Contracted but effective: production of enantiopure 2,3-butanediol by thermophilic and GRAS Bacillus licheniformis

Abstract

The production of chemicals with chiral groups through chemical methods is attractive but difficult to perform. Optically active 2,3-butanediol (2,3-BD) can provide two chiral groups and has special applications in asymmetric synthesis. However, the chemical production of 2,3-BD from nonrenewable resources is expensive to perform and results in a mixture of three stereoisomeric forms. Enantiopure 2,3-BD production through green biotechnological routes is thus rather desirable. In this work, we introduced a “SAME” (Screening, Analysis, Mutation, and Evaluation) process to achieve efficient microbes for chiral chemicals’ production using 2,3-BD as a target molecule. Bacillus licheniformis MW3, a GRAS and thermophilic strain, was firstly selected as a promising host. The mechanism of stereoisomer formation of 2,3-BD in B. licheniformis MW3 was then investigated. Two stereospecific 2,3-BD dehydrogenases (BDHs), 2R,3R-BDH (encoded by gdh) and meso-BDH (encoded by budC), were identified to be responsible for the production of (2R,3R)-2,3-BD and meso-2,3-BD. Two metabolically engineered strains, B. licheniformis MW3 (ΔbudC) and B. licheniformis MW3 (Δgdh), were constructed for the production of (2R,3R)-2,3-BD and meso-2,3-BD, respectively. After 42 h of fermentation, 123.7 g L⁻¹ (2R,3R)-2,3-BD was synthesized using B. licheniformis MW3 (ΔbudC); while 90.1 g L⁻¹meso-2,3-BD was synthesized after 32 h using B. licheniformis MW3 (Δgdh). Because of their high levels of production and biosecurity, these engineered B. licheniformis strains might have potential in the commercial production of enantiopure (2R,3R)-2,3-BD and meso-2,3-BD. The contracted but effective “SAME” process might serve as an alternative method for other chiral chemicals’ production.