Codon-Optimized PAL Expressed in With Enhanced Activities.

PAL (phenylalanine ammonia lyase) is important for secondary metabolite production in plants and microorganisms. There is broad interest in engineering PAL for its biocatalytic applications in industry, agriculture, and medicine. The production of quantities of high-activity enzymes has been explored by gene cloning and heterogeneous expression of the corresponding protein. Here, we cloned the cDNA of PAL (PAL) and introduced codon optimization to improve protein expression in and enzyme activities . The PAL gene was cloned by reverse transcription and named -wt. It had a full-length of 2,121 bp and encoded a 706-amino-acid protein. The -wt was inefficiently expressed in , even when the expression host and physical conditions were optimized. Therefore, codon optimization was used to obtain the corresponding gene sequence, named -opt, in order to encode the same amino acid for the PAL protein. The recombinant protein encoded by -opt, named PAL-opt, was successfully expressed in and then purified to detect its enzymatic activity . Consequently, 55.33 ± 0.88 mg/L of PAL-opt protein with a specific activity of 1,219 ± 147 U/mg and value of 609 μM for substrate L-phenylalanine was easily obtained. The enzyme protein also displayed tyrosine ammonia lyase (TAL)-specific activity of 80 ± 2 U/mg and value of 13.3 μM for substrate L-tyrosine. The bifunctional enzyme PAL/TAL (PAL-opt) and its easy expression advantage will provide an important basis for further applications.