Heterologous expression of stearoyl-acyl carrier protein - QIBEBT-IR

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Heterologous expression of stearoyl-acyl carrier protein - QIBEBT-IR
Protein Expression and Purification 69 (2010) 209–214
Contents lists available at ScienceDirect
Protein Expression and Purification
journal homepage: www.elsevier.com/locate/yprep
Heterologous expression of stearoyl-acyl carrier protein desaturase (S-ACP-DES)
from Arabidopsis thaliana in Escherichia coli
Yujin Cao, Mo Xian *, Jianming Yang, Xin Xu, Wei Liu, Liangzhi Li
Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, 266101 Qingdao, China
a r t i c l e
i n f o
Article history:
Received 21 July 2009
and in revised form 16 August 2009
Available online 27 August 2009
Keywords:
GC–MS
Heterologous expression
ssi2/fab2
Stearoyl-ACP desaturase
a b s t r a c t
Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains, among which stearoyl-acyl carrier protein desaturase (S-ACP-DES) was widely distributed in the plant kingdom. We
cloned the cDNA coding for fab2/ssi2, an S-ACP-DES from Arabidopsis thaliana, into the vector pET30a
and heterologously expressed this fatty acid desaturase in Escherichia coli BL21 (DE3). After being induced
with IPTG, the fusion protein was efficiently expressed in a soluble form. The SSI2 desaturase was purified
by nickel ion affinity chromatography and the product obtained showed a single band by SDS–PAGE analysis. The expression of ssi2 modified the fatty acid composition of the recombinant strain. The ratio of
palmitic acid (16:0) decreased from 45.2% (the control strain) to 35.2% while palmitoleate (16:1D9)
and cis-vaccenate (18:1D11) levels were enhanced to some extent. The desaturase enzymatic activity
was measured in vivo when the enzyme substrate stearic acid was provided in the culture medium. A
new fatty acid, oleic acid (18:1D9) was found in the recombinant strain which did not exist in wild-type
E. coli. These results demonstrated that the cofactors of the host system can complement the requirement
of the SSI2 desaturase.
Ó 2009 Elsevier Inc. All rights reserved.
Introduction
Fatty acid desaturases are enzymes that introduce double bonds
into the hydrocarbon chains of fatty acids. They are present in all
groups of organisms and play a key role in the maintenance of
the proper structure and function of biological membranes [1].
There are three types of fatty acid desaturases: acyl-CoA, acylACP, and acyl-lipid desaturases, among which acyl-acyl carrier protein (ACP)1 desaturases are soluble enzymes that catalyze the
insertion of a double bond into saturated fatty acids bound to
ACP. The most widely occurring member of this family is the D9stearoyl (18:0)-ACP desaturase (S-ACP-DES, EC 1.14.99.6). S-ACPDES mediated conversion of stearic acid to oleic acid (18:1) is the
key step that regulates the levels of unsaturated fatty acids in plant
cells [2]. The S-ACP-DES has been purified from several plants and
the encoding genes were characterized from several different spe-
* Corresponding author. Fax: +86 0532 80662765.
E-mail address: [email protected] (M. Xian).
1
Abbreviations used: S-ACP-DES, stearoyl-acyl carrier protein desaturase; ACP, acyl
carrier protein; LB, Luria–Bertani; IPTG, isopropylthiogalactoside; SDS–PAGE, sodium
dodecyl sulfate–polyacrylamide gel electrophoresis; FAMEs, fatty acid methyl esters;
GC, gas chromatography; GC–MS, gas chromatography–mass spectrometry; CFA,
cyclopropane fatty acid; KAS, b-keto-acyl-ACP synthase; UFAs, unsaturated fatty
acids.
1046-5928/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.pep.2009.08.011
cies, i.e., castor bean [3], cat’s claw [4], English ivy [5] and soybean
[6].
Genes involved in unsaturated fatty acids synthesis have been
described in Arabidopsis by many former researchers. The Arabidopsis genome carries seven S-ACP-DES like genes, including the
ssi2/fab2 [7]. The mutation at the fab2 locus of Arabidopsis caused
increased levels of stearate in leaves [8] and the fab2 plants are
profoundly affected in growth and development when grown at
22 °C [9]. With the use of a map-based approach, the ssi2 gene
was cloned from this mutant and shown to encode an S-ACP-DES
[10]. The desaturation of long-chain fatty acids in Arabidopsis begins with the conversion of stearoyl-ACP to oleoyl-ACP by this stearoyl-ACP desaturase. The SSI2 desaturase is of great importance in
controlling fatty acid composition of membrane lipids. The ssi2
mutant plant is dwarf, spontaneously develops lesions containing
dead cells [11] and the altered oleic acid content in the ssi2 mutants has an impact on salicylic acid- and jasmonic acid-mediated
defense signaling.
In the present study, the ssi2/fab2 gene was cloned from Arabidopsis thaliana. With the aim to study the stearoyl-ACP desaturase
biochemically, this gene was thus heterologously expressed in
Escherichia coli BL21 (DE3). In addition, we measured the enzymatic activity of this fatty acid desaturase in order to demonstrate
that the gene products were in their active form by detecting the
enzyme catalysis product using gas chromatography–mass spectrometry analysis.
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Y. Cao et al. / Protein Expression and Purification 69 (2010) 209–214
Material and methods
Strains, plasmids and chemicals
Escherichia coli DH5a was used for gene cloning and E. coli BL21
(DE3) was used as a host for the expression of the recombinant
proteins. The pUCm-T vector for cloning of PCR products was purchased from Sangon Biotechnology (Shanghai, China). The expression vector pET30a was obtained from Novagen (Madison, WI).
Restriction enzymes and T4 DNA ligase were purchased from
MBI Fermentas (Hanover, MD). Pfu polymerase and protein marker
were obtained from TaKaRa (DaLian, China). All chemicals were
purchased from Sigma–Aldrich unless otherwise specified.
Plasmid construction and cloning
Arabidopsis was grown on half-strength Murashige and Skoog
medium (1/2MS) supplemented with 3% sucrose (pH 5.8). Total
RNA was isolated from young leaves using hot phenol method described by Verwoerd et al. [12]. First strand cDNA was synthesized
from the total RNA using PrimeScript RT-PCR Kit (TaKaRa). The
ssi2/fab2 (GenBank Accession No. AF395441) gene was amplified
using cDNA of Arabidopsis as template and with primers ssi2-F
(50 -CCATGGCTCTAAAGTTTAACCC-30 ) and ssi2-R (50 -GTCGACTTAGA
GCTGCACTTCTCTG-30 ) containing the start and stop codons as well
as the restriction sites of NcoI and SalI. The amplified PCR products
were analyzed by agarose gel electrophoresis and directly cloned
to pUCm-T Vector. The plasmid pUCm-T-ssi2 was double-digested
with NcoI and SalI, and the digested ssi2 fragment was withdrawn
and ligated to pET30a expression vector predigested with the same
restriction enzymes and generated pET-ssi2 (Fig. 1).
Protein expression and purification
A single colony of recombinant E. coli strain carrying pET-ssi2
was inoculated into Luria–Bertani (LB) medium containing 50 lg/
ml kanamycin and cultured at 37 °C. The overnight culture was
100-fold diluted to inoculate into fresh liquid LB medium, grown
to an OD600 of about 0.6 and then induced with isopropylthiogalactoside (IPTG). The culture was subsequently incubated to express
the fusion protein [13]. About 120 mg wet cells were collected
from 10 ml of bacteria culture by centrifugation, suspended in
1 ml Tris–HCl buffer (pH = 8.0) and subject to ultrasonication.
The mixture was centrifuged at 12,000 rpm and 4 °C for 10 min,
and the supernatant obtained was purified by Ni–NTA purification
system (Invitrogen). Protein concentrations of different fraction
were determined by coomassie brilliant blue staining. Both the
crude and purified protein fractions were boiled for 5 min and analyzed by 12% sodium dodecyl sulfate–polyacrylamide gel electro-
phoresis (SDS–PAGE). Proteins were visualized by Coomassie
brilliant blue staining. Quantitative evaluation of protein bands
was carried out using the Universal Hood II gel imaging system
equipped with Quantity One software (Bio-Rad).
Lipid extraction and FAME preparation
For fatty acid analysis, E. coli strains were cultured using M9
minimal medium with 2% glucose as the carbon source. Lipids
were extracted from pelleted cells according to the method of Oursel et al. [14] with some modifications. Approximately 150 mg
E. coli cells were harvested from 400 ml fermentation culture
(0.42 g/L per OD600). Cell pellets were resuspended in 1 ml of distilled water and vortex mixed with 5 ml of CHCl3/CH3OH (2:1 by
v/v) for 3 min. The resulting mixture was left over night. After centrifugation, the lower phase (chloroform phase) was evaporated
with nitrogen. Two milliliters of 6% (w/v) NaOH in methanol/water
(4:1, by vol) was added to the dried lipids and saponified in 60 °C
water bath for 2 h. Then 2 ml of BF3/CH3OH (1:4, by vol) was added
to the saponification solution and heated for another 30 min at
60 °C to generate fatty acid methyl esters (FAMEs). Esterified fatty
acids were extracted twice using 2 ml of hexane.
GC and GC–MS analysis
The FAMEs were analyzed by gas chromatography (GC; GC-450,
Varian) and gas chromatography–mass spectrometry (GC–MS;
Trace GC Ultra-ITQ1100, Thermo Fisher). The GC was equipped
with a 30 m HP-5 column (internal diameter 0.32 mm, film thickness 0.25 lm) using nitrogen as carrier gas with a linear velocity of
1 ml/min. The column temperature program was composed of an
initial hold at 100 °C for 2 min, ramping at 20 °C per min to
180 °C, followed by heating until 250 °C with 10 °C per min. The
injector temperature was 250 °C and the FID detector temperature
was 300 °C. The GC–MS conditions were as follows: a 30 m HP5 ms column (internal diameter 0.25 mm, film thickness
0.25 lm); an oven temperature program composed of an initial
hold at 100 °C for 2 min, ramping at 10 °C per min to 280 °C, and
a final hold at 280 °C for 5 min; an ion source temperature of
220 °C and EI ionization at 70 eV.
Enzyme activity assays in vivo
The optimal substrate stearic acid for S-ACP-DES was not present as the major component fatty acid in E. coli. In order to assay
desaturase activity in vivo, both the recombinant strain and the
control strain were cultured using M9 minimal medium containing
2% glucose as carbon source, and supplemented with 50 mg/L of
stearic acid or eicosanoic acid as the substrate for the SSI2 desaturase, respectively. Bacterial cells were harvested in logarithmic
growth phase and used immediately or stored at 20 °C and used
later. Total lipids were extracted and the fatty acids were analyzed
using the procedure above.
Results
Expression and purification of ssi2 gene in E. coli
Fig. 1. Construct for recombinant plasmid expressing ssi2. The ssi2 gene was
inserted into the expression vector of pET30a between NcoI and SalI.
To express SSI2 desaturase in E. coli, we cloned the coding region of ssi2 into pET30a under the T7 promoter and His-tag, yielding pET-ssi2. The expression construct was checked by restriction
enzyme digestion and DNA sequencing. The recombinant plasmid
pET-ssi2 was transformed into E. coli BL21 (DE3) and transformed
E. coli cells carrying pET-ssi2 were grown in liquid LB medium. Initially, the expression of fusion protein was induced with 1 mM
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Y. Cao et al. / Protein Expression and Purification 69 (2010) 209–214
IPTG at 37 °C for 2 h and we noted a distinct band of the expected
size (50.5 kDa). No band was observed in the extract from the control pET30a strain (Fig. 2, lane 1). In order to determine whether
the SSI2 protein was expressed in the form of inclusion bodies,
the bacteria cells were sonicated and separated into soluble and
insoluble fractions by centrifugation. Luckily, most of the induced
protein was present in the soluble fraction. The fusion SSI2 protein
contains 445 amino acids and only 37.5% of these residues are
hydrophobic. Therefore, most of the SSI2 proteins were expressed
in a soluble form in the recombinant strain. We then optimized
the expression conditions to growth at 37 °C for 3 h and induced
with 0.1 mM IPTG. SDS–PAGE analysis revealed that the target protein accumulated up to 10.3% of the total cell protein (Fig. 2, lane
2). To purify the putative SSI2 desaturase, the cell lysates were subjected to His-tag purification using a Ni2+–NTA column. After nickel
ion metal affinity chromatography, the final elute gave a single distinct band (Fig. 2, lane 3). And about 74% of the target protein was
recovered from the cell lysates (Table 1).
Table 1
Purification table for the SSI2 protein heterologously expressed in E. coli BL21 (DE3).
Modification of fatty acid content in E. coli
The activity of SSI2 desaturase was measured in vivo by providing the substrate in the culture medium. Fig. 4 shows the result of
GC–MS analysis of the fatty acid methyl esters of transformed
E. coli strains and the control strain after 3 h induction. An additional peak is apparent in the trace obtained from induced SSI2
grown in the presence of stearic acid compared to the empty-vector control. Mass spectrum of this fatty acid methyl derivative
demonstrated that the novel peak was oleic acid methyl ester
(Fig. 4 C) and this fatty acid accounted for 2.7% of the total fatty
acids in the recombinant cells. These data supported that the recombinant desaturase was actively expressed in E. coli, had the
ability to catalyze the conversions from stearic acid to oleic acid
and that the reactions could take place using cofactors present in
E. coli.
However, when eicosanoic acid was provided in the culture
medium, seldom 20:0 and no 20:1 fatty acids were detected by
GC–MS analysis (data not shown). This result might not be due
to the inability of SSI2 desaturase to convert eicosanoic acid but
to low level of eicosanoyl-ACP in E. coli. Little eicosanoic acid was
incorporated into the cell phospholipids of E. coli. If the amount
of eicosanoyl-ACP was not limited to the desaturation reaction, this
enzyme could probably use eicosanoic acid as its potential substrate in vivo.
Palmitate, palmitoleate and cis-vaccenate make up the bulk of
the fatty acids found in E. coli membranes [15]. However, the
two kinds of unsaturated fatty acids were modified by cyclopropane fatty acid (CFA) synthase and formed cyclopropane ring during the transition from log phase to stationary phase [16]. In order
to avoid CFA formation, the E. coli strains were induced at an OD600
of 0.4 and harvested when OD600 reached about 1.0 (3 h after
induction).
The expression of the SSI2 desaturase did not alter the fatty acid
constituents of E. coli. As shown in Fig. 3, the GC chromatograms of
E. coli strains transformed with pET30a and pET-ssi2 both showed
three main peaks. Based on GC retention times and mass spectra,
these three main fatty acids were identified as 16:0, 16:1(D9)
and 18:1(D11). This result is likely not due to an inability of the
desaturase to function in E. coli. Instead, the lack of oleic acid production in the recombinant strain expressing SSI2 desaturase may
be a result of the absence of substrate stearoyl-ACP for this enzyme. However, the great changes in the pattern of palmitic acid
contents between the pET-ssi2 transformant and the control strain
indicated that the SSI2 desaturase was capable of using palmitoylACP as an alternative substrate. In the recombinant strain, palmi-
Fraction
Volume
(ml)
Protein
concentration
(mg/ml)
Total
protein
(mg)
Target
protein
(mg)
%
Yield
Wet weight of cells
Lysate
His-tag purified
protein
10
1
2
0.54
2.9
0.11
5.4
2.9
0.21
0.29
0.21
100
74
tate level decreased from 45.2% (the reference strain) to 35.2%.
Meanwhile, the ratio of palmitoleate and cis-vaccenate increased
to 26.9% and 31.7%, respectively (Table 2). We also noticed that
the palmitic acid level remained stable after the expression of
the SSI2 protein. The ratio of palmitic acid was 34.9% after 6 h
induction.
Characterization of recombinant ssi2 enzyme activity in vivo
Discussion
Fig. 2. SDS–PAGE analysis of SSI2 expression in E. coli after IPTG induction. IPTG at
0.1 mM was added to the growth medium for induction of ssi2, and then the culture
was incubated at 37 °C for 3 h before analysis of cellular proteins. Lane M,
prestained protein molecular weight marker; lane 1, control strain harboring
pET30a; lane 2, total protein of pET-ssi2; lane 3, purified recombinant SSI2 (arrow
indicates recombinant SSI2).
During the past few years, many of the genes that are responsible for fatty acid desaturases have been cloned from various organisms, including bacteria [17], algae [18], yeast [19], plants [20] and
mammals [21]. The expression of genes for desaturase is very
important since it provides the molecular basis for the acclimation
of organisms to changing environmental temperatures [22]. However, characterization of fatty acid desaturase, which is required for
a full understanding of its physiological role, has been hampered
by the difficulty of expressing it in a common heterologous host
such as E. coli. Most of the fatty acid desaturases are integral membrane proteins. The toxic effects of the overexpression of a particular membrane protein and unsuitable lipid composition of E. coli
may limit the utility of this most widely used vehicle for eukaryotic
membrane protein production [23]. On the other hand, wild-type
E. coli does not contain any fatty acid desaturase. It possesses an
anaerobic pathway to synthesize unsaturated fatty acids. The fatty
acid desaturase enzymes employ ferredoxin [24] or cytochrome b5
[25] as the electron donor, whereas E. coli does not have ferredoxin
and the ferredoxin:NADP+ oxidoreductase system. Thus, many
fatty acid desaturases heterologously expressed in E. coli were in
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Y. Cao et al. / Protein Expression and Purification 69 (2010) 209–214
Fig. 3. Gas chromatograms of FAMEs obtained from different E. coli transformants. (A) The control strain harboring pET30a; (B) the pET-ssi2 transformants. 16:0, palmitic
acid; 16:1(D9), palmitoleic acid; 18:1(D11), cis-vaccenic acid.
Table 2
Fatty acid composition of E. coli strains carrying control and recombinant plasmids.
Transformants
Fatty acid methyl esters
16:1
16:0
17:0D
18:1
19:0D
3 h after induction
pET30a (%)
5.9
pET-ssi2 (%)
6.2
21.2
26.9
45.1
35.2
—
—
27.8
31.7
—
—
6 h after induction
pET30a (%)
5.8
pET-ssi2 (%)
4.5
16.6
18.6
43.1
34.8
8.2
8.9
26.3
33.2
—
—
14:0
inactive form and their functions in vivo were suppressed, as the
electron carriers of the host system cannot complement the system
of the donor species.
Plant stearoyl (18:0)-ACP desaturase catalyzes the conversion of
stearic acid to oleic acid through the eukaryotic pathway of plant
lipid biosynthesis. This fatty acid desaturase is unique to the plant
kingdom in that all other known desaturases are integral membrane proteins [26]. SSI2 was the first cloned stearoyl-ACP desaturase in the model plant Arabidopsis. In this study, the ssi2/fab2 gene
from A. thaliana was successfully expressed in E. coli BL21 (DE3).
We found that this overexpressed S-ACP-DES was in soluble form
under the pET expression system. The desaturation activity of recombinant protein can be markedly detected when assayed
in vivo with added substrate stearic acid. Although the coupled ferredoxin and ferredoxin–NADP+ oxidoreductase were the optimal
electron donor for the S-ACP-DES family in the plastids of plants,
the SSI2 desaturase could also obtain electrons from the cytochrome b5 and cytochrome b5 reductase system [27] which exists
in the cytoplasm of E. coli. This result was in accordance with many
other fatty acid desaturases expressed in E. coli [28–30].
The composition of E. coli membrane lipid is lacking in stearic
acid. In the type II fatty acid synthesis system of E. coli, fatty acid
Y. Cao et al. / Protein Expression and Purification 69 (2010) 209–214
213
Fig. 4. Identification of oleic acid methyl ester in E. coli transformants by GC–MS analysis. (A) E. coli transformed with control vector pET30a; (B) E. coli transformed with
recombinant plasmid pET-ssi2. The arrow indicates the novel peak of oleic acid methyl ester; (C) mass spectra of the novel peak. NIST-library searching program indicated
that the novel peak was oleic acid methyl ester.
elongation is catalyzed by the action of b-keto-acyl-ACP synthase
(KAS). However, palmitoyl-ACP was not a suitable substrate for
both KASI (FabB) and KASII (FabF) [31]. Stearoyl-ACP desaturase,
which acts mainly on stearic acid, could also use palmitic acid as
an alternative substrate to produce palmitoleic acid. In this work,
the engineered strains of E. coli expressing SSI2 protein showed sig-
Fig. 5. Biosynthesis of unsaturated fatty acids (UFAs) in E. coli. FabA catalyzes the key step in UFAs production, whereas FabB and FabF are required for the elongation of these
unsaturated acyl-ACP intermediates. In the recombinant strain carrying pET-ssi2, the SSI2 desaturase catalyze the conversion of palmitoyl-ACP to palmitoleoyl-ACP.
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Y. Cao et al. / Protein Expression and Purification 69 (2010) 209–214
nificant decreasing in palmitic acid content. Meanwhile, the proportion of palmitoleic acid and cis-vaccenate was enhanced to
some extent. The pathway for E. coli to synthesize unsaturated
fatty acids is shown in Fig. 5. In the recombinant strain carrying
pET-ssi2, the SSI2 desaturase converted palmitoyl-ACP to palmitoleoyl-ACP and palmitoleoyl-ACP was further elongated by FabF.
Therefore, the recombinant strain showed increased palmitoleate
and cis-vaccenate levels.
Our results also indicated that the palmitic acid level remained
stable even when the SSI2 desaturase was induced for a much
longer time. The fatty acid composition exerts a major influence
on fluidity of biological membranes [32]. Extreme saturated fatty
acid constituents seem to hamper the regular function of cell
membrane. The palmitic acid content is tightly regulated in
E. coli and hence the palmitic acid could not decrease to a much
lower level by prolonging the induction time.
In conclusion, we have shown that the ssi2 gene expressed in
E. coli BL21 was in its active form. The cofactors present in E. coli
host cells can fulfill the requirement for desaturation of SSI2 enzymes. The desaturation reaction can thus be carried out in vivo
where no exogenously added cofactors are present. These results
indicate that the aerobic acyl-ACP desaturation system of plants
can be imposed on the anaerobic pathway of unsaturated fatty acid
synthesis of E. coli. Furthermore, the fatty acid composition was
significantly modified in E. coli strains heterologously expressing
SSI2 desaturase compared to the control strain. Research on stearoyl-ACP desaturase may enable us to further evaluate its potential
role in altering the ratio of unsaturated to saturated fatty acids.
Acknowledgments
This work was financially sponsored by CAS 100 Talents Program (KGCX2-YW-801). We are grateful to Dr. Yun Fa for GC analysis; Dr. Wenna Guan and Cong Wang for GC–MS analysis.
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