Team:UNITN-Trento/Safety

When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the plants ethylene producing pathway, because we wanted to work on fruit ripening and we knew that ethylene is a plant hormone able to accelerate this process.

After a careful analysis we stumbled upon a problem: the pathway produces hydrogen cyanide as byproduct, an highly toxic gas that inhibits the cytochrome C oxydase enzyme. It can kill a human within 10 minutes even at very low concentration (around 300 ppm). Plants however have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!

Fruit renders hydrogen cyanide harmless thanks to the β-cyanoalanine synthase enzyme, which catalyzes the synthesis of β-cyanoalanine from cystein and hydrogen cyanide. The issue is that this reaction produces also hydrogen sulfide, which is toxic and inflammable. We weren't able to find a biological way to remove this compound, so we tried to look further into alternative pathways.

The plant pathway would have been a convenient way to produce ethylene. It already contains SAM synthetase, that our team was planning to exploit also for methyl salycilate production. However, we tried to look further into alternative pathways.

We found three alternative pathways: one very complicated and involving superoxide species, and the remaining two exploiting the same enzyme derived from Pseudomonas syringae. Ps is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. It takes 2-Oxoglutarate as substrate to transform it into ethylene, water and carbon dioxyde (Goto M. Plant and Cell Physiology (2012) 26, 141-150).

Ethylene is the simplest unsatured hydrocarbon. Like all hydrocarbons, it is asphyxiating and flammable. In the ripen facility it is stored in high pressure cylinders that can be very dangerous.

Using our system can avoid this issue. In fact, our transformed strain cannot produce ethylene in a concentration high enough to be explosive (from 2.7% to 34% v,v is needed). With an air/culture volume ratio equal to 4 we detected and quantified around 200 ppm of ethylene.

We carefully checked the MSDS for both ethylene and MeSA. We always worked with our producing ethylene bacteria under the hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed the connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk when ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts carachterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold.

We used some strains of E. coli (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM29) and one strain of B. subtilis (168). All these strains belong to the risk group 1. In our project we used only one biobrick coming from organisms of the second risk group: BBa_J45319: catalyses the production of salycilate from chorismate and comes from Pseudomonas aeruginosa. If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up, anyway our vending machine will not contain sufficient ethylene to be risky. Besides, most of our parts are under control of inducible promoters. Furthermore the strain of B. subtilis that we used is auxotrophic for thriptofane (and also threonine if transformed).

Safety forms were approved on September 23, 2013 by Evan Appleton.