Microbial biocatalysis in organic solvents such as ionic liquids (ILs) is attractive for making high-value chemicals. However, IL toxicity at a level of 0.5% ~1% (v/v) can drastically reduce microbial activity. In this study, we engineered a mutant Yarrowia lipolytica YlCW001 through adaptive evolution engineering that can thrive in up to 18% (v/v) 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), lethal to almost all microorganisms. Remarkably, YlCW001 also exhibits universal tolerance in most commonly used ILs such as 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]) and bromide ([BMIM][Br]), [EMIM][Cl], [EMIM][Br], and 1-allyl-3-methylimidazolium chloride ([AMIM][Cl])). Scanning electron microscopy reveals that ILs significantly damage cell wall and/or membrane of wildtype Y. lipolytica while YlCW001 maintains healthy cellular morphology even in high concentration of ILs up to 18% (v/v). Through comprehensive metabolomics, lipidomics, and transcriptomics study, we elucidate the underlying mechanisms of how the mutant reconfigures membrane composition and structure during evolutionary engineering and hence exhibits high IL tolerance. To further identify the genotypes conferring the robustness of Y. lipolytica, we develop a novel gene expression classifier (GEC) algorithm to analyze the dynamic transcriptional data of strains under various conditions integrating differential gene expression classification, gene co-expression network analysis, and scaled degree centrality. Using GEC, we discover a synergistic set of novel genes conferring the robustness of Y. lipolytica and validate these genotypes in wildtype Yarrowia strains of different origins.

Discovery of novel genotypes conferring robustness of Yarrowia lipolytica in organic solvents for novel biotransformation processes

ACS Spring 2022

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