtain pellets. The pellets was resuspended in 1 mQ
of a TSMC buffer solution (pH 7.5) consisting of 10 mM magnesium
chloride, 30 mM calcium chloride, 50 mM Tris and 400 mM sucrose
and centrifuged. The protoplasts were resuspended in 0.1 mQ of
the TSMC buffer solution. Then, 100 ~Q of a mixture of a two-
fold concentrated TSMC buffer solution and the above-described
DNA mixture treated with ligase (1:1) was added to the
suspension and 0.8 mQ of a TSMC buffer solution containing 20%
PEG 6,000 was added. After 3 minutes, 2 mQ of the RCGP medium
(pH 7.2) was a~ded and the mixture was centrifuged at 2,500 x g
for 5 minutes. The supernatant fluid was removed and the
precipîtated protoplasts were suspended in 1 mQ of the RCGP
medium. The suspension was slowly shaken at 30C for 2 hours.
Then, 0.1 mQ of the suspension was spread on RCGP agar
medium (pH 7.2), the RCGP medium supplemented b~y 1.4% agar,
- 16 ~ 1 2 2 8 O~ 9
containing 400 ~g/mQ kanamycin, and culturing was carried out at
30C for 6 days to regenerate the transformants resistant to
kanamycin. Cells grown over the whole surface of the agar
medium were scraped, washed with physiological saline solution
and subjected to centrifugation. The cells were spread on a
minimal agar medium Ml (pH 7.2) consisting of 10 g/Q glucose,
l g/Q NH4H2PO4, 0.2 g/Q KCl, 0.2 g/Q ~gSO4 7H2O, 10 mg/Q FeSO4
7H20, 0.2 mg/Q MnSO4 (4-6)H20, 0.9 mg/Q ZnSO4 7H2O, 0.4 mg/Q
CuSO4-5H2O, 0.09 mg/Q Na2B4O7 10H2O, 0.04 mg/Q
(NH4) 6Mo7O24 4H2O, 50 ~g/Q biotin, 2.5 mg/Q p-amino-benzoic
acid, l mg/Q thiamine hydrochloride and 16 g/Q agar and
containing 50 ~g/mQ leucine. Culturing was carried out at 30C
for 3 days. Among colonies developed, those able to grow on the
NB agar medium containing 20 ~g/mQ kanamycin and 100 ~g/mQ
15 spectinomycin were obtained. Three strains selected at random
were grown in 400 m~ of the NB medium to an OD value of about
0.8. Cells were recovered and the plasmids were isolated from
the cells by ethidium bromide-cesium chloride density gradient
centrifugation described in Example l (2) whereby 40 to 55 ~g of
20 plasmid DNA were recovered from each strain.
These plasmid DNAs were digested with restriction
endonucleases and analyzed by agarose gel electrophoresis to
determine the molecular weights and cleavage sites for PstI,
EcoRI and XhoI. The plasmid obtained from one strain is named
pEthrl and the structure is illustrated in Fig. l. It was
confirmed that pEthrl has the structure wherein a BamHI fragment
containing pGH2 threonine operon is inserted into pCGll at its
BglII site. One of the remaining strains has the same plasmid
as pEthrl and the other has a plasmid wherein the BamHI fragment
containing pGH2 threonine operon is combined at the opposite
orientation.
Corynebacterium qlutamicum LA201 strain was again
transformed with these plasmid DNAs as mentioned above. As a
result, strains which do not require homoserine were obtained at
35 high frequency, about 10-3 cell/regenerated cell. All of them
are endowed with the phenotypes of the resistance to kanamycin
and spectinomycin and have the same plasmid as the donor plasmid
characterized by the cleavage pattern for various restriction
endonucleases.
- 17 - 12 2 8039
(4) Production of L-isoleucine by the pEthrl-carrying
strain:
Corynebacterium qlutamicum ~40 and Brevibacterium
flavum ATCC 14067 were transformed with pEthrl. The thus
obtained transformants were cultured with shaking in NB medium
at 30C for 16 hours, and 0.1 mQ of the seed culture was
inoculated into 10 mQ of SSM medium (pH 7.2) comprising 10 g/Q
glucose, 4 g/Q NH4Cl, 2 g/Q urea, 1 g/Q yeast extract, 1 g/Q
KH2PO4~ 3 g/Q K2HPO4, 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
5H20, 0.09 mg/Q Na2B4O7-10H2O~ 0.04 mg/Q (NH4)6Mo7O24-4H2O,
30 ug/Q biotin and 1 mg/Q thiamine hydrochloride in an L-tube.
Culturing was carried out at 30C in a Monod-type culture bath,
and penicillin G was added at an OD value of 0.15 to a
concentration of 0.5 unit/mQ. Culturing was continued to an OD
value of about 0.6. Cells were harvested and suspended in 2 mQ
of RCGP medium (pH 7.6) containing 1 mg/mQ lysozyme. The
suspension was put in an L-tube and stirred slowly at 30C for
14 hours to obtain protoplasts.
Then, 1 mQ of the protoplast suspension was put in a
small test tube and centrifuged at 2,500 x g for 15 minutes.
The protoplasts were resuspended in 1 mQ of TSMC buffer and
centrifuged at 2,500 x g. 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-fold
concentrated TSMC buffer and the pEthrl DNA described above was
added to the protoplast suspension. Transformation was carried
out using PEG 6,000 by the same method described in Example 1
~3) for expression of the desired gene. Then, ~ Q of the
mixture was spread on RCGP agar medium containing 400 ~g/mQ
spectinomycin and incubated at 30C for 10 days. From the
colonies developed, the transformants which grow on N8 agar
medium containing 100 ug/mQ spectinomycin and 20 ug/mQ kanamycin
were obtained. The strains resistant to spectino~ycin and
kanamycin were cultured with shaking in 400 mQ of SSM medium,
and penicillin G was added at an OD value of 0.15 to a
concentration of 0.5 unit/mQ. Culturing was continued to an CD
value of 0.55, and cells were harvested. From the cells,
- 18 - 1228039
plasmids were isolated by the same method as the isolation
method of pCGll in Example 1 (2). These plasmids were digested
with restriction endonucleases and analyzed by agarose gel
electrophoresis. The analysis showed that some of the plasmids
have the same physical structure as pEthrl characterized by the
cleavage pattern for various restriction endonucleases. Such
transformants are CorYnebacterium qlutamicum K41 (FERM P-7161)
~FERM BP-456) and Brevibacterium flavum K42 (FERM BP-355).
Corynebacterium qlutamicum K40, Brevibacterium flavum
ATCC 14067 and their pEthrl-carrying strains were tested for L-
isoleucine production as follows.
The strain was cultured in NB medium at 30C ror 16
hours and 0.5 mQ of the culture liquor was inoculated in a
production medium adjusted to pH 7.2 consisting of 100 g/Q
glucose, 20 g/Q (NH4)2SO4, 0.5 g/Q KH2PO4, 0.5 g/Q K2HPO4, 1 g/Q
MgSO4 7H20, la mg/Q FeSO4 7H2O, 10 mg/Q MnSO4 (4-6)H2O,
100 ~g/Q biotin and 30 g/Q CaCO3. Culturing was carried out
with shaking at 30C for 72 hours. The culture filtrate was
subjected to paper chromatography, color reaction with ninhydrin
and the amount of L-isoleucine formed was determined
colorimetrically. The results are shown in Table 1.
Table 1
Amount of L-isoleucine
Strain (mq/mQ)
CorYnebacteriu~ qlutamicum K40 1.2
CorYnebacterlum qlutamicum K41 2.7
Brevibacterium flavum ATCC 14067 0
Brevibacterium flavum K42 0.8
