Difference between revisions of "Team:MIT/BiodieselProduction"

Using the method given by Steen et al. (2010), we worked on engineering E. coli to produce fatty acid ethyl esters (FAEEs) directly from sugars. The metabolic pathway for this is given by the diagram:
In order to get E. coli to produce FAEEs from simple sugars, one must ensure overproduction of both fatty acyl-coA’s and ethanol. These are the substrates of the reaction, catalyzed by the wax ester synthase atfA, that produces FAEEs.

Steen et al. (2010) have made the following modifications to overproduce fatty acyl-coAs in E. coli:

1. Overexpression of a leaderless thioesterase A (‘tesA): E. coli naturally produces fatty acyl-ACPs from sugars via fatty acid biosynthesis (FAB) pathways. E. coli uses a native thioesterase A (tesA) enzyme to convert these fatty acyl-ACPs into free fatty acids. Others have shown that overexpressing a leaderless version of tesA, designated ‘tesA, which is targeted to the cytosol instead of the periplasm, causes a buildup of free fatty acids by disrupting regulation of fatty-acid synthesis (Cho and Cronan 1995; Liu et al. 2012).
2. Overexpression of fatty acid degradation D (fadD): in E. coli, fatty acid degradation D (fadD) enzyme catalyses the first step in E. coli beta-oxidation, the conversion of free fatty acids into fatty acyl-coA’s. By overexpressing this gene, one can increase the production of fatty acyl-coAs from the free fatty acids produced by ‘tesA (Steen et al. 2010).
3. Knockout of fatty acid degradation E (ΔfadE): fadE catalyzes the second step in fatty acid degradation and uses fatty acyl-CoA’s as substrates. In order to prevent the to prevent the degradation of fatty acyl-CoAs, the β-oxidation pathway was blocked by deleting the fadE gene (Steen et al. 2010).

Next, Steen et al. (2010) have made the following modifications to overproduce ethanol in E. coli:

1. Insertion of pyruvate decarboxylase (pdc): pdc is an enzyme from Zymomonas mobilis that converts pyruvate into acetaldehyde. E. coli naturally produces pyruvate from simple sugars.
2. Insertion of alcohol dehydrogenase (adhB): adhB is an enzyme from Zymomonas mobilis that converts acetaldehyde into ethanol.

Finally, insertion of wax ester synthase (atfA) from Acinetobacter baylyi catalyzes the reaction that combines acyl-coA’s with ethanol to produce FAEEs.

We obtained the A2A DH1 strain from the Keasling Laboratory, which had the fadE knockout and contained the following plasmids, with the corresponding genes, promoters and selection markers:

• pKS1: p15a PlacUV5: ‘tesA Cam
• pKS17 pBBR PlacUV5: pdc, adhB, atfA Tet
• pKS104: ColE1 PlacUV5: fadD (M335I), atfA Amp