Part:BBa_K4081085

pGAP-FKP-tADH1

Biology and Usage

A lot of attention has been drawn to Glyceraldehyde-3-Phosphate Dehydrogenase (GAP) promoter. GAP mRNA accounts for 2–5% of the poly(A) RNA in yeasts, suggesting that it can be effectively used in the high-efficiency expression of transgenes. In our project, we use GAP promoter to promote the expression of FKP, and use ADH1 terminator to terminate transcript in Saccharomyces cerevisiae BY4741 (figure 1). The genes coding for FKP ,a bifunctional enzyme, L-fucokinase/GDP-L-fucose phosphorylase, which can make biosynthesis of GDP‐L‐fucose from exogenous fucose(figure 2).

Production of 2-FL requires α-1,2-fucosyltransferase which transfers the fucosyl residue from guanosine 5′-diphosphate-L-fucose (GDP-L-fucose) into lactose. GDP-L-fucose can be generated through two distinct metabolic pathways: the de novo or salvage pathway. The alternative salvage pathway requires L-fucose as the substrate for producing GDP-L-fucose. This pathway is catalyzed by a bifunctional enzyme, L-fucokinase/GDP-L-fucose phosphorylase (FKP). The salvage pathway was assumed to exist only in eukaryotes until a bacterial FKP was discovered from Bacteroides fragilis 9343. Intracellular GDP-l-fucose level has been considered as a bottleneck in 2-FL production.Thus, genes coding for FKP from Bacteroides species other than B. fragilis 9343 were tested for their efficacies for the production GDP-l-fucose. FKP genes from B. thetaiotaomicron and B. ovatus were introduced into S. cerevisiae, and subsequent production of GDP-l-fucose was confirmed, too. Nonetheless, the overexpression of B. fragilis 9343 FKP led to the highest production of GDP-L-fucose in the engineered yeast.

Figure 1. Using GAP promoter to promote the expression of FKP.

[[Image:T--Jianhua--fkp11111.png|300px|thumb|center|Figure2. The catalytic mechanism of L-fucokinase/GDP-L-fucose phosphorylase (FKP).]

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 GAP promoter(BBa_K4081994) to promote the expression of FKP(BBa_K4081992), and use ADH1 terminator(BBa_K4081823) to terminate transcript in Saccharomyces cerevisiae BY4741 (figure 1).

We tested the ADH1 promoter: we transform the gene "GAP promoter- FKP - 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 FKP(figure3).

Figure 3. 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.