Designing a genome engineering platform for microalgae – SynDia

Among the microalgae that have so far attracted attention, the diatom P. tricornutum stands out because of its lipid abundance and its robustness in industrial process engineering demonstrated by its industrial exploitation for EPA production. In 2014, we were able to establish the proof of concept of metabolic engineering by creating a high lipid producer strain resulting from the inactivation of a key gene involved in the storage of energy on sugar form (Daboussi et al, Nature Communications, 2014). In this SynDia project we go a step further.This project is divided in 3 workpackages. In the first work package, we aim to develop strategies to achieve rapid and efficient genome editing in the diatom Phaeodactylum tricornutum for rewriting its metabolism. To achieve this goal, we plan to develop the CRISPR/Cas9 system and its derivatives as tools for allowing multiple gene modifications. After monitoring the impact of these nucleases on genome integrity, we will set up alternative genome editing methodologies without introducing DNA into the cells to avoid genome instability due to the random integration of vectors into the genome or the toxicity (off-target) effects due to the persistence of this nuclease into diatom cells.
The second work package is dedicated to the development of methodologies to control and fine tune gene expression. This work package is separated in two distinct tasks, the first aims to define one or several appropriate loci for sustainable transgene expression. The second task will be focused on the development of methodologies to modulate transgene expression of existing and artificial metabolic pathways.
Altogether, these tools and methodologies will be integrated to rewrite the lipid metabolism both quantitatively and qualitatively (workpackages 3), as well as to create microalgae strains with potential high industrial value.

We have recently developed a new method to modify the genome of P. tricornutum with ease and prediction. This method is based on the delivery of the CRISPR/Cas9 molecular scissors in the protein form (RNPs). The power of this methodology was confirmed by creating strains in which three genes were simultaneously inactivated (triple knock-out) without introducing any selection marker or DNA into the cells (Serif et al., Nature Communications, 2018).

These results represent a significant step forward on several levels: (i) improving the specificity of genome engineering methodologies since no vector is randomly integrated into the genome; (ii) reducing the time of exposure of cells to nucleases and thus reducing potential non-specific cuts; (iii) identifying and validating two new positive selection markers, avoiding the use of antibiotic resistance genes. This development makes it possible to use production processes that are compatible with the specifications of industrial biotechnology and that limit health and environmental risks; (iv) the establishment of the first proof of concept of the genetic modification of several genes simultaneously in microalgae, which should open the way to the study of putative redundant functions of multiple gene family members.
This exceptional result allows microalgae to catch up with other industrial chassis (yeasts, bacteria). Another attractive perspective relies on the fact that the counter-selectable markers are well conserved within the microalgae phylogenetic tree and among other eukaryotic groups, makingthis strategy extendable to other organisms.
This is particularly important for hard-to-transfect species or those for which there is no known regulatory system (no biobricks :promoters, terminators).

By developing metabolic engineering methodologies to increase the productivity of a desired metabolite and
to produce new compounds, SynDia enables the end-users to build microalgae as an economically viable
platform for pharmacology, cosmetic, green chemistry and energy markets. The success of SynDia project
will improve the economic value of microalgae for these markets and open the way for the production of
chemicals and biofuels, for a potential market value estimated at 100 billion and 1 trillion dollars, respectively.

One-step generation of multiple gene knock-outs in the diatom Phaeodactylum tricornutum by DNA-free genome editing. Manuel Serif, Gwendoline Dubois, Anne Laure Finoux, Marie-Ange Teste, Denis Jallet, and Fayza Daboussi*. (2018). Nature Communications. DOI: 10.1038/s41467-018-06378-9

In the period of unprecedented expansion of energy demand and the desire to reduce greenhouse gas emissions, industrial biotechnology is expected to provide solutions to contribute to a more sustainable society, using for example renewable resources and waste as raw materials for manufacturing of bulk and fine chemicals and energy. By their abilities to combine plants properties (CO2 as substrate for the production of complexes molecules) and microorganism’s properties (rapid growth), microalgae are undoubtedly attractable for transition towards renewable energy. Despite of the high biotechnological potential of microalgae in nutrition, cosmetic, nutraceutical markets, there are a number of barriers to overcome to make them economically viable for mass markets such as energy and green chemistry.

To meet this challenge, several national and european initiatives aim at creating a partnership between academic scientific communities and industry in which industrial specifications and constraints are taking into account by academics to identify leverage actions to enhance the competitiveness. The SynDia project in line with this approach will generate considerable added value and provide the necessary impetus to significantly accelerate the development of industrial biomanufacturing processes. My double experience in academic laboratories and in a biotechnology company enables me to structure my research around the development of knowledge and methodologies to circumvent industrial bottlenecks.

Synthetic biology is emerging as an important sub-area of industrial biotechnology. This new field deals with the development of biocatalysts using an engineering approach to both improve productivity of natural compounds and design and construct novel biological parts, devices and systems to perform new functions. Synthetic biology requires the creation of microalgae chassis platform, robustness in challenging processes straightforward genome engineering and with an efficient regulatory structure. The diatom, Phaeodactylum tricornum is one of them. This species able to produce huge amount of lipids is already exploited for the production of long chain fatty acid such as EPA.

Regarding the metabolic engineering of Phaeodactylum tricornutum, the production of engineered strain is still far from straightforward process and there is a need for faster and more effective genome engineering methodologies. In this research program we plan to develop microalgae as industrial biocatalysts for the production of fuels and chemical and to achieve this goal, we propose:

1. To develop a new class of genome-specific nucleases and modulate double-strand break mechanisms in order to achieve improved genome modification frequencies adapted to study of gene function and/or to create strains with novel genetic properties

2. To identify optimal loci suitable for transgene expression in order to ensure the efficacy of their expression in terms of level as well as stability over time and to maximize the safety of genome editing

3. To generate artificial transcriptional modulators and synthetic promoters which will provide the means to tune gene expression in metabolic pathways and thus strains cable of high efficiency conversion of natural resources into target industrial commodities

4. To exemplify the power of the genomic tools to harness P. tricornutum for biofuel production by engineering lipid metabolism quantitatively as well as qualitatively

Altogether, SynDia will deliver a microalgae platform adapted for the industrial production, leading microalgae among the key enablers of the bioeconomy transition. This project will open new doors for biotechnological applications, notably as regards to the reengineering of diatoms’ lipid metabolism for biofuel production and the creation of artificial metabolic pathway for energy and green chemistry.