We designed two plasmids: the DNA fragments of the genes bktB and ydiI were cloned into the pETDeut1 vector, and the genes fadB and ter were inserted into the pCDFD vector as shown in Figure 1.
Figure 1. The map of recombinant plasmids. 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 into E. coli BL21(DE3), expanded the culture in the LB culture medium, and added IPTG to induce protein expression when the OD600reached 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 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 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, 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, and Panel C represented the successful production of 1.36 g/L MCFAs by strain BL21_ bktB- ydiI- fadB-ter-acs. So that our platform for producing MCFAs was successfully established.
In order to improve the yield of MCFAs, we optimized the speed we used when culturing the engineered strain. As shown in Figure 7, we can conclude that with the decrease in shaking speed, MCFAs production increased first and then decreased, and the end cell concentration showed a decreasing trend. When the shaking speed was 100 RPM, the MCFAs production reached 1.08 g/L which is the most efficient speed (Figure 7).
Figure 7. Effect of supplying limited amounts of oxygen on MCFAs titers and final OD600.
What’s more, we also detected the yield of MCFAs when the engineered strain was co-expressed with Acetyl-CoA. We set up a series of sodium acetate of razor concentration to optimize the ingredient of the culture medium. In figure 8, we can conclude that with the increase of the sodium acetate, MCFA production increased first and then kept in balance. When the concentration of the sodium acetate was 2g/L, the MCFAs production reached 1.35 g/L.
Figure 8. Effect of sodium acetate content on MCFAs titer
In this project, we successfully set up an MCFAs fermentation platform in BL21(DE3) and improved the yield of MCFAs by both changing the speed during incubation and the concentration of sodium acetate. However, there were still some elements could be optimized, such as the promoter we used.
EGFP is used as a reporter gene to study gene expression, regulation, cell differentiation, and protein localization and transport in organisms. In order to construct the plasmid, We used the pETD-EGFP plasmid as the PCR template and amplified gene GFP through PCR. The pET28a plasmid was digested with NcoI and BamHI enzymes, and the fragment GFP was inserted into the NcoI and BamHI sites of the pET28a vector through a one-step clone method. Then we transformed the recombinant plasmid into E. coli DH5α competent cells and coated on the LB solid plate. We inoculated the correct colony into the LB culture medium and extracted the plasmid.
In order to introduce mutations into the T7 promoter, we set four different mutants, which were named C1, C2, C3, and C4. We amplified the optimal T7 point mutant plasmid by PCR using pET28a-GFP as the PCR template (Figure 9). DpnI was used to digest templates, then transformed the plasmids to E. coli DH5α competent cells and coated on LB medium plates. Next, we inoculated a mono-colony in LB medium overnight, extracted plasmids, and used Sanger sequencing to verify the successful construction of point mutations.
Figure 9. Gel electrophoresis results of target gene fragments. We transformed the plasmids into BL21(DE3), and measure OD600 and the fluorescence intensity of GFP to determine which type of T7 promotor mutant produces the most. With this method, we can easily find out which mutant T7 promoter is the most efficient to express target proteins. We incubated those four different kinds of bacteria at 37°C for 2.5h. Then we measured the intensity of the EGFP fluorescence (Figure 10). Take the average of each type of T7 promotor mutant as a group, collecting the following data.
Figure 10. validation of the four mutant T7 promoters. 
Figure 11. the intensity of the EGFP fluorescence of the strains
We calculate the A600 of different samples by dividing the average lightness by the average density. From figure 11, we can clearly find that the T7-C1 mutant promotor was the most efficient promoter. So that the promoter T7-C1 could be used to improve the yield of MCFAs in future research on producing MCFAs or other projects.
