PYR1 biosensor-driven genome-wide CRISPR screens for improved monoterpenoid production in Kluyveromyces marxianus

Robertson, N. R.; Lenert-Mondou, C.; Leonard, A. C.; Tafrishi, A.; Carrera, S.; Lee, S.; Aguilar, Y.; Zamora, L. S.; Nguyen, T.; Beltran, J.; Li, M.; Cutler, S. R.; Whitehead, T. A.; Wheeldon, I.

doi:10.1101/2024.11.14.623641

bioRxiv · 10.1101/2024.11.14.623641

PYR1 biosensor-driven genome-wide CRISPR screens for improved monoterpenoid production in Kluyveromyces marxianus

Robertson, N. R.·Lenert-Mondou, C.·Leonard, A. C.·Tafrishi, A.·Carrera, S.·Lee, S.·Aguilar, Y.·Zamora, L. S.·Nguyen, T.·Beltran, J.·Li, M.·Cutler, S. R.·Whitehead, T. A.·Wheeldon, I.

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Abstract

Monoterpenoids are valued for their roles as flavors, fragrances, insecticides, and energy-dense fuels. Microbial biosynthesis offers sustainable biosynthesis routes for these important molecules, but production levels remain limited. Here, we introduce a biosensor-driven microbial engineering strategy to enhance monoterpenoid production, specifically targeting geraniol. Using mutagenized libraries of the PYR1 receptor--a versatile biosensor from plant ABA signaling pathways with a malleable binding pocket--we screened 24 monoterpenoids and identified PYR1 variants responsive to eight, including geraniol. A low background, highly selective geraniol-sensitive PYR1 variant was expressed in the thermotolerant yeast Kluyveromyces marxianus as a growth-based biosensor circuit, allowing for rapid strain engineering. By coupling the geraniol-sensitive PYR1 sensor with a genome-wide CRISPR-Cas9 mutagenesis approach, we identified six gene knockouts that enhance geraniol production, achieving up to a 2-fold increase in titer. This study demonstrates the power of the PYR1 biosensor platform to enable rapid strain engineering and the identification of mutants that improve titer of a desired metabolite.

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Robertson, N. R., Lenert-Mondou, C., Leonard, A. C., Tafrishi, A., Carrera, S., Lee, S., Aguilar, Y., Zamora, L. S., Nguyen, T., Beltran, J., Li, M., Cutler, S. R., Whitehead, T. A., Wheeldon, I.. 2024-11-14. PYR1 biosensor-driven genome-wide CRISPR screens for improved monoterpenoid production in Kluyveromyces marxianus. https://doi.org/10.1101/2024.11.14.623641

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