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Increasing fucoxanthin production in Phaeodactylum tricornutum using genetic engineering and optimization of culture conditions

Catarina Perfeito1, 2, Miguel Ambrósio1, Rita B. Santos1, Clélia N. Afonso2 and Rita Abranches1*
  • 1 Instituto de Tecnologia Química e Biológica (ITQB-NOVA), Portugal
  • 2 Escola Superior de Turismo e Tecnologia do Mar, Politécnico de Leiria, MARE - Marine and Environmental Sciences Centre, Portugal

In the last two decades, the use of plant-based systems for the production of recombinant proteins and high-value natural compounds has opened the path for alternative cost-effective platforms. Recently, our team has been working with plant cell suspension cultures for the production of valuable compounds for human health [1,2]. Although these systems offer many advantages, plant suspension cells are undifferentiated and do not live in their natural environment. On the contrary, photosynthetic microalgae are unicellular eukaryotic organisms that naturally thrive in liquid salt medium, and their maintenance in laboratory conditions is easy and inexpensive. They can be grown, similarly to microorganisms such as bacteria and yeast, as well as plant cell cultures, in controlled and contained environments. Additionally, they have the advantage of growing in autotrophic conditions since they are photosynthetic, making them more sustainable promising platforms for biotechnology. Moreover, strain improvement in these organisms gives them a strong potential to be productive cell factories. Mutagenesis, adaptive laboratory evolution and genetic engineering are established strategies for developing algal cell factories, together with systems biology and synthetic biology approaches [3,4,5]. In this work we are exploring microalgae as platforms for the production of high-value compounds, namely carotenoids, using the model diatom Phaeodactylum tricornutum. Diatoms are unicellular photosynthetic eukaryotes considered as the most important group of eukaryotic phytoplankton, responsible for more than 20% of global carbon fixation and 40% of marine primary productivity [6]. Diatoms have diversified their genome very rapidly along evolution, as they contain genes from both eukaryotes and prokaryotes, due to endosymbiotic processes and extensive horizontal gene transfer. Their enormous plasticity allows for the environmental surroundings to influence their biology and metabolism, which is then reflected in their evolutionary success. They are able of synthesizing and accumulating lipids, carbohydrates and secondary metabolites such as pigments and vitamins, especially when growing under abiotic stress conditions. There is evidence showing they use chromatin mediated regulation of gene expression to adapt to changing environmental conditions, such as light, temperature, or nutrient depletion. These environmental conditions can be manipulated to produce multiple value-added products such as photosynthetic pigments. Fucoxanthin is the main carotenoid produced in brown algae as a component of the light harvesting complex for photosynthesis and photoprotection, and it does not exist in red or green algae. In plants, carotenoids are precursors of the hormone abscisic acid, and also of the recently identified hormone strigolactone. However, in brown algae, the fucoxanthin biosynthetic pathway is not yet fully understood, especially the last steps of the pathway. The remarkable biological properties of this pigment are based on its unique molecular structure, responsible for the high antioxidant activity [7], and many of its biological effects are related to the ability to scavenge reactive oxygen species. This is the basis for the high commercial value of this pigment as a potential health promoter. Fucoxanthin has attracted significant interest not only due to its antioxidant activity, but also because of anticancer, anti-inflammatory and anti-obesity effects, among other useful properties [8]. Animals do not have pathways for de novo synthesis of carotenoids, so they obtain them from food and further modify them. Phaeodactylum tricornutum is a potential commercial source of fucoxanthin, since it contains at least ten times more fucoxanthin per gram of dry weight than brown algae [8]. Our goal is to combine microalgae engineering with physiological approaches to enhance the production of fucoxanthin in the brown microalgae Phaeodactylum tricornutum. To this end, we are engineering P. tricornutum to overexpress the gene encoding lycopene B cyclase (LCYB), a key enzyme in the biosynthetic pathway of this pigment, under the control of the fcpB promoter. P. tricornutum strain CCAP1055/1 was used in this study. The LCYB sequence was amplified from cDNA synthesized from total RNA, and cloned into plasmid pPha-T1 [9]. P. tricornutum transformation was carried out using a simple electroporation protocol described by Zhang and colleagues [10]. For the analysis of growth conditions effects on fucoxanthin production, the basal medium F/2 with silica was modified by adding sodium nitrate or glycerol in different concentrations. Growth was monitored for 8 days and compared with the basal medium conditions. Quantitative analysis of fucoxanthin was carried out using high performance liquid chromatography (HPLC) with a C18 reverse phase column. The mobile phase consisted of acetonitrile and water with a flow rate of 0.5 ml/min. Fucoxanthin standards and samples were detected at 445 nm. Our results show that the amount of fucoxanthin doubles when supplementing the basal medium with 10 fold concentration of sodium nitrate, at day 4 of the growth curve. This increase is also observed when analyzing the amount of fucoxanthin produced per cell. On the contrary, supplementation with glycerol did not lead to a significant effect on fucoxanthin production. Cultivation conditions of both wild type and genetically engineered strains will be optimized for maximum production. Since epigenomic reprogramming is involved in the morphological and physiological changes of P. tricornutum in response to environmental stimuli, epigenetic mechanisms which may influence the production levels of fucoxanthin will also be investigated in order to further optimize fucoxanthin yields. With this work, we aim at optimizing fucoxanthin production in Phaeodactylum tricornutum, so that it will become a commercially sustainable source of this high valuable compound.

Acknowledgements

This work was supported by Portuguese national funds through Fundação para a Ciência e a Tecnologia (FCT, Portugal) grant Bioresources 4 Sustainability UID/Multi/04551/2013.

References

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[2] Santos, R.B., Chandra, B., Mandal, M.K., Kaschani, F., Kaiser, M., Both, L., van der Hoorn, R.A.L., Schiermeyer, A., Abranches, R. (2018). Low protease content in Medicago truncatula cell cultures facilitates recombinant protein production. Biotechnology Journal. doi: 10.1002/biot.201800050. [Epub ahead of print] 

[3] Daboussi, F., Leduc, S., Maréchal, A., Dubois, G., Guyot, V., Perez-Michaut, C., Amato, A., Falciatore, A., Juillerat, A., Beurdeley, M., Voytas, D.F., Cavarec, L., Duchateau, P. (2014). Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology. Nat Commun. 5:3831. 

[4] Weyman, P.D., Beeri, K., Lefebvre, S.C., Rivera, J., McCarthy, J.K., Heuberger, A.L., Peers, G., Allen, A.E., Dupont, C.L. (2015). Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis. Plant Biotechnol J. 13, 460-470. 

[5] Nymark, M., Sharma, A.K., Sparstad, T., Bones, A.M., Winge, P. (2016). A CRISPR/Cas9 system adapted for gene editing in marine algae. Scientific Reports 6:24951. 

[6] Falciatore, A. and Bowler, C. (2002). Revealing the Molecular Secrets of Marine Diatoms. Annual Review of Plant Biology 53, 109-130. 

[7] Sachindra, N.M., Sato, E., Maeda, H., Hosokawa, M., Niwano, Y., Kohno, M., Miyashita, K. (2017). Radical scavenging and singlet oxygen quenching activity of marine carotenoid fucoxanthin and its metabolites. Journal of Agricultural and Food Chemistry 55, 8516–8522. 

[8] Stonik, V. and Stonik, I. (2015). Low-Molecular-Weight Metabolites from Diatoms: Structures, Biological Roles and Biosynthesis. Marine Drugs 13, 3672-3709. 

[9] Zaslavskaia, L.A. and Lippmeier, C.J. (2000). Transformation of the diatom Phaeodactylum tricornutum (bacillariophyceae) with a variety of selectable marker and reporter genes. Journal of Phycology 36, 379–386. 

[10] Zhang, C., Hu, H. (2014). High-efficiency nuclear transformation of the diatom Phaeodactylum tricornutum by electroporation. Marine Genomics 16, 63-66.

Keywords: Microalgae, Genetic Engineering, Culture conditions, phaeodactylum tricornutum, fucoxanthin

Conference: IMMR'18 | International Meeting on Marine Research 2018, Peniche, Portugal, 5 Jul - 6 Jul, 2018.

Presentation Type: Poster Presentation

Topic: Blue Biotech

Citation: Perfeito C, Ambrósio M, Santos RB, Afonso CN and Abranches R (2019). Increasing fucoxanthin production in Phaeodactylum tricornutum using genetic engineering and optimization of culture conditions. Front. Mar. Sci. Conference Abstract: IMMR'18 | International Meeting on Marine Research 2018. doi: 10.3389/conf.FMARS.2018.06.00082

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Received: 24 Apr 2018; Published Online: 07 Jan 2019.

* Correspondence: Dr. Rita Abranches, Instituto de Tecnologia Química e Biológica (ITQB-NOVA), Oeiras, Portugal, ritaa@itqb.unl.pt