deliverable d7.1 transgenic lines with traits of interest ... d7.1 transgenic lines with traits of...

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

EMBRIC – D7.1: Transgenic lines with traits of interest available to the blue biotech industry,

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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

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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.

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Table of Contents 1. Introduction...........................................................................................................................5

2. MetabolicEngineeringApproaches......................................................................................7

3. Identificationandcharacterizationofdiatomstrainswithbiotechnologicalapplications..9

4. Moleculartoolsfornon-modelspecies..............................................................................11

5. Conclusion...........................................................................................................................13

6. References...........................................................................................................................14

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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

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still need to be significantly improved. New and modified protocols including methods for microalgal transformation recently reported in literature are currently being tested.

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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).

Figure 1: Thin Layer Chromatography, a resolutive method for quick and cheap

profiling of carotenoids in mutants.

Mix

O. tauri extracts (5 to 30µL)

β-caroteneChlorophyll aChlorophyll b

Prasinoxanthin

Violaxanthin

Neoxanthin

Systemsolvents Xanthophylls β-carotene/

chlorophylls

Methanol/CH Cl3(50/50) - -

Methanol/CH Cl3 (5/95) ++ -

Diethylether/petroleumether (50/50) - ++

Petroleum ether100% - +

Ethyl acetate/cyclohexane (70/30)

+++ -

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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).

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.

A

wt

lov-hkcounts

B

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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

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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).

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.

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.

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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 Pseudo-

nitzschia 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:

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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.

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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 François-

Yves 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]).

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6. References

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Century, K., Reuber, T. L. & Ratcliffe, O. J. Regulating the Regulators: The Future

Prospects for Transcription-Factor-Based Agricultural Biotechnology Products.

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De Riso, V., Raniello, R., Maumus, F., Rogato, A., Bowler, C. & Falciatore, A. Gene

silencing in the marine diatom Phaeodactylum tricornutum. Nucleic Acids

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Gomi, T., Sidle, R.C. & Richardson, J.S. Headwater and channel network:

understanding processes and downstream linkages of headwater systems.

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Wilson, W.H., Xu, Y., Zingone, A. & Worden, A.Z. The Marine Microbial Eukaryote

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