Our composite part BBa_K4248005 was improved based on the existing part BBa_K4085015, wcaG, and BBa_K4081996, lac12. In 2021, iGEM21_Jianhua designed a basic part BBa_K4081996,lac12. In 2021, iGEM21_Ulink-SIP constructed BBa_K4085015, wcaG. The team used the BBa _ K4085015, wcaG for mass production of 2-FL sugar by biological method to replace the normal presence of white sugar.
Compared to the team, our experimental results are better than the experimental data already uploaded in the iGEM library, which also proves the advantages of our experimental design scheme. We hope that our experiments can provide reference for other igem teams and provide guidance for subsequent industrial production improvements.

There is a relatively abundant source of GDP-mannose in the cytoplasm of Saccharomyces cerevisiae, which can serve as a precursor for 2'-FL synthesis. Studies have shown that the expression of heterologous gmd and wcaG genes in Saccharomyces cerevisiae can convert yeast endogenous GDP-mannose to GDP-fucose. At the same time, the heterologous lactose transporter gene lac12 is expressed to transport lactose into the cell. Finally, the protein encoded by the wbgL gene transfers the fucosyl group from GDP-fucose to lactose, resulting in 2'-FL (Fig. 1).
In this study, the industrial diploid Saccharomyces cerevisiae CCTCC M94055 was used as the host, the 2'-FL pathway gene was expressed by chromosomal integration, and the molecular construction was successfully completed. Fermentation experiments with synthetic medium and sweet potato pomace mash showed that engineered yeast could produce about 1 g/L of 2'-FL into the medium.

We constructed two plasmids including four genes WbgL, lac12, gmd, and wacG which are key genes to produce 2'-Fucosyllactose(2'-FL) in a yeast cellular factory. In addition, we need to add 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.

Then, we used CRISPR-Cas9 technology to integrate the target four genes WbgL, lac12, gmd, and wacG into the genome of S. cerevisiae. And colony PCR was used to verify the integration of exogenous genes.

Colony PCR was used to verify whether the X-3 and XI-2 loci gene fragments were integrated into S. cerevisiae strains. The results are shown in Figure 3. The integrated copy number of the four transformants was verified using four different primer pairs. If the internal primers (X-3 inner-primer pairs and XI-2 inner-primer pairs) can amplify the target band, and the outer primers (X-3 outer-primer pairs and XI-2 outer-primer pairs) cannot amplify the target band with the size of the integration homology arm (4346bp and 4880bp), it means that 2 copies have been integrated. If the primers outside the site amplify the target band of the size of the integration homology arm, it means that 1 copy of the target gene has been integrated. Based on the above analysis, we judged that the middle transformants 2 and 3 have clearly integrated one copy of the target gene, and can be tested for subsequent experiments.

The productivity of 2'-FL by recombinant yeast in the synthetic medium YPD30L2 was tested. As shown in Figure 4, 30 g/L glucose was completely consumed within the initial 4 h, and then the strain began to use the produced ethanol as a carbon source, showing a secondary growth state (Figure 4A). The initial addition of 2 g/L of lactose was undetectable after 48 h, resulting in about 0.7 g/L of 2'-FL (Figure 4B). The theoretical conversion rate of lactose to 2'-FL was 100%. Part of 2'-FL accumulated intracellularly and failed to be effluxed into the medium.

General model:

Coefficients (with 98% confidence bounds):

The increase first and then stabilized yield of 2' -FL of synthetic medium L2FL was attributed to an increase in the fermentation time.

The productivety of 2'-FL by recombinant yeast from sweet potato residues was tested, as shown in Figure 6. Generally, the growth of the strain was slightly worse than that of the synthetic medium (Figure 6A), which may be due to the presence of some inhibitor for yeast growth in the sweet potato residues. The presence of lactose was not detected at 48 h, and the final yield was about 0.6 g/L 2'-FL (Figure 6B).

General model:

Coefficients (with 98% confidence bounds):

The yield of 2' -FL in sweet potato residue medium L2FL increased first, and then stabilized with the increase of fermentation time.

The experimental results show that we successfully constructed the metabolic pathway of 2'-FL in S. cerevisiae. The test results prove to show that the yield of 2'-FL was increased, and the goal of constructing a high-yield 2'-fucosyllactose S. cerevisiae cell factory was achieved. Our products not only have a high yield of 2 ' -FL, but also solve the problem of food safety. Meanwhile, the model can predict the ability of fermented sweet potato residue to produce 2'-FL, which is of great significance for the high-value utilization of sweet potato residue and the production of 2'-FL by the biological method. In the future, high-yield 2'-fucosyl lactose S. cerevisiae cells can be used in milk powder, the market demand is enormous, broad application prospects.