Codon optimization of feruloyl esterase gene of Aspergillus niger and its high expression in Pichia pastoris

Abstract:

[Objective] Gene cloning and codon optimization of the feruloyl esterase from Aspergillus niger (AnfaeA) for its inducible expression in Pichia pastoris X-33. [Methods] The AnfaeA gene was amplified by overlap extension PCR using the genome of A. niger as template. At the same time, the AnfaeA gene was optimized by ‘one amino acid one codon’ and ‘codon randomization’ and then synthesized. The three kinds of ferulic acid esterase gene were cloned into the expression vector pPICZαA, respectively, obtaining the recombinant expression vectors pPICZαA-AnfaeA, pPICZαA-opAnfaeA I and pPICZαA-opAnfFaeA II. The plasmids were then linearized by Sac I, and transformed into P. pastoris X-33. The positive transformants of each gene type were identified by PCR, and further screened by determination of feruloyl esterase activity in fermentations using high performance liquid chromatography (HPLC). [Results] The feruloyl esterase activity of FaeA-ori, FaeA-opt I and FaeA-opt II were 6.8±0.1 U/mL, 5.2±0.1 U/mL, and 39.9±0.1 U/mL, respectively. By ‘codon randomization’ optimization, the activity of recombinant enzyme was 6 times higher than that coded by original AnfaeA gene. However, the feruloyl esterase coded by ‘one amino acid one codon’ optimized gene was only 76.5% of the original enzyme. The optimal temperature and pH for recombinant AnfaeA (reAnfaeA) was 50 °C and 5.5，respectively．In addition, reAnfaeA showed stability when incubated in 45?50 °C and pH 4.5?7.0. [Conclusions] By ‘codon randomization’ optimization, the resultant recombinant feruloyl esterase expressed in P. pastoris X-33 was 6 times higher than that coded by original AnfaeA, reaching the highest activity among existed recombinant feruloyl esterase until now. The results provided large-scale application potential of feruloyl esterase in industrial, and laid the experimental foundation for the further improvement the enzyme activity.