Humanity has been harnessing microbes since Neolithic times, e.g. brewing beer or wine using yeast. Following the discovery of the DNA structure, scientists have endeavored to genetically reprogram some of these microbes to enhance the production of various compounds beneficial for our society, e.g. insulin, monoclonal antibodies, and biofuels, as well as finding new ways for producing them. Engineering strategies are commonly performed by introducing genetic modifications into the genome of the host, e.g. gene deletions and regulations, and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology has recently been revolutionizing our ability to perform such genetic manipulations. In contrast to other methods, this technology works like a molecular pair of scissors (Cas9) which is guided by a programmable RNA (a guide-RNA or gRNA) molecule binding at a specific location in the DNA.
In this thesis, I present different examples where the technological diversity provided by CRISPR can be applied to engineer Saccharomyces cerevisiae (baker’s yeast) into an efficient producer of oleochemicals - sustainable substitutes for petrochemicals and fuels. Since these compounds are derived from vegetable and animal oil, the presented strategies throughout the thesis focus on fatty acid metabolism and its precursors. Firstly, we exploited CRISPR/Cas9 to remove several genes involved in the yeast lipid metabolism to ultimately rewire it towards the accumulation of free fatty acids and triacylglycerols, two important precursors utilized for production of oleochemicals. Secondly, we harnessed the ability to precisely regulate genes using a deactivated form of the Cas9 (dCas9) protein coupled to transcription factors for controlling the expression of different genes involved in lipid biogenesis. We also propose a computational framework for dCas9-based strategies, which allows predicting genes to regulate in order to rewire the yeast metabolism towards the production of specific metabolites. Finally, we expanded the CRISPR repertoire by building new tools developed to facilitate yeast engineering.
The work presented in this thesis offers various ways to efficiently engineer yeast metabolism by exploiting the diversity of CRISPR technologies, and also provides new tools to the community for future engineering strategies.