(228d) Title: Metabolic Engineering of the Non-Conventional Yeast, Kluyveromyces Marxianus, for Enhanced Biosynthesis of a Platform Biochemical

Kluyveromyces marxianus is a non-conventional yeast commonly found in fermented dairy products that is exceptionally fast-growing, thermo- and pH-tolerant, metabolizes a wide range of low-cost carbon sources, and has great potential for biosynthesis of acetyl-CoA-based products. Despite these advantageous characteristics, K. marxianus has not been widely engineered, in part, due to the high strength of the non-homologous end joining pathway which greatly hinders genomic modification; although, the recent emergence of CRISPR-Cas9 genome editing technology has alleviated some of these roadblocks. After constructing our own CRISPR-Cas9 expression system and disrupting the non-homologous end joining pathway, we have created a robust and efficient platform of metabolic engineering tools for use in K. marxianus that routinely enables a high rate of targeted editing including genomic integrations with as little as 40 base pair homologies. Using these tools, we have conducted rationally and computationally guided metabolic engineering to significantly improve the biosynthesis of a heterologous polyketide, triacetic acid lactone (TAL), an acetyl-CoA-derived platform chemical that serves as a precursor to several prominent high-value and commodity products. Our computational analysis of the K. marxianus metabolism invoked use of the first available genome-scale model for this yeast as well as the COBRA toolbox algorithms OptKnock and OptForce. Use of these computational tools largely validated our rational pathway analysis in that disruption of key byproduct accumulation pathways (e.g. glycogen) is a prominent strategy toward improved precursor accumulation. Other metabolic engineering strategies employed upregulations of reactions directly leading to TAL precursors and modifications to compartmental cofactor balances. Combining the best performing modifications led to a several-fold increase in TAL biosynthesis over the unengineered strains using defined media supplemented with xylose or lactose, sugars typically treated as waste products. Altogether, our work demonstrates effective strategies for targeted metabolic engineering, as well as the great potential for high-titer biosynthesis of acetyl-CoA-based heterologous products in this under-studied and promising industrial yeast species. As an alternative to this bottom-up, genotype-to-phenotype approach to metabolic engineering, we have pursued application of high-throughput CRISPR-Cas9 genome-scale guide RNA libraries which instead enable phenotype-to-genotype discovery. Such methods have expedited the unbiased and direct identification of essential genes in our K. marxianus strains of interest and in prescribed growth environments, thus avoiding the limitations of rational pathway analysis and computational models in a species that is under-characterized.