Engineering Escherichia coli with a symbiotic plasmid for the production of phenylpyruvic acid†

Abstract

Plasmid-based microbial systems have become a major avenue for the production of pharmaceutical and chemical products; however, antibiotics are often required to maintain the stability of the plasmid. To eliminate the need for antibiotics, we developed a symbiotic system between plasmids and hosts by knocking out the essential gene of folP on the chromosome and placing it on the same plasmid as L-amino acid dehydrogenase (aadL); the resulting strain was named E. coli A06ΔfolP. To increase the copy number of aadL, different strengths of promoters were used for the expression of folP, resulting in the creation of a mutant E. coli A17ΔfolP. The yield of phenylpyruvic acid (PPA) from E. coli A17ΔfolP (4.1 ± 0.3 g L⁻¹) was 1.9-fold that of E. coli A06ΔfolP (2.1 ± 0.2 g L⁻¹). Next, the stability of plasmids was tested, and results showed that the plasmids could be maintained stably for 10 transfer numbers under antibiotic-free conditions. Finally, E. coli A17ΔfolP was used to produce PPA; the yield of PPA was 18.7 g L⁻¹ within 14 h.