Genome Engineering to Support Rational Design for Metabolic Engineering in Bacillus Subtilis

Genome engineering to support rational design for metabolic engineering in Bacillus subtilis Anne-Gaëlle Planson, Michael Mangan, Philippe Noirot, Etienne Dervyn, Matthieu Jules Micalis Institute, INRA, AgroParisTech, Jouy-en-Josas, France
Genome engineering is a rapidly growing field of microbial research but still remains limiting when
developing Bacillus subtilis-based cell factories. B. subtilis is the model organism for the Gram- positive bacteria, is GRAS (Generally Regarded As Safe) and is heavily used in industry. We successfully designed and developed B. subtilis chassis strains lacking functions such as sporulation, antibiotic resistance, mobile DNA elements, prophages, isozymes while retaining deletion making functions. This latter work represents a proof-of-concept for strain development for biotechnologies. To develop adapted chassis strains it is critical to be able to modify the genome "à la carte". We are currently developing a set of novel recombineering methodologies based on single strand annealing proteins (SSAP) in B. subtilis. Such recombinases mediating oligonucleotide-targeted mutagenesis will serve to introduce point mutations in multiple loci, enabling simultaneous modification of several chromosomal targets. We are also developing a CRISPR-Cas system for B. subtilis using the inactivated Cas9 as an interference system for gene expression modulation. Altogether this will result in an integrated toolbox for synthetic biology and metabolic engineering. These new methods for rapid introduction of multiple point mutations combined with the interference system should lead to the development of robust and high-performing bacterial cell factories.