Part:BBa_K4292021

X-3-gmd-wcaG

Assembly Compatibility:

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

  Illegal AgeI site found at 5089
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 5346
  Illegal BsaI.rc site found at 5506
  

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 gmd, wcaG into S. cerevisiae genome using CRISPER-cas9. As shown in Figure1. The genes, gmd, wcaG , are encoding the important enzymes involving the 2'-FL synthesis. gmd and wcaG are converted the abundant intracellular GDP-mannose in S. cerevisiae into GDP-fucose. Further, 2'-FL can be produced from GDP-fucose by expression of Wbgl and metabolization. In this work, we used sweet potato residues as main carbon source.

Figure 1. The metabolic pathway of biosynthesis 2’-FL in S. cerevisiae. The purple part represents the heterologous pathway gene.

Engineering Success

We constructed two plasmids including two genes, gmd and wacG which are key genes to produce 2'-Fucosyllactose(2'-FL) in a yeast cellular factory. In addition, we need to added the promoter and terminator to flanking regions of these exogenous genes in order to facilitate expression in the engineered yeast. The components were incorporated into the integration backbone plasmid with NotI and XhoI sites. Firstly, we constructed the integration backbone plasmids XI-2 and X-3. These colonies were verified by colony PCR and Sanger sequencing and the results were shown as follows.