Enhanced production of (R,R)-2,3-butanediol by metabolically engineered Klebsiella oxytoca

Abstract

Microbial fermentation produces a racemic mixture of 2,3-butanediol ((R,R)-BD, (S,S)-BD, and meso-BD), and the compositions and physiochemical properties vary from microorganism to microorganism. Although the meso form is much more difficult to transport and store because of its higher freezing point than those of the optically active forms, most microorganisms capable of producing 2,3-BD mainly yield meso-2,3-BD. Thus, we developed a metabolically engineered (R,R)-2,3-BD overproducing strain using a Klebsiella oxytoca ΔldhA ΔpflB strain, which shows an outstanding 2,3-BD production performance with more than 90 % of the meso form. A budC gene encoding 2,3-BD dehydrogenase in the K. oxytoca ΔldhA ΔpflB strain was replaced with an exogenous gene encoding (R,R)-2,3-BD dehydrogenase from Paenibacillus polymyxa (K. oxytoca ΔldhA ΔpflB ΔbudC::PBDH strain), and then its expression level was further amplified with using a pBBR1MCS plasmid. The fed-batch fermentation of the K. oxytoca ΔldhA ΔpflB ΔbudC::PBDH (pBBR-PBDH) strain with intermittent glucose feeding allowed the production of 106.7 g/L of (R,R)-2,3-BD [meso-2,3-BD, 9.3 g/L], with a yield of 0.40 g/g and a productivity of 3.1 g/L/h, which should be useful for the industrial application of 2,3-BD.