Overexpression of a designed 2.2 kb gene of eukaryotic phenylalanine ammonia-lyase in Escherichia coli

Abstract

Phenylalanine ammonia-lyase (EC 4.3.1.5) is a key enzyme in the secondary metabolism of higher plants catalyzing the non-oxidative conversion of L-phenylalanine into transcinnamate. The nucleotide sequence of its 2.2 kb gene was designed for expression in Escherichia coli and synthesized in a single reaction from 108 oligonucleotides using assembly PCR. After amplification, the gene was cloned into the expression vector pT7-7 and coexpressed with the chaperone HSP-60 system. The expression system yielded 70 mg of fully active enzyme per liter culture.