Wednesday, june 29, 2022 - Thesis defense - Gabrielle GUESDON

"Development of in-yeast genome cloning methods for the construction of semi-synthetic Bacillus subtilis-derived chassis strains"

Gabrielle GUESDON

Team Mollicutes

Speciality : Microbiology-Immunology

Wednesday, june 29, 2022 - 2:00 p.m. - ISVV Amphitheater - Campus INRAE Villenave d'Ornon

Abstract :

One of the major challenges in the synthetic biology (BS) field, is to provide new solutions to global issues (therapeutic/sanitary or climatic), in particular through the construction of useful, efficient and environmentally friendly production strains.
The well-characterized, non-pathogenic, Gram+ bacterium Bacillus subtilis (Bsu), is widely used in industry as a biotechnological workhorse. Recent studies have established that mutant strains with modified genomes are able to produce larger amounts of recombinant proteins. This suggests that the production of rationally designed Bsu chassis could be an important step in the improvement of valuable strains for industrial purposes.
This work was performed within the Bacillus 2.0's ANR project, which aims at applying SB tools for Bsu, and at developing an effective pipeline for the high-throughput construction of versatile Bsu chassis strains. Selected SB technologies for the pipeline include (i) the synthetic genome design, (ii) the in-yeast DNA assembly methods using Saccharomyces cerevisiae, (iii) the from-yeast whole genome isolation and transplantation (GT) to a recipient bacteria cell and, (iv) the characterization of recombinant strains.

The objectives of this thesis were to ensure the feasibility of these methods using a Gram+ bacterium, by showing, in particular, that it was possible to clone and maintain in S. cerevisiae the genome of a minimal Bsu strain, MPG192 (2.86 Mbp) and to modify it using the large repertoire of yeast genetic tools. Our first attempts to clone the entire Bsu genome into yeast using already described methods failed. Using a TAR-Cloning approach, we then attempted to clone large DNA fragments obtained by restriction of the Bsu genome. In a first experiment, five out of seven fragments were cloned. Difficulties to clone the largest fragment (1.50 Mbp), are presumably related to its size, and/or the lack of ARS elements. Concerning the other fragment, several factors have been proposed to explain the cloning failure: again, an insufficient number of ARS elements, but also, the presence of many repeated sequences (7 ribosomal operons), and/or the deleterious expression of these genes. Finally with other experiments, the whole 2.86 Mb genome was cloned in 21 pieces ranging from 6 kbp to 515 kbp. As TAR-Cloning imposes constraints in the choice of restriction sites, a new cloning method, called CReasPy-Fusion, was developed. This method allows the simultaneous cloning and engineering of mega-sized genome in yeast using the CRISPR-Cas9 system, after direct bacterial cell to yeast spheroplast cell fusion. As a proof of concept, we demonstrated that the method can be used to capture a piece of genome, or to clone and edit the whole genome from six different Mycoplasma species. This method was then adapted to Bsu, showing for the first-time yeast spheroplast and Gram+ protoplast cell fusion.  A fragment of ~150 kb has been successfully cloned in yeast.

Even if, the entire Bsu genome has not yet been cloned in yeast, several critical elements have been identified. First of all, this work underlines the importance of the cloning method to be adopted depending on the organism of interest. Then, it emphasizes the existence of both biological and technical factors that explain current difficulties and that will have to be taken into account in subsequent experiments. Finally, it enabled the development of the new in-yeast cloning method called CReasPy-Fusion which expands the catalog of technics already described. Through its versatility, it opens up prospects for the capture of large genome fragments, the suppression of problematic loci, and to support the assembly of synthetic fragments.

Jury :

  • M. JULES Matthieu
    Professeur à INRAE, AgroParisTech et Université Paris-Saclay - Président
  • M. RODRIGUE Sébastien
    Professeur agrégé à l’Université de Sherbrooke (QC Canada) - Rapporteur
  • Mme DABOUSSI Fayza
    Directrice de recherche à INRAE-INSA de Toulouse - Rapportrice
  • M. LABROUSSAA Fabien
    Docteur à l’Université de Bern (Suisse) - Examinateur
  • Mme LARTIGUE Carole
    Chargée de Recherche à INRAE de Bordeaux - Directrice de thèse
  • M. BLANCHARD Alain
    Professeur à l’Université de Bordeaux - Directeur de thèse

Modification date : 17 October 2023 | Publication date : 13 June 2022 | Redactor : M. Gauthier