Terpenoids are the largest class of specialized metabolites and are widely used as pharmaceuticals, flavors, fragrances, etc. The biosynthesis of terpenoids is restricted by the isoprene rule, according to which all terpenoids are synthesized by assembling the five-carbon prenyl (or isoprenyl) units, IPP and DMAPP. This is called the “isoprene rule”. As a result, all terpenoids have carbon atoms that are multiples of five, which severely constrains their chemical diversity. By establishing cell factories that can bypass the isoprene rule will enable the sustainable biological synthesis of non-canonical terpenoids. This will allow to significantly broaden the chemical diversity of terpenoid structures, thereby expanding the range of their potential commercial applications, which otherwise could be achieved only through organic synthesis.
In this work, I aimed to develop a viable approach for synthetically generating non-canonical terpenoids by engineering yeast cells. This research was driven by three primary goals: first, to establish a production platform for the generation of canonical and non-canonical terpenoid building blocks by supplying yeast with external isopentenol and isopentenol-like alcohols respectively. The supplied alcohols should be converted to canonical and non-canonical isoprenoid building blocks, and eventually to isoprenoids, by generated yeast engineered strains; second, to produce a range of non-canonical mono-, sesqui- and triterpenes, as well as high-value compounds containing GPP prenyl units (cannabinoids); and third, to assess the bioactivity of produced non-canonical compounds, thereby demonstrating the potential of this established approach.
To achieve the first goal, the idea was to establish a yeast system that can utilize externally supplied isopentenol (with C5) and isopentenol-like (non-C5) alcohols for terpenoids biosynthesis, independently of the yeast endogenous terpenoid biosynthesis pathway. The first step was to identify efficient phosphokinases that could accept isopentenol alcohols as substrates and convert them to the terpenoid building blocks, IPP and DMAPP. After I identified specific phosphokinases that could perform this task, I expressed them in yeast together with various plant monoterpene synthases. By feeding isopentenols to the corresponding yeast cells, I achieved a substantial improvement in the yield of a number of studied monoterpenes (Chapter 3). Subsequently, I screened for different isopentenol-like alcohols that could serve as substrates for the selected phosphokinases, enabling the generation of non-canonical terpenoid building blocks (Chapter 4, paragraph 4.1).
For the second goal, I engineered yeast cells that were able to convert these non-canonical terpenoid building blocks to non-canonical terpenoids, by the catalytic activity of terpene synthases. This effort resulted in the production of a diverse array of non-canonical mono-, sesqui- and triterpenes (Chapter 4 and Chapter 5). Moreover, I employed protein engineering to develop a dedicated limonene synthase that facilitated the efficient conversion of GPP analogs to non-canonical limonene (paragraph 4.3). These results confirm the successful engineering of yeast cells capable of producing non-canonical terpenoids, by bypassing the endogenous terpenoid pathway. Furthermore, I continued by demonstrating the utility of this established approach by two proof-of-concept studies: the biosynthesis of the high-valued aroma ingredient ethyllinalool, and the production of pharmacologically significant non- canonical cannabinoids cannabigerolic acid (CBGA, Chapter 6).
To realize my third goal, I isolated and purified six non-canonical CBGA compounds. Utilizing a yeast cell-based cannabinoid receptor activation assay developed in our lab, I observed that several non-canonical CBGA compounds exhibited improved activation of the cannabinoid receptor CB2 (paragraph 6.3). These findings suggest potential new pharmacological or industrial applications for these compounds.
This approach established in this thesis enables the unprecedented expansion of the chemical diversity of terpenoids synthesized by engineered organisms. With this approach, we can embark on an exploration of terpenoid structures that were previously beyond our grasp, transcending the boundaries of what was once considered impossible.