Evolutionary Engineering Improves Tolerance for Replacement Jet Fuels in Saccharomyces cerevisiae

• Published in: Applied and Environmental Microbiology 2015-05, Vol.81 (10), p.3316-3325
• Main Authors: Brennan, Timothy C R, Williams, Thomas C, Schulz, Benjamin L, Palfreyman, Robin W, Krömer, Jens O, Nielsen, Lars K
• Format: Article
• Language: English
• Subjects: Biological Evolution; Biosynthesis; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - metabolism; Cyclohexenes - metabolism; Genomics; Hydrocarbons; Hydrocarbons - metabolism; Membrane Proteins - genetics; Membrane Proteins - metabolism; Metabolic Engineering; Monoterpenes - metabolism; Mutation; Physiology; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Terpenes - metabolism; Toxicity; Yeast
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/AEM.04144-14

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 (β-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.