Team:TP CC-SanDiego

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<li id="Link0" class="SlideLink">Introduction</li>
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<h1>The Project</h1>
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Our project focuses on two main points:</div>
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1) The breakdown of deadly mycotoxins into harmless byproducts through the use of  various enzymes.</p>
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2) Maximizing production rates of said detoxifying enzymes . </p>
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Mycotoxins are a type of harmful, and sometimes lethal, toxin produced by fungi. These fungi readily colonize improperly stored crops like maize, coffee beans, peanuts, pistachios, and tobacco. Consequently, mycotoxicosis, or poisoning from the consumption of mycotoxins, has become a prevalent problem in third world countries where crops are commonly improperly stored in moist conditions. Mycotoxicosis is also spread through the consumption of infected animals, including but not limited to, pigs, cows, and birds. </p>
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One notable mycotoxin is Aflatoxin B1 (AFB1), a potent natural carcinogen and an acutely toxic agent synthesized by Aspergillus flavus. 5-50 mg of AFB1 per kilogram of body mass will kill a median member of the human population, as grimly proved in Kenya, where the development of A. flavus on maize killed 120 local people and affected hundreds more in 2003. </p>
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Another widespread mycotoxin is Zearalenone, which is secreted by fungi of the Fusarium genus, including F. graminearum and F. cerealis. Zearalenone affects many cereal plants, including barley, oats, wheat, rice, and maize. Although the Zearalenone toxin (ZEN) is not as lethal as AFB1, it also functions as an oestrogen. Research has shown high levels of ZEN causes birth defects and interference with ovulation, implantation, and fetal development.</p>
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Thankfully, there is a solution to this dangerously extensive problem. Through research we found multiple mycotoxin-degrading organisms that secrete various detoxifying enzymes. These organisms, Armillariella tabescens and Clonostachys rosea, produce Aflatoxin-Detoxifizyme (ADTZ) and Zearalenone Hydrolase (ZHD101), respectively. ADTZ’s method of degradation of AFB1 is as follows: AFB1 is first converted into AFB1-epoxide and then hydrolyzed to form dihydrodiol. The final hydrolysis opens the difuran ring in dihydrodiol, ending the conversion of AFB1 into a vastly less dangerous form. The method of degradation used by ZHD101 on ZEN is unclear to researchers as of now, but it is hypothesized that the ZEN molecule is converted into a less oestrogenic form through a simple cleavage. </p>
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While these detoxifying enzymes are successfully synthesized by the fungi, secretion could be vastly improved by producing these enzymes in high-secretion bacteria such as E Coli. By creating a bacterial plasmid containing the signaling sequence (ß-lactamase or α-amylase), the gene of interest (ZHD101 or ADTZ), and a HIS tag (for protein detection), the synthesized protein would be easily secretable, detectable, and isolatable. The increased production system using E. Coli will allow large quantities of detoxifying enzymes to be harvested for agricultural application. These mass-produced enzymes can then be used to destroy AFB1 and ZEN present on crops, reducing cancer incidences and deaths from mycotoxicosis. Perhaps, with the detoxifying enzymes that will be produced in our experimentation, the deadly outbreaks of these deleterious mycotoxins will be an incidence of the past.</p>
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Latest revision as of 22:44, 19 June 2014

iGEM San Diego

Statistics
Statistics that are quite relevant to the nature of this experiment

Toxicity of aflotoxin is 10 times that of hydrocyanic acid and 68 times of arsenic.

This disease is the third-leading cause of cancer death globally according to WHO (2008), with about 550,000–600,000 new cases each year.

Mycotoxins affect nearly 15-20% of all crops.
Engineering
E. Coli Capable of Extracelluar Secretion of Mycotoxin Detoxifying Enzymes

Microfungi produces harmful mycotoxins that flourish on improperly-stored nuts, grains, meat, and dairy. They especially thrive in developing countries, where the lack of advanced food storage and mycotoxin exposure cause 40% of the diseases. To lessen the problem, our team engineered E. coli strains using synthetic biology tools to produce chimeric mycotoxin-degrading fungal enzymes, Aflatoxin-Detoxifizyme (ADTZ) and Zearalenone Hydrolase (ZHD101), which are designed to secrete extracellularly by fusing with secretion signal peptides from alpha-amylase and beta-lactamase. In this study, we have successfully generated synthetic genetic materials to produce four chimeric mycotoxin-detoxifying enzymes. The levels of extracellular secretion is also characterized and analyzed. The project will allow a mass production of detoxification enzymes in a cost effective way, preventing the squandering of harvested crops, and limiting mycotoxin-related diseases. Increased access to these proteins will have immense commercial, industrial, health, and agricultural impacts.

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