Results
1. Gene integration plasmid construction: Wbgl and lac12; gmd and wacG
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 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.
Figure1. XI-2 site integration backbone plasmid by colony PCR verification
XI-2 site target gene integration backbone plasmid, containing 200 bp upstream and downstream homology arms of chromosome XI-2 site. Randomly pick 10 transformants from the LB-Amp plate, and use the upstream and downstream primer pairs of the homology arm to verify whether the fragment inserted. The target band is about 450 bp. It’s possible that 2, 3, 4, 5, 9 and 10 transformants has inserted the DNA fragments as shown in Figure 1.
Figure 2 Validation of XI-2 site target gene integration backbone plasmid by digestion
The plasmids of transformants 2, 3, 4, 5, 9, and 10 were extracted and further verified by NotI digestion (Figure 2). If the cut band size is correct as shown in Figure 2, we picked No. 10 to perform Sanger sequencing.
Figure 3 The details of sequence alignment of No. 10
The sequence of the transformant plasmid No. 10 is no mutation and mismatch as shown in Figure 3, indicating we successfully constructed XI-2 site integration backbone plasmid.
Figure 4. X-3 site target gene integration backbone plasmid by colony PCR verification
The X-3 site contains 200 bp of homology arms, which on the upper and lower sides of the chromosome X-3 site. 10 transformants were randomly picked from the LB-Amp plate, and the upstream and downstream primer pairs of the homology arms were used to verify whether the fragment inserted. The target band was about 450 bp. As shown in Figure 4, except for 6 and 7, the rest of the transformants might insert fragments.
Figure 5. X-3 site target gene integration backbone plasmid by digestion verification
The plasmid of transformants 2, 3, 5, and 8 were extracted and further verified by by Not1 digestion (Figure 5). The target band was 3762+430 bp. Thus, we picked No. 5 for sequencing, and the results are shown in Figure 6.
Figure6. Sequencing of the transformant No. 5
The sequence alignment well matched, indicating that the X-3 site integrated backbone plasmid was constructed successfully.

Secondly, the wbgL-lac12 and gmd-wcaG incorporated into the integration plasmids XI-2 and X-3, respectively. The verification results were shown as follows.
Figure 7 Validation of XI-2-wbgL-lac12 plasmid BamH1+Xho1 digestion
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.

Plasmids of 10 transformants were extracted and verified by Xho1+BamH1 double-enzyme digestion (Fig. 7). The positive transformant band was 6007+2635 bp, and the correct NO.11 was selected for sequencing. The results were shown as Figure 8.
Figure 8 Sequencing of plasmid 11 correctly digested
The sequence alignment results showed well matched, indicating that the XI-2-wbgL-lac12 plasmid was constructed successfully.
Figure 9 X-3-gmd-wcaG plasmid Xho1+Mss1 digestion verification
In the X-3-gmd-wcaG plasmid, the gmd and wcaG gene expression cassettes are inserted between the upstream and downstream of the X-3 homology arm, in the same direction. The gmd+wcaG gene for X-3 site integration was constructed by a two-step digestion cloning method. After the gmd gene expression cassette was integrated, the wcaG was introduced using Xho1 digestion site.

plasmids of 12 transformants were extracted and verified by Xho1+Mss1 double-enzyme digestion (Fig. 9). The positive transformant band was 6220+1896 bp, and the No.11 plasmid with correct digestion was randomly selected and sent for sequencing.
Figure10. Sequencing of the correct transformant plasmid of No. 11
The details of sequence alignment showed there is no mutation, indicated that the X-3-gmd-wcaG plasmid was constructed successfully.
2. Using CRISPR Cas-9 technology to integrate the target genes into the genome of S. cerevisiae
The constructed X-3-gmd-wcaG and XI-2-wbgL-lac12 plasmids were digested with Not1, respectively, and the large fragments were extracted from the gel. Using the lithium acetate transformation method, together with the gRNA, they were introduced into the yeast competent cell that already contains the Cas9 expression plasmid. In yeast CCTCC M94055, colony PCR was used to verify the integration of exogenous genes.
Figure11. Validation of exogenous gene integration by colony PCR
From left to right, No.1, 6, 7, 10 transformants.
Primer pairs we used were shown as follows.
X-3 outer-primer: h-x-3u-bb-f1/h-x-3d-bb-r1.
XI-2 outer primer: h-xi-2u-bb-f1/h-xi-2d-bb-r1.
X-3 inner-primer pairs: h-x-3u-bb-f1/h-gap-gmd-r1.
XI-2 inner-primer pairs: h-xi-2u-bb-f1/h-wbgL-cyc1t-r1.
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 11. The integrated copy number of the four transformants was verified using different primer pairs. If the internal primers (inner-primer pairs) can amplify the target band, and the outer primers (outer-primer pairs) cannot amplify the target band with the size of the integration homology arm, 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 6 and 7 have clearly integrated one copy of the target gene, and can be tested for subsequent experiments.
3. The engineered strain utilizes synthetic medium to produce 2'-FL
Figure 12 Fermentation of recombinant yeast in YPD30L2 medium to produce 2'-FL
The productivity of 2'-FL by recombinant yeast in the synthetic medium YPD30L2 was tested. As shown in Figure 12, 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 (Fig. 12A). The initial addition of 2 g/L of lactose was undetectable after 48 h, resulting in about 0.7 g/L of 2'-FL (Fig. 12B). 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.
4. Production of 2'-FL from sweet potato residues by engineered strains
Figure 13 Production of 2'-FL from sweet potato residues in recombinant yeast
The productivety of 2'-FL by recombinant yeast from sweet potato residues was tested, as shown in Figure 18. Generally, the growth of the strain was slightly worse than that of the synthetic medium (Figure 13A), 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 (Fig. 13B).