Single-step in vivo assembly of synthetic chromosomes with a modular design for engineering yeast metabolism

Microorganisms have been used by humanity since ancient times to produce fermented food and beverages such as wine, bread and beer. With the current molecular biology techniques, it is not only possible to increase microbial performance for the production of native products, but also to adapt microorganism to make heterologous or even novel-to-nature compounds. Hereby, microbial cell factories can produce fuels and chemicals to replace the polluting and unsustainable fossil fuel industry. Saccharomyces cerevisiae, also known as baker’s yeast has been used since decades as a preferred host to produce a range of industrial relevant compounds and as model organism in fundamental research. Next to its simple nutritional requirements and tractability, the popularity of S. cerevisiae also stems from its remarkable ability to efficiently and precisely stitch DNA molecules together via homologous recombination (HR). While S. cerevisiae uses this native mechanism to repair deleterious breaks in its genetic material while remaining faithful to the original sequence, HR has been harnessed by genetic engineers to seamlessly introduce or delete specific DNA sequences. The combination of CRISPR/Cas genome editing ability with HR precision repair enables genome remodelling for the construction of cell factories. A current drawback of this approach is that only a limited number of DNA modifications can be achieved in one transformation round, making extensive remodelling a time-consuming process. The goal of this thesis was to design and evaluate a strategy for extensive and efficient engineering of metabolism in S. cerevisiae...