Improving Fructooligosaccharide Production via sacC Gene Deletion in Zymomonas mobilis: A Novel Approach for Enhanced Prebiotic Production

Fructooligosaccharides (FOS) are promising prebiotics in the relevant and increasing market of functional food. Industrially, these compounds are produced from sucrose by the action of fructosyltransferase or b-fructofuranosidase enzymes. However, this process often yields low conversion rates and results in impure mixtures due to the release of high levels of glucose. Zymomonas mobilis is a well-known ethanol-producing bacterium with native levansucrase enzymes able to convert sucrose into FOS. This study aimed to use synthetic biology tools to eliminate invertase (sacC) activity in Z. mobilis, reducing substrate competition and maximizing FOS production. Additionally, we explored the potential use of agro-industrial by-products, such as sugarcane molasses (M) and corn step liquor (CSL), as nutrients for FOS production using Z. mobilis in an in vivo bioprocess strategy. Invertase deletion from the Z. mobilis ZM4 genome was accomplished by homologous recombination of an engineered suicide plasmid. Using Z. mobilis sacC⁻, we observed a 70% reduction in monosaccharide production and a 9.0-fold increase in levan formation compared to the wild-type strain. Implementation of a fed-batch approach with CSL and molasses (CSLM) medium at flask-scale allowed to produce 41.9 g L⁻¹ of FOS (0.25 g_FOS g_sucrose⁻¹). To our knowledge, this work describes for the first time the production of FOS from agro-waste residues using a genetically modified Z. mobilis strain in a one-step fermentation. Through this innovative approach, we aim to contribute to the advancement of biotechnological strategies for prebiotic production, offering insights into genetic engineering techniques for improving the efficiency and sustainability of FOS synthesis in Z. mobilis.