Mol. Cells, Vol. 4. pp.

295-299

Cloning and Functional Expr~ssion of the ddh Gene Involved

in the Novel Pathway of Lysine Biosynthesis from
Brevibacterium lactofermentum

Jung Hyeob Roh, Ok Mi Kim, Dong Chul Park, Hyun Jeong Kim, Hyon Kyong Yun,
Sang Dal Kim I, In-Seon Lee 2 and Kap Rang Lee*
IDepartment of Food & Nutrition and Department of Applied Microbiology, Yeungnam University,
Kyongsan 712-749, Korea; 2Department of Food Technology and Science, Keimy ung University,
Taegu 704-701, Korea

(Received on May 24, 1994)

The Brevibacterium lactoJermentum ddh gene encoding meso-diaminopimelate (meso-DAP)-dehy-

drogenase (DDH) was cloned by complementation of the Escherichia coli dapD mutation. Sub-
cloning experiments and complementation tests indicated that the region for DDH activity
was within the ~2.5 kb XhoI fragment. Southern hybridization analysis confirmed that the
cloned DNA fragment originated from B. lactoJermentum genomic DNA. The activity staining
showed that a specific band of DDH activity was observed in the strains carrying each subclone.

L-Iysine biosynthesis proceeds in procaryotes th-
rough three different pathways (Schrumpf et aI., 1991)
(Fig. 1). The diaminopimelate (DAP) pathway invol-
ving succinylated intennediates requires four steps
from tetrahydrodipicolinate (THOP) to meso-diamino-
pimelate (meso-OAP) and has been extensively studied
in E coli (Aida et aI., 1986). In Bacillus sphaericus,
THOP is converted by a si ngle enzym atic step to
meso-OAP, catalyzed by meso-OAP-dehydrogenase
(OOH) (White, 1983). OOH has been purified and
characterized to the extent that it consists of two iden-
tical subunits (80,000 MW) of B. sphaericus (Misono
and Soda, 1980a). We refer to this step as the OOH
pathway. It is presumed that the DDH pathway has
more merit by not requiring acetyl-CoA or succinyl-
CoA and abbreviating the enzymatic reaction of four
steps as found in the OAP pathway. The pathway
involving acetylated intennediates is employed in cer-
tain Bacillus species (Schrumpt et al., 1991). Corynebac-
terium glutamicum, a gram-positive bacteria, has both
the OAP pathway and OOH pathway (Misono et aI.,
1986b; Cremer et al., 1988). This organism is widely
used for the industrial production of amino acids be-
cause it produces higher levels of the lysine than do
the strains utilizing only one pathway (Yeh et al.,
1988).
In Brevibacterium lactofermentum , a lysine producer,
there is considerable confusion about the lysine bio-
synthetic pathway. Tosaka and Takinami (1978) have
reported that lysine biosynthesis in B. lactofermentum
proceeds through the OAP pathway' as found in E
coli. In contrast, Misono et al. (1986a) have reported
* To whom correspondence should be addressed.
that OOH activity occurs in Brevibacterium sp. IC R
7000. W e presumed that the OOH pathway in addi-
tion to the OAP pathway is utilized by B. lactofermen -
tum , and then we measured the OOH activity, confir-
ming the existence of the OOH pathway (unpublished
data).
Therefore, characterizing the expression of genes in-
volved in the DOH pathway is an important first step
toward understanding th e lysine pathway of B. laClofer-
mentum a nd metabolic control for high productivity
of lysine. Functional expression in E coli of genes
(lysA a nd trp4) from B. IactoJermentum has been done
in several cases (Oel Real et al., 1985; M arquez et
aI., 1985). Because the ddh gene is a ble to bypass the
block in the OAP pathway by expressing OOH acti-
vity (Fig. 1), we used E coli dapD mutant to isolate
the B. lactofermentum ddh gene.
In this paper, we report the cloning of the B. lac-
toJermentum ddh gene by complementation of the E
coli dapD mutation. We also describe the expression
of the ddh clone by measuring DOH activity.
Materials and Methods
Chemicals and enzymes
Restriction enzymes were purchased fro m Promega,
Kosco and Boehringer Mannheim. T4 ONA ligase
and calf-intestinal alkaline phosphatase were from
Promega. The nick translation kit and [a-32P]CTP
The abbreviations used are: DAP, diaminopimelate; THDP,
tetra hydrodipicolinate; meso-DAP, meso-diaminopimelate;
DDH, meso-DAP-dehydrogenase; Ap, ampicillin; Tc, tetra-
cycline.

© 1994 The Korean Society for Molecular Biology

296 Cloning and Expression of the B. lactojelmentum ddh Gene Mol. Cells

L-Aspa rtate
concentration of 10 mg/ml. Large-scale preparation of
L-'8-Aspart)'~ phospha t e plasmid DNA and rapid analysis of recombinant pla-
L-Asparta te O!emi3Ideh)'de smids were carried out by the alkaline lysis procedure
L-2. 3-0ihYdr!i pi ca l ina le (Birnboim and Doly, 1979). E. coli strains were trans-
L-;l. ' - Tetrah~rodipicOI inate~ formed by either the CaCh (Sambrook et al., 1989)
Acetylase ~ B1 Succiny l ase(dap 0)
or the hexaminecobalt chloride method (Hanahan,
1985). By employing the phenotypic complementation
L-N-AcetY l - z-fmin: L- N- Succ inyl - 2-amino- C
6- ketoPime r te 6- ketop imelate test, the DAP + and ApR transformants obtained from
Transaminas.e !T ransaminase(d8P C) the E. coli dapD mutant (AT986) were selected from
L, L- N-Acely -2 .6- L. L-N-Succi nyi - 2. 6- meso-Di amino-
their growth on supplemented M9/Ap plates without
diaminopimelate diaminop ime iale pilt21ate
Deacetyl ase" . l Desucc i nyl ase( dap £) dehydrogenase ( ddh)
DAP after 48 hs incubation.
L'L-2.6-Di8minOPimelat~
! Epimerase(dap F) Construction of a B. lactofermentum genomic library
meso-Oi ami nopi me I ale B. lactojermentum ATCC13869 DNA was partially
1 Decarboxy l ase ( Jys A)
L- Lysine
.
.
digested with Sau3A., and large restriction fragments
(> 4 kb) were isolated by sucrose gradient centrifuga-
Figure L Biosynthetic pathway of L-Iysine in procaryotes. tion. The Sau3A fragments were then ligated with pBR
The pathway A involving acetylated intermediates and the 322 DNA which was digested with BamHI and calf
pathway B (DAP pathway) involving succinylated interme- intestinal alkaline phosphatase (Sambrook et al., 1989).
diates operate in certain Bacillus species and E. coli, respecti- The ligation mixture was used to transform E. coli
vely. The pathway C (ddh pathway), in which meso-diamino- JM83. The library obtained consisted of approximately
pimelate is directely formed from L, d '-tetrahydrodipicoli- 5,800 independent colonies of which 99% contained
nate, is used by B. sphaericus, and C gluta~icum uses both
a B. lactojermentum insert. Analysis of several represen-
pathway B and pathway C.
tative clones showed that the average size of the plas-
mid inserts was 10 kb.

were from Amersham. Thiamine, DAP (mixture of Nick translation and Southern hybridization
LL, DD-and meso-isomers), ampicillin (Ap) and tetra- An insert fragment of recombinant plasmid was la-
cycline (Tc) were from Sigma. beled by nick translation to a specific activity of 2
X 108 cpm/ flg with [a-32P]CTP. The labeled DNA
Bacterial strains and plasmids was used for hybridization at 42 t with a blotted
A wild-type strain, B. lactojermentum ATCC13869 DNA fragment on a nylon membrane by the proce-
(KCTC 1844), was the DNA donor for the genomic dure of Southern (1975).
library. E. coli JM83 [ara, rspL, ll(lac-proAB) <1>80, lac-
ZllM15] and E. coli JM109 [recAl, supE44, endAI, hsdR A ctivity staining of DDH
17, gyrA96, relA I, thill(lac-proAB) F(traD36 proA +B + 1a- Activity staining for DDH using the crude extracts
cIq lacZllMI5)] were used as the host for the B. lacto- of E. coli and B. lactojermentum was done according
jermentum ATCC13869 genomic library and the host to the methods of Misono and Soda (1980b) and
for propagating plasmids, respectively. E. coli AT986 Ishino et al. (1984). The gel after polyacrylamide gel
(dapD8, relAI, spoTl, thi-I , A. - ), obtained from Dr. B. electrophoresis was stained for the location of DDH
Bachmann, Yale University, U.SA. (Bachmann, 1983), with staining solution, composed of 4.3 mM NADP +,
was the recipient for transfornlation with library DNA. 0.56 mM tetrazolium salt, 0.28 mM phenazine meso-
Plasmids pBR322 and pBluescriptlI SK + were used sulfate (PMS), 50 mM Tris' HCI buffer (PH 7.4) and
as the cloning vector. 43 mM meso-DAP.

Media and growth condition Enzyme assay

LB medium (Sambrook et al., 1989) was used to DDH activity was determined at 25 °C by measu-
grow E. coli and B. lactojermentum ATCC13869. M9 ring the rate of an increase in the absorbance at 340
medium (Sambrook et al., 1989) was used as minimal nm, essentially as described by Yeh et al. (1988). The
medium for the growth of E. coli. DAP (50 flg/ml), reaction mixture contained 10 mM DAP, 0.1 mM
thiamine (0.1 mM), Ap (50 flg/ml) and Tc (15 flg/ml) NADP +, 0.5 M glycine-KCI-KOH buffer (PH 10.5)
were added to culture media when appropriate. E. coli and the crude extract in a final volume of 1.0 ml.
and B. lactojermentum ATCC13869 were grown under One unit of enzyme was defined as the amount of
aerobic conditions at 37 °C and 30 °c , respectively. enzyme that catalyzes the formation of 1 f..!M of NA-
DPH per min. Specific activity was expressed as units
Isolation of DNA and transformation per milligram of total protein. The amounts of protein
Chromosomal DNA from B. lactojermentum ATCC in the crude extracts were measured by the method
13869 was obtained by the method of Hintermann of Lowery et al. (1951) with bovine serum albumin
et at. (1981), except that lysozyme was added to a final as a standard.
Vol. 4 ( 1994) Jung Hyeob Roh er al. 297

X
~3S ~ 82
(kb)
... 23.0
... 9.4
"'6.6
"'4.4
p8l2
11kb
~2 . 3
2 .0
~

P 81 E P P
s{ xl 83 s

Figure 2. Restriction pattern of pB12. Plasmid pBl2 was di-

gested with various restriction endonucleases and identified X
the restriction sites in the insert. Each lane revealed restric-
tion fragments as follows: Lane I, ADNA digested with Hin-
dIII; lane 2, pBR322 digested with BamHI; lane 3-9. pBI2
digested with Bam HI, BamHI +XhoI. XhoI. £Co RI, PSfI.
EcoRI + Bam HI. PstI + EcoRI. respectively; lane 10. pBR322
digested with BamHI; lane II, DNA digested with HindIII .

Results

Isolation of B. lactofennentum ddh gene

Competent cells of DAP deficient mutant E. coli
AT986 (dapD) were transformed with a B. lactofermen -
tum genomic library in pBR322 and plated on supple-
mented M9 plates without DAP. After two days of
incubation at 37 t, the eight ApR and DAP + transfo-
rmants were selected. These transformants were rest-
rea ked o n the M9 plates and the results were positive.
The specific activity of DOH was measured for eight
ApR and DAP + transform a nts, resulting in the detec-
tion of DOH activity in all eight. From the eight ApR
a nd DAP + transformants, plasmids were isolated and
digested with Bam HI. Restriction enzyme analysis of
plasm ids DNA revealed identical restriction patterns
indicating the presence of an identical plasmid in
these tran sformants.
One of the clones with DOH activity designated
pB 12 was selected for further studies. A restriction
map of pBI2 is shown in Figures 2 and 4. This plas-
mid was approximately II kb in length, consisting
of the vector and 6.6 kb Bam H I insert.
Subcloning of the ddh gene
. We subcloned the 6.6 kb fragment from pBI2 and
obtained four plasm ids pBBI. pESL pPL20 and pXX
14 (Fig. 3). As shown in Figure 4. the plasmids pESL
pPL20 and pXXl4 showed DOH activity. However.
the plasmid pBB I containing the BamHI 1-Bam H I2 fra -
gment of the pB12 did not express the DOH activity.
Subcloning experiments and complementation tests
indicate that the region for DOH activity is within
the -2.5 kb XhoI fragment (Fig. 4).
X
Figure 3. Schematic diagram showing the construction of
plasm ids. The pESI and pBBI were derived from pBI2 after
deletion of the EcoRI fragment and deletion of the BamHh
IBamHI.1 fragment respectively. The pPL20 was constructed
by Pstl/Sall fragment insertion to the pBluescriptII SK + and
pXXl4 by XhoI fragment insertion into the pBluescriptII
SK +. B. Bam HI; E. EcoRI: P. PsrI: X. XhoI: pBS SK + .
pBluescriptII SK + .
Enzyme actiVity and actIVIty staining of DDH
The specific activity of the DOH was determined
from crude extracts of the B. lactoJermentum ATCC
13869 and E. coli ddh transformants. The data show
that E. coli ddh transformants have a level of DOH
activity equal to that of activity found in the wild
type of B. lactoJermentum ATCC 13869. On the other
hand, no activity was detected in E. coli 1M 109 har-
boring pBluescriptII SK + or pBR322 (Table 1).
The activity staining was done for the plasm ids pB
12, pESI, pPL20 and pXX14. B. lactoJermentum ATCC
13869 and E. coli AT986 were used as a positive and
298 Cloning and Expressio n of the B. lactore/mentum ddh Ge ne Mol. Cells

Comple-
mentation
B1 P E P X B2 X83
I I I I I I I I +
6 .6kb pB12

B1 82
I
5.2kb pBB1

E 83
I I I
+
Skb pES1

P 83
1 I
3.7kb I pPL26 +

X
I
X
I
pXX14 +
2.5kb

Figure 4. Physical map a nd de letion analysis of B. lactofer-
m entum DNA containing the ddh gene. The subclones of
pBI 2 a re designated pBBI . pES I. pPL20 and pXX1 4. Comp-
lementation via transfonnation of the E. coli dapD mutant
by each subclone is designated positive (+) or negative ( - ).
B. BamHI; E. Eco RI: P. PstL X X ho L +. growth; - . no
growth .
Figure 5. Activity sta ining fo r DOH of strains. The cru de
extracts of E. coli and B. lactofennentum we re used fo r activity
staining. The gel was stained with staining solution a fter
polyacrylamide gel electrophoresis. Lane 1. E. coli AT986
with pB 12; la ne 2. E. coli AT986 with pES I; la ne 3, E. coli
AT986 with pPL20; la ne 4, E. coli AT986 with pXX14; lane
5. B. lactofermentum ATCC I 3869; lane 6. E. coli AT986.

Table I. Ex pressio n of the B. lactofermelltum ddh ge ne in A B

E. coli
123 4 123 4
Strain/plasmid Releva nt Specific activity
ge notype (U"/mg of
total protein) (kb)
Brevibacrerium lacr~fermenrum wild type 6.7
ATCC13869 23. 0
E. coli 1M 109 (pBR322) Ap ' NO" 9 .4
6 ·6

--
E. coli 1M 109 (pBluescriptlI SK + ) Ap i NO 4 .4
E. coli 1M I09 (pBI 2) Ap ' ddh ' 5.0
E. coli 1M I09 (pES I) Ap - ddh - 6.3 2 .3
E. coli 1M I09 (pP L20) Ap ddh ' 7.3 2 .0
E. coli 1MI 09 (pXX I4) Ap - ddh " 6.2
" I unit. the amo unt of enzy me that catalyzes the fo rnlatio n
of I 11M of NAOPH per min.
"N O. not detected.

n egative co ntro ls. respecti vely. As sh own in Figure 5.

a specific b a nd of DOH activity was o b served in the
stra in s carrying each subclo ne.
Southern blot analysis
T o confirm w hethe r th e insert fragm e nt o f pXX I4
h a d o rigina ted from th e c hro m oso m a l DNA of B. lac-
toJermentum A T ee 13869. a hy bridizatio n expe rime nt
w as p e rfo rmed . B. lactoJermentum c hro mosom a l DNA
was digested with X h o I a nd h ybridized with [a-.1~P J
Figure 6. Ide ntification of the plasmid pXX l4 wi th Southern
blot hybridization. The chro mosomal DNA of B. lactofermen·
tum was digested with X hoI. The a _32 p labeled 2.5 kb insert
fragment from pXX 14 was hybrid ized to chromosomal
DNA. A) Agarose gel electro photogram; Lane I, DNA diges-
ted with Hind III ; lane 2. pXX l4 d igested wi th Xho l: lane
3. B. lactofermentum ch ro mosomal DNA digested with X hoI;
la ne 4. E. coli AT986 chromosomal DNA d igested with XhoI.
B) Autoradiogram.
Vol. 4 (1994) lung Hyeob Ro h el 01.
CTP labeled ~2.5 kb Xho l fragment of pXX14. As
shown in Figure 6, a singJe band which was found
in hybridiza tion with Xhol-digested B. lacLOJermenLum
chromosomal 0 A fragments corresponded to the
size of the insert fragment of pXX14. The result confi-
rmed that the cloned DNA fragment o riginated from
B. lactoJermentum genomic DNA
Discussion
To clarify that the DOH pathway in addition to
the DAP pathway is involved in lysine biosynthesis
of B. lactoJermentum, we detected the DOH activity
a nd cloned the ddh gene encoding meso-DAP-dehyd-
rogenase (DOH) from B. lacLOJermentum.
From a genomic library of B. lactoJermentum, we
isolated eight ApR and DAP + transfo rmants by hete-
rologous complementation of an E. coli dapD muta-
tion. These eight ApR a nd DAP + transformants all
contained DOH activity a nd the insert fragment of
an identical size. It was strange that no dapD gene
could be cloned. In th e cloning experiment of the
C glutamicum ddh gene repo rted by Ishino et al. (1988),
no activity for the dapD gene was detected in twenty
clones. This is probably due to the disruption of the
dapD gene by the digestion of restliction en zyme. W e
a re now performing DNA sequencing for ddh gene
and will analyze a nd compare it with the DNA se-
quence of the C glutamicum ddh gene.
Schrumpt el al. (1991) reported that the inactivation
of the DOH pathway in C glutarnicum resulted in
a n accumulation of N-succinyl-diaminopimelate and
reduction of lysine production. Production of high
amounts of lysine in glutamic acid-producing bacteria
is probably due to the existence of a n additional
DOH pathway because the DOH pathway is essential
for converting intermediates to m eso-DAP when meta-
bolic flux is increased.
We have found here th at the DOH pathway in ad-
dition to the DAP pathway can operate in B. lactoJer-
m entum [o r m eso-OAP and L-lysine production. W e
have an interest in the role and function of the ddh
gene for lysine production in the DOH pathway of
B. lactoJermentum and will perform fUlther studies on
it.
Acknowledgments
This work was supported by grant 921-0400-035-2
from th e Korea Science and Engineeling Foundation.
We thank Dr. Moon-Hee Sung and Dr. Ki-Hong
299
Yoon, Applied Microbiology La boratory, G enetic
Engineering Resea rch Institute, KIST, for helpful di s-
cussions.
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