UC Irvine

It is widely accepted that aggressive development of the bio-economy is needed to mitigate the effects of imminent resource shortages and climate change. The employment of microorganisms, such as yeasts, as biorefineries is one feasible route to meet this demand. 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. In this work we demonstrate the high native capacity of K. marxianus to produce triacetic acid lactone (TAL), a member of the diverse polyketide class of bioactive molecules, from waste substrates, achieving a yield comparable to other industrially relevant yeasts that required extensive engineering. Despite these advantageous characteristics, K. marxianus has not been widely engineered; this is due in part to the strong non-homologous end joining pathway which greatly hinders targeted genomic modification. The recent emergence of CRISPR-Cas9 genome editing technology has alleviated this roadblock, allowing metabolic engineering of K. marxianus to be more accessible. By constructing a highly efficient CRISPR-Cas9 system and disrupting the non-homologous end joining pathway, we have created a robust and efficient platform that routinely enables a high rate of targeted editing in K. marxianus, including genomic integrations with as little as 40 base pair homologies. Using this tool, we have conducted rationally and computationally guided metabolic engineering to significantly improve the biosynthesis of TAL and an additional polyketide, 6-methylsalisylic acid (6-MSA). Combining the best performing modifications led to 1.66- and 2.5-fold increases in TAL and 6-MSA biosynthesis, respectively, over the unengineered strains in defined media containing xylose or lactose. 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 implemented high-throughput CRISPR-Cas9 genome-scale guide RNA libraries which instead enable phenotype-to-genotype gene discovery. Optimizing the library composition and expression system resulted in a final library with exceptionally high cutting activity. Application of this library has enabled the unbiased and direct identification of essential genes in our K. marxianus strains under prescribed growth environments. Pairing these libraries with a growth-coupled metabolite sensor also enabled direct identification of metabolic engineering targets that increase accumulation of critical polyketide precursors. This approach bypasses the current limitations of rational pathway analysis and use of computational models in less-characterized yeasts, by providing precise insights into the best interventions for optimal biosynthesis of heterologous products.