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

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.Terpenes, being a large family of natural products with interesting biological and chemical properties, have found a multitude of applications ranging from flavors and fragrances to medicines and advanced biofuels (1, 2). Microbial production presents an attractive route for industrial terpene biosynthesis, as evidenced by the successful engineering of yeast to provide a stable supply of the antimalarial drug artemisinin (2,–4). We have recently investigated the sesquiterpene olefin α-bisabolene (Fig. 1) as a candidate biodiesel, achieving moderate titers through expression of a codon-optimized bisabolene synthase (BIS) gene from Abies grandis in an engineered strain of Saccharomyces cerevisiae (5). Our ability to further increase bisabolene titers through engineering of metabolic pathways or individual enzymes is most limited by the growth and product quantitation phase of this iterative process. To facilitate high-throughput engineering of metabolic networks, pathways, and enzymes for improvement of terpene titers, we undertook a search for a chemical agent that could act as a selection agent for higher sesquiterpene levels in yeast cells.Open in a separate windowFIG 1Structures of the nonionic surfactant, Tween 20, yeast membrane component, ergosterol, and sesquiterpenes α-bisabolene and β-farnesene.Considering that sesquiterpenes such as bisabolene are predominantly cell associated in yeast when cultured in the absence of an organic overlay such as dodecane (6), we reasoned that an altered membrane composition may result in an increased tolerance of membrane-disrupting agents. Modification of yeast sterol or fatty acid composition has led to differences in growth rates and has also altered susceptibility to drugs or ethanol as a result of changes in membrane fluidity (7,–10). Of various groups of candidates evaluated as agents for selection of higher bisabolene levels in S. cerevisiae, nonionic surfactants were found to be the most promising.Although surfactants may be used to improve extraction of olefins and pigments from microbial cultures, their use as a selective agent has not to our knowledge been investigated previously (11, 12). To initially evaluate candidates, we utilized two strains of S. cerevisiae, one of which makes twice as much bisabolene as the other, initially comparing growth levels of these strains in the presence of inhibitory concentrations of nonionic surfactants. Tween 20 (T20) was the most promising of the surfactants tested and is this focus of this work, but other candidates such as Brij 35 were also found to be effective. A proof-of-concept study was undertaken to investigate if T20 could be used to enrich for a strain that makes more bisabolene in a mixed population. Following this, we targeted the bisabolene synthase gene from A. grandis, taking various approaches to improve the kinetics or stability of the enzyme though mutagenesis with a goal of enhancing bisabolene production in yeast.In addition to demonstrating that T20 could be used as a selection agent for higher terpene production in a mixed population, we also investigated whether it might be used to improve production titers as well as strain stability in a monoculture. We considered that an agent which selects for the metabolic product of an engineered pathway, rather than just for the biosynthetic genes, may help to address strain stability issues associated with the larger-scale and longer-duration industrial production process. Overall, the use of nonionic surfactants such as T20 presents an attractive approach for both pathway and enzyme engineering and also for stabilizing and enhancing industrial production.