Acute Limonene Toxicity in Escherichia coli Is Caused by Limonene Hydroperoxide and Alleviated by a Point Mutation in Alkyl Hydroperoxidase AhpC

Abstract

f Limonene, a major component of citrus peel oil, has a number of applications related to microbiology. The antimicrobial properties of limonene make it a popular disinfectant and food preservative, while its potential as a biofuel component has made it the target of renewable production efforts through microbial metabolic engineering. For both applications, an understanding of microbial sensitivity or tolerance to limonene is crucial, but the mechanism of limonene toxicity remains enigmatic. In this study, we characterized a limonene-tolerant strain of Escherichia coli and found a mutation in ahpC, encoding alkyl hydroperoxidase, which alleviated limonene toxicity. We show that the acute toxicity previously attributed to limonene is largely due to the common oxidation product limonene hydroperoxide, which forms spontaneously in aerobic environments. The mutant AhpC protein with an L-to-Q change at position 177 (AhpC L177Q ) was able to alleviate this toxicity by reducing the hydroperoxide to a more benign compound. We show that the degree of limonene toxicity is a function of its oxidation level and that nonoxidized limonene has relatively little toxicity to wild-type E. coli cells. Our results have implications for both the renewable production of limonene and the applications of limonene as an antimicrobial. L imonene, the major component of citrus peel oil, has a variety of industrial and microbiological applications. Its antimicrobial properties make it a popular component of disinfectants and food preservatives and an environmentally friendly solvent used at the industrial scale (1-4). More recently, limonene or its hydrogenated forms have been identified to be potential jet fuel components (5-7). As such, the anticipation of a greater global demand for limonene has provided significant motivation for the renewable production of this compound from plant biomass through a microbial process (8-10), and recent efforts to optimize production have resulted in titers of over 400 mg/liter at the bench scale (11). In this context, the toxicity of limonene to the microbial host presents a major challenge, as the accumulation of toxic products limits growth and metabolic activity.Curiously, the toxicity of limonene has been reported to be significantly higher than that of other monoterpenes or solvents with similar hydrophobicities (12, 13), suggesting that this acute toxicity is due to something other than its solvent-like properties. However, molecular-level studies of limonene toxicity in microbes are limited. While several studies in Escherichia coli have noted an impact of limonene on lipid composition (14) and suggested a role for reactive oxygen species (ROS) (15), no specific hypothesis for how limonene causes these cellular perturbations has been proposed.In this work, we investigated the basis of limonene toxicity in the model Gram-negative bacterium E. coli. We identified a mutant with significantly enhanced tolerance to limonene and show that the majority of the toxicity in wild-type (WT) cells is due no...

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“…d Toxicity is reported using very different assays in the literature [15,20,23,65,75,86,101,102]: % values represent v/v levels sufficient to cause decrease in growth by at least half of optimal growth in the given media. e Acute limonene toxicity is caused by products of limonene oxidation such as limonene hydroperoxide [132].…”

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“…Finally, exogenous addition can introduce unintended artifacts or impurities to the experimental system. In the case of limonene, for instance, extended chemical storage forms hydroperoxide derivatives that are significantly more toxic than limonene itself (Chubukov et al, 2015).…”

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