Constitutive expression of feruloyl esterase from Aspergillus niger in Pichia pastoris

Abstract:

[Objective] This study is aimed to constitutively express feruloyl esterase gene (AnfaeA) from Aspergillus niger in Pichia pastoris GS115. [Methods] The AnfaeA gene was amplified from A. niger by overlap extension PCR and cloned into the expression vector pGAP9K.The recombinant expression vector (pGAP9KAnfaeA) was linearized by Sal I, then transformed into P. pastoris GS115. Feruloyl esterase activity of the recombinant strain was determined by high-performance liquid chromatography (HPLC) and we optimized the fermentation conditions. [Results] We cloned AnfaeA gene (783 bp) and constitutively expressed AnfaeA gene in P. pastoris GS115. Feruloyl esterase activity reached its peak (5.72±0.10 U/mL) after 84 h fermentation, its specific activity was 59.75 U/mg, and the molecular weight of reAnfaeA was about 40 kD. The optimal fermentation conditions were as follows: glucose 40.0 g/L, tryptone 10.0 g/L, yeast extract 30.0 g/L, CaCO3 0.2 g/L, inoculum age 28 hours, inoculation level 3% (v/v), medium volume 50 mL in 250 mL flask. Under these conditions, feruloyl esterase activity in fermentation supernatant was 15.60±0.23 U/mL. [Conclusion] We constitutively expressed feruloyl esterase in P. pastoris GS115. It was helpful to study the constitutive expression system in P. pastoris and improve the fermentation production of feruloyl esterase.

Key words:Aspergillus niger, Feruloyl esterase, GAP promoter, Constitutive expression, Fermentation optimization