Part:BBa_K4081333

ADH1 promoter- FucT2 - ADH1 terminator

Biology and Usage

ADH1 promoter is the yeast promoter for Alcohol Dehydrogenase I. It can be suppressed by ethanol. The full-length version is strong and promotes high expression. Truncated promoters are constitutive and have low expression. It can be effectively used in the high-efficiency expression of transgenes. In our project, we use ADH1 promoter to promote the expression of FucT2, and use ADH1 terminator to terminate transcript in Saccharomyces cerevisiae BY4741 (figure 1). The genes can code for α-1,2-fucosyltransferase, which catalyzes fucosylation of lactose into 2-FL using GDP-l-fucose needs to be introduced into S. cerevisiae. Several α-1,2-fucosyltransferases have been verified to facilitate the synthesis of 2-FL, which included FucT2 from Helicobacter pylori,WcfB from B. fragilis 9343, and WbgL from E. coli O126. Among those, FucT2 has been most frequently used for the microbial production of 2-FL.

Figure1.Using ADH1 promoter to promote the expression of FucT2.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design and Properties

In our project, we use ADH1 promoter(BBa_K4081990) to promote the expression of FucT2(BBa_K4081998), and use ADH1 terminator(BBa_K4081823) to terminate transcript in Saccharomyces cerevisiae BY4741 (figure 1).

We transform the gene "ADH1 promoter- FucT2 - ADH1 terminator” into Saccharomyces cerevisiae BY4741 by lithium acetate conversion method to integrate the genes into the genome of BY4741 for expression.The result of SDS-PAGE showed that we successfully expressed FucT2(figure2).

Figure 2. The result of SDS-PAGE. Line 1: Marker; line 2: Control; line 3: Express FKP(106KD), LAC12(65KD), FucT2(35KD).

Experimental approach

1.Construct recombinant plasmids. Get GAP promoter from vector PML104. Get TEF1 promoter from the genome of Saccharomyces cerevisiae BY4741. Get ADH1 promoter from vector pAUR123. Company synthetic genes of FKP, LAC12 and FucT2. Use vector pAUR123 to construct our plasimd “pAUR123-pGAP-FKP-pADH1-FucT2-pTEF1-LAC12”.

2.Transform the product (2.5μL) into DH5α competent cells (50μL), grow cells on agar plates (containing Ampicillin). Incubate plates at 37°C overnight. Colonies were screened by colony PCR and then grown at 37℃, 200rpm. Plasmids were extracted and sent for sequencing.

3.PCR the genes “pGAP-FKP-pADH1-FucT2-pTEF1-LAC12” and the resistance gene AurR from the plasmid with homology arms of BY4741. Transform it into BY4741 by lithium acetate conversion method to integrate genes into the genome of BY4741 for expression. Screen for transformants by AbA-YPD selection medium.

4.Extract yeast total protein. Use SDS-PAGE to test whether the three proteins(FKP, LAC12, FucT2) are successfully expressed.

References

[1]Yu, S. , Liu, J. J. , Yun, E. J. , Kwak, S. , Kim, K. H. , & Jin, Y. S. . (2018). Production of a human milk oligosaccharide 2′-fucosyllactose by metabolically engineered saccharomyces cerevisiae. Microbial Cell Factories, 17.