Evolutionary Engineering Improves Tolerance for Replacement Jet Fuels in Saccharomyces cerevisiae.

Monoterpenes are liquid hydrocarbons with applications ranging from flavor and fragrance to replacement jet fuel. Their toxicity, however, presents a major challenge for microbial synthesis. Here we evolved limonene-tolerant Saccharomyces cerevisiae strains and sequenced six strains across the 200-generation evolutionary time course. Mutations were found in the tricalbin proteins Tcb2p and Tcb3p. Genomic reconstruction in the parent strain showed that truncation of a single protein (tTcb3p(1-989)), but not its complete deletion, was sufficient to recover the evolved phenotype improving limonene fitness 9-fold. tTcb3p(1-989) increased tolerance toward two other monoterpenes (ß-pinene and myrcene) 11- and 8-fold, respectively, and tolerance toward the biojet fuel blend AMJ-700t (10% cymene, 50% limonene, 40% farnesene) 4-fold. tTcb3p(1-989) is the first example of successful engineering of phase tolerance and creates opportunities for production of the highly toxic C10 alkenes in yeast.

Analysis of the biomass composition of the demosponge Amphimedon queenslandica on Heron Island Reef, Australia.

Marine sponges are a potential source of important pharmaceutical drugs, the commercialisation of which is restricted by the difficulties of obtaining a sufficient and regular supply of biomass. One way to optimize commercial cell lines for production is the in-depth characterization and target identification through genome scale metabolic modeling and flux analysis. By applying these tools to a sponge, we hope to gain insights into how biomass is formed. We chose Amphimedon queenslandica as it has an assembled and annotated genome, a prerequisite for genome scale modeling. The first stepping stone on the way to metabolic flux analysis in a sponge holobiont, is the characterization of its biomass composition. In this study we quantified the macromolecular composition and investigated the variation between and within sponges of a single population. We found lipids and protein to be the most abundant macromolecules, while carbohydrates were the most variable. We also analysed the composition and abundance of the fatty acids and amino acids, the important building blocks required to synthesise the abundant macromolecule types, lipids, and protein. These data complement the extensive genomic information available for A. queenslandica and lay the basis for genome scale modelling and flux analysis.

Physiological and transcriptional responses of Saccharomyces cerevisiae to d-limonene show changes to the cell wall but not to the plasma membrane.

Monoterpenes can, upon hydrogenation, be used as light-fraction components of sustainable aviation fuels. Fermentative production of monoterpenes in engineered microorganisms, such as Saccharomyces cerevisiae, has gained attention as a potential route to deliver these next-generation fuels from renewable biomass. However, end product toxicity presents a formidable problem for microbial synthesis. Due to their hydrophobicity, monoterpene inhibition has long been attributed to membrane interference, but the molecular mechanism remains largely unsolved. In order to gain a better understanding of the mode of action, we analyzed the composition and structural integrity of the cell envelope as well as the transcriptional response of yeast cells treated with an inhibitory amount of d-limonene (107 mg/liter). We found no alterations in membrane fluidity, structural membrane integrity, or fatty acid composition after the solvent challenge. A 4-fold increase in the mean fluorescence intensity per cell (using calcofluor white stain) and increased sensitivity to cell wall-degrading enzymes demonstrated that limonene disrupts cell wall properties. Global transcript measurements confirmed the membrane integrity observations by showing no upregulation of ergosterol or fatty acid biosynthesis pathways, which are commonly overexpressed in yeast to reinforce membrane rigidity during ethanol exposure. Limonene shock did cause a compensatory response to cell wall damage through overexpression of several genes (ROM1, RLM1, PIR3, CTT1, YGP1, MLP1, PST1, and CWP1) involved with the cell wall integrity signaling pathway. This is the first report demonstrating that cell wall, rather than plasma membrane, deterioration is the main source of monoterpene inhibition. We show that limonene can alter the structure and function of the cell wall, which has a clear effect on cytokinesis.

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

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: ß-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.