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.
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.
Firstly, we need to amplify the DNA fragments of ACC1, FAS1 and FAS2 by using Polymerase Chain Reaction (PCR) technology, then put the amplified fragments into the electrophoresis system, and cut the target DNA fragments after the electrophoresis process is finished.
Secondly, the target DNA fragment was extracted with the quick Gel Extraction Kit, then combined with carrier and ligase to form the recombinant.
Thirdly, after the recombination is completed, transferred it to the competent cells of E. coli. The competent cells with the recombinant products are amplified using an oscillator. Then the amplified competent cells are coated on the solid LB medium and placed in the incubator overnight.
Fourthly, after the bacteria grow out of the culture dish, the plasmid is put into the liquid LB culture medium. In this part, the OD600 value is detected to check whether the concentration of cell expansion is in a suitable range —— about 0.6 ~ 1.0.
Fifthly, prepare the transformation solution using PEG, LiAC, and Single-stranded carrier DNA. The plasmid was transferred into the competent state of yeast cells to increase the FAEEs production capacity of yeast cells. The competent cells were amplified again in the oscillator, then coated the cell on the solid culture medium and placed in the incubator overnight.
Lastly, add the S. cerevisiae to the liquid culture medium, and use PCR to measure the BP value to see whether it is consistent with the expected BP value; If it’s consistent, it means that S. cerevisiae with plasmid can be sent to the brewery to determine the output of FAEEs.
1. Successfully constructed FAS1, FAS2, and ACC1 plasmids, respectively.
2. Successfully transferred the recombinant plasmids into yeast strain.
3. Successfully produced alcohol containing ethyl caproate by fermented.
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