Advancing CRISPR technologies to engineer yeast metabolism

Abstract: The advent of genetic engineering tools has initiated an era of manipulating microorganisms for the production of valuable compounds for our society. Precise engineering of these microbes commonly requires introducing genetic modifications such as gene deletion, overexpression, and accurate regulation in order to enhance the production of the compound of interest. In this context, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology, adapted from the prokaryotic adaptive immune system, has revolutionized our ability to manipulate a broad range of living organisms. In contrast to other methods, this technology works like a molecular pair of scissors (Cas9) which is guided by a programmable RNA (gRNA) molecule binding at a specific location in the DNA. The programmability and time-efficiency offered by this technology have in the recent years been successfully exploited in rewiring the metabolic network to enhance the production of metabolites used in various areas of industrial biotechnology.   In this thesis, I present several studies applying the technological diversity provided by CRISPR in the context of building efficient yeast cell factories for the production of oleochemicals -sustainable substitutes for plant derived lipids. Since oleochemicals derive from lipid products, the main engineering strategies presented essentially focus on fatty acid metabolism and its precursors. First, we exploited CRISPR/Cas9 endonuclease capacity to extensively remodel yeast lipid metabolism. We showed that the disruption of several metabolic fluxes allows to overcome the main limiting steps in fatty acid biosynthesis and favors the production of free fatty acids and triacylglycerols, two important precursors for the production of oleochemicals. Second, we harnessed the ability to precisely regulate genes using the catalytically deactivated form of the Cas9 protein (dCas9) coupled to transcription factors for fine-tuning the expression of genes involved in lipid biogenesis. Additionally, we proposed a framework for dCas9-based applications based on computational techniques for predicting key genes potentially favoring the production of yeast endogenous metabolites. Finally, we expanded the CRISPR repertoire by building new tools to accelerate yeast cell factory design. We exploited a Type I CRISPR-associated endoribonuclease for multiplex genome engineering and transcriptional regulation via processing an RNA transcript into multiple gRNAs, and we developed a computational tool for designing gRNAs targeting multiple loci at once. In summary, the work presented in this thesis provides various ways to efficiently engineer yeast metabolism by exploiting the diversity of CRISPR technologies, as well as new tools to the community for future engineering strategies.