Friday, December 13, 2024 - Thesis defense - Julien Barret

Julien Barret

Thesis presented to obtain the degree of Doctor of the University of Bordeaux - Specialization : Microbiology - Immunology

Team Mollicutes

Friday, December 13, 2024 - 14:pm - Amphithéâtre ISVV - Campus INRAE Villenave d'Ornon
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Abstract : Genome engineering of microorganisms has become a standard in microbial biotechnology. In 2010, promising synthetic biology technologies using yeast as a platform for the assembly and engineering of synthetic bacterial genomes followed by their transplantation into a recipient cell have emerged. These technologies have led to the creation of the first synthetic cells and opened new avenues towards the construction of cells with fully controlled biological properties. Transferring these tools to microorganisms of industrial interest such as the Gram+ bacterium Bacillus subtilis (Bsu), a model in the biotechnology sector, would be a major step forward. This is precisely the aim of the ANR "Bacillus 2.0" project, which brings together two INRAE teams and aims to adapt all these synthetic biology tools to Bsu so as to be able to go from computer-aided design of semi-synthetic Bsu genomes to the production of new industrial strains. However, initial work on this project showed that the entire Bsu genome could not be cloned and maintained in yeast in its current state. These results threatened to call into question the feasibility of the entire project and, in particular, the relevance of using yeast as a platform for assembling the semi-synthetic Bsu genome.
The goal of my thesis was to demonstrate that yeast remained a relevant host for the Bacillus 2.0 project. It was divided into 3 parts. In the first part, a genome cloning method recently developed in the laboratory, called CReasPy-Fusion, was progressively adapted to Bsu. The results obtained showed (i) the possible transfer of plasmid DNA between bacterial protoplasts and yeast spheroplasts, (ii) the efficiency of a CRISPR-Cas9 system carried by yeast cells to capture/modify this plasmid DNA during Bsu/yeast fusion, and then (iii) the efficiency of the same system to capture genomic fragments of about a hundred kb from three different strains. Fluorescence microscopy observations were also carried out revealing two types of interaction that would enable the transition from protoplast/spheroplast contact to cloned bacterial DNA in yeast. In the second part of my thesis, the CReasPy-Fusion method was used in an attempt to clone large Bsu genome fragments in yeast. Genomic fragments of up to ~1 Mb could be cloned in yeast, but their capture required the prior addition of a large number of ARS to the Bsu genome to stabilize the genetic constructs. The final part was the adaptation of the RAGE method to Bsu. This method allow the transfer, not of a whole genome, but of portions of bacterial genomes from yeast to the bacteria to be edited. Proof of concept was achieved by exchanging a 155 kb genome fragment with a reduced 44 kb version.
In conclusion, the work carried out during this thesis has shown the relevance of using yeast as an engineering platform for large-scale modifications of the Bsu genome. On the one hand, we have shown that fragments of around 100 kb can be cloned in yeast, modified and transferred into a recipient cell to generate Bsu mutants. This strategy offers a real alternative to genome transplantation. On the other hand, we have shown that large fragments of the Bsu genome (up to 1 Mb) can also be cloned in yeast, provided they contain numerous ARS in their sequences. Thanks to these results, cloning a reduced Bsu genome in yeast has once again become an achievable goal.

Direction : Mme Lartigue Carole, Chargée de Recherche, INRAE, Villenave d'Ornon

Jury members :

M. JULES Matthieu, Professeur, INRAE, AgroParisTech, Univ. Paris-Saclay - Président
M. DANELON Christophe, Professeur, INSA, Toulouse - Rapporteur
M. CAMBRAY Guillaume, Chargé de Recherche, CBS, Montpellier - Rapporteur
M. DOIGNON François, Professeur, Université de Bordeaux - Examinateur
Mme LE RHUN, Chargée de Recherche, CNRS, Bordeaux - Examinatrice
Mme PLANSON Anne-Gaëlle, Chargée de Recherche, INRAE, AgroParisTech, Univ. Paris-Saclay - Examinatrice