Fed-batch production of l-phenylalanine from glycerol and ammonia with recombinant Escherichia coli

• A specific rationally designed multi-phase fed-batch process was applied.
• Therein recombinant Escherichia coli strains convert lactic acid and glycerol to biomass.
• During production phase glycerol and ammonia are converted to l-phenylalanine.
• l-Phenylalanine concentrations of up to 13.4 g L−1 can be achieved.
• Exchange of ampicillin for kanamycin resistance leads to plasmid stabilization.

Glycerol was used as carbon source for l-phenylalanine production with recombinant Escherichia coli. In contrast to glucose, no consumption of the precursor phosphoenolpyruvate (PEP) is necessary for glycerol uptake. Additional lactic acid feeding was necessary for growth because the genes encoding the PEP consuming pyruvate kinase isoenzymes have been deleted. Thus a fed-batch process was developed with feeding of lactic acid and glycerol for biomass formation followed by feeding of glycerol and ammonia for l-phenylalanine production. Unfortunately, plasmid instability was observed in the first process. Plasmid stability could be successfully assured by replacing an ampicillin resistance gene by a kanamycin resistance gene cassette. The resulting maximum l-phenylalanine concentration of 13.4 g L−1 was improved by 26% and biomass specific productivity (22 mgL-phe gCDW−1 h−1) was raised by 69%. The final l-phenylalanine concentration of 13.4 g L−1 was thus improved by a factor of 2.4 compared to earlier reports.