Modular Metabolic Engineering of Bacillus licheniformis for Efficient 2,3-Butanediol Production by Consolidated Bioprocessing of Jerusalem Artichoke Tubers

Consolidated bioprocessing (CBP) has emerged as a promising strategy for green synthesis of 2,3-butanediol (BDO) from Jerusalem artichoke tubers (JAT). Here, modular metabolic engineering of Bacillus licheniformis MW3 was implemented, in which the overall BDO synthesis process was divided into four modules (inulin hydrolysis, sporulation elimination, carbon flux redistribution, and enantiopure meso-BDO synthesis). First, endo-inulinase was introduced to boost inulin utilization. Second, sporulation was blocked by spoIIE knockout, thus reducing the leakage risk of the genetically engineered strain from the fermentation plant. Third, more carbon flux was shunted into BDO production via exchanging the alsS start codon, blocking acetoin catabolism, and competing pathways. Finally, enantiopure meso-BDO was synthesized by gdh knockout. Encouragingly, the final engineered strain, MWO-8, produced meso-BDO with 99.3% purity at a titer, productivity, and yield of 82.51 g/L, 1.38 g/L/h, and 0.439 g/g, respectively. To our knowledge, this is the highest titer of BDO from JAT by CBP so far. Collectively, this work provides a highly promising alternative approach for sustainable and cost-effective production of BDO.