One of the main goals of the relatively new field of synthetic biology is to develop new, artificial biochemical routes starting from standardized parts or building blocks, such as
promoters, genes, etc. that allow the modulation of de novo designed biochemical routes. These new routes (or pathways) can lead to the production of biochemicals that are used in pharma and food applications, and that are cumbersome to make via classical organic chemistry. By expressing multiple non-native genes in an organism, a new biochemical pathway can be built. There are however two main challenges: 1) the activity of these new enzyme has to be balanced in concert with the rest of the metabolism and 2) the various intermediates have to easily find the corresponding enzyme in the crowded intracellular environment. Nature solves this problem via a wide variety of regulatory systems and protein-protein interactions, but these types of mechanisms are not present in synthetic biochemical routes. The goal of this project is to fine-tune the pathway and to build up protein-protein interactions, so that the different enzymes involved in the pathway can form a protein complex or scaffold. Then, the pathway intermediates are channeled through this scaffold or “metabolon”, allowing locally increased substrate concentrations and increased reaction velocities. In this project we want to design and optimize such a protein scaffold or synthetic metabolon for the synthesis of 2-fucosyllactose, and oligosaccharide found in mother’s milk with nutraceutical properties and with applications in infant formula.