Autolysin Gene Deletion on Clostridium Saccharoperbutylacetonicum N1-4 Increase Strain Stability and Production for Biobutanol Fermentation

Biobutanol production is one of the most promissory alternatives to replace fossil fuels, because of its superior properties, such as: higher energy content, miscibility and hydrophobicity. Bio-butanol is an important chemical as well, used in the industry of paintings, farmaceuticals, cosmetics, food and others. Biotechnological production of butanol generate lower concentration, productivity and yield than ethanol production. Metabolic engineering on butanol-producer microorganisms is an opportunity to create a bigger economical stimulus for butanol industry. C. saccharoperbutylacetonicum N1-4 is a hyper-butanol producer strain, and generates a high titer (17 g/L of butanol). Besides the strain benefits, its applicability at industrial scale is limited by the strong autolytic behavior, which conducts to poor stability on continuous processes. The autolysis process makes cells self-destroy, and reduce the number of active microorganism, with a negative impact on the production. Also, the autolysins (enzymes responsible of autolysis) make the long-term continuous fermentation difficult and unstable. In this work, we have identified two autolysins and deleted them from the genome, in order to increase the strains stability, and production. Putative autolysins were identified from bioinformatic comparison of aminoacid homology in related strains. Then, putative genes had been overexpressed and biomass curves were compared with a control, to identify autolytic activity. Those genes fund autolytic were deleted using our customized CRISPR-Cas9 gene-editing tool, finding a better performance of the mutants, during fermentations.