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.

This project aims at studying the role of gene expression variability (due to the stochastic fluctuations at the molecular level) in stress response and genetic instability. The impact of this variability on population dynamics is now well-studied, and increase of stochasticity (or noise) in gene expression is considered as a relevant evolutionary strategy in fluctuating environments. Here we want to determine if such an increase for some genes has been a way for technological yeast strains to adapt to the stressful fluctuating conditions they have to deal with. Indeed these strains are well-adapted to many environmental stress compared to laboratory strains. In the first part of this project, we will focus on the recently sequenced oenological strain of Saccharomyces cerevisiae EC1118 (NOVO et al. 2009) to detect promoters that are noisier in this strain compared to the standard non-adapted laboratory strain S288c. If such differences of noise are detected, we will study their impact on stress response and adaptation in stressful environments, especially in terms of fitness. This original stragety should enable the identification of new determinants of stress resistance and tolerance. At the moment no study has linked noise in gene expression to genetic variability. But, like any other phenotype, maintenance of genome integrity is under the influence of genes expressed with stochastic fluctuations. The rate of genetic-variant generation (RGVG) could be variable as a consequence of stochastic fluctuations in the expression of DNA repair and maintenance genes from cell-to-cell. High noise in the expression of genes involved in Double-Strand Break repair or DNA replication for instance, could confer a broad range RGVG from cell-to-cell in the population, and favour the emergence of sub-populations with higher genetic variability in times of stress, thanks to a second-order selection process (indirect selection of mutator strains along with favourable mutations they generate which counterbalance possible deleterious mutations) (CAPP, 2010). The aim of this project is to determine if industrial strains have evolved towards such a high noise in the expression of genes involved in DNA repair and maintenance. If this is the case, we will study the impact of different noise levels in the xepression of these genes on genetic variability under selective conditions. Capp, J. P. (2010). Noise-driven heterogeneity in the rate of genetic-variant generation as a basis for evolvability. Genetics 185, 395-404 Novo, M., et al. (2009). Eukaryote-to-eukaryote gene transfer events revealed by the genome sequence of the wine yeast Saccharomyces cerevisiae EC1118. Proc Natl Acad Sci U S A 106, 16333-16338.

The performance of many processes such as sewage treatment plants is highly related to the oxygen transfer from air bubbles, usually injected through diffusers, to microorganisms able to degrade pollutants contained in wastewaters. However, characterizing accurately the oxygen mass transfer in such processes is still a challenging issue mainly because of the liquid phase complexity. The aim of this project is to develop specific techniques and rigorous models to better estimate the various mechanisms that locally govern the gas/liquid mass transfer process. New and efficient visualization techniques are proposed to visualize and estimate the surfactant migration, structure and layer thickness. A new modelisation will permit to adapt and reduce the energy consumption of aeration that represents 80% of the global process energy.

With a worldwide production capacity of more than 100 million tons and a decreasing commodity price, methanol is regarded as a highly attractive alternative non-food raw material for biotechnology sector. The supply of methanol comes from both fossil and renewable resources, rendering it a highly flexible and sustainable raw material. C1 compounds are used by specialized groups of microorganisms i.e. the methylotrophs as their sole source of carbon and energy. While progress to use natural methylotrophs in biotechnology is on-going, ECOMUT propose to launch an alternative strategy by integrating methylotrophy into the established bacterial production host Escherichia coli. The synthetic biology approach we plan will be combined with a systems level understanding of methylotrophy. This will not only generate new a fundamental knowledge of C1 metabolism but will also provide a biological platform capable of transforming methanol in any molecule of interest. This research will make a significant contribution towards unleashing the potential of methanol as a raw material in a vast range biotechnological applications in any industrialised location.

In the context of climate change and increasing energy needs of the world population, the global interest for sustainable sources to produce energy is growing. One promising resource for biofuel production is microalgae, although their industrial use is limited by the lack of efficient harvesting techniques. Assisted flotation represents a promising harvesting technique that consists in air dispersed into microbubbles rising through a microalgae suspension. As a result, microalgae cells get attached to gaz-liquid interfaces and are carried out and accumulated on the surface, without being damaged. Flotation is thus a relatively rapid operation that needs low space, has moderate operational costs, and that could thus overcome the bottleneck of feasible microalgal biofuel production. However, the efficiency of this method is limited by the fact that the interaction between the bubbles and the cells is generally repulsive, due to the negative surface charge of the cells and the bubbles in water, and the low hydrophobicity of the algal cells. The goal of this project is to improve the efficiency of flotation, in order to better exploit the potential of the microalgal bioressource. Fundamental knowledge at the molecular and cellular scales will be acquired on the cell wall of microalgae and on the molecular mechanisms underlying its adhesion to gaz/liquid interfaces, using advanced force spectroscopy techniques such as optical tweezers and FluidFM technology. These data will then be further used to identify adhesive components promoting cell aggregation at the cells interface, and functionalize them at the surface of bubbles, thus improving flotation efficiency without altering the cells. Finally the overall evaluation of the efficiency of the functionalized bubbles for microalgae flotation will be evaluated and compared to other harvesting techniques. The results obtained in this project will allow to generate fundamental knowledge on the cell wall of microalgae and on the molecular mechanisms underlying their adhesion to gaz-liquid interfaces. These are not the only benefits of this project, as it will also provide a new technological solution to measure the interactions between fluid and biological interfaces, as well as a way to increase the efficiency of flotation process. Therefore, understanding the biophysics of microalgae flotation will open up new strategies to transform the microalgal biomass into 3rd generation biofuels.