Toward sustainable production of bulnesol and elemol enabled by synthetic biology in Saccharomyces cerevisiae

Guaiac oil obtained from the heartwood of the Bulnesia sarmentoi tree native to South America is prized for its characteristic woody-rosy aroma. The aroma is primarily attributed to bulnesol, which makes up ~ 40% of the oil. B. sarmentoi is classified as an endangered species by the International Union for Conservation of Nature (IUCN) Red List, its sensitivity to climate change and challenges associated with its cultivation hinder the production of guaiac oil. We had previously characterized a unique sesquiterpene synthase (StBUS/ELS) from potato that produced a mixture of bulnesol and elemol when incubated with farnesyl pyrophosphate. It was shown that elemol was a thermal rearrangement product of bulnesol. The present study sought to engineer yeast, Saccharomyces cerevisiae, for the sustainable production of bulnesol using StBUS/ELS. Yeast expressing StBUS/ELS was found to produce a mixture of elemol, γ-eudesmol, α-eudesmol, β-eudesmol and bulnesol with pH of the fermentation medium having a significant influence on the product profile. A pH of 7.5 favored the production of bulnesol/elemol, whereas an acidic pH (2.5 − 5.5) formed eudesmols. Further metabolic engineering for enhanced precursor and co-factor supply while also eliminating the need for galactose inducers resulted in a 1.85-fold improvement in bulnesol/elemol titers. Finally, fed-batch fermentation with the best engineered strain resulted in a combined bulnesol/elemol titer of 103.92 ± 7.41 mg/L, while total sesquiterpene titers exceeded 460 mg/L. This study, for the first time, demonstrates the influence of pH on the product profile of a terpene synthase in vivo and provides a foundation toward the sustainable production of bulnesol in microbial host platforms.