Template:NAU-CHINA/mycotoxin
Degradation and
Acetylation
- Background
-
Mycotoxins are secondary metabolites produced by filamentous fungi that can contaminate agricultural crops in the field as well as during harvest, transportation, processing, or storage.
Zearalenone (ZEN) along with deoxynivalenol (DON) are two major residues in crop processed food. Eating contaminated food can cause severe even fatal symptoms to human or livestock. Increasing attention has been paid to the development of an effective strategy for ZEN decontamination. Thus, the decontamination of ZEN in foods and feeds is an important issue. In this module, we use ZHD101, a lactonohydrolase produced by the fungal species Clonostachys rosea, to convert ZEN into 1-(3,5-dihydroxy-phenyl)-10-hydroxy-1-undecen- 6-one, a significantly less toxic product.
In terms of DON, a toxin than belongs to trichothecenes. We try to use Trichothecene 3-O-acetyltransferase(AYT1)，an indispensable enzyme for the biosynthesis of trichothecenes, to catalyzes the transfer of an acetyl group from acetyl coenzyme A to the C3 hydroxyl moiety which will result in a switch from DON to 3ADON, so the toxicity will be reduced by 100-fold.
Goals:
In degradation and acetylation part ,we aim to establish the gene pathway to degrade ZEN and acetylate DON, respectively. In our project, we want to find two enzymes to degrade ZEN and acetylate DON. So we have found that the gene zhd101 from Clonostachysrosea can degrade ZEN and the gene of ayt1 from Saccharomyces cerevisiae can acetylate DON. Also, we want to use a genetic switch which control by bule light to regulation the expression of target genes.
Design
Pathway design：
We want to construct a switchable system with excellent dark-off/light-on properties are obtained with the light-activatable VVD domain and its variants as regulatory domains and we want to express two domains which are T7 RNA polymerase (RNAP) domains N4564 and C565 with VVD fragments, respectively.
The VVD domain can connect spontaneously by light induction. Thus, the two domains N564 and C565 can connect by connection of VVD domains. After connection, the complete T7 RNA polymerase will possess its activity and bind to T7 promoter to activate the expression of downstream gene ZHD101 or AYT1.
Experimental design:
light-operated switch
Construction expression vector
We got the plasmid which has sequences N564 and C565 from University of Science and Technology of China. N564 and C565 sequences were amplified by PCR from plasmid. Amplified products were digested with restriction enzymes (Takara) and ligated into vectors which contain Ampicillin and Kanamycin resistance gene.
Fluorescence data assay
Transformed cells were cultured in LB medium containing Ampicillin and Kanamycin under dark conditions to mid log phase. Then cells were continuously grown in the dark or switched to light exposure for gene expression measurement in dark or light state, respectively. To investigate the process of activity changing from the light condition to the dark condition, cells were placed in the dark after 5 h of light treatment. Samples were collected once per hour over a 14 h period. The fluorescence values at each time point were normalized by OD600 to provide fluorescence strength per cell.
ZEN degradation
Construction of eukaryotic expression vector:
We got the degrading enzyme sequence ZHD101 from an article (GenBank accession number AB076037 ) and carried out the codon optimization for the expression in the Pichia pastoris. Then we synthesized this gene sequence into the expression vector of Pichia pastoris which called as ppiczαA, choosing ECOR1 and Xba1 as enzyme sites. That means our sequence can be activated by AOX promoter and be secreted out of the cell.
The recombinant plasmid is transferred to the yeast by electrical transfer:
First, we expanded the monoclonal Pichia pastoris containing recombinant plasmid, and then used the bacteria fluid to verify the transfer was successful by PCR.
Functional assay:
We used BMGY to develop the basic culture. 12 hours later, we started to induction culture for adding a certain amount of methanol every 12 hours and it was keeped for 3 days before we ended it. Then we centrifuged induced liquid at 4℃, 12000RPM, and collected centrifugal supernatant as our reaction fluid. Then we put a certain amount of zearalenone into our reaction fluid for reacting. After some minutes, we ended the reaction and used dichloromethane to extract the organic phase. Then we put them in super clean bench for dryness, and then measured the effects of our ZEN-degrading enzyme by HPLC
HPLC Measures:
Our flowing phase is acetonitrile/water by 50/50, and our samples were dissolved by methanol. Our target product appeared at about 245nm.
DON acetylation
Construction of prokaryotic expression vector
AYT1 sequence from Saccharomyces cerevisiae was downloaded from GenBank and aligned using Snapgene 2.3.2. Amplification primers were designed based on AYT1 sequence on Primer 5. We added restriction sites BamhI and SalI on forward primer and reverse primer respectively and also with protection bases. AYT1 genes were amplified by PCR from genome of Saccharomyces cerevisiae. Amplified products were digested with BamHI and SalI restriction enzymes (Takara) and ligated into a modified pET28a (National Center for Protein Science, Shanghai) vector containing an N-terminal His6 tag and suom tag
Expression of AYT1 enzymes in E. coli.
The AYT1 orthologs were expressed in E. coli strain BL21(DE3). Starter cultures from a single colony were grown overnight in Luria broth (LB) supplemented with 100 g/ml kanamycin. Five milliliters of the starter culture was used to inoculate 1 liter of LB medium including kanamycin. Cultures were grown to mid-log phase (OD600 0.6) at 37°C, cooled to 16°C, and induced with 1 mM β-D-thiogalactopyranoside (IPTG; Sigma Chemical Co.). Cells were harvested after 20 h by centrifugation at 4000rpm, washed with a buffer containing 10 mM HEPES and 100 mM NaCl at pH 7.6, and flash frozen in liquid nitrogen. Cells were stored at -80°C.
Purification of AYT1 protein
All purification steps were carried out on ice or at 4°C. Seven grams of transformed E. coli cells were resuspended in 50 ml lysis buffer and cells were lysed by sonicator. The lysate was clarified by centrifugation and the supernatant was loaded onto a column of nickel-nitrilotriacetic acid-agarose (Ni-NTA). After the protein was eluted in a linear gradient, the fractions containing the AYT1 protein were identified by SDS-PAGE and Coomassie blue staining. Then protein was drop-frozen in liquid nitrogen and stored at -80°C.
Enzyme function assays
First, we want to analyse enzyme function of this protein in E.coli. We use 20 μL of the starter culture inoculate 4 mL of LB medium including kanamycin. Cultures were grown to mid-log phase (OD600 0.6) at 37°C, cooled to 16°C, and induced with 1 mM β-D-thiogalactopyranoside (IPTG; Sigma Chemical Co.) and deoxynivalenol (DON; Sigma Chemical Co.). Cells were harvested after 20 h by centrifugation at 12000rpm and the supernatant was removed into a new EP tube. After extracting by dichloromethane, we use UV spectrometer to assay acetylation ability of AYT1 protein preliminarily. We also use the AYT1 protein to do extracellular reaction. Sample 1(a, b, c), as contrast groups and Sample 2(a, b, c) as test groups. Each group has three repetitive samples.
| trichothecene 3-O-Acetyltransferase(40 μL ,5 μg/mL) | Acetyl-CoA(2μL,10mg/mL) | deoxynivalenol(DON)(3μL,2mg/mL) | H2O(5μL) | |
| Sample 1a | × | × | √ | √ |
| Sample 1b | × | × | √ | √ |
| Sample 1c | × | × | √ | √ |
| Sample 2a | √ | √ | √ | √ |
| Sample 2b | √ | √ | √ | √ |
| Sample 2c | √ | √ | √ | √ |
√ = exist ×=do not exist
Analysis by High Performance Liquid
Chromatography(HPLC)
We use the High Performance Liquid Chromatography(HPLC) to analyze the ability of acetylation of AYT1 protein. Column: Eclipse XDB-C18 column(250mm × 4.6mm i.d.，5μm ), the condition of Chromatography: flowing phase:
Acetonitrile: H2O is 1:4; wave at 219 nm; 10 minutes.
Data and Result
light-operated switch
In the dark state, the fluorescence intensity under 2500A.U. In the light state,it is very clear that the fluorescence intensity is much higher than that in the dark state.According to these datas, the light-operated switch was functional.
fig.1 Fluorescence strengths of the two-fragment systems associated with split positions.
ZEN degradation
Fig2. is our Uv mapping result. For this result we set four time point, Z2 as our time point 1 which was taken after 2 hours’culturation, shown in pink colour line on the figure and Z4 as time point 2 which was taken after 4 hours’culturation, shown in dark blue line while Z6 as our time point 3 which was taken after 6 hours’culturation shown in pretty blue line. The bottom of the figure is time point 4 which was taken after 8 hours’culturation.
We added zearalenone by 1 ug/ml and then the degrading enzyme started to work. The ZEN was degraded as time went by.The obvious degrading effect is proved by the height of Uv mapping.
We use the High Performance Liquid Chromatography(HPLC) to analyze the ability of zearalenone(ZEN) degrading enzyme.And from articles we know that the absorption peak is in about 245 nm. The following figure shows : Data1 representing the sample only with ZEN as contrast group, the peak appears at 2.5 min represents ZEN and the peaks appear at 3.5min represent the isomers of ZEN. Its peak area is about 27570 uV·min.
Data 2 from our test groups(sample 2 which reacts for 15minutes.). As we can see from the figure, there are about two peaks. The peak which appears at 3min represents ZEN and the peak which appears at 0.8min represents the degrading products of ZEN. The other folling lines appear from 5min represent the degrading products of the ZEN and its isomer. And compared with data1 we can see an obvious decrease at 2.5min and increase at 0.8min.
Data3 from our test groups(sample 3 which reacts for 30 minutes.). As we can see from the figure, there are about two peaks. The peak which appears at 2.5min represents ZEN and the peak which appears at 6min represents the degrading products of ZEN and its isomer. Compared with data2, we can see a decrease at 2.5min and a little increase at 6min. It shows that with the reaction time going, more and more ZEN and its isomer are degraded.
Data4 from our test groups(sample 4 which reacts for 60 minutes.) As we can see from the figure, there are about two peaks. The peak which appears at 2.5min represents ZEN and the peak which appears at 6min represents the degrading products of ZEN and its isomer. Compared with data3, we can not see a decrease at 2.5min and increase at 6min. It shows that the reaction has nearly ended at 30 min.
So, we can give the conclusion that the best reacting time for degrading is about 30minutes, by calculate the peak area of data1 and data3. We preliminarily proved that ZEN-degrading enzyme from the sequence (which comes from GenBank AB076037 and then optimized by us) works.
DON acetylation
The result of construction of prokaryotic expression vector. From fig4, we can identify that colony 1 is recombinant strain.
From fig5, we can identify that nearby 66KDa, a band was displayed on test sample but wasn’t displayed on control sample. So the expression of AYT1 protein was successful.
Fig6 shows that we use the MSD to analyze the degrading effects of our DON degrading enzyme.In theory, the higher one of the two absorption peaks for BL21+DON group and DON group should be the same. In fact, the result in the figure qualifies our inference.(dont in view of the extraction efficiency).Compared with our control groups(BL21+DON and DON), there is a peak valley in DON’s absorption peak for our test group BL21+DON+IPTG, from which the DON degrading enzyme can be induced and then expressed. It proves that the degrading enzyme expressed in BL21 has a significant degrading effect. Download
We use the High Performance Liquid Chromatography(HPLC) to analyze the ability of acetylation of trichothecene 3-O-Acetyltransferase from Saccharomyces cerevisiae. The figure above shows data 1 representing the sample only with deoxynivalenol(DON) as contrast groups(sample 1a, 1b, 1b) and data 2 from our test groups(sample 2a, 2b, 2c). As we can apparantly see from the figure, there are three peaks. The peaks which appear at 2min and 4min represents the isomers of DON while the peak which appears at 3min represents DON. Due to the specifity of trichothecene 3-O-Acetyltransferase, its efficiency of acetylation on the isomers of DON is quite low. So we can not see an obvious decrease at 2min and 4min. However, at 3min the peak area of data1 is 4400 uV·min and that of data2 is 1486 uV·min and the ability of acetylation can reach as high as 66.23%. We preliminarily proved that trichothecene 3-O-Acetyltransferase from Saccharomyces cerevisiae works in the transformation from DON to 3ADON.
Key Achievements
◎Successful verification of light-operated switch activate the transcription of downstream gene under the induction of blue LED lights.
◎The expression of ZHD101 was successfully expressed in Pichia pastoris expression system and BL21 prokaryotic expression system, and it was proved that ZHD101 had a significant effect on the degradation of ZEN toxin.
◎The expression of AYT1 was successfully expressed BL21 prokaryotic expression system, and it was proved that ZHD101 had a significant effect on the degradation of DON toxin.
What’s Next
◎Try to transform light-operated switch and AYT1 into eukaryotic expression vector for expression, to achieve the ultimate goal: make the whole bioreactor function in the eukaryotic expression system.
◎The structure and stability of ZEN and DON toxins after being modified and degraded by ZHD101 and AYT1 need to be further verified by mass spectrometry and other means.
◎The physiological activity and cytotoxicity of ZEN and DON toxins after being modified and degraded by ZHD101 and AYT1 need to be further explored.
Reference
1.Successful verification of light-operated switch activate the transcription of downstream gene under the induction of blue LED lights.
2. Alexander NJ, McCormick SP, Hohn TM. 1999b. TRI12, a trichothecene efflux pump from Fusarium sporotrichioides: gene isolation and expression in yeast. Mol Gen Genet 261: 977–984.
3. Alexander, N. J., T. M. Hohn, and S. P. McCormick. 1998. The TRI11 gene of Fusarium sporotrichioides encodes a cytochrome P-450 monooxygenase required for C-15 hydroxylation in trichothecene biosynthesis. Appl. Envi- ron. Microbiol. 64:221–225.
4. Chatchai KOSAWANG,Magnus KARLSSON, David B. COLLINGE,Birgit JENSENa,Dan Funck JENSENb. Zearalenone detoxification by zearalenone hydrolase is important for the antagonistic ability of Clonostachys rosea against mycotoxigenic Fusarium graminearum[J]. fungal biology 118 (2014) 364 -373
5. Daniel A. Sousa, Octávio L. Franco. Influence of Cysteine and Tryptophan Substitution on DNA-Binding Activity on Maize α-Hairpinin Antimicrobial Peptide[J]. Molecules 2016, 21, 1062
6. Graeme S. Garvey , Susan P. McCormick , and Ivan Rayment1．Structural and Functional Characterization of the AYT1 Trichothecene 3-O-Acetyltransferase from Fusarium sporotrichioides and Fusarium graminearum[J]. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 3, pp. 1660 –1669, January 18, 2008
7.Long M，Liu Y，Chen X L，et al. Construction of zhd101 gene expressing vector and its expression in Escherichia coli [J]. Animal Husbandry & Veterinary Medicine，2016，48 ( 1) : 1 - 4
8. Mitsuyuki Ichinose, and Masashi Sawada．A Flow Cytometric Assay Reveals a Suppression of Phagocytosis by Rabbit Defensin NP-3A in Mouse Peritoneal Macrophages[J]. Microbiol. Immunol., 39(5), 365-367, 1995
9. Naoko Takahashi-Ando . Takeshi Tokai . Efficient decontamination of zearalenone, the mycotoxin of cereal pathogen, by transgenic yeasts through the expression of a synthetic lactonohydrolase gene[J]. Appl Microbiol Biotechnol (2005) 67: 838–844
10. Nancy J. Alexander, Susan P. McCormick and Thomas M. Hohn2.The identification of the Saccharomyces cerevisiae gene AYT1(ORF-YLL063c) encoding an acetyltransferase. Yeast 2002; 19: 1425–1430.
11. Piyum A Khatibi, Justin Montanti, Nhuan P Nghiem. Conversion of deoxynivalenol to 3- acetyldeoxynivalenol in barley-derived fuel ethanol co-products with yeast expressing trichothecene 3-O-acetyltransferases. Biotechnology for Biofuels 2011, 4:26.
12. Piyum A. Khatibi, Sean A. Newmister, Ivan Rayment. Bioprospecting for Trichothecene 3-O-Acetyltransferases in the Fungal Genus Fusarium Yields Functional Enzymes with Different Abilities To Modify the Mycotoxin Deoxynivalenol. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2011, p. 1162–1170
13. SUSAN P. MCCORMICK, NANCY J. ALEXANDER, SUSAN E. TRAPP. Disruption of TRI101, the Gene Encoding Trichothecene 3-O-Acetyltransferase, from Fusarium sporotrichioides. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1999, p. 5252–5256.
14. Tiyun Han, Quan Chen and Haiyan Liu. Engineered Photoactivatable Genetic Switches Based on the Bacterium Phage T7 RNA Polymerase. ACS Synth. Biol. 2017, 6, 357−366
15. WenChun Yang, TsuiChun Hsu, KuanChen Cheng. Expression of the Clonostachys rosea lactonohydrolase gene by Lactobacillus reuteri to increase its zearalenone-removing ability [J]. Microbial Cell Factories, (2017) 16:69
16. Yuichi NAKAJIMA, Tatsuro KAWAMURA, Kazuyuki MAEDA．Identification and Characterization of an Inhibitor of Trichothecene 3-O-Acetyltransferase, AYT1, by the Chemical Array Approach[J]. Biosci. Biotechnol. Biochem., 77 (9), 1958–1960, 2013
17. Yuqian Tang, Junmei Xiao, Xinglong Xiao, Hui Wua. Secretory expression and characterization of a novel peroxiredoxin for zearalenone detoxification in Saccharomyces cerevisiae[J]. Microbiological Research 168 (2013) 6–11
