Propane pathway

There is still plenty of room for improvement in the propane pathway. The identified bottlenecks, enzymes CAR and ADO could be studied further to find out whether there are more efficient enzymes with the same function available in the nature. If not, it might be worth the effort to try and engineer the existing CAR and ADO to be more efficient, as has already been once done for ADO. The idea of having different enzymes of the pathway close together, by fusion to each other or by using different kinds of scaffolds, including our amphiphilic proteins, could also be studied further.

In out pathway model, we also identified the propane output to be sensitive to NADPH/NADH concentration. Therefore, it might be that NADPH/NADH is a limiting factor, if its generation is insufficient. This is something worth studying, and if NADPH/NADH regeneration is indeed verified to be a bottleneck, then it could be studied whether this regeneration could somehow be enhanced.

Even though it was not possible within our timeframe, one could try knocking out more endogenous aldehyde reductases and alcohol dehydrogenases that compete with ADO for butyraldehyde. This approach has been tried by Pauli Kallio and his associates with success: knocking out two endogenous aldehyde reductases Ahr and YqhD resulted in significant improvement in propane output.

Propane out of sunlight, water and thin air

One significant benefit of the pathway is that it can operate in the presence of ​oxygen. This is required to incorporate the pathway in oxygenic, photosynthetic organisms like cyanobacteria. Cyanobacterial propane production could have a tremendous effect on the way energy is produced and consumed in the society. Fuel production would essentially require only sunlight, water and CO\(_2\), and would thus be completely renewable.

Improving safety

As propane is a highly flammable liquid, large-scale microbial production could pose a fire and/or explosion hazard. The production would most likely happen in closed containers with nothing to ignite the gas, easing the problem. However, leaks are always possible: the propane or the bacteria themselves could leak from the microbial container or pipelines to enclosed spaces where ignition is possible. By replacing air, propane could also cause a suffocation danger. To help avoid these problems, it would be beneficial if the propane could be detected. However, propane itself cannot be seen and it has no odour, making detection difficult. Gas molecules with an odour (e.g. ethyl mercaptan) could be added to the purified product and production container. However, this does not allow us to detect the gas if the bacteria leak to produce propane in an enclosed space where no such safety measures are taken.

To help detect microbial propane production, it might be thus reasonable to have the propane-producing bacteria also produce a certain scent when propane production is active. This could be achieved by for instance incorporating a banana smell generator device to the same bacterium which is producing propane. Another, perhaps even better option would be to modify the bacteria so that they need to be given certain nutrients not widely available in the environment to survive.