High-level Expression Of A ZEN-detoxifying Gene By Codon Optimization And Bio-brick In Pichia Pastoris

Zearalenone(ZEN)is a nonsteroidal oestrogenic mycotoxin,which is produced by some Fusarium fungi.ZEN was first isolated from the moldy com by Stob et al.In cereal plants that are infected by the toxigenic species of Fusarium in many countries,high level of ZEN are frequently detected in the cereal and its by-products.ZEN usually enters human or animal body through the food chains.When ZEN is taken into the human or animal body,it can lead to hyperestrogenism symptoms,and infertility problems in extreme case,especially in pigs.What's even worse is that,ZEN has strong genetic toxicity and carcinogenicity.Because of the economic losses engendered by ZEN and its impact on human and animal health,the ZEN detoxification technology is of great importance.However,the traditional physical chemistry methods are not effective to detoxify ZEN,and it is likely to cause loss of nutrients and decline in sensory quality of food.One promising strategy for reducing ZEN contaminaton is enzymatic degradation.Enzyme has excellent properties of high selectivity and specificity,reaction conditions are moderate.Enzyme degradation can efficiently degrade ZEN to non-toxic products without destroying the nutrients.In 2002,Takahashi-Ando et al.isolated the ZEN-detoxifying gene,zhd101,from Clonostachys rosea.The recombinant E.coli carrying the detoxifying gene was able to remove ZEN.However,the degradation efficiency of ZHD was relatively low.At present zhd101 gene has not been expressed in P.Pastors.In our study,based on the native zhd101 gene,a new zhd gene was synthesized,which was used in optimal codon for high expression in Pichia pastoris.Meanwhile,to further improve the expression of recombinant protein ZHD,the multi-copy expression cassettes were constructed in vitro by using the biological brick method.The recombinant P.pastoris carrying one,two,three,four copies of zhd gene were generated and named as X1c,X2c,X3c,X4c,respectively.With the increase of copy number,ZHD protein expression increased gradually.But X4c with four copies brought about a decrease in expression levels.In shake culture,the highest ZHD yield of X1c,X2c,X3c,X4c was 0.03 mg/mL,0.062 mg/mL,0.083 mg/mL,0.058 mg/mL,respectively.What's more,the enzyme activity of degradation ZEN was defined for the first time.The ZEN degradation activity of ZHD purified from shake flask fermentation was 42.3 U/mL with specific activity of 4976.5 U/mg.And the enzyme activity of ZHD in the shake flask fermentation supernatant of recombinant P.pastoris X3c was calculated as 22.5 U/mL,it could degrade 6.34 ?g/mL ZEN only in 15 min.While for the ZEN-JJM and ZLHY-6,which were 98%homology with ZHD,the degradation rates are 9 h for 1 ?g/mL ZEN and 4 h for 1.6 ?g/mL ZEN,respectively.Also the high-density fermentation of P.pastoris X3c strain was performed in 5 L fermenter for the first time.And the maximum enzyme activity in the supernatant was 150.1 U/mL,which was 6.7 folds higher than that of shake flask fermentation.