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Synthetic platforms for ad libitum remodelling of yeast central metabolism

Periodic Reporting for period 4 - AdLibYeast (Synthetic platforms for ad libitum remodelling of yeast central metabolism)

Reporting period: 2020-03-01 to 2020-08-31

Replacement of petrochemistry by bio-based processes is key to sustainable development and requires microbes equipped with outstanding, novel-to-nature capabilities. Constructing such advanced ‘cell factories’ requires large-scale remodelling of their core machinery, and therefore of their genome. However, microbes often harbour mosaic genomes in which thousands of genes are scattered over dozens of chromosomes. This absence of a modular organization tremendously restricts genetic accessibility and defies current technologies. To tackle this limitation we develop microbial platforms with specialized, interchangeable synthetic chromosomes that will enable the core machinery to be remodeled at will. Access to a sheer endless variety of configurations of core metabolism offers unique, new possibilities for fundamental understanding and rational engineering of microbes. Our research paves the way for a modular approach to engineering of microbial cells.
This project focused on the industrial and model yeast Saccharomyces cerevisiae, most commonly known as baker’s yeast. We developed new, CRISPR-based tools for fast and easy modification (editing) of the yeast genome, and for one-step addition of new functions using newly synthetized and specialized chromosomes. The major outcome of this project can be summarized along three key achievements. We focused on a set of ca 120 biochemical reactions that are essential for life and for the production of all industrially-relevant molecules, called central carbon metabolism (CCM). CCM converts sugars into bioethanol, organic acids and many other interesting bulk and fine chemicals. Out of these 120 reactions we demonstrated that a substantial fraction (ca. 35%) is not required for yeast growth in a broad range of environments. Thanks to this fundamental discovery the first achievement was the construction of yeast strains with simplified CCM, that can be more easily engineered. Secondly, we demonstrated the amazing ability of bakers’ yeast for one-step assembly of linear and circular chromosomes from pieces of DNA. Based on this technology, the second achievement was the implementation of synthetic chromosomes for fast and easy construction of new yeast cell factories equipped with novel capabilities. As demonstration, standard yeast was turned into an anthocyanin producer. Anthocyanins have many applications in the pharmaceutical and food industry compounds and are naturally produced by plants. Last but not least, our project focused on a metabolic pathway essential for all forms of life, including human, called glycolysis. This project developed tools for fast and easy swapping of yeast glycolysis by other variants. This work lead to the humanization of yeast entire glycolytic pathway. Demonstrating the remarkable ability of human glycolytic enzymes to replace their yeast homologue as individual enzyme and as entire pathway, this work provides a new humanized yeast model to study human glycolysis and its connection to many known diseases.
This project therefore produced new molecular tools, new yeast (model) strains and new fundamental insight, spurred by technological advances in DNA and chromosome engineering. This research paves the way for a modular approach to engineering of microbial cells.
Assembly of modular, specialized chromosomes for the construction of yeast cell factories