FadD is one of a regulatory enzyme in fatty acid biosynthesis in E. coli. , which is in charge of acetylating the fatty acid and produce a molecule, acetylated-fatty acid, that initiates promoter, pFadBA, to initiate the whole gene expression. We obtain the sequence of FadD directly by cell disruption of E. coli MG1655. After obtaining FadR, we further combine it with the promoter R0010 and a strong RBS B0030.
Characterization of fadD & fadL by BUCT 2021
Characterization of fadD & fadL:
Our project wants to enhance the decomposition of fatty acids by beta oxidation in intestinal tract, so we need to enhance the involved in translocation of long-chain fatty acids across the outer membrane. This composite part consists of lac promoter and two genes which are FadL and FadD. The FadL can help long-chain fatty acids involved in translocation across the outer membrane. FadL may form a specific channel. FadD catalyzes the esterification, concomitant with transport, of exogenous long-chain fatty acids into metabolically active CoA thioesters for subsequent degradation or incorporation into phospholipids. FadD is involved in the aerobic beta-oxidative degradation of fatty acids, which allows aerobic growth of E. coli on fatty acids as a sole carbon and energy source.
PCR amplification technique was used to obtain the endogenous fadL and fadD of E. coli, then cut the two genes from the double strands. We use EcoRⅠand HindⅢ to cut the FadD, using BamHⅠand Bpu10Ⅰto cut the FadL. Last, we link the FadL and FadD by enzyme ligation method.
We configure the Gas Chromatograph test group: 200µl fermentation broth (obtain 20g/l palm oil), 2000µl petroleum ether-diethyl ether, 100µl methanol-KOH. From the chart, we can see the amount of palm oil is decreasing, and it proves this composite part can enhance the decomposition of fatty acids by beta oxidation in E. coli.
Characterization of FadD WHU-China 2021
FadD, which is also called acyl-CoA synthetase , catalyze the formation of a thiol ester bond between the fatty acid and the thiol group of coenzyme A. This condensation with CoA activates the fatty acid for reaction in the b-oxidation pathway. The reaction is accompanied by the hydrolysis of ATP to form AMP and pyrophosphate. The overall reaction has a net DG°9 of about 20.8 kJ/mol, so the reaction is favorable but easily reversible. However，the pyrophosphate produced in this reaction is rapidly hydrolyzed by inorganic pyrophosphatase to two molecules of phosphate, with a net DG°9 of about 233.6 kJ/mol. Thus, pyrophosphate is maintained at a low concentration in the cell (usually less than 10 mM), and the synthetase reaction is strongly promoted. The mechanism of the acyl-CoA synthetase reaction is shown in Figure 1 and involves attack of the fatty acid carboxylate on ATP to form an acyladenylate intermediate, which is subsequently attacked by CoA, forming a fatty acyl-CoA thioester.
Figure1. The FadD catalytic principle
In our experiment, we hope to improve β-oxidation capacity of our engineered bacteria by overexpressing FadD protein . As shown in the figure below, we constructed a recombinant plasmid containing FadD gene and introduced it into our engineered bacteria.
Figure2.Schematic diagram of recombinant vector containing Fad gene
After confirming that we correctly constructed and transferred the recombinant plasmid into the engineering strain E. coli DH5 α, we used IPTG to induce the expression of FadD protein and tested its improvement on the fatty acid decomposition ability of engineered bacteria. Our experimental results showed that induced overexpression of FadE did not significantly improve the fatty acid decomposition ability of engineered bacteria, and did not reproduce the experimental results in references.
Figure3. Changes of fatty acid decomposition ability of engineering bacteria overexpressing FadE protein.
In order to explore the reasons for the failure of the experiment, we detected the protein expression more carefully. After inducing the expression of FadD proteins, they were purified by Ni2+ affinity chromatography column. After purification, SDS-PAGE results showed that the molecular weight of our target band was about 20 kDa lower than our predicted value. It is speculated that the engineering strain we used is E. coli DH5 α. The endogenous protease system of this strain has not been artificially knocked out, so the overexpressed protein is easy to be degraded. Considering this possibility, we replaced our engineered strain with E. coli BL21 strain. Then we purified the protein by Ni2+ affinity chromatography column and detected it by SDS-PAGE. The results showed that the bands of FadD protein were in line with our prediction.
Fig 3 . The result of Ni-resin purification. (A) Detection of protein expression in E. coli DH5α strain . Lane 1-4. Ni-resin purification result of FadD protein. Lane 4. The purpose bond was about 20 kDa lower than our predicted value. Lane 5-8 Ni-resin purification result of FadE protein. Lane 8. The purpose bond was about 20 kDa lower than our predicted value. (B) Detection of FadD protein expression in E. coli BL21strain . Lane 1-3. Ni-resin purification result of FadD protein. Lane 3 The FadD protein bond was is basically the same as we expected.
While conducting our own experiments to explore the optimal protein expression condition, we sought the help of Ailurus biotechnology and asked them to help us design the best expression vector of FadD protein. Their scalable pipeline enables an ultra-high throughput assay of soluble protein yields under different vectors, with much lower costs than typical lab works.
The construction results of the best FadD protein expression vector are as follows：
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