Part:BBa_K4124007

YEP2K-FAS2

YPF2K + FAS2

contribution

Fatty acid synthase (FAS), as a key enzyme for fatty acid synthesis, has rich enzyme system functions. It exists in different forms in high and low animals, and plays a great role in affecting the energy metabolism of organisms. In recent years, there are more and more research achievements on fatty acid synthase.

Because the fragrant flavor of Baijiu is produced by FAEEs, the concentration of this substance is also the standard for the quality of Baijiu, thus the output of FAEEs from S. cerevisiae is important; However, due to the single strain fermentation process of Baijiu, the production of FAEEs is not enough causing the flavor becoming insufficient. Therefore, if the FAEEs produced by yeast are increased, the flavor of Baijiu can be more abundant and fragrant.

The content of this project is to improve the FAEEs production ability of S.cerevisiae cells by constructing FAS1, FAS2 and ACC1 plasmids.

Figure 1. An overview of the experimental principle.

In this experiment, we will construct three plasmids ACC1, FAS1, and FAS2. Under the function of Malonyl-CoA, ACC1, FAS1, and FAS2 synthesize fatty acids, fatty Acyl-CoAs produce FAEEs; Therefore, as long as we successfully construct one of the three plasmids, the production of FAEEs can be significantly increased.

Engineering Success

Establish the plasmid of YPF2K-FAS2

We first try to construct the yep plasmid, the plasmid map is shown below Figure 2.

Figure 2. Establish the plasmid of YPF2K-FAS2.

After PCR amplification of the exogenous DNA, the vector and the exogenous DNA fragment were cut by restriction enzyme respectively, and the two were ligated by DNA ligase, and then transferred into the host bacteria. The recombinant clones were obtained by screening and identification. After a series of operations, the plasmid completes the task of presenting the target fragment.

Figure 3. Results of PCR amplification of FAS2 fragments.

DH10B(Yep-FAS2) were obtained using single-fragment recombination. The sequencing results showed that the constructed Yep-FAS2 contained multiple synonymous mutations, but all of them also contained individual mutation sites, which would lead to changes in the corresponding amino acids of the protein. Pick clones with fewer mutations in them (Yep-FAS2-13), The mutation site was then backmutated using site-directed mutagenesis

Figure 4. Sequencing results for corrected mutations of Yep-FAS2.

After recombination is complete, it is transferred into competent cells of E. coli. Use a shaker to expand competent cells with recombination products. The expanded competent cells were then coated on solid LB medium and placed in the incubator overnight.

After the bacteria have grown out of the petri dish, place the plasmid into liquid LB medium. This part checks the OD 600 value to check if the cell expansion concentration is in the proper range - about 0.6~1.0.

Prepare transformation solutions using PEG, LiAC, and single-stranded carrier DNA. The plasmid was transferred into competent yeast cells to improve the FAEEs production capacity of yeast cells. Competent cells were re-expanded in a shaker, then cells were coated on solid medium and placed in an incubator overnight.

Finally, S. cerevisiae was added to the liquid medium, and the BP value was measured by PCR to see if it was consistent with the expected BP value; if it was consistent, it meant that S. cerevisiae with plasmids could be sent to the brewery to determine the yield of FAEE.

Fermentation Effect Assay

1. Add 1 mL of yeast (Yep, Yep-FAS2) to 110 mL of LYPD liquid medium

In each group, 2 parallels were set, and the fermentation was carried out at 30°C and 220rpm for 60h;

2. After fermentation, centrifuge at 4000rpm at 4°C for 10min to collect the supernatant, discard the precipitate, and seal it with parafilm.

Avoid alcohol volatilization, send it to the company to measure gc ms, The fermentation time statistics are shown in the table below

Table 1. Fermentation time statistics table

Figure 5. Fermentation effect comparison..

After the fermentation time of each group continues data processing and drawing, it can be clearly seen from the figure below that after the optimization and improvement of our experiment, through the characterization of OD600, the brewing yield is significantly higher than that of the control group(Figure 5.), indicating that our experiment has been successful and also It is hoped that our experiments can provide reference for other igem teams and provide certain guidance for subsequent industrial production.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 260
  Illegal BglII site found at 3393
  Illegal BglII site found at 3980
  Illegal BglII site found at 4252
  Illegal BglII site found at 4702
  Illegal BamHI site found at 5721
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2824
  Illegal AgeI site found at 3070
  Illegal AgeI site found at 3223
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 11321
  Illegal BsaI.rc site found at 7935
  Illegal SapI site found at 8087
  Illegal SapI.rc site found at 4444
  Illegal SapI.rc site found at 12403