Development of a CRISPR-based tool for localized random mutagenesis of biosynthetic pathways

Yeast is one of the most frequently used microbes in today’s biotechnology and fermentation industry. But, despite its domestication and adaptation to man-made environments, new applications (such as the production of bioethanol or high-value chemicals) confront it with new challenges and unfamiliar, harsh environments. This leaves many opportunities to generate new, superior variants optimized for specific applications. Current approaches based on classic genetic modification often only partly succeed in meeting these goals and are very time-consuming, especially for multigenic phenotypes. Therefore, in this project, we address these limitations by developing a novel, powerful strategy for industrial strain engineering. This strategy, referred to as ‘confined mutagenesis’, is founded on the most recent developments in CRISPR-based gene editing tools, such as Type-I CRISPR systems and ‘base editors’, which we will repurpose for microbe engineering. It will allow for a rapid and inducible introduction of mutations within a user-defined part of the genome. We will develop such systems for a wide array of industrially-relevant yeasts. As a proof-of-concept, we will use this technology for the optimization of 1) xylose conversion (for the production of biofuels) in Kluyveromyces marxianus and 2) astaxanthin production (a carotenoid widely used as food colorant and dietary supplement) in Saccharomyces cerevisiae. The proposed tool will address an unmet need in synthetic biology.