Development of a new constitutive expression system for the transformation of the diatom Phaeodactylum tricornutum

Seo, Seungbeom; Jeon, Hancheol; Hwang, Seongbin; Jin, EonSeon; Chang, Kwang Suk

doi:10.1016/j.algal.2015.05.012

Cited by 59 publications

(62 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…To enhance expression of single transgenes in Nannochloropsis species, engineers have used several endogenous promoters including the lipid droplet surface protein (LDSP) (Kaye et al ., ; Vieler et al ., ), β‐tubulin, heat‐shock protein 70 and ubiquitin extension promoters (UEP) (Kang et al ., ; Radakovits et al ., ). We have tested an elongation factor promoter that displays high and stable expression throughout light:dark cycles (Poliner et al ., ), and has been utilized in diverse organisms (Gill et al ., ; Kim et al ., ), including diatoms (Seo et al ., ). A variety of promoters allow gene expression at different levels and in response to different environmental conditions.…”

Section: Discussionmentioning

confidence: 97%

A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long‐chain polyunsaturated fatty acid production

Poliner

Pulman

Zienkiewicz

et al. 2017

Plant Biotechnology Journal

125

105

View full text Add to dashboard Cite

Summary Nannochloropsis oceanica is an oleaginous microalga rich in ω3 long‐chain polyunsaturated fatty acids (LC‐PUFAs) content, in the form of eicosapentaenoic acid (EPA). We identified the enzymes involved in LC‐PUFA biosynthesis in N. oceanica CCMP1779 and generated multigene expression vectors aiming at increasing LC‐PUFA content in vivo. We isolated the cDNAs encoding four fatty acid desaturases (FAD) and determined their function by heterologous expression in S. cerevisiae. To increase the expression of multiple fatty acid desaturases in N. oceanica CCMP1779, we developed a genetic engineering toolkit that includes an endogenous bidirectional promoter and optimized peptide bond skipping 2A peptides. The toolkit also includes multiple epitopes for tagged fusion protein production and two antibiotic resistance genes. We applied this toolkit, towards building a gene stacking system for N. oceanica that consists of two vector series, pNOC‐OX and pNOC‐stacked. These tools for genetic engineering were employed to test the effects of the overproduction of one, two or three desaturase‐encoding cDNAs in N. oceanica CCMP1779 and prove the feasibility of gene stacking in this genetically tractable oleaginous microalga. All FAD overexpressing lines had considerable increases in the proportion of LC‐PUFAs, with the overexpression of Δ12 and Δ5 FAD encoding sequences leading to an increase in the final ω3 product, EPA.

show abstract

Section: Discussionmentioning

confidence: 97%

A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long‐chain polyunsaturated fatty acid production

Poliner

Pulman

Zienkiewicz

et al. 2017

Plant Biotechnology Journal

125

105

View full text Add to dashboard Cite

show abstract

“…However, fcp promoters may not be strong enough to overexpress introduced activities due to the presence of lightresponsive cis-regulatory elements [29] and stability under low nutrient conditions. Seo et al, showed that the endogenous elongation factor 2 (EF2) promoter isolated from P. tricornutum drove the expression of a transgene 1.2-fold stronger than that driven by the fcp promoter in light conditions and was stable throughout light and dark cycles [30]. However, recently we have shown that the expression of the Δ5-elongase (OtElo5) gene with an EF2 promoter resulted in comparable levels of EPA and DHA to that in transgenic strains in which the OtElo5 gene was under control of the fcpA promoter [26].…”

Section: Cell Growth and Transgene Expression: The Impact Of Ptdgat2bmentioning

confidence: 99%

Overexpression of an endogenous type 2 diacylglycerol acyltransferase in the marine diatom Phaeodactylum tricornutum enhances lipid production and omega-3 Long Chain Polyunsaturated Fatty Acid content

Haslam

Hamilton

Economou

et al. 2020

Preprint

View full text Add to dashboard Cite

Background: Oleaginous microalgae represent a valuable resource for the production of high value molecules. Considering the importance of omega-3 long chain polyunsaturated fatty acids (LC-PUFAs) for human health and nutrition the yields of high value eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) require significant improvement to meet demand, however, the current cost of production remains high. A promising approach is to metabolically engineer strains with enhanced levels of triacylglycerols (TAGs) enriched in EPA and DHA.Results: Recently we have engineered the marine diatom Phaeodactylum tricornutum to accumulate enhanced levels of DHA in TAG. To further improve the incorporation of omega-3 LC-PUFAs in TAG we focused our effort on the identification of a type 2 acyl-CoA:diacylglycerol acyltransferase (DGAT) capable of improving lipid production and the incorporation of DHA in TAG. DGAT is a key enzyme in lipid synthesis. Following a diatom based in vivo screen of candidate DGATs, a native P. tricornutum DGAT2B was taken forward for detailed characterisation. Overexpression of the endogenous P. tricornutum DGAT2B was confirmed by qRT-PCR and the transgenic strain grew successfully in comparison to wildtype. PtDGAT2B has broad substrate specificity with preferences for C16 and LC-PUFAs acyl groups. Moreover, the overexpression of an endogenous DGAT2B resulted in higher lipid yields and enhanced levels of DHA in TAG. Furthermore, a combined overexpression of the endogenous DGAT2B and ectopic expression of a Δ5-elongase showed how iterative metabolic engineering can be used to increase DHA and TAG content, irrespective of nitrogen treatment.Conclusion: This study provides further insight into lipid metabolism in P. tricornutum and suggests a metabolic engineering approach for the efficient production of EPA and DHA in microalgae.

show abstract

“…We previously constructed the pPhatT-EF2 expression vector harboring the elongation factor 2 (EF2) promoter from P. tricornutum for the generation of an overexpressing transgenic target gene [21]. The EF2 promoter triggers constitutive gene expression regardless of light/dark cycles [21]. To construct a transgenic transformant that causes continuous PTP gene overexpression using the EF2 promoter, a transformation vector (pPhaT-EF2-PtPTP) capable of expressing an exogenous PtPTP coding sequence, PtPTP::EYFP (enhanced yellow uorescence protein, EYFP, is fused to the C-terminus of PtPTP) or EYFP were constructed between the EF2 promoter and FcpA terminator of the pPhaT-EF vector (Fig.…”

Section: Subcellular Localization Of Ptptp and Generation Of Transformentioning

confidence: 99%

“…P. tricornutum is cultivated photoautotrophically and accumulates a high content of lipids [17]. Its whole-genome and transcriptome data are readily available [18,19], and methods for its transformation and various molecular tools for its analysis have been developed [20][21][22]. Recently genome editing tools have been established for P. tricornutum to modify speci c target genes with transcription activator-like effector nucleases (TALENs) and CRISPR/Cas9 [23,24].…”

Section: Introductionmentioning

confidence: 99%

Enhanced pyruvate metabolism in plastids by overexpression of putative plastidial pyruvate transporter in Phaeodactylum tricornutum

Seo

Kim

Lee

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Background: The development of microalgal strains for enhanced biomass and biofuel production has received increased attention. Moreover, strain development via metabolic engineering for commercial production is being considered as the most efficient strategy. Pyruvate is an essential metabolite in the cells and plays an essential role in amino acid biosynthesis and de novo fatty acid biosynthesis in plastids. Although pyruvate can be a valuable target for metabolic engineering, its transporters have rarely been studied in microalgae. In this study, we aimed to identify the plastidial pyruvate transporter of Phaeodactylum tricornutum and utilize it for strain development.Results: We identified putative pyruvate transporter localized in the plastid membrane of Phaeodactylum tricornutum. Transformants overexpressing the pyruvate transporter were generated to increase the influx of pyruvate into plastids. Overexpression of a plastidial pyruvate transporter in P. tricornutum resulted in enhanced biomass (13.6% to 21.9%), lipid contents (11% to 30%), and growth (3.3% to 8.0%) compared to those of wild type during one-stage cultivation. Conclusion: To regulate the pyruvate influx and its metabolism in plastids, we generated transformants overexpressing the putative plastidial pyruvate transporter in P. tricornutum. They showed that its overexpression for compartmentalizing pyruvate in plastids could be an attractive strategy for the effective production of biomass and lipids with better growth, via enhanced pyruvate metabolism in plastids.

show abstract

Development of a new constitutive expression system for the transformation of the diatom Phaeodactylum tricornutum

Cited by 59 publications

References 18 publications

A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long‐chain polyunsaturated fatty acid production

A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long‐chain polyunsaturated fatty acid production

Overexpression of an endogenous type 2 diacylglycerol acyltransferase in the marine diatom Phaeodactylum tricornutum enhances lipid production and omega-3 Long Chain Polyunsaturated Fatty Acid content

Enhanced pyruvate metabolism in plastids by overexpression of putative plastidial pyruvate transporter in Phaeodactylum tricornutum

Contact Info

Product

Resources

About