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Background  Glycerol is a desirable alternative substrate for 2,3-butanediol (2,3-BD) production for sustainable development in biotechnological industries and non-food competitive feedstock.  B. subtilis , a “generally recognized as safe” organism that is highly tolerant to fermentation products, is an ideal platform microorganism to engineer the pathways for the production of valuable bio-based chemicals, but it has never been engineered to improve 2,3-BD production from glycerol. In this study, we aimed to enhance 2,3-BD production from glycerol in  B. subtilis  through in silico analysis. Genome-scale metabolic model (GSM) simulations was used to design and develop the metabolic pathways of  B. subtilis . Flux balance analysis (FBA) simulation was used to evaluate the effects of step-by-step gene knockouts to improve 2,3-BD production from glycerol in  B. subtilis .    Results   B. subtilis  was bioengineered to enhance 2,3-BD production from glycerol using FBA in a published GSM model of  B. subtilis , iYO844. Four genes,  ackA, pta, lctE,  and  mmgA , were knocked out step by step, and the effects thereof on 2,3-BD production were evaluated. While knockout of  ackA  and  pta  had no effect on 2,3-BD production,  lctE  knockout led to a substantial increase in 2,3-BD production. Moreover, 2,3-BD production was improved by  mmgA  knockout, which had never been investigated. In addition, comparisons between in silico simulations and fermentation profiles of all  B. subtilis  strains are presented in this study.    Conclusions  The strategy developed in this study, using in silico FBA combined with experimental validation, can be used to optimize metabolic pathways for enhanced 2,3-BD production from glycerol. It is expected to provide a novel platform for the bioengineering of strains to enhance the bioconversion of glycerol into other highly valuable chemical products.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12934-021-01688-y.

Metabolic engineering design to enhance (R,R)-2,3-butanediol production from glycerol in Bacillus subtilis based on flux balance analysis