Biotransformation of L-lysine to L-pipecolic acid catalyzed by L-lysine 6-aminotransferase and pyrroline-5-carboxylate reductase

Article abstract of DOI:10.1271/bbb.66.622

The enzyme involved in the reduction of Δ¹-piperideine-6- carboxylate (P6C) to L-pipecolic acid (L-PA) has never been identified. We found that Escherichia coli JM109 transformed with the lat gene encoding L-lysine 6-aminotransferase (LAT) converted L-lysine (L-Lys) to L-PA. This suggested that there is a gene encoding "P6C reductase" that catalyzes the reduction of P6C to L-PA in the genome of E. coli. The complementation experiment of proC32 in E. coli RK4904 for L-PA production clearly shows that the expression of both lat and proC is essential for the biotransformation of L-Lys to L-PA. Further, We showed that both LAT and pyrroline-5-carboxylate (P5C) reductase, the product of proC, were needed to convert L-Lys to L-PA in vitro. These results demonstrate that P5C reductase catalyzes the reduction of P6C to L-PA. Biotransformation of L-Lys to L-PA using lat-expressing E. coli BL21 was done and L-PA was accumulated in the medium to reach at an amount of 3.9 g/l after 159 h of cultivation. It is noteworthy that the ee-value of the produced pipecolic acid was 100%.

Full text of DOI:10.1271/bbb.66.622

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Biotransformation of L-Lysine to L-Pipecolic Acid
Catalyzed by L-Lysine 6-Aminotransferase and
Pyrroline-5-carboxylate Reductase
Tadashi FUJII, Manabu MUKAIHARA, Hitosi AGEMATU & Hiroshi TSUNEKAWA
To cite this article: Tadashi FUJII, Manabu MUKAIHARA, Hitosi AGEMATU & Hiroshi
TSUNEKAWA (2002) Biotransformation of L-Lysine to L-Pipecolic Acid Catalyzed by L-Lysine
6-Aminotransferase and Pyrroline-5-carboxylate Reductase, Bioscience, Biotechnology, and
Biochemistry, 66:3, 622-627, DOI: 10.1271/bbb.66.622
To link to this article: http://dx.doi.org/10.1271/bbb.66.622
Published online: 22 May 2014.
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Date: 22 October 2015, At: 18:30

Biosci. Biotechnol. Biochem., 66 (3), 622–627, 2002
L
L
L
Biotransformation of -Lysine to -Pipecolic Acid Catalyzed by -Lysine
6-Aminotransferase and Pyrroline-5-carboxylate Reductase
Tadashi FUJII,† Manabu MUKAIHARA, Hitosi AGEMATU, and Hiroshi TSUNEKAWA
Bioresource Laboratories, Mercian Corp., 4-9-1, Johnan, Fujisawa 251-0057, Japan
Received November 7, 2001; Accepted November 9, 2001
1)
The enzyme involved in the reduction of D1
piperideine-6-carboxylate (P6C) to -pipecolic acid
-PA) has never been identiˆed. We found that
Escherichia coli JM109 transformed with the lat gene
encoding -lysine 6-aminotransferase (LAT) converted
-lysine ( -Lys) to -PA. This suggested that there is a
gene encoding ``P6C reductase'' that catalyzes the
reduction of P6C to -PA in the genome of E. coli. The
complementation experiment of proC32 in E. coli
RK4904 for -PA production clearly shows that the ex-
pression of both lat and proC is essential for the
biotransformation of -Lys to -PA. Further, We
-
L
L
deaminative cyclization of -Lys to -PA. The rapL
gene encoding lysine cyclodeaminase is located in the
gene cluster for rapamycin.4) In Pseudomonas puti-
L
(
L
da
lized to P2C, and then P2C is converted to
P2C reductase.5) P6C, which is the reversible cyclic
form of -aminoadipic semialdehyde, was identiˆed
,
L
-Lys is initially converted to -lysine, metabo-
D
L
L
-PA by
L
L
L
a
L
as the product of the deamination of -Lys that was
L
catalyzed by -lysine 6-aminotransferase (LAT,
L
E.C.2.6.1.36) in Flavobacterium lutescens IFO30846)
L
and b
-lactam-producing actinomycetes7) (Fig. 1). In
L
L
addition, P6C is also produced from -Lys by lysine
L
6-dehydrogenase in Agrobacterium tumefaciens8)
showed that both LAT and pyrroline-5-carboxylate
(P5C) reductase, the product of proC, were needed to
and from saccharopine by saccharopine oxidase (EC
9)
convert
strate that P5C reductase catalyzes the reduction of P6C
to -PA. Biotransformation of -Lys to -PA using lat
expressing E. coli BL21 was done and -PA was
accumulated in the medium to reach at an amount of
3.9 g l after 159 h of cultivation. It is noteworthy that
L
-Lys to
L
-PA in vitro. These results demon-
1.5.1.10) in Rhizoctonia leguminicola
.
The activity
of the reduction of P6C to -PA was found in cell-
free enzyme systems of microorganisms.9) However,
no information about the enzyme involved in the
reduction of P6C has been ever obtained, because the
substrate, P6C, is chemically unstable and the lack of
available P6C precluded the characterization of the
enzyme.
L
L
L
L
-
L
W
the ee-value of the produced pipecolic acid was 100
%.
Key words: lat
;
L
-pipecolic acid ( -PA); proC; D
1-pyr-
L
We have investigated the biosynthesis of
L
-a-
roline-5-carboxylate (P5C) reductase; D1
-
L
L
aminoadipic acid ( -AAA) from -Lys in F. lutescens
IFO3084 and cloned the lat gene encoding LAT and
the pcd gene encoding P6C dehydrogenase from F.
lutescens IFO3084.10,11) Our next attention was
focused on the ``P6C reductase'' that catalyzes the
piperideine-6-carboxylate (P6C)
L
-Pipecolic acid ( -PA) is an amino acid not ob-
L
served in proteins, and is an important component
and a precursor of many microbial and plant secon-
dary metabolites such as immunosuppressants,1) pep-
tide antibiotics,2) and piperideine alkaloids.3) The
reduction of P6C to
L
-PA to produce
L
-PA from -
L
Lys. We found that ``P6C reductase'' exists in
Escherichia coli. Further our works in vivo and in
vitro demonstrate that the enzyme is pyrroline-5-
carboxylate (P5C) reductase (EC 1.5.1.2) encoded by
biosynthesis of
of -lysine ( -Lys) metabolism for a long time. It has
been known that -PA is synthesized biologically by
the deaminative cyclization of -Lys, by the reduc-
tion of
1-piperideine-2-carboxylate (P2C), or by the
reduction of
1-piperideine-6-carboxylate (P6C).
In Streptomyces hygroscopicus, which produces
L
-PA has been investigated in terms
L
L
L
proC, which catalyzes the reduction of D
1-pyrroline-
L
5-carboxylate to
L
-proline and is found in almost all
D
organisms.12,13)
D
In this paper we describe the reduction of P6C to
-PA catalyzed by P5C reductase and the biotran-
L
rapamycin, an immunosuppressant containing
L
-PA
sformation of
coli
L
-Lys to -PA using lat-expressing E.
L
as a component, lysine cyclodeaminase catalyses the
.
†
To whom correspondence should be addressed. Tel: +81-466-35-1519; Fax: +81-466-35-1524; E-mail: fujii-td
-AAA, -aminoadipic acid; LAT, -lysine 6-aminotransferase; -Lys, -lysine; -PA,
-pipecolic acid; P2C, D1
Piperideine-2-carboxylate; P5C,
1-pyrroline-5-carboxylate; P6C, 1-Piperideine-6-carboxylate
＠
mercian.co.jp
Abbreviations
:
L
L-
a
L
L
L
L
L
-
D
D

Biotransformation of
L
-Lys to
L
-PA Catalyzed by LAT and P5C Reductase
623
with T4 ligase.
A
proC-expressing plasmid
pTrcP5CR was constructed as follows: two primers,
primer EX1 and primer EX2, were prepared for PCR
to obtain proC. Primer EX1 was designed on the ba-
sis of the N-terminal sequence of proC and contained
a
5
?
BamHI
AAAATCGGTTTTATTGGCT-3
was designed on the basis of the C-terminal sequence
of proC and contained a 5 EcoRI site (5 -TCGAAT-
TCCAGGATTTGCTGAGTTTTTCT-3 ). Using E.
site
(5
?-ATGGATCCG-
?
). Primer EX2
?
?
?
coli JM109 chromosomal DNA as a template, an am-
pliˆcation by PCR was done. The PCR product was
digested with BamHI and EcoRI and then ligated
into pTrcHis2A (Invitrogen) digested with BamHI
and EcoRI. The resulting plasmid was designated as
pTrcP5CR.
Expression of lat in E. coli JM109. E. coli JM109
harboring pRH124 was cultivated at 32
9
C with
, yeast extract
, pH 7.2) containing
g of ampicillin sodium per ml. Thirty l of over-
night-grown culture was added to 3 ml of L-broth
containing 50 g ampicillin sodium per ml. The cul-
ture was grown at 32 C with shaking, and the expres-
sion of LAT was induced by the addition of
isopropyl- -thiogalactopyranoside (IPTG) (1 m
shaking in L-broth (polypeptone 1.0
0.5 , NaCl 0.5 , glucose 0.1
50
z
Fig. 1. Conversion of -Lys to -PA by LAT and P5C Reduc-
L L
tase.
z
m
z
z
m
m
Materials and Methods
9
Bacterial strains. E. coli JM109 was used for the
isolation of chromosomal DNA and the production
b
-
D
M
,
of
L
-PA. E. coli RK4904 (CGSC
#
: 6404), which is a
ˆnal concentration) when the absorbance at 660 nm
of the culture reached 1.0. After 4 h of cultivation,
proC32 mutant obtained from the E. coli Genetic
Stock Center (Yale University, USA), was used as the
host strain for a complementation experiment of
z z
30 ml of 50 glycerol and 30 ml of 50 L-lysine HCl
were added to the culture, and the culture was culti-
vated for 15 h more. The supernatants of these cul-
tures were analyzed by TLC and HPLC to estimate
proC32
protein expression. E. coli BL21 was used as the host
strain for the production of -PA.
. E. coli TOP10 (Invitrogen) was used for
L
the amount of -PA.
L
Construction of plasmids.
A
lat-expressing plas-
Complementation experiment of proC32. E. coli
mid pRH124 was constructed as follows: using F. lu-
tescens IFO3084 chromosomal DNA as a template, a
1571-bp fragment containing lat was ampliˆed by
PCR. The primers were designed on the basis of the
RK4904 harboring pUC19, pRH124, pRH134, or
pRH135 was cultivated at 32
broth containing 50
Two hundred and seventy ˆve
ded to 25 ml of TB (polypeptone 3.2
2.4 , glycerol 0.4 , 100 m phosphate buŠer (pH
7.4)) containing 50 g ampicillin sodium per ml.
After 8 h of cultivation, 500 l of 50 glycerol and
500 l of 50 -lysine HCl were added to the culture
9
C with shaking in L-
m
g of ampicillin sodium per ml.
l of the broth was ad-
, yeast extract
m
sequence of lat and contained a 5
primer (5 -ATAAGCTTGTCCCTTCTTGCCC-
CGCTCGC-3 ) and a 5 BamHI site in the primer II
(5 -GCGGATCCTGTTGCCGCTGGTGCCGGGC-
AG-3 ). The ampliˆed PCR product was digested
?
HindIII site in
z
I
?
?
z
z
M
?
m
?
m
z
?
m
z
L
with HindIII and BamHI, and ligated into HindIII-
BamHI-digested pUC19. A lat and proC expressing
plasmid pRH134 was constructed as follows: using
E. coli JM109 chromosomal DNA as a template, a
1443-bp fragment containing proC was ampliˆed by
broth and the cultivation was continued for 40 h
more. The supernatants of these cultures were
analyzed by TLC and HPLC to estimate the amount
of -PA.
L
PCR with the primers that contained a 5
each (5 - CCGGTACCATAAAATCGCGCATCGT-
CAGGC-3 and 5 -CCGGTACCGCCACAGGTAA-
CTTTACGGATT-3 ). The Kpn digested PCR
?
KpnI site in
Fermentation experiment. E. coli BL21 harboring
?
pRH124 was cultured in the same manner as the
?
?
cultivation of E. coli RK4904 described above. One
?
I
hundred ml of each culture was collected after 15, 39,
product was ligated into the KpnI site of pRH124. A
proC-expressing plasmid pRH135 was also construct-
ed as follows: pRH134 was digested with HindIII and
BamHI, ˆlled with T4 polymerase, and self-ligated
63, 87, 111 and 159 h of cultivation.
Expression and puriˆcation of recombinant
proteins. Recombinant LAT and recombinant P5C

624
T. FUJII et al.
reductase were expressed and puriˆed using E. coli
TOP10 pTrcLAT and E. coli TOP10 pTrcP5CR,
shown). This result suggested that there is the gene in
the genome of F. lutescens IFO3084 encoding ``P6C
W
W
respectively, by the method described previously.10)
reductase'' that catalyzes the reduction of P6C to -
L
PA. To clone the gene encoding ``P6C reductase'',
we ˆrst tried to construct the gene library of F.
lutescens IFO3084, introduce the gene library into
Cooperative enzymatic transformation of
-PA in vitro. Reaction mixture contained 0.2 mmol
of sodium phosphate buŠer (pH 7.2), 1 mol of
lysine HCl, 10 mol of 2-ketoglutarate, 37.5 pmol of
pyridoxsal phosphate, 2 mol of NADPH, 1 g of
LAT, and 5 g of P5C reductase in a ˆnal volume of
1 ml. The reaction mixture was incubated at 32 C. At
regular times, a sample (100 l) of the reaction mix-
ture was collected and added 900 l of acetonitrile
water (5:4) to stop the enzymatic reactions. The
L
-Lys to
L
m
L
-
lat-expressing E. coli JM109 pRH124, and isolate
W
m
the clone in which
L
-Lys is converted to
L
-PA.
m
m
However, to our surprise, E. coli JM109 pRH124
W
m
itself converted
L
-Lys to
L
-PA (Fig. 2A). Since LAT
9
catalyzes the conversion of
L
-Lys to P6C,6,10) this
m
result implies that there is a gene encoding another
``P6C reductase'' also in the genome of E. coli
JM109.
m
W
amounts of
L
-Lys and -PA in the reaction mixture
L
were measured by ESI LC MS.
Complementation of proC32 for -PA production
L
W
The structural similarity between P6C and P5C
brought us an idea that ``P6C reductase'' may be
similar to P5C reductase. However, we failed to ˆnd
any additional protein with high similarity to P5C
reductase in E. coli by a BLAST search (data not
shown), suggesting that P5C reductase itself cata-
lyzes the reduction of P6C as well as P5C. To exa-
mine whether P5C reductase catalyzes the reduction
TLC analysis. The culture broth was centrifuged
and the resulting supernatant was used for quantita-
tive analysis with TLC as described previously.11)
HPLC analysis. Pipecolic acid and lysine were
labeled
leucineamide (
tives of pipecolic acid and lysine were analyzed by
with
Na-(5-‰uoro-2,4-dinitrophenyl)-
-
L
L
-FDLA),14) and the
-FDLA deriva-
L
of P6C to
ment of proC32, a mutation of proC encoding P5C
reductase. We transformed E. coli RK4904 ( proC32
L
-PA, we did a complementation experi-
×
HPLC on a YMC-Pack s-5 60A ODS (4.6 150 mm)
column maintained at 35 C using a acetonitrile con-
centration gradient in 1
tion from 40 to 80
9
)
z
acetic acid aqueous solu-
with pUC19, a lat expression plasmid pRH124, a lat
and proC expression plasmid pRH134, or a proC ex-
pression plasmid pRH135, and cultured these trans-
z
z for 20 minutes with a ‰ow
rate of 1.0 ml per minute. The derivatives were de-
tected by UV-monitoring at 340 nm. The enantio-
meric purity of pipecolic acid was also determined by
this HPLC method.
formants in TB added with
L
-Lys. -PA was not de-
L
tected by TLC and HPLC analysis in the culture
broth of E. coli RK4904 pUC19 (lat„
,
proC „), E.
W
coli RK4904 pRH124 (lat+
,
proC „), and E. coli
W
LC MS analysis. The cooperative enzymatic trans-
RK4904 pRH135 (lat„
,
proC +) (Fig. 2(B)). On the
W
W
formation from
L
-Lys to
L
-PA by LAT and P5C
other hand,
L
-PA was detected in the culture broth of
reductase was monitored by liquid chromatography
mass spectrometry (LC MS). HPLC conditions were
E. coli RK4904 pRH134 (lat+
,
proC +) (Fig. 2(B)).
W
W
These results clearly show that the expression of both
W
×
as follows: column, YMC J's phere ODS (4.5
75 mm); mobile phase, methanol 2 m ammonium
nona‰uorovaleric
acid (30:70); ‰ow rate, 0.5 ml minute; column tem-
lat and proC is essential for the biotransformation of
M
L
-Lys to -PA.
L
W
acetate solution containing 5 m
M
Reduction of P6C to
To verify that P5C reductase, the proC gene
product, catalyzes the reduction of P6C to -PA in
vitro, we tried the cooperative enzymatic transforma-
tion of -Lys to -PA by LAT and P5C reductase.
L
-PA by P5C reductase
W
perature, 35
Finnigan AQA (Thermo Quest, UK). The protonated
molecules [M+H]+ of
-Lys and -PA were detected
in positive ion mode to give peaks at m z 147 and
130, respectively. Under these LC MS conditions,
the retention times of
6.72 min, respectively.
9
C. The mass spectrometer used was a
L
L
L
L
L
W
First, proC of E. coli JM109 was ampliˆed and ligat-
ed into pTrcHis2A to construct a recombinant P5C
reductase expression plasmid of pTrcP5CR. The ex-
pressed and puriˆed recombinant P5C reductase gave
a single band on SDS-PAGE, corresponding to a
molecular mass of about 32 kDa (Fig. 3), which is in
good agreement with the value (31.4 kDa) estimated
from the deduced amino acid sequence. By using the
recombinant P5C reductase and the recombinant
W
L
-PA and -Lys were 4.01 and
L
Results
Production of
lat gene
L
-PA by E. coli transformed with
In the course of our studies on pcd, we found that
the pcd deˆcient mutant of F. lutescens IFO3084
LAT,10)
PA in vitro. Figure 4 shows that the number of moles
of consumed -Lys was almost equal to that of
L
-Lys was stoichiometrically converted to -
L
slightly converted
L
-Lys to
L
-PA in phosphate buŠer
(pH 7.0) containing
L
-Lys and glycerol (data not
L

Biotransformation of
L
-Lys to
L
-PA Catalyzed by LAT and P5C Reductase
625
Fig. 2. Biotransformation of
(A) TLC analysis of the culture broth of lat-expressing E. coli JM109. Lane 1,
JM109 pRH124.
-Lys to -PA.
L L
L
-PA (500 mg ml); lane 2, JM109 pUC19; lane 3,
W W
W
(B) TLC and HPLC analysis of the culture broth of lat-expressing E. coli RK4904. Lane 1 and 7,
(500 g ml); lane 3, RK4904 pUC19; lane 4, RK4904 pRH124; lane 5, RK4904 pRH135; lane 6, RK4904 pRH134. N.D., not
detected.
L
-PA (500 mg ml); lane 2, -Lys
L
W
m
W
W
W
W
W
Fig. 3. SDS-PAGE Analysis of Recombinant LAT and Recom-
binant P5C Reductase.
Lane 1, molecular mass standards (sizes in kilodaltons are
indicated at the left); lane 2, recombinant LAT; lane 3, recom-
binant P5C reductase.
Fig. 4. Cooperative Enzymatic Transformation of
L
-Lys to
L
-PA
by LAT and P5C Reductase.
Cooperative enzymatic transformation of
L
-Lys to
L
-PA by
-Lys (
L

)
LAT and P5C reductase was done and the amounts of
L
-PA
(

) in the reaction mixture were measured as
and
described in Materials and Methods.

626
T. FUJII et al.
ble, tends to undergo polymerization, and is reversi-
bly converted to
a-aminoadipic semialdehyde in
aqueous solution.15,16) However, our data suggest that
following reaction steps are involved in the biotran-
sformation of
L
-Lys to
-Lys+2-ketoglutarate
-aminoadipic semialdehyde+
-aminoadipic semialdehyde
-PA+NADP
L
-PA:
(i)
L
ª
a
a
L
-glutamate
(ii)
ªP6C+H2O
(iii) P6C+NADPH+H+ª
L
Step (i) is the known transamination by LAT.6,10)
Step (ii) is a spontaneous chemical reaction. Step (iii)
is the novel reduction by P5C reductase. The instabil-
ity of P6C at pH above 5.0 may be the reason why
the number of moles of produced -PA was not com-
L
pletely equal to the number of moles of added
L
-Lys
at the incubation time of 23 h.15,16)
It is currently unclear why E. coli BL21 pRH124
W
produced a larger amount of
L
-PA than E. coli BL21
Fig. 5. Fermentation Experiment for L-PA Production.
E. coli BL21 pRH124 was cultured and the amount of L-PA
pRH134. It seems possible that in this biotransfor-
W
W
in the culture broth was measured as described in Materials and
Methods.
mation system of
to -PA is not the rate-limiting step and the excess ex-
pression of proC has a bad in‰uence.
-PA is an important compound as a component
L
-Lys to
L
-PA the reduction of P6C
L
L
produced
the formation of
reaction mixture (data not shown).
L
-PA in each incubation time. In addition,
of many drugs. Recently, much attention has been
focused on the importance of chirality as the key to
the e‹cacy of many drug products. The current route
to the pure enantiomer of pipecolic acid is the classi-
cal resolution of the racemate by fractional crystalli-
zation of diastereomeric salts17) or the stereoselective
transformation of the racemate by biocatalytical
L
-glutamate was observed in the
Fermentation experiment for
L
-PA production
-PA was accumu-
We have described above that
L
lated in the culture broth of lat-expressing E. coli
JM109. Further, we transformed E. coli JM109,
C600, and BL21 with pRH124 or pRH134 and exa-
process.18) However, it is di‹cult to obtain the 100
z
ee of
sformation system was (
and its ee-value was 100
biotransformation of
L
-PA. Pipecolic acid produced by our biotran-
)-pipecolic acid ( -PA),
. It is noteworthy that our
-Lys to -PA by the lat
mined
L
-PA production using these transformants.
S
L
We found that E. coli BL21 pRH124 produced the
z
L
W
largest amount of
(data not shown). Then, a fermentation experiment
for -PA production using E. coli BL21 pRH124
was done. E. coli BL21 pRH124 was cultured and
L
-PA of these six transformants
L
-
expressing E. coli preserved the chirality. On the
L
basis of this biotransformation, highly e‹cient
W
production of
future.
L
-PA will become possible in the near
W
excessive
ture medium.
L
-Lys and glycerol were added to the cul-
L
-PA was accumulated in the medium
and reached at an amount of 3.9 g l after 159 h of
Acknowledgments
W
cultivation (Fig. 5). The ee-value of the produced
pipecolic acid was 100
z.
The authors would like to thank Ms. Konomi
Iguchi, Mercian Corp., for carrying out the ESI LC
MS analysis of the reaction mixture.
W
Discussion
We here demonstrated for the ˆrst time that the
References
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L
(Fig. 1). This ˆnding suggests that in the microorgan-
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a strain of Streptomyces
isms that produce
L
-PA via P6C from -Lys, such as
L
R. leguminicola9) and F. lutescens, P5C reductase is
,
responsible, at least in part, for the reduction of P6C
to -PA, because P5C reductase is present in almost
L
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a
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Biotransformation of
L
-Lys to
L
-PA Catalyzed by LAT and P5C Reductase
627
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