Our composite components, BBa_K3521022 and BBa_K4284031, are used to develop the platform to produce MCFAs for future use. As early as 2019, the iGEM19_Ionis_Paris team was committed to providing an Acetyl-coenzyme A synthetase, ACS (BBa_K3102021) transforms acetate to acetyl-CoA in an ATP-dependent manner. Based on their work, by reading literature and consulting experts in related fields, we found that acetyl-CoA also plays an important role in the r-BOX cycle. In order to improve the yield of MCFAs, we further overexpressed this protein in our engineered strain and detected the yield of MCFAs.
In order to further optimized our MCFAs-producing platform, we also changed the speed while the fermentation is performed. Then, by detecting the yield of MCFAs, it was further confirmed that when the shaking speed was 100 rpm, the yield of MCFAs and cell concentration achieved a comprehensive best state.
Acetyl-coenzyme A synthetase (ACS) is an enzyme whose activity is central to the metabolism of prokaryotic and eukaryotic cells. The physiological role of this enzyme is to activate acetate to acetyl-coenzyme A (Ac-CoA). Because of the importance of acetyl-CoA in r-BOX cycle and related to the production of MCFAs, we optimized our MCFAs-producing platform by overexpress the genes bktB, ydiI, fadB, ter, and ACS (Figure 1).
Figure 1. Construction of fatty acid exocytosis system (Junjun Wu ,et
al., 2019)
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 pCDFDeut1 vector as shown in Figure 2.
Figure 2. The map of recombinant plasmids. In order to construct our plasmids, we amplify all four enzymes with corresponding templates by PCR. Then we obtained the target DNA fragments through agarose electrophoresis and gel extraction. Afterward, we cloned the corresponding plasmids and used DNA sanger sequencing to verify the construction. Then, we extracted the recombinant plasmids from E. coli DH5α and transferred them into BL21(DE3), so that can be used as an MCFAs-producing platform.
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 3).
Figure 3. HPLC profiles of BL21(DE3) strain expressing r-BOX As shown in Figure 3, 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 4, 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 4).
Figure 4. 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 5, 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 5. 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 that could be optimized, such as the promoter we used.
