Building synthetic cells is extremely interesting from a fundamental perspective, as the ability to rationally build viable, dividing, and self-maintaining cells provides knowledge on the minimal requirements to sustain life. This should enhance our understanding about which biological parts are minimally required for a cell to live, and how different cellular functions work and interact with each other. Besides a fundamental understanding of life, synthetic cells can also be applied as synthetic biology tools, for example for synthesis and delivery of therapeutics, or the production of compounds that cannot be produced with currently available organisms used as cell factories. The overarching goal of the research described in this thesis was to devise a strategy for building genomes for synthetic cell, using baker’s yeast Saccharomyces cerevisiae. Two methods were explored, the first being building a genome de novo using yeast in vivo assembly, and secondly, it was investigated whether the preexisting minimal genome of S. cerevisiae mitochondria could be expanded. To this end, yeast mitochondrial DNA and RNA was characterized using novel methods and various strategies for engineering the yeast mitochondrial genome were tested.

Saccharomyces cerevisiae, also known as baker’s yeast, is a robust microorganism frequently used in industrial biotechnology. The scale of its applications ranges from several millilitres for research and process development in the lab to hundreds of cubic meters for cultivation in industrial production processes. In large-scale reactors mixing limitations inherently lead to physiochemical gradients in substrate and oxygen concentrations, pH or temperature. Such inhomogeneous environment in production processes can cause a reduced yield or titer compared to the small-scale development processes. Such scale performance differences can lead to significant worse process economics and increase costs and development time.
The scope of this thesis is to study and understand the regulation of Saccharomyces cerevisiae metabolism under dynamic substrate conditions, using both experimental and modelling approaches.

One of the most conserved and central parts of metabolism is the pathway of glycolysis, that breaks down hexose sugars. While multiple variants of glycolytic pathway exist, the Emden-Meyerhof-Parnas pathway which converts glucose to two molecules of pyruvate with the net gain of two ATP and two reduced NADH moieties is the one predominantly found in eukaryotes. This well-studied metabolic pathway is important for industrial biotechnology, because sugars are often used as substrate for microbial production of fuels and chemicals, as well as human health, because of the involvement of glycolytic dysfunction and deregulation in diseases such as cancer. The analysis of glycolytic enzyme function is complicated by the presence in many organisms of a large range of secondary ‘moonlighting’ functions besides the main glycolytic function. Because of its robustness, genetic accessibility, ease of culture and highly expressed glycolytic pathway, baker’s yeast, Saccharomyces cerevisiae has been key to research on glycolysis. Besides its role in food and beverage production and as industrial host for e.g. bioethanol and insulin production, this yeast has been intensively used as a model organism to study fundamental processes and pathways...

Already for millennia, microbial fermentation is used for the production of dairy products, alcoholic beverages and bread. In the last decades, the field of biotechnology has tremendously expanded and nowadays, a wide range of compounds ranging from biofuels to chemicals and pharmaceuticals is produced using microbial cell factories. The development of genetic engineering tools has greatly contributed to this rapid development. Catalysing the conversion of renewable carbohydrate feedstocks into fuels and chemicals, microbial cell factories offer a sustainable alternative to fossil resources-based production, and thereby contribute to reduce greenhouse gas emissions. The yeast Saccharomyces cerevisiae plays an important role in industrial biotechnology. Its popularity for applied research and industrial production can be attributed to several factors as its fast fermentative metabolism, its tolerance to low pH, high sugar and alcohol concentrations and its genetic tractability. S. cerevisiae possesses one of the best furbished molecular toolboxes, which makes it possible to assemble complex heterologous pathways, as was recently illustrated by the successful biosynthesis of opioids in yeast. Despite this great progress, extensive genetic remodelling of native pathways remains challenging. This can largely be explained by the high genetic redundancy present in the yeast genome, in which multiple genes encode proteins with redundant functions, and by the fact that the genes belonging to a pathway are scattered over the entire genome. The goal of this thesis was to design, set up and validate a strategy aiming at facilitating the remodelling of (essential) pathways, based on simplifying and reorganizing the yeast genome. The starting point of this research is the central carbon metabolism and in particular, as proof of concept, the glycolytic pathway.