This project attempts to produce the infant nutritional supplements lactoferrin and N-acetylneuraminic acid in the Saccharomyces cerevisiae BY4741. We hope to reduce the cost of these two substances to reduce the financial strain on many low and middle income families. We also validated the viability of the protein expression system using a fluorescent reporter gene, and may be able to provide a viable option for others to validate protein expression in Saccharomyces cerevisiae in the future.

Our team aims to address the issue of nutritional deficiencies and the high cost of formula. We propose that essential nutrients that need to be added to formula can be produced in large quantities at low cost by microorganisms, such as lactoferrin and N-acetylneuraminic acid, two nutrients that are intimately related to immune health and mental development in infancy and early childhood.

Chassis Cell Selection
To achieve our goals, we intend to use genetic engineering techniques to introduce genes for the production of lactoferrin and N-acetylneuraminic acid into Saccharomyces cerevisiae in order to produce these two essential nutrients.

Saccharomyces cerevisiae BY4741 is considered a safe (GRAS) organism and is widely used in the food and pharmaceutical industries. As a more well-studied model organism, yeast cells are easy to genetically manipulate, can efficiently insert multiple genes, and are suitable for large-scale fermentation cultures, which facilitates the industrial production of target products.

In addition, yeast, as a eukaryote, can undergo post-translational modifications similar to those of human cells, which is important for the expression of functional human proteins. And we are in need of such chassis to express human lactoferrin gene.

We chose pESC-URA, a protein expression plasmid commonly used in Saccharomyces cerevisiae, as a vector to introduce the human lactoferrin gene into cells. The plasmid is an E. coli and Saccharomyces cerevisiae shuttle plasmid, which is convenient for editing. It uses a nutrient-deficient screening marker in eukaryotes, avoiding the addition of antibiotics.

Besides, the expression of this plasmid in Saccharomyces cerevisiae is regulated by the galactose promoter, which avoids the addition of toxic inducers and provides a guarantee for the safety of our products.

Our experimental project is divided into three parts: synthesis and validation of lactoferrin and N-acetylneuraminic acid, as well as examination of protein expression systems in Saccharomyces cerevisiae.

Considering that our products will ultimately be used to supplement the nutrition of infants and young children, we chose human-derived lactoferrin as our target product, rather than sources such as cow's milk or goat's milk. In this way, we can ensure that our products are better absorbed and utilised without antigenic reactions.

We turned to Gene Synthesis to synthesise the human lactoferrin gene HsLF, which was then ligated into the pESC-URA plasmid vector using the Gibson assembly method. Colony PCR and sequencing were used to confirm the correctness of the recombinant plasmids.

After the recombinant plasmid pESC-HsLF was constructed, we transferred it into Saccharomyces cerevisiae BY4741 and screened it using URA-deficient medium. Subsequent expanded cultures of engineered yeast and induced fermentations were carried out using SS-URA medium.

The synthesis of N-acetylneuraminic acid in Saccharomyces cerevisiae was engineered based on the glycolytic pathway. After glucose is taken up by the cell, it is converted to glucose-6-phosphate, then to fructose-6-phosphate, which is converted to N-acetylneuraminic acid by the action of five genes: gfal, gnal, yqaB, age, and neuB. One of them, the glucose-6-phosphate synthase gene gfal, is endogenous to Saccharomyces cerevisiae.

In our original design, we were looking to introduce four additional genes to complete the de novo synthesis of N-acetylneuraminic acid in Saccharomyces cerevisiae.

We first focused on the N-acetylglucosaminidase gene (age) from Anabaena sp. CH1 and the N-acetylneuraminic acid synthase gene (neuB) from Escherichia coli K1. Similarly, we constructed the plasmid pESC-neuB-age by inserting these two synthetic genes into pESC-URA. If these two genes are successfully expressed in Saccharomyces cerevisiae, they will be able to convert N-acetylglucosaminide to N-acetylneuraminic acid in cells.

Similar to pESC-HsLF, we constructed the plasmid pESC-neuB-age using the Gibson assembly method. After colony PCR and sequencing verified that the recombinant plasmid was correct, it was transferred into Saccharomyces cerevisiae BY4741 for the following step.

Since the cells of Saccharomyces cerevisiae are difficult to fragment, SDS-PAGE may have the problem of not detecting all proteins. Therefore, we would like to design a system to verify whether the proteins in the recombinant plasmid can be expressed properly.

Two plasmids, pESC-mCherry and pESC-HsLF-mCherry, were constructed for this purpose using Gibson assembly, the latter of which was used to examine lactoferrin expression. After sequencing, the two plasmids were transferred into Saccharomyces cerevisiae for subsequent amplification culture.

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