Exeter/4 June 2013

Today we had a discussion about potential project ideas with Dr. John Love and Dr. Tom Howard. We mainly focussed on BioFuels and potential aspects of the BioFuel industry that could work with an iGEM project. Our notes are below.

BioFuels

Biofuels fall into 2 categories:

• Alcohols, mainly ethanol

• Biodiesel (FAMES and FAEES), similar to fatty acids

Both must be blended with petrol before combustion.

Diesel is just a chain of hydrocarbons. They therefore contain no oxygen until they are burnt. The addition of oxygen means fuels can take up water which corrodes engines.

There are many examples of hydrocarbons in nature, but few pathways have been characterised (Arabidopsis and cyanobacteria). Can we makes these hydrocarbons naturally?

Cyanobacterial hydrocarbon biosynthesis

• Takes fatty acyl-ACP and converts to a fatty aldehyde, then to an alkane/alkene

• Uses common initial substrates (Acetyl CoA and Malonyl-ACP)

• Can insert pathway into E. coli.

• Want to control output and only produce required chain length (<C17) and branched alkanes (fuel is less viscous due to difficulty stacking, different fuels used for different conditions)

• In cyanobacteria pathway, could also utilise fatty acids which are produced in abundance (higher yield) and input into the alkane/alkene area of the pathway (fatty acid to fatty aldehyde to alkane/alkene)

Bacterial luciferase system

• Artificial pathway uses 3 lux genes and the red enzyme

• Change alkane output by changing nature of fatty acids (third trace graph). Eg. put in C14 chain with a branch, produced C13 alkanes with a branch

• This can’t be done in cyanobacterial pathway, have to use artificial path

• Change slr1609 and cyanobacteria CAN do this

Combine thioesterase gene with the various lux genes to produce alkanes/alkenes of required length

• Now have to try and make branched chains

• E. coli doesn’t do branching but others to (branched fatty acids) although can be bad model organisms

• Construct 5 gene operon (in green) to produce branched primers

Improving productivity and yield

• Yeast (currently to makes ethanol) converts ~90% of its glucose to ethanol

• Paper: determining promoters and ribosome binding sites

• Promoters on LHS of grid. If same square is red on both grids = good. Both blue = bad. One red, one blue, can use for other analysis

Use BioBricks to induce artificial variation

Using crops is NOT LONGER SUSTAINABLE

• Waste biomass (cellulose, waste water from chemical manufacturing processes, animal waste)

Make it really clear how a profit can be made from the process

• remember that fuel is not meant to stick around, we burn it and then it’s gone

Can we radicalise E. coli? Allow bacteria to function in difficult circumstances? High heat levels, high waste metabolite levels, etc.

Can we use heat as an energy source? Can this heat be concentrated?

Can we use endothermic reactions as a heat sink to remove “waste” heat from more important reactions? Activate a solution and convert biotic energy…

Papers to look at:

Engineered reversal of the beta-oxidation cycle for the synthesis of fuels and chemical, Dellomonaco et al, 2011 Integrated electromicrobial conversion of CO2 to higher alcohols, Li et al, 2012

Take me back to the notebook.