Part:BBa_K4278723

pYES2-ATF1

pYES2-ATF1

Profile

Name: pYES2-ATF1

Base Pairs: 7832 bp

Origin: Saccharomyces cerevisiae, genome

Properties: the composite plasmid containing alcohol acetyltransferase ATF1

Usage and Biology

This part is a composite part named pYES2-ATF1, and it is built up of the pYES2 shuttle vector (BBa_K4278716) and TEF1 pro-ATF1-CYC1 ter (BBa_K4278719). The pYES2 vector is one of the most commonly used Saccharomyces cerevisiae vectors, which can shuttle in E. coli and S. cerevisiae. The vector is a high-copy-number plasmid. When expressed in the prokaryotic system, the Amp+ resistance can be used to screen the right colony, while transformed into the S. cerevisiae, the strain should be cultured at 28-30℃. This plasmid backbone can be used to express different proteins in the future.

The ATF1 gene encodes a single alcohol acetyl-CoA transferase (AATase) enzyme, because of the importance of the ATF1 in the aroma profile of the wine, its regulation during fermentation has been investigated in detail. The yeast with a constitutively overexpressed ATF1 gene produces more isoamyl acetate and ethyl acetate compared to a wild-type strain [1-5]. The ATF1 enzyme contains transmembrane domains which fix the protein in the membrane of lipid droplets that bud out from the endoplasmic reticulum (ER). This enzyme widely exists in different varieties of yeast.

Construct design

We designed the program by inserting the ATF1 gene into SpeI and SalI sites of the pYES2 vector. In order to build our plasmids, we amplified the gene fragments from the genome of S. cerevisiae by PCR (Figure 1), double-enzyme digestion, and ligase to pYES2 carrier.

Figure 1. the schematic diagram of the composite part BBa_K4278723.

Experimental approach

1. Construct the plasmid

Figure 2. The PCR gel electrophoresis result of the Kana gene fragment. M. DNA marker, A. 1: The ENA fragment of ATF1, B. 2: The overlap TEF1pro-ATF1-CYC1ter DNA fragment.

In order to obtain our target genes, we amplified the target gene ATF1 from the S. cerevisiae genome. We did gel electrophoresis to verify if we successfully amplified the target fragment. Next, we extracted the target genes and fused them by overlap PCR. In figure2, a clear and single DNA band could be seen, indicating that we successfully amplified our target genes. Then, we digested the fused gene fragment and the pYES2 plasmid with SpeI and SalI and extracted the digested fragments, ligated them by T4 DNA ligase, and transformed them into the E. coli competent cells.

Figure 3. verification of the recombinant plasmids by colony-PCR.

We verified the colonies through colony-PCR (Figure 3), and then we inoculated single colonies (1, 2, 5, and 7) and we send the constructed recombinant plasmid to a sequencing company for sequencing.

2. construct the engineered S. cerevisiae

We extracted the correct pYES2-ATF1plasmid and transformed it into the S. cerevisiae competent cells through the Lithium acetate transform method. Next, We verified the colonies through colony-PCR (Figure 4).

Figure 4. verification of the engineered S. cerevisiae by colony-PCR.

we inoculated single engineered S. cerevisiae colonies (SFA-1) in 5 mL YEPD medium and grew at 30℃ and 180 r/min for 12 h. The above culture 1:100 culture liquid was connected to three bottles of 50mL liquid YEPD medium and incubated under the same conditions. The absorbance value at 600 nm was measured at 2,4,8,16,24 and 32 h after culture (Table 1). This result indicates that the genetically modified S. cerevisiae SFA-1 showed no harmful effects on the growth of the organism itself. Therefore, giving support for applications in the wine fermentation industry (Table 1)

Table 1. Raw data were obtained at each time period 2, 4, 7, 16, 24, and 32 hours, respectively. Each strain of WT and SFA-1 was tested with Spectrophotometer three times, to obtain an OD600 absorbance level.

References

1.Zhang J., Zhang C., Wang J., Dai L., Xiao D. (2014) Expression of the Gene Lg-ATF1 Encoding Alcohol Acetyltransferases from Brewery Lager Yeast in Chinese Rice Wine Yeast. In: Zhang TC., Ouyang P., Kaplan S., Skarnes B. (eds) Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012). Lecture Notes in Electrical Engineering, vol 249. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-37916-1_5.

2.Ma L, Huang S, Du L, Tang P, Xiao D. Reduced Production of Higher Alcohols by Saccharomyces cerevisiae in Red Wine Fermentation by Simultaneously Overexpressing BAT1 and Deleting BAT2. J Agric Food Chem. 2017 Aug 16;65(32):6936-6942. doi: 10.1021/acs.jafc.7b01974.

3.Lilly M, Bauer FF, Styger G, Lambrechts MG, Pretorius IS. The effect of increased branched-chain amino acid transaminase activity in yeast on the production of higher alcohols and on the flavour profiles of wine and distillates. FEMS Yeast Res. 2006 Aug;6(5):726-43. doi: 10.1111/j.1567-1364.2006.00057.x.

4.Dai L., Zhang C., Zhang J., Qi Y., Xiao D. (2014) Effects of IAH1 Gene Deletion on the Profiles of Chinese Yellow Rice Wine. In: Zhang TC., Ouyang P., Kaplan S., Skarnes B. (eds) Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012). Lecture Notes in Electrical Engineering, vol 249. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-37916-1_42.

5.Choi YJ, Lee J, Jang YS, Lee SY. Metabolic engineering of microorganisms for the production of higher alcohols. mBio. 2014 Sep 2;5(5):e01524-14. doi: 10.1128/mBio.01524-14.

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 6097
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 335
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 5763
  Illegal SapI site found at 1153
  Illegal SapI.rc site found at 6524