The global flavor and fragrance market was worth $29 billion in 2021 and is expected to reach $37.3 billion by the year 2026 at a compounded annual growth rate of 5.1% (Marketsandmarkets, 2023). To accomplish this high demand in an appealing market, microbial production of Flavours and Fragrance compounds is an attractive route (Zhang et al., 2018).

“(-)-Geosmin”, a word derived from two ancient Greek words “geo” meaning “earth” and “osme” meaning “sense of smell”(Gerber, 1967) is the chemical compound that has an earthy fragrance. This earthy aroma typically arises when the first rain falls on sun-baked soil, also known as a petrichor (IJ Bear, 1964). The pleasing aroma of (-)-geosmin makes it one of the most sought-after compounds in the perfumery industry, as it is added to cosmetic products and essential oils as middle note perfume (Finato et al., 1992). (-)-Geosmin is a degraded sesquiterpene that is naturally produced by a diverse class of microorganisms such as actinomycetes, myxobacteria, cyanobacteria, various fungi, and algae (Dickschat et al., 2005, Gerber and Lechevalier, 1965, Guerche et al., 2005)

The (-)geomsin enantiomer is naturally produced solely through microbial synthesis, and it exhibits a higher smell threshold when compared to the (+) geosmin form (Darriet et al., 2001, Jüttner et al., 2007, Xu and Dickschat, 2023). The enzyme, geosmin synthase and its corresponding gene for the biosynthesis of (-)-geosmin in actinomycetes has been identified and well characterized (Cane et al., 2006, Ghimire et al., 2008, Jiang et al., 2006). In-vitro studies with recombinant geosmin synthase enzyme have shown that it is responsible for multi-step conversion of farnesyl diphosphate (FPP) to (-)-geosmin (Jiang et al., 2007, Jiang et al., 2006). This geosmin synthase is a bifunctional enzyme of about 700 amino acids and consists of two catalytic domains. The N-terminal domain of this enzyme catalyzes the Mg2+ dependent conversion of FPP to germacradienol and germacrene D whereas C-terminal domain is responsible for the Mg2+ dependent conversion of germacradienol to octalin and (-)-geosmin (Cane et al., 2006, Jiang et al., 2007, Jiang et al., 2006). The aspartate-rich motif followed by a NSE triad motif are essential for the binding of the Mg2+ cofactor (Jiang et al., 2007). Such motifs have been identified in the N-terminal domain – the strictly conserved aspartate-rich motif (DDHFL(D, E)), the NSE triad (ND(L, I)FSY(Q, E)RE)and an unusual repeat of NSE triad (NDVLTSRLHQFE). Similarly, the C-terminal sequence exhibits less conserved aspartate-rich motifs of (DDYYP) and the variant of NSE triad (ND(L, I, V)FSYQKE) (Jiang et al., 2007). Both N and C-terminal domains are joined by a linker sequence of amino acids (Citron et al., 2012). Geosmin synthase of Streptomyces coelicolor origin, SCO6073 (CAB41566), has been shown to produce germacradienol, germacrene D, octalin and (-)-geosmin in a ratio of 74:10:3:13 (Jiang et al., 2007) whereas the ratio of enzyme of Streptomyces avermitilis origin, SAV2163, is shown to be 66:24:2:8 (Cane et al., 2006). Single nucleotide polymorphism targeting aspartate-rich motif in SCO6073 led little to no geosmin production, emphasizing the importance of highly conserved motifs (Jiang et al., 2007).

Currently, production of (-)-geosmin is achieved by three methods viz, distillation of sun-baked soil (Vittal et al., 2019), fermentation using naturally producing micro-organisms (Gerber and Lechevalier, 1965) and by chemical synthesis (FUHSHUKU and SUGAI, 2002, Saito and Tanaka, 1996). Each of these production methods has its own disadvantages. Distillation of soil is a seasonal process (Vittal et al., 2019), hence continuous production is not possible throughout the year. In microbial fermentation only a trace amount of (-)-geosmin can be produced using natural host (Gerber and Lechevalier, 1965). The limited set of genetic manipulation tools and insufficient knowledge of genetic information available for natural hosts make (-)-geosmin production challenging. Chemical synthesis of (-)-geosmin is a multistep, complex, and expensive process (Vittal et al., 2019). Presence of three chiral centres makes it difficult to produce enantiomerically pure (-)-geosmin (Engels et al., 2008, Saito and Tanaka, 1996). These (routes are neither environmentally friendly nor efficient.

To overcome these difficulties, heterologous production of (-)-geosmin by Saccharomyces cerevisiae is being proposed in this study. Yeast (S. cerevisiae) has been extensively used for industrial production of terpenes (Meadows et al., 2016). It has native chassis to produce isoprenoid precursors (Dejong et al., 2005). Terpenes are inherently biosynthesized in S. cerevisiae through the mevalonate pathway (Fig.1) where acetyl CoA is a central metabolite. Isopentenyl diphosphate (IPP) and its isomers dimethylallyl pyrophosphate (DMAPP) are condensed to generate geranyl pyrophosphate (GPP) and farnesyl diphosphate (FPP). FPP serves as a major precursor for sesquiterpene biosynthesis (Zhou et al., 2020).

S. cerevisiae has several inherent properties that make it a preferred host for heterologous sesquiterpene production. It has the ability to grow on a wide variety of carbon sources (Dijken et al., 2000). S. cerevisiae has high transformation efficiency and the tools for metabolic engineering of S. cerevisiae are readily available and well-studied (Hoek et al., 2000). As a eukaryotic host, S. cerevisiae is widely used for the production of heterologous proteins (Dijken et al., 2000). S. cerevisiae has also been classified as GRAS (generally regarded as a safe) organism by the US FDA (Apel et al., 2017). This study explores the expression of various geosmin synthases in S. cerevisiae. The results indicated that the transformants successfully produced (-)-geosmin which is a step forward in the feasible and sustainable production of (-)-geosmin by fermentation.