GLYCINE is one of the twenty-two genetically encoded amino acids required for protein synthesis in all living systems. Although not considered as an essential amino acid, it is of increasing interest in the nutraceutical, cosmetic and pharmaceutical industry with a production > 22000 tons in 2018 and a estimated compound annual growth rate (CGAR) of 6,8% between 2020 and 2030. Commercialized glycine is currently exclusively produced by chemical means from fossil precursors. The aim of the G-BIOFERM project is to establish the foreground of a glycine production by microbial fermentation from renewable carbon sources, as a green and economically viable alternative. While we recently established the proof of concept of glycine bioproduction routes, we identified three major bottlenecks that must be unlocked in order to make this biotechnological process of glycine industrially attractive and economically viable. These bottlenecks which will be addressed in this G-BIOFERM are (i) the low catalytic efficiency of a key enzyme whose is critically important to reach optimal yield from sugars, (II) the apparent lack of glycine exporter and (iii) the high toxicity of glycine to the microbial cells. These bottlenecks will be addressed by combining systems and synthetic approaches, involving expertise in enzyme modelling, metabolic and strains engineering and fermentation process, with the full commitment of young French SME whose business model is to sell exclusively bio-based food supplements to its customers. The G-BIOFERM is therefore strategic and in full adequacy with the axis H.7 of the AAPG22 call: “Bioéconomie, de la biomasse aux usages” and will also perfectly meet society's expectations in terms of carbon neutrality, with a clean green product in the spirit of the French industrial revival supporting its small and medium-sized enterprises..

Synthetic microbiology is among the most promising approaches for getting more at lower cost and in the respect of the environment. Directed evolution is recognized as a key approach to obtain biobricks for synthetic biology. In this context there is a considerable interest in the development of continuous systems for directed evolution of biomolecules based on “orthogonal” evolution vector on which accumulation of mutations can be uncoupled from accumulation of mutations on the host genome. This project aims at developing such a system for the gram-positive bacterium Bacillus subtilis. An important step towards biotechnological applications will also be made by using the proposed system for: the evolution of new transcription factors for genetic circuit engineering in B. subtilis; and the evolution of new proteins binding inorganic ions such as heavy metals that might serve as biosensors and in bioextraction systems. The work program decomposes into three work-packages : development of a system for directed evolution in B. subtilis ; in silico analyses for the optimization of the system ; application to biobrick production. B. subtilis is a totally harmless bacterium of considerable biotechnological interest: it stands as the second model bacterium after Escherichia coli and is as such a natural chassis for synthetic biology; it is also a soil dweller (and probably a normal gut commensal in humans) with highly diverse physiological capabilities, and an ability to survive extreme conditions in the form of spores. B. subtilis and several of its close relatives of the Bacillus genus (notably B. licheniformis and B. amyloliquefaciens) exhibit a remarkable capacity of biological compound production that can be scaled-up to industrial levels are widely used in the industry for enzyme production.

Monoclonal antibodies (mAb) used to treat cancers, immune and infectious diseases represent a multibillion dollar industry. Chinese Hamster Ovary cells are currently the gold standard. However, high expense, potential virus contamination and mAb heterogeneity represent major drawbacks that drive scientists to seek alternatives. Among these, the diatom P. tricornutum represent an option because of (i) its robustness in industrial process, (ii) its ability to be cultivated in mixotrophy, (iii) its genome availability and possibility to be genetically engineered. The proof of concept in P. tricornutum has already been exemplified through the production of a glycosylated mAb. A human-like N-glycosylation is now required for mAb optimal safety and efficacy when used in human therapies. Therefore, we propose to engineer the N-glycosylation pathway of P. tricornutum by gene silencing and expressing heterologous enzymes, thus allowing galactosylated algae-made mAb production.