Characterization of a novel thioesterase (PtTE) from Phaeodactylum tricornutum

666

Journal of Basic Microbiology 2011, 51, 666 – 672

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Short Communication

Characterization of a novel thioesterase (PtTE)
from Phaeodactylum tricornutum

Yangmin Gong, Xiaojing Guo, Xia Wan, Zhuo Liang and Mulan Jiang

Oil Crops Research Institute of Chinese Acadamy of Agricultural Sciences, Wuhan, Hubei, China

The Phaeodactylum tricornutum (P. tricornutum) thioesterase PtTE was encoded by a 648 bp open
reading frame. The deduced 216 amino acids showed no similarity with plant acyl-acyl carrier
protein (ACP) thioesterases and bacterial thioesterases. Southern blot analysis revealed that one
copy of PtTE was present in the P. tricornutum genome, and Real-time quantitative PCR showed
that PtTE was up-regulated upon nitrogen deprivation. Thioesterase activity of PtTE was
established by heterologous expression of PtTE cDNA in Escherichia coli (E. coli) XL1-Blue and
K27fadD88, a mutant strain of fatty acid β-oxidation pathway. The substrate specificity of PtTE
was determined by fatty acid profile analyses of the culture supernatant and membrane lipid
of recombinant strains. Recombinant PtTE in E.coli enhanced total fatty acid content of XL1-
Blue by 21%, and also changed the fatty acid compositions of membrane lipid and culture
supernatant. These changes were directed predominantly towards C18:0 and C18:1 fatty acids.
Overexpression of PtTE alone in P. tricornutum did not alter the fatty acid composition of
P. tricornutum, but enhanced total fatty acid content by 72%. This novel thioesterase gene shows
its potential in metabolic engineering for enhancing lipid yield of microalgae. This is so far the
first report of thioesterase from eukaryotic microalgae.

Abbreviations: Phaeodactylum tricornutum, P. tricornutum; acyl carrier protein, ACP; coenzyme A, CoA;

triacylglycerol, TAG; polyunsaturated fatty acids, PUFAs; eicosapentaenoic acid, EPA; fatty acid methyl

esters, FAME;

isopropylthiogalactoside, IPTG;

Gas Chromatography, GC

Supporting Information for this article is available from the authors on the WWW under
http://www.wiley-vch.de/contents/jc2248/2011/201000520_s.pdf

Keywords: P. tricornutum / Thioesterase / Fatty acid

Received: December 20, 2010; accepted: February 17, 2011

DOI 10.1002/jobm.201000520

Introduction

Eukaryotic microalgae and plants have similar fatty
acid biosynthesis pathway, which generally take places
in the plastids and is compromised of a repeated cycle
of reactions involving the sequential condensation of
two-carbon units onto a growing fatty acyl-ACP deriva-
tives or an acetyl-coenzyme A (CoA) precursor [1]. The
successive elongation of acyl-ACP chain can be termi-
nated by acyl-ACP thioesterases (E.C. 3.1.2.14) that hy-

Correspondence: Mulan Jiang, Oil Crops Research Institute of Chinese
Acadamy of Agricultural Sciences, Xudong 2nd Road, Wuhan, Hubei
430062, People’s Republic of China
E-mail: mljiang@oilcrops.cn
Phone: 86-27-86838791
Fax: 86-27-86822291

drolyze the thioester bond of acyl-ACP. The released
free fatty acids are subsequently exported out of the
plastid to the endoplasmic reticulum (ER) and re-
esterified to CoA, which are required for glycerolipid
synthesis. Although these glycerolipids are generated
from ER, they may be shuttled between ER and plastid
during membrane lipid biosynthesis in almost all plant
cells [2]. However, in developing seeds, most of ER glyc-
erolipids are converted into storage triacylglycerols
(TAGs). Thus, acyl-ACP thioesterases play a crucial role
in determining the fatty acid composition of storage
TAGs and membrane polar lipids [3, 4].
Plants acyl-ACP thioesterases are encoded by nuclear
genes, but soluble mature enzymes are targeted into
plastid. Based on sequence identity and substrate (acyl-

Journal of Basic Microbiology 2011, 51, 666 – 672

Thioesterase from Phaeodactylum tricornutum 667

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ACP) specificity, these enzymes have been classified
into two distinct but related families termed FatA and
FatB [4, 5]. The FatA orthologs exhibit the highest activ-
ity towards unsaturated oleoyl (C18:1)-ACP. In contrast,
FatB thioesterases have predominant activity towards
saturated acyl-ACP ranging from C8:0 to C18:0-ACP
[3, 6–8]. Different from plants, E.coli contains two sepa-
rable thioesterases that hydrolyze the thioester bond of
acyl-CoA molecules. Both enzymes can hydrolyze acyl-
ACP thioesterases, but at much slower rates than the
analogous acyl-CoA substrates [9]. Thioesterase I en-
coded by the tesA gene shows broad specificity for
C12 to C18 acyl-CoA esters [10]. Thioesterase I was re-
ported to mediate acyl-ACP intermediates from the
fatty acid de novo biosynthesis pathway to fatty acid

β-oxidation in E. coli  [11]. On the other hand, thio-
esterase II encoded by the tesB gene has broader sub-
strate specificity, and is capable of hydrolysis of C6 to
C18 acyl-CoA esters [9].
  Although both groups of thioesterases FatA and FatB
in plants, thioesterase I and II in E. coli have been char-
acterized in these organisms, there is almost no report
on the study of thioesterases from photosynthetic mi-
croalgae. Microalgae offer great potential for biofuel
production because some microalgal species or strains
can accumulate substantial quantities of lipids, and
metabolic engineering plays an important role in bio-
fuel production with microalgae as feedstock [12].
Phaeodactylum tricornutum is a silica-less diatom and has
a high content of lipid and polyunsaturated fatty acids
(PUFAs), especially eicosapentaenoic acid (EPA) [13]. In
the  P. tricornutum genome, there are two genes anno-
tated for thioesterases that are predicted thioesterase
and palmitoyl protein thioesterase (http://genome.jgi-
psf.org/Phatr2/Phatr2.home.html). However, the two
thioesterases are not identified and the possible roles in
fatty acid biosynthesis remain unknown. In this study,
the putative thioesterase PtTE was characterized, and
heterologous expression of PtTE cDNA in E. coli, overex-
pression of PtTE cDNA in P. tricornutum were carried out.
This work can help us to understand the role of thio-
esterase PtTE in fatty acid metabolism of P. tricornutum,
and might also lead to its future application in meta-
bolic engineering for enhancing lipid yields or modify-
ing the fatty acid profile of microalgae.

Materials and methods

Strains and culture conditions
E. coli Top10 was used for gene cloning, and E. coli XL1-
Blue, Rosetta (DE3), K27 fadD88 mutant (E. coli Genetic

Stock Center, Yale University, CGSC #5478) were used
as hosts for expression of PtTE encoding P. tricornutum
thioesterase. All cultures were grown in Luria-Bertani
(LB) media at 37 °C or 28 °C (fadD88 mutant). For
plasmid selection, 100 μg ml

–1

of ampicillin and/or

30 μg ml

–1

of chloramphenicol were added. For fatty

acid analyses, recombinant strains were cultured using
M9 minimal salts medium supplemented with 1 mM
MgSO

and 2% glucose as the carbon source. All strains

and plasmids used in this work were listed in Supple-
mentary Table S1. P. tricornutum was from Freshwater
Algal Culture Collection of the Institute of Hydrobiol-
ogy, China. It was grown at 22 °C and continuously
illuminated with 75 μmol photons m

–2

–1

on LDM me-

dium [14] containing 1.2% agar. Liquid cultures were
grown in f/2 medium [15].

Plasmid construction
To construct the expression plasmid  with  PtTE driven
by  lac promoter, the 648-bp PtTE  coding sequence
of  P. tricornutum was obtained by Reverse Transcrip-
tion-PCR (RT-PCR) using primer pair PtTE-1 (5′-
GCGCGAATTCGATGGGACTGATTCACACTCC-3′) and
PtTE-2 (5′-GAGAAGCTTTCACTCCTTATTTGCCTCCT-3′).
The amplified product was digested with EcoRI and
HindIII, purified and ligated into EcoRI and HindIII-
digested pUC18, resulting in pOC64. To construct the
expression plasmid with PtTE driven by T7 promoter, the
coding region of PtTE was generated by RT-PCR with
primers PtTEex-1 (5′-ACATATGGGACTGATTCACACTCC-3′)
and PtTEex-2 (5′-ACTCGAGCTCCTTATTTGCCTCCTT-3′).
The purified product was cloned into pMD18-T, result-
ing in pOC60. The NdeI-XhoI PtTE fragment excised from
pOC60 with NdeI and XhoI, was cloned into NdeI and
XhoI-digested pET21b, resulting in pOC66. To construct
plasmid overproducing PtTE in P. tricornutum, pOC64
was digested with EcoRI and HindIII, and the EcoRI-
HindIII PtTE fragment was cloned into the EcoRI-HindIII
sites of pPha-T1, resulting in transformation plasmid
pOC37.

Preparation of nucleic acids and Southern blot
analysis
The preparation of total DNA from P. tricornutum was
performed following to Falciatore et al. [16]. DNA probe
labeling and hybridization were performed with DIG
High Prime DNA Labelling and Detection Starter Kit I as
described in the manufacturer’s instruction manual
(Roche Applied Science, USA). DNA fragment used to
probe the blot was generated by PCR using pair of
primers PtTE-1 and PtTE-2.

668 Yangmin

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qRT-PCR analysis
Real Time-PCR amplification mixtures (20 μl) contained
2 μl of cDNA obtained after the reverse transcription,
200 nM forward and reverse primers, and 2 × SYBR Green
I PCR Master Mix (Bio-Rad). PtTE was amplified with
primer pair Q-PtTE-1 (5′-CGAAAGTTCCAGGTTGACAC-3′)
and Q-PtTE-2 (5′-TGAACTGCCACTCCTTGAAC-3′). As an
internal standard, constitutively expressed gene for TBP
(TATA box binding protein) mRNA was amplified with
primer pair Q-TBP-1 (5′-ACCGGAGTCAAGAGCACACAC-3′)
and Q-TBP-2 (5′-CGGAATGCGCGTATACCAGT-3′). Gene ex-
pression analysis was performed with the method of
2

–ΔΔCt

[17], where 0 h treatment was used as calibrator

sample.

Recombinant PtTE expression in E. coli
For the expression of PtTE under lac promoter of pUC18
in frame with the N-terminal of lacZ α-fragment, the
correct reading frame of the fusion gene with the ex-
pected tag of seven amino acids from lacZ α-fragment
was confirmed by DNA sequencing. The plasmid pOC64
carrying P

lac

-PtTE and control plasmid (pUC18) were

transformed into E. coli strains XL1-Blue and K27fadD88,
a fatty acid β-oxidation mutant. The strains E. coli XL1-
Blue bearing pUC18 and pOC64 were grown in LB broth
supplemented with 100 mg ampicillin/ml with shaking
at 37 °C. The mutants bearing pUC18 and pOC64 were
grown at same conditions except at 30 °C. Cells were
induced at OD

600

0.6 with 0.4 mM IPTG and growth was

continued for additional 4 h.

Transformation of P. tricornutum
The plasmid pOC37 and control vector were trans-
formed into P. tricornutum using microparticle bom-
bardment as described by Zaslavskaia et al. [18]. Cells
were bombarded using the Biolistic PDS-1000/He Parti-
cle Delivery System (BioRad). Approximately 10

cells

(200 μl) were spread on fresh LDM agar medium con-
taining 75 μg/ml Zeocin (Invitrogen). Resistant colonies
were obtained after 2 to 3 weeks of incubation at 20 °C
under constant illumination (75 μmol m

–2

–1

). Putative

transformants were restreaked on plates containing
100 μg/ml Zeocin.

Lipid extraction and analysis
Cultures of 200 ml E. coli carrying pUC18 (as control) or
pOC64 were grown to OD

600

0.6, induced as described

above, and the total lipids were extracted and trans-
esterified [2, 19]. The fatty acid methyl esters (FAME)
were analyzed and quantified by gas chromatography-
flame ionization detection (GC-FID, Agilent). Fatty

acids were identified by comparison with a combi-
nation of known standards (Sigma). Lipid from E. coli
mutant K27fadD88 culture supernatant was extracted
according to the protocol of Voelker and Davies [20].
Free fatty acids were separated from polar lipids by
thin-layer chromatography (TLC) with hexane/diethyl-
ether/acetic acid (60:40:1). Free fatty acids were ex-
tracted from the appropriate regions of separate TLC
plates and transesterified [21]. FAME analysis was
performed as described above. Data presented in this
study are the average of three experiments in each
case.
Fatty

acids

P. tricornutum were extracted and trans-

methylated according to the method by Lepage and Roy
[22]. Heptadecanoic acid (C17:0, 0.2 mg) was used as
internal standard, and fatty acid methylesters were
identified and quantified by GC.

Results and discussion

Sequence analysis of PtTE from P. tricornutum
The obtained 648-nucleotide PtTE ORF encoded a
predicted polypeptide of 216 amino acid residues of
calculated molecular mass 24 kDa. The N-terminal se-
quences of PtTE had a putative mitochondrial tar-
geting peptide. The neighbor-joining phylogenetic tree
(Fig. S1) showed that thioesterases were plotted out two
major groups including plant FatA, FatB and other
several independent groups, with P. tricornutum thio-
esterase PtTE distributed in a novel group and show-
ing no similarity with other thioesterases. These analy-
ses demonstrated that P. tricornutum PtTE was clearly
different in amino acids sequence from plant acyl-
ACP thioesterases and other prokaryotic thioestera-
ses.

Genomic organization and expression pattern
of PtTE
Southern blot analysis of P. tricornutum genome digested
with four different restriction enzymes followed by
hybridization with full-length PtTE gene revealed the
presence of distinct single band in each lane (Fig. 1A).
This suggested that only one copy of PtTE gene was
present in the P. tricornutum genome. Quantitative real
time RT-PCR showed that PtTE was gradually up-
regulated after P. tricornutum was deprived of nitrogen
source, and two-fold increase in the expression level of
PtTE was observed at 60 h after nitrogen deprivation
(Fig. 1B). This expression pattern was correlated with
the remarkable increase of triacylglycerols content
under nitrogen deficiency [23].

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Thioesterase from Phaeodactylum tricornutum 669

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(A)

(B)

Figure 1. Genomic organization and expression pattern of the
P. tricornutum PtTE gene. (A) Southern blot analysis of PtTE from
the P. tricornutum genome. Thirty micrograms of genomic DNA was
digested by overnight incubation at 37

°C with HindIII (lane 1), PstI

(lane 2), EcoRV (lane 3), PvuII (lane 4), separated in 0.8% agarose
gel and transferred to a nylon membrane (GE) by capillary transfer.
The migration position of the

λ-HindIII marker was indicated. (B)

Expression of PtTE under nitrogen deprivation at different times
(given as relative % abundance of mRNA level normalized to TBP
mRNA). The real-time PCRs were performed to quantify the relative
amounts of PtTE transcripts at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h after
nitrogen deprivation. Data was presented as the mean ± SD (n = 3).

Expression of PtTE in E. coli
E. coli strain Rosetta (DE3) was initially chosen for ex-
pressing P. tricornutum PtTE driven by T7 promoter of

pET21b. Recombinant thioesterase (6×His tagged PtTE at
the C-terminus) was remarkably expressed within 3 h
upon 1 M

isopropylthiogalactoside

(IPTG) induction.

However, almost all expressed proteins were inclusion
bodies (Fig. S2). Further optimization of growth tem-
perature and IPTG concentration did not improve the
solubility of PtTE thioesterase in E. coli Rosetta cells.
Nevertheless, we purified this recombinant thioesterase
under denaturing condition and used to raise poly-
clonal antiserum in rabbit.
  To obtain functionally active PtTE thioesterase and
minimize the toxicity of the bacterial cells expressing
P. tricornutum  PtTE, the coding sequence of PtTE was
recloned in frame with the N terminal of lacZ

α-fragment of pUC18 and under the control of lac pro-
moter. The resulting plasmid pOC64 and control pUC18
were transformed into E. coli XL1-Blue (fadD

) and K27

fadD88 mutant, a strain defective in the fatty acid deg-
radation pathway that lacks acyl CoA synthetase. West-
ern blot analysis of IPTG-induced E. coli samples re-
vealed that the expression levels of PtTE were consistent
in all E. coli strains used for GC analysis (Figs. S3 & S4).
The E. coli XL1-Blue strain expressing PtTE showed mod-
erate increase in C16:1 and C18:1 contents  by 19%
(mol/mol) and 16% respectively, as compared to the
control strain (Fig. 2A). We also found that expression
of  PtTE cDNA led to 21% increase in total fatty acid
content of membrane lipids. This is similar to that ex-
pression of plant FatA-type thioesterase also increased
total bacterial fatty acid content [24]. E. coli XL1-Blue
strain has the capacity to incorporate the free fatty
acids available in the medium (released by the action of
thioesterase) into the membrane lipid [20]. In order to
eliminate the impact of fatty acid degradation in XL1-
Blue, the fatty acid profiles were also analyzed through
GC-FID using the FAMEs prepared from cell membrane
and the culture supernatants of the recombinant E. coli
K27 fadD88 clones. When PtTE was expressed in fadD88
mutant (grown at 30 °C) C16:1, C18:0 and C18:1 were
increased in the membrane lipid by ∼72%, 30% and
76%, respectively, along with decrease in other fatty
acids, primarily C17 and C19 derived cyclopropane fatty
acids (Fig. 2B). However, Fig. 2C showed that only a
moderate increase (12%) was observed in C16:1 free
fatty acid content in the medium of PtTE-transformed
fadD88 cells when compared with pUC18-transformed
cells (equivalent number of cells and medium). In addi-
tion, although PtTE activity was unable to change the
total fatty acids content, there were 78% and 16% in-
crease in C18:1 and C18:0 free fatty acid contents in
the medium, respectively. These data indicate that the
P. tricornutum PtTE cDNA expressed in E. coli encodes a

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(A)

(B)

(C)

Figure 2. Changes of fatty acid profiles in the membrane lipid and
culture supernatant of E. coli expressing P. tricornutum thioesterase
PtTE. Gray and black bars represent for E. coli cells harboring
pUC18 and recombinant plasmid with the PtTE gene, respectively.
Individual fatty acids are represented by standard symbol. The
‘others’ include C14 : 0 and low amounts of cyclopropane fatty acids
(C17 and C19 derived). (A) membrane lipid of E.coli fadD

(XL1-

Blue) cells grown at 37

°C with the expression of PtTE driven by lac

promoter; (B) membrane lipid of E. coli K27 fadD88 cells grown at
30

°C with the expression of PtTE driven by lac promoter; (C) re-

lease of free fatty acids (

μg) into the culture medium of E. coli K27

fadD88 cells containing control and recombinant plasmids.

functional acyl-ACP thioesterase with primary activity
on C18:1 and C18:0 fatty acids. The P. tricornutum thio-
esterase PtTE is different from previously reported bac-
terial thioesterases that exhibit broad substrate speci-
ficities ranging from C6 to C18 acyl-ACP or acyl-CoA
derivatives [9, 25]. PtTE thioesterase is more similar to
plants FatA thioesterases, which are preferentially spe-
cific for unsaturated acyl-ACPs with greater specificity
for C18:1-ACP [24]. Moreover, it must be noted that
in the fatty acid profile of P. tricornutum, C16 (83%,
mol/mol) and EPA (9%) are the major fatty acids,
whereas oleic acid (C18:1) and all the intermediates of
the EPA biosynthetic pathway (C18:2, C18:3 and C20
derivatives) take up no more than 5% (Fig. S5). PtTE
thioesterase with relatively low activity towards C18
fatty acid substrates might be responsible for control-
ling the C18 fatty acids in the lipids of P. tricornutum to
low levels, and thus more C18-acyl-ACPs can be used for
EPA synthesis through further elongation and desat-
uration.

Overexpression of PtTE in P. tricornutum
The establishment of P. tricornutum transformation
method makes it possible to implement further charac-
terization of gene function [18]. In order to overexpress
PtTE in P. tricornutum, the construct carrying coding
sequence of PtTE under control of fcpA (encoding the
fucoxanthin-chlorophyll binding protein) promoter was
introduced into P. tricornutum cells by microparticle
bombardment. Surprisingly, overexpression of PtTE
alone did not lead to significant change of fatty acid
composition of P. tricornutum (data not shown), but the
total fatty acids content increased by 72% (from 78 to
133 mg/g dry cell weight) compared with that of control
strain (Table 1). We speculated that besides PtTE, other
genes or factors might also involve controlling the fatty
acid composition of P. tricornutum. As major fatty acids,
C16:0 and C16:1 together accounted for the primary
increase of total fatty acids content. It has been shown
that several enzymes involving fatty acid synthesis are
feedback-inhibited by long-chain fatty acyl-ACPs, and
expression of endogenous acyl-ACP thioesterases can
relieve this inhibitory mechanism [26, 27]. The increase
in total fatty acids content by expressing PtTE in P. tri-
cornutum might be achieved through this mechanism.
What deserves to be mentioned is that overexpression
of endogenous thioesterase has been used in metabolic
engineering toward enhancing fatty acid yields in E. coli
[28]. In genetic engineering of microalgae for biofuel
production, thioesterases are interesting candidates for
overexpression strategies since overexpression of an
endogenous thioesterase from microalgae could en-

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Table 1. Major fatty acid content (mg/g dry cell weight) of
P. tricornutum strains overexpressing the PtTE thioesterase.
Total fatty acids of wild type and three independent trans-
formants were analyzed.

Microalgal strain

Fatty acid group

Wild type

PtTE

C14:0

2.92 ± 0.27

3.92 ± 0.10

C16:0

16.89 ± 0.31

35.93 ± 1.19

C16:1n–7

38.03 ± 0.28

68.22 ± 1.37

C18:0

0.51 ± 0.05

0.92 ± 0.03

C18:1n–9

2.59 ± 0.01

4.18 ± 0.09

C18:2n–6

0.94 ± 0.01

1.12 ± 0.04

C20:5n–3

9.05 ± 0.14

10.57 ± 0.09

C22:6n–3

0.61 ± 0.01

0.52 ± 0.01

Total fatty acids

78.50 ± 0.09

133.50 ± 2.29

hance lipid biosynthesis, and expression of acyl-ACP
thioesterases from other organisms that are specific for
certain fatty acid chain lengths could improve the suit-
ability of microalgae-derived feedstock for biofuel pro-
duction [1].

Acknowledgements

We thank Prof. Peter Kroth, University of Konstanz
(Germany), for kindly providing the P. tricornutum trans-
formation vector pPha-T1. This work was supported
by the Major S&T Projects on the Cultivation of New
Varieties of Genetically Modified Organisms (Grant
2009ZX08009-120B), and the National Natural Science
Foundation of China (Grant No. 30970262, 31000778).

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• Major S&T Projects on the Cultivation of New Varie-
ties of Genetically Modified Organisms; grant number:
2009ZX08009-120B
• National Natural Science Foundation of China; grant
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