Construction and transplantation of semi-synthetic genomes of Bacillus subtilis: towards the development of the next generation of bacterial chassis – Bacillus2-0

Preliminary data showed that with a repertoire of dispensable chromosomal regions, genetic modifications can be designed and combined to rationally streamline the biotechnology workhorse, Gram-positive model bacterium, Bacillus subtilis. This project will take advantage of cutting-edge procedures for the rapid construction of improved Bacillus-derived strains, including in-yeast genome assembly, cloning and engineering before back-transplantation of redesigned genomes or genome parts into B. subtilis. The most promising transplants will be characterized and evolved towards better fitness as a proof-of-concept and as a prerequisite to future development of strains valuable in industry, medicine or biotechnology.

A first step consisted in cloning all or part of the B. subtilis genome in yeast to validate that the yeast can effectively propagate B. subtilis genomes. The second step consisted in the design of the SynBsu2.0 reduced semi-synthetic genome and the implementation of an automated pipeline applying a series of algorithms that, by taking into account the essentiality, quasi-essentiality or non-essentiality of the genes, as well as their genetic environment and level of expression, generate in silico the fragments of the chromosome to be reassembled, as well as the primers to be synthesized. The third step consisted in optimizing the conditions of in-yeast assembly of synthetic fragments to generate a first set of megachunks of ~100 kb.

Comparing the genomes of SynBsu2.0 with JCVI-Syn3.0 will bring unique insight on the minimal set of genes required to maintain streamlined but still efficient machineries for the biosynthesis of the cell wall, lipids, amino acids and nucleotides and more generally for gene expression.

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Synthetic genetic circuits often fail to function as designed because of unwanted interactions between circuit components and the host system. A promising strategy to foster innovation in biotechnology relies on the construction of adequately streamlined cellular chassis. Ideally, a valuable chassis should (1) include the minimal set of genes required for rapid growth and efficient production of proteins/chemicals of interest, and (2) possess a physically robust cell envelope to allow high-density, large-scale culture. Previous studies by the J.C. Venter Institute on minimal synthetic cell led to the construction of JCVI-Syn3.0, a mycoplasma-derived cell with a genome of 0.53 Mb and 473 genes. While this bacterium currently stands as the best approximation of a minimal free-living organism, its growth requires complex and expensive culture medium, in accordance with mycoplasma inability to synthesize cell wall, amino acids, lipids and nucleic acids precursors. Here, we propose to build a semi-synthetic, minimal and flexibly programmable bacterial chassis for biotechnology able to grow in basic and inexpensive media. Preliminary data showed that with a repertoire of dispensable chromosomal regions, genetic modifications can be designed and combined to rationally streamline the biotechnology workhorse, Gram-positive model bacterium, Bacillus subtilis. Using state-of-the-art genome design and reengineering tools, Bacillus 2.0 aims at developing SynBsu2.0, a minimal chassis derived from B. subtilis. This project will take advantage of cutting-edge procedures for the rapid construction of improved Bacillus-derived strains, including in-yeast genome cloning and engineering before back-transplantation of redesigned genomes into B. subtilis protoplasts. The most promising transplants will be characterized and evolved towards better fitness as a proof-of-concept and as a prerequisite to future development of strains valuable in industry, medicine or biotechnology. Moreover, comparing the genomes of SynBsu2.0 with JCVI-Syn3.0 will bring unique insight on the minimal set of genes required to maintain streamlined but still efficient machineries for the biosynthesis of the cell wall, lipids, amino acids and nucleotides and more generally for gene expression.