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Letter
Delta 5 fatty acid desaturase upregulates the synthesis of polyunsaturated fatty acids in marine diatom Phaeodactylum tricornutum Kun-Tao Peng, Cun-Ni Zheng, Jiao Xue, Xiao-Yan Chen, WeiDong Yang, Jie-Sheng Liu, Weibin Bai, and Hongye-Ye Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5031086 • Publication Date (Web): 11 Aug 2014 Downloaded from http://pubs.acs.org on August 17, 2014
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Journal of Agricultural and Food Chemistry
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Delta 5 Fatty Acid Desaturase Upregulates the Synthesis of
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Polyunsaturated Fatty Acids in the Marine Diatom Phaeodactylum
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tricornutum
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Kun-Tao Peng†, Cun-Ni Zheng†, Jiao Xue†, Xiao-Yan Chen†, Wei-Dong Yang†, Jie-Sheng Liu†, Weibin
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Bai§, Hong-Ye Li*,
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†
†
Key Laboratory of Eutrophication and Red Tide Prevention of Guangdong Higher Education Institutes,
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College of Life Science, Jinan University, Guangzhou 510632, China.
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§
Department of Food Science, Jinan University, Guangzhou 510632, China.
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Corresponding Author
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*(H.L.) E-mail:
[email protected]. Phone: 86-20-85228470. Fax: 86-20-85222720.
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ABSTRACT: Microalgae are important primary producers in the marine ecosystem and excellent
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sources of lipids and other bioactive compounds. The marine diatom Phaeodactylum tricornutum
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accumulates eicosapentaenoic acid (EPA, 20:5n-3) as its major component of fatty acids. To improve the
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EPA production, delta 5 desaturase, which plays a role in EPA biosynthetic pathway, was characterized
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in P. tricornutum. An annotated delta 5 desaturase PtD5b gene was cloned and overexpressed in P.
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tricornutum. The transgene was integrated into the genome demonstrated by Southern blot, and the
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overexpression of PtD5b was verified by qPCR and western blot analysis. Fatty acid composition
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exhibited a significant increase in the unsaturated fatty acids. Monounsaturated fatty acids (MUFA) and
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polyunsaturated fatty acids (PUFA) showed an increase of 75% and 64%, respectively. In particular,
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EPA showed an increase of 58% in engineered microalgae. Meanwhile, neutral lipid content showed an
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increase up to 65% in engineered microalgae. More importantly, engineered cells showed similar growth
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rate with the wild type, thus keeping high biomass productivity. This work provides an effective way to
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improve the production of microalgal value-added bioproducts by metabolic engineering.
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KEYWORDS: Microalga, polyunsaturated fatty acid, desaturase, lipid
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INTRODUCTION
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To meet the increasing demand for PUFA (polyunsaturated fatty acid) consumption from human
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nutrition and animal feed, microalgae, as the main natural producer of PUFAs, have been attracting
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much attention. The marine diatom Phaeodactylum tricornutum, which accumulates eicosapentaenoic
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acid (EPA, 20:5n-3) as its major component of fatty acids, has been tested for large scale production of
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EPA1. The content of PUFA is a critical criterion in industrial production, and an effective way is to
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create ideal strains by genetic modification of the biosynthetic pathway.2 The desaturase and elongase
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were demonstrated by radiolabeled precursors to be involved in long chain PUFA biosynthetic pathways
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in the marine microalga Pavlova lutheri.3 ∆5 desaturase, as a rate-limiting enzyme in the synthesis of
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PUFA, has been studied in some bacteria, fungi and humans. A human ∆5 desaturase was expressed in ACS Paragon Plus Environment
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Chinese hamster ovary cells and enabled the cells to convert 20:3(n-6) to 20:4(n-6).4 Saccharomyces
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Journal of Agricultural and Food Chemistry
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cerevisiae expressing a ∆5 desaturase from Paramecium tetraurelia successfully produced EPA.5
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Though ∆5 desaturase has been studied a lot, however, so far few studies have focused on its role in
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PUFA accumulation in microalgae. The current study attempted to effectively improve PUFA
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accumulation in the diatom by overexpressing endogenous ∆5 desaturase.
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MATERIALS AND METHODS
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Diatom cultures
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Phaeodactylum tricornutum Bohlin (CCMP2561) was used in this study. The diatom was grown in
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f/2-Si medium which was prepared as f/2 medium by omitting Si and sterilized with 0.22-µm filters
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(Millipore, USA). Cultures were grown at (21±0.5°C) in 15h/9h (light/dark). To detect the growth of
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microalgae, cells were counted with a hemacytometer.
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Cloning of PtD5b and generation of transgenic diatoms
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Amino acid sequence homologous with putative PtD5b were searched by BLAST at NCBI
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(http://blast.ncbi.nlm.nih.gov/Blast/)
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(http://www.ebi.ac.uk/Tools/msa/clustalo/). Phylogenetic tree of PtD5b was constructed by using
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software MEGA. Genomic DNA of P. tricornutum was extracted with a HP Plant DNA Kit (Omega,
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USA). The full-length cDNA of ∆5 desaturase gene in P. tricornutum (PtD5b) (GenBank accession:
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XM_002182822.1)
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(5′-TTACAATCCAGTGGTACCATGGACGTGTCGTTACGGA-3′)
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(5′-TTTTGTTCCAGGTGTTCCAATTTGGTAATTGCGTCC-3′). The PtD5b was cloned into plasmid
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pHY18 by infusion ligation and the resultant plasmid was introduced into P. tricornutum by
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electroporation using Bio-Rad GenePulserXcell apparatus (Bio-Rad, USA) according to Niu et al.6
was
and
amplified
aligned
by
PCR
with
using
Clustal
primers: and
Omega
Pt61inf Pt62inf
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Neutral lipid content and fatty acid composition analysis
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Neutral lipid content in diatom cells was analyzed by Nile red staining according to Yang et al. with
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modifications.1 Cell cultures in the stationary phase were first treated with 20% DMSO for 20 min at
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room temperature. Then 30 µL of Nile red (0.1 mg/mL in acetone) was added to a 3 mL aliquot of
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pretreated cell culture in triplicates, mixed by rapid inversion and incubated in darkness for 20 min at
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room temperature. The stained cells were transferred to a 96-well plate for the detection of fluorescence
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intensity with a F4600 microplate reader (Hitachi, Japan). The wavelengths used were: excitation at 530
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nm and emission at 580 nm. The relative fluorescence intensity values provided a quantitative
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comparison of neutral lipid contents between samples. Total lipids of microalgae were extracted and
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analyzed by gas chromatography–mass spectrometry (GC-MS) according to Yang et al.1. To determine
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the content of EPA and DHA at dry cell weight (DCW), 150 µL N-nonadecyl ester (1mg/mL) was added
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as an internal standard to the samples for analysis of fatty acid composition by gas
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chromatography-mass spectrometry (GC-MS). Fatty acid concentration (CFA,mg/g) was calculated by
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comparing the peak area of fatty acid in the sample with the peak area of internal standard (N-nonadecyl
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ester) according to the following equation: CFA=AS/AIS×CIS/WS, where A indicates peak area; C,
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concentration; W, weight; IS, internal standard; S, sample.7 Three parallely grown cultures were used for
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the above assays and results were tested by one-way ANOVA analysis and T-test.
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Detection of PtD5b expression in transgenic microalgae by molecular approaches
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PtD5b mRNA expression was verified by quantitative real-time PCR (qPCR), and relative expression
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levels of PtD5b gene were calculated by normalization against the expression of β-actin. Primers used
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for qPCR were as follows: PTD5b (Forward primer: CATCACGGACCCAATCAATAC, Reverse
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primer:
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AGGCAAAGCGTGGTGTTCTTA,
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the foreign gene integration into the genome, Southern blot was performed with a fragment of CAT to
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prepare a DIG-labelled probe. PtD5b protein expression was detected by western blot analysis using an
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anti-Myc antibody (Invitrogen). GAPDH was used as internal housekeeping control. Algal cell cultures
CGACGGACAATCTGGAAGAC
）
;
(Forward
primer:
Reverse primer: TCTGGGGAGCCTCAGTCAATA）. To verify
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β-actin
in the stationary phase were used in the above assays.
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RESULTS AND DISCUSSION
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Prediction and subcellular localization of ∆5 desaturase
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Phylogenetic tree of retrieved sequences homologous to the annotated P. tricornutum ∆5 desaturase
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(PtD5b) was initially constructed to verify its functional annotation. As shown in Figure 1, PtD5b had a
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close relationship with the other annotated ∆5 fatty acid desaturase (PtD5p) from P. tricornutum and that
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from the marine diatom Thalassiosira pseudonana,8 and they were clustered into one group. PtD5b also
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exhibited a high homology with those ∆5 fatty acid desaturases from protozoa including Trypanosoma
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and Leishmania, and microalgae species including Chlamydomonas and Ostreococcus.
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Proteins are targeted to specific subcellular locations for their proper function.9 According to
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software Target P ver1.1 (http://www.cbs.dtu.dk/services/TargetP/), a signal peptide was predicted in
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PtD5b
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(http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/), the subcellular localization of PtD5b was predicted
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to be the endoplasmic reticulum (ER). The most likely location was also predicted to be the ER
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(possibility of 77.8%) by software PSORT II, followed by the cytoplasm (11.1%) and the plasma
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membrane (11.1%). Taken together, PtD5b could be predicted to target to the ER.
(with
a
possibility
of
0.318).
According
to
software
Cell-PLoc
2.0
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Growth and neutral lipid analysis of transgenic Phaeodactylum tricornutum
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The neutral lipid content was determined by Nile red fluorescence staining and indicated as the relative
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fluorescence intensity. As shown in Figure 2A, a significant increase of 24% in neutral lipid content was
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detected in transgenic line PtD5b-1 compared to the wild type (P
