Understanding and Overcoming Monoterpene Toxicity in Yeast for the Production of Renewable Jet Fuels

Monoterpenes are ten-carbon (C₁₀) liquid hydrocarbons that can serve as immediate precursors for replacement jet fuels. In a demonstration flight in June 2012, Amyris Inc. showed that their biojet fuel, AMJ-700, met engine performance requirements and required no changes to the aircraft. Monoterpenes make up 60% of this jet fuel mixture and are essential for meeting the strict physiochemical specifications. Fermentative production of monoterpenes in industrially relevant microbial hosts, such as Saccharomyces cerevisiae, is severely hindered by product toxicity. The first part of this work describes the physiological impact of limonene on the yeast cell envelope. Membrane fluidity, composition and structural integrity were found undisturbed during limonene stress. Surprisingly, limonene caused damage to the cell wall lattice structure. The data is the first report demonstrating that cell wall, rather than the plasma membrane, deterioration is the source of toxicity ¹. Secondly, we describe two strategies to overcome limonene toxicity: (1) extractive fermentation employing farnesene as a co-product and extractant ²and (2) adaptive laboratory evolution (ALE). For ALE, strains were evolved through serial passage achieving a 5-fold improvement in solvent tolerance. Strains collected across the evolution process were sequenced and mutations identified. The original strain was engineered to carry one or more of these mutations, and it was found that a single mutation could confer the tolerant phenotype. We are currently characterizing this mutation using a full complement of systems biology tools.

1. Brennan, T.C.R., Krömer, J.O. & Nielsen, L.K. Physiological and transcriptional responses of Saccharomyces cerevisiae to d-limonene show changes to the cell wall but not to the plasma membrane. Appl Environ Microbiol 79, 3590-3600 (2013).
2. Brennan, T.C.R., Turner, C.D., Krömer, J.O. & Nielsen, L.K. Alleviating monoterpene toxicity using a two-phase extractive fermentation for the bioproduction of jet fuel mixtures in Saccharomyces cerevisiae. Biotechnol Bioeng 109, 2513-2522 (2012).