Metabolic Engineering of Saccharomyces cerevisiae for Production of Fragrant Terpenoids from Agarwood and Sandalwood

Abstract

Sandalwood and agarwood essential oils are rare natural oils comprising fragrant terpenoids that have been used in perfumes and incense for millennia. Increasing demand for these terpenoids, coupled with difficulties in isolating them from natural sources, have led to an interest in finding alternative production platforms. Here, we engineered the budding yeast Saccharomyces cerevisiae to produce fragrant terpenoids from sandalwood and agarwood. Specifically, we constructed strain FPPY005_39850, which overexpresses all eight genes in the mevalonate pathway. Using this engineered strain as the background strain, we screened seven distinct terpene synthases from agarwood, sandalwood, and related plant species for their activities in the context of yeast. Five terpene synthases led to the production of fragrant terpenoids, including α-santalene, α-humulene, δ-guaiene, α-guaiene, and β-eudesmol. To our knowledge, this is the first demonstration of β-eudesmol production in yeast. We further improved the production titers by downregulating ERG9, a key enzyme from a competing pathway, as well as employing enzyme fusions. Our final engineered strains produced fragrant terpenoids at up to 101.7 ± 6.9 mg/L. We envision our work will pave the way for a scalable route to these fragrant terpenoids and further establish S. cerevisiae as a versatile production platform for high-value chemicals.

A synthetic biology and green bioprocess approach to recreate agarwood sesquiterpenoid mixtures

“…Using more complex mixtures of terpenes obtained by additional metabolic engineering will undoubtedly increase the complexity achievable in synthetically functionalised mixtures. Here, we did not express specific α-humulene, β-eudesmol 14 and vetispiradiene synthases, which could broaden the complexity of the starting mixtures, as would expression of mono and di-terpene synthases. The combination of possibilities for these mixtures is immense, offering wide scope for additional combinations of microbial engineered terpene production and synthetic chemistry to yield fragrant mixtures.…”

“…So far, only guaiene STP synthases (STPSs) have been identified from Aquilaria crassna or A. sinensis and enzymatic routes to other primary STPs skeletons found in agarwood are not yet identified. 11,14–16…”

A green alternative to fragrant agarwood sesquiterpenoid production

“…1, Supplemental File 1), using more complex mixtures of STPs obtained by additional metabolic engineering will undoubtedly increase the complexity achievable in synthetically functionalised mixtures. Here, we did not express specific α-humulene, β-eudesmol 14 and vetispiradiene synthases, which could broaden the complexity of the starting mixtures. The combination of possibilities for these mixtures is immense, offering wide scope for additional combinations of microbial engineered terpene production and synthetic chemistry to yield fragrant mixtures.…”

“…Here, we did not express specific α-humulene, β-eudesmol 14 and vetispiradiene synthases, which could broaden the complexity of the starting mixtures. The combination of possibilities for these mixtures is immense, offering wide scope for additional combinations of microbial engineered terpene production and synthetic chemistry to yield fragrant mixtures.…”

“…Both abiotic and biotic factors govern their creation in situ where oxygenation of STPs to derivatives occurs, further shaping their individual scent profiles. From a metabolic-engineering standpoint, the tree itself serves only as the vehicle and matrix for chemical biosynthesis and resin accumulation, such that STP biosynthesis by metabolic engineering could be an alternative means for production of fragrant chemicals 4,14 . However, biochemical pathways leading to agarwood STPs are complex, poorly understood, and can be non-enzymatic in nature.…”