Cloning of the pyridoxine 5′-phosphate phosphatase gene (pdxP) and vitamin B6 production in pdxP recombinant Sinorhizobium meliloti

Article abstract of DOI:10.1271/bbb.70539

A novel gene (pdxP) encoding a pyridoxine 5′-phosphate (PNP) phosphatase involved in the last step of pyridoxine biosynthesis was cloned from Sinorhizobium meliloti IFO 14782 on the basis of the peptide sequences of the natural enzyme. The pdxP gene is an open reading frame (708 bp) encoding 235 amino acid residues with a calculated molecular weight of 26,466. From its deduced amino acid sequence, it was predicted that the enzyme belongs to the haloacid dehalogenase superfamily. Transformants of Escherichia coli and S. meliloti by pdxP gene expression plasmids showed stimulated PNP phosphatase activities. When pdxP was over-expressed together with the PNP synthase gene (pdxJ) in S. meliloti, the recombinant strain produced 149 mg/l of pyridoxine, 46% and 16% higher than the host strain and the pdxJ recombinant of S. meliloti respectively.

Full text of DOI:10.1271/bbb.70539

Biosci. Biotechnol. Biochem., 72 (2), 421–427, 2008
Cloning of the Pyridoxine 50-Phosphate Phosphatase Gene (pdxP)
and Vitamin B6 Production in pdxP Recombinant Sinorhizobium meliloti
y
*
*
Yoshie NAGAHASHI, Masaaki TAZOE, and Tatsuo HOSHINO
Department of Applied Microbiology, Nippon Roche Research Center,
Kamakura, Kanagawa 247-8530, Japan
Received August 22, 2007; Accepted November 2, 2007; Online Publication, February 7, 2008
[doi:10.1271/bbb.70539]
A novel gene (pdxP) encoding a pyridoxine 50-
phosphate (PNP) phosphatase involved in the last step
of pyridoxine biosynthesis was cloned from Sinorhizobi-
um meliloti IFO 14782 on the basis of the peptide
sequences of the natural enzyme. The pdxP gene is an
open reading frame (708 bp) encoding 235 amino acid
residues with a calculated molecular weight of 26,466.
From its deduced amino acid sequence, it was predicted
that the enzyme belongs to the haloacid dehalogenase
superfamily.
pharmaceuticals, and cosmetics, because it is converted
to the active form of the vitamin by pyridoxine (PN)
kinase and pyridoxine 50-phosphate (PNP) oxidase,
which exist in many organisms.
In the process of screening for vitamin B6 over-
producers, we found that Sinorhizobium meliloti IFO
14782 produced large amounts of PN in culture broth.5)
To improve the production of PN using metabolic
engineering techniques, we studied the biosynthetic
pathway of vitamin B6 in S. meliloti.6,7) In de novo
synthesis of vitamin B6 in Gram-negative bacteria,
including S. meliloti and Escherichia coli,8) PNP is
thought to be the first vitamin B6 synthesized from two
precursors, 1-deoxy-D-xylulose 5-phosphate and 4-phos-
phohydroxy-L-threonine, by two enzymes, 4-phospho-
hydroxy-L-threonine dehydrogenase (PdxA) and PNP
synthase (PdxJ) (Fig. 1). We purified PNP phosphatase,
which catalyzes the last-step reaction in the biosynthetic
pathway of PN, from the cytoplasm in S. meliloti.9) It
has a unique substrate specificity that acts on PNP and
PLP, but not on PMP or other physiologically phos-
phorylated compounds. An enzyme having such proper-
ties has been reported to be PLP phosphatase in human
erythrocytes. It is thought to regulate the level of PLP
in blood in cooperation with pyridoxal kinase.10) The
characteristics of Sinorhizobium PNP phosphatase are
very similar to those of human phosphatase, except for
the molecular size.
Transformants of Escherichia coli and S. meliloti
by pdxP gene expression plasmids showed stimulated
PNP phosphatase activities. When pdxP was overex-
pressed together with the PNP synthase gene (pdxJ)
in S. meliloti, the recombinant strain produced 149 mg/l
of pyridoxine, 46% and 16% higher than the host
strain and the pdxJ recombinant of S. meliloti respec-
tively.
Key words: vitamin B6; pyridoxine 50-phosphate phos-
phatase gene; pyridoxine production; Sino-
rhizobium meliloti recombinant
Vitamin B6 is the generic name for six compounds:
pyridoxine (PN), pyridoxal, pyridoxamine (PM), and
their 50-phosphate esters. One type of vitamin B6,
pyridoxal 50-phosphate (PLP), is well known as the
cofactor of enzymes involved in amino acid metabolism.
Recently it has been reported that the antioxidant
potency of vitamin B6 is comparable to that of vitamins
C and E,1,2) and that there is the possibly of a preventive
effect against Parkinson’s and Alzheimer’s diseases.3,4)
Vitamin B6 is indispensable to humans and other
animals, but it is not synthesized de novo. PN is used
mainly as a source of vitamin B6 in foods, animal feed,
Here we report the cloning of a novel gene (pdxP)
encoding a PNP phosphatase from S. meliloti, a
comparison of the amino acid sequences of PdxP
with those of other phosphatases, and the contribution
of overexpression of PdxP on PN production in
S. meliloti.
y
To whom correspondence should be addressed. Present address: Pharmaceutical Technology Department, Kamakura Research Laboratories,
Chugai Pharmaceutical Co., Ltd., 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan; Tel/Fax: +81-467-47-2226;
E-mail: nagahashiyse@chugai-pharm.co.jp
*
Present address: Mycology and Metabolic Diversity Research Center, Tamagawa University Research Institute, Machida, Tokyo 194-8610,
Japan
Abbreviations: CFE, cell-free extract; ORF, open reading frame; PCR, polymerase chain reaction; PLP, pyridoxal 50-phosphate; PMP,
pyridoxamine 50-phosphate; PM, pyridoxamine; PN, pyridoxine; PNP, pyridoxine 50-phosphate; SDS–PAGE, sodium dodecyl sulfate-polyacrylamide
gel electrophoresis

422
Y. NAGAHASHI et al.
CHO
CH2OPi
OH
H3C
H
N
Pyridoxal 5'P
(PLP)
PNP oxidase
CH2OH
CH2OPi
CH2OH
CH2OH
OH
OH
4-Phospho
hydroxy-L-
threonine
PdxA
PdxJ
PNP phosphatase
H3C
H
H3C
H
N
N
Pyridoxine
PN kinase
Pyridoxine 5'P
(PNP)
1-Deoxy-D-
xylulose 5P
(PN)
Fig. 1. Pyridoxine Synthesis from 1-Deoxy-D-xylulose 5P and 4-Phosphohydroxy-L-threonine in Sinorhizobium meliloti IFO 14782.
Table 1. Key Plasmids Used in This Study
kit was from Clontech (Mountain View, CA), and PCR
2.1-TOPO and TOPO TA cloning kits were from
Invitrogen Japan (Tokyo). E. coli recombinant clones
were grown at 37 ꢀC in Luria-Bertani (LB) medium with
10 mg/ml of tetracycline or 100 mg/ml of ampicillin.
When the tac promoter-driven PNP phosphatase gene
cassette was constructed in E. coli, LB was supple-
mented with 1 mg/ml of PM to obtain recombinant
E. coli efficiently. Chromosomal and plasmid DNA
were isolated using Qiagen genomic-tips and Qiagen
plasmid mini (Qiagen, Hilden, Germany) respectively.
Plasmid
pVK100
pRK2013
Characteristics
Reference
cos, mob, Kmr, Tcr, 23 kb
mob, Kmr, ColE1 replicon, 48 kb
pKK223-3 containing 0.9 kb
tac promoter driven pdxP
expression cassette
22)
23)
pKKpdxP
This study
This study
14)
pVK100 containing 0.9 kb
tac promoter driven pdxP
expression cassette
pVKptacpdxP
pSHT
pVK100 containing native
promoter driven
pdxJ expression cassette
pSHT containing native
promoter driven
Cloning of the PNP phosphatase gene.
pSHTptacpdxP
This study
Isolation of a partial fragment of the PNP phospha-
tase gene. Polymerase chain reaction (PCR) was
performed with ExTaq polymerase using 100 pmol of
degenerate oligonucleotides as primers and 100 ng of
the chromosomal DNA of S. meliloti IFO 14782 as a
template. PCR conditions were 94 ꢀC for 5 min, fol-
lowed by 30 cycles of 94 ꢀC for 30 s, 50 ꢀC for 30 s,
72 ꢀC for 30 s, and 72 ꢀC for 7 min. The reaction mixture
was analyzed by agarose gel electrophoresis, and
amplified fragments were purified using QIAEXII
(Qiagen) and cloned into pCR2.1-TOPO. Insertion of
the resulting plasmid was sequenced using a Cy5
Autocycle Sequencing kit and an ALFred DNA se-
quencer (Amersham Biosciences).
pdxP expression cassette
Materials and Methods
Bacterial strains and plasmids. S. meliloti IFO 14782
was used as the source of chromosomal DNA in cloning
the PNP phosphatase gene and as a host strain in gene
expression. E. coli JM109 and HB101 were obtained
from Takara Shuzo (Shiga, Japan). Plasmid pKK223-3
was purchased from Amersham Biosciences. (Piscat-
away, NJ). Key plasmids used in this study are listed in
Table 1.
Southern blot hybridization analysis. Chromosomal
DNA (3.4 mg) or plasmid DNA (0.5 mg) was digested
with restriction enzymes and subjected to agarose gel
electrophoresis. The DNA was transferred to a nylon
membrane (Hybondꢀ-Nþ, Amersham Biosciences) in a
buffer containing 87.65 g NaCl and 44.1 g sodium citrate
in one liter with a vacuum blotter (Bio-Rad, Hercules,
CA). A partial fragment of the PNP phosphatase gene
was labeled with digoxigenin using a DIG DNA labeling
kit (Roche Diagnostics, Basel, Switzerland), and was
used as a probe in Southern blot hybridization. Hybri-
Chemicals. PNP was synthesized by reducing PLP
with sodium borohydride.11) Other chemicals were
purchased from Wako Pure Chemical Industries (Osaka)
or Sigma Chemical (St.Louis, MO).
DNA manipulation. Recombinant DNA techniques
were performed as described by Maniatis et al.12)
Restriction enzymes, bacterial alkaline phosphatase, a
ligation kit, a blunting kit, and Ex Taq polymerase were
purchased from Takara Shuzo. An Advantage-HF PCR

Pyridoxine 50-Phosphate Phosphatase Gene of S. meliloti
423
.
dized DNA fragments were detected using a DIG
detection kit (Roche Diagnostics).
0.036% CaCl2 2H2O. S. meliloti IFO 14782 having
pVKPtacpdxP was obtained by tri-parental conjugal
mating.15) The transconjugants obtained were grown in
glucose-polypeptone medium containing 4% glucose,
Isolation of clones having the complete PNP phos-
phatase gene. The genomic library of S. meliloti IFO
14782 was constructed using partially digested EcoRI
fragments (15 to 30 kb) of chromosomal DNA with
pVK100 as a vector, as described previously.13) The
PNP phosphatase gene was screened from the library as
follows: colonies grown on LB with tetracycline were
transferred to a nylon transfer membrane and subjected
to colony hybridization with the same probe used in the
Southern blot hybridization analysis.
.
2% polypeptone, 0.2% yeast extract, 0.05% MgSO4
.
7H2O, 0.05% MnSO4 5H2O, and 0.001% FeSO4
.
7H2O for 7 d at 30 ꢀC, and the cells were used in the
preparation of the CFE.
PNP phosphatase activity in CFE. E. coli and
S. meliloti cells were harvested by centrifugation,
washed with saline, and suspended in 20 mM Tris–HCl
(pH 7.5) containing 15% sucrose, 1 mM dithiothreitol,
1 mM MnCl2, 1 mM MgCl2, and 0.1 mM phenylmethyl-
sulfonyl fluoride. The cell suspension was subjected to
a French pressure cell, and then the homogenate was
centrifuged at 35;000 ꢁ g to remove cell debris. The
supernatant was dialyzed against the same buffer and
used as the CFE. The reaction mixture contained 50 mM
Tris–HCl buffer (pH 7.5), 2 mM PNP or PMP, 1 mM
MnCl2, and CFE (0.4 mg protein) in 125 ml. The reaction
was done at 37 ꢀC for 30 min, and the amounts of
PN and PM in the reaction mixture were measured
by high pressure liquid chromatography as described
previously.5)
Construction of PNP phosphatase gene expression
plasmids. The PNP phosphatase gene was amplified
from the chromosomal DNA of S. meliloti IFO 14782
with an Advantage-HF PCR kit (Clontech) using
primers P101 (50-GAAGCTTCCCGGGCCGTGTCA-
TAAACCCGCCCATG-30) and P102 (50-CAAGCTT-
CCCGGGATCATCGCCGGGTTTTACG-30). Reaction
conditions were as follows: 94 ꢀC for 15 s, followed by
30 cycles of 94 ꢀC for 15 s and 68 ꢀC for 4 min. The
reaction mixture was subjected to agarose gel electro-
phoresis, and a 0.86-kb DNA fragment was recovered.
The fragment was cloned in a pCRII-TOPO vector to
obtain TOPOpdxP. A 0.86-kb SmaI fragment from
TOPOpdxP was ligated into the SmaI site of pKK223-3
in an orientation that allowed transcription of the PNP
phosphatase gene from the tac promoter. The resulting
plasmid was named pKKpdxP. An expression plasmid
of the PNP phosphatase gene in S. meliloti was prepared
as follows: Mobilizable cosmid pVK100 was digested
with BglII, and then a fragment of about 21.3 kb was
recovered. After the fragment was treated with bacterial
alkaline phosphatase, a 1.1-kb BamHI fragment from
pKKpdxP was ligated into the BglII-digested, dephos-
phorylated fragment to give plasmid pVKPtacpdxP.
Construction of pSHT carrying pdxJ gene was described
previously.14) pSHT was digested with BglII, and a
fragment of about 22.2 kb was recovered. After the
fragment from pSHT was treated with bacterial alkaline
phosphatase, a 1.1-kb BamHI fragment from pKKpdxP
was ligated into the BglII-digested, dephosphorylated
fragment to give plasmid pSHTPtacpdxP.
PN production by S. meliloti recombinant. S. meliloti
recombinants were grown in a seed medium and
transferred into flasks containing glucose-CSL medium
composed of 6% glucose, 3% corn steep liquor,
0.8% yeast extract, 0.175% NH4Cl, 0.05% MgSO4
7H2O, 0.025% MnSO4 5H2O, and 1% allophosite
(Shinagawa Kasei, Tokyo), and cultivated at 30 ꢀC for
9 d. The amount of PN in the supernatant of culture
broth was measured by high pressure liquid chromatog-
raphy.
.
.
Results and Discussion
Cloning of the PNP phosphatase gene
PNP phosphatase was purified from S. meliloti, as
described previously.9) Amino acid sequences of the
N-terminal position and three tryptic fragments of
the PNP phosphatase were determined to be as follows:
N-terminal, MKKLDRMPTH; Fr 60, AHAIDYSVV-
PADPALGEAIK; Fr 64, IDTANAVMFEDLPR; and Fr
70, DHGTTLQGLMLHHGIDPNDFLER. Degenerate
primers were designed on the basis of these sequences,
and a partial fragment of the PNP phosphatase gene was
amplified. Two primers (50-ATGAARAARYTIGAYM-
GIATG-30 and 50-TCYTCRAACATNCANGCRTT-30),
were designed based on the N-terminal and Fr-64
internal peptide sequences respectively, and a 0.53-kb
fragment was amplified from the chromosomal DNA of
S. meliloti IFO 14782. The fragment was cloned in
pCR2.1TOPO, and was named phoC28. The cloned
DNA fragment was sequenced, and it was confirmed
that an amino acid sequence deduced from the DNA
Expression of the PNP phosphatase gene. E. coli
JM109 having pKKpdxP (JM109/pKKpdxP) was grown
in LB with ampicillin and PM. After 1.5 h of cultivation,
expression of the PNP phosphatase gene was induced
by adding isopropyl-ꢀ-D-thiogalactopyranoside. After
cultivation for another 4.5 h, cells were harvested and
used in preparation of the cell-free extract (CFE). A
portion of the CFE was subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS–
PAGE) on 12.5% (w/v) gels, and stained with Coo-
massie Brilliant Blue.
S. meliloti strains were grown at 30 ꢀC in LB
.
supplemented with 0.061% MgSO4 7H2O and

424
Y. NAGAHASHI et al.
C101
C102
Fig. 2. DNA Sequence and Deduced Amino Acid Sequence of Sinorhizobium PNP Phosphatase.
Amino acid sequences corresponding to the sequences obtained by peptide sequencing are shown in bold letters. Sequences corresponding to
the sites of restriction enzymes are in italics. Arrows show primers C101 and C102. *1 Start codon in SMc01730. *2 Ala changed to Thr in
SMc01730.
sequences contained not only the N-terminal and partial
Fr-64 internal amino acid sequences of PNP phospha-
tase, but also the other two internal amino acid
sequences (Fr 60 and Fr 70) (Fig. 2). A partial fragment
of PNP phosphatase gene was then amplified with
ExTaq polymerase using primer C101; 50-GCCG-
AATTCGCCCATGTCACC-30 and primer C102; 50-
CGCCGTGTCGATGCGGTGAAG-30, which were de-
signed based on the DNA sequence of an insert fragment
in plasmid phoC28 using the chromosome DNA of
S. meliloti IFO 14782 as the template. An amplified
0.48-kb fragment was cloned in the pCRII-TOPO vector
to obtain plasmid pC1. An EcoRI fragment (0.5 kb) of
pC1 was labeled with digoxigenin, and Southern blot
hybridization was performed with the digoxigenin-
labeled DNA probe. On the other hand, chromosomal
DNA of S. meliloti was completely digested with SalI or
EcoRI, and then subjected to agarose gel electrophoresis
on 0.8% (w/v) gels. It was found that the probe
hybridized to 2.3-kb SalI and 3.5-kb EcoRI DNA
fragments of S. meliloti chromosomal digests (data not
shown). Approximately 3,000 clones of the genomic
library of S. meliloti IFO 14782 were screened by
colony hybridization with the same probe, resulting in
isolation of seven positive colonies. The plasmids were
recovered from these clones using Qiagen-midi tip,
digested with SalI or EcoRI, and subjected to agarose
gel electrophoresis on 1% (w/v) gels, and Southern blot
hybridization analysis was done. It was confirmed that
the 2.3-kb SalI and 3.5-kb EcoRI fragments of positive
clones were hybridized. One of the plasmids of positive
clones with an EcoRI fragment of about 24 kb named
pVEC1, was selected as a candidate having the entire
PNP phosphatase gene. The 2.3-kb SalI and 3.5-kb
EcoRI fragments of pVEC1 were ligated into pUC19,
and the two resulting plasmids were named pUCEC1
and pUCEC19 respectively. The nucleotide sequences
of the fragments inserted in both plasmids were
determined. It was found that pUCEC1 contained the
entire open reading frame (ORF) of PNP phosphatase
gene. But pUCEC19 lacked the N-terminal region of the
gene (Fig. 3). The PNP phosphatase gene was identified
as an open reading frame (ORF) of 708 bp and 235
amino acid residues (Fig. 2). We designated the gene
pdxP (accession no. AB353726).
Amino acid sequence of PdxP
All four amino acid sequences of the N-terminal
position and tryptic fragments of native PNP phospha-
tase of S. meliloti were found in the deduced amino acid
sequence of the ORF (Fig. 2, bold letters). The genome
sequence of S. meliloti strain 1021 has been deter-
mined.16,17) We conducted an NCBI BLAST search, and
the DNA sequence of the ORF was found to be very
closely related to the coding sequence region of
SMc01730 (positions 212,349 to 213,105, complement;

Pyridoxine 50-Phosphate Phosphatase Gene of S. meliloti
425
1 Kb
SalI
KpnI EcoRI
SalI
EcoRI
KpnI
pdxP
pUCEC1
pUCEC19
Fig. 3. Restriction Map of Sinorhizobium Chromosomal DNA Including the pdxP Gene.
Fig. 4. Alignment of PdxP with Human and Mouse PLPase.
Amino acid sequences, of PdxP with human and mouse PLPase were aligned using the ClustalW program. Conserved residues are indicated
by *, strongly similar residues by :, and weakly similar residues by ., Motif I, DXDX[T/V][L/V]; Motif III, [K/R]-[D/G].19,20)
GenBank accession no. AL591783). There are, however,
two differences between SMc01730 and pdxP gene: (i)
the start codon of pdxP is 68 bp downstream of that of
SMc01730, and (ii) Ala residue (*2 in Fig. 2) in the
deduced amino acid sequence of PdxP was Thr in
SMc01730 due to one base substitution (271G!A). As
described in the previous section, we determined the
N-terminal amino acid sequence of PNP phosphatase of
S. meliloti IFO 14782 using the natural enzyme. The
sequence strongly suggests that the original start codon
of pdxP is 68 bp downstream of SMc01730 (*1 in
Fig. 2). The predicted molecular mass of PdxP was
26,466. This was slightly different from the molecular
size (29 kDa) of the PNP phosphatase estimated by
SDS–PAGE analysis.9) A Blast search predicted that the
PdxP protein belonged to the haloacid dehalogenase
(HAD) superfamily, and the Conserved Domain Data-
base (CDD)18) was searched for the conserved domains
of the PdxP protein. PdxP was homologous to HAD
proteins that include putative hydrolase and phospho-
glycolate phosphatase in many microorganisms, such as
Brucella melitensis, Caulobacter crescentus, Neisseria
meningilidis, and Mesorhizobium loti, but the enzyme
function of those proteins has not been confirmed yet.
Hence a multiple alignment of the PdxP protein
sequence with human and mouse PLP-specific phospha-
tases10) with known functions was performed using the
ClustalW program (version 1.83). As shown in Fig. 4,
motifs I and III, which are conserved in the HAD
superfamily,19,20) are partially conserved in PdxP,
human, and mouse PLP phosphatases. When more
PNP/PLP-specific phosphatases are identified and char-
acterized, the conserved domain of PNP/PLP-specific
phosphatases should become more obvious and will

426
Y. NAGAHASHI et al.
Table 2. Pyridoxine Production by Sinorhizobium meliloti Recombi-
nants
1
2
3
M
Expressed Growth Pyridoxine Ratio
pdx gene (OD600
Recombinant
A
B
)
(mg/l)
(%)
IFO 14782
IFO 14782/pVKPtacpdxP
IFO 14782/pSHT
none
pdxP
pdxJ
46 ꢂ 4:7 102 ꢂ 6:0
51 ꢂ 3:8 96 ꢂ 7:7
53 ꢂ 2:8 129 ꢂ 0:1
100
94
C
126
IFO 14782/pSHTPtacpdxP pdxJ, pdxP 52 ꢂ 3:0 149 ꢂ 12:8 146
D
E
PdxP
assayed. CFE of S. meliloti IFO 14782/pVKPtacpdxP
showed PNP phosphatase activity of 28:3 ꢂ 9:3 nmol/
min/mg-protein, 3.5 times higher than that of the parent
strain (8:0 ꢂ 1:1 nmol/min/mg-protein), when 2 mM
PNP was used as the substrate. When PMP was used
as substrate, the phosphatase activity in the CFE of
S. meliloti IFO 14782/pVKPtacpdxP was almost the
same as that of the parent (0:5 ꢂ 0:1 and 0:4 ꢂ 0:1
nmol/min/mg-protein respectively). The PNP phospha-
tase of S. meliloti is unique in that it acts on PNP but not
on PMP,9) and the overexpressed PdxP in S. meliloti
showed an expected substrate specificity.
F
Fig. 5. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS–PAGE) of Cell Extracts of E. coli JM109/pKKpdxP and
Purified PdxP protein.
Lane 1, 10 mg of CFE of JM109/pKK223-3 (vector); lane 2,
10 mg of CFE of JM109/pKKpdxP; lane 3, 10 mg of purified PdxP;
lane 4, molecular mass markers (Bio-Rad). A, phosphorylase B
(101 kDa); B, bovine serum albumin (79 kDa); C, ovalbumin
(50.1 kDa); D, carbonic anhydrase (34.7 kDa); E, soybean trypsin
inhibitor (28.4 kDa); F, lysozyme (20.8 kDa).
Production of vitamin B6 in S. meliloti recombinants
To determine the effect of overexpression of the PNP
phosphatase gene on the production of PN, S. meliloti
pdxP recombinants were cultivated in glucose-CSL
medium (see ‘‘Materials and Methods’’). As shown in
Table 2, PN titer of the parent strain after cultivation for
9 d was 102 ꢂ 6:0 mg/l. When the pdxP gene was
overexpressed in S. meliloti IFO 14782, PN titer of the
pdxP recombinant was almost the same as that of the
parent (96 ꢂ 7:7 mg/l), although overexpression of pdxP
did not influence cell growth and showed potential for
production of PN. While the pdxP gene was overex-
pressed together with the PNP synthase gene (pdxJ), PN
titer increased to 149 ꢂ 12:8 mg/l respectively, 46% and
16% higher than that of the parent strain and the pdxJ
recombinant (IFO 14782/pSHT). These results suggest
that the dephosphorylation step in the PN biosynthesis of
S. meliloti is not a bottleneck in the IFO 14782 strain.
But overexpression of the pdxP gene contributed to PN
production in S. meliloti together with expression of the
pdxJ gene. This suggests that overproduction of PdxP
protein improves the PN production of S. meliloti if
enough PNP synthase is expressed. These results suggest
the possibility of development of the PN production
process by a metabolic engineering approach. In the
future, overexpression of the pdxP gene in combination
with another pyridoxine gene will be necessary to
develop even more efficient processes for production
of vitamin B6.
perhaps be differentiated from other phosphatase mono-
esterases.
Expression of pdxP gene in E. coli and S. meliloti
and PNP phosphatase activity
E. coli JM109 with S. meliloti pdxP expression
plasmid pKKpdxP was constructed, and CFE was
prepared as described in ‘‘Materials and Methods.’’
When PNP phosphatase gene was overexpressed in
E. coli, recombinant colonies were not obtained on an
LB agar plate, but were obtained on agar supplemented
with a high concentration of PM, but not with PN or
pyridoxal. Supplementation with PM might compensate
for a shortage of PLP, because it is converted to PMP,
which is not a substrate of PNP phosphatase, and
subsequently is converted to PLP by a salvage pathway.8)
On SDS–PAGE, overproduction of a polypeptide with
an expected molecular size was observed in E. coli
JM109/pKKpdxP (Fig. 5, lane 2). PNP phosphatase
activity of the CFE was assayed by quantifying the
amount of PN formed. It is possible that E. coli has
cytoplasmic PLP phosphatase activity,21) and so CFE
of E. coli JM109/pKK223-3 (vector control) had its
own native phosphatase activity (7:1 ꢂ 0:8 nmol/min/
mg-protein), but that of E. coli JM109/pKKpdxP
showed a slight increase in activity (8:8 ꢂ 2:2 nmol/
min/mg-protein) even though overexpression of PdxP
was observed by SDS–PAGE.
Acknowledgments
CFE of S. meliloti
IFO 14782 recombinant with pVKPtacpdxP was
prepared (see ‘‘Materials and Methods’’), and phospha-
tase activity on PNP and on PMP as substrate was
We are grateful to Ms. Keiko Ichikawa for helpful
suggestions, and we thank DSM Nutritional Products
Ltd. for kind support.

Pyridoxine 50-Phosphate Phosphatase Gene of S. meliloti
427
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