Biosynthesis of phenylpyruvic acid from l-phenylalanine using chromosomally engineered Escherichia coli

The efficiency of whole-cell biotransformation is often affected by the genetic instability of plasmid-based expression systems, which require selective pressure to maintain the stability of the plasmids. To circumvent this shortcoming, we constructed a chromosome engineering strain for the synthesis of phenylpyruvic acid (PPA) from l-phenylalanine. First, l-amino acid deaminase (pmLAAD) from Proteus myxofaciens was incorporated into Escherichia coli BL21 (DE3) chromosome and the copy numbers of pmLAAD were increased by chemically induced chromosomal evolution (CIChE). Fifty-nine copies of pmLAAD were obtained in E. coli BL8. The PPA titer of E. coli BL8 reached 2.22 g/L at 6 h. Furthermore, the deletion of lacI improved PPA production. In the absence of isopropyl-beta-d-thiogalactopyranoside, the resulting strain, E. coli BL8orecAolacI, produced 2.65 g/L PPA at 6 h and yielded a 19.37% increase in PPA production compared to E. coli BL8orecA. Finally, the engineered E. coli BL8orecAolacI strain achieved 19.14 g/L PPA at 24 h in a 5-L bioreactor.

Automated summary

This study constructed a chromosome engineering strain for whole-cell biotransformation to synthesize phenylpyruvic acid (PPA) from l-phenylalanine. Results showed that increasing the copy number of l-amino acid deaminase (pmLAAD) and deleting the lacI gene could enhance PPA production. The engineered E. coli strain achieved a high PPA titer of 19.14 g/L within 24 hours in a 5-L bioreactor.