Part:BBa_K4292020

XI-2-wbgL-lac12

XI-2-wbgL-lac12

contribution

Our team aims to produce 2'-FL in S. cerevisiae for providing the economic strategy. In terms of raw material, there are two pathways to produce GDP-L-fucose from GDP-D-mannose (called de novo pathway) or L-fucose (called salvage pathway). In the de novo pathway, the GDP-D-mannose converts 2'-FL and secreted into medium involving three enzymes gmd, wcaG, and wbgL, as well as transporter lac12. Our team aims to produce 2'-FL in S. cerevisiae for providing the economic strategy. In terms of raw material, there are two pathways to produce GDP-L-fucose from GDP-D-mannose (called de novo pathway) or L-fucose (called salvage pathway). In the de novo pathway, the GDP-D-mannose converts 2'-FL and secreted into medium involving three enzymes gmd, wcaG, and wbgL, as well as transporter lac12.

In order to obtain the high yield of 2'-FL, we introduced the four exogenous gene including Lac12 and Wbgl into S. cerevisiae genome using CRISPER-cas9. As shown in Figure1, expression of Lac12 (lactose permease) from Kluyveromyces lactis is a transporter to facilitate lactose transporting out of the cytoplasm. Other three gene，Wbgl, is encoding the important enzymes involving the 2'-FL synthesis.

Figure1. The metabolic pathway of biosynthesis 2’-FL in S. cerevisiae. The purple part represents the heterologous pathway gene.

Engineering Success

pHCas9-Nours plasmid serves as a DNA template and amplified PCR to obtain the XI-2 and X-3 site target gene integration plasmid. The lac12 and wbgl gene fragments were amplified by PCR using the XI-2-wbgL-lac12 plasmid as template.

The DNA fragment wbgL-lac12, as well as XI-2 integration plasmids were digested with NotI and XhoI to form the cohesive ends, respectively. Then, the DNA fragments and vector were joined together by the ligase. The recombinant plasmid transformed into the competent cells and verified by colony PCR and Sanger sequencing. After that, the corrected wbgL-lac12 DNA fragments, which were digested with NotI, and gRNA were introduced into the yeast that already contains the Cas9 expression plasmid using lithium acetate transformation method. These colonies were used colony PCR to verify whether the colony’s genome carried recombinant DNA fragments, and the positive transformants further confirmed through Sanger sequencing.

Figure 2. Gene integration plasmid construction: Wbgl and lac12.

In the XI-2-wbgL-lac12 plasmid, the wbgL and lac12 gene expression cassettes are inserted between the XI-2 homology arm, in different orientations. The wbgL+lac12 gene expression plasmid for XI-2 site integration was constructed by a two-step digestion cloning method. After wbgL gene expression cassette was integrated, lac12 inserted though Xho1 and BamH1 digestion sites.

Figure 3 Validation of XI-2-wbgL-lac12 plasmid BamH1+Xho1 digestion.

Plasmids of 10 transformants were extracted and verified by Xho1+BamH1 double-enzyme digestion (Figure. 3). The positive transformant band was 6007+2635 bp, and the correct NO.11 was selected for sequencing. The results were shown as Figure 4.

Figure 4. Sequencing of plasmid 11 correctly digeste.

The sequence alignment results showed well matched, indicating that the XI-2-wbgL-lac12 plasmid was constructed successfully.

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 5510
  Illegal BglII site found at 7078
  Illegal BglII site found at 7252
  Illegal BglII site found at 7669
  Illegal BglII site found at 7687
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]