Our team aims to construct engineered S. cerevisiae strains to produce a high yield of 2’-FL. To do that, we specially searched the iGEM Biological Parts library for 2-FL synthesis genes and related transporters, and picked BBa_K4085012, gmd, and BBa_K4081996, lac12, which were submitted by iGEM21_Ulink-SIP and iGEM21_Jianhua, respectively, with only DNA sequence information and little protein expression results.

Based on those teams’ work, we designed an experiment of introducing the four exogenous gene including Lac12, gmd, wcaG, and Wbgl into S. cerevisiae genome to obtain a yeast strain that produce high 2'-FL. We added new experimental data to the existing part BBa_K4085012, gmd, and BBa_K4081996, lac12.

In addition, the sites located at XI-2 and X-3 are suitable for integrating long gene fragments in S. cerevisiae genome. And we have submitted the DNA sequence of the XI-2 integration backbone plasmid (BBa_K4292022) and X-3 integration backbone plasmid (BBa_K4292023) for future iGEM teams. And the protocol of genome-editing technology we used can be found in our experiment page on the wiki for future iGEM teams.

The lac12 and wbgl gene fragments were amplified by PCR. And 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. In the recombinant XI-2-wbgL-lac12 plasmid, the wbgL and lac12 gene expression cassettes are inserted between the XI-2 homology arm, in different orientations. Next, the recombinant plasmid was transformed into the E.coli DH5α and verified by colony PCR (figure 2) and Sanger sequencing (figure 3).

There were ten transformants extracted and verified by Xho1+BamH1 double-enzyme digestion (Figure 2). The positive transformant band was 6007+2635 bp, and the correct NO.11 was selected for sequencing. The results were shown as Figure 3. The sequence alignment results showed well matched, indicating that the XI-2-wbgL-lac12 plasmid was constructed successfully.

After that, we used CRISPR-Cas9 technology to integrate the target genes WbgL and lac12 into the genome of S. cerevisiae. The corrected XI-2-wbgL-lac12 plasmid, which was digested with NotI, and gRNA were introduced into the yeast that already contains the Cas9 expression plasmid using lithium acetate transformation method. These yeast colonies were used colony PCR to verify whether the colony’s genome carried wbgL-lac12 expression cassettes.After that, we used CRISPR-Cas9 technology to integrate the target genes WbgL and lac12 into the genome of S. cerevisiae. The corrected XI-2-wbgL-lac12 plasmid, which was digested with NotI, and gRNA were introduced into the yeast that already contains the Cas9 expression plasmid using lithium acetate transformation method. These yeast colonies were used colony PCR to verify whether the colony’s genome carried wbgL-lac12 expression cassettes.

Colony PCR was used to verify whether the XI-2 loci gene fragments were integrated into S. cerevisiae strains. The results are shown in Figure 4. The integrated copy number of the four transformants was verified using different primer pairs. If the internal primers (XI-2 inner-primer pairs) can amplify the target band, and the outer primers (XI-2 outer-primer pairs) cannot amplify the target band with the size of the integration homology arm ( 4880bp), it means that two 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 a copy of the target gene has been integrated. Based on the above analysis, we judged that the middle transformants No.2 and No.3 have clearly integrated a copy of the target gene successfully.

BBa_K4292009 is the coding sequence of E. coli Gmd. The Gmd is made up of 374 aa. Gmd is GDP-D-mannose 4,6-dehydratase. Gmd forms the first step in the biosynthesis of GDP-alpha-D-rhamnose and GDP-alpha-L-fucose [1]. In Aneurinibacillus thermoaerophilus L420-91T, this enzyme acts as a bifunctional enzyme, catalyzing the above reaction as well as the reaction catalyzed by EC 1.1.1.281 [2]. Recently, it has been shown that Gmd have the significant impact in decoration (modification) the main structure of nod factors [3-4]. Protein sequences in the National Center of Biotechnology Information (NCBI) databases showed that GedM are from both prokaryotic and eukaryotic organisms [5]. The recent co-overexpression of gmd, wcaG, manB, and manC contributes to the major flux toward GDP-fucose biosynthesis and a combinatorial modular pathway engineering remarkably enhances the GDP-fucose biosynthesis [6].

Pichia pastoris is an efficient host for the expression and secretion of heterologous proteins and the most important feature of P. pastoris is the existence of a strong and tightly regulated promoter from the alcohol oxidase I (AOX1) gene. The AOX1 promoter (pAOX1) has been used to express foreign genes and to produce a variety of recombinant proteins in P. pastoris. However, some efforts have been made to develop new alternative promoters to pAOX1 to avoid the use of methanol. The glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) has been used for constitutive expression of many heterologous proteins. The pGAP-based expression system is more suitable for large-scale production because the hazard and cost associated with the storage and delivery of large volume of methanol are eliminated. Some important developments and features of this expression system will be summarized in this review.

The sites located at XI-2 and X-3 are suitable for integrating long gene fragments in S. cerevisiae genome. And we have submitted the DNA sequence of the XI-2 integration backbone plasmid (BBa_K4292022) and X-3 integration backbone plasmid (BBa_K4292023) for future iGEM teams.

Take the gene wbgL-lac12 integration by our project as an example, XI-up and XI-down were the upstream and downstream homologous arm of genome site XI-2. And the X-3 is the same as XI-2. First of all, the genes wbgL and lac12 need to add promoter and terminator to constitute full expression cassettes. Then the gene expression cassettes were inserted between XI-up and XI-down in the XI-2 integration backbone plasmid. After the recombinant plasmid XI-2-Wbgl-lac12 (figure 1) was constructed successfully in E.coli DH5α, it was extracted from E.coli DH5αand digested with NotI in vitro to obtain linearized fragments XI-2up-GAPpromoter-wbgl-CYC1terminator-TEFterminator-lac12-GAPpromoter-XI-2down (XI-2-Wbgl-lac12) (figure7).

Next, the linearized fragments XI-2-Wbgl-lac12, the plasmid pYES2-XI-2 of the sgRNA targeting the XI-2 site, and the Cas9 expression plasmid pHCas9 were introduced into the yeast by using lithium acetate transformation method. These yeast colonies were used colony PCR to verify whether the colony’s genome carried wbgL-lac12 expression cassettes. This is the complete method flow for editing the yeast genome using the CRISPR-Cas9 system.

[1] Kanda Y, Imai-Nishiya H, Kuni-Kamochi R et al (2007) Establishment of a GDP-mannose 4,6-dehydratase (GMD) knockout host cell line: a new strategy for generating completely non-fucosylated recombinant therapeutics. J Biotechnol
[2] Sraphet, S., Javadi, B. (2021) Computational characterizations of GDP-mannose 4,6-dehydratase (NoeL) Rhizobial proteins. Curr Genet 67, 769-784.
[3] Sutherland IW (2001) Microbial polysaccharides from gram-negative bacteria. Int Dairy J 11:663–674
[4]. Szklarczyk D, Gable AL, Lyon D, Junge A, Wyder S, Huerta-Cepas J, Simonovic M, Doncheva NT, Morris JH, Bork P, Jensen LJ, Mering CV (2019) STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res 47:D607-D613.
[5]. Li W, Zhu Y, Wan L, Guang C, Mu W. (2021) Pathway Optimization of 2'-Fucosyllactose Production in Engineered Escherichia coli. J Agric Food Chem. Feb 10;69(5):1567-1577.
[6] Wan, L.; Zhu, Y.; Li, W.; Zhang, W.; Mu, W. (2020) Combinatorial Modular Pathway Engineering for Guanosine 5′-Diphosphate-l-fucose Production in Recombinant Escherichia coli. J. Agric. Food Chem. 68, 5668-5675.