Continuous production of d-lactic acid from cellobiose in cell recycle fermentation using β-glucosidase-displaying Escherichia coli.

The present study demonstrates continuous production of d-lactic acid from cellobiose in a cell recycle fermentation with a hollow fiber membrane using recombinant Escherichia coli constructed by deleting its pyruvate formate-lyase activating enzyme gene pflA and expressing a heterologous β-glucosidase on its cell surface. The β-glucosidase gene bglC from Thermobifida fusca YX was cloned into a cell surface display vector pGV3, resulting in pGV3-bglC. Recombinant E. coli JM109 harboring the pGV3-bglC showed β-glucosidase activity (18.9 ± 5.7 U/OD), indicating the cell surface functioning of mutant β-glucosidase. pH-stat cultivation using d-lactic acid producer E. coli BW25113 (ΔpflA) harboring pGV3-bglC in minimum medium with 10 g/L cellobiose in a jar fermentor under anaerobic condition resulted in 5.2 ± 0.1 g/L of d-lactic acid was obtained after 84 h cultivation, indicating that the engineered E. coli produced d-lactic acid directly from cellobiose. For continuous d-lactic acid production, cell recycle fermentation was conducted under anaerobic condition and the culture was continuously ultrafiltrated with a hollow fiber cartridge. The permeate was drawn to the reservoir and a minimum medium containing 10 g/L cellobiose was fed to the fermentor at the same rate (dilution rate, 0.05 h). Thus, this system maintained the d-lactic acid production (4.3-5.0 g/L), d-lactic acid production rate (0.22-0.25 g/L/h), and showed no residual cellobiose in the culture during 72 h operation. Interestingly, the d-lactic acid production rate in cell recycle fermentation was more than 3 times higher than that in the batch operation (0.06 ± 0.00 g/L/h).