Engineering

Introduction

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 to become insufficient. Therefore, if the FAEEs produced by yeast is 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 overexpressing FAS1, FAS2 or ACC1. Under the function of Malonyl-CoA, ACC1, FAS1, and FAS2 synthesize fatty acids, fatty Acyl-CoAs produce FAEEs (fig.1). Therefore, as long as we successfully construct one of the three overexpression plasmids, and transferred it into yeast, the production of FAEEs can be significantly increased.

Figure 1. The metabolic pathway of producing FAEEs.

Design

In this experiment, we designed two plasmids YPF1K-FAS1 and YPF2K-FAS2. Because the PGK promoter have strong ability in expression in yeast, and the URA3 usually be used as an auxotrophic screening tag in uracil-deficient yeast, we choose Yep352-pPGK1 and BY4741 as plasmid-vector and strain host respectively. The DNA sequences of FAS1 and FAS2 were inserted into the XhoI site of the Yep352-pPGK1 vector, so that produce two recombinant plasmids YPF1K-FAS1 (fig.2A) and YPF2K-FAS2 (fig.2B).

Figure 2. The map of plasmid YPF1K-FAS1 and YPF2K-FAS2.

Build

Firstly, the exogenous gene FAS1 and FAS2 were amplified by PCR, and the Yep352-pPGK1 was linearized by XhoI restriction enzyme digestion(fig.3). Then, the DNA fragments and plasmid-vector were ligated by homologous recombination, and then transferred into E.coli DH10b.

Figure 3. Results of PCR amplification of FAS1 and FAS2 fragments.

DH10B(YPF1K-FAS1 and YPF2K-FAS2) colonies were obtained using single-fragment recombination. The sequencing results showed that the constructed YPF1K-FAS1 and YPF2K-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. The mutation site was then back-mutated using site-directed mutagenesis

Figure 4. Sequencing results for corrected mutations of YPF1K-FAS1 and YPF2K-FAS2.

After back-mutating, it is transferred into competent cells of E. coli DH10b. The correct recombination plasmids YPF1K-FAS1 and YPF2K-FAS2 were extracted from E. coli DH10b with concentrations up to 2μg/μL. Then, the plasmid was transferred into competent yeast cells BY4741 to improve the FAEEs production capacity of yeast cells by using LiAc/SS carrier DNA/PEG method[1]. Finally, yeast clones were identified by direct PCR. The result showed that there were 6147bp (FAS1) and 5685bp (FAS2) bands indicated that transformation success (fig.5).

Figure 5. Results of PCR identification of yeast clones.

Test

In order to test the ability of our engineered yeast strain we constructed to produce Luzhou-flavor baijiu, the engineered yeast strain YPF1K-FAS1/BY4741(YEP-FAS1) and YPF2K-FAS2/BY4741(YEP-FAS2) was placed in 110 mL of YP20D liquid medium and fermented at 30 °C and 220 rpm for 60 h. Two parallels were set for each strain, and 1 ml of fermentation broth was taken out every 10 hours to measure the growth of the strain. The growth curve of the strain during the fermentation process is shown in figure 6. It can be seen that the growth of our engineered yeast strain after overexpressing the FAS1 or FAS2 gene is comparable to that of the negative control yeast strain (YEP), indicating that the genetic engineering modification will not affect the growth of the strain, which is in line with expectations.

Table 1. Fermentation time statistics table

Figure 6. Fermentation effect comparison

According to the results of detection of ethyl caproate content(fig.9), compared with the control group, YEP-FAS1 and YEP-FAS2 engineering bacteria can produce more ethyl caproate, and the fermentation content of YEP-FAS1 engineering bacteria is significantly higher. From the peak graph of ethyl caproate, our engineering bacteria really ferment to produce ethyl caproate. This is also a supplement to the wiki results. Our experiment can provide reference for other iGEM teams, and provide some guidance for subsequent industrial production, which verifies the engineering success.

Figure 7. The ethyl caproate peak of YEP-FAS1 detectedby GC-MS.

Figure 8. Partial enlarged view of ethyl caproate peak of YEP-FAS1 detected by GC-MS.

Figure 9. Relative value of ethyl caproate fermented of three strains by GC-MS detection.

Learn

After the fermentation time of each group continues data processing and drawing, it can be clearly seen from the figure 6 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(Fig.6), 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.

Reference

[1] R Daniel Gietz & Robert H Schiestl Nat. Protoc. 2, 1–4 (2007); doi:10.1038/nprot.2007.17; published online: 31 January 2007; corrected online 4 December 2008.