Team:Arizona State/science

Biodiesel Production in a Multi-Strain System

Current system of biodiesel production in E. coli
When attempting to produce biodiesel in E. Coli, certain problems emerge, especially surrounding the metabolic use of certain intermediate products. E. coli has to use the pyruvate from glycolysis to produce both intermediates of wax ethyl ester, ethanol and acyl-COAs. In order to circumvent this metabolic competition, we decided that specialization in a multi strain system would be our solution. Two colonies of E. Coli will create the intermediates individually, therefore avoiding competition for use of resources inside of the individual cells. With the two colonies growing together, a greater titer of the final product (FAEE's) can be accomplished.

Our Solution
The first intermediate, ethanol, is formed using a two enzyme plasmid. Pdc and adhB take pyruvate from glycolisis and convert it into first, acetaldehyde, and then into ethanol.

The second intermediates, acyl coa's, are formed with a thioesterase combined with the four components of the Acc plasmid (A,B,C,D). The two intermediates are combined with a wax-synthase and a coa-ligase to produce fatty acid ethyl esters.

Plasmid Designs
The two strains of E. Coli will both have one plasmid, containing all of the necessary genes to code for an entire intermediate path, with one of the plsmids also containing the genes for the final wax synthase/coa ligase step. Both plasmids would need resistance to the same antibiotic or set of antibiotics. Both strains will be placed in a culture together, and glucose will be fed.

Plasmid 1: Fatty Acid Producer
Promotor-RBS-TesA-RBS-ACCabcd-terminator

Plasmid 2: Ethanol Producer
Promotor-RBS-Pdc-RBS-adhB-fadD-atfA

Future plans for this project
There are some future objectives that can expand upon this project. Maximizing the production of ethanol and acyl-COAs without stopping normal cellular metabolism can optimize the production of the fatty ethyl esters. Another consideration is the balance between the ethanol and fatty acid populations. It is possible that more of one population is required to optimize production. Quorum sensing techniques could be used to track the two populations and reach an equilibrium with high production.