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Title: PYR1 Biosensor-Driven Genome-Wide CRISPR Screens for Improved Monoterpene Production in Kluyveromyces marxianus
Monoterpenes are valued for their roles as flavors, fragrances, insecticides, and energy-dense fuels. Microorganisms provide sustainable biosynthesis routes for these important molecules, but production levels remain limited. Here, we introduce a biosensor-driven microbial engineering strategy to enhance monoterpene 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 monoterpenes 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 the titer of a desired metabolite.  more » « less
Award ID(s):
2225878 2128016
PAR ID:
10663668
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ; ; ;
Publisher / Repository:
American Chemical Society, ACS Synthetic Biology
Date Published:
Journal Name:
ACS Synthetic Biology
Volume:
14
Issue:
8
ISSN:
2161-5063
Page Range / eLocation ID:
2972 to 2978
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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