Fed-batch fermentation of recombinant Citrobacter freundii with expression of a violacein-synthesizing gene cluster for efficient violacein production from glycerol

The extensive prospects of violacein in the pharmaceutical industry have attracted increasing interest. However, the fermentation levels of violacein are currently inadequate to meet the demands of industrial production. This study was undertaken to develop an efficient process for the production of violacein by recombinant Citrobacter freundii. The effects of dissolved oxygen (DO) and pH on cell growth and violacein production in batch cultures were investigated first. When the DO and pH of the medium were controlled at around 25% and 7.0, respectively, the biomass and concentration of violacein were maximized. Based on the consumption of nutrients in the medium observed during batch culture, a fed-batch fermentation strategy with controlled DO and pH was implemented. By continuously feeding glycerol, NH4Cl, and l-tryptophan at a constant feeding rate of 16 mL h−1, the final concentration of violacein reached 4.13 g L−1, which was 4.09-fold higher than the corresponding batch culture, and the maximal dry cell weight (DCW) and average violacein productivity obtained for the fed-batch culture were 3.34 g DCW L−1 and 82.6 mg L−1 h−1, respectively. To date, this is the first report on the efficient production of violacein by genetically engineered strains in a fermentor.

► A violacein productivity of 82.6 mg L−1 h−1 was obtained by the implementation of a fed-batch fermentation strategy.
► It is the first time that an efficient production of violacein by genetically engineered strains was studied with the highest productivity ever reported.
► Violacein could be produced by recombinant Citrobacter freundii economically and efficiently using glycerol as the sole carbon source and endogenous l-tryptophan as the precursor.
► We found that the production of violacein partly leads to the increase of intracellular pH.