Robertson Nicholas R, Lenert-Mondou Chase, Leonard Alison C, Tafrishi Aida, Carrera Stephanie, Lee Sangcheon, Aguilar Yuna, Sanchez Zamora Leonardo, Nguyen Trinity, Beltrán Jesús, Li Mengwan, Cutler Sean R, Whitehead Timothy A, Wheeldon Ian
Bioengineering, University of California, Riverside, Riverside, California 92521, United States.
Biochemistry and Molecular Biology, University of California, Riverside, Riverside, California 92521, United States.
ACS Synth Biol. 2025 Jul 10. doi: 10.1021/acssynbio.4c00797.
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 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.
单萜类化合物因其作为香料、香水、杀虫剂和高能量密度燃料的作用而备受重视。微生物为这些重要分子提供了可持续的生物合成途径，但产量仍然有限。在这里，我们引入了一种生物传感器驱动的微生物工程策略来提高单萜类化合物的产量，特别是针对香叶醇。使用PYR1受体的诱变文库——一种来自植物ABA信号通路的通用生物传感器，具有可塑的结合口袋——我们筛选了24种单萜类化合物，并鉴定出对包括香叶醇在内的8种化合物有反应的PYR1变体。一种低背景、高选择性的香叶醇敏感型PYR1变体在耐热酵母中作为基于生长的生物传感器电路表达，从而实现快速的菌株工程改造。通过将香叶醇敏感型PYR1传感器与全基因组CRISPR-Cas9诱变方法相结合，我们鉴定出6个提高香叶醇产量的基因敲除，使产量提高了2倍。这项研究证明了PYR1生物传感器平台在实现快速菌株工程改造以及鉴定提高所需代谢物产量的突变体方面的强大作用。
