Kluyveromyces marxianus is a non-conventional yeast with great potential for industrial production of biobased chemicals due to its high thermo-tolerance (&gt;45oC), exceptionally fast growth rate (twice that of Saccharomyces cerevisiae), high TCA cycle flux, and ability to assimilate a broad range of carbon sources including C5, C6, and C12 sugars. After constructing a host of CRISPR-Cas9 tools tailored for use in K. marxianus, we have conducted rationally and computationally guided metabolic engineering to significantly improve biosynthesis of the heterologous polyketides triacetic acid lactone (TAL) and 6-methylsalisylic acid (6-MSA); these acetyl-CoA-derived chemicals can serve as precursors to several prominent high-value and commodity products. Our computational analysis of the K. marxianus metabolism utilized the first available genome-scale model for this yeast as well as the COBRA toolbox algorithms OptKnock and OptForce. Combining the best performing modifications led to a significant increase in polyketide biosynthesis over the unengineered strains. While our work has demonstrated effective strategies for targeted metabolic engineering in this promising non-conventional yeast, as well as its great potential for high-titer biosynthesis of acetyl-CoA-based products, rational and predicted interventions were not as successful as in other more-studied yeast species. As an alternative to this bottom-up, genotype-to-phenotype approach to metabolic engineering, we have now implemented high-throughput CRISPR-Cas9 genome-scale guide RNA libraries which instead enable phenotype-to-genotype gene discovery. Such methods have expedited the unbiased and direct identification of essential genes in our particular K. marxianus strains of interest and in prescribed growth environments. Pairing these libraries with a growth-coupled metabolite sensor also enabled direct identification of metabolic engineering targets that increase accumulation of a critical polyketide precursor, thus avoiding the limitations of rational pathway analysis and computational models in a species that is under-characterized.

Metabolic engineering of the thermotolerant yeast Kluyveromyces marxianus for high yield biosynthesis of polyketides

ACS Spring 2022

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