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Deliverable D7.1 Transgenic lines with traits of interest available to the blue biotech industry Date: 25/05/2017 HORIZON 2020 - INFRADEV Implementation and operation of cross-cutting services and solutions for clusters of ESFRI H2020-GRANT NO 654008 Grant Agreement number: 654008 Project acronym: EMBRIC Contract start date: 01/06/2015 Project website address: www.embric.eu Due date of deliverable: 31/05/2017 / month 24 Dissemination level: Public EMBRIC – D7.1: Transgenic lines with traits of interest available to the blue biotech industry, page 2 of 16 H2020-GRANT NO 654008 Document properties Partner responsible UPMC Author(s)/editor(s) Angela Falciatore, François-Yves Bouget, Mariella Ferrante Version 1 Abstract Microalgae are emerging as potentially significant, renewable and sustainable source of biomass for food, energy and other natural compounds that could be beneficial for human health and wellness. Task 7.2 is focused on the amelioration of microalgal strains using genetic engineering. For these studies, we selected three microalgae that can be used as model systems in the laboratory because they are easy to grow and because of the availability of genome-enabled molecular tools. These resources are providing foundations for the development of metabolically engineered strains. In particular, we used the green alga Ostreococcus tauri to engineer metabolic pathways involved in the synthesis of carotenoids and lipid derivatives. For this purpose, we have used gene knock-out, knock-in by homologous recombination or overexpression. We also used the diatom Phaeodactylum tricornutum, the established diatom species for molecular and cellular studies (e.g., genetic transformation, gene silencing, genome editing tools). Finally, we used another diatom belonging to the Pseudo-nitzschia genus which has specific metabolic pathways that can be a target for the regulation of cell growth and therefore, indirectly, of biomass production. EMBRIC – D7.1: Transgenic lines with traits of interest available to the blue biotech industry, page 3 of 16 H2020-GRANT NO 654008 Table of Contents 1. Introduction...........................................................................................................................5 2. MetabolicEngineeringApproaches......................................................................................7 3. Identificationandcharacterizationofdiatomstrainswithbiotechnologicalapplications..9 4. Moleculartoolsfornon-modelspecies..............................................................................11 5. Conclusion...........................................................................................................................13 6. References...........................................................................................................................14 EMBRIC – D7.1: Transgenic lines with traits of interest available to the blue biotech industry, page 4 of 16 H2020-GRANT NO 654008 1. Introduction Task 7.2, Genomics resources and toolboxes for strain improvement, is led by the SZN (Stazione Zoologica Anton Dohrn of Naples) and includes the participation of UPMC (Université Pierre et Marie Curie, Jussieu and Banyuls-sur-Mer laboratories), MBA (Marine Biological Association), and UGENT (University of Gent). In this task pilot studies are being conducted on selected model species for which post-genomic tools and -omics information are available (the model chlorophyte Ostreococcus tauri, the diatoms Phaeodactylum tricornutum, Seminavis robusta and Pseudo-nitzschia multistriata, and the coccolithophore Emiliania huxleyi) to demonstrate the feasibility and interest of producing lines over-expressing molecules of interest, knockout/silenced strains to minimize production of compounds that hinder the extraction process/decrease yield, and sterile strains (to avoid uncontrolled sexual reproduction). In this deliverable, a report on the metabolic engineering that was deployed through reverse genetic approaches such as genetic targeting by homologous recombination as well as RNA interference or overexpression technologies in O. tauri and P. tricornutum, (UPMC) is presented. Genetic transformation of species of the genus Pseudo-nitzschia is also included (SZN). Moreover, examples of protocols developed for the screening of mutant collections that allow identification of strains of interest (flow cytometry cell sorting, photochemical quantum yield, PAM technology) are also explained. Partners in this task have shared tools and techniques, such as screening assays. As a result of the scientific discussion and of the exchanges among partners in Task 1 of this work package, it appeared useful to involve UNS (University of Nice) in the development of a fast and reliable method to screen for carotenoids, given the relevant expertise in suitable analytical techniques. Efforts were conducted to set up transformation protocols for the marine benthic diatom Seminavis robusta. Although successful, transformation efficiency and reproducibility EMBRIC – D7.1: Transgenic lines with traits of interest available to the blue biotech industry, page 5 of 16 H2020-GRANT NO 654008 still need to be significantly improved. New and modified protocols including methods for microalgal transformation recently reported in literature are currently being tested. EMBRIC – D7.1: Transgenic lines with traits of interest available to the blue biotech industry, page 6 of 16 H2020-GRANT NO 654008 2. Metabolic Engineering Approaches UPMC- Banyuls-sur-Mer (LOMIC, Laboratoire d'océanographie microbienne) aims to identify, using metabolic engineering approaches, new strains of the picoeukaryote Ostreococcus tauri of interest for blue biotechnologies. LOMIC has established industrial partnerships (Microphyt SME) who managed to grow Ostreococcus tauri in 5000 liters industrial photobioreactors. Ostreococcus tauri contains high-added value compounds of potential interest for biotechnologies such as polyunsaturated fatty acids, carotenoids or exo-polysaccharides. We use both forward genetic approaches by phenotyping a 10 000 insertion mutant collection as well as reverse genetic approaches including knock-out or knock-in of genes of interest by homologous recombination, or overexpression of heterologous proteins to overexpress or modify endogenous metabolites of interest. We use imaging PAM technology (Pulse Amplitude Modulated fluorometry) to screen candidate lines and, in collaboration with Mohamed Mehiri (UNS), we have implemented a low-cost approach for profiling Ostreococcus pigments based on TLC (Thin Layer Chromatography). β-carotene Chlorophyll a Chlorophyll b Prasinoxanthin Violaxanthin Neoxanthin System solvents Xanthophylls β-carotene/ chlorophylls Methanol/CH Cl3 (50/50) - - Methanol/CH Cl3 (5/95) ++ - Diethyl ether/petroleum ether (50/50) - ++ Petroleum ether 100% - + +++ - Mix Ethyl acetate/ cyclohexane (70/30) O. tauri extracts (5 to 30µL) Figure 1: Thin Layer Chromatography, a resolutive method for quick and cheap profiling of carotenoids in mutants. EMBRIC – D7.1: Transgenic lines with traits of interest available to the blue biotech industry, page 7 of 16 H2020-GRANT NO 654008 Using a reverse genetic approach on a key enzyme of the alpha/beta carotenoid biosynthesis pathway (knock-out of specific domain combined with knock-in of a strong promoter to overexpress another domain of interest), we have obtained lines with modified carotenoid contents, hence of potential interest for blue biotech. We have also generated lines with modified carotenoid contents by knocking out a sensor kinase involved in blue light sensing. This line displays lower chlorophyll and carotenoid contents (Figure 2). Although this line has no interest for blue biotech, this approach demonstrates the capacity of modifying carotenoid contents using genetic engineering approaches (Figure 2). B counts A wt lov-hk Figure 2: An Ostreococcus tauri knock-out (KO) line in the blue light photoreceptor LOV histidine kinase with altered pigments content. A. Two cultures of Wild type and KO LOV-HK line at the same cellular density. B. Red fluorescence of Wt and KO LOV-HK line. C and D. Carotenoid composition under low light (LL) and high light (HL) in Wt and KO LOV-HK cells. EMBRIC – D7.1: Transgenic lines with traits of interest available to the blue biotech industry, page 8 of 16 H2020-GRANT NO 654008 3. Identification and characterization of diatom strains with biotechnological applications UPMC-Paris (Jussieu) aims to identify new diatom strains showing novel traits of interest (e.g., altered growth, pigments and biomass productivity). To this aim, UPMC is characterizing an important number of P. tricornutum strains genetically modified by a reverse genetic approach. In particular, the analyses are performed on the collection of Transcription Factors (TFs) mutants that the Falciatore’s team is generating by RNA interference in P. tricornutum (De Riso et al., 2009). TFs are master regulators of cellular processes and have been shown to be excellent candidates both for fundamental research approaches (global understanding of biology) and for applied research (modification of complex traits) (Century et al., 2008; Rabara et al., 2014). In the context of the Task 7.2, UPMC-Paris has set up conditions for the screening of the P. tricornutum TF RNAi collection to identify strains showing altered photosynthesis. Photosynthesis is a key process for life on Earth, but also a crucial target for metabolic engineering of algae. In these last years, routes to improve photosynthesis performance and the production of bio-derived compounds from plants and algae have been identified, by improving light capture and antenna composition, optimizing C fixation or decreasing photosynthesis feedback inhibition (Zhu et al., 2010; Wobbe et al., 2016; Kromdijk et al., 2016). High photosynthetic efficiency is also crucial for a viable large-scale cultivation of microalgae, normally cultivated in photobioreactors or outdoor ponds (Stephens et al., 2010; Simionato et al., 2013). In collaboration with the laboratory of Prof. Tomas Morosinotto (University of Padova, IT), UPMC-Paris has used a systematic non-invasive fluorescence screening procedure previously developed to screen collection of ≈ 12000 random mutants from Nannochloropsis gaditana (Perin et al., 2015). In particular, different tests have been done to optimize P. tricornutum screening conditions and to limit numbers of false positive. Finally, strong and reproducible phenotypes have been obtained on cells grown on plates (Figure 3). Some chlorophyll fluorescence-based parameters (Maxwell et al., 2000) have been identified as the most EMBRIC – D7.1: Transgenic lines with traits of interest available to the blue biotech industry, page 9 of 16 H2020-GRANT NO 654008 informative to identify modifications of the photosynthetic apparatus: the photochemical quantum yield (ФPSII, which estimates the proportion of absorbed photons used for photosynthesis), the NPQ (which estimates ability to activate photoprotection mechanisms) and fluorescence intensity/colony area that allows identifying strains with altered pigmentation and/or growth. By using this approach, few independent transgenic lines with a TF down-regulated expression and showing altered fluorescence levels have been identified (Figures 3 and 4). Figure 3: Example of screen of RNAi lines. P. tricornutum wild type (top) and various RNAi TF strains (bottom), spotted on plate at a same cellular concentration (Optical Density= 0,2) are shown. Measures of fluorescence intensity (F0/colony area) (right), recorded after 10 days, allows identifying strains with altered pigmentation and/or growth to be retained for further investigation. Fluorescence signals are detected with a video-imaging apparatus available at UPMC-Paris. Figure 4: Evaluation of the maximum quantum yield of PSII in the wild type, transgenic control line (ZEO), and 4 independent KD TF lines. These modifications can derive from alteration in pigmentation due to alterations of photosynthetic apparatus and/or altered strain growth. Other strains were instead found to have altered PSII quantum yield, indicating an alteration of photosynthetic apparatus. Molecular and biochemical characterization of these lines is in progress. New products or the improved content of specific metabolites will be potentially identified by in-depth metabolic analyses in the framework of the EMBRIC consortium. EMBRIC – D7.1: Transgenic lines with traits of interest available to the blue biotech industry, page 10 of 16 H2020-GRANT NO 654008 4. Molecular tools for non-model species SZN aims at developing molecular tools to allow genetic manipulation in non-model diatoms, using mostly species of the genus Pseudo-nitzschia. This will boost exploitation of novel species that show interesting properties. We recently developed genetic transformation for Pseudo-nitzschia (Sabatino et al., 2015), enabling in this way several approaches including gene silencing via RNAi. With this method, we targeted a key gene in the production of oxylipins, important lipid derivatives that have been proposed as mediators of bloom termination (Vardi et al., 2006). Oxylipins are not produced in the traditional diatom model P. tricornutum whereas Pseudo-nitzschia species have been shown to possess a rich oxylipin metabolism (Lamari et al., 2014). We reasoned that, if oxylipins mediate bloom termination, downregulation of the pathway should affect growth rates, possibly leading to a delay in reaching the stationary phase, resulting in increased biomass. We used the available Pseudonitzschia arenysensis transcriptome (Keeling et al., 2014) to identify one of the key enzymes involved in oxylipin production, the lipoxygenase (LOX). LOXs are a large family of non-heme iron-containing dioxygenases that catalyze the insertion of molecular oxygen into the (1Z,4Z)- pentadiene system of polyunsaturated fatty acids (PUFAs) to produce the corresponding dienyl hydroperoxides (Brash 1999). LOX proteins play important roles in lipid peroxidation under biotic and abiotic stress, and in plants are required during different developmental stages (Siedow 1991; Kolomiets et al. 2001). Some of them were shown to be involved in plant defence reactions such as a pathogen infection (Gomi et al. 2002) or wounding (Kim et al. 2003). We targeted the 3’ region of the P. arenysensis LOX to obtain silenced strains using RNAi (De Riso et al., 2009), and characterised the silenced strain properties in collaboration with the team of Dr Angelo Fontana (CNR, Italy), expert in the chemistry of natural products. Experimental evidence showed that RNAi was effective in reducing LOX levels: EMBRIC – D7.1: Transgenic lines with traits of interest available to the blue biotech industry, page 11 of 16 H2020-GRANT NO 654008 Western blot showed a reduction of the protein, the Fox assay (Orefice et al., 2015) a reduction of the enzyme activity and LC/MS a reduction of the products (data not shown and Figure 5). Strikingly, the silenced diatom cells revealed a reduced growth compared to wild type cells and showed a phenotype with a marked photoinhibition, confirming a regulatory role of LOX pathways in diatoms but linking oxylipins to healthy growth rather than to growth inhibition. Further manipulations of the pathway, in this and in other oxylipin producing species, will be exploited as a strategy to control growth. Figure 5: Reduction of oxylipins (HEpETE and HEPE) in an interfered sample (red) compared to the wild type (blue) measured by LC/MS analyses. EMBRIC – D7.1: Transgenic lines with traits of interest available to the blue biotech industry, page 12 of 16 H2020-GRANT NO 654008 5. Conclusion The work conducted in WP7 task 7.1 led to the generation and preliminary characterization of several valuable microalgal transgenic strains. These lines have been cryopreserved and access to them can be discussed contacting Dr FrançoisYves Bouget at UPMC LOMIC ([email protected]) or Dr Angela Falciatore at UPMC Jussieu Paris ([email protected]). Selected strains with encouraging properties (such as putative higher or more varied content in antioxidants) will be included in the pipeline devised in Task 7.1 for metabolic profiling and possible isolation of pure compounds from microalgal strains. Access to the toolboxes and methods devised will be granted through the Transnational Access program (see WP10, [email protected]). EMBRIC – D7.1: Transgenic lines with traits of interest available to the blue biotech industry, page 13 of 16 H2020-GRANT NO 654008 6. References Brash, A. R. Lipoxygenases: Occurrence, Functions, Catalysis, and Acquisition of Substrate. The Journal of Biological Chemistry, 274(34), 23679–23682 (1999). Century, K., Reuber, T. L. & Ratcliffe, O. J. Regulating the Regulators: The Future Prospects for Transcription-Factor-Based Agricultural Biotechnology Products. Plant Physiology 147, 20–29 (2008). De Riso, V., Raniello, R., Maumus, F., Rogato, A., Bowler, C. & Falciatore, A. Gene silencing in the marine diatom Phaeodactylum tricornutum. Nucleic Acids Research 37, e96–e96 (2009). Gomi, T., Sidle, R.C. & Richardson, J.S. Headwater and channel network: understanding processes and downstream linkages of headwater systems. BioScience 52 (10): 905-916 (2002). Keeling, P.J., Burki, F., Wilcox, H.M., Allam, B., Allen, E.E., Amaral-Zettler, L.A., Armbrust, E.V., Archibald, J.M., Bharti, A.K., Bell, C.J., Beszteri, B., Bidle, K.D., Cameron, C.T., Campbell, L., Caron, D.A., Cattolico, R.A., Collier, J.L., Coyne, K., Davy, S.K., Deschamps, P., Dyhrman, S.T., Edvardsen, B., Gates, R.D., Gobler, C.J., Greenwood, S.J., Guida, S.M., Jacobi, J.L., Jakobsen, K.S., James, E.R., Jenkins, B., John, U., Johnson, M.D., Juhl, A.R., Kamp, A., Katz, L.A., Kiene, R., Kudryavtsev, A., Leander, B.S., Lin, S., Lovejoy, C., Lynn, D., Marchetti, A., McManus, G., Nedelcu, A.M., Menden-Deuer, S., Miceli, C., Mock, T., Montresor, M., Moran, M.A., Murray, S., Nadathur, G, Nagai, S, Ngam, PB, Palenik, B, Pawlowski, J, Petroni, G, Piganeau, G, Posewitz, MC, Rengefors, K., Romano, G., Rumpho, M.E., Rynearson T., Schilling, K.B., Schroeder, D.C., Simpson, A.G.B., Slamovits, C.H., Smith, D.R., Smith, G.J., Smith, S.R., Sosik, H.M., Stief, P., Theriot, E., Twary, S.N., Umale, P.E., Vaulot, D., Wawrik, B., Wheeler, G.L., Wilson, W.H., Xu, Y., Zingone, A. & Worden, A.Z. The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): Illuminating the functional diversity EMBRIC – D7.1: Transgenic lines with traits of interest available to the blue biotech industry, page 14 of 16 H2020-GRANT NO 654008 of eukaryotic life in the oceans through transcriptome sequencing. PLoS Biol 12(6): e1001889 (2014). Kim, E-S., Choi, E., Kim, Y., Cho, K., Lee, A., Shim, J., Rakwal, R., Agrawal, G.K. & Han, O. Dual positional specificity and expression of nontraditional lipoxygenase induced by wounding and methyl jasmonate in maize seedlings. Plant Mol Biol 52: 1203–1213 (2003). Kolomiets, M.V., Hannapel, D.J., Chen, H., Tymeson, M. & Gladon, R.J. Lipoxygenase is involved in the control of potato tuber development. Plant Cell 13:613–626 (2001). Kromdijk, J., Głowacka, K., Leonelli, L., Gabilly, S. T., Iwai, M., NiyogI, K. K. & Long S. P. Improving photosynthesis and crop productivity by accelerating recovery from photoprotection. Science 354, 857–861 (2016). Lamari, N., Ruggiero, M. V., d’Ippolito, G., Kooistra, W. H. C. F., Fontana, A., & Montresor, M. Specificity of Lipoxygenase Pathways Supports Species Delineation in the Marine Diatom Genus Pseudo-nitzschia. PLoS ONE, 8(8), 1–10 (2013). Maxwell, K. & Johnson, G. N. Chlorophyll fluorescence--a practical guide. J. Exp. Bot. 51, 659–668 (2000). Orefice, I., Gerecht, A., d'ippolito, G., Fontana, A., Ianora, A. & Romano, G. Determination of lipid hydroperoxides in marine diatoms by the FOX2. Assay. Mar. Drugs 13, 5767–5783 (2015). Perin, G., Bellan, A., Segalla, A., Meneghesso, A., Alboresi, A. & Morosinotto, T. Generation of random mutants to improve light-use efficiency of Nannochloropsis gaditana cultures for biofuel production. Biotechnol Biofuels 8, 161 (2015). Rabara, R. C., Tripathi, P. & Rushton, P. J. The Potential of Transcription Factor-Based Genetic Engineering in Improving Crop Tolerance to Drought. OMICS: A Journal of Integrative Biology 18, 601–614 (2014). EMBRIC – D7.1: Transgenic lines with traits of interest available to the blue biotech industry, page 15 of 16 H2020-GRANT NO 654008 Sabatino, V., Russo, M.T., Patil, S., d’Ippolito, G., Fontana, A. & Ferrante M.I. Establishment of Genetic Transformation in the Sexually Reproducing Diatoms Pseudo-nitzschia multistriata and Pseudo-nitzschia arenysensis and Inheritance of the Transgene. Mar Biotechnol 17: 452 (2015). Siedow, J.N. Plant lipoxygenase: structure and function. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:145–188 (1991). Simionato, D., Basso, S., Giacometti, G. M. & Morosinotto, T. Optimization of light use efficiency for biofuel production in algae. Biophys. Chem. 182, 71–78 (2013). Stephens, E., Ross, I. L., King, Z., Mussgnug, J. H., Kruse, O., Posten, C., Borowitzka, M. A. & Hankamer, B. An economic and technical evaluation of microalgal biofuels. Nat. Biotechnol. 28, 126–128 (2010). Vardi, A., Formiggini, F., Casotti, R., De Martino, A., Ribalet, F., Miralto, A. & Bowler, C. A stress surveillance system based on calcium and nitric Oxide in marine diatoms. PLoS Biology, 4(3), 0411–0419 (2006). Wobbe, L., Bassi, R. & Kruse, O. Multi-Level Light Capture Control in Plants and Green Algae. Trends in Plant Science 21, 55–68 (2016). Zhu, X. G., Long, S. P. & Ort, D. R. Improving Photosynthetic Efficiency for Greater Yield. Annual Review of Plant Biology 61, 235–261 (2010) EMBRIC – D7.1: Transgenic lines with traits of interest available to the blue biotech industry, page 16 of 16