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:

'TesA Module

In Escherichia coli, tesA codes for the thioesterase I enzyme which is expressed in the periplasm. Although the actual physiological role of thioesterase I is not known, it has been demonstrated that thioesterase I has the ability to cleave the thioester bond in fatty acyl-CoA (Cho & Cronan, 1993). It has been previously shown that deleting the leader sequence of the TesA protein results in the enzyme being expressed as a cytosolic enzyme (Cho & Cronan, 1995). This altered form of the enzyme without the leader sequence was named ‘TesA and overexpression of the cytosolic ‘TesA has been used to increase the yield of free fatty acids (FFAs) in biotechnology as a feedstock for biofuel production (Janßen & Steinbüchel, 2014).

During the transport of long chain fatty acid (LFCA), the FadD protein in the inner membrane activates LFCA to form fatty acyl-CoA, which may be broken down to acetyl-CoA through β-oxidation. To retard the β-oxidation of fatty acyl-CoA, the ‘tesA gene is cloned and overexpressed in E. coli. It is predicted that overexpression of ‘TesA in E. coli would favor the conversion of fatty acyl-CoA to free fatty acids (FFAs) in the cytosol (Figure 1). By co-transforming the ‘tesA plasmid with the Δ9- Δ12-Δ15 desaturase gene cluster plasmid into E. coli cells, increased conversion of FFAs to α-linolenic acid (ALA) will be observed.

Figure 1. Conversion of fatty acyl-CoA to free fatty acid.

To create the ‘tesA gene construct, the coding sequence (without the leader sequence) of tesA was amplified by PCR and the PCR amplicon ligated to the RBS (BBa_B0034) and PBAD promoter (BBa_I13453) fragments.

References:
Cho, H. & Cronan, J. E. (1993). Escherichia coli thioesterase I, molecular cloning and sequencing of the structural gene and identification as a periplasmic enzyme. Journal of Biological Chemistry 268(13), 9238-9245.

Cho, H. & Cronan, J.E. (1995). Defective export of a periplasmic enzyme disrupts regulation of fatty acid synthesis. Journal of Biological Chemistry 270(9), 4216-4219

Janßen, H. J. & Steinbüchel, A. (2014). Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnology for biofuels 7(1), 7.

Lee, L., Lee, Y., Leu, R. & Shaw, J. (2006). Functional role of catalytic triad and oxyanion hole-forming residues on enzyme activity of Escherichia coli thioesterase I/protease I/phospholipase L1. Biochem. J 397, 69-76.

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.