Mining And Heterologous Expression Of Microbial Aflatoxin B₁ Degrading Enzyme Gene

Aflatoxin（AFT）is a carcinogenic fungal toxin produced by Aspergillus fungi under high temperature and humidity conditions which is widely distributed in nature.Among them,aflatoxin B₁（AFB₁）is considered to be the most common and harmful AFT,with toxic effects such as carcinogenicity,DNA mutation,immunotoxicity and hepatotoxicity in mammals and poultry.Different crops such as peanuts,maize,cotton and peppers are highly susceptible to AFB₁contamination during growth,storage and processing and pose a serious threat to human and animal health through food and agricultural by-products,as well as costing the world economy billions of dollars.A variety of strategies have been reported for the removal of AFB₁,which can be broadly classified as physical,chemical and biological methods.Biological methods are considered to be the most promising detoxification methods due to their mild degradation conditions and minimal impact on the nutritional value of the crop.Based on the Pac Bio sequencing platform,we sequenced and assembled the whole genome of Aspergillus terrus HNGD-TM15,a highly efficient AFB₁-degrading fungus,to systematically analyze its AFB₁-degrading genes and heterologously express them in E.coli and Pichia pastoris to expand the aflatoxin-degrading microbial resource base.We also investigated the degradation mechanism of AFB₁by the recombinant bacteria and the hepatotoxic and mutagenic effects of the degradation products.The specific studies are summarized as follows.1.Firstly,the whole genome of A.terres HNGD-TM15,which was screened in the laboratory in the early stage,was sequenced for three generations.The coding gene and protein sequence of the strain were obtained,and its gene were compared and annotated with the commonly used database by BLAST.The whole gene sequencing results showed that the total gene size of the strain was 34.39 Mbp,the total length was 24,983,996 bp,and the sequence GC content was 52.23%.In addition,five AFB₁degrading enzyme genes were obtained by comparing with the reported AFB₁degrading enzyme genes by BLASTp and screening by gene function annotation.2.Secondly,all the excavated genes were amplified,vector constructed,expressed and purified in E.coli,and the degradation function of AFB₁was verified.By extracting A.terres RNA,the c DNA obtained by reverse transcription was used as a template,and all candidate genes were amplified by PCR,and the genes were recovered and purified.After purification,the gene concentrations were 87.0,84.2,98.2,107.3 and 93.1 ng/μL,respectively,and the plasmid concentration of p ET-28a vector was 179.3 ng/μL.Using p ET-28a as the expression vector,five candidate genes were connected with p ET-28a by homologous recombination,and the expression vector was successfully constructed by plasmid PCR,double restriction endonuclease digestion and sequencing.The constructed plasmids with candidate genes were transferred into E.coli BL21 for protein expression,and four candidate genes were successfully expressed after IPTG induction.Among them,the recombinant FET3,DDP3 and Cpo P were expressed as inclusion bodies with molecular weights of 65,36 and 36 k Da,respectively.The recombinant Frs A enzyme is soluble and its molecular weight is 32 k Da.The recombinant Frs A enzyme is soluble and its molecular weight is 32 k Da.When the purified recombinant protein was mixed with AFB₁,the degradation rates of AFB₁by FET3,DDP3,Frs A and Cpo P were 8.6%,55.18%,20.58%and11.6%,respectively.In the presence of medium,the degradation rate of FET3 was only21.45%.3.The verified degradation enzyme gene DDP3 was heterologous expressed in P.pastoris and named ATADE.Firstly,the codon of ATADE gene sequence was optimized.After optimization,the GC content of the sequence decreased from 53.93%to 41%,and the codon adaptation index（CAI）increased from 0.68 to 0.94.Using homologous recombination technology,p PIC9K-His was selected as the expression vector for heterologous expression of ATADE,and the optimized ATADE gene was connected with the selected vector to obtain the recombinant expression vector p PIC9K-His-ATADE.The constructed recombinant plasmid was transformed into P.pastoris GS115,and the recombinant GS115（p PIC9K-His-ATADE）was constructed.Because p PIC9K-His is a secretory expression vector,the fermentation supernatant was separated and purified,and the target protein was obtained.SDS-PAGE electrophoresis showed that the molecular weight was 36 k Da,and the enzyme activity test showed that the recombinant protein had the activity of AFB₁degrading enzyme.In addition,the small-scale shake flask fermentation conditions of recombinant strain GS115 were optimized,and the best induction conditions were as follows:inoculation amount was 2.5%,methanol concentration was kept at 4%,and induction temperature was 30℃for 72 h.Through the degradation of AFB₁by recombinant ATADE at different p H and temperature,it was found that the enzyme was sensitive to p H,and the degradation rate was higher under alkaline conditions.In addition,it is more suitable for degradation of AFB₁at 30℃～40℃.4.The degradation products of the degradation enzymes were assayed to obtain the structures of the degradation products and the putative degradation pathways,and to demonstrate the toxicity and mutagenic effects of the degradation products by zebrafish assays and Ames assays.By UPLC-MS assay,we identified AFB₁and four possible degradation products based on the determined parent ion and fragment masses,as well as the standard molecular weight of AFB₁.The m/z of these components were 299.0914(C₁₇H₁₄O₅),343.0812(C₁₈H₁₄O₇),287.0914(C₁₆H₁₄O₅),375.1074(C₁₉H₁₈O₈).The molecular structures of the degradation products were obtained from the secondary mass spectrometry fragmentation profiles of the products and from the relevant literature.The pathways of production of the degradation products were inferred from the structures of the products,mainly lactone ring breakage and the substitution reaction of the furan ring.In addition,the hepatotoxicity and mutagenicity of the degradation products were assessed by zebrafish assay and Ames assay.The results showed that when the degradation products treated zebrafish larvae at 96 hpf,the survival rate of zebrafish was 100%,no liver atrophy and other deformities were observed,and the serum enzyme activities of ALT,AST and LDH were not significantly different from those of the blank control group（P>0.05）,indicating that the degradation products did not have toxic effects on zebrafish.The results of the Ames experiment showed that the spontaneous gyration of the degradation products was not greater than twice the spontaneous gyration of the test strains themselves,but was significantly different compared to the positive control（P<0.05）.Therefore,the degradation products were not judged to be mutagenic.