Cloning, characterization and prokaryotic expression analysis of two phenylalanine ammonia-lyase genes from Peucedanum praeruptorum Dunn

Peucedanum praeruptorum Dunn is a well-known Chinese medicinal plant that mainly contains coumarins derived from the phenylpropanoid pathway. Phenylalanine ammonia-lyase (PAL) is the first key enzyme in the phenylpropanoid pathway and catalyzes the formation of trans-cinnamic acid (t-CA) by L-phenylalanine (L-Phe). Two PALs (PpPAL1 and PpPAL2) were cloned and verified in this study, demonstrating that there is a multi-gene family of PAL in P. praeruptorum. PpPAL1 (GenBank accession number: MZ355581) and PpPAL2 (GenBank accession number: MZ355582) contained open reading frame of 2157 bp encoding 718 amino acids and 2118 bp encoding 705 amino acids, respectively. BLAST analysis showed that the amino acid sequences of two novel PpPALs were highly similar to other known plant PAL proteins. To confirm their functions, the two PpPALs were cloned into the pET-30a vector and expressed in Escherichia coli Transetta (DE3). The enzymatic activity of the two PAL recombinant proteins was subsequently detected. They both catalyzed the conversion of L-Phe to t-CA, but the activity of PpPAL2 was higher than that of PpPAL1. Phylogenetic results revealed that PpPAL1 and PpPAL2 were classified into different clusters. In addition, tissue-specific expression of the two PpPALs was different of both non-bolting and bolting plants. These results might be useful for future studies regarding phenylpropanoid biosynthesis and improving the content of medicinal components in P. praeruptorum.