Part:BBa_K4124112
YEP-FAS1
Contribution
Our team aims to improve the ability of saccharomyces cerevisiae to produce ester in saccharomyces to improve the lack of flavoring caused by single flavor saccharomyces of l. Our main content is to study a major substance: by yeast culture, mores ethyl hexanoate to enrich the spirit's flavor.
Ethyl Hexanoate is a colorless liquid, and it has an apple smell. And it is fatty acid ethyl hexanoate. Ethyl Hexanoate is always used to brew spirit. And it can affect the taste of the spirit. Thus, the subject of our team is to use Ethyl Hexanoate to adjust the spirit's flavor.
Our project was inspired by the fact that Ethyl Caproate is one of the most important matters that directly determines the flavor of the Chinese baijiu. During the transition between Acetyl-CoA and Malonyl-CoA, the production rate rises significantly with the increase of Acetyl-CoA carboxylase (ACC1), and during the production of Fatty Acyl-CoAs, Fatty acid synthase (FAS1 FAS2) would improve the production rate as well.
To increase the quantity of Ethyl Caproate in baijiu, we need to transfer both ACC1 and Fatty acid synthase to saccharomycetes through the progress of transformation.
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 .
In this experiment, we will construct two plasmids, FAS1 and FAS2. Under the function of Malonyl-CoA, 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
Firstly, we need to amplify the DNA fragments of 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.
Establish the plasmid of YPF2K-FAS1 and YPF2K-FAS2
We first try to construct the yep plasmid, the plasmid map is shown below Figure 2.
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.
DH10B(Yep-FAS1 and Yep-FAS2) were obtained using single-fragment recombination. The sequencing results showed that the constructed Yep-FAS1/2 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-FAS1/2), The mutation site was then backmutated using site-directed mutagenesis.
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-FAS1, 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
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.
Functional Improvement
Compared with iGEM Team 21_ Beijing_ United, which simply increased the content of ethanol. This year, in addition to ethanol production, SuZhou_Union determined the formation and consumption path of fatty acid ethyl ester in Saccharomyces cerevisiae. They constructed a high-yield yeast strain producing ethyl caproate to improve the quality of Luzhou flavor liquor, increasing the content of aroma substances for taste, and lay a foundation for subsequent quality improvement research.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3931
Illegal BglII site found at 4324
Illegal BglII site found at 5240
Illegal BglII site found at 5284
Illegal BglII site found at 5536
Illegal BamHI site found at 6189 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 811
Illegal AgeI site found at 1567
Illegal AgeI site found at 2473 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 11789
Illegal BsaI.rc site found at 3024
Illegal BsaI.rc site found at 5263
Illegal BsaI.rc site found at 8403
Illegal SapI site found at 4211
Illegal SapI site found at 8555
Illegal SapI.rc site found at 12871
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