Use of Nonionic Surfactants for Improvement of Terpene Production in Saccharomyces cerevisiae

Abstract

dTo facilitate enzyme and pathway engineering, a selection was developed for improved sesquiterpene titers in Saccharomyces cerevisiae. ␣-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 ␣-bisabolene and ␤-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.

Rhodosporidium toruloides:A new platform organism for conversion of lignocellulose into terpene biofuels and bioproducts

“…Both of these features reduce OPEX in a biorefinery. In comparison, the bisabolene titer from an engineered strain of S. cerevisiae grown under similar conditions was found to decline by more than 75% over 14 days [29]. The strain stability we observed in engineered R. toruloides is an important industrial phenotype and a critical factor for large-scale economical production of any bioproduct.

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Enhancement of eritadenine production using three carbon sources, immobilization and surfactants in submerged culture with shiitake mushroom (Lentinula edodes) (Berk.) Singer)

“…Tween 20 did not enhance biomass production. Similarly, Kirby et al (2014) found higher production of terpene in surviving yeast cells (Saccharomyces sp. ), after being submitted to an inhibitory concentration of Tween 20 (up to 10%).…”

“…Accordingly, in Saccharomyces sp. occurs the same: Tween 80 has no growth inhibitory effect, but tween 20 inhibits it, although the tolerant cells are at the same time good terpene producers (Kirby et al, 2014).…”

“…Furthermore, the addition of surfactants has promoted higher yields of terpenes in Saccharomyces sp. (Kirby et al, 2014), has enhanced significant laccases activity in Armillaria sp. (Hadibarata and Kristanti, 2013), and have improved yields of monakolin K in Monascus (Zhang et al, 2014).…”

“…Nutritional conditions, fungal immobilization and surfactants could increase eritadenine production in shiitake mycelium under submerged cultivation, as well as some reports have demonstrated improved yields of metabolites in many filamentous fungi (Kirby et al, 2014;Noreen et al, 2016;Hameed et al, 2017). For example, nitrogen and carbon sources influence positively the production of the antioxidant ergothioneine in shiitake (Tepwong et al, 2012a;, as well as the production of the anticancer alkaloid chaetominine with Aspergillus fumigatus (Zhang et al, 2016).…”