title: Use of nonionic surfactants for improvement of terpene production in Saccharomyces cerevisiae.
abstract: To facilitate enzyme and pathway engineering, a selection was developed for improved sesquiterpene titers in Saccharomyces cerevisiae. α-Bisabolene, a candidate advanced biofuel, was found to protect yeast against the disruptive action of nonionic surfactants such as Tween 20 (T20). An experiment employing competition between two strains of yeast, one of which makes twice as much bisabolene as the other, demonstrated that growth in the presence of T20 provided sufficient selective pressure to enrich the high-titer strain to form 97% of the population. Following this, various methods were used to mutagenize the bisabolene synthase (BIS) coding sequence, coupled with selection by subculturing in the presence of T20. Mutagenesis targeting the BIS active site did not yield an improvement in bisabolene titers, although mutants were found which made a mixture of α-bisabolene and β-farnesene, another candidate biofuel. Based on evidence that the 3' end of the BIS mRNA may be unstable in yeast, we randomly recoded the last 20 amino acids of the enzyme and, following selection in T20, found a variant which increased specific production of bisabolene by more than 30%. Since T20 could enrich a mixed population, efficiently removing strains that produced little or no bisabolene, we investigated whether it could also be applied to sustain high product titers in a monoculture for an extended period. Cultures grown in the presence of T20 for 14 days produced bisabolene at titers up to 4-fold higher than cultures grown with an overlay of dodecane, used to sequester the terpene product, and 20-fold higher than cultures grown without dodecane.
authors.affiliation: California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USA Joint BioEnergy Institute, Emeryville, California, USA.
authors.forename: James
authors.initials: J
authors.lastname: Kirby
authors.affiliation: California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USA Joint BioEnergy Institute, Emeryville, California, USA.
authors.forename: Minobu
authors.initials: M
authors.lastname: Nishimoto
authors.affiliation: California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USA.
authors.forename: Ruthie W N
authors.initials: RW
authors.lastname: Chow
authors.affiliation: California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USA.
authors.forename: Venkata N
authors.initials: VN
authors.lastname: Pasumarthi
authors.affiliation: California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USA.
authors.forename: Rossana
authors.initials: R
authors.lastname: Chan
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, USA.
authors.forename: Leanne Jade G
authors.initials: LJ
authors.lastname: Chan
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Petzold
authors.affiliation: California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USA Joint BioEnergy Institute, Emeryville, California, USA Department of Chemical Biomolecular Engineering, University of California, Berkeley, Berkeley, California, USA Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, Berkeley, California, USA keasling@berkeley.edu.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
mesh_terms: D060847:Metabolic Engineering; D016296:Mutagenesis; D011136:Polysorbates / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012641:Selection, Genetic; D013501:Surface-Active Agents / Q000378:metabolism*; D013729:Terpenes / Q000378:metabolism*
chemical_list: D011136:Polysorbates; D029701:Saccharomyces cerevisiae Proteins; D013501:Surface-Active Agents; D013729:Terpenes