Medium-chain fatty acids (MCFAs) have a favorable safety profile and can be used to treat a variety of conditions, such as benefits for skin care, weight loss, and cardiovascular use. However, most of the medium-chain fatty acids in use today are extracted from tropical plants, which is time-consuming and limits the yield of medium-chain triglycerides. As a result, its yield is much lower than that of long-chain fatty acids.
In order to overcome this limitation, we used synthetic biology strategies to conduct experiments on the four core enzymes of the reverse β-oxidation cycle, namely fadB, ydiI, ter, and bktB. This was then applied to the MCFAs synthesis pathway in E. coli to increase the production of MCFAs.
The production of medium-chain fatty acids (MCFAs) can be increased by overexpressing key enzymes in the reversal of the β-oxidation cycle. We use the four key enzymes, bktB, fadB, ter, and ydiI to construct the reverse β-oxidation cycle and form the MCFAs synthesis pathway in E. coli. In this way, we can improve the yield of the production of MCFAs.
Because the T7 promoter has a strong ability in translation and usually be used as protein expression, we choose the pETDuet-1 vector and pCDFDuet-1 vector, with T7 promoter respectively, to express our target enzymes bktB, fadB, ter, and ydiI to develop r-BOX cycle. To achieve this, we inserted genes fadB and ter into pCDFDuet-1vector, and inserted genes bktB and ydiI into pCDFDuet-1vector, transferred the recombinant plasmid into E. coli BL21(DE3) for protein expression (Figure 1).
Figure 1. the workflow of this project (Junjun Wu , et al., 2019)
In order to construct our plasmids, we amplify all four enzymes with corresponding templates by PCR. Salmonella enterica genome was used as the template to amplify gene fadB, the plasmid pUC57-ter was used as the template to amplify gene ter, the plasmid pUC57-bktb was used as the template to amplify gene bktB, and the genome of E. coli MG1655 was used as the template to amplify gene ydiI (Figure 2).
Figure 2. Gel electrophoresis results of target gene fragments. In Figure 2 we can find that there were four clear DNA bands, indicating that genes bktB, ydiI, fadB and ter were successfully amplified by PCR.
Then we used a one-step cloning method to ligate the gene fadB into the NcoI and BamHI sites of the pETDuet-1 vector. Then we transformed the recombinant plasmid into E. coli DH5α competent cells. We inoculated the correct strain and extracted the plasmid pETDuet1-fadB. Then we inserted gene ter into the NdeI and XhoI sites of pETDuet1-fadB through a one-step cloning method. The recombinant plasmid pETDuet1-fadB-ter was verified by NcoI/HindIII and NdeI/XhoI, respectively (Figure3 line2, 4).
Meanwhile, we also used a one-step cloning method to ligate the gene bktB into the NcoI and BamHI sites of the pCDFDuet-1 vector. Then we transformed the recombinant plasmid into E. coli DH5α competent cells. We inoculated the correct strain and extracted the plasmid pCDFDuet1-bktB. Then we inserted gene ydiI into the NdeI and XhoI sites of pCDFDuet1-bktB through a one-step cloning method. The recombinant plasmid pCDFDuet1-bktB-ydiI was verified by NcoI/HindIII and NdeI/XhoI, respectively (Figure3 line1, 3).
Figure 3. Digestion verification of pETDuet1-fadB-ter and pCDFDuet1-bktB-ydiI. We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 4.), and the plasmids were successfully constructed. So far, we have successfully obtained the two recombinant plasmids.
Figure 4. The sequencing blast results of the recombinant plasmids. In order to obtain the four enzymes, we transferred the recombinant plasmids, pETDuet1-fadB-ter and pCDFDuet1-bktB-ydiI, into E. coli BL21(DE3), inoculated the correct colony in the LB culture medium and added IPTG to induce protein expression when the OD600 reached 0.5. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used nickel column purification to purify the enzymes we wanted, and detected the proteins by SDS-PAGE (Figure 5).
Figure 5. SDS-PAGE analysis of bktB, ydiI, fadB, ter. Four clear protein bands can be seen in figure 5, while there is no corresponding band in the control, indicating the successful expression of bktB, ydiI, fadB and ter.
To confirm the ability of the platform we developed in BL21(DE3) with the genes r-BOX cycle (bktB, ydiI, fadB and ter), can produce MCFAs, we detected the yield of MCFAs through HPLC.
We co-transformed the recombinant plasmids, pETDuet1-fadB-ter and pCDFDuet1-bktB-ydiI, into BL21(DE3), and inoculated the successfully transformed strain into LB medium overnight at 37℃. Then transferred the cultured medium into 25 mL fresh LB culture medium and make the initial OD600 equal to 0.1 and incubated at 37℃ 200rpm. Added Glucose to the medium when OD600 was about 0.5, IPTG was also added to induce the expression of R-box at a final concentration of 1mM. After culturing for 40h, we collected 6 mL of the culture medium, collected the cells, and ultrasonic crushing was performed, centrifuged, and collect the supernatant. Mixed the supernatant with 2-Bromoacetophenone and Triethylamine, incubated at 50℃ for 4h, and then use HPLC to detect the yield of MCFAs (Figure 6).
Figure 6. HPLC profiles of BL21(DE3) strain expressing r-BOX. As shown in Figure 6, After the HPLC analysis of C4-C12 fatty acid standards and BL21_bktB-ydiI-fadB-ter, at the same location of the peak as the standard MCFAs, B represented the successful production of 1.08 g/L MCFAs by strain BL21_ bktB- ydiI- fadB-ter. So that our platform for producing MCFAs was successfully established.
The results showed that the overexpression of the β-oxidation reversal composed of four enzymes, fadB, ydiI, ter, and bktB, can effectively increase the production of MCFAs in E. coli. In this case, a higher yield of products can be made through our genetic engineering. We believed that our engineered strain could be used for future research or even be applied for industrial production.
Because of the important effect on human daily life, if we can produce more MCFAs in the future, we believe that it will provide people to live in a healthier way.
