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 ethylene producing pathway from plants, 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 plant pathway produces hydrogen cyanide as byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins. Cyanide can kill a person within 10 minutes even at very low concentrations (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 HCN removing reaction produces hydrogen sulfide, which is also toxic and inflammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.

The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.

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 unsaturated hydrocarbon and one of the chemical most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.

Whatever the final employment, ethylene is distributed, stored and consumed in airtight systems always at high pressure. The fact that ethylene is asphyxiating, extremely flammable and explosive at 27.000 ppm renders the usage of ethylene quite dangerous. (Sigma-Aldrich MSDS sheet) (R A Strehlow, INT SYMP ON COMB, 14(1):1189-1200, 1973)

Ethylene handling requires many precautions: the employees of every manufacturing facility must attend an advanced training program, unnecessary ethylene exposure must be limited and safety equipment have to be present in every facility to this purpose. Because of ethylene asphyxiating characteristic, the American Conference of Governmental Industrial Hygienists adopted a Threshold Limit Value of 200 ppm for ethylene as an 8-hour time-weighted average. (Ethylene Product Safety Assessment of The Dow Chemical Company, 8 Dec 2007, LINK)

A very serious issue is the possibility of explosion that can cause injuries and, in some cases, deaths.

On 3 July 1987 an explosion occurred inside an ethylene oxide purification column at a chemical factory at Antwerp, Belgium. The explosion was due to decomposition of ethylene oxide. It was accompanied by a fire ball, which started a number of secondary fires. These, together with blasts and missiles, caused extensive damage. Fourteen people were injured. (F I Khan and S. A Abbasi, J LOSS PREVENT PROC, 5(12):361-78, 1999)

Even in the field of fruit ripening some cases of explosion can be found. (United Press International, 20 Jan 2013, LINK) (George Ramos, Los Angeles Times, 7 Jul 1999, LINK)

Using our system can avoid this issue. In fact, our transformed strain cannot produce ethylene in a concentration high enough to be explosive. 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.