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

<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>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. <jats:italic>B. subtilis</jats:italic>, 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 <jats:italic>B. subtilis</jats:italic> through in silico analysis. Genome-scale metabolic model (GSM) simulations was used to design and develop the metabolic pathways of <jats:italic>B. subtilis</jats:italic>. 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 <jats:italic>B. subtilis</jats:italic>.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p><jats:italic>B. subtilis</jats:italic> was bioengineered to enhance 2,3-BD production from glycerol using FBA in a published GSM model of <jats:italic>B. subtilis</jats:italic>, iYO844. Four genes, <jats:italic>ackA, pta, lctE,</jats:italic> and <jats:italic>mmgA</jats:italic>, were knocked out step by step, and the effects thereof on 2,3-BD production were evaluated. While knockout of <jats:italic>ackA</jats:italic> and <jats:italic>pta</jats:italic> had no effect on 2,3-BD production, <jats:italic>lctE</jats:italic> knockout led to a substantial increase in 2,3-BD production. Moreover, 2,3-BD production was improved by <jats:italic>mmgA</jats:italic> knockout, which had never been investigated. In addition, comparisons between in silico simulations and fermentation profiles of all <jats:italic>B. subtilis</jats:italic> strains are presented in this study.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>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.</jats:p> </jats:sec>