Inactivation of sacB Gene Allows Higher 2,3-Butanediol Production by Bacillus licheniformis from Inulin

<i>Bacillus licheniformis</i> 24 (BL24) is an efficient, non-pathogenic producer of 2,3-butanediol (2,3-BD). However, during inulin fermentation, the strain produces large amounts of exopolysaccharides (EPS), which interfere with the process' performance. The present study aims to investigate the effect that inactivation of the <i>sacB</i> gene, encoding levansucrase in BL24, has on 2,3-BD production efficiency. Knockout of the <i>sacB</i> gene was accomplished via insertional inactivation. The <i>sacB</i>-knockout variant formed 0.57 g/L EPS from sucrose and 0.7-0.8 g/L EPS from glucose and fructose, a 15- and 2.5-fold reduction relative to the wild type, respectively. Likewise, during batch fermentation with soluble inulin Frutafit^® CLR, the mutant BLΔ<i>sacB</i> produced significantly less EPS than the wild type, allowing the maintenance of pH at values favoring 2,3-BD synthesis. At pH 6.50, BLΔ<i>sacB</i> reached a record titer of 128.7 g/L 2,3-BD, with productivity of 1.65 g/L/h, and a yield of 85.8% of the theoretical maximum. The obtained concentration of 2,3-BD is two-fold higher compared to that of the wild type. Subsequent RT-qPCR assays confirmed a successful <i>sacB</i> knockout. Three of the genes involved in inulin hydrolysis (<i>sacA</i>, <i>sacC</i>, and <i>fruA</i>) maintained their expression levels compared to the wild type, while that of <i>levB</i> increased. Although total EPS accumulation could not be completely eliminated via <i>sacB</i> gene knockout alone, the overall reduction in EPS content has enabled the highest yield of 2,3-BD from inulin to date, a promising result for the industrial production from inulin-rich substrates.