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Applied and Environmental Microbiology, July 2001, p. 3064-3070, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.3064-3070.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Overproduction of L-Lysine from Methanol by
Methylobacillus glycogenes Derivatives Carrying a
Plasmid with a Mutated dapA Gene
Hiroaki
Motoyama,*
Hiroshi
Yano,
Yoko
Terasaki, and
Hideharu
Anazawa
Tokyo Research Laboratories, Kyowa Hakko
Kogyo Co. Ltd., Asahi-machi, Machida-shi, Tokyo 194, Japan
Received 21 December 2000/Accepted 10 April 2001
 |
ABSTRACT |
The dapA gene, encoding dihydrodipicolinate synthase
(DDPS) partially desensitized to inhibition by L-lysine,
was cloned from an L-threonine- and
L-lysine-coproducing mutant of the obligate methylotroph
Methylobacillus glycogenes DHL122 by complementation of
the nutritional requirement of an Escherichia coli dapA
mutant. Introduction of the dapA gene into DHL122 and
AL119, which is the parent of DHL122 and an L-threonine
producing mutant, elevated the specific activity of DDPS 20-fold and
L-lysine production 2- to 3-fold with concomitant reduction
of L-threonine in test tube cultures. AL119 containing the
dapA gene produced 8 g of L-lysine per
liter in a 5-liter jar fermentor from methanol as a substrate. Analysis
of the nucleotide sequence of the dapA gene shows that
it encodes a peptide with an Mr of 30,664 and that the encoded amino acid sequence is extensively homologous to
those of other organisms. In order to study the mutation that occurred in DHL122, the dapA genes of the wild type and AL119
were cloned and sequenced. Comparison of the nucleotide sequences of
the dapA genes revealed that the amino acid at residue
88 was F in DHL122 whereas it was L in the wild type and AL119,
suggesting that this amino acid alteration that occurred in DHL122
caused the partial desensitization of DDPS to the inhibition by
L-lysine. The similarity in the amino acid sequences of
DDPS in M. glycogenes and other organisms suggests that
the mutation of the dapA gene in DHL122 is located in
the region concerned with interaction of the allosteric effector,
L-lysine.
| |
INTRODUCTION |
Methanol, a compound easily
synthesized from natural gas, is an attractive raw material for
microbial industries. Using methanol as a carbon source, production
costs could be greatly reduced and purification and waste treatment
processes could be simplified. A number of production processes for
useful compounds with methylotrophs (methanol-utilizing microorganisms)
have been studied. Production of single-cell protein by a gram-negative
methylotroph, Methylophilus methylotrophus, was extensively
studied in the 1970s and finally industrialized (1).
Efficient production systems for recombinant proteins were also
constructed with the methanol-utilizing yeast Pichia
pastoris (4).
Many attempts have also been made to use methanol in amino acid
production; however, successful studies were limited. Lee et al.
(9) reported the production of 47 g of
L-lysine per liter by a gram-positive methylotroph,
Bacillus methanolicus. Izumi et al. (7)
reported efficient conversion of glycine to L-serine by a gram-negative methylotroph,
Hyphomicrobium methylovorum. Breeding of amino
acid-producing mutants requires isolation of mutants desensitized in
the feedback regulation of the biosynthetic enzymes for a desired amino
acid and blocked in metabolic pathways of by-products. Isolation of
mutants from methylotrophs is usually difficult, due to unknown
reasons, preventing the use of methanol in amino acid production.
We isolated L-glutamic acid-hyperproducing mutants from the
obligate methylotrophs Methylobacillus glycogenes ATCC 21276 and 21371 and subsequently derived L-lysine- and
L-threonine-producing mutants from
L-glutamic acid-producing mutants
(13). An L-threonine-producing mutant, AL119, was isolated among mutants resistant to both
DL-
-amino-
-hydroxy-valeric acid and
L-lysine, and an L-lysine- and
L-threonine-coproducing mutant, DHL119, was
isolated among 2,6-diamino-4-hexenoic acid hydrochloride-resistant
mutants derived from AL119. Enzymatic analysis revealed that
aspartokinase, the common regulatory enzyme in the biosynthesis of
L-lysine and L-threonine,
was insensitive to the feedback inhibition by
L-lysine in AL119 and DHL122 and that
dihydrodipicolinate synthase (DDPS), the key regulatory enzyme located
at the branch point of L-lysine and
L-threonine biosynthesis, was partially
insensitive to the feedback inhibition by
L-lysine in DHL122 (14). We
concluded that desensitization of these regulatory enzymes led to the
production of L-threonine and
L-lysine.
As is the case for other gram-negative methylotrophs, isolation of
mutants from M. glycogenes was not easy. In order to
efficiently enhance amino acid production, we tried to employ a
recombinant technology. The hom and thrC genes,
which encode homoserine dehydrogenase and threonine synthase in the
L-threonine biosynthesis pathway, respectively,
were cloned from M. glycogenes (15) and
introduced into L-threonine-producing mutants.
The strains amplified with these genes produced more
L-threonine than their parents (16). We reasoned that amplification of the dapA gene of DHL122,
which encodes desensitized DDPS in DHL122, might greatly elevate
L-lysine production. We report here the effects
of the dapA gene of DHL122 on amino acid production from
methanol by M. glycogenes and the analysis of the nucleotide
sequence of the dapA gene.
| |
MATERIALS AND METHODS |
Bacterial strains and plasmids.
The bacterial strains and
plasmids used in this study are listed in Table
1.
Media and culture methods.
M. glycogenes ATCC
21276 and the derived mutants were cultivated as described by us
previously (13). Methanol was used as a sole carbon
source. Escherichia coli cells were cultivated as described
previously (10). M9S5 medium (M9 medium
[10] supplemented with 50 mg each of 19 amino acids
[except L-lysine] per liter) was used for
complementation testing of E. coli AT997. Tetracycline (10 mg/liter) or ampicillin (100 mg/liter) was used to supplement liquid or
agar media to cultivate M. glycogenes and E. coli
strains containing plasmids.
DNA manipulation.
DNA restriction enzyme digestion,
separation of DNA fragments by gel electrophoresis, and transformation
of E. coli strains were performed by standard methods as
described previously (10). Restriction enzymes and DNA
ligase were supplied by Takara Shuzo Co. Ltd. (Kyoto, Japan). Southern
hybridization and colony hybridization were done with a DNA labeling
and detection kit (Boehringer Mannheim) according to the method
recommended by the supplier. Chromosomal DNAs of M. glycogenes strains were prepared by the method described by Marmur
(11).
Cloning of the dapA gene.
The dapA
gene of DHL122 was isolated by complementation of E. coli
AT997 with a lesion in dapA as follows. The chromosomal DNA
of M. glycogenes DHL122 was partially digested with
Sau3AI and separated by agarose gel electrophoresis. The 2- to 6-kb DNA fragments purified from the gel were ligated into the
BamHI site of pUC19 to construct the gene library and
transform E. coli AT997. The cells were plated onto M9S5
medium supplemented with 100 mg of ampicillin per liter and incubated
overnight at 37°C. Plasmids isolated from the resulting colonies were
further analyzed. The dapA genes of the wild type and AL119
were isolated by colony hybridization with the dapA gene of
DHL122 as a probe. The chromosomal DNAs of M. glycogenes
ATCC 21276 and AL119 were completely digested with EcoRI and
separated by 10 to 40% sucrose density gradient centrifugation. The 2- to 4-kb DNA fragments were collected, ligated into the EcoRI
site of pMW119, and transformed into E. coli DH5. The cells
were plated onto L broth (10) supplemented with 100 mg of
ampicillin per liter, and the resulting colonies were examined by
colony hybridization with the 1.7-kb EcoRI fragment
containing the dapA gene of DHL122. Plasmids isolated from
the positive colonies were analyzed.
Subcloning of plasmids.
The 1.7-kb EcoRI fragment
of pDYO1 was introduced into the EcoRI site of pUC19 to
construct pDYO2. The 2.4-kb PstI fragment of pDYO1 was
inserted into the PstI site of pUC19 to form pDYO4-1 and
pDYO4-2, in both orientations. pDYO6 was formed by the self-ligation of
the 6.3-kb EcoRV fragment of pDYO1. The 2.4-kb
PstI fragment of pDYO4-2 was inserted into the
PstI site of pMFY42 to construct pDYOM4-2.
Conjugation.
Introduction of plasmids from E. coli S17-1 to M. glycogenes by conjugation was done as
described previously (16).
DNA sequencing and analysis.
DNA sequencing was performed by
the dideoxy chain termination method on both strands with an Applied
Biosystems model 373A sequencer. DNA sequences analyses and homology
alignments of amino acid sequences were carried out using GENETYX MAC
version 9.0 (Software Development Co. Ltd., Tokyo, Japan).
Analysis.
Bacterial growth was measured by the increase in
absorbance at 660 nm, and amino acids in culture supernatants were
measured by high-pressure liquid chromatography as described previously (13). Cell extracts of the M. glycogenes
transconjugants were prepared and DDPS activities were measured as
described previously (14).
Nucleotide sequence accession number.
The nucleotide
sequence of the 1.7-kb EcoRI fragment encoding DDPS of
M. glycogenes ATCC 21276 will appear in the EMBL, GenBank, and DDBJ nucleotide sequence data libraries under accession no. AB038266.
| |
RESULTS |
Cloning of the dapA gene of M.
glycogenes DHL122.
We attempted to isolate the
dapA gene of M. glycogenes DHL122 by
complementation of an E. coli DDPS-deficient mutant,
E. coli AT997, which has a lesion in dapA and
requires L-diaminopimelic acid for growth
(20). This strain was transformed by the gene library of M. glycogenes DHL122 in a vector plasmid, pUC19,
and the cells were inoculated onto M9S5 medium that did not contain L-diaminopimelic acid. A plasmid with a 3.9-kb
insert, designated pDYO1, was obtained from a colony that appeared on
the M9S5 medium. It complemented the nutritional requirement of AT997
by retransformation and was analyzed further. Subcloning of pDYO1 and
complementation analysis with AT997 revealed that the dapA
gene was localized in the 1.7-kb EcoRI fragment (Fig.
1). Both pDYO4-1 and pDYO4-2, which had
the same 2.4-kb PstI fragment in both orientations, complemented E. coli AT997, suggesting that the promoter of
the dapA gene was included in the fragment and was
functional in E. coli.
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FIG. 1.
Restriction maps of the subcloned DNA fragments
containing the dapA gene isolated from M.
glycogenes DHL122. The arrow indicates the direction of the ORF
of the dapA gene as determined by the nucleotide
sequence. Complementation activities conferred by each plasmid in
E. coli dapA mutant AT997 are indicated by + or  .
P lac and P km indicate the locations of
the lac promoter of pUC19 and the promoter of the
kanamycin resistance gene of pMFY42, respectively. B,
BamHI; EI, EcoRI; EV,
EcoRV; Ps, PstI; Sp,
SphI.
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|
Effects of the dapA gene of M.
glycogenes DHL122 on amino acid production from methanol.
In order to examine the effect of the dapA gene on amino
acid production from methanol, the 2.4-kb PstI fragment of
pDYO4-2 was inserted into the PstI site of a
broad-host-range plasmid, pMFY42. The plasmid thus constructed,
pDYOM4-2, was introduced into E. coli S17-1, a strain which
could mobilize plasmids from E. coli to other gram-negative
microorganisms (19), and then E. coli S17-1
containing the plasmid was conjugated with M. glycogenes DHL122 and its parental L-threonine producer,
AL119, to transfer the plasmid. The transconjugants containing the
vector were constructed in the same manner. Restriction analysis of the
plasmids isolated from the M. glycogenes transconjugants
showed the same structures as in the E. coli transformants
from which the plasmids were transferred (data not shown).
Introduction of the
dapA gene elevated the DDPS activities
about 20-fold and reduced the growth of DHL122 and AL119 in test
tube
cultures (Table
2). The
L-lysine in the culture supernatants
increased
two- to threefold, whereas the accumulation of
L-threonine
and
L-glutamic
acid was reduced. We thought that the growth reduction
of the strains
with the
dapA genes might be caused by a shortage
of
cofactors, such as ATP and NADPH, the compounds required for
both
L-lysine biosynthesis and microbial growth. The
enhanced
L-lysine biosynthesis caused by the
elevation of the DDPS activity
might consume a considerable amount of
cofactors. As sufficient
amounts of the cofactors might not be
generated due to the limited
oxygen supply in test tube cultures, this
might cause the growth
to be reduced.
To circumvent the drawback of test tube cultures, we examined the
effect of the
dapA gene by cultivating the strains in
5-liter
jar fermentors, where the oxygen supply was more favorable than
in test tube cultures. Figure
2 shows the
time courses of cultivation
of the AL119 and DHL122 strains with or
without the
dapA gene.
The growth of the strains with the
dapA gene was comparable to
that of the strains without the
dapA gene in 5-liter jar fermentors.
L-Lysine accumulation was greatly enhanced by the
introduction
of the
dapA gene and was accompanied by the
reduction of
L-threonine
accumulation as in test
tube cultures. DHL122 containing pDYOM4-2
and AL119 harboring pDYOM4-2
accumulated 5.3 and 8 g of
L-lysine
per
liter, respectively, after 72 h of cultivation. All of the
transconjugants constructed produced more than 30 g of
L-glutamic
acid per liter, which was much higher
than the accumulation of
L-lysine and
L-threonine, in 5-liter jar fermentors.
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FIG. 2.
Time courses of amino acid production in 5-liter jar
fermentors. (A) DHL122/pMFY42; (B) DHL122/pDYOM4-2; (C) AL119/pMFY42;
(D) AL119/pDYOM4-2.  , optical density (OD) at 660 nm;  , Lys;  ,
Thr; ×, Glu.
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|
Analysis of the nucleotide sequence of dapA and the
location of the mutation in DHL122.
The 1.7-kb EcoRI
fragment of pDYO2 was sequenced on both strands, and a single open
reading frame (ORF) was found (Fig. 3). The ORF encodes a predicted peptide with an
Mr of 30,664, initiating at the ATG
codon (nucleotides 472 to 474) and terminating at the TGA codon
(nucleotides 1342 to 1344). Neither distinct promoter-like nor
distinct terminator-like sequences were found in the upstream and downstream regions of the ORF, respectively. To identify the mutation in DHL122, the 1.7-kb EcoRI fragments containing
the dapA gene were cloned from the wild type, ATCC 21276, and AL119 and sequenced. The nucleotide sequences of the 1.7-kb
EcoRI fragments from ATCC 21276 and AL119 were identical to
that from DHL122 except at nucleotide 733. Nucleotide 733 is C in ATCC
21276 and AL119, whereas it is T in DHL122, which altered the amino
acid residue L88 in ATCC 21276 and AL119 to F88 in DHL122. The mutation
in this amino acid residue was considered to cause the partial
desensitization of DDPS of DHL122 to feedback inhibition by
L-lysine (14).
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FIG. 3.
Nucleotide sequence of the 1.7-kb EcoRI
fragment containing the dapA gene of M.
glycogenes ATCC 21276. The putative amino acid sequence is
shown below the nucleotide sequence. The start codon, ATG, and the stop
codon, TGA, are underlined. The location of the mutation found in
DHL122 (nucleotide 733) is indicated.
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|
Comparison of the predicted amino acid sequences of the
dapA genes.
The predicted amino acid sequence of
the dapA gene of M. glycogenes was compared with
those from other organisms (Fig. 4). Extensive amino acid sequence homology was found between M. glycogenes and other organisms. The amino acid sequence of
M. glycogenes has identities of 52.4% (153 of 292 amino
acid residues), 47.2% (137 of 290 amino acid residues), 35.5% (107 of
301 amino acid residues), 29.4% (96 of 326 amino acid residues), and
25.3% (96 of 380 amino acid residues) with those from E. coli, Bacillus subtilis, Corynebacterium
glutamicum, Nicotiana sylvestris, and Zea
mays, respectively.
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FIG. 4.
Homology between the amino acid sequences of the DDPSs
of M. glycogenes ATCC 21276 and those of other organisms.
Conserved amino acid residues found in more than four species are
shaded. Asterisks indicate the putative active sites of the DDPS. Amino
acid residue 88, which was found to be mutated in DHL122, is indicated
as F(DHL122) above the sequence. The amino acid residues whose
mutations were reported to cause the desensitization of DDPSs of other
organisms are indicated by plus signs: E. coli, A81 V
and H118Y (8); Z. mays, S157N,
E162K, and A166T,V (17); and N.
sylvestris, N104I (6). MG, M.
glycogenes; EC, E. coli; BS, B.
subtilis; CG, C. glutamicum; NS, N.
sylvestris; ZM, Z. mays.
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| |
DISCUSSION |
In our previous study we derived an L-lysine and
L-threonine coproducer, DHL122, from an
L-threonine producer, AL119 (13). However,
DHL122 could produce only a small amount of L-lysine. We
report here that the introduction of the dapA gene into
DHL122 and AL119 greatly elevated the specific activity of DDPS and
L-lysine production with concomitant reduction of
L-threonine accumulation. This suggests that the
carbon flow from L-aspartic acid to
L-lysine was limited at the conversion of
L-aspartic acid--semialdehyde to
dihydrodipicolinate in both strains and was liberated by the amplification of the dapA gene with the redirection of the
carbon flow from toward L-threonine to toward
L-lysine. We found that DDPS of DHL122,
from which the dapA gene was cloned, was partially desensitized to the feedback inhibition by
L-lysine (14). The altered
regulatory property of the enzyme was thought to contribute to the
increased production of L-lysine. However, we
believe that this effect was partial. DHL122, which had mutated
DDPS, produced more L-lysine than AL119, which
had wild-type DDPS, but the production of
L-lysine by DHL122 was less than that of
L-threonine, suggesting that the partial
desensitization of DDPS was not enough to change the carbon flow from
toward L-threonine to toward
L-lysine (13) (Fig. 2A and
C). The introduction of the dapA gene-containing plasmid
drastically enhanced the enzyme activity, and the production of
L-lysine greatly exceeded that of
L-threonine (Fig. 2B and D). We speculate that
the elevated DDPS activity mainly caused the overproduction of
L-lysine in the strains with the
dapA-containing plasmid.
AL119 and DHL122 were derived from an L-glutamic acid
producer, iA111 (13). The transconjugants bearing the
dapA gene produced much more
L-glutamic acid than
L-lysine and L-threonine.
The metabolic flow from methanol is possibly limited at some steps in
the biosynthesis of L-aspartic acid and directed
toward the formation of L-glutamic acid. To
further enhance L-lysine production, it is
necessary to reduce the metabolic flow toward
L-glutamic acid and direct it toward
L-aspartic acid. In the course of breeding
L-lysine-producing mutants from a C. glutamicum L-glutamic acid producer,
pyruvate kinase deficiency (18), reduction of citrate
synthase activity, and desensitization of phosphoenolpyruvate
carboxylase (21) were reported to be effective to reduce
the production of L-glutamic acid and enhance
that of L-aspartic acid and
L-lysine. These strategies may also be applicable
for M. glycogenes. The isolation of mutants with lesions in
the enzymes of the tricarboxylic acid cycle and those altered in the
regulation of the enzymes of the biosynthesis of
L-aspartic acid, together with the amplification
of biosynthetic genes of L-aspartic acid, should
be considered.
The dapA gene was cloned from M. glycogenes and
sequenced, and the amino acid sequence was found to have extensive
homology with those from other organisms. In E. coli
(12) and N. sylvestris (2), DDPS
was shown to consist of homotetramers by X-ray crystallography studies.
The homology throughout the amino acid sequence between DDPS from
M. glycogenes and those from E. coli and N. sylvestris suggests that DDPS of M. glycogenes has a
similar structure. Blickling and Knäblein (3)
proposed that in E. coli and other organisms K161, Y133, and
R138 (numbering refers to the E. coli and M. glycogenes sequences) are involved in significant roles in
catalysis
the formation of Schiff bases with pyruvate, proton
shuttling during imine formation and transimination, and interaction
with the carboxy group of L-aspartatic
acid--semialdehyde, respectively. These residues are
conserved in M. glycogenes and might have the same roles in
catalysis as in other organisms. It was also suggested that in E. coli the residues H53, H56, Y106, Y107, N80, and E84 were involved
in the interaction with an allosteric effector, L-lysine (3). Most of the mutations
that have been reported to cause the desensitization of E. coli and plant DDPSs (marked by plus signs in Fig. 4) were found
in the region between amino acid residues 79 and 88. The mutation in
the DDPS of DHL122 (F88) is also located in this region, suggesting
that the mutation that occurred in this region might alter the
structure of DDPS, prevent the efficient interaction between
L-lysine and other amino acid residues (possibly
N80 and E84), and lead to the partial desensitization of
DDPS to L-lysine. DDPS of DHL122 was shown to be
inhibited by high concentrations of L-lysine
(14). The DDPS might be further desensitized by
alteration of other amino acid residues that were found to be effective
for the desensitization of other DDPSs. The availability of more
desensitized DDPSs constructed in such ways will be an important tool
to improve L-lysine production from methanol by
M. glycogenes.
| |
ACKNOWLEDGMENTS |
We thank Y. Yonetani for analyzing the nucleotide sequence and K. Hasegawa, K. Honma, and K. Maki for their skillful work in DNA
manipulation and cultivation of microorganisms.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Development
Department, Bio-Chemicals Company, Kyowa Hakko Kogyo Co. Ltd., 1-6-1 Ohtemachi, Chiyoda-ku, Tokyo 100-8185, Japan. Phone: 81-3-3282-0995. Fax: 81-3-3284-1839. E-mail: hmotoyama{at}kyowa.co.jp.
| |
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Applied and Environmental Microbiology, July 2001, p. 3064-3070, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.3064-3070.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
