fadD & fadL Module

The ultimate goal of our project is to genetically engineer an E. coli strain to increase its fatty acid uptake efficiency and ability to convert the absorbed fatty acid into α-linolenic acid (ALA).

In order to achieve this, we have sub-divided our project into three parts:
a. Enhanced uptake of long chain fatty acids (LCFAs) by E. coli
b. Increased conversion of fatty acyl-CoA into free fatty acid
c. Enhanced conversion of free fatty acid into ALA

The uptake of exogenous long chain fatty acids is controlled by the fadL and fadD genes.

Figure 1. Importation of long-chain fatty acids by FadL and FadD.

Long chain fatty acids (LCFAs) are protonated in the periplasmic space and FadD facilitates its transpoprt into the cytosol.

The fadL gene codes for an outer membrane-bound fatty acids transport protein that facilitates the import of long chain fatty acids into the periplasmic space where they are protonated and then partitioned at the inner membrane (with the ionic head pointing towards the periplasmic space). Protonated fatty acids are unable to pass through the inner membrane into the cytosol and requires the assistance of the FadD protein. The fadD gene codes for an inner membrane-associated long chain acyl-CoA synthetase enzyme in E. coli which catalyses the addition of a CoA moiety to the fatty acid and its subsequent transport across the inner membrane into the cytosol. FadD is therefore required both for the transport of LCFAs through the inner membrane into the cytosol and their activation for metabolism via the β-oxidation pathway. By overexpressing FadL and FadD, we aim to enhance LCFA uptake to boost the subsequent conversion of long chain acyl-CoA to ALA.

References:

Desaturase Module

After absorption of exogenous fatty acids from the environment, the enzymes, Δ9, Δ12 and Δ15 desaturases, catalyze the conversion of fatty acids (stearic acid) into α-linolenic acid, ALA (Qiu, 2002). Because E. coli originally lacks the genes encoding the desaturases, we have to construct a recombinant DNA plasmid containing them and transform it into E. coli.

The gene sequences of the three desaturases can be obtained from Synechocystis, and optimized for expression in E. coli. After transformation of the constructed plasmid, the bacterium is able to produce the corresponding enzymes. Δ9 desaturase removes two hydrogen atoms from carbon 9 and carbon 10 of the fatty acid and a double bond will be formed there. Similarly, Δ12 desaturase works between carbon 12 and carbon 13 while Δ15 desaturase works between carbon 15 and carbon 16.

After the completion of reactions, the fatty acid (stearic acid) will be converted into ALA (Figure 1). When ALA is released from E. coli and absorbed by human cells, ALA will be further converted into docosahexaenoic acid, DHA, since human cells contain genes encoding the remaining desaturases and elongases that are necessary for the conversion of ALA into DHA (Innis, 2007), which is our final target product.

Figure 1. Conversion of stearic acid into α-linolenic acid by three desaturases.

References:
Innis, S. M. (2007). Dietary (n-3) fatty acids and brain development. The Journal of Nutrition. 137: 855.
Qiu, X. (2003). Biosynthesis of docosahenaenoic acid (DHA, 22:6-4, 7, 10, 13, 16, 19): Two distinct pathways. Prostaglandins, Leukotrienes and Essential Fatty Acids. 68(2003): 181-182.

'TesA Module

Coming soon!

References: