Download Deliverable D7.1 Transgenic lines with traits of interest

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
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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).
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.
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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).
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.
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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).
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.
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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 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:
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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 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]).
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6. References
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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.,
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Wilson, W.H., Xu, Y., Zingone, A. & Worden, A.Z. The Marine Microbial Eukaryote
Transcriptome Sequencing Project (MMETSP): Illuminating the functional diversity
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