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 (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.
Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Queensland, Australia.
Timothy C R
TC
Brennan
Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Queensland, Australia.
Thomas C
TC
Williams
School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland, Australia.
Benjamin L
BL
Schulz
Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Queensland, Australia.
Robin W
RW
Palfreyman
Centre for Microbial Electrochemical Systems (CEMES), Advanced Water Management Centre (AWMC), University of Queensland, Brisbane, Queensland, Australia j.kromer@uq.edu.au.
Jens O
JO
Krömer
Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Queensland, Australia.
Lars K
LK
Nielsen
D005075:Biological Evolution; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D053138:Cyclohexenes / Q000378:metabolism; D006838:Hydrocarbons / Q000378:metabolism*; D000077222:Limonene; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D039821:Monoterpenes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013729:Terpenes / Q000378:metabolism
D002135:Calcium-Binding Proteins; D053138:Cyclohexenes; D006838:Hydrocarbons; D008565:Membrane Proteins; D039821:Monoterpenes; D029701:Saccharomyces cerevisiae Proteins; C487549:TCB2 protein, S cerevisiae; C487550:TCB3 protein, S cerevisiae; D013729:Terpenes; C029662:JP5 jet fuel; D000077222:Limonene