# Proteome-Level Responses ofEscherichia coli to Long-Chain Fatty Acids and Use of Fatty Acid Inducible Promoter in  Protein Production

Authors: Mee-Jung Han; Jeong Wook Lee; Sang Yup Lee; Jong Shin Yoo
Journal: Journal of Biomedicine and Biotechnology
 (2008)

---

## Abstract

InEscherichia coli, a
long-chain acyl-CoA is a regulatory
signal that modulates gene expression
through its binding to a transcription
factor FadR. In this study,
comparative proteomic analysis of
E. coli in the presence
of glucose and oleic acid was
performed to understand cell
physiology in response to oleic acid.
Among total of 52 proteins showing
altered expression levels with oleic
acid presence, 9 proteins including
AldA, Cdd, FadA, FadB, FadL, MalE,
RbsB, Udp, and YccU were newly
synthesized. Among the genes that were
induced by oleic acid, the promoter of
the aldA gene was used
for the production of a green
fluorescent protein (GFP). Analysis of
fluorescence intensities and confocal
microscopic images revealed that
soluble GFP was highly expressed under
the control of the aldA
promoter. These results suggest that
proteomics is playing an important
role not only in biological research
but also in various biotechnological
applications.


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## Body

## 1. Introduction

Exogenous fatty acids and their
derivatives influence a wide variety of cellular processes including fatty acids
and phospholipids synthesis, organelle inheritance, vesicle fusion, protein
export and modification, enzyme activation or deactivation, cell signaling,
membrane permeability, bacterial pathogenesis, and transcriptional control [1, 2].
The process governing the transport of fatty acids from environmental
conditions across the membrane is distinct from the transport of hydrophilic
substrates such as sugars and amino acids. In a number of cell types, the
process of fatty acid transport is inducible and commensurate with the
expression of specific sets of proteins [2]. In wild-type Escherichia coli, growth on fatty acids requires specific transport
system (FadL), acyl-CoA dehydrogenase (FadD), enzymes of the β-oxidation cycle (FadA,
FadB, FadE, FadF, FadG, and FadH), and glyoxylate shunt (AceA, AceB, and AceK),
and these genes are negatively regulated by a transcriptional factor, FadR. Supply
of long-chain fatty acids that contain 12 or more carbons results in the derepression of the genes negatively controlled by FadR but leads to the decreased expression
of the genes (e.g., fabA and fabB) activated by FadR, indicating that
long-chain acyl-CoA esters are the effector molecules that regulate fatty acids
metabolism and thereby mediate inductions [3]. Therefore, E. coli cells can grow on minimal
medium containing long-chain fatty acids but it cannot grow on short- and medium-chain
fatty acids due to no induction of the enzymes associated with fatty acids
metabolism. So far, genes involved in fatty acids metabolism (i.e., fad regulon) of E. coli have been reported at the transcriptional level by
biochemical and genetic analyses [3–6]. Therefore, in this study, we looked at the
effects of long-chain fatty acids at the translational level of E. coli.Proteomics has changed the way to study cellular
physiology. Previously, one or more proteins were chosen as models for
understanding local physiological phenomena. Nowadays, proteomic studies allow
researchers to identify large members of stimulons, a set of proteins whose
amount or synthesis rate changes in response to a certain stimulus, and to
obtain information that indicates which specific proteins should be studied
further. Comparative proteome profiling under various environmental conditions also
reveal new regulatory circuits and the relative abundances of protein sets at
the system-wide level. Such analyses of every protein induced or repressed by the
stimulus may provide the necessary information to understand a response in the
cell. Furthermore, proteome profiles can prove
invaluable when used in conjunction with various molecular biological tools
including recombinant DNA technology [7]. For example, conditional
promoters activated by the specific stimulus, such as stationary phase, pH,
temperature, and nutrient limitation have been used for efficient production of
heterologous proteins in bacteria [8].In this study, proteomic studies
that compared global translational differences betweenE. coli W3110 cells in the presence of glucose and oleic acid (C18)
were conducted. The present study has three goals: (i) to identify the stimulon
of the oleic acid; (ii) to select target proteins from the stimulon to utilize
them as the oleic acid-inducible promoter; and (iii) further to apply it for
the production of recombinant proteins or other biotechnological systems.


## 2. Materials and Methods

### 2.1. Bacterial Strains and Plasmids

The bacterial strains and plasmids used in this
study are shown in Table1. E. coli XL1-Blue was used as a strain forcloning and maintenance of plasmids. E. coli W3110 was used as a
host strain for proteomic studies and the production of a recombinant protein. PCR
primers used in this study are listed in Table 2. Primers for the amplification
of the promoter regions of aldA and udp genes were designed based on the genome sequence of E. coli K-12 W3110 (AC_000091).
The promoter region of aldA gene was
amplified by PCR using primers 1 and 2, and was cloned into the EcoRV
and EcoRI sites of pTac99A to make pAD99A
(Table 1). In fact, pTac99A is a derivative of pTrc99A (Pharmacia Biotech.,
Uppsala, Sweden), which was constructed by replacing the trc promoter of pTrc99A with the tac promoter from pKK223-3 (Pharmacia Biotech) digested by PvuII and EcoRI [9]. Also, the promoter region of udp gene was amplified by PCR using primers 3 and 4, and was cloned
into the EcoRV and EcoRI sites of the high-copy-number plasmid
pTac99A to make pUP99A (Table 1). Both promoters were constructed with the ribosome
binding sites consisting of the AGGA sequence having an optimal distance length
of 8 bases from a start codon [10].Table 1
Bacterial strains and plasmids used in this study.
Strain or plasmidRelevant characteristicsReference or sourceE. coli strainsXL1-BluerecA1, endA1, gyrA96, thi, hsdR17, suppE44, relA1,
l-, lac-, F’[proAB laclq lacZ ΔM15, Tn10 (tet)r]StratageneaW3110F-mcrA mcrB IN(rrnD-rrnE)1λ-KCTCbPlasmidspTac99ApTrc99A derivative;tac promoter, cloning vehicle; AprPark and Lee [9]pAD99ApTac99A derivative; aldehyde dehydrogenase (aldA) promoter; AprThis
studypUP99ApTac99A derivative; uridine phosphorylase (udp) promoter; AprThis
studypGFPuvApr, lac promoter, gfpClontechcpAD99GFPpAD99A derivative;gfpThis studypTac99GFPpTac99A derivative;gfpThis
studypUP99GFPpUP99A derivative;gfpThis
studyaStratagene Cloning System (La Jolla, Calif, USA).bKorean Collection for Type Cultures, (Daejeon, Korea).cBD Biosciences Clontech (Palo Alto, Calif, USA).Table 2
List of primers used in PCR experiments.
PrimerPrimer sequenceaGene to be amplifiedTemplatePrimer 1aaaaccgttgatatctttgcaaacgggcatgactcctgacttttaldA promoterE. coli W3110 chromosomePrimer 2aaaaccgttgaattcctcctgtgatttatatgtttgttttcPrimer
3aaaaccgttgatatctgcagaatgaagggtgatttatgtgatttgudp promoterE. coli W3110 chromosomePrimer 4aaaaccgttgaattcctcctctgtgaatcggtttagtcagaPrimer 5ggaattcatgagtaaaggagaagaactttGFPpGFPuvPrimer 6cccaagcttttatttgatgagctcatccaRestriction enzyme sites are shown in bold.PCR was performed in the PCR Thermal Cycler MP (Takara Shuzo
Co., LTD., Shiga, Japan) using the Expand High Fidelity PCR System (Roche
Molecular Biochemicals, Mannheim, Germany). DNA sequencing was carried out
using the Bigdye terminator cycle sequencing kit (Perkin-Elmer Co., Boston, Mass,
USA), Taq polymerase and the ABI Prism 377 DNA sequencer (Perkin-Elmer Co.,
Mass, USA). All DNA manipulations were carried out according to standard
procedures [11].


### 2.2. Cell Growth Conditions and Analytical Procedure

Cells were cultivated at
37°C and 250 rpm in 100 mL of Luria-Bertani (LB) medium (10 g/L of tryptone,
5 g/L of yeast extract, and 5 g/L of NaCl), or R/2 medium plus 10 g/L glucose
or 5 g/L oleic acid (Daejung Chemicals & Metals
Co., Gyeonggi-do, Korea) as a carbon source. The R/2 medium (pH 6.8) contains per liter: 2 g of (NH4)2HPO4,
6.75 g of KH2PO4, 0.85 g of citric acid, 0.7 g
of MgSO4·7H2O, and 5 mL of a trace metal solution. The
trace metal solution contains per liter of 5 M HCl : 10 g of FeSO4·7H2O,
2.25 g of ZnSO4·7H2O, 1 g of CuSO4·5H2O, 0.5 g of MnSO4·5H2O, 0.23 g of Na2B4O7·10H2O,
2 g of CaCl2·2H2O, and 0.1 g of (NH4)6MO7O24.
For the cultivation of
recombinant E. coli strains, ampicillin (Ap, 50 μg/mL) was added. Cell growth was
monitored by measuring the absorbance at 600 nm (OD600; DU Series
600 Spectrophotometer, Beckman, Fullerton, Calif, USA). At an OD600 of 0.7 or 1.2, isopropyl-β-D-thiogalactopyranoside
(IPTG, Sigma Chemical Co., St. Louis, Mo, USA) was added at a final
concentration of 1 mM. For induction by oleic acid, the
defined medium supplemented with 10 g/L glucose was changed into the medium plus
5 g/L oleic acid after cells were collected by centrifugation at the same OD600 of 0.7 or 1.2. Then, cells were further cultivated for 5, 10, and 20 hours,
and harvested by centrifugation at 3,500 × g for 5 minutes at 4°C. Protein samples were analyzed by
electrophoresis on 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) as described by Laemmli [12]. The gels were stained
with Coomassie brilliant blue R250 (Bio-Rad, Hercules, Calif, USA), and the
protein bands were quantified by a GS-710 Calibrated Imaging Densitometer
(Bio-Rad).


### 2.3. Two-Dimensional Gel Electrophoresis (2DE)

Proteome analysis was performed by 2DE using
the IPGphor IEF system (GE Healthcare, Chalfont St. Giles,
UK) and Protean II xi Cell
(Bio-Rad) as described previously [13]. In brief, E. coli W3110 cells grown in the presence of glucose and oleic acid
were
harvested at the exponential and stationary phases, respectively, by
centrifugation for 5 minutes at
3,500 × g and 4°C, and washed four
times with low-salt washing buffer. The pellet was then resuspended in 600 μL
of a buffer containing 10 mM Tris-HCl (pH 8.0), 1.5 mM MgCl2, 10 mM
KCl, 0.5 mM DTT, 0.1% (w/v) SDS, and 1% (v/v) cocktail protease inhibitor (Complete Mini EDTA-free; Roche Diagnostics GmbH,
Germany). One μL of this sample was mixed with 60 μL of a solution consisting
of 8 M urea, 4% (w/v) CHAPS, 40 mM Tris, 65 mM DTT, and a trace of bromophenol
blue. Proteins (200 μg) quantified by Bradford
assay [14] were resuspended in 350 μL of IEF denaturation buffer
composed of 8 M urea, 2% (w/v) CHAPS, 20 mM DTT, and 0.8% (v/v) IPG buffer (pH
3–10 NL;  GE Healthcare). The samples were carefully loaded on the IPG
strips (18 cm, pH 3–10 NL; GE Healthcare).
The loaded IPG strips were rehydrated for 12 hours and focused at 20°C for 15
minutes at 250 V, followed by 8,000 V until a total of 60,000 V·h was reached. The
strips were equilibrated in two equilibration buffers as described previously [15]
and then placed on 12%
(w/v) SDS-PAGE gels prepared by the standard protocol [12]. Protein spots were visualized using a
silver staining kit (GE Healthcare), and the stained gels were scanned by a GS-710
Calibrated Imaging Densitometer (Bio-Rad). ImageMaster 2D Platinum Software (version
5.0; GE Healthcare) was used to identify spots, to match gels,
and to quantify spot densities on a volume basis (i.e., integration of spot
optical intensity over the spot area).


### 2.4. Fractionation of Outer Membrane Proteins

Culture broth (3 mL) was centrifuged at 3,500× g for 5 minutes at 4°C, and the pellet was washed with 1 mL of 10 mM Na2HPO4 buffer (pH 7.2), followed by centrifugation at 3,500 × g for 5 minutes at 4°C.
The cell pellet was resuspended in 0.5 mL of 10 mM Na2HPO4 buffer (pH 7.2). Crude extracts of E. coli cells were prepared by five cycles
of sonication (each for 15 seconds at 20% of maximum output; High-intensity
ultrasonic liquid processors; Sonics & Material Inc., Newtown, Conn, USA).
Partially disrupted cells were first removed by centrifugation of sonicated
samples at 12,000 × g for 2 minutes at room temperature. Membrane
proteins and lipid layers were isolated by centrifugation at 12,000 × g for
30 minutes at 4°C, followed by resuspension in 0.5 mL of 0.5% (w/v) sarcosyl in
10 mM Na2HPO4 buffer (pH 7.2). After incubation at 37°C
for 30 minutes, the insoluble pellet containing membrane proteins was obtained
by centrifugation at 12,000 × g for 30 minutes at 4°C. Membrane proteins
were obtained by washing the insoluble pellet with 10 mM Na2HPO4 buffer (pH 7.2), followed by resuspending in 50 μL of Tris-EDTA buffer (pH
8.0).


### 2.5. Protein Identification by LC-MS/MS Analysis

Samples
for the MS/MS analysis were prepared as described previously [16]. Briefly,
protein spots were excised and destained by incubating in 30 mM potassium
ferricyanide and 65 mM sodium thiosulfate for 10 minutes. Gel pieces were
washed in Milli-Q water until they became colorless and transparent, and then vacuum-dried. These
pieces were proteolysed with 0.02 μg/μL of modified trypsin (Promega, Madison, Wis,
USA) in 40 mM ammonium bicarbonate for overnight at 37°C. Tryptic peptides (10 μL
aliquots) were analyzed by a nano-LC/MS system consisting of an Ultimate
HPLC system (LC Packings, Amsterdam, Netherlands) and a quadrupole-time-of-flight (Q-TOF) MS (Micromass, Manchester, UK) equipped
with a nano-ESI source as
described previously [15]. The MASCOT search server  (version 1.8; http://www.matrixscience.com) was used
for the identification of protein spots by querying sequence of the tryptic
peptide fragments. Reference databases used for the identification of target
proteins were UniProt Knowledgebase (Swiss-Prot and TrEMBL; http://kr.expasy.org) and NCBI (http://www.ncbi.nlm.nih.gov).


### 2.6. Fluorescence Microscopy and Intensity of GFP

For
fluorescence imaging, cells were harvested by centrifugation for 5 minutes at
3,500× g and 4°C,
washed with and resuspended in phosphate-buffered saline (PBS) solution. The
samples were mounted on microscopic slide glasses and examined by confocal
microscopy (Carl Zeiss, Jena,
Germany).
Photographs were taken with a Carl Zeiss LSM 410 instrument. Samples were
excited by a 364-nm argon laser, and images were filtered by a longpass 505-nm
filter. Three-dimensional images were constructed from 5–10 serial images (each
1-Am thick) made by automatic optical sectioning. Fluorescence
intensities were measured at 395 nm (excitation) and 509 nm (emission) using
the SpectraMax M2 multi-detection system (Molecular Devices, Sunnyvale, Calif,
USA), and a 96-well black and clear flat-bottom plate (Coastar, Los Angeles, Calif,
USA).


## 2.1. Bacterial Strains and Plasmids

The bacterial strains and plasmids used in this
study are shown in Table1. E. coli XL1-Blue was used as a strain forcloning and maintenance of plasmids. E. coli W3110 was used as a
host strain for proteomic studies and the production of a recombinant protein. PCR
primers used in this study are listed in Table 2. Primers for the amplification
of the promoter regions of aldA and udp genes were designed based on the genome sequence of E. coli K-12 W3110 (AC_000091).
The promoter region of aldA gene was
amplified by PCR using primers 1 and 2, and was cloned into the EcoRV
and EcoRI sites of pTac99A to make pAD99A
(Table 1). In fact, pTac99A is a derivative of pTrc99A (Pharmacia Biotech.,
Uppsala, Sweden), which was constructed by replacing the trc promoter of pTrc99A with the tac promoter from pKK223-3 (Pharmacia Biotech) digested by PvuII and EcoRI [9]. Also, the promoter region of udp gene was amplified by PCR using primers 3 and 4, and was cloned
into the EcoRV and EcoRI sites of the high-copy-number plasmid
pTac99A to make pUP99A (Table 1). Both promoters were constructed with the ribosome
binding sites consisting of the AGGA sequence having an optimal distance length
of 8 bases from a start codon [10].Table 1
Bacterial strains and plasmids used in this study.
Strain or plasmidRelevant characteristicsReference or sourceE. coli strainsXL1-BluerecA1, endA1, gyrA96, thi, hsdR17, suppE44, relA1,
l-, lac-, F’[proAB laclq lacZ ΔM15, Tn10 (tet)r]StratageneaW3110F-mcrA mcrB IN(rrnD-rrnE)1λ-KCTCbPlasmidspTac99ApTrc99A derivative;tac promoter, cloning vehicle; AprPark and Lee [9]pAD99ApTac99A derivative; aldehyde dehydrogenase (aldA) promoter; AprThis
studypUP99ApTac99A derivative; uridine phosphorylase (udp) promoter; AprThis
studypGFPuvApr, lac promoter, gfpClontechcpAD99GFPpAD99A derivative;gfpThis studypTac99GFPpTac99A derivative;gfpThis
studypUP99GFPpUP99A derivative;gfpThis
studyaStratagene Cloning System (La Jolla, Calif, USA).bKorean Collection for Type Cultures, (Daejeon, Korea).cBD Biosciences Clontech (Palo Alto, Calif, USA).Table 2
List of primers used in PCR experiments.
PrimerPrimer sequenceaGene to be amplifiedTemplatePrimer 1aaaaccgttgatatctttgcaaacgggcatgactcctgacttttaldA promoterE. coli W3110 chromosomePrimer 2aaaaccgttgaattcctcctgtgatttatatgtttgttttcPrimer
3aaaaccgttgatatctgcagaatgaagggtgatttatgtgatttgudp promoterE. coli W3110 chromosomePrimer 4aaaaccgttgaattcctcctctgtgaatcggtttagtcagaPrimer 5ggaattcatgagtaaaggagaagaactttGFPpGFPuvPrimer 6cccaagcttttatttgatgagctcatccaRestriction enzyme sites are shown in bold.PCR was performed in the PCR Thermal Cycler MP (Takara Shuzo
Co., LTD., Shiga, Japan) using the Expand High Fidelity PCR System (Roche
Molecular Biochemicals, Mannheim, Germany). DNA sequencing was carried out
using the Bigdye terminator cycle sequencing kit (Perkin-Elmer Co., Boston, Mass,
USA), Taq polymerase and the ABI Prism 377 DNA sequencer (Perkin-Elmer Co.,
Mass, USA). All DNA manipulations were carried out according to standard
procedures [11].


## 2.2. Cell Growth Conditions and Analytical Procedure

Cells were cultivated at
37°C and 250 rpm in 100 mL of Luria-Bertani (LB) medium (10 g/L of tryptone,
5 g/L of yeast extract, and 5 g/L of NaCl), or R/2 medium plus 10 g/L glucose
or 5 g/L oleic acid (Daejung Chemicals & Metals
Co., Gyeonggi-do, Korea) as a carbon source. The R/2 medium (pH 6.8) contains per liter: 2 g of (NH4)2HPO4,
6.75 g of KH2PO4, 0.85 g of citric acid, 0.7 g
of MgSO4·7H2O, and 5 mL of a trace metal solution. The
trace metal solution contains per liter of 5 M HCl : 10 g of FeSO4·7H2O,
2.25 g of ZnSO4·7H2O, 1 g of CuSO4·5H2O, 0.5 g of MnSO4·5H2O, 0.23 g of Na2B4O7·10H2O,
2 g of CaCl2·2H2O, and 0.1 g of (NH4)6MO7O24.
For the cultivation of
recombinant E. coli strains, ampicillin (Ap, 50 μg/mL) was added. Cell growth was
monitored by measuring the absorbance at 600 nm (OD600; DU Series
600 Spectrophotometer, Beckman, Fullerton, Calif, USA). At an OD600 of 0.7 or 1.2, isopropyl-β-D-thiogalactopyranoside
(IPTG, Sigma Chemical Co., St. Louis, Mo, USA) was added at a final
concentration of 1 mM. For induction by oleic acid, the
defined medium supplemented with 10 g/L glucose was changed into the medium plus
5 g/L oleic acid after cells were collected by centrifugation at the same OD600 of 0.7 or 1.2. Then, cells were further cultivated for 5, 10, and 20 hours,
and harvested by centrifugation at 3,500 × g for 5 minutes at 4°C. Protein samples were analyzed by
electrophoresis on 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) as described by Laemmli [12]. The gels were stained
with Coomassie brilliant blue R250 (Bio-Rad, Hercules, Calif, USA), and the
protein bands were quantified by a GS-710 Calibrated Imaging Densitometer
(Bio-Rad).


## 2.3. Two-Dimensional Gel Electrophoresis (2DE)

Proteome analysis was performed by 2DE using
the IPGphor IEF system (GE Healthcare, Chalfont St. Giles,
UK) and Protean II xi Cell
(Bio-Rad) as described previously [13]. In brief, E. coli W3110 cells grown in the presence of glucose and oleic acid
were
harvested at the exponential and stationary phases, respectively, by
centrifugation for 5 minutes at
3,500 × g and 4°C, and washed four
times with low-salt washing buffer. The pellet was then resuspended in 600 μL
of a buffer containing 10 mM Tris-HCl (pH 8.0), 1.5 mM MgCl2, 10 mM
KCl, 0.5 mM DTT, 0.1% (w/v) SDS, and 1% (v/v) cocktail protease inhibitor (Complete Mini EDTA-free; Roche Diagnostics GmbH,
Germany). One μL of this sample was mixed with 60 μL of a solution consisting
of 8 M urea, 4% (w/v) CHAPS, 40 mM Tris, 65 mM DTT, and a trace of bromophenol
blue. Proteins (200 μg) quantified by Bradford
assay [14] were resuspended in 350 μL of IEF denaturation buffer
composed of 8 M urea, 2% (w/v) CHAPS, 20 mM DTT, and 0.8% (v/v) IPG buffer (pH
3–10 NL;  GE Healthcare). The samples were carefully loaded on the IPG
strips (18 cm, pH 3–10 NL; GE Healthcare).
The loaded IPG strips were rehydrated for 12 hours and focused at 20°C for 15
minutes at 250 V, followed by 8,000 V until a total of 60,000 V·h was reached. The
strips were equilibrated in two equilibration buffers as described previously [15]
and then placed on 12%
(w/v) SDS-PAGE gels prepared by the standard protocol [12]. Protein spots were visualized using a
silver staining kit (GE Healthcare), and the stained gels were scanned by a GS-710
Calibrated Imaging Densitometer (Bio-Rad). ImageMaster 2D Platinum Software (version
5.0; GE Healthcare) was used to identify spots, to match gels,
and to quantify spot densities on a volume basis (i.e., integration of spot
optical intensity over the spot area).


## 2.4. Fractionation of Outer Membrane Proteins

Culture broth (3 mL) was centrifuged at 3,500× g for 5 minutes at 4°C, and the pellet was washed with 1 mL of 10 mM Na2HPO4 buffer (pH 7.2), followed by centrifugation at 3,500 × g for 5 minutes at 4°C.
The cell pellet was resuspended in 0.5 mL of 10 mM Na2HPO4 buffer (pH 7.2). Crude extracts of E. coli cells were prepared by five cycles
of sonication (each for 15 seconds at 20% of maximum output; High-intensity
ultrasonic liquid processors; Sonics & Material Inc., Newtown, Conn, USA).
Partially disrupted cells were first removed by centrifugation of sonicated
samples at 12,000 × g for 2 minutes at room temperature. Membrane
proteins and lipid layers were isolated by centrifugation at 12,000 × g for
30 minutes at 4°C, followed by resuspension in 0.5 mL of 0.5% (w/v) sarcosyl in
10 mM Na2HPO4 buffer (pH 7.2). After incubation at 37°C
for 30 minutes, the insoluble pellet containing membrane proteins was obtained
by centrifugation at 12,000 × g for 30 minutes at 4°C. Membrane proteins
were obtained by washing the insoluble pellet with 10 mM Na2HPO4 buffer (pH 7.2), followed by resuspending in 50 μL of Tris-EDTA buffer (pH
8.0).


## 2.5. Protein Identification by LC-MS/MS Analysis

Samples
for the MS/MS analysis were prepared as described previously [16]. Briefly,
protein spots were excised and destained by incubating in 30 mM potassium
ferricyanide and 65 mM sodium thiosulfate for 10 minutes. Gel pieces were
washed in Milli-Q water until they became colorless and transparent, and then vacuum-dried. These
pieces were proteolysed with 0.02 μg/μL of modified trypsin (Promega, Madison, Wis,
USA) in 40 mM ammonium bicarbonate for overnight at 37°C. Tryptic peptides (10 μL
aliquots) were analyzed by a nano-LC/MS system consisting of an Ultimate
HPLC system (LC Packings, Amsterdam, Netherlands) and a quadrupole-time-of-flight (Q-TOF) MS (Micromass, Manchester, UK) equipped
with a nano-ESI source as
described previously [15]. The MASCOT search server  (version 1.8; http://www.matrixscience.com) was used
for the identification of protein spots by querying sequence of the tryptic
peptide fragments. Reference databases used for the identification of target
proteins were UniProt Knowledgebase (Swiss-Prot and TrEMBL; http://kr.expasy.org) and NCBI (http://www.ncbi.nlm.nih.gov).


## 2.6. Fluorescence Microscopy and Intensity of GFP

For
fluorescence imaging, cells were harvested by centrifugation for 5 minutes at
3,500× g and 4°C,
washed with and resuspended in phosphate-buffered saline (PBS) solution. The
samples were mounted on microscopic slide glasses and examined by confocal
microscopy (Carl Zeiss, Jena,
Germany).
Photographs were taken with a Carl Zeiss LSM 410 instrument. Samples were
excited by a 364-nm argon laser, and images were filtered by a longpass 505-nm
filter. Three-dimensional images were constructed from 5–10 serial images (each
1-Am thick) made by automatic optical sectioning. Fluorescence
intensities were measured at 395 nm (excitation) and 509 nm (emission) using
the SpectraMax M2 multi-detection system (Molecular Devices, Sunnyvale, Calif,
USA), and a 96-well black and clear flat-bottom plate (Coastar, Los Angeles, Calif,
USA).


## 3. Results and Discussion

### 3.1. Proteome Analysis

To understand physiological changes triggered by the long-chain fatty
acid, we analyzed the proteome profiles ofE. coli K-12 W3110 grown in the presence of glucose and oleic acid,
respectively. The final concentration of cells cultured in oleic
acid as a carbon source was 4-fold higher than that of cells grown in glucose,
although the former took a longer lag-period for induction of the fad regulon (see Figure 1). Samples of proteome were taken at the exponential
and stationary phases in two different media (see Figure 1): when the OD600 of E. coli reached 0.57 and
1.25 in the presence of glucose, named G1 and G2, respectively; and when the OD600 reached 0.56 and 5 in the presence of oleic acid, named O1 and O2, respectively.
The proteome profiles of the four samples, G1, G2, O1, and O2, were analyzed by
2D PAGE using a strip of 3–10 pI range and 12% polyacrylamide gel for subsequent comparisons (see Figure 2). The
overall profiles of whole cellular proteins were reproducible. From over 2,000
spots on each 2D gel shown in Figure 2, we identified 92 proteins by comparing
with our in-house E. coli proteome database or by conducting LC-MS/MS
analysis. Functions and fold changes of individual proteins are shown in Table 3.Table 3
Proteins identified from 2DE.
Spot no.Protein nameMethod for identityAccession no.pI/Mwa (kDa)Protein descriptionFold changebO1/G1O2/G21AcnBGel matchP366835.24/75.9Aconitate hydratase 2ΔΔ2AsnSGel matchP172425.64/92.8Asparaginyl-tRNA synthetase——3AceFGel matchP069595.01/77.5Dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex∇—4DnaKGel matchP044754.81/69.6Chaperone protein DnaK∇∇5PtsIGel matchP088394.78/59.8Phosphoenolpyruvate-protein
phosphotransferase∇3×∇6AldAMS/MSP255535.07/52.2Aldehyde dehydrogenase AAppearedAppeared7MopA (GroEL)Gel matchP061394.85/56.760 kDa chaperonin (GroEL protein)∇∇8TigGel matchP222574.83/51.0Trigger factor (TF)——9HtpGGel matchP104135.06/65.6Chaperone protein HtpG (Heat shock protein HtpG)——10AtpDGel matchP008244.90/47.7ATP synthase beta chain——11Icd (IcdA)Gel matchP082005.02/46.0Isocitrate dehydrogenase—Δ12AceA1MS/MSP053135.19/44.1 5.12/44.1Isocitrate
lyase5×Δ7×ΔAceA2MS/MS5.01/33.8Isocitrate lyase fragment6×Δ7×Δ13GlnAGel
matchP067115.25/53.8Glutamine synthetase——14IlvCGel matchP057935.26/52.0Ketol-acid reductoisomerase——15GlpKGel
matchP088595.30/50.6Glycerol kinase (Glycerokinase)——16EnoGel matchP083245.34/46.5   5.29/46.2Enolase
(2-phosphoglycerate dehydratase)—∇17TufA (EF-Tu)Gel matchP029905.32/44.6Elongation factor Tu (EF-Tu)——18FabD (TfpA)Gel matchP257155.37/44.8Malonyl CoA-acyl carrier protein transacylase∇—19LeuCGel matchP301275.42/44.4  5.95/51.63-isopropylmalate
dehydratase large subunit∇—20FadBMS/MSP211775.84/79.5Fatty oxidation complex alpha subunitAppearedAppeared21SdhAGel matchP104445.74/63.7Succinate dehydrogenase flavoprotein subunit——22OppAGel matchP238435.93/56.1Periplasmic oligopeptide-binding protein∇—23TrpDGel matchP009046.08/55.9Anthranilate synthase component II; Anthranilate——24GuaB (GuaR)Gel matchP069816.01/55.0Inosine-5′-monophosphate
dehydrogenase∇∇25AtpAGel matchP008225.84/53.1ATP synthase alpha chain—Δ26DppAGel matchP238475.69/52.1Periplasmic dipeptide transport protein——27GlyAGel matchP004776.04/45.9  5.94/46.1Serine hydroxymethyltransferase (Serine methylase)——28CarA (PyrA)Gel matchP009075.91/44.0Carbamoyl-phosphate synthase small chain——29UnknownMS/MS———Disappeared—30UnknownMS/MS———DisappearedDisappeared31FadAMS/MSP211516.31/40.9Fatty oxidation complex beta subunitAppearedAppeared32FbaGel matchP116045.55/40.6Fructose-bisphosphate aldolase class II——33SerC (PdxF)Gel matchP237215.34/40.2Phosphoserine aminotransferase∇∇34SucCGel matchP074605.30/42.3Succinyl-CoA synthetase beta
chainΔΔ35LivJMS/MSP029175.28/41.9Leu/Ile/Val-binding protein——36PgkGel matchP116655.07/41.9  5.02/41.7Phosphoglycerate kinase—∇37MalEMS/MSP029285.08/41.1Maltose-binding periplasmic proteinAppearedAppeared38LivKGel matchP048165.00/41.4Leucine-specific binding protein——39RfaD (HtrM)Gel matchP179634.85/36.8ADP-L-glycero-D-manno-heptose-6-epimerase——40PotDGel matchP238614.77/35.8Spermidine/putrescine-binding periplasmic protein——41TalBGel matchP301485.01/35.8Transaldolase B—∇42Tsf (EF-Ts)Gel matchP029975.15/33.6Elongation factor Ts (EF-Ts)—∇43MdhMS/MSP069945.55/35.5Malate
dehydrogenaseΔ—44CysKGel matchP110965.81/36.0Cysteine synthase A——45ManX (PtsL)Gel matchP081865.17/26.1PTS system, mannose-specific IIAB component∇—46UnknownMS/MS———2×∇3×∇47AroGGel matchP008866.12/39.4Phospho-2-dehydro-3-deoxyheptonate
aldolase2×∇∇48SbpGel matchP069976.49/49.5Sulfate-binding protein2×∇∇49GapAGel matchP069776.58/36.3Glyceraldehyde 3-phosphate dehydrogenase A——50PyrBGel
matchP004796.13/35.3Aspartate carbamoyltransferase catalytic chain——51FkpAGel
matchP455237.08/33.2FKBP-type peptidyl-prolylcis-trans isomerase FkpA——52SucDMS/MSP074596.31/29.6Succinyl-CoA synthetase alpha chainΔΔ53GapAMS/MSP069776.58/23.0No. 49 fragmentDisappearedDisappeared54GlnHMS/MSP103446.87/24.9Glutamine-binding periplasmic protein——55SodAGel matchP004486.44/22.9Superoxide dismutase [Mn] (MnSOD)——56RbsBMS/MSP029255.92/29.1D-ribose-binding periplasmic proteinAppearedAppeared57UdpGel matchP127585.86/27.9Uridine phosphorylase (UDRPase)AppearedAppeared58YadKGel matchP370165.55/28.4Protein YadK——59TpiA (Tpi)Gel matchP047905.57/26.9Triosephosphate isomerase——60CddMS/MSP136525.08/31.5Cytidine
deaminaseAppearedAppearedMS/MS5.42/31.5AppearedAppeared61TrpAGel matchP009285.30/28.7Tryptophan synthase alpha chain——62SspA (Ssp)Gel matchP058385.24/26.6Stringent starvation protein A——63HisJGel matchP391825.05/28.6Histidine-binding periplasmic protein——64FliYGel matchP391745.01/26.2  5.11/25.8Cystine-binding periplasmic protein∇3×∇65HdhA (HsdH)Gel matchP255295.17/25.07-alpha-hydroxysteroid dehydrogenase∇∇66Upp (UraP)Gel matchP255325.29/23.8Uracil phosphoribosyltransferase——67GrpEGel matchP093724.68/25.5GrpE protein (HSP-70 cofactor)—2×∇68AccB (FabE)Gel matchP029054.57/22.0Biotin carboxyl carrier protein of acetyl-CoA carboxylase2×∇2×∇69YfdXMS/MSP765205.38/23.0Protein yfdXDisappearedDisappeared70AhpCGel matchP264275.01/21.5Alkyl hydroperoxide reductase C22 protein∇∇71CrrGel matchP088374.57/20.0
4.68/18.9PTS system, glucose-specific IIA component——72DksAGel matchP182744.90/18.7DnaK suppressor protein—∇73AroKGel matchP241675.30/17.9Shikimate kinase I——74SodBGel matchP091575.53/22.1Superoxide dismutase [Fe]2×∇∇75PpiBGel matchP238695.51/17.7Peptidyl-prolyl cis-trans isomerase B—∇76RplIGel matchP024186.20/19.8
6.17/15.750S ribosomal protein L9∇∇77YbdQGel matchP391776.08/15.5Unknown protein from 2D-page——78RbfAGel matchP091706.00/15.6Ribosome-binding factor A——79RplUGel matchP024226.71/10.350S ribosomal protein L212×∇3×∇80HnsGel matchP089365.45/15.6DNA-binding protein H-NS (Histone-like protein HLP-II)—Δ81NdkGel matchP242335.59/15.2Nucleoside diphosphate kinase (NDP kinase)——82AtpCGel matchP008325.48/14.8ATP synthase epsilon chain——83RpsFGel matchP023585.31/15.8
5.15/15.8
5.26/15.830S ribosomal protein S6——84BcpGel matchP234805.02/15.8Bacterioferritin comigratory protein∇2×∇85GreAGel
matchP213464.68/15.9Transcription elongation factor GreA2×∇Disappeared86GroES (MopB)Gel matchP053805.15/15.610 kDa chaperonin (GroES protein)——87YfiDMS/MSP336335.09/14.3Protein YfiD——88UspAGel matchP282425.14/15.1Universal stress protein A——89YjgFGel
matchP393305.29/13.0Protein YjgF∇∇90TrxA (TsnC)Gel matchP002744.67/11.5Thioredoxin 1——91HdeBGel matchP266054.85/11.2Protein HdeB (10K-L protein)—3×∇92YccUMS/MSP758746.72/14.7Protein YccU; Predicted CoA-binding proteinAppearedAppearedaUnit of the molecular weight (MW) is kDa.bFold change: 0 ~0.3-fold, 3×∇; 0.3 ~0.5-fold, 2×∇; 0.5 ~0.6-fold, ∇; 1.5-fold, Δ; 2-fold, 2×Δ; fold change, fold
number×Δ.Figure 1
Time profiles of the concentrations ofE. coli cells.
The cell densities (OD600) of E.
coli W3110 in the presence of glucose (•) or oleic acid  (∘)
are shown. Gl, G2, O1, and O2 are the sampling points for proteome analyses.The 2DE maps ofE. coli W3110 cells
at the exponential (left panels; A, C) and stationary phases (right panels; B,
D) in the presence of glucose (A, B) and oleic acid (C, D), respectively. Identified
proteins shown in numbers are listed in Table 3. Boxes
further highlight specific corresponding regions of the 2D gel images, which
are compared at higher resolution in the bottom of (E). Arrow lines indicate individual
spots of AldA and Udp.
(a)(b)(c)(d)(e)The outer membrane proteins were enriched by fractionation, and
separated on 12% SDS-PAGE (see Figure3). Membrane proteins are typically difficult
to be resolved in the IEF denaturation buffer used commonly for 2D gels because
of their hydrophobic property. The highly abundant porin, OmpF whose expression
level was regulated by osmolarity [17], was observed at the exponential phase
of E. coli in the presence of glucose.
As expected, the long-chain fatty acid transporter protein, FadL, was newly
synthesized in the presence of oleic acid. This result proves that E. coli requires the specific transport
system (fadL) on the growth of fatty
acids.Figure 3
SDS-PAGE of the outer membrane proteins taken from samples of proteome. Identified
proteins are shown on the right side. Size markers (in kDa) are indicated on
the left.


### 3.2. Identification of the Proteins Stimulated by Oleic Acid

To examine the
influence of oleic acid on the proteome profile variation, we compared the
proteomes obtained from the exponential phase (O1 versus G1) and the stationary
phase (O2 versus G2), as shown in Table3. At the exponential growth phase, the
levels of 41 identified
proteins were altered in the presence of oleic acid. Among them, 9 proteins including AldA, Cdd, FadA, FadB, FadL, MalE, RbsB, Udp, and YccU
were newly synthesized in response to oleic acid, while GapA (the fragment), hypothetical
protein YfdX, and two unidentified proteins were not detectable. As expected,
the levels of proteins involved in fatty acid degradation (FadA and FadB), long-chain
fatty acid transport system (FadL), glyoxylate shunt (AceA). and TCA cycle (Mdh,
SdhA, SucC, and SucD) were significantly increased to replenish the
dicarboxylic acid intermediates consumed in amino acid biosynthesis. Particularly,
isocitrate lyase (AceA) in the aceBAK operon was significantly synthesized by more than five folds in the presence of
oleic acid, making it the most abundant protein. Concurrently, there were decreased
levels of proteins involved in the biosynthesis of fatty acids (FabD and FabE)
and amino acids (AroG, LeuC, and SerC). These results showed that the variation
patterns of most proteins identified as a fad regulon were in agreement with their corresponding transcriptional levels previously
reported [2–6].Furthermore, the growth ofE. coli on oleic acid involves a significant contribution of the
pyrimidine salvage pathway (Cdd and Udp) and specific binding-protein-dependent
transport system (MalE and RbsB) because the levels of these proteins highly increased by oleic acid. The salvage pathway of E. coli functions to reutilize free bases and nucleosides produced
intracellularly from nucleotide turnover [18]. Also, the pyrimidine salvage
pathway has been reported to recycle the pentose moieties of exogenous
nucleosides to use them as carbon and energy sources and the amino groups of
cytosine compounds as a nitrogen source. The D-ribose-binding periplasmic
protein, RbsB in the rbsACBK operon,
mediates the entry of D-ribose across the cell membrane in the form of D-ribose
5-phosphate, which is an intermediate of the pentose phosphate cycle [19]. Therefore,
long-chain fatty acids seem to influence the status of the pyrimidine salvage
pathway and its associated transport system.Interestingly, aldehyde dehydrogenase (AldA), which oxidize diverse
aldehydes throughout the cellular metabolism, received a special attention in
this study because of its possible applications in gene expression system with
oleic acid as an inducer. It has been reported that the expression ofaldA gene was induced on growth on
fucose, rhamnose, arabinose, glutamate, or 2-oxoglutarate during aerobic
condition, while that is repressed by glucose [20]. Our observation found in
this study demonstrated that oleic acid is another inducer of the aldA gene. Its application as an
inducible promoter is demonstrated in the next two sections.At the stationary phase, the levels of 45 identified proteins
were altered in response to oleic acid (Table3). Sixteen proteins including AceA,
AcnB, AldA, AtpA, Cdd, FadA, FadB, FadL, Hns, Icd, MalE, RbsB, SucC, SucD, Udp,
and YccU were significantly increased or newly synthesized, while 29 proteins
were reduced or disappeared on 2D gels. Although the variations of proteins at
the stationary phase were more complex, most proteins with altered levels on 2D
gels at this phase showed patterns similar to those at the exponential growth
phase.


### 3.3. Construction of the Expression System with Oleic Acid-Inducible Promoters

Among proteins highly inducible by oleic acid, we
selected two target proteins, AldA and Udp according to the following two
criteria for their utilization as promoters: (i) they are only induced in the
presence of oleic acid to be strictly controlled; (ii) they are strongly and
highly expressed for the enhanced bioproducts production. AldA and Udp were synthesized
in response to oleic acid with relatively high abundance from the exponential
to stationary phases, and were not synthesized in the presence of glucose,
suggesting that the native promoters of these proteins could be used as an oleic
acid-inducible promoter inE. coli W3110.For the construction of the expression
systems controlled byaldA or udp promoter, the promoter regions of
these genes were amplified as described in Section 2. The representative
schematic plasmid map under the control of aldA promoter is illustrated in Figure 4. This is a high-copy-number plasmid with replication
origin of pBR322 (ATCC 37017).Figure 4
Map of plasmid pAD99A. Its characteristics include pBR322 ori, origin of
replication of pBR322;bla,
ampicillin-resistance gene; PaldA, aldA promoter; a target gene to be
inserted in multiple cloning sites (MCSs).


### 3.4. Comparison between the Expression Efficiency of Oleic Acid- and IPTG-Inducible Promoters

Various cultivation strategies employing different
host strains and expression systems have been employed for the production of
recombinant proteins [10, 21]. One of the most popular approaches is the use of
different promoters to regulate expression levels [10]. In E. coli, many inducible promoters have been developed, which can be
induced by various mechanisms such as temperature upshifting, pH fluctuation,
nutrient starvation, and addition of chemical inducers. Among these inducible
systems, T7 or lac-based promoters (tac, trc, lac, lacUV5-T7 hybrid, etc.), which can be effectively induced by the
addition of IPTG, are the most frequently used ones.In order to evaluate the effectiveness of oleic acid-inducible promoters discovered
in this study, we chose the IPTG-inducibletac promoter as a control. Green fluorescent protein (GFP) from the jellyfish Aequorea
Victoria was employed as a model recombinant protein to examine its expression
under the control of aldA or udp promoter. For the induction of GFP
by oleic acid, the defined medium supplemented with glucose was transferred
into oleic acid medium at the OD600 of 0.7 or 1.2. For the control, cells
harboring the plasmid containing tac promoter were added with 1 mM IPTG at the same values of OD600 in
the defined medium supplemented with glucose. After induction by IPTG or oleic
acid,  cells were further cultured, harvested at each time, and analyzed by 12%
SDS-PAGE (see Figure 5). When GFP is induced at the OD600 of 1.2 in
recombinant E. coli W3110 harboring pTac99GFP, pAD99GFP, and pUP99GFP,
its contents were approximately 27%, 42%, and 25% of the total proteins at 10 hours,
and 28%, 50%, and 25% at 20 hours, respectively. The GFP content induced by aldA promoter was about 2-fold higher
than that obtained by IPTG-inducible tac or udp promoter. Under the
IPTG-inducible promoter, the expression of GFP was low even in LB medium
compared to the oleic acid-inducible aldA promoter (data not shown). Additionally, the final cell concentration of
recombinant E. coli W3110 cells in
the presence of oleic acid was 4-fold higher than that of recombinant E. coli cultured in glucose as a carbon
source. This result was further confirmed by fluorescence intensity
measurements and confocal microscopy (see Figure 6). Strong fluorescence was uniformly
detected in recombinant E. coli cells
under the control of aldA promoter.
The fluorescence intensity of GFP obtained from W3110 harboring pAD99GFP was
more than 30-fold higher than that obtained from W3110 harboring pTac99GFP, indicating
that the aldA promoter efficiently enhances
recombinant protein production compared to the tac promoter. Since the aldA promoter was not activated by glucose, GFP was not produced in the presence of
glucose in accordance with the proteome profiles. GFP was only produced under
the aldA promoter along with the
supply of exogenous oleic acid. However, the tac promoter was not tightly controlled, leaking the recombinant protein
even without IPTG induction. These results manifest that the aldA promoter is very efficient for the production
of recombinant proteins in E. coli as
an inducible promoter. The maximum productivity can be achieved when the growth
and production phases are separated as conducted in this study. Separation of
the two phases is often achieved by delaying induction time until the cell
density reaches a suitable value.The effect of the recombinant protein production by oleic acid-inducible
promoter in recombinantE. coli W3110. The cells are induced by
exchanging medium supplemented with glucose into the one with oleic acid at the
OD600 of 0.7 (a) or 1.2 (b). For the control, cells harboring the
plasmid containing tac promoter were
added with 1 mM IPTG at the same values of OD600 in the defined
medium supplemented with glucose. After induction by IPTG or oleic acid, cells
were further cultured for 5, 10, 20 hours, and harvested for 12% (w/v) SDS-PAGE.
The arrows indicate the green fluorescent protein (GFP; 26.9 kDa). Size markers
(in kDa) are also indicated.
(a)(b)The fluorescence intensities (a) and confocal microscopic images (b) ofE. coli
W3110 cells by induction with IPTG (middle panels) or oleic
acid (bottom panels). As a control, the E. coli strain without plasmid
is also shown (top panels). Shown in (b) are immunofluorescence
micrographs (left panels), differential interference micrographs (middle panels),
and merged images (right panels) of wild-type E. coli W3110 and its
recombinant cells harboring pTac99GFP and pAD99GFP.
(a)(b)Until
now, the IPTG-inducible promoterstac or trc have been widely used for basic
research. However, the use of IPTG for the large-scale production of human
therapeutic proteins is undesirable because of its toxicity and relatively high
cost; the high concentration of IPTG can inhibit cell growth and recombinant
protein production [22, 23]. Therefore, determination of optimal induction time
point as well as the inducer concentration is crucial to increase the overall
productivity of recombinant protein. In this regard, there have been
significant efforts to overcome such problems by using different inducible or even
constitutive expression systems [24]. Thus, the aldA promoter found in this study can be effectively used for the
enhanced production of recombinant proteins with the aforementioned problems
largely resolved.In summary, thealdA promoter satisfies
requirements for its utilization as a promoter. First, it is tightly controllable
with an appropriate inducer, oleic acid in this case. Tight regulation of the
promoter is essential for the synthesis of proteins which may be detrimental to
the host cell. For example, the toxic rotavirus VP7 protein effectively kills
cells, and must be produced under tightly regulated conditions [25]. Second, its
expression is strong and long lasting, resulting in the accumulation of the
target protein constituting up to 50% of the total cellular proteins. The third
important characteristic of aldA promoter is its inducibility in a cost-effective manner by using exogenous
oleic acid as an inducer.


## 3.1. Proteome Analysis

To understand physiological changes triggered by the long-chain fatty
acid, we analyzed the proteome profiles ofE. coli K-12 W3110 grown in the presence of glucose and oleic acid,
respectively. The final concentration of cells cultured in oleic
acid as a carbon source was 4-fold higher than that of cells grown in glucose,
although the former took a longer lag-period for induction of the fad regulon (see Figure 1). Samples of proteome were taken at the exponential
and stationary phases in two different media (see Figure 1): when the OD600 of E. coli reached 0.57 and
1.25 in the presence of glucose, named G1 and G2, respectively; and when the OD600 reached 0.56 and 5 in the presence of oleic acid, named O1 and O2, respectively.
The proteome profiles of the four samples, G1, G2, O1, and O2, were analyzed by
2D PAGE using a strip of 3–10 pI range and 12% polyacrylamide gel for subsequent comparisons (see Figure 2). The
overall profiles of whole cellular proteins were reproducible. From over 2,000
spots on each 2D gel shown in Figure 2, we identified 92 proteins by comparing
with our in-house E. coli proteome database or by conducting LC-MS/MS
analysis. Functions and fold changes of individual proteins are shown in Table 3.Table 3
Proteins identified from 2DE.
Spot no.Protein nameMethod for identityAccession no.pI/Mwa (kDa)Protein descriptionFold changebO1/G1O2/G21AcnBGel matchP366835.24/75.9Aconitate hydratase 2ΔΔ2AsnSGel matchP172425.64/92.8Asparaginyl-tRNA synthetase——3AceFGel matchP069595.01/77.5Dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex∇—4DnaKGel matchP044754.81/69.6Chaperone protein DnaK∇∇5PtsIGel matchP088394.78/59.8Phosphoenolpyruvate-protein
phosphotransferase∇3×∇6AldAMS/MSP255535.07/52.2Aldehyde dehydrogenase AAppearedAppeared7MopA (GroEL)Gel matchP061394.85/56.760 kDa chaperonin (GroEL protein)∇∇8TigGel matchP222574.83/51.0Trigger factor (TF)——9HtpGGel matchP104135.06/65.6Chaperone protein HtpG (Heat shock protein HtpG)——10AtpDGel matchP008244.90/47.7ATP synthase beta chain——11Icd (IcdA)Gel matchP082005.02/46.0Isocitrate dehydrogenase—Δ12AceA1MS/MSP053135.19/44.1 5.12/44.1Isocitrate
lyase5×Δ7×ΔAceA2MS/MS5.01/33.8Isocitrate lyase fragment6×Δ7×Δ13GlnAGel
matchP067115.25/53.8Glutamine synthetase——14IlvCGel matchP057935.26/52.0Ketol-acid reductoisomerase——15GlpKGel
matchP088595.30/50.6Glycerol kinase (Glycerokinase)——16EnoGel matchP083245.34/46.5   5.29/46.2Enolase
(2-phosphoglycerate dehydratase)—∇17TufA (EF-Tu)Gel matchP029905.32/44.6Elongation factor Tu (EF-Tu)——18FabD (TfpA)Gel matchP257155.37/44.8Malonyl CoA-acyl carrier protein transacylase∇—19LeuCGel matchP301275.42/44.4  5.95/51.63-isopropylmalate
dehydratase large subunit∇—20FadBMS/MSP211775.84/79.5Fatty oxidation complex alpha subunitAppearedAppeared21SdhAGel matchP104445.74/63.7Succinate dehydrogenase flavoprotein subunit——22OppAGel matchP238435.93/56.1Periplasmic oligopeptide-binding protein∇—23TrpDGel matchP009046.08/55.9Anthranilate synthase component II; Anthranilate——24GuaB (GuaR)Gel matchP069816.01/55.0Inosine-5′-monophosphate
dehydrogenase∇∇25AtpAGel matchP008225.84/53.1ATP synthase alpha chain—Δ26DppAGel matchP238475.69/52.1Periplasmic dipeptide transport protein——27GlyAGel matchP004776.04/45.9  5.94/46.1Serine hydroxymethyltransferase (Serine methylase)——28CarA (PyrA)Gel matchP009075.91/44.0Carbamoyl-phosphate synthase small chain——29UnknownMS/MS———Disappeared—30UnknownMS/MS———DisappearedDisappeared31FadAMS/MSP211516.31/40.9Fatty oxidation complex beta subunitAppearedAppeared32FbaGel matchP116045.55/40.6Fructose-bisphosphate aldolase class II——33SerC (PdxF)Gel matchP237215.34/40.2Phosphoserine aminotransferase∇∇34SucCGel matchP074605.30/42.3Succinyl-CoA synthetase beta
chainΔΔ35LivJMS/MSP029175.28/41.9Leu/Ile/Val-binding protein——36PgkGel matchP116655.07/41.9  5.02/41.7Phosphoglycerate kinase—∇37MalEMS/MSP029285.08/41.1Maltose-binding periplasmic proteinAppearedAppeared38LivKGel matchP048165.00/41.4Leucine-specific binding protein——39RfaD (HtrM)Gel matchP179634.85/36.8ADP-L-glycero-D-manno-heptose-6-epimerase——40PotDGel matchP238614.77/35.8Spermidine/putrescine-binding periplasmic protein——41TalBGel matchP301485.01/35.8Transaldolase B—∇42Tsf (EF-Ts)Gel matchP029975.15/33.6Elongation factor Ts (EF-Ts)—∇43MdhMS/MSP069945.55/35.5Malate
dehydrogenaseΔ—44CysKGel matchP110965.81/36.0Cysteine synthase A——45ManX (PtsL)Gel matchP081865.17/26.1PTS system, mannose-specific IIAB component∇—46UnknownMS/MS———2×∇3×∇47AroGGel matchP008866.12/39.4Phospho-2-dehydro-3-deoxyheptonate
aldolase2×∇∇48SbpGel matchP069976.49/49.5Sulfate-binding protein2×∇∇49GapAGel matchP069776.58/36.3Glyceraldehyde 3-phosphate dehydrogenase A——50PyrBGel
matchP004796.13/35.3Aspartate carbamoyltransferase catalytic chain——51FkpAGel
matchP455237.08/33.2FKBP-type peptidyl-prolylcis-trans isomerase FkpA——52SucDMS/MSP074596.31/29.6Succinyl-CoA synthetase alpha chainΔΔ53GapAMS/MSP069776.58/23.0No. 49 fragmentDisappearedDisappeared54GlnHMS/MSP103446.87/24.9Glutamine-binding periplasmic protein——55SodAGel matchP004486.44/22.9Superoxide dismutase [Mn] (MnSOD)——56RbsBMS/MSP029255.92/29.1D-ribose-binding periplasmic proteinAppearedAppeared57UdpGel matchP127585.86/27.9Uridine phosphorylase (UDRPase)AppearedAppeared58YadKGel matchP370165.55/28.4Protein YadK——59TpiA (Tpi)Gel matchP047905.57/26.9Triosephosphate isomerase——60CddMS/MSP136525.08/31.5Cytidine
deaminaseAppearedAppearedMS/MS5.42/31.5AppearedAppeared61TrpAGel matchP009285.30/28.7Tryptophan synthase alpha chain——62SspA (Ssp)Gel matchP058385.24/26.6Stringent starvation protein A——63HisJGel matchP391825.05/28.6Histidine-binding periplasmic protein——64FliYGel matchP391745.01/26.2  5.11/25.8Cystine-binding periplasmic protein∇3×∇65HdhA (HsdH)Gel matchP255295.17/25.07-alpha-hydroxysteroid dehydrogenase∇∇66Upp (UraP)Gel matchP255325.29/23.8Uracil phosphoribosyltransferase——67GrpEGel matchP093724.68/25.5GrpE protein (HSP-70 cofactor)—2×∇68AccB (FabE)Gel matchP029054.57/22.0Biotin carboxyl carrier protein of acetyl-CoA carboxylase2×∇2×∇69YfdXMS/MSP765205.38/23.0Protein yfdXDisappearedDisappeared70AhpCGel matchP264275.01/21.5Alkyl hydroperoxide reductase C22 protein∇∇71CrrGel matchP088374.57/20.0
4.68/18.9PTS system, glucose-specific IIA component——72DksAGel matchP182744.90/18.7DnaK suppressor protein—∇73AroKGel matchP241675.30/17.9Shikimate kinase I——74SodBGel matchP091575.53/22.1Superoxide dismutase [Fe]2×∇∇75PpiBGel matchP238695.51/17.7Peptidyl-prolyl cis-trans isomerase B—∇76RplIGel matchP024186.20/19.8
6.17/15.750S ribosomal protein L9∇∇77YbdQGel matchP391776.08/15.5Unknown protein from 2D-page——78RbfAGel matchP091706.00/15.6Ribosome-binding factor A——79RplUGel matchP024226.71/10.350S ribosomal protein L212×∇3×∇80HnsGel matchP089365.45/15.6DNA-binding protein H-NS (Histone-like protein HLP-II)—Δ81NdkGel matchP242335.59/15.2Nucleoside diphosphate kinase (NDP kinase)——82AtpCGel matchP008325.48/14.8ATP synthase epsilon chain——83RpsFGel matchP023585.31/15.8
5.15/15.8
5.26/15.830S ribosomal protein S6——84BcpGel matchP234805.02/15.8Bacterioferritin comigratory protein∇2×∇85GreAGel
matchP213464.68/15.9Transcription elongation factor GreA2×∇Disappeared86GroES (MopB)Gel matchP053805.15/15.610 kDa chaperonin (GroES protein)——87YfiDMS/MSP336335.09/14.3Protein YfiD——88UspAGel matchP282425.14/15.1Universal stress protein A——89YjgFGel
matchP393305.29/13.0Protein YjgF∇∇90TrxA (TsnC)Gel matchP002744.67/11.5Thioredoxin 1——91HdeBGel matchP266054.85/11.2Protein HdeB (10K-L protein)—3×∇92YccUMS/MSP758746.72/14.7Protein YccU; Predicted CoA-binding proteinAppearedAppearedaUnit of the molecular weight (MW) is kDa.bFold change: 0 ~0.3-fold, 3×∇; 0.3 ~0.5-fold, 2×∇; 0.5 ~0.6-fold, ∇; 1.5-fold, Δ; 2-fold, 2×Δ; fold change, fold
number×Δ.Figure 1
Time profiles of the concentrations ofE. coli cells.
The cell densities (OD600) of E.
coli W3110 in the presence of glucose (•) or oleic acid  (∘)
are shown. Gl, G2, O1, and O2 are the sampling points for proteome analyses.The 2DE maps ofE. coli W3110 cells
at the exponential (left panels; A, C) and stationary phases (right panels; B,
D) in the presence of glucose (A, B) and oleic acid (C, D), respectively. Identified
proteins shown in numbers are listed in Table 3. Boxes
further highlight specific corresponding regions of the 2D gel images, which
are compared at higher resolution in the bottom of (E). Arrow lines indicate individual
spots of AldA and Udp.
(a)(b)(c)(d)(e)The outer membrane proteins were enriched by fractionation, and
separated on 12% SDS-PAGE (see Figure3). Membrane proteins are typically difficult
to be resolved in the IEF denaturation buffer used commonly for 2D gels because
of their hydrophobic property. The highly abundant porin, OmpF whose expression
level was regulated by osmolarity [17], was observed at the exponential phase
of E. coli in the presence of glucose.
As expected, the long-chain fatty acid transporter protein, FadL, was newly
synthesized in the presence of oleic acid. This result proves that E. coli requires the specific transport
system (fadL) on the growth of fatty
acids.Figure 3
SDS-PAGE of the outer membrane proteins taken from samples of proteome. Identified
proteins are shown on the right side. Size markers (in kDa) are indicated on
the left.


## 3.2. Identification of the Proteins Stimulated by Oleic Acid

To examine the
influence of oleic acid on the proteome profile variation, we compared the
proteomes obtained from the exponential phase (O1 versus G1) and the stationary
phase (O2 versus G2), as shown in Table3. At the exponential growth phase, the
levels of 41 identified
proteins were altered in the presence of oleic acid. Among them, 9 proteins including AldA, Cdd, FadA, FadB, FadL, MalE, RbsB, Udp, and YccU
were newly synthesized in response to oleic acid, while GapA (the fragment), hypothetical
protein YfdX, and two unidentified proteins were not detectable. As expected,
the levels of proteins involved in fatty acid degradation (FadA and FadB), long-chain
fatty acid transport system (FadL), glyoxylate shunt (AceA). and TCA cycle (Mdh,
SdhA, SucC, and SucD) were significantly increased to replenish the
dicarboxylic acid intermediates consumed in amino acid biosynthesis. Particularly,
isocitrate lyase (AceA) in the aceBAK operon was significantly synthesized by more than five folds in the presence of
oleic acid, making it the most abundant protein. Concurrently, there were decreased
levels of proteins involved in the biosynthesis of fatty acids (FabD and FabE)
and amino acids (AroG, LeuC, and SerC). These results showed that the variation
patterns of most proteins identified as a fad regulon were in agreement with their corresponding transcriptional levels previously
reported [2–6].Furthermore, the growth ofE. coli on oleic acid involves a significant contribution of the
pyrimidine salvage pathway (Cdd and Udp) and specific binding-protein-dependent
transport system (MalE and RbsB) because the levels of these proteins highly increased by oleic acid. The salvage pathway of E. coli functions to reutilize free bases and nucleosides produced
intracellularly from nucleotide turnover [18]. Also, the pyrimidine salvage
pathway has been reported to recycle the pentose moieties of exogenous
nucleosides to use them as carbon and energy sources and the amino groups of
cytosine compounds as a nitrogen source. The D-ribose-binding periplasmic
protein, RbsB in the rbsACBK operon,
mediates the entry of D-ribose across the cell membrane in the form of D-ribose
5-phosphate, which is an intermediate of the pentose phosphate cycle [19]. Therefore,
long-chain fatty acids seem to influence the status of the pyrimidine salvage
pathway and its associated transport system.Interestingly, aldehyde dehydrogenase (AldA), which oxidize diverse
aldehydes throughout the cellular metabolism, received a special attention in
this study because of its possible applications in gene expression system with
oleic acid as an inducer. It has been reported that the expression ofaldA gene was induced on growth on
fucose, rhamnose, arabinose, glutamate, or 2-oxoglutarate during aerobic
condition, while that is repressed by glucose [20]. Our observation found in
this study demonstrated that oleic acid is another inducer of the aldA gene. Its application as an
inducible promoter is demonstrated in the next two sections.At the stationary phase, the levels of 45 identified proteins
were altered in response to oleic acid (Table3). Sixteen proteins including AceA,
AcnB, AldA, AtpA, Cdd, FadA, FadB, FadL, Hns, Icd, MalE, RbsB, SucC, SucD, Udp,
and YccU were significantly increased or newly synthesized, while 29 proteins
were reduced or disappeared on 2D gels. Although the variations of proteins at
the stationary phase were more complex, most proteins with altered levels on 2D
gels at this phase showed patterns similar to those at the exponential growth
phase.


## 3.3. Construction of the Expression System with Oleic Acid-Inducible Promoters

Among proteins highly inducible by oleic acid, we
selected two target proteins, AldA and Udp according to the following two
criteria for their utilization as promoters: (i) they are only induced in the
presence of oleic acid to be strictly controlled; (ii) they are strongly and
highly expressed for the enhanced bioproducts production. AldA and Udp were synthesized
in response to oleic acid with relatively high abundance from the exponential
to stationary phases, and were not synthesized in the presence of glucose,
suggesting that the native promoters of these proteins could be used as an oleic
acid-inducible promoter inE. coli W3110.For the construction of the expression
systems controlled byaldA or udp promoter, the promoter regions of
these genes were amplified as described in Section 2. The representative
schematic plasmid map under the control of aldA promoter is illustrated in Figure 4. This is a high-copy-number plasmid with replication
origin of pBR322 (ATCC 37017).Figure 4
Map of plasmid pAD99A. Its characteristics include pBR322 ori, origin of
replication of pBR322;bla,
ampicillin-resistance gene; PaldA, aldA promoter; a target gene to be
inserted in multiple cloning sites (MCSs).


## 3.4. Comparison between the Expression Efficiency of Oleic Acid- and IPTG-Inducible Promoters

Various cultivation strategies employing different
host strains and expression systems have been employed for the production of
recombinant proteins [10, 21]. One of the most popular approaches is the use of
different promoters to regulate expression levels [10]. In E. coli, many inducible promoters have been developed, which can be
induced by various mechanisms such as temperature upshifting, pH fluctuation,
nutrient starvation, and addition of chemical inducers. Among these inducible
systems, T7 or lac-based promoters (tac, trc, lac, lacUV5-T7 hybrid, etc.), which can be effectively induced by the
addition of IPTG, are the most frequently used ones.In order to evaluate the effectiveness of oleic acid-inducible promoters discovered
in this study, we chose the IPTG-inducibletac promoter as a control. Green fluorescent protein (GFP) from the jellyfish Aequorea
Victoria was employed as a model recombinant protein to examine its expression
under the control of aldA or udp promoter. For the induction of GFP
by oleic acid, the defined medium supplemented with glucose was transferred
into oleic acid medium at the OD600 of 0.7 or 1.2. For the control, cells
harboring the plasmid containing tac promoter were added with 1 mM IPTG at the same values of OD600 in
the defined medium supplemented with glucose. After induction by IPTG or oleic
acid,  cells were further cultured, harvested at each time, and analyzed by 12%
SDS-PAGE (see Figure 5). When GFP is induced at the OD600 of 1.2 in
recombinant E. coli W3110 harboring pTac99GFP, pAD99GFP, and pUP99GFP,
its contents were approximately 27%, 42%, and 25% of the total proteins at 10 hours,
and 28%, 50%, and 25% at 20 hours, respectively. The GFP content induced by aldA promoter was about 2-fold higher
than that obtained by IPTG-inducible tac or udp promoter. Under the
IPTG-inducible promoter, the expression of GFP was low even in LB medium
compared to the oleic acid-inducible aldA promoter (data not shown). Additionally, the final cell concentration of
recombinant E. coli W3110 cells in
the presence of oleic acid was 4-fold higher than that of recombinant E. coli cultured in glucose as a carbon
source. This result was further confirmed by fluorescence intensity
measurements and confocal microscopy (see Figure 6). Strong fluorescence was uniformly
detected in recombinant E. coli cells
under the control of aldA promoter.
The fluorescence intensity of GFP obtained from W3110 harboring pAD99GFP was
more than 30-fold higher than that obtained from W3110 harboring pTac99GFP, indicating
that the aldA promoter efficiently enhances
recombinant protein production compared to the tac promoter. Since the aldA promoter was not activated by glucose, GFP was not produced in the presence of
glucose in accordance with the proteome profiles. GFP was only produced under
the aldA promoter along with the
supply of exogenous oleic acid. However, the tac promoter was not tightly controlled, leaking the recombinant protein
even without IPTG induction. These results manifest that the aldA promoter is very efficient for the production
of recombinant proteins in E. coli as
an inducible promoter. The maximum productivity can be achieved when the growth
and production phases are separated as conducted in this study. Separation of
the two phases is often achieved by delaying induction time until the cell
density reaches a suitable value.The effect of the recombinant protein production by oleic acid-inducible
promoter in recombinantE. coli W3110. The cells are induced by
exchanging medium supplemented with glucose into the one with oleic acid at the
OD600 of 0.7 (a) or 1.2 (b). For the control, cells harboring the
plasmid containing tac promoter were
added with 1 mM IPTG at the same values of OD600 in the defined
medium supplemented with glucose. After induction by IPTG or oleic acid, cells
were further cultured for 5, 10, 20 hours, and harvested for 12% (w/v) SDS-PAGE.
The arrows indicate the green fluorescent protein (GFP; 26.9 kDa). Size markers
(in kDa) are also indicated.
(a)(b)The fluorescence intensities (a) and confocal microscopic images (b) ofE. coli
W3110 cells by induction with IPTG (middle panels) or oleic
acid (bottom panels). As a control, the E. coli strain without plasmid
is also shown (top panels). Shown in (b) are immunofluorescence
micrographs (left panels), differential interference micrographs (middle panels),
and merged images (right panels) of wild-type E. coli W3110 and its
recombinant cells harboring pTac99GFP and pAD99GFP.
(a)(b)Until
now, the IPTG-inducible promoterstac or trc have been widely used for basic
research. However, the use of IPTG for the large-scale production of human
therapeutic proteins is undesirable because of its toxicity and relatively high
cost; the high concentration of IPTG can inhibit cell growth and recombinant
protein production [22, 23]. Therefore, determination of optimal induction time
point as well as the inducer concentration is crucial to increase the overall
productivity of recombinant protein. In this regard, there have been
significant efforts to overcome such problems by using different inducible or even
constitutive expression systems [24]. Thus, the aldA promoter found in this study can be effectively used for the
enhanced production of recombinant proteins with the aforementioned problems
largely resolved.In summary, thealdA promoter satisfies
requirements for its utilization as a promoter. First, it is tightly controllable
with an appropriate inducer, oleic acid in this case. Tight regulation of the
promoter is essential for the synthesis of proteins which may be detrimental to
the host cell. For example, the toxic rotavirus VP7 protein effectively kills
cells, and must be produced under tightly regulated conditions [25]. Second, its
expression is strong and long lasting, resulting in the accumulation of the
target protein constituting up to 50% of the total cellular proteins. The third
important characteristic of aldA promoter is its inducibility in a cost-effective manner by using exogenous
oleic acid as an inducer.


## 4. Conclusion

Proteome analysis of the cells with focus on proteins
induced or repressed by the stimulus provides clues to the understanding of
cellular responses. This study revealed that 52 proteins showed significantly
altered levels inE. coli grown with oleic
acid compared to the glucose. Based on the resulting proteome profiles, the
promoter of aldA gene was found to be
strongly activated by oleic acid and subsequently to be demonstrated useful as
an inducible promoter for the enhanced production of desirable targets. Thus,
this study demonstrates that E. coli proteome profiles not only provide invaluable information for physiological
status of the organism under specific conditions but also propose its biotechnological
applications.



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*Source: 735101-2007-12-17.xml*