L-alanine production in recombinant Escherichia coli with thermo-regulated genetic switch

Abstract:

[Objective] Specific growth rate of Escherichia coli could be significantly decreased by L-alanine, which would result in reduction in L-alanine volumetric productivity. Therefore, the λpR-pL promoter was used as a thermo-controllable genetic switch to coordinate the processes of cell growth and L-alanine production in E. coli. [Methods] Synthetic routes for acetate, formate, ethanol, succinate and lactate as well as for L-alanine recemization were inactivated by deleting the corresponding genes (ackA-pta, pflB, adhE, frdA, ldhA, dadX) in the wild-type E. coli B0016 to generate B0016-060B. Subsequently, alanine dehydrogenase derived from Geobacillus stearothermophilus was cloned downstream of the pL promoter and expressed in B0016-060B to produce B0016-060B/pPL-alaD. Shake-flask and bioreactor experiments were conducted to investigate the properties of cell growth and L-alanine fermentation. [Results] Deletions of the competing pathways significantly decreased by-products accumulation with formation of low levels of acetate, succinate and ethanol. Strain B0016-060B/pPL-alaD hardly produced L-alanine during cell growth phase at 28 °C, which facilitated high growth rate. Meanwhile, efficient L-alanine production was obtained when cultured at 42 °C under oxygen-limited conditions. In bioreactor experiment, strain B0016-060B/pPL-alaD produced 67.2 g/L L-alanine, with a productivity of 2.06 g/(L·h). [Conclusion] Efficient cell growth and L-alanine production were realized simply by switching the cultivation temperature.

Key words:L-Alanine, Genetic switch, Escherichia coli, Fermentation, Metabolic engineering