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. An overview of the experimental principle
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 FAS2 were inserted into the XhoI site of the Yep352-pPGK1 vector, so that produce two recombinant plasmids YPF2K-FAS2 (fig.2).
Figure 2. Establish the plasmid of YPF2K-FAS2.
We firstly constructed the plasmid YPF2K-FAS2 which can overexpressing FAS2 in yeast. The correct recombination plasmids 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. Finally, yeast clones were identified by direct PCR. The result showed that there were 5685bp (FAS2) bands indicated that transformation success (fig.3).
Figure 3. Results of PCR identification of yeast clones.
In order to test the ability of our engineered yeast strain we constructed to produce Luzhou-flavor baijiu, the engineered yeast strain 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 4. It can be seen that the growth of our engineered yeast strain after overexpressing the FAS2 gene is comparable to that of the wild-type yeast strain, indicating that the genetic engineering modification will not affect the growth of the strain, which is in line with expectations.
Figure 4. Fermentation effect comparison
We collected samples that have been fermented for 60 hours and send them to the company to measure the content of ethyl hexanoate. We first used conventional gas chromatography (GC) methods to detect ethyl hexanoate in the sample, and found that the peaks of the ester and alcohol crossed and could not be completely separated or the content of ethyl hexanoate could not be detected. Subsequently, through interviews with experts and research literature, we learned that alcohols and esters can be separated using the gas chromatography-mass spectrometry (GC-MS) method. So we re-fermented, collected 60 hours of fermentation samples, and sent them to the company to measure GC-MS.
Figure 5. Relative value of ethyl caproate fermented of YEP-FAS2 strain by GC-MS detection.
YEP strain was the negative control.
According to the results of detection of ethyl caproate content(fig.5), compared with the control group, YEP-FAS2 engineering bacteria can produce more ethyl caproate. Our experiment can provide reference for other iGEM teams, and provide some guidance for subsequent industrial production, which verifies the engineering success.
