Efficient biotransformations using Escherichia coli with tolC acrAB mutations expressing cytochrome P450 genes

Article abstract of DOI:10.1271/bbb.80627

We report here some efficient biotransformations using Escherichia coli strains with disruptions for the AcrAB-TolC efflux pump system. Biotransformations of compactin into pravastatin (6α-hydroxy-iso- compactin) were performed using E. coli strains with tolC and/or acrAB mutations expressing a cytochrome P450 (P450) gene. The production levels of pravastatin using strains with acrAB, tolC, and tolC acrAB mutations increased by 3.7-, 7.0-, and 7.1-fold, respectively. Likewise, the production levels of 25-hydroxy vitamin D₃ and 25-hydroxy 4-cholesten 3-one using tolC acrAB mutant strains expressing an individual P450 gene increased by 2.2- and 16-fold, respectively. The enhancement of this biotransformation efficiency could be explained by increases in the intracellular amounts of substrates and the concentrations of active P450s. These results demonstrate that we have achieved versatile methods for efficient biotransformations using E. coli strains with tolC acrAB mutations expressing P450 genes.

Full text of DOI:10.1271/bbb.80627

Biosci. Biotechnol. Biochem., 73 (4), 805–810, 2009
Efficient Biotransformations Using Escherichia coli
with tolC acrAB Mutations Expressing Cytochrome P450 Genes
y
Tadashi FUJII, Yoshikazu FUJII, Kazuhiro MACHIDA, Atsushi OCHIAI, and Masashi ITO
Bioresource Laboratories, Mercian Corporation, 1808 Nakaizumi, Iwata, Shizuoka 438-0078, Japan
Received September 5, 2008; Accepted December 18, 2008; Online Publication, April 7, 2009
[doi:10.1271/bbb.80627]
We report here some efficient biotransformations
kinetics theory, the enzymatic reaction rate increases as
the concentration of substrate increases when enzyme
quantity is kept constant and the substrate concentration
is low enough. An enhancement of the ability to uptake
L-lysine by overexpressing lysP (encoding lysine-
specific permease) led to L-lysine accumulation in
E. coli cells, and that caused efficient bioproduction of
L-pipecolic acid.
using Escherichia coli strains with disruptions for the
AcrAB-TolC efflux pump system. Biotransformations of
compactin into pravastatin (6ꢀ-hydroxy-iso-compactin)
were performed using E. coli strains with tolC and/or
acrAB mutations expressing a cytochrome P450 (P450)
gene. The production levels of pravastatin using strains
with acrAB, tolC, and tolC acrAB mutations increased
by 3.7-, 7.0-, and 7.1-fold, respectively. Likewise, the
production levels of 25-hydroxy vitamin D3 and 25-
hydroxy 4-cholesten 3-one using tolC acrAB mutant
strains expressing an individual P450 gene increased
by 2.2- and 16-fold, respectively. The enhancement of
this biotransformation efficiency could be explained by
increases in the intracellular amounts of substrates and
the concentrations of active P450s. These results dem-
onstrate that we have achieved versatile methods for
efficient biotransformations using E. coli strains with
tolC acrAB mutations expressing P450 genes.
Several physiological and biochemical approaches
have been applied with the aim of elucidating the
mechanisms of microbial resistance to solvents and
6
)
antibiotics. Genetic evidence suggests that the AcrAB-
TolC efflux pump is involved in the solvent resistance
of E. coli cells, and the AcrAB-TolC pump reduces the
intracellular solvent concentration in E. coli cells ex-
7
)
posed to solvents. The antibiotic susceptibility profiles
of E. coli strains lacking multidrug efflux pump genes
revealed that deletions in acrAB or tolC definitely
resulted in increased susceptibility to the majority of
8
)
compounds tested.
The P450 enzymes constitute a large superfamily of
heme proteins involved in the metabolism of a wide
Key words: biotransformation; cytochrome P450; pra-
vastatin; tolC
9
)
variety of both exogenous and endogenous compounds.
There is considerable interest in the development of
technology for the production of commercially valuable
chemicals from hydrophobic or amphiphilic compounds
using biocatalysts. In particular, regio-specific oxidation
of compounds has attracted much interest because many
important chemicals can be produced by these reac-
tions.1 The enzymatic oxidation reactions require
regeneration of the redox cofactor, for example NADH,
and thus biotransformation using the whole cell is
favored as a method for this biocatalysis. The above
necessitates the use of biotransformation systems using
Escherichia coli expressing several oxidases, including
cytochrome P450 (P450), for the development of
bioproduction processes.
Usually, they act as the terminal oxidase in multi-
component electron transfer chains. All the multicom-
ponent electron transfer chains which contain P450 are
termed here P450-containing systems. P450-containing
systems can be classified according to the number of their
protein components. Mitochondrial and most bacterial
P450-containing systems have three components: an
FAD-containing flavoprotein (ferredoxin reductase), an
iron-sulphur protein (ferredoxin), and a P450.
One of the most famous biotransformations using
P450 expressed by microorganisms is that of compactin
into pravastatin, which is 6ꢀ-hydroxy-iso-compactin and
a 3-hydroxy-3-methyl glutaryl CoA reductase inhibitor.
Pravastatin is produced by two-step fermentation. First
compactin is produced by Penicillium citrinum, and then
1
0)
)
2
)
Biotransformations using E. coli expressing enzy-
matic genes have been performed for some other
it is oxidized by biotransformation using Streptomyces
11,12)
3
,4)
industrial bioproduction processes, and it is commer-
cially important to enhance the biotransformation
efficiency. We previously showed that L-pipecolic acid
can be produced by the biotransformation of L-lysine
using lat-expressing E. coli, and the efficiency of
L-pipecolic acid production was enhanced by lysP and
carbophilus to form pravastatin.
The P450 enzyme
responsible for this oxidation, cytochrome P450 105A3
(P450sca-2), was identified and its coding gene was
1
3)
cloned. However, no study about the biotransforma-
tion of compactin into pravastatin using a recombinant
strain was reported except that using Streptomyces
5
)
13)
yeiE amplification. As propounded by the enzymatic
lividans expressing the P450sca-2 gene.
y
To whom correspondence should be addressed. Tel: +81-538-21-1134; Fax: +81-538-21-1135; E-mail: fujii-td@mercian.co.jp
Abbreviations: BRM, biotransformation reaction mixture; 4C3O, 4-cholesten 3-one; OMF, outer membrane factor; P450, cytochrome P450;
PMCD, 2,3,6-partially methylated-ꢁ-cyclodextrin; VD3, vitamin D3

8
06
T. FUJII et al.
DNA fragment containing the SD-sequence and the NdeI site was
We report here versatile methods for efficient bio-
amplified using primer 7 and primer 8, digested with BglII and BamHI,
and ligated into the BglII-BamHI site of the plasmid. The resulting
plasmid was named pEN1122.
transformations of hydrophobic and amphiphilic com-
pounds to produce their regio-specific oxidized products
using E. coli strains with tolC acrAB mutations ex-
pressing P450 genes.
A DNA fragment of the N-terminal 150 bp of aciA was amplified
from pDolABC using primer 9 and primer 10, and digested with NdeI
and SpeI. A DNA fragment of dvbA was amplified from Dactylospor-
angium variesporum IFO 14104 chromosomal DNA using primer 11
and primer 12, and digested with SpeI and BamHI. The resulting
fragments were then ligated into the NdeI-BamHI site of pEN1122, and
the resulting plasmid was named pEN1123.
Materials and Methods
Strains. E. coli BLstarTol (tolC210::Tn10) was constructed by P1
transduction of the tolC210::Tn10 allele from CAG12184 (provided by
the E. coli Genetic Stock Center, Yale University) into BL21star(DE3)
A DNA fragment of vdh was amplified from Pseudonocardia
autotrophica NBRC 12743 chromosomal DNA using primer 13 and
primer 14, and digested with SpeI and BglII. This fragment was then
ligated into the SpeI-BamHI site of pEN1123, and the resulting plasmid
was named pEN7373.
(Invitrogen, Carlsbad, CA). E. coli BLstarAcr (acrAB::cat) and
BLstarTolAcr (tolC210::Tn10, acrAB::cat) were constructed by P1
transduction of the acrAB::cat allele from JA300A into BL21star(DE3)
and BLstarTol, respectively.7) The mutations were confirmed by DNA
sequencing.
Streptomyces sp. TM-7 was used for the isolation of boxAB
Accession no. AB180845). Pseudonocardia autotrophica NBRC
Biotransformation of compactin. E. coli BL21star(DE3),
BLstarAcr, BLstarTol, and BLstarTolAcr were transformed with
(
ꢀ
12743 was used for the isolation of vdh (Accession no. AB456955).
pCP101, and each strain was cultivated at 25 C with shaking in
P450M9-SEED broth (6.78 g/l Na2HPO4, 3 g/l KH2PO4, 0.5 g/l NaCl,
1 g/l NH4Cl, 10 g/l casamino acids, 4 g/l D-glucose, 0.1 mM CaCl2,
Deactylosporangium variesporum IFO 14104 was used for the
isolation of dvbA (Accession no. AB442014).
1
1
mM MgCl2, 0.1 mM FeSO4, and 20 mg/l thymine) containing
00 mg/l carbenicillin. After 24 h of cultivation, 200 ml of each culture
DNA manipulation. Chromosomal DNAs from Streptomyces sp.
TM-7, Pseudonocardia autotrophica NBRC 12743, and Deactylospor-
angium variesporum IFO 14104 were prepared using Isoplant (Nippon
Gene, Tokyo). Plasmids were prepared using a QIAprep Spin Miniprep
Kit (Qiagen, Hilden, Germany). DNA ligation was performed using the
ligation kit (Takara, Kyoto). All restriction enzymes were obtained
from Takara. All amplifications by PCR were performed using KOD-
Plus-(Toyobo, Osaka). DNA sequencing analysis was done using the
BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems,
Foster City, CA). The primers used in this study were synthesized by
Sigma Aldorich Japan (Tokyo) and are shown in Table 1. The
plasmids used in this study were constructed as follows.
was added into 25 ml of P450M9-Main broth (6.78 g/l Na2HPO4, 3 g/l
KH2PO4, 0.5 g/l NaCl, 1 g/l NH4Cl, 10 g/l casamino acids, 0.1 mM
CaCl2, 0.1 mM FeSO4, 20 mg/l thymine, and 80 mg/l 5-aminolevulinic
acid) containing 100 mg/l of carbenicillin and the Overnight Expres-
sion System (20 ml of solution I, 50 ml of solution II, and 1 ml of
solution III per ml, Merck, Darmstadt, Germany). 5-Aminolevulinic
acid, a precursor of heme, was added because P450 enzymes are heme-
containing proteins and require heme to be expressed. After 24 h of
ꢀ
cultivation at 25 C with shaking at 220 rpm, the cells were collected
(
(
about 0.66 g of wet cells) and suspended in 5 ml of CV2 buffer
50 mM sodium phosphate buffer, pH 7.4, 2% (v/v) glycerol, 50 mg/l
carbenicillin, and 0.1 mM isopropyl-ꢁ-D-thiogalactopyranoside). This
mixture was defined as biotransformation reaction mixture (BRM).
Biotransformations were started when 30 ml of 25 mg/ml of sodium
A fragment of aciC derived from pDolABC was ligated into the
BamHI-HindIII site of pETduet-1 (Novagen, San Diego, CA).14) Then,
a DNA fragment of boxB was amplified from Streptomyces sp. TM-7
chromosomal DNA using primer 1 and primer 2. Next, a DNA
fragment of the boxA was amplified from Streptomyces sp. TM-7
chromosomal DNA using primer 3 and primer 4, digested with NdeI
and XhoI, and ligated into the NdeI-XhoI site of this plasmid. The
resulting plasmid was named pCP101.
compactin in water were added into 1 ml of BRM in test tubes. After 0,
ꢀ
2
, 5, 9, or 24 h of incubation at 28 C with shaking at 220 rpm, 1 ml of
acetonitrile and 1 ml of methanol were added to each test tube and
mixed. For the sample of 24 h of incubation, an additional 30 ml of the
compactin solution was added after 9 h of incubation. The samples
were centrifuged at 20;000 ꢁ g for 5 min, and 1 ml of the supernatant
was recovered. Then HPLC analyses were performed as described
below.
A DNA fragment of aciB from pDolABC was digested with NcoI
and BamHI and ligated into the NcoI-BamHI site of pETduet-1. The
aciC fragment was amplified from pDolABC using primer 5 and
primer 6, digested with BamHI and HindIII, and ligated into the
BamHI-HindIII site of this plasmid. Then the 2nd T7-promoter and the
NdeI site in this plasmid were removed by digestion with EcoRV and
NotI, and this DNA fragment was blunted and self-ligated. Then the
Biotransformation of vitamin D3. E. coli BL21star(DE3),
BLstarAcr, BLstarTol, and BLstarTolAcr were transformed with
pEN7373, and BRM was prepared using these cells. Then 25 ml of
10 mg/ml of vitamin D3 (VD3) in DMSO and 30 ml of 25% 2,3,6-
partially methylated-ꢁ-cyclodextrin (PMCD) were added to 1 ml of
BRM in test tubes. PMCD was added for solubilization of VD3. After
ꢀ
Table 1. Primers Used in This Study
0
, 2, 4, 6, 8, or 24 h of incubation at 28 C with shaking, 2 ml of
Primer
number
methanol was added into each test tube and mixed. These samples were
centrifuged at 20;000 ꢁ g for 5 min, and 1 ml of the supernatant was
recovered. Then HPLC analyses were performed as described below.
0
0
Sequence (5 ! 3 )
primer 1 CAGCCATGGGTGTGACGGCCGACCGGGAGGT
primer 2 CACGGATCCTACTCGACGACGCGTACCGCGCC-
GGA
Biotransformation of 4-cholesten 3-one. E. coli BL21star(DE3),
BLstarAcr, BLstarTol, and BLstarTolAcr were transformed with
pEN1123, and BRM was prepared using these cells. Then 40 ml of
10 mg/ml of 4-cholesten 3-one (4C3O) in methanol and 30 ml of 25%
PMCD were added to 1 ml of BRM in test tubes. PMCD was added for
primer 3 GGACATATGACCGAGACCGTTACGACGCCC
primer 4 TCGCTCGAGTCACCAGGTGACCGGGAGTTC
primer 5 CATGGATCCTGAACTGAGTGAATTGATATGCAA
primer 6 CCCAAGCTTCTACCCCATCAACGCCTGTACC
primer 7 GTAAGATCTAAATAAGGAGGAATAACATATGG-
CGCTGACCACCACCGGCA
solubilization of 4C3O. After 0, 2, 4, 6, 8, or 24 h of incubation at
ꢀ
2
8 C with shaking, 2 ml of methanol was added to each test tube and
this was mixed. These samples were centrifuged at 20;000 ꢁ g for
min, and 1 ml of the supernatant was recovered. Then HPLC analyses
were performed as described below.
primer 8 CCCGGATCCCGGTGCCGGTGGTGGTCAGCGCCA
primer 9 TAACATATGAACTCAGTCGCAGAAATTTTTGA
primer 10 CGAACTAGTGGTCACTTGACCATGCAAAAACT
primer 11 CCGACTAGTACCGAAACGCTGTACCCCGAG
primer 12 CCTGGATCCTCATGAGAACGTCACCGGCAG
primer 13 ACCACTAGTGCGCTGACCACCACCGGCACCG
primer 14 GGGAGATCTTCAGGCGCTGCGCGGCCCCATC
5
Determination of the intracellular amounts of substrates. The
intracellular amounts of substrates were determined by method
similar to that described previously.7) Briefly, E. coli BL21star(DE3),
ꢀ
BLstarAcr, BLstarTol, and BLstarTolAcr were cultivated at 25 C with

Efficient Biotransformations Using E. coli with tolC acrAB Mutations
807
shaking in P450M9-SEED broth. After 24 h of cultivation, 200 ml of
the culture was added to 25 ml of same media. After 24 h of cultivation
formed with pCP101. About 237, 867, 1,670 and
,700 mg/l of pravastatin were produced from compac-
1
ꢀ
at 25 C with shaking at 220 rpm, the cells were collected (about 0.64 g
of wet cells) and suspended in 5 ml of CV buffer (50 mM sodium
tin after 24 h of incubation by biotransformation using
E. coli BL21star(DE3), BLstarAcr, BLstarTol, and
BLstarTolAcr, respectively (Fig. 2A). Accordingly, the
production levels of pravastatin using strains with
acrAB, tolC, and tolC acrAB mutations increased by
phosphate buffer, pH 7.4, and 2% (v/v) glycerol). Then 30 ml of 25 mg
/ml of sodium compactin in water, 100 ml of 10 mg/ml of VD3 in
DMSO and 80 ml of PMCD, or 20 ml of 10 mg/ml of 4C3O in methanol
and 30 ml of 25% PMCD were added to 1.0 ml of this cell suspension in
a test tube. After 4.5 h of incubation at 28 C with shaking at 220 rpm,
ꢀ
3.7, 7.0, and 7.1-fold, respectively. Almost all the
samples were centrifuged at 2;000 ꢁ g for 5 min, and the precipitates
were recovered. After these precipitates were washed twice, 1 ml of
CV buffer was added and the substrates were extracted as described
above. Then, HPLC analyses were performed as described below.
compactin consumed in these biotransformations were
converted into pravastatin without loss.
Biotransformation of VD3 into 25-hydroxy VD3
In order to confirm the superiority of the E. coli strains
with tolC and/or acrAB mutations for bioproduction
using P450 genes, we attempted biotransformation of
VD3, a hydrophobic compound, into 25-hydroxy VD3
using a P450 gene vdh belonging to the CYP107 family
CO difference spectral analysis. To quantitate the concentration
of P450s in the E. coli cells, CO difference spectral analyses were
performed. The CO difference spectrum of each BRM diluted by 5-fold
was measured with a UV-visible spectrophotometer U-3310 (Hitachi,
Tokyo) as described previously.15) In order to calculate the concen-
ꢂ1
ꢂ1
trations of active P450s, an extinction coefficient of 91 mM cm
was used (concentration of active P450 (nM) = CO difference at
(Fujii Y, et al., unpublished results). We constructed
pEN7373 expressing P450-containing system genes
composed of vdh, aciB (encoding a ferredoxin), and
ꢂ1
ꢂ1
6
4
50 nm/91 (mM cm ) ꢁ 10 .
1
4)
HPLC analyses. Pravastatin and compactin were analyzed by HPLC
a
aciC (Fig. 1B), because no gene encoding ferredoxins
or ferredoxin reductases were in the neighborhood of
vdh. Then we performed biotransformation of VD3 into
on
Chromolith Performance RP-18e (4.6 mm I.D. ꢁ 100 mm)
ꢀ
column maintained at 40 C using a methanol concentration gradient
in water aqueous solution (containing 0.1% triethylamine and 0.1% of
acetic acid) from 50% to 90% for 3 min, and 90% to 90% for 0.5 min,
followed by 50% to 50% for 2.5 min at a flow rate of 2.0 ml per min.
The derivatives were detected by UV-monitoring at 238 nm. Pravas-
tatin and compactin had retention times of 1.8 min and 3.2 min,
2
5-hydroxy VD3 using E. coli strains with tolC and/or
acrAB mutations transformed with pEN7373. About
6.2, 135, 187, and 216 mg/l of 25-hydroxy VD3
9
was produced from VD3 after 24 h of incubation with
E. coli BL21star(DE3), BLstarAcr, BLstarTol, and
BLstarTolAcr, respectively (Fig. 2B). Accordingly, the
production levels of 25-hydroxy VD3 using strains with
acrAB, tolC, and tolC acrAB mutations increased by 1.4,
respectively.
0
2
5-hydroxy VD3 and VD3 were analyzed by HPLC on a J sphere
ꢀ
ODS-H80 (4.6 mm I.D. ꢁ 75 mm) column maintained at 40 C using
an acetonitrile concentration gradient in water aqueous solution from
3
7
0% to 30% for 13 min, 30% to 100% for 1 min, 100% to 100% for
min, and 100% to 70% for 1 min, followed by 70% to 70% for 3 min
1.9, and 2.2-fold, respectively. Almost all the VD3
consumed in these biotransformations were converted
into 25-hydroxy VD3 without loss.
at a flow rate of 1.0 ml per min. The derivatives were detected by
UV-monitoring at 265 nm. 25-hydroxy VD3 and VD3 had retention
times of 3.0 min and 9.3 min, respectively.
2
5-hydroxy 4C3O and 4C3O were analyzed by HPLC on a Inertsil
Biotransformation of 4C3O into 25-hydroxy 4C3O
We also attempted biotransformation of 4C3O, a
hydrophobic compound, into 25-hydroxy 4C3O using a
P450 gene dvbA belonging to the CYP105 family. We
constructed pEN1123 expressing P450-containing sys-
tem genes composed of dvbA, aciB, and aciC (Fig. 1C).
Then, we performed biotransformation of 4C3O into 25-
hydroxy 4C3O using mutant strains transformed with
pEN1123. About 26.4, 79.7, 398, and 415 mg/l of 25-
hydroxy 4C3O was produced from 4C3O after 24 h of
incubation with E. coli BL21star(DE3), BLstarAcr,
BLstarTol, and BLstarTolAcr, respectively (Fig. 2C).
Accordingly, the production levels of 25-hydroxy 4C3O
using strains with acrAB, tolC, and tolC acrAB
mutations increased by 3.0-, 15-, and 16-fold, respec-
tively. Almost all the 4C3O consumed in these bio-
transformations were converted into 25-hydroxy 4C3O
without loss.
ꢀ
ODS-3 (4.6 mm I.D. ꢁ 50 mm) column maintained at 40 C using an
acetonitrile concentration gradient in 0.85% phosphate aqueous solu-
tion from 40% to 100% for 5 min, 100% to 100% for 3 min, and 100%
to 40% for 0.5 min, followed by 40% to 40% for 2.5 min at a flow rate
of 1.2 ml per min. The derivatives were detected by UV-monitoring at
235 nm. 25-hydroxy 4C3O and 4C3O had retention times of 5.3 min
and 8.1 min, respectively.
Results
Biotransformation of compactin into pravastatin
Previously, we isolated the boxAB gene from Strep-
tomyces sp. TM-7. The boxA gene encodes the protein
belonging to CYP105 family that showed high similarity
to P450sca-2 with 76% identity. This gene was followed
by boxB encoding a protein showing high similarity to
many ferredoxins from Streptomyces. In order to utilize
the P450-containing system including BoxA for bio-
production, we constructed pCP101 expressing P450-
containing system genes composed of boxA, boxB, and
aciC (Fig. 1A). We adopted aciC as the gene encoding a
ferredoxin reductase, because no gene encoding ferre-
doxin reductases was in the neighborhood of boxAB, and
E. coli expressing P450-containing system genes com-
posed of aciABC showed efficient biotransformation
Intracellular amounts of substrates
We exposed E. coli cells with tolC and/or acrAB
mutations to compactin and analyzed the intracellular
amounts of them. The intracellular amounts of compac-
tin in E. coli cells with acrAB, tolC, and tolC acrAB
mutations increased by 7.0, 75, and 77-fold, respectively
(Fig. 3A). The intracellular amounts of VD3 and 4C3O
similarly increased in the tolC and tolC acrAB mutant
strains, whereas decreased in the acrAB mutants
(Fig. 3B and C). These results suggest that E. coli
1
4)
activity of n-octane.
Biotransformation of compactin, an amphiphilic
compound, into pravastatin was performed using the
E. coli strains with tolC and/or acrAB mutations trans-

808
T. FUJII et al.
A
B
pCP101
T7 promoter boxB
aciC
T7 promoter
boxA
pEN7373
T7 promoter aciB
vdh
aciC
C
pEN1123
T7 promoter aciB
dvbA
aciC
Fig. 1. P450-Containing System Genes on Plasmids.
Plasmids were constructed composed of P450-containing system genes for the biotransformation of compactin (A), VD3 (B), and 4C3O (C),
respectively. Triangles show T7 promoters. Arrows show the genes belonging to P450-containing systems. boxA, a P450 gene (CYP105 family)
isolated from Streptomyces sp. TM-7; vdh, a P450 gene (CYP107 family) isolated from P. autotrophica NBRC 12743; dvbA, a P450 gene
(CYP105 family) isolated from D. variesporum IFO 14104; boxB, a ferredoxin gene isolated from Streptomyces sp. TM-7; aciB, a ferredoxin
gene isolated from Acinetobacter sp. OC4; aciC, a ferredoxin reductase gene isolated from Acinetobacter sp. OC4.
A
B
C
2
1
1
000
500
000
250
500
400
300
200
100
0
2
1
1
00
50
00
500
5
0
0
0
0
5
10
15
20
25
0
5
10
15
20
25
0
5
10
15
20
25
Time (h)
Time (h)
Time (h)
Fig. 2. Biotransformation Using E. coli Strains with tolC and/or acrAB Mutations Expressing P450-Containing System Genes.
Biotransformation of compactin into pravastatin (A), VD3 into 25-hydroxy VD3 (B), and 4C3O into 25-hydroxy 4C3O (C) was performed
using E. coli BL21star(DE3) (triangles), BLstarAcr (squares), BlstarTol (open circles), and BLstarTolAcr (closed circles) individually
ꢀ
transformed with pCP101(A), pEN7373 (B), and pEN1123 (C). After incubation at 28 C, the amounts of products in 1 ml of reaction solution
were determined. Control experiments using E. coli cells transformed with pETduet-1 did not convert the substrates at all. Three independent
experiments were done, and the average is shown.
strains with tolC mutation reduced the abilities to release
substrates (hydrophobic or amphiphilic compounds) and
accumulated them inside the cells, and that acrAB
mutation in the tolC mutant strain had additive effects.
Only in the case of compactin, the acrAB mutation
added into the wild-type strain seemed to contribute to
an additional accumulation of the substrate.
Discussion
Biotransformations using E. coli have been performed
for some industrial bioproductions, and it is commer-
cially important to enhance their biotransformation
efficiency. We demonstrated here that the production
level of pravastatin using the strain with tolC acrAB
mutations increased by 7.1-fold, and about 1,700 mg/l
of pravastatin was produced.
CO difference spectral analyses of the P450s
In order to quantitate the concentrations of active
BoxA, we analyzed the spectral characteristics of BoxA
in E. coli cells with tolC and/or acrAB mutations
transformed with pCP101. The intracellular concentra-
tions of BoxA in the E. coli cells with acrAB, tolC, and
tolC acrAB mutations increased by 11, 18, and 27-fold,
respectively (Fig. 4A). Similarly, the intracellular con-
centrations of VDH and DvbA in E. coli cells increased
a little with acrAB mutation and largely with tolC
mutation (Fig. 4B and C). These results suggest that
E. coli strains with tolC mutation efficiently accumu-
lated P450s expressed their own. In the cases of BoxA
and DvbA, the acrAB mutation added into the tolC
mutant strain contributed to additional accumulation of
P450s.
One explanation for this enhancement could be
derived by the result that the intracellular amount of
compactin in the tolC acrAB mutant strain increased by
77-fold. As propounded by the enzymatic kinetics
theory, the enzymatic reaction rate increases as the
concentration of substrate increases. The disruption of
AcrAB-TolC efflux pump system reduces the ability to
release substrates and accumulates them inside the cells,
and thus enhanced oxidation by P450 might be achieved.
In a water-organic solvent two-phase system, it was
reported that organic solvent resistant E. coli showed
higher biotransformation ability than the wild-type
1
6)
strain. Other solvent-tolerant bacteria able to grow
in the presence of organic solvents are also thought to be
good candidates for the biotransformation of hydro-
1
7)
phobic substrates. Contrary to these general assump-

Efficient Biotransformations Using E. coli with tolC acrAB Mutations
809
A
B
C
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160
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Fig. 3. Entry of Substrates into E. coli Cells with tolC and/or acrAB Mutations.
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E. coli BL21star(DE3) (wild type), BLstarAcr (acrAB ), BlstarTol (tolC ), and BLstarTolAcr (tolC acrAB ) were incubated in 1 ml of
media with compactin (A), VD3 (B), and 4C3O (C) individually, and the amounts of these substrates in these cells were determined.
A
B
3000
2500
2000
1500
1000
500
C
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Fig. 4. CO Difference Spectral Analyses.
The CO difference spectrum of E. coli BL21star(DE3) (wild type), BLstarAcr (acrAB ), BlstarTol (tolC ), and BLstarTolAcr (tolC
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acrAB ) transformed with pCP101 (A), pEN7373 (B), and pEN1123 were analyzed individually. Then the concentrations of active P450s (BoxA
(A), Vdh (B), and DvbA (C)) in cells were calculated. Three independent experiments were done, and the average is shown.
tions, our strains with tolC acrAB mutations showed
higher biotransformation ability than the wild-type strain
in water-phase systems.
converted into pravastatin without loss, possibly because
E. coli had no endogenous P450s. Thus our E. coli-
system has an advantage over other Streptomyces-
systems.
Another explanation for the enhanced biotransforma-
tion of compactin is that the intracellular concentration
of BoxA in E. coli cells with tolC acrAB mutation
increased by 27-fold. Recently, it was reported that the
TolC-dependent efflux system is involved in the ex-
clusion of porphyrin(ogen)s, including heme.1 The
tolC mutant strains reduce the ability to release not only
substrates but also heme and accumulate them inside
the cells. Because P450 enzymes are heme-containing
proteins, accumulation of heme in the cells might lead to
increased expression of P450s and thereby enhanced
oxidation might be achieved.
We also report here that the production levels of
25-hydroxy VD3 and 25-hydroxy 4C3O using strains
with acrAB, tolC, and tolC acrAB mutations increased,
and especially those using the tolC acrAB mutant strains
increased by 2.2 and 16-fold, respectively. These results
demonstrate the versatility of our methods for efficient
biotransformations using E. coli strains with tolC acrAB
mutations expressing P450 genes. The biotransformation
efficiencies of VD3 and 4C3O using the acrAB mutant
strain were higher than those using the wild-type strain
in spite of decreases in the intracellular amounts of
substrates, suggesting that the increased accumulation of
P450s in the acrAB mutant contribute to enhanced
biotransformation. The biotransformation efficiency of
VD3 using the tolC acrAB mutant strain was higher than
that using tolC mutant in spite of decreases in the
intracellular amounts of the P450, suggesting that the
increased accumulation of the substrate in the tolC acrAB
mutant contributes to enhanced biotransformation.
Several different multidrug resistance efflux systems
in E. coli have been identified. All of them consist of an
outer membrane factor (OMF) and a membrane fusion
8)
Some studies have reported on the biotransformation
of compactin into pravastatin using Streptomyces. The
highest production level of pravastatin was 340 mg/l
1
9)
from 750 mg/l of compactin for 24 h. In the Strepto-
myces-system, only about 60% of compactin was
converted into pravastatin and about 40% of compactin
was metabolized into other chemicals, possibly due to
oxidation activities by endogenous P450s. This is one of
the most serious problems in the industrial production of
pravastatin, because compactin is the most expensive
substance in the material cost. On the other hand, almost
the compactin consumed in our E. coli-system was
8
)
protein. About OMFs, it was reported that TolC is a

810
T. FUJII et al.
major example in E. coli, and that other OMFs (Yjcp,
YohG, and YlcB) hardly contribute to efflux pump
systems.8 We demonstrated that E. coli strains with
tolC mutation accumulated substrates and showed more
efficient biotransformation efficiency, and that acrAB
mutation into the tolC mutant had additive effects. These
additive effects suggest possibilities of AcrAB-cou-
plings with other OMFs and export by these complexes.
That is, inactivation of other OMFs-AcrAB efflux
pumps could have some additive effects on the accu-
mulation of substrates. Indeed, a previous study pro-
posed the possibility that two putative members of the
OMF also affect intrinsic resistance in addition to TolC,
based on the fact that deletion of yjcP and yohG resulted
in a 4-fold increase in susceptibility to some limited
antibiotics (puromycin, acriflavin, and tetraphenylarso-
nium chloride of 35 antibiotics). We identified that the
vast majority of products oxidized by biotransformations
using the tolC and/or acrAB mutant strains did not
localize in intracellular domains but in the extracellular
media (data not shown). These results also suggest the
existence of TolC-independent efflux systems to export
these products. Fortunately, this localization of products
in extracellular media prevented the decline of our
biotransformation efficiency.
using E. coli strains with tolC acrAB mutations ex-
pressing P450 genes. We have realized that our methods
probably improve the efficiency of biotransformations
using enzymatic genes other than P450s.
)
Acknowledgment
We are very grateful to Dr. Wanyoike George
(Mercian corporation) for critical reading of the manu-
script and for helpful comments. We thank Professor
Noriyuki Doukyu (Toyo university) for E. coli JA300A.
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Here we demonstrated versatile methods for efficient
biotransformations of hydrophobic and amphiphilic
compounds to produce regio-specific oxidized products
1