High-level expression of a ZEN-detoxifying gene by codon optimization and biobrick in Pichia pastoris.
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.
Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, The College of Life Sciences, Hubei University, Wuhan 430062, China.
La
Xiang
Tianjin institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Qinhong
Q
Wang
Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, The College of Life Sciences, Hubei University, Wuhan 430062, China.
Yuling
Y
Zhou
Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, The College of Life Sciences, Hubei University, Wuhan 430062, China.
Lifeng
L
Yin
Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, The College of Life Sciences, Hubei University, Wuhan 430062, China. Electronic address: zhangguimin@hubu.edu.cn.
Guimin
G
Zhang
Tianjin institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Yanhe
Y
Ma
D001711:Biotransformation; D003062:Codon; D018628:Gene Dosage; D015870:Gene Expression; D005822:Genetic Vectors; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D015025:Zearalenone / Q000037:antagonists & inhibitors* / Q000378:metabolism
D003062:Codon; D011994:Recombinant Proteins; D015025:Zearalenone; D006867:Hydrolases
Biobrick; High-density fermentation; Protein expression; Zearalenone degradation