- Top of page
- Materials and methods
- Bacterial strains used
- Media and culture conditions
- Plasmid construction and transformation
- Analytical procedure
- Preparation of cell-free extracts
- Polyacrylamide gel electrophoresis
- Results and discussion
- Production and expression of a construct for l-BD formation
- Examination of culture conditions
- Preparation of l-BD
Aims: A metabolic pathway for l-2,3-butanediol (BD) as the main product has not yet been found. To rectify this situation, we attempted to produce l-BD from diacetyl (DA) by producing simultaneous expression of diacetyl reductase (DAR) and l-2,3-butanediol dehydrogenase (BDH) using transgenic bacteria, Escherichia coli JM109/pBUD-comb.
Methods and Results: The meso-BDH of Klebsiella pneumoniae was used for its DAR activity to convert DA to l-acetoin (AC) and the l-BDH of Brevibacterium saccharolyticum was used to reduce l-AC to l-BD. The respective gene coding each enzyme was connected in tandem to the MCS of pFLAG-CTC (pBUD-comb). The divided addition of DA as a source, addition of 2% glucose, and the combination of static and shaking culture was effective for the production.
Conclusions: l-BD (2200 mg l−1) was generated from 3000 mg l−1 added of DA, which corresponded to a 73% conversion rate. Meso-BD as a by-product was mixed by 2% at most.
Significance and Impact of the Study: An enzyme system for converting DA to l-BD was constructed with a view to using DA-producing bacteria in the future.