Abstract:[Background] Having special fragrance and various biological properties, linalool has become an important feedstock for food, pharmaceutical and cosmetics industries. With the development of synthetic biology, metabolic engineering of microorganisms has become an influential alternative for biosynthesis of linalool. However, the strong toxicity of linalool to host cells is a key bottleneck for microbial production of linalool and other monoterpenes. [Objective] This paper aimed to develop effective strategies for improving the tolerance of microbial host cells to linalool. [Methods] In this study, the ATP-binding cassette (ABC) transporters, reactive oxygen species (ROS)-related enzymes and transcription factors were overexpressed in Saccharomyces cerevisiae BY4741 to identify their roles in the tolerance of S. cerevisiae to linalool. In addition, adaptive laboratory evolution was adopted to obtain the S. cerevisiae strains with increased fitness towards linalool. [Results] Individual overexpression of ABC transporters (Yor1, Snq2, Pdr5, Pdr15 and Pdr18), ROS-related enzymes (Gre2, Ctt1, Yhb1, Gpx2, Trr1, Trx2 and Gsh2) and transcription factors (Ino2, Yap1, Yap5 and Stb5) in S. cerevisiae BY4741 failed to improve the tolerance of S. cerevisiae. Furthermore, S. cerevisiae with improved tolerance (lethal concentration of linalool was increased from 430 mg/L to 645 mg/L) were obtained via adaptive evolution and the SNV/InDel genes were analyzed by whole-genome resequencing. Mutations were found in YBR074W, YBR172C, YHR007C and YMR275C, which enhanced the tolerance to linalool. [Conclusion] The tolerance of S. cerevisiae to linalool was improved by evolutionary engineering, which laid a foundation for analyzing the mechanism of S. cerevisiae to tolerate monoterpenes and provided an excellent chassis cell for heterologous synthesis of monoterpenes.
