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 n-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 OA and expressing a heterologous I-glucosidase on its cell surface. The beta-glucosidase gene bg1C from Thermobifida fusca YX was cloned into a cell surface display vector pGV3, resulting in pGV3-bg1C. Recombinant E. coli JM109 harboring the pGV3-bglC showed 13-glucosidase activity (18.9 +/- 5.7 U/OD600), indicating the cell surface functioning of mutant beta-glucosidase. pH-stat cultivation using n-lactic acid producer E. coil BW25113 (JAM) 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 o-lactic acid was obtained after 84 h cultivation, indicating that the engineered E. coil produced n-lactic acid directly from cellobiose. For continuous n-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(-1)). Thus, this system maintained the n-lactic acid production (4.3-5.0 g/L), n-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 n-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). (C) 2018, The Society for Biotechnology, Japan. All rights reserved.