Alleviating monoterpene toxicity using a two‐phase extractive fermentation for the bioproduction of jet fuel mixtures in Saccharomyces cerevisiae

Abstract

Monoterpenes are a diverse class of compounds with applications as flavors and fragrances, pharmaceuticals and more recently, jet fuels. Engineering biosynthetic pathways for monoterpene production in microbial hosts has received increasing attention. However, monoterpenes are highly toxic to many microorganisms including Saccharomyces cerevisiae, a widely used industrial biocatalyst. In this work, the minimum inhibitory concentration (MIC) for S. cerevisiae was determined for five monoterpenes: β-pinene, limonene, myrcene, γ-terpinene, and terpinolene (1.52, 0.44, 2.12, 0.70, 0.53 mM, respectively). Given the low MIC for all compounds tested, a liquid two-phase solvent extraction system to alleviate toxicity during fermentation was evaluated. Ten solvents were tested for biocompatibility, monoterpene distribution, phase separation, and price. The solvents dioctyl phthalate, dibutyl phthalate, isopropyl myristate, and farnesene showed greater than 100-fold increase in the MIC compared to the monoterpenes in a solvent-free system. In particular, the MIC for limonene in dibutyl phthalate showed a 702-fold (308 mM, 42.1 g L(-1) of limonene) improvement while cell viability was maintained above 90%, demonstrating that extractive fermentation is a suitable tool for the reduction of monoterpene toxicity. Finally, we estimated that a limonane to farnesane ratio of 1:9 has physicochemical properties similar to traditional Jet-A aviation fuel. Since farnesene is currently produced in S. cerevisiae, its use as a co-product and extractant for microbial terpene-based jet fuel production in a two-phase system offers an attractive bioprocessing option.

Physiological and Transcriptional Responses of Saccharomyces cerevisiae to d -Limonene Show Changes to the Cell Wall but Not to the Plasma Membrane

“…Increasing membrane rigidity through changes in fatty acid content or by actively pumping monoterpene compounds from the cell (46) was not found to increase tolerance. Our results underscore the position that monoterpene inhibition is not at the molecular level (e.g., membrane interference effects) (3) and that the mechanism of action must stem from the physical interaction between an insoluble monoterpene phase and the surface of a cell. To this end, we have demonstrated here that limonene can alter the properties of yeast cell walls while triggering a compensatory transcriptional response to cell wall damage.…”

“…Like limonene, terpinenes are cyclic monoterpenes. Terpinene was used at a higher concentration (170 mg/liter rather than 107 mg/liter) but is also slightly less toxic; the terpinene concentration was chosen as the 50% inhibitory concentration (IC 50 ) in the previous study (15), while our study used nearly two times the IC 50 for limonene (3). The monoterpenes were added to cells in mid-exponential phase, i.e., 5 h in culture in our study and overnight in the terpinene study.…”

“…Monoterpenes, such as limonene, pinene, and cymene, have traditionally been used as flavors and fragrances (2) but recently have gained attention as potential light-component precursors for drop-in jet fuels (3)(4)(5). In June 2012, a demonstration flight by Amyris Inc. proved that their biojet fuel, AMJ-700, successfully met engine performance requirements and required no changes to the aircraft (6).…”

“…While solvent stress in microorganisms has been studied extensively over the past 40 years, these reports have focused primarily on short-chain alcohols and organic acids (13), and although monoterpenes are well-known antifungal agents, only a few quantitative studies exist (3,(14)(15)(16)(17). Due to their lipophilicity (log P ϭ 4.5), monoterpene inhibition has been attributed to molecular toxicity, i.e., the direct interference of solvent molecules with membrane function (17)(18)(19).…”

Evolutionary Engineering Improves Tolerance for Replacement Jet Fuels in Saccharomyces cerevisiae

“…Traditionally used in the flavor and fragrance industry, monoterpenes, such as d-limonene, are now also sought after as precursors for light end components of "dropin" jet fuels (2). With the ability to convert simple sugars into a variety of enantiomerically pure monoterpene products (3)(4)(5), microbial synthesis has the potential to overcome the low yields, impurities, and high costs associated with conventional chemical synthesis or extraction from biological tissues (6).…”