US008062869B2

(12) United States Patent (10) Patent No.: US 8,062,869 B2

Nakanishi et al. (45) Date of Patent: *Nov. 22, 2011

(54) METHOD FOR PRODUCING L-LYSINE 2002/0160461 A1 10, 2002 Nakai et al.
2003.0049804 A1 3/2003 Pompeius et al.
O O fly. 2004/O126854 A1 7/2004 Hanke et al.
(75) Inventors: SG N.E. E. 2004/0265956 All 12/2004 Takikawa et al.
oshimi Kikuchi, Kawasaki (JP), 2006/00 19367 A1 1/2006 Umezawa et al.
Junichiro Kojima, Kawasaki (JP); 2006/0205043 A1 9/2006 Tsujimoto et al.
Tomoko Suzuki, Kawasaki (JP); 2007. O184525 A1 8, 2007 Date et al.
Yasushi Nishimura, Kawasaki (JP): 2007,0254345 A1 11/2007 Fukui et al.
s s 2009/01489.15 A1 6/2009 Van Dien et al.
Hiroyuki Kojima, Kawasaki (JP)
FOREIGN PATENT DOCUMENTS
(73) Assignee: Ajinomoto Co., Inc., Tokyo (JP) DE 38 23 451 1, 1990
EP O 318663 6, 1989
(*) Notice: Subject to any disclaimer, the term of this EP O 723 O11 T 1996
patent is extended or adjusted under 35 EP O 733 710 9, 1996
U.S.C. 154(b) by 0 days. EP O 733 712 9, 1996
WO 95/16042 6, 1995
This patent is Subject to a terminal dis OTHER PUBLICATIONS
claimer.
Chatterjee, M., et al., “Microbial Production of L-lysine: A Review.”
(21) Appl. No.: 12/721,813 Hind. Antibiot. Bull. 1997, vol.39, pp. 20-49.
Jetten, M. S. M., et al., “Recent Advances in the Physiology and
(22) Filed: Mar 11, 2010 Genetics of Amino Acid-Producing Bacteria.” Critical Rev.
Biotechnol. 1995; 15(1): 73-103.
(65) Prior Publication Data Vauterni, M., et al., “Functional rescue of a bacterial dapA auxotroph
US 2010/O173368A1 Jul. 8, 2010 with a plant cDNA library selects for mutant clones encoding a
feedback-insensitive dihydrodipicolinate synthase.” The Plant Jour
Related U.S. Application Data nal 2000:21(3):239-248.
English translation of Third Party Letter (Degussa) to European
(63) Continuation of application No. 10/149.450, filed as Patent Office concerning the patentability of the invention, dated Jul.
application No. PCT/JP00/00298 on Jan. 21, 2000, 26, 2007, pp. 1-7.
now Pat. No. 7,723,081. Office Communication from EP Patent App. No. 00900872.3 (Sep.
17, 2007).
Supplementary European Search Report for EP Patent App. No.
(51) Int. C.
00900872.3 (Jun. 23, 2004).
CI2PI3/08 (2006.01)
(52) U.S. C. .......... 435/115; 435/252:435/325; 435/7.1 Primary Examiner — Hope Robinson
(58) Field of Classification Search ........................ None (74) Attorney, Agent, or Firm — Shelly Guest Cermak;
See application file for complete search history. Cermak Nakajima LLP
(56) References Cited (57) ABSTRACT
U.S. PATENT DOCUMENTS An Escherichia bacterium having dihydrodipicolinate Syn
4,346,170 A 8, 1982 Sano et al.
thase and aspartokinase, both of which are desensitized to
5,827,698 A 10, 1998 Kikuchi et al. feedback inhibition by L-lysine. The intracellular activity of
5,830,716 A 11/1998 Kojima et al. dihydrodipicolinate reductase in this bacterium can also be
5,876,983 A 3/1999 Sugimoto et al. enhanced. Furthermore, a diaminopimelate dehydrogenase
5,919,694 A 7/1999 Sugimoto et al. gene can be introduced into this bacterium, or intracellular
5,932,453 A 8, 1999 Kikuchi et al. activities of tetrahydrodipicolinate Succinylase and Succinyl
6,040,160 A 3/2000 Kojima et al. diaminopimelate deacylase can be enhanced. Finally, the
6,200,785 B1 3, 2001 Kreutzer et al.
7,090,998 B2 8/2006 Ishikawa et al. intracellular activities of aspartate-semialdehyde dehydroge
7,097.999 B2 8/2006 Tsujimoto et al. nase orphosphoenolpyruvate carboxylase can be enhanced in
7,252,972 B2 8, 2007 Kikuchi et al. this bacterium. The bacterium can be cultured in a suitable
7.306,933 B2 12/2007 Van Dien et al. medium to produce and accumulate L-lysine in culture, and
7,399.617 B1 7/2008 Livshits et al.
7,524,656 B2 4/2009 Livshits et al. the L-lysine is collected from the culture.
7,527,950 B2 5, 2009 Livshits et al.
2002fO110876 A1 8/2002 Miyata et al. 4 Claims, 20 Drawing Sheets
U.S. Patent Nov. 22, 2011 Sheet 1 of 20 US 8,062,869 B2

BahnhI&Dracut EcoRI&Kpm cut

T4 polymerase treatment T4 polymerase treatment

Ban.H.EcoR}

(Dra/Kpn))
U.S. Patent Nov. 22, 2011 Sheet 2 of 20 US 8,062,869 B2

EcoR
Banfeco
Not
ySC-80
(Sima Ban.H) - ra
Dra eles, Small/Ase)
(8 Saci
pLLC+80
Acc 3.7kb

&s
Acc (Dral/Kipni) Še
Sapi Pst
st digestion Drai&Saci cui
T4 polymerase treatment T4 polymerase treatment

EcoR
(Ban.Hi? EcoR)

(Dra/Kpni)
Psi Dral
Acc Smal/Ban 3)

SNY (Snatasel)
Sap) Psil/Sach)
U.S. Patent Nov. 22, 2011 Sheet 3 of 20 US 8,062,869 B2

Spel P
(Small Ase2sW Snel/Dra)
Fu

Ps (Dra.
Acc (Sinai Banl)

Sapi (PsitSad)
EcoRI cat Pyu cut
T4 polymerase treatment

Smeliaset)
Sna/Drs) dap (EcoR1/Pvt.)
(Earl/EcoR)

tPst IOrs)
(Sinai/Barna)

Easts (SmalAse
Acc sa Psil/Sac)
U.S. Patent Nov. 22, 2011 Sheet 4 of 20 US 8,062,869 B2

StatDrai daps
(EcoRIP: at

Sir Ava
Psora O - Fw

s PsFSc)

Sap cut
T4 polymerase treatment Pty cut

Singise
(SmallRandape
EcoRIPvil W

(Orli Kpril)
sire

Staff
A.

Srialise
Acc Psi Sad

Sup/Pvu}
(Spiff) Snew
Sanaecol2

Fig. 4
U.S. Patent Nov. 22, 2011 Sheet 5 of 20 US 8,062,869 B2

is “ . . Small Ase)
c Sai (PsAFSed)

Sap cut Phull cut

T4 polymerase treatment

(Smalaise) (EcoRiply
(Small Drai) a BarthER)
EcoR/F'i)

Now

Psafra.)
Acc Saa/FanH)
S.
5
Acc s

(Similasa)
(PSAISsc)
Sapid
36 (Snai/Ecool09)
Sira Sat)
4Sinatigli Fix}
Snaithful

Fig. 5
U.S. Patent Nov. 22, 2011 Sheet 6 of 20 US 8,062,869 B2

PvE Snai/Sinal

"(Sua/Sca)
Pt

Fig. 6
U.S. Patent Nov. 22, 2011 Sheet 7 of 20 US 8,062,869 B2
U.S. Patent Nov. 22, 2011 Sheet 8 of 20 US 8,062,869 B2

E. coli chromosome DNA

essamarm
Synthetic primers

PCR reaction

BamHI&HindIII digestion
pntA pntb
Hindi BamH&HindIII digestion

DNA ligase

Fig. 8
U.S. Patent Nov. 22, 2011 Sheet 9 of 20 US 8,062,869 B2

5'-reATCAGCGAAACACTTFA-3 (SEQID NO. 5)

5'-cAGCAAACTATGATGAGAA-3 ' (SEQID NO.: 6)
pMW 118 Primers for E. coli asp? amplification
PCR
Blunt-ending

pMW 8:aspA lac pro.

Sac

Hird
Xibal
Ban H aspa

Fig. 9
U.S. Patent Nov. 22, 2011 Sheet 10 of 20 US 8,062,869 B2

Hird
Xibal
Bath

ppc
Ear
Xiba
Blunt-ending lac pro,
al
Hic
Sph N
Xa---
- Kipni
Bamh-f
Hid

Kpn
Sph
Blunt-ending

Hird

Hindi
Ban

Fig. 10
U.S. Patent Nov. 22, 2011 Sheet 11 of 20 US 8,062,869 B2

Hird
Xa
Barn

ppc
Earth
Xtra
Blunt-ending
Hind

Sinal
Fird

had pro,
pMW18 N Sac
Sna
Hid -

Bay
Earl

Fig. 11
U.S. Patent Nov. 22, 2011 Sheet 12 of 20 US 8,062,869 B2

Sail
Sinal
Hid
Xa
Earl

PC

Xbal
Blunt-ending
Hinds

find
Xibal
Earth
Sac
Blunt-ending
Hindl

Sac
Sria i

Baml Earl
U.S. Patent Nov. 22, 2011 Sheet 13 of 20 US 8,062,869 B2

lac pro,
pMW118 N Sac
Sina

Earl
Earl

Hird
Blunt-ending
Dephosphorylation pMW i 18

Air

Kpn
Sph
Blunt-ending
U.S. Patent Nov. 22, 2011 Sheet 14 of 20 US 8,062,869 B2

fact pro.
find --
Xia m Sac
Atari
-Sira
Bah

Aa
Earth

Sina
Xiba Hird
Blunt-ending
Hird

id

-Bath

Bat- Barr

Fig. 14
U.S. Patent Nov. 22, 2011 Sheet 15 of 20 US 8,062,869 B2

Bari Bantil

Hird
Blunt-ending pMW2f 8
Sna ECOR
Dephosphorylation
Blunt-ending

Hird
Xtal pMW218 lac pro.
Bari
Siria Sa
Sac

Ea

Ban
EH Earl

Fig. 15
U.S. Patent Nov. 22, 2011 Sheet 16 of 20 US 8,062,869 B2

pMW i8

find ac
Sph Rpni
Bay

Hird pSTV29
Pst
Kon Blunt-ending
Blunt-ending
Ban
Ban.H

d
Soft

Fig. 16
U.S. Patent Nov. 22, 2011 Sheet 17 of 20 US 8,062,869 B2

Hird
Bal MW28 fact pro.
Earth p Na
Smal
Sa:
a pro,

HindIII
Sph I

Hid
Earl
ar Barth

Sph
Sao Blunt-ending
Biunt-ending
Xiba
Xta

pMW218
Xiba

Sal
ppc

Fig. 17
U.S. Patent Nov. 22, 2011 Sheet 18 of 20 US 8,062,869 B2

PCR amplification of DNA fragment containing

pucepn promoter site of Tet resistance gene in pRR322

ac pro.
EcoR

Hid
Hid
Earth
Fon teoro

Fig. 18
U.S. Patent Nov. 22, 2011 Sheet 19 of 20 US 8,062,869 B2

facpio.
EcoR

Hid
Hird I
Earl
April teoro PCR-amplified fragment of Brew dapA

pUC18

Xibal
Ban

Hird

Apni Hird

Fig. 19
U.S. Patent Nov. 22, 2011 Sheet 20 of 20 US 8,062,869 B2

Brev, dapA
tepo,
EcoR
EcoRI
Spe Xta
Blunt-ending Blunt-ending

(Nhe I/EcoRI)
pCABD (B)
Brew.dapA
- (Xba I/Spe I)

Fig. 20
 US 8,062,869 B2
1. 2
METHOD FOR PRODUCING L-LYSINE yield by several percent may provide significant industrial
value, and thus improving the yield, even if only a small
This application is a continuation under 35 U.S.C. S 120 of amount, is desirable.
U.S. patent application Ser. No. 10/149.450, filed on Jun. 27.
2002 now U.S. Pat. No. 7,723,081, which was a national SUMMARY OF THE INVENTION
phase filing under 35 U.S.C. S371 of PCT Patent Application
No. PCT/JP00/00298, filed Jan. 21, 2000, which are incorpo It is an aspect of the present invention is to provide an
rated in their entireties by reference. The Sequence Listing in improved method for producing L-lysine by fermentation
electronic format filed herewith is also hereby incorporated compared with the conventional methods.
by reference in its entirety (File Name: 2010-03-11T US 10 The present inventors assiduously studied in order to
134C Seq List: File Size: 2 KB: Date Created: Mar. 11, achieve the aforementioned object. As a result, they found
that, if the activity of aspartate-semialdehyde dehydrogenase
2010). or phosphoenolpyruvate carboxylase was enhanced in an
BACKGROUND OF THE INVENTION Escherichia bacterium having a specified property, and if
15 activity or activities of a specific enzyme or enzymes were
1. Field of the Invention enhanced in addition to the aforementioned enzymes in Such
an Escherichia bacterium, the productivity of the bacterium
The present invention relates to the microbial industry. for L-lysine could be improved.
More specifically, the present invention relates to a method That is, the present invention provides:
for producing L-lysine by fermentation, and a microorganism in a first aspect, an Escherichia bacterium in which (1)
used in the production method. intracellular activities of dihydrodipicolinate synthase, aspar
2. Brief Description of the Related Art tokinase and dihydrodipicolinate reductase are enhanced, and
In the production of L-lysine by fermentation, Strains iso (2) intracellular activity of diaminopimelate dehydrogenase
lated from nature or artificial mutants thereof have conven or intracellular activities of tetrahydrodipicolinate succiny
tionally been used to improve the productivity. Many artificial 25 lase and Succinyl diaminopimelate deacylase is/are enhanced,
mutant strains that produce L-lysine are known, and include wherein intracellular activity of aspartate-semialdehyde
S-2-aminoethylcysteine (AEC) resistant strains and those dehydrogenase or phosphoenolpyruvate carboxylase is
that belong to the genus Brevibacterium, Corynebacterium, enhanced;
Bacillus or Escherichia. Furthermore, various techniques in a second aspect, an Escherichia bacterium in which (1)
have been disclosed for increasing the amino acid production, 30 intracellular activities of dihydrodipicolinate synthase, aspar
for example, the use of a transformant obtained by using tokinase and dihydrodipicolinate reductase are enhanced, and
recombinant DNA. (2) intracellular activity of diaminopimelate dehydrogenase
As for Escherichia bacteria, for example, methods for pro or intracellular activities of tetrahydrodipicolinate succiny
ducing L-lysine by using a strain in which dihydrodipicoli lase and Succinyl diaminopimelate deacylase is/are enhanced,
nate synthase (DDPS) activity is enhanced have been dis
35 wherein intracellular activity of phosphoenolpyruvate car
closed in Japanese Patent Application Laid-open (Kokai) No. boxylase and intracellular activity of nicotinamide adenine
dinucleotide transhydrogenase or aspartate-semialdehyde
56-18596, U.S. Pat. No. 4,346,170 and Applied Microbiol dehydrogenase are enhanced; and
ogy and Biotechnology, 15, pp. 227-331 (1982). Further in a third aspect, an Escherichia bacterium in which (1)
more, a method for producing L-lysine by using an Escheri 40 intracellular activities of dihydrodipicolinate synthase, aspar
chia bacterium into which DDPS derived from, and native to, tokinase and dihydrodipicolinate reductase are enhanced, and
a Corynebacterium bacterium has been introduced is dis (2) intracellular activity of diaminopimelate dehydrogenase
closed in Korean Patent Publication No. 92-8382. Further or intracellular activities of tetrahydrodipicolinate succiny
more, a method for producing L-lysine using a strain which lase and Succinyl diaminopimelate deacylase is/are enhanced,
has been transformed with a plasmid containing DNA that 45 wherein intracellular activities of phosphoenolpyruvate car
codes for dihydrodipicolinate synthase derived from, and boxylase and nicotinamide adenine dinucleotide transhydro
native to, an Escherichia bacterium which has a mutation to genase and intracellular activity of aspartate-semialdehyde
desensitize feedback inhibition by L-lysine, DNA that codes dehydrogenase or aspartase are enhanced (hereinafter, the
for aspartokinase which is desensitized to feedback inhibition bacteria according to the aforementioned three aspects are
by L-lysine, DNA that codes for dihydrodipicolinate reduc 50 also collectively referred to as the “bacteria of the present
tase, and DNA that codes for diaminopimelate dehydroge invention').
nase derived from, and native to, a coryneform bacterium is The intracellular activities of aspartate-semialdehyde
disclosed in International Publication No. WO95/16042. dehydrogenase and aspartase are enhanced in the bacteria.
As for Brevibacterium bacteria, International Publication Furthermore, aspartokinase, dihydrodipicolinate reduc
No. WO95/11985 discloses that L-lysine productivity can be 55 tase, tetrahydrodipicolinate Succinylase, Succinyl diami
improved by enhancing the activity of intracellular nicotina nopimelate deacylase, phosphoenolpyruvate carboxylase and
aspartate-semialdehyde dehydrogenase are each derived
mide adenine dinucleotide transhydrogenase. Furthermore, a from an Escherichia bacterium, nicotinamide adenine
method for producing L-lysine using a strain in which phos dinucleotide transhydrogenase and aspartase, if present, are
phoenolpyruvate carboxylase activity is solely enhanced and 60 each derived from an Escherichia bacterium, dihydrodipi
a method for producing L-lysine using a strain in which colinate synthase is derived from an Escherichia bacterium or
aspartate-semialdehyde dehydrogenase activity is solely a Brevibacterium bacterium, and diaminopimelate dehydro
enhanced are disclosed in Japanese Patent Application Laid genase is derived from a Brevibacterium bacterium.
open No. 60-87788 and Japanese Patent Publication The intracellular activity of an enzyme can be enhanced by
(Kokoku) No. 6-102028, respectively. 65 any of the following methods, or any combination thereof.
The industrial production of amino acids by fermentation (1) Introduction of a plasmid having a gene encoding the
is performed on a large scale. Therefore, even improving the enzyme.
 US 8,062,869 B2
3 4
(2) Increase of copy number of agene encoding the enzyme colinate synthase, aspartokinase and dihydrodipicolinate
on the chromosome. reductase are enhanced, (2) intracellular activity of diami
(3) Modification of a promoter sequence of a gene encod nopimelate dehydrogenase, or intracellular activities of tet
ing the enzyme on the chromosome. rahydrodipicolinate Succinylase and Succinyl diaminopime
Furthermore, the intracellular activities of dihydrodipicoli 5 late deacylase is/are enhanced, and (3) the intracellular
nate synthase and aspartokinase are enhanced by harboring activities of the following enzymes are further enhanced:
dihydrodipicolinate synthase and aspartokinase, both of (a) aspartate-semialdehyde dehydrogenase or phospho
which are desensitized to feedback inhibition by L-lysine, enolpyruvate carboxylase,
and the intracellular activity of diaminopimelate dehydroge (b) phosphoenolpyruvate carboxylase, and nicotinamide
nase is enhanced by introduction of a diaminopimelate dehy 10 adenine dinucleotide transhydrogenase (hereinafter also
drogenase gene. referred to as “transhydrogenase') or aspartate-semialdehyde
It is a further aspect of the present invention to provide a dehydrogenase, or
method for producing L-lysine, which comprises culturing (c) phosphoenolpyruvate carboxylase and transhydroge
any of the bacteria as described herein in a suitable medium to nase, and aspartate-semialdehyde dehydrogenase or aspar
produce and accumulate L-lysine in the culture, and collect 15 tase.
ing the L-lysine from the culture.
The bacteria of the present invention can include Escheri
BRIEF DESCRIPTION OF THE DRAWINGS chia bacteria in which intracellular activities of phospho
enolpyruvate carboxylase, transhydrogenase, aspartate
FIG. 1 shows a process of producing the plasmid RSF24P semialdehyde dehydrogenase and aspartase are further
from RSF1010, which contains dap A*24. enhanced.
FIG. 2 shows a process of producing the plasmid RSFD80 The bacteria of the present invention can include strains of
containing dap A*24 and lysC*80. Escherichia coli (E. coli).
FIG. 3 shows a process of producing the plasmid pCAB1 The expression “intracellular activity is enhanced’ can
containing dap A*24, lysC*80, and dapB. mean that the intracellular enzymatic activity is increased as
FIG. 4 shows a process of producing the plasmid pCABD2 25 compared with a wild-type strain (for example, an E. coli
containing dap A*24, lysC*80, dapB, and ddh. W3110 strain), or a parent strain (a strain with no enhanced
FIG. 5 shows a process of producing the plasmid intracellular activities), and also can mean that a bacterium
pCABDE1 containing dap A*24, lysC*80, dapB, dapD, and has an enzymatic activity not typically present in the wild
daph. type or parent strain. The methods for measuring the activities
FIG. 6 shows the structure of plasmid pppc containing ppc. 30 of the aforementioned enzymes are known, and the increase
FIG. 7 shows the structure of plasmid pasd containing asd. in their intracellular activities can be easily confirmed by
FIG. 8 shows a process of producing the plasmid pMW: those skilled in the art.
THY containing pntAB (pntA and pntB). Methods for enhancing the intracellular activities include
FIG. 9 shows a process of producing the plasmid the following, but are not limited to these.
pMW 118:aspA containing asp.A. 35 Specifically, the following are methods for increasing the
FIG. 10 shows a process of producing the plasmid ppcd expression of enzymes.
containing ppc and asd. (1) Introduction of Plasmid Containing Gene of Enzyme
FIG. 11 shows a process of producing the plasmid pPTS As the plasmid, a vector that is autonomously replicable in
containing ppc and pntAB. an Escherichia bacterium cell can be used. It can be intro
FIG. 12 shows a process of producing the plasmid pAPW 40 duced by a known method. That is, the gene of interest can be
containing ppc and asp.A. inserted into the vector, and the vector can be used to trans
FIG. 13 shows a process of producing the plasmid pPTd form an Escherichia bacterium. This vector can be a multi
containing ppc, pntAB, and asd. copy type plasmid.
FIG. 14 shows a process of producing the plasmid paPT The genes can be on the same plasmid or on different
containing ppc, pntAB, and asp.A. 45 plasmids. Some of the genes can be on the same plasmid.
FIG. 15 shows a process of producing the plasmid pAPTK When two or more kinds of plasmids are used, plasmids can
containing ppc, pntAB, and asp.A. be used that have stable partitioning systems so that they are
FIG. 16 shows a process of producing the plasmid pSd able to stably co-exist in a cell. The order in which the genes
containing asd. are introduced is not particularly limited.
FIG. 17 shows a process of producing the plasmid pKD 50 (2) Increasing the Copy Number of the Gene Encoding the
containing ppc, pntAB, asp.A, and asd. Enzyme on the Cellular Chromosome
FIG. 18 shows a process of producing the plasmid pluTES The copy number can be increased by amplifying the DNA
containing tet'. on the chromosomal DNA using Mu phage or the like.
FIG. 19 shows a process of producing the plasmid DNA on the chromosomal DNA can be that which is native
pUEBL3 containing tet', and dap A from Brevibacterium 55 to Escherichia bacteria, or DNA which is incorporated into
lactofermentum, which is located downstream from tet'. the chromosome of the host microorganism by a method
FIG. 20 shows a process of producing the plasmid pCABD using transduction, transposon (Berg, D. E. and Berg C. M.,
(B) containing dapA from Brevibacterium lactofermentum Bio/Technol. 1, 417 (1983)), Mu phage (Japanese Patent
(Brev. dap A), lysC, dapB, and ddh. Application Laid-open No. 2-109985), or homologous
60 recombination (Experiments in Molecular Genetics and Cold
DESCRIPTION OF THE PREFERRED Spring Harbor Lab. (1972)).
EMBODIMENTS (3) Modification of the Promoter Sequence of the Gene
Encoding the Enzyme
<1> Bacteria of the Present Invention A promoter sequence can be modified to increase tran
65 Scription of a gene and thereby increase the amount which is
The bacteria of the present invention include Escherichia expressed. For example, the promoter can be enhanced by
bacteria in which (1) intracellular activities of dihydrodipi introducing a mutation into the promoter to increase the tran
 US 8,062,869 B2
5 6
Scription amount of a gene that is located downstream from A Succinyl diaminopimelate deacylase gene (dapE) can be
the promoter. Other methods include introducing a promoter obtained by amplification by PCR using the E. coli chromo
that is able to function in Escherichia bacteria, Such as lac, trp, somal DNA as a template and two kinds of oligonucleotide
tac, trc, and PL. Alternatively, gene transcription can be primers (for example, SEQ ID NOS: 17 and 18 reported in
increased by introducing an enhancer. The promoter can be 5 International Publication No. WO95/16042) prepared based
on the chromosome or a plasmid. Introducing genes and/or on the known nucleotide sequence of dapE (Bouvier, J. et al.,
promoters into chromosomal DNA is described in Japanese J. Bacteriol., 174,5265 (1992)).
Patent Application Laid-open No. 1-2 15280, for example. An aspartase gene (aspA) can be obtained by amplification
The origins of the genes which enocode the aforemen by PCR using the E. coli chromosomal DNA as a template
tioned enzymes are not particularly limited, and genes 10 and two kinds of oligonucleotide primers (for example, SEQ
obtained from various origins can be used so long as the genes ID NOS: 5 and 6 reported in the Sequence Listing of the
can be expressed and the genetic products can function in present specification) prepared based on the known nucle
Escherichia bacteria. otide sequence of aspA (Woods, S.A. et al., Biochem. J., 237
Hereinafter, methods which can be used to obtain the (2), 547-557 (1986)).
L-lysine biosynthesis system genes and transhydrogenase 15 A transhydrogenase gene (pntAB) can be prepared based
gene of E. coli, as well as the dihydrodipicolinate synthase on the known nucleotide sequence of the transhydrogenase
and diaminopimelate dehydrogenase genes of Brevibacte gene (D. M. Clarke et al., Eur. J. Biochem., 158, 647-653
rium lactofermentum are exemplified. (1986)). In E. coli, transhydrogenase is composed of two
A phosphoenolpyruvate carboxylase gene (ppc) can be subunits, which are encoded by pntA and pnths (D. M. Clarke
obtained from the plasmid pS2 (Sabe, H. et al., Gene, 31, 279 et al., Supra). Therefore, Both gene Subunits can be prepared
(1984)) or pT2, both of which contain this gene. A DNA (see, for example, International Publication No. WO95/
fragment that contains ppc can be obtained by digesting pS2 11985).
with Aati I and AflII. Furthermore, a DNA fragment that con The genes can also be obtained from a plasmid containing
tains ppc can also be obtained by digesting pT2 with SmaI and pntAB, such as pMW:THY (International Publication No.
Scal. An E. coli F15 strain harboring pT2 (AJ12873) was 25 WO95/11985). This plasmid is obtained by ligating a 3.0 kb
deposited at the National Institute of Bioscience and Human DNA fragment from the E. coli K-12 MC1061 strain, which
Technology, Agency of Industrial Science and Technology, contains pntA and pnt3, as well as a BamHI and HindIII
Ministry of International Trade and Industry (postal code fragment from the plasmid vector pMW 118. An Escherichia
305-8566, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, coli JM109 strain harboring pMW::THY was designated as
Japan) on Jul. 15, 1993, and received an accession number of 30 AJ12929, and deposited at the National Institute of Bio
FERMP-13752. Then, it was converted to an international Science and Human-Technology, Agency of Industrial Sci
deposit under the provisions of the Budapest Treaty on Jul. ence and Technology, Ministry of International Trade and
11, 1994, and received an accession number of FERM Industry (postal code 305-0046, 1-3 Higashi 1-chome,
BP-4732. Tsukuba-shi, Ibaraki-ken, Japan) on Oct. 4, 1993, and
An aspartokinase gene (lysC) can be obtained by amplifi 35 received an accession number of FERMP-13890. Then, it
cation by PCR using the E. coli chromosomal DNA as a was converted to an international deposit under the provisions
template and two kinds of oligonucleotide primers prepared of the Budapest Treaty on Sep. 11, 1994, and received an
based on the known nucleotide sequence of lysC (Cassan, M., accession number of FERM BP-4798.
Parsot, C., Cohen, G. N., and Patte, J. C., J. Biol. Chem., 261, The dihydrodipicolinate synthase gene (dapA) from Brevi
1052 (1986) (for example, see SEQ ID NOS: 5 and 6 in 40 bacterium lactofermentum can be obtained by amplification
International Publication No. WO95/16042). by PCR using the chromosomal DNA of Brevibacterium
An aspartate-semialdehyde dehydrogenase gene (asd) can lactofermentum as a template and two kinds of oligonucle
be obtained from the plasmid pAD20 (Haziza, C. et al., otide primers (for example SEQID NOS: 3 and 4 reported in
EMBO, 1,379 (1982)), which contains this gene. If p AD20 is the Sequence Listing of the present specification) prepared
digested with Asel and Clal, a DNA fragment containing asd 45 based on the known nucleotide sequence of dapA (Bonassie,
will be obtained. S. et al., N.A.R., 18 (21), 6421 (1990)).
A dihydrodipicolinate synthase gene (dapA) can be The diaminopimelate dehydrogenase gene (ddh) from
obtained by amplification by PCR using the E. coli chromo Brevibacterium lactofermentum can be obtained by amplifi
somal DNA as a template and two kinds of oligonucleotide cation by PCR using the chromosomal DNA of Brevibacte
primers (for example, SEQ ID NOS: 1 and 2 reported by 50 rium lactofermentum as a template and two kinds of oligo
International Publication No. WO95/16042) prepared based nucleotide primers (for example, SEQ ID NOS: 11 and 12
on the known nucleotide sequence of dapA (Richaud, F. et al., reported in International Publication No. WO95/16042) pre
J. Bacteriol., 297 (1986)). pared based on the known nucleotide sequence of ddh from
A dihydrodipicolinate reductase gene (dapB) can be Corynebacterium glutamicum (Ishino, S. et al., Nucleic Acids
obtained by amplification by PCR using the E. coli chromo 55 Res., 15, 3917 (1987)).
somal DNA as a template and two kinds of oligonucleotide To enhance intracellular activities of the enzymes, the spe
primers (for example, SEQ ID NOS: 9 and 10 reported in cific activities of the enzymes can also be increased. This
International Publication No. WO95/16042) prepared based method may be combined with the methods described herein
on the known nucleotide sequence of dapB (Bouvier, J. et al., for increasing the expression of the genes encoding the
J. Biol. Chem., 259, 14829 (1984)). 60 enzymes.
A tetrahydrodipicolinate Succinylase gene (dapD) can be To increase the specific activities of the enzymes, a muta
obtained by amplification by PCR using the E. coli chromo tion can be made in the enzyme to desensitize the feedback
somal DNA as a template and two kinds of oligonucleotide inhibition by the produced metabolite, and so forth.
primers (for example, SEQ ID NOS: 15 and 16 reported in Examples of enzymes in which feedback inhibition has
International Publication No. WO95/16042) prepared based 65 been desensitized include dihydrodipicolinate synthase
on the known nucleotide sequence of dapD (Richaud, C. et (DDPS) and aspartokinase (AK), both of which have been
al., J. Biol. Chem., 259, 14824 (1984)). desensitized to L-lysine.
 US 8,062,869 B2
7 8
The phrase “feedback inhibition by L-lysine is desensi those recited above will result in a DNA coding for a desen
tized' means that substantial desensitization of the inhibition sitized DDPS. Furthermore, slight differences in the amino
is Sufficient, and it is not required that the inhibition is com acid sequences of DDPS among bacterial species and strains
pletely desensitized. Furthermore, desensitized enzymes are expected. However, when these differences result in
derived from organisms other than Escherichia bacteria can 5 replacement, deletion or insertion of amino acid residues at
also be used, irrespective of whether it is a wild-type or positions that do not affect the activity of enzyme. Such
mutant type enzyme, if the degree of the feedback inhibition sequences fall within the scope of the desensitized DDPS
by L-lysine is lower than that of a wild-type enzyme derived gene.
from an Escherichia bacterium. Therefore, an enzyme that, in A desensitized DDPS gene can be obtained, for example,
its native form, is not subject to feedback inhibition by 10
as follows. First, DNA containing a wild-type DDPS gene, or
L-lysine, such as DDPS derived from Brevibacterium bacte another DDPS gene having a mutation, is subjected to invitro
ria, is also included. mutation, and this mutated DNA is ligated with a vector DNA
The degree of the feedback inhibition by L-lysine can be compatible with the chosen host to prepare a recombinant
evaluated by known methods such as those described in Inter DNA. The recombinant DNA is introduced into the host
national Publication No. WO95/16042. Examples 1 and 2. 15
DDPS and AK which have been desensitized to feedback microorganisms to obtain transformants, and a transformant
inhibition by L-lysine are disclosed in International Publica that is able to express a desensitized DDPS is selected. Alter
tion No. WO95/16042 and Japanese Patent Application Laid natively, an in vitro mutation treatment can be performed on
open No. 10-113183. the vector DNA containing the wild-type DDPS gene, or
That is, examples of desensitized DDPS include DDPS another DDPS gene having a mutation. Then, the mutated
enzymes that have been mutated to obtain the desensitization DNA can be introduced into host microorganisms to obtain
to the feedback inhibition by L-lysine. The DDPS from transformants, and a transformant that is able to express a
Escherichia bacteria, and E. coli, are exemplary. Examples of desensitized DDPS can be selected from the transformants.
the mutation that desensitizes the feedback inhibition by Such a transformant also harbors the desensitized DDPS
L-lysine in DDPS include: 25 gene.
(1) replacing the alanine residue at position 81 with another Furthermore, a microorganism that produces wild-type
amino acid residue (Valine, for example), DDPS may be subjected to a mutation treatment which results
(2 replacing the histidine residue at position 118 with in a mutant strain that produces a desensitized DDPS, and
another amino acid residue (tyrosine, for example), and then a desensitized DDPS gene can be obtained from the
(3) replacing the alanine residue at position 81 with another 30 mutant strain. Alternatively, if a microorganism which has
amino acid residue (valine, for example), and replacing the been transformed with a recombinant DNA ligated with a
histidine residue at position 118 with another amino acid wild-type gene is subjected to a mutation treatment to prepare
residue (tyrosine, for example) in the DDPS of E. coli (see a mutant strain producing a desensitized DDPS and then a
SEQ ID NO: 4 in International Publication No. WO95/ recombinant DNA is collected from the mutant strain, a
16042), as counted from N-terminus. It is well known that 35 desensitized DDPS gene is created on that DNA.
differences may occur among amino acid sequences from Examples of agents for the in vitro mutation treatment of
different species or Strains, although these differences may DNA include hydroxylamine and so forth. Hydroxylamine is
not affect activity, and anamino acid residue corresponding to a chemical mutation treatment agent that causes replacement
the aforementioned specific amino acid residues can be easily of cytosine with thymine by changing cytosine into N'-hy
recognized by those skilled in the art. 40 droxycytosine. When a microorganism itself is subjected to a
Other examples of a desensitized DDPS include DDPS mutation treatment, the treatment is performed with ultravio
derived from coryneform bacteria, for example, Brevibacte let irradiation or a typically-used mutagenizing agent Such as
rium lactofermentum (Cremer J. et al., J. Gen. Microbiol. N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and nitrous
134, 3221-3229 (1988)). acid.
In order to obtain an Escherichia bacteria which contains a 45 Bacteria from which the DNA containing a wild-type
desensitized DDPS, for example, DNA that encodes the DDPS gene, or another DDPS gene containing a mutation, is
desensitized DDPS can be introduced. derived, any of microorganisms belonging to the genus
Examples of the DNA coding for desensitized DDPS Escherichia may be used. Specifically, those mentioned in the
include DNA coding for a wild-type DDPS which includes a literature of Neidhardt et al. (Neidhardt, F. C. et al., Escheri
mutation which results in a DDPS which is desensitized to 50 chia coli and Salmonella Tiphimurium, American Society for
feedback inhibition by L-lysine. Microbiology, Washington D.C., 1208, Table 1) can be used.
Hereinafter, a method for obtaining DNA coding for desen For example, E. coli JM109 strain and MC1061 strain can be
sitized DDPS (desensitized DDPS gene) will be explained by used. When a wild-type strain is used as the donor of DNA
exemplifying DDPS derived from Escherichia bacteria, containing a DDPS gene, DNA containing a wild-type DDPS
although DNA encoding DDPS from other organisms can be 55 gene can be obtained.
similarly obtained. Furthermore, ifa wild-type DDPS derived Examples of the desensitized AK include AK enzymes that
from another organism is a desensitized DDPS, DNA coding have been mutated to obtain the desensitization to the feed
for it can be used as it is. back inhibition by L-lysine. The AK derived from Escheri
The DNA coding for wild-type DDPS is not particularly chia bacteria, and the AKIII derived from E. coli are exem
limited so long as it codes for DDPS derived from an Escheri 60 plary. Examples of the mutation that desensitizes the
chia bacterium. Specifically, DNA coding for the amino acid feedback inhibition by L-lysine in AKIII include:
sequence shown in International Publication No. WO95/ (a) replacing the glycine residue at position 323 with
16042, SEQ ID NO: 4, can be used. More specifically, the another amino acid residue (aspartic acid, for example),
sequence of nucleotide numbers 272-1147 within the nucle (b) replacing the glycine residue at position 323 with
otide sequence shown in SEQ ID NO: 3 of International 65 another amino acid residue (aspartic acid, for example), and
Publication No. WO95/16042 can be used. In these replacing the glycine residue at position 408 with another
sequences, mutations of the nucleotide sequence such as amino acid residue (aspartic acid, for example),
 US 8,062,869 B2
9 10
(c) replacing the arginine residue at position 34 with tional Publication No. WO95/16042 can be used. AKIII from
another amino acid residue (cysteine, for example), and E. coli is encoded by the lysC gene.
replacing the glycine residue at position 323 with another Mutations such as those disclosed above will result in a
amino acid residue (aspartic acid, for example), DNA coding for a mutant-type AKIII Furthermore, slight
(d) replacing the leucine residue at position 325 with 5 differences in the amino acid sequences of AKIII among
another amino acid residue (phenylalanine, for example), bacterial species and strains are expected. However, when
(e) replacing the methionine residue at position 318 with these differences result in replacement, deletion or insertion
another amino acid residue (isoleucine, for example), of amino acid residues at positions that do not affect the
(f) replacing the methionine residue at position 318 with activity of enzyme. Such sequences fall within the scope of the
another amino acid residue (isoleucine, for example), and 10 mutant AKIII gene. For example, the nucleotide sequence of
replacing the valine residue at position 349 with another the wild-type lysC gene obtained in Example 2 mentioned
amino acid residue (methionine, for example), hereinafter (International Publication No. WO95/16042,
(g) replacing the serine residue at position 345 with another SEQID NO: 7) has differences in the sequence at 6 positions
with respect to the already published nucleotide sequence of
amino acid residue (leucine, for example), 15 lysC of E. coli K-12 JC411 strain (Cassan M. Parsot, C.,
(h) replacing the valine residue at position 347 with another Cohen, G. N., and Patte, J. C., J. Biol. Chem., 261, 1052
amino acid residue (methionine, for example), (1986)), among which two of the differences provide differ
(i) replacing the threonine residue at position 352 with ent encoded amino acid residues (lysC of the JC411 strain
another amino acid residue (isoleucine, for example), provides replacement of the glycine residue at position 58
() replacing the threonine residue at position 352 with with a cysteine residue and replacement of the glycine residue
another amino acid residue (isoleucine, for example), and at position 401 with an alanine residue in the amino acid
replacing the serine residue at position 369 with another sequence encoded by lysC shown in SEQID NO: 8 of Inter
amino acid residue (phenylalanine, for example), national Publication No. WO95/16042 wherein the position
(k) replacing the glutamic acid residue at position 164 with is counted from the N-terminus thereof). It is expected that
another amino acid residue (lysine, for example), and 25 even lysC having the same sequence as lySC of the E. coli
(1) replacing the methionine residue at position 417 with K-12 JC411 strain may provide lysC having a mutation that
another amino acid residue (isoleucine, for example), and desensitizes the feedback inhibition by L-lysine, if it is intro
replacing the cysteine residue at position 419 with another duced along with any of mutations mentioned in the above (a)
amino acid residue (tyrosine, for example). The positions are to (1), or a mutation for replacing an amino acid residue
those in the E. coli AKIII amino acid sequence (see SEQID 30 corresponding to the glycine residue at position 323 with
NO: 8 in International Publication No. WO95/16042), as another amino acid residue (for example aspartic acid), and
counted from the N-terminus. Furthermore, a mutation which replacing an amino acid residue corresponding to the
results in replacing the glycine residue at position 323 with methionine residue at position 318 with another amino acid
residue.
another amino acid residue, for example aspartic acid, and 35 A DNA coding for a mutant-type AKIII that is desensitized
replacing the methionine residue at position 318 with another to feedback inhibition by L-lysine can be obtained, for
amino acid residue, for example isoleucine can also be used example, as follows. First, DNA containing a wild-type AKIII
(Japanese Patent Application Laid-open No. 10-113183). It is gene, or another AKIII gene having a mutation, is Subjected to
well known that differences may occur among amino acid in vitro mutation, and this mutated DNA is ligated to a vector
sequences from different species or strains, although these 40 DNA compatible with a chosen host to prepare a recombinant
differences may not affect activity, and an amino acid residue DNA. The recombinant DNA can be introduced into the host
corresponding to the aforementioned specific amino acid microorganisms to obtain transformants, and a transformant
residues can be easily recognized by those skilled in the art. that is able to express a mutant type AKIII can be selected.
Other examples of the desensitized AK include a mutant Alternatively, an in vitro mutation treatment can be per
type AK derived from coryneform bacteria (Japanese Patent 45 formed on the vector DNA containing the wild-type AKIII, or
Application Laid-open No. 6-62866). another AKIII gene having a mutation. Then, the mutated
In order to obtain an Escherichia bacterium which contains DNA can be introduced into the host microorganisms to
a desensitized AK, for example, DNA that encodes the desen obtain transformants, and a transformant that is able to
sitized AK can be introduced into the Escherichia bacteria. express a mutant-type AKIII can be selected from the trans
Examples of the DNA coding for a desensitized AK 50 formants. Such a transformant also harbors the mutant type
gene.
include DNA coding for a wild-type AK which includes a Furthermore, a microorganism that produces a wild-type
mutation which results in an AK which is desensitized to
feedback inhibition by L-lysine. enzyme may be subjected to a mutation treatment which
Hereinafter, a method for obtaining DNA coding for a results in a mutant strain that produces a mutant-type enzyme,
desensitized AK will be explained by exemplifying AKIII
55 and then the mutant-type gene can be obtained from the
mutant strain. Examples of agents for directly subjecting
derived from Escherichia bacteria, although DNA encoding DNA to a mutation treatment include hydroxylamine and so
AK from other organisms can be similarly obtained. Further forth. Hydroxylamine is a chemical mutation treatment agent
more, if a wild-type AK derived from another organism is a that causes replacement of cytosine with thymine by chang
desensitized AK, DNA coding for it can be used as it is. 60 ing cytosine into N'-hydroxycytosine. When a microorgan
The DNA coding for wild-type AKIII is not particularly ism itself is subjected to a mutation treatment, the treatment is
limited. For example, DNA coding for AKIII derived from an performed with ultraviolet irradiation or a mutagenizing
Escherichia bacterium, such as E. coli, can be used. Specifi agent usually used for artificial mutation Such as N-methyl
cally, DNA coding for the amino acid sequence shown in N-nitro-N-nitrosoguanidine (NTG).
International Publication No. WO95/16042 (see specifically 65 Bacteria from which the DNA containing a wild-type
SEQ ID NO: 8 of this publication), and the sequence of AKIII gene, or another AKIII gene having a mutation, is
nucleotide numbers 584-1930 of SEQ ID NO: 7 in Interna derived, any of microorganisms belonging to the genus
 US 8,062,869 B2
11 12
Escherichia may be used. Specifically, those mentioned in the EXAMPLES
literature of Neidhardt et al. (Neidhardt, F. C. et al., Escheri
chia coli and Salmonella Tiphimurium, American Society for The present invention will be further specifically explained
Microbiology, Washington D.C., 1208, Table 1) can be used. hereinafter with reference to the following examples.
For example, E. coli JM109 strain, MC1061 strain and so 5
forth can be used. Example 1
Aspartokinase, dihydrodipicolinate reductase, tetrahy
drodipicolinate Succinylase, Succinyl diaminopimelate <1> Preparation of Escherichia Bacteria Having
deacylase, phosphoenolpyruvate carboxylase, and aspartate Various Properties
10
semialdehyde dehydrogenase each can be derived from
Escherichia bacteria; nicotinamide adenine dinucleotide The plasmids shown below were introduced into E. coli
transhydrogenase and aspartase, if present, can each be W3110 (tyrA).
derived from Escherichia bacteria; dihydrodipicolinate syn
thase can be derived from an Escherichia bacterium or Brevi 15
bacterium bacterium, and diaminopimelate dehydrogenase Name of plasmid gene(s) on plasmid
can be derived from a Brevibacterium bacterium.
RSF24P dapA*
Examples of the Brevibacterium bacteria include Brevi pCAB1 dapA*, lysC*, dapB
bacterium lactofermentum, Brevibacterium flavum, Brevi pCABD2 dapA*, lysC*, dapB, ddh
bacterium divaricatum, Corynebacterium glutamicum, pCABD(B) Brev. dap A, lysC*, dapB, ddh
Corynebacterium lilium, and so forth. pCABDE1 dapA*, lysC*, dapB, dapD, dapF.
Furthermore, the intracellular activities of dihydrodipicoli
nate synthase and aspartokinase are enhanced by the use of The abbreviations used for the genes encode the following
feedback-desensitized dihydrodipicolinate synthase and 25 proteins.
aspartokinase, and the intracellular activity of diaminopime ppc: Phosphoenolpyruvate carboxylase
late dehydrogenase can be enhanced by introduction of a lysC: Aspartokinase III
diaminopimelate dehydrogenase gene. Such bacteria may be lysC*: Aspartokinase III desensitized to inhibition
obtained by introducing the plasmid pCABD2 or pCABDE1 asd: Aspartate-semialdehyde dehydrogenase
(see International Publication No. WO95/16042) into an 30 dapA: Dihydrodipicolinate synthase
Escherichia bacterium. dapA*: Dihydrodipicolinate synthase desensitized to inhi
bition
Brev. dap A: Dihydrodipicolinate synthase desensitized to
<2> Production Method of the Present Invention inhibition (derived from Brevibacterium lactofermentum)
35 dapb: Dihydrodipicolinate reductase
L-Lysine can efficiently be produced by culturing the bac dap): Tetrahydrodipicolinate succinylase
teria as described above in a suitable medium to produce and dapF: Succinyl diaminopimelate deacylase
accumulate L-lysine in the culture, and collecting the ddh: Diaminopimelate dehydrogenase (derived from
L-lysine from the culture. Brevibacterium lactofermentum)
The medium used for the culture of the bacteria may be a
40 The plasmids RSF24P, RSFD80, pCAB1, pCABD2, and
typical medium which includes a carbon Source, a nitrogen pCABDE1 are described in International Publication No.
WO95/16042. The constructions thereofare also described in
Source, inorganic ions, and other organic trace nutrients as International Publication No. WO95/16042, and outlined
required. below.
As the carbon Source, Sugars Such as glucose, lactose, 45 (1) RSF24P
galactose, fructose and starch hydrolysate; alcohols such as Based on the known dapA nucleotide sequence of E. coli
glycerol and Sorbitol; or organic acids such as fumaric acid, (J. Bacteriol. 166,297 (1986)), a fragment containing the SD
citric acid and Succinic acid can be used. sequence and open reading frame (ORF) of dap A was ampli
As the nitrogen Source, inorganic ammonium salts such as fied by PCR. The amplified fragment was ligated to the clon
ammonium sulfate, ammonium chloride or ammonium phos 50 ing vector pCR1000 to obtain the plasmid pdap A2, in which
phate; organic nitrogen Such as Soybean hydrolysate; ammo dapA was ligated so that the transcription direction of dap A
nia gas; or aqueous ammonia can be used. was reverse to the transcription direction by the lacZ pro
As for the organic trace nutrients, required Substances such moter in pCR1000. The plasmid pdap A2 was subjected to a
as vitamin B and L-isoleucine, yeast extract and so forth can mutagenesis treatment by using hydroxylamine, and pdap A2
be added in a suitable amount. In addition to these, a small 55 Subjected to the mutagenesis treatment was introduced into E.
amount of potassium phosphate, magnesium Sulfate, iron coli W31 10. From the transformants, those exhibiting AEC
ions, manganese ions and so forth can be added. resistance were selected. Furthermore, the degree of the inhi
Culture can be carried out under aerobic conditions for
bition by L-lysine of DDPS encoded by the plasmids har
bored by the selected resistant strains was measured, and a
16-72 hours. The culture temperature can be controlled to be 60 strain that was desensitized to the inhibition by L-lysine was
20° C. to 45° C., and the pH can be controlled to be 5 to 8 selected. The plasmidpdap A24, which was confirmed to have
during the culture. Inorganic or organic, acidic or alkaline the change at 597th C to T by sequencing, was ligated to
Substances as well as ammonia gas and so forth can be used to pVIC40 at a position downstream from the tetracycline resis
adjust the pH. tance gene promoter to obtain RSF24P (FIG. 1).
Collection of L-lysine from the fermented liquor is usually 65 An E. coli JM109 Strain into which RSF24P was intro
carried out by a combination of an ion exchange resin duced was designated as AJ12395, and it was deposited at the
method, a precipitation method, and other known techniques. National Institute of Bioscience and Human-Technology,
 US 8,062,869 B2
13 14
Agency of Industrial Science and Technology, Ministry of with EcoO1091 and Sad, and the obtained fragment was
International Trade and Industry (postal code 305-8566, 1-3 blunt-ended and inserted into the SmaI site of pMW 118 to
Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on Oct. obtain a plasmid pdapD. Furthermore, based on the known
28, 1993 and received an accession number of FERM dapF nucleotide sequence (Bouvier, J. et al., J. Bacteriol.
P-13935. Then, it was converted to an international deposit 174, 5265 (1992)), dapF was amplified from chromosome
under the provisions of the Budapest Treaty on Nov. 1, 1994, DNA of E. coli W3110 strain by PCR. The obtained amplified
and received an accession number of FERM BP-4858. DNA fragment was digested with MunI and BglII, and an
(2) RSFD80 obtained fragment was blunt-ended and inserted into the
Based on the known lysC nucleotide sequence of E. coli (J. SmaI site of pMW 118 to obtain a plasmid pdapE. Further
Biol. Chem..., 261, 1052 (1986)), a fragment containing the SD 10 more, dapE was excised from pdapE and inserted into pdapD
sequence and ORF of lysC was amplified by PCR. The ampli to obtain a plasmid pMWclapDE1 containing both of dapF.
fied fragment was ligated to the multi-copy vector puC18 to and dapD. As shown in FIG. 5, a fragment containing dapF.
obtain the plasmid pLYSC1, in which lysC was ligated so that and dapD was excised from pMWclapDE1, and inserted into
the transcription direction of lysC was reverse to the tran pCAB1 to obtain pCABDE1.
scription direction by the lacz promoter in puC18. The plas 15 A plasmid pCABD(B) was constructed as follows.
mid plYSC1 was subjected to a mutagenesis treatment by First, a DNA fragment containing the promoter site of Tet
using hydroxylamine, and pLYSC1 subjected to the mutagen resistance gene was amplified from pBR322 by using primers
esis treatment was introduced into E. coli GT3. From the having the following sequences:
transformants, those exhibiting AEC resistance and L-lysine
resistance were selected. Furthermore, pIYSC1 was intro
duced into E. coli MC1061, then the cells were subjected to a s" - TCAAGAATTCTCATGTTTGA-3' (SEQ ID NO: 1)
mutagenesis treatment by using hydroxylamine, and those 5'-GTTAGATTTGGTACCCGGTGCCTGACTGCGTTA (SEO ID NO : 2)
exhibiting AEC resistance and L-lysine resistance were
selected. Furthermore, the degree of the inhibition by
L-lysine and thermal stability of AK encoded by the plasmids 25 The amplified DNA fragment was digested with KpnI and
harbored by the selected resistant strains were measured, and EcoRI, and inserted between KpnI and EcoRI cleavage sites
a strain which was desensitized to the inhibition by L-lysine of puC18 to obtain puTES (FIG. 18).
and which exhibited superior stability was selected. The plas Then, the Brev. dap A gene was amplified by using chro
mid plYSC 180, which was confirmed to have the change at mosomal DNA of Brevibacterium lactofermentum Ysr strain
352nd C to T by sequencing, was ligated to pHSG399 at a 30 as a template and the primers having the following
position downstream from the lacZ promoter to obtain Sequences.
pLLC*80. From plLC*80 and RSF24P RSFD80 was con
structed as shown in FIG. 2.
An E. coli JM109 Strain into which RSFD80 was intro 5'-GGTTGTGGTACCCCCAAATGAGGGAAGAAG-3" (SEQ ID NO : 3)
duced was designated as AJ12396, and it was deposited at the 35
s" -TGGAACCTCTGTTGCTGCAG-3' (SEQ ID NO : 4)
National Institute of Bioscience and Human-Technology,
Agency of Industrial Science and Technology, Ministry of The amplified Brev. dap A gene was digested with KpnI and
International Trade and Industry (postal code 305-8566, 1-3 BamHI, and inserted between KpnI and BamHI cleavage
Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on Oct. sites of pUTES to obtain puEBL3 (FIG. 19).
28, 1993 and received an accession number of FERM 40 Then, puEBL3 was digested with EcoRI and Xbal, and
P-13936. Then, it was converted to an international deposi blunt-ended for the both ends to obtain a fragment containing
tion under the provisions of the Budapest Treaty on Nov. 1, Brev. dapA. Thereafter, pCABD2 (see International Publica
1994, and received an accession number of FERM BP-4859. tion No. WO95/16042) was digested with Nheland Speland
(3) pCAB1 blunt-ended for the both ends. A fragment containing lysC.
Based on the known dapB nucleotide sequence (Bouvier, J. 45 dapb and ddh was collected, and then the previously obtained
et al., J. Biol. Chem..., 259, 14829 (1984)), dapB was amplified fragment containing Brev. dap A was inserted thereto to
from E. coli W3110 strain chromosomal DNA by PCR. The obtain pCABD(B) (FIG. 20)
obtained amplified DNA fragment was digested with Asel While E. coli W3110 (tyr A) is detailed in European Patent
and DraI, and the obtained fragment was blunt-ended and Publication No. 488-424, the preparation method therefor
inserted into the SmaI site of pMW 119 to obtain a plasmid 50 will be briefly explained below. The E. coli W3110 strain was
pdapB. Subsequently, dapR was introduced into RSFD80 as obtained from the National Institute of Genetics (Shizuoka
shown in FIG.3 to obtain pCAB1. ken, Mishima-shi). This strain was inoculated on an LB plate
(4) pCABD2 containing Streptomycin, and a strain that formed a colony
Based on the known ddh nucleotide sequence of Coryne was selected to obtain a streptomycin resistant strain. The
bacterium glutamicum (Ishino, S. et al., Nucleic Acids Res., 55 selected Streptomycin resistant Strain and E. coli K-12
15, 3917 (1987)), ddh was amplified from chromosomal ME8424 strain were mixed and cultured in a complete
DNA of Brevibacterium lactofermentum ATCC13869 by medium (L-Broth: 1% Bacto trypton, 0.5% Yeast extract,
PCR. The obtained amplified DNA fragment was digested 0.5% NaCl) as stationary culture at 37° C. for 15 minutes to
with EcoT22I and Aval, and the obtained fragment was blunt induce conjugation. The E. coli K-12 ME8424 strain has
ended and inserted into the SmaI site of pMW 119 to obtain a 60 genetic traits of HfrPO45, thi, relA1, tyr A::Tnl0, ung-1 and
plasmidpddh. Subsequently, ddh was introduced into pCAB1 nadB, and can be obtained from the National Institute of
as shown in FIG. 4 to obtain a plasmid pCABD2. Genetics.
(5) pCABDE1 Then, the culture was inoculated to a complete medium
Based on the known dapD nucleotide sequence (Richaud, (L-Broth: 1% Bacto trypton, 0.5% yeast extract, 0.5% NaCl,
C. et al., J. Biol. Chem., 259, 14824 (1984)), dapD was 65 1.5% agar) containing streptomycin, tetracycline and L-ty
amplified from chromosomal DNA of E. coli W3110 strain rosine, and a strain that formed a colony was selected. This
by PCR. The obtained amplified DNA fragment was digested strain was designated as E. coli W3110 (tyra) strain.
 US 8,062,869 B2
15 16
Many strains formed by introducing a plasmid into the described in International Publication No. WO95/16042. The
W3110 (tyrA) strain are disclosed in European Patent Publi specific procedure was as follows.
cation No. 488-424. For example, a strain obtained by intro The cells were cultured in 20 ml of a medium having the
ducing a plasmid pHATerm was designated as E. coli W3110 following composition contained in a 500-ml Sakaguchi flask
(tyra)/pHATerm strain (E. coli AJ12662 strain), and it was at a temperature of 37°C. for 30 hours with stirring at 114-116
deposited at the National Institute of Bioscience and Human rpm.
Technology, Agency of Industrial Science and Technology, Medium Composition:
Ministry of International Trade and Industry (postal code
305-8566, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken,
Japan) on Nov. 16, 1991 as an international deposit and 10
received an accession number of FERM BP-3653. The Glucose 40 g/l
W3110 (tyrA) strain can also be obtained by eliminating the MgSO 7H2O 1 g/l
(NH4)2SO 16 g/l
plasmid pHATerm from this E. coli W3110 (tyrA)/pHATerm KH2PO 1 g/l
strain. The elimination of the plasmid can be performed in a FeSO4·7HO 0.01 g/l
conventional manner. 15 MnSO 5H2O 0.01 g/l
Yeast Extract (Difco) 2 g/l
L-Tyrosine 0.1 g/l
<2> Introduction of Aspartate-Semialdehyde
Dehydrogenase Gene (asd), Phosphoenolpyruvate
Gene (ppc) or Aspartase Gene (aspa), and Evaluation Adjusted to pH 7.0 with KOH, and autoclaved at 115° C.
of L-Lysine Productivity for 10 minute (glucose and MgSO4.7HO were separately
sterilized).
As a plasmid containing asd and a plasmid containing ppc,
pasd and pppc (see International Publication No. WO95/
16042) were used. Constructions of these plasmids were CaCO 25 g/l
detailed in International Publication No. WO95/16042. The 25
outlines are as follows.
(1) pasd (according to the Pharmacopoeia of Japan, Sterilized by dry
The plasmid asd was obtained from a plasmid paD20 heat at 180° C. for 2 days)
(Haziza, C. et al., EMBO, 1,379 (1982)), which contained the Antibiotic (20 mg/l of streptomycin, 50 mg/l of amplicillin
gene. The plasmid pAD20 was digested with Asel and ClaI, 30 or 25 mg/l of kanamycindepending on the kind of the plasmid
blunt-ended and inserted into the SmaI site of pMW 118 to to be introduced)
obtain a plasmid pasd (FIG. 8). L-Lysine in the culture broth (medium after the culture)
(2) ppp.c was quantified by using Biotech Analyzer AS210 produced
The plasmid pppc was obtained from a plasmid pT2 that by Asahi Chemical Industry Co., Ltd.
contained the gene. The plasmid pT2 was digested with SmaI 35 The results are shown in Table 1. In the table, the amount of
and Scal, blunt-ended, and inserted into the SmaI site of L-lysine is indicated in terms of mg per dl of the medium.
pMW118 to obtain the plasmid pppc (FIG.9). An E. coli F15
strain (AJ12873) harboring pT2 was deposited at the National TABLE 1
Institute of Bioscience and Human-Technology, Agency of
Industrial Science and Technology, Ministry of International 40 Lys accumulation (mg/dl
Trade and Industry (postal code 305-8566, 1-3 Higashi pMW118 pasd ppp.c pMW 118:aspA
1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on Jul. 15, 1993
and received an accession number of FERMP-13752. Then, 40 50 60 60
RSF24P 350 340 360 350
it was converted to an international deposit under the provi RSFD8O 960 790 990 960
sions of the Budapest Treaty on Jul. 11, 1994, and received an 45
pCAB1 1120 1140 11SO 1130
accession number of FERM BP-4732. PCABD2 1230 1320 1380 1240
A plasmid containing aspA was constructed as follows. pCABD(B) 1230 1320 1370 n.d.
The aspA gene of E. coli was amplified by using primers PCABDE1 1210 1310 1350 n.d.
having the following sequences: n.d.; Not determined
50
As clearly seen from the results shown in Table 1, when asd
5'-TGATCAGCGAAACACTTTTA-3 (SEO ID NO. 5) or ppc was enhanced each alone or together with dap A
5 - CAGCAAACTATGATGAGAA-3' (SEQ ID NO : 6) (RSF24P), dapA+lysC(RSFD80) or dapA+lysC+dapB
(pCAB1) in E. coli, the production amount of L-lysine (accu
Then, the obtained amplified fragment was inserted into 55 mulated amount) was not changed or only slightly changed
the SmaI cleavage site of pMW 118 (Nippon Gene) to obtain and, as for asd, it might be reduced compared with the case
pMW 118:aspA (FIG. 9). where asd or ppc was not enhanced (-180 to 20 mg/dl as for
Each of pMW 118 (control plasmid), pasd, pppc and asd, 10 to 30 mg/dl as for ppc). In contrast, if they were
pMW 118:aspA (comparative plasmid) was introduced into enhanced together with dapA+lysC+dapB+ddh (pCABD2)
each of E. coli W3110 (tyra) and the transformants obtained 60 or dap A+lysC+dapB+dapD+dapE (pCABDE1), a marked
in the aforementioned <1>. The obtained transformants, increase of the L-lysine production amount was observed
except for those obtained by introducing pMW 118, pasd, compared with when asdor ppc was not enhanced (70 mg/dl
pppc orpMW 118::aspA into E. coli W3110 (tyr A), contained as for asd, 90 mg/dl as for ppc). However, when aspA was
two kinds of plasmids, i.e. one of pMW 118, pasd, pppc and enhanced with dap A+lysC+dapB+ddh (pCABD2), a marked
pMW118:asp A and one of RSF24P, RSFD80, pCAB1, 65 increase of the L-lysine production amount was not observed.
pCABD2, pCABD(B) and pCABDE1. These transformants Furthermore, even when dap A derived from Brevibacterium
were examined for L-lysine productivity by the method lactofermentum was used instead of dap A derived from
 US 8,062,869 B2
17 18
Escherichia coli (pCABD (B)), the same effect was obtained (reference plasmid), ppcd, pPTS and pAPW (comparative
as when dap A derived from Escherichia coli was used plasmid) was introduced. The obtained transformants con
(pCABD2). Therefore, the origin of the genes is not consid tained two kinds of plasmids, i.e. one of pppc, ppcd, pPTS and
ered important, but the combination thereof is important. pAPW, and pCABD2. These transformants were examined
Example 2 for the L-lysine productivity in the same manner as in
Example 1 <2>.
The results are shown in Table 2.
<1> Construction of Plasmids Containing
Phosphoenolpyruvate Carboxylase Gene (ppc) and
Aspartate-Semialdehyde Dehydrogenase Gene (asd), TABLE 2
Transhydrogenase Gene (pntab) or Aspartase Gene Lysaccumulation (mg/dl)
(aspA)
pCABD2 + ppp.c 1380
A plasmid containing ppc and asd, a plasmid containing pCABD2 + ppcd 1460
ppc and pntAB, and a plasmid containing ppc and aspA were pCABD2 + pPTS 1450
constructed as follows. 15 pCABD2 + p.APW 1390
(1) Plasmid Containing ppc and asd (ppcd)
The plasmid pasd disclosed in International Publication As clearly seen from the results shown in Table 2, when asd
No. WO95/16042 was digested with KpnI and SphI, and the or pntAB was enhanced together with dap A+lysC+dapB+
DNA fragment containing asd was blunt-ended for the both ddh+ppc (ppcd or pPTS), marked increase of the L-lysine
ends. Then, pppc disclosed in International Publication No. production amount was observed (80 mg/dl as for asd, 70
WO95/16042 was digested with Xbaland blunt-ended for the mg/dl as for pntAB). As for asp.A., however, even when aspA
both ends, and the previously obtained asd fragment was was enhanced together with dap A+lysC+dapB+ddh+ppc
inserted thereto to obtain ppcd (FIG. 10). (pAPW), marked increase of the L-lysine production amount
(2) Plasmid Containing ppc and pntAB (ppTS) was not observed.
The plasmid plMW:THY disclosed in International Publi- is
cation No. WO95/11985 was digested with SmaI and HindIII,
and the DNA fragment containing pntAB was collected. Example 3
Then, pppc disclosed in International Publication No. WO95/
16042 was digested with Xbal, blunt-ended for the both ends
and further digested with HindIII, and the previously <1> Construction of Plasmids Containing
obtained pntAB fragment was inserted at the cleaved site to Phosphoenolpyruvate Carboxylase Gene (ppc),
obtain pPTS (FIG. 11). Transhydrogenase Gene (pntAB) and
Construction of the plasmid pMW::THY is detailed in Aspartate-Semialdehyde Dehydrogenase Gene (asd)
International Publication No. WO95/11985. It will be out or Aspartase Gene (aspA)
lined below.
Based on the known pntA and pnth nucleotide sequences 35
A plasmid containing ppc, pntAB and asd genes and a
of E. coli (D. M. Clarke et al., Eur. J. Biochem., 158,647-653 plasmid containing ppc, pntAB and aspA were constructed as
(1986)), a fragment containing both genes including regions follows.
having promoter activity was amplified by PCR. The ampli (1) Plasmid Containing ppc, pntAB and asd (pPTd)
fied DNA fragment was digested with BamHI and HindIII,
and the obtained DNA fragment was ligated to the plasmid 40 The plasmid pasd (see International Publication No.
vector pMW 118 (Nippon Gene) digested with BamHI and WO95/16042) was digested with KpnI and SphI, and the
HindIII to obtain pMW:THY (FIG. 8). DNA fragment containing asd was blunt-ended for the both
The E. coli JM109 strain into which pMW 118::THY was ends. Then, pPTS described in the aforementioned Example
introduced was designated as AJ12929, and it was deposited 2 was digested with HindIII and blunt-ended, and the previ
at the National Institute of Bioscience and Human-Technol 45 ously obtained asd fragment was inserted into the HindIII
ogy, Agency of Industrial Science and Technology, Ministry cleavage site to obtain pPTd (FIG. 13).
of International Trade and Industry (postal code 305-8566, (2) Plasmid Containing ppc, pntAB and aspA (pAPT)
1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on The plasmid pMW::THY (see International Publication
Oct. 4, 1993, and received an accession number of FERM No. WO95/11985) was digested with SmaI and HindIII to
P-13890. Then, it was transferred to an international deposit 50
obtain a DNA fragment containing pntAB. Then, p.APW
under the provisions of the Budapest Treaty on Sep. 14, 1994, described in the aforementioned Example 2 was digested
and received an accession number of FERM BP-4798. with Xbal, blunt-ended for the both ends, and further digested
(3) Plasmid Containing ppc and aspA (pAPW) with HindIII. The previously obtained fragment containing
The plasmid pMW 118::asp.A described in the aforemen pntAB was inserted into the HindIII cleavage site to obtain
tioned Example 1 was digested with SacI, blunt-ended at both 55
pAPT (FIG. 14).
ends, and further digested with HindIII to obtain a DNA
fragment containing aspA. Then, pppc (see International <2> Introduction of Three Kinds of Genes and
Publication No. WO95/16042) was digested with Xbal, Evaluation of L-Lysine Productivity
blunt-ended for the both ends, and further digested with Hin
dIII, and the previously obtained aspA fragment was inserted 60
into the HindIII cleavage site to obtain paPW (FIG. 12). To a pCABD2-introduced transformant which was
obtained in the aforementioned Example 1, pPTS (reference
<2> Introduction of Two Kinds of Genes and plasmid), pPTdorp APT was introduced. The obtained trans
Evaluation of L-Lysine Productivity formants contained two kinds of plasmids, i.e. one of pPTS,
65 pPTd and pAPT, and pCABD2. These transformants were
To a pCABD2-introduced transformant which was examined for the L-lysine productivity in the same manner as
obtained in the aforementioned Example 1, each of pppc in Example 1 <2>.
 US 8,062,869 B2
19 20
The results are shown in Table 3. mentioned Example 1, p.APT (Reference plasmid 1), pAPTK
(Reference plasmid 2) or pKD was introduced. The obtained
TABLE 3 transformants contained two kinds of plasmids, i.e. one of
Lysaccumulation (mg/dl)
pAPT, paPTK and pKD, and one of pCABD2, pCABD(B)
and pCABDE1. These transformants were examined for the
pCABD2 + pPTS 1450 L-lysine productivity in the same manner as in Example 1
pCABD2 + pPTd 1510 <2>.
pCABD2 + p.APT 1SOO
The results are shown in Table 4.
As clearly seen from the results shown in Table 3, when asd 10
TABLE 4
or aspA was enhanced together withdapA+lysC+dapB+ddh+
ppc-pntAB (pPTdorpAPT), marked increase of the L-lysine
production amount was observed (60 mg/dl as for asd, 50 Lysaccumulation (mg/dl)
mg/dl as for aspA).
15 pCABD2 + p.APT 1SOO
Example 4 pCABD2 + p.APTK 1SOO
pCABD2 + pKD 1600
<1> Construction of Plasmids Containing pCABD(B) + pKD 1590
Phosphoenolpyruvate Carboxylase Gene (ppc),
Transhydrogenase Gene (pntAB), pCABDE1 + pKD 1580
Aspartate-Semialdehyde Dehydrogenase Gene (asd)
and Aspartase (aspA) Gene
As clearly seen from the results shown in Table 4, when asd
A plasmid containing ppc, pntAB, asd and aspA was con was enhanced together with dap A+lySC+dapB+ddh+ppc--
structed as follows. pntAB+asp A, marked increase of the L-lysine production
25
The plasmid paPT described in the aforementioned amount was observed. A similar result was obtained when
Example 3 was digested with HindIII, blunt-ended for the pCABD(B) or pCABDE1 was used instead of pCABD2.
both ends, and further digested with SmaI to obtain a DNA
fragment containing ppc, asp.A and pntAB. Then, pMW218 The plasmidpAPTK mentioned in Table 4 corresponded to
(Nippon Gene) was digested with EcoRI and blunt-ended for pAPT of which drug resistance gene was changed from that
the both ends, and the previously obtained DNA fragment 30 for amplicillin to that for kanamycin (because the vector was
containing ppc, aspA and pntAB was inserted into the blunt changed from pMW 118 to pMW218). Since pKD was pre
ended EcoRI cleavage site to obtain paPTK (FIG. 15). pared by inserting asd into pAPTK, it was considered that
Then, pasd described in International Publication No. pAPTK was more suitable than pAPT as control of pKD.
WO95/16042 was digested with KpnI, blunt-ended for the Therefore, the data of p APTK is also shown. It was also
both ends, and further digested with BamHI to obtain a DNA 35
confirmed that the L-lysine production amount was not influ
fragment containing asd. Then, pSTV29 was digested with
Pst, blunt-ended for the both ends, and inserted with the enced even if the drug resistance gene was changed.
previously obtained asd fragment at the BamHI cleavage site
to obtain pSd (FIG. 16). INDUSTRIAL APPLICABILITY
Then, pSq was digested with SphI, blunt-ended for the both 40
ends, and further digested with Xbal to obtain a DNA frag
ment containing asdagain. Then, pAPTK was digested with The present invention provides Escherichia bacteria with
Sad and blunt-ended for the both ends, and the previously high L-lysine productivity, and L-lysine can be obtained with
obtained asd fragment was inserted at the Xbal cleavage site a high yield by using these bacteria.
to obtain pKD (FIG. 17). 45 While the invention has been described in detail with ref
<2> Introduction of Four Kinds of Genes and erence to preferred embodiments thereof, it will be apparent
Evaluation of L-Lysine Productivity to one skilled in the art that various changes can be made, and
equivalents employed, without departing from the scope of
To a transformant into which pCABD2, pCABD(B) or the invention. Each of the aforementioned documents is
pCABDE1 was introduced, which were obtained in the afore incorporated by reference herein in its entirety.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 6

<21 Oc SEO ID NO 1
<211 LENGTH: 2O
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<22 Os FEATURE;
<223> OTHER INFORMATION: Primer for amplification of a promoter portion
of tet

<4 OOs SEQUENCE: 1

tdaagaattic to atgtttga
 US 8,062,869 B2
21 22
- Continued

<210s, SEQ ID NO 2
&211s LENGTH: 35
&212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence
22 Os. FEATURE:
<223> OTHER INFORMATION: Primer for amplification of a promoter portion
of tet

<4 OOs, SEQUENCE: 2

gttagatttg gtaccc.ggtg cct gactg.cg ttagc 35

<210s, SEQ ID NO 3
&211s LENGTH: 30
&212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence
22 Os. FEATURE:
<223> OTHER INFORMATION: Primer for amplification of dapA.
<4 OOs, SEQUENCE: 3

ggttgttggta CCC ccalaatgagggaagaag 3O

<210s, SEQ ID NO 4
&211s LENGTH: 2O
&212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence
22 Os. FEATURE:
<223> OTHER INFORMATION: Primer for amplification of dapA.
<4 OOs, SEQUENCE: 4

tggalacct ct gttgctgcag 2O

<210s, SEQ ID NO 5
&211s LENGTH: 2O
&212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence
22 Os. FEATURE:
<223> OTHER INFORMATION: Primer for amplification of asp.A.
<4 OOs, SEQUENCE: 5

tgat cagoga aac acttitta 2O

<210s, SEQ ID NO 6
&211s LENGTH: 19
&212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence
22 Os. FEATURE:
<223> OTHER INFORMATION: Primer for amplification of asp.A.
<4 OOs, SEQUENCE: 6

Cagcaaacta tatgagaa 19

What is claimed is: dihydrodipicolinate synthase prior to said mutation(s)

1. A method of producing L-lysine comprising: is the amino acid sequence of wild-type dihydrodipi
A)Syring in a medium an Escherichia coli bacterium in colinate synthase of Escherichia coli,
60
(a) the intracellular activities of all of the following aspartokinase,
enzymes are enhanced: dihydrodipicolinate reductase,
dihydrodipicolinate synthase which comprises a muta
tion replacing at least one amino acid selected from phosphoenolpyruvate carboxylase,
the group consisting of the alanine at the 81st position 65 nicotinamide adenine dinucleotide transhydrogenase,
and the histidine at the 118th position with another and
amino acid, wherein the amino acid sequence of said aspartate-semialdehyde dehydrogenase; and
 US 8,062,869 B2
23 24
(b) the intracellular activities of at least one of the follow (m) replacement of the glycine at the 323rd position and the
ing enzymes are enhanced: i) diaminopimelate dehydro methionine at the 318th position with another amino
genase, or acid; and
ii) tetrahydrodipicolinate Succinylase and Succinyldiami (n) combinations thereof,
nopimelate deacylase, wherein said activities are wherein the amino acid sequence of said aspartokinase
enhanced by a method selected from the group consist prior to said mutation(s) is the amino acid sequence of
ing of wild-type aspartokinase III of Escherichia coli.
a) introducing a promoter or promoters which are operably 3. The method of claim 1, wherein the alanine at the 81
linked to the gene encoding the enzymes into the chro position is replaced with valine, and the histidine at the 118"
mosome of said bacterium, position is replaced with tyrosine.
b) increasing the copy number of the genes encoding the 4. The method of claim 2, wherein
enzymes by introducing one or more expression vectors (a) the glycine at the 323" position is replaced with aspar
into said bacterium, and
c) combinations thereof; tic acid;
B) allowing L-lysine to accumulate in said medium or said 15 (b) the glycine at the 323" and 408" positions is replaced
bacterium; and with aspartic acid;
C) collecting L-lysine from the medium or the bacterium. (c) the arginine at the 34" position is replaced with cysteine
2. The method according to claim 1, wherein said aspar and the glycine at the 323" position is replaced with
tokinase comprises a mutation selected from the group con aspartic acid;
sisting of: (d) the leucine at the 325" position is replaced with phe
(a) replacement of the glycine at the 323rd position with nylalanine;
another amino acid; (e) the methionine at the 318" position is replaced with
(b) replacement of the glycine at the 323rd and 408th isoleucine;
positions with another amino acid; (f) the methionine at the 318" position is replaced with
(c) replacement of the arginine at the 34th position and the 25 isoleucine and the valine at the 349" position is replaced
glycine at the 323rd position with another amino acid; with methionine;
(d) replacement of the leucine at the 325th position with (g) the serine at the 345" position is replaced with leucine;
another amino acid; (h) the valine at the 347" position is replaced with methion
(e) replacement of the methionine at the 318th position 1ne,
with another amino acid; 30 (i) the threonine at the 352" position is replaced with
(f) replacement of the methionine at the 318th position and isoleucine;
the valine at the 349th position with another amino acid; (j) the threonine at the 352" position is replaced with
(g) replacement of the serine at the 345th position with isoleucine and the serine at the 369' position is replaced
another amino acid; with phenylalanine;
(h) replacement of the valine at the 347th position with 35 (k) the glutamic acid at the 164" position is replaced with
another amino acid; lysine;
(i) replacement of the threonine at the 352nd position with (1) the methionine at the 417" position is replaced with
another amino acid; isoleucine and the cysteine at the 419' position is
() replacement of the threonine at the 352nd position and replaced with tyrosine;
serine at the 369th position with another amino acid; 40 (m) the glycine at the 323" position is replaced with aspar
(k) replacement of the glutamic acid at the 164th position tic acid and the methionine at the 318" position is
with another amino acid; replaced with isoleucine.
(1) replacement of the methionine at the 417th position and
the cysteine at the 419th position with another amino
acid;