High-level expression of a ZEN-detoxifying gene by codon optimization and biobrick in Pichia pastoris

Abstract

The mycotoxin zearalenone (ZEN) can be degraded by a lactone hydrolase ZHD, which was derived from Gliocladium roseum. Here, based on the native ZHD encoding gene zhd101, a codon optimized zhd gene was synthesized, which was used for high expression of ZHD in Pichia pastoris GS115. Meanwhile, to further improve the expression of recombinant ZHD, the plasmids containing 1 to 4 copies of the zhd expression cassette were constructed, respectively, using the biobrick method. The protein expression in the recombinant P. pastoris X3c, which was transformed with the plasmid containing 3 copies of zhd expression cassette, was the highest. In addition, the enzymatic activity of ZHD against ZEN was defined for the first time based on a standard curve of peak area vs ZEN concentration. The ZEN degradation activity of ZHD from shake flask fermentation was calculated as 22.5U/mL with the specific activity of 4976.5U/mg. Furthermore, the high-density fermentation of P. pastoris X3c strain was also performed in 5L fermenter. The maximum enzyme activity of the supernatant was 150.1U/mL, which were 6.7-fold higher than that of the shake flask fermentation.

Keywords: Biobrick; High-density fermentation; Protein expression; Zearalenone degradation.