Employed was a combinatorial assembly of yeast toolkit parts and iterative design-build-learn-test cycles with strain selection guided by a mathematical model relating genetic design to monoterpene flux. To be functionally useful, the engineered strain needed to retain its ability to convert sugars to ethanol, and have precise, stable expression of flavor-determining monoterpenes linalool and geraniol. This work was in contrast to most metabolic engineering efforts which are commonly enlisted to maximize product titers. Multiple state of the art engineering techniques and iterative improvement schemes were employed to tune production of multiple commercially important metabolites without major collateral metabolic changes. For cannabinoids from C. sativa, an aromatic prenyltransferase catalyzes the formation of cannabigerolic acid from olivetolic acid and geranyl pyrophosphate . The pathway then branches again toward different cyclized products, such as tetrahydrocannabinolic acid , cannabidiolic acid , and cannabichromenic acid . Unnatural cannabinoid variants with tailored alkyl chains could also be obtained via feeding the engineered strain with hexanoic acid analogs, rolling grow trays demonstrating the substrate promiscuity of olivetolic acid pathway enzymes. Most notably, cannabinoid variants with an alkyne moiety were synthesized, paving the way for future click derivatization.
It has been shown that the cannabinoid alkyl side chain is a critical pharmacophore and may be a promising target for pharmaceutical discovery. Another study successfully reconstructed the entire β-bitter acid pathway by heterologous expression of two CoA ligases, a polyketide synthase, and a prenyltransferase complex in an optimized yeast system. A metabolon composed of two aromatic prenyltransferases was elucidated. Another key tool for increasing transgene expression and function for terpenoid biosynthesis is mutagenesis analysis, particularly for prenyltransferases given the plasticity and promiscuity of their active sites. Prenylated flavonoids are another subclass of plant phenolics, which combine a flavonoid skeleton with a prenyl side chain. Unlike other flavonoids, they have a narrow distribution in plants, limited to only several plant families, including Cannabaceae. Recent studies have demonstrated that hop terpenophenolics exhibit diverse bio-activities with a high potential for pharmaceutical applications 208. A prenylated flavonoid with a very potent phytoestrogen activity is 8-prenylnaringenin, produced in Humulus lupulus . 8-Prenylnaringenin was recently produced de novo as a proof of concept for yeast as a platform for biosynthesis of prenylated flavonoids . Recently, the importance of non-catalytic foldases and chaperones for terpenoid production in trichomes has been elucidates. THCA and CBDA are unstable and will be non-enzymatically converted to the decarboxylated forms, Δ9-tetrahydrocannabinol and cannabidiol respectively. It is hypothesized that CsaCHIL, a chalcone isomerase-like protein lacking catalytic activity, potentially binds THCA and/or CBDA for stabilization in hemp glandular trichomes and limits negative feedback to upstream enzymes. It has also been shown that upregulation of multiple foldases and chaperones resulted in a 20-fold improvement of THCA synthase functionality in yeast and poses a promising avenue for optimizing microbial production 210 .
The progression of terpenoid biosynthesis in microorganisms is limited by the dearth of characterized terpene synthases as well as the CYPs and GTs that modify these terpenes. Computational biology has enabled the discovery of new enzymes, as demonstrated by the identification of 55 predicted terpene synthases from C. sativa. CYPs, in particular, are hypothesized to be a main driving force of terpenoid diversification in plants through hydroxylation, sequential oxidations of specific positions , as well as catalyzing ring closure and rearrangement reactions that significantly increase terpenoid complexity. Most CYPs react with a distinct carbon on the terpene backbone, reactions that are challenging for synthetic chemistry, making biosynthesis of oxidized terpenoids a preferable option for production. These CYPs are generally localized to the ER of the native host in close proximity to the terpene synthase producing the substrate for the reaction. Often included on the ER are GTs required for the glycosylation of the oxidized terpenoid, forming potential metabolons on the ER membrane. There are many inherent challenges with transferring into microorganisms CYPs optimized by nature to work in plant systems. This is a major hurdle when working in prokaryotic cell factories due to their lack of an ER and cytochrome P450 reductases responsible for transferring electrons between the CYPs and electron carriers in eukaryotes. Groups have successfully engineered E. coli with functionally reconstructed plant-derived CYPs by generating fusion proteins with membrane anchors suitable for prokaryotic cells along with the co-expression of a CPR. A major advantage of working in yeast systems like S. cervasiae and Yarrowia lipolytica for the production of decorated terpenoids is the endogenous ER system. This has been successfully demonstrated in S. cerevisiae engineered to produce oxidized casbenes, a medically important diterpenoid derivative, that required the optimization of six CYPs, achieving titers of over 1 g/L, building upon techniques initially demonstrated in the landmark paper producing artemisinic acid, a plant-derived sesquiterpene, in yeast.
The terpenoid target space can be further expanded through the introduction of GTs from plants into microorganisms for the glycosylation of oxidized terpenoids. Beyond adding new functionality, plants natively produce glycosylated volatile or toxic terpenes for long-distance transport as well as storage of “disarmed” molecules. Saponins, modified triterpenoids synthesized through varying oxiditions and glycosylations of a β-amyrin backbone, have garnered recent interest in both the industrial and human health spaces 221. The biosynthesis of β- amyrin has been achieved in both E. coli and S. cerevisiae, but the production of its oxidized and glycosylated derivatives has been limited to yeast. Recently, Wang et al. achieved 2.25 g/L production of ginsenoside Rh2, an oxidized and glycosylated triterpene generally harvested from Panax spp., by the directed evolution of UGTPg45. This was the highest titer reported to date for an in vivo production system. Advances in cell-free platforms have enabled the interrogation of GT function in vitro and was recently deployed for the production of novel cannabinoid glycosides. This method allows for the characterization of GTs that can then be introduced to a production host for large scale biosynthesis. A challenge for future engineering will be the availability of substrate, nucleotide sugars, for glycosylation reactions in heterologous hosts. Limited work has been done in microbes aimed at producing various nucleotide sugars, but the formation, interconversion, and salvage of these substrates has been extensively studied in plants, providing a framework for future microbial engineering efforts. A new paradigm of modifying the subcellular morphology of production cells rather than optimizing metabolic flux has successfully increased oxidized terpenoid production titers in yeast. Kim et al. overexpressed INO2, an ER size regulation factor,horticulture trays which resulted in an increase in ER biogenesis, ER protein abundance, protein-folding capacity, and cell growth while limiting ER stress response. This resulted in a 71-fold increase in squalene production and an 8-fold increase in the CYP-mediated production of protopanaxadiol compared to control strains. A similar goal was achieved by knocking-out PAH1, which generates neutral triglycerides from phosphatidic acid. This strategy also enlarged the ER and boosted production of β-amyrin, medicagenic acid , and medicagenic-28-O-glucoside by eight-, six- and 16-fold, respectively, over the control strain. These strategies will prove to be pivotal advances in terpenoid engineering and may be applied to any yeast chassis engineered for maximizing the biosynthesis of terpenoids derivatives. A potential hindrance of terpenoid biosynthesis in microorganisms is the potential for product or intermediate toxicity preventing the accumulation of high levels of a desired molecule. Achieving maximum accumulation will be essential when commercializing next-generation bio-fuel alternatives like the sesquiterpene bisabolene. Groups have engineered synthetic hydrophobic droplets within the cell that allow for the storage and accumulation of lipophilic compounds like terpenes while circumventing growth or toxicity issues. While this work was done in plants, there is potential to transfer these technologies to microorganisms. Lipid engineering in yeast was accomplished through the overproduction of triacylglycerol and a knock-out of FLD1, which regulates lipid droplet size, resulting in oversized lipid droplets that accumulate and store lycopene, an acyclic tetraterpene, resulting in record titers of 2.37 g/L 234 . These challenges have brought recent attention to Yarrowia as a production host for plant derived terpenes due to its capacity to accumulate lipophilic compounds and the potential to utilize technology developed for S. cerevisiae in this new host. A recent pivotal study harnessed peroxisomes to produce squalene at an unprecedented titer through dual cytoplasmic peroxisomal engineering. This study indicates that peroxisomes can function analogously to trichomes due to their pathway compartmentalization.
While there has been little exploration thus far of the capability of yeast peroxisomes to mimic the trichome metabolic environment specifically, they are a promising avenue for the optimization of heterologous production of terpenoids in yeast. Utilizing microbial biosynthesis to produce economically relevant terpenoids limits the need to grow, harvest, and extract plant material. This provides an environmentally friendly synthesis platform for specialized terpenoids and permits their production at high concentration and purity. Advances in technologies and strategies for the identification and heterologous expression of terpenoid biosynthesis pathways in microorganisms will provide numerous opportunities for future research. While there has been recent success in engineering prokaryotes for terpene production, yeast will prove to be the optimum production host for more complex terpenoid derivatives and should be a cornerstone for future efforts. The progression of metabolic engineering for terpenoid production is only limited by the identification and application of plant-derived terpene synthases, prenyltransferases, CYPs, and GTs for the biosynthesis and decoration of natural terpenoid scaffolds. By implementing techniques previously described there is potential to expand the latent target space beyond the natural/known terpenome, enabling the biosynthesis of synthetic terpenoids. Achieving this goal will require new breakthroughs in host engineering along with optimizing the expression and function of heterologous pathways. Additionally, generating host strains that produce various or specialized nucleotide sugars for glycosylated terpenoids will provide a chassis for the production of terpenoid glycosides, allowing for the microbial biosynthesis of compounds with altered and enhanced bio-active properties.The difficulty sourcing medicinal plant terpenes is exemplified by the Taxol story: clinical development of Taxol was an agonizingly slow progress due to supply shortages of the natural producer Taxus brevifolia in the 1980s and 1990s. The concentration of Taxol in the plant is very low , and harvesting of yew for extraction is not sustainable, since T. brevifolia is now endangered. As is the case for all complex plant terpenes, full chemical synthesis is also not currently a viable economic option as it requires many steps , gives low yield, and it not scalable for production. Taxol is currently manufactured either by semisynthesis from 10-deacetylbaccatin III extracted from the needles of Taxus spp., or by extraction from plant cell suspension cultures grown with elicitors to improve production. Both methods still rely on a plant source, resulting in a low and unstable yield, high production costs, and unwanted byproducts. There are many examples of medicinally relevant plant diterpenes that are currently facing similar sourcing issues, with Taxol and cyclopamine as lead example. This is particularly regrettable because plant terpenes can have unique mechanisms of action not demonstrated by any other class of compounds. For example, Taxol stabilizes microtubules by binding at a unique and specific site resulting in cell cycle arrest making it an effective cancer treatment. There are two major challenges that historically have limited the production of complex plant terpenes in yeast, low yields for the first step in the pathway and optimizing complex pathways for the elaboration of the terpene scaffold requiring multiple tailoring enzymes. Previous work with Taxol indicates that multiple products are produced in early stages of the pathway, a major cause of low yields observed in yeast. Additionally, enzymes such as P450s are a notorious challenge for yeast heterologous expression, especially when required to act in series, resulting in diminishing yields of products, thus limiting both pathway discovery efforts as well as the reconstitution of multistep pathways. Despite these challenges, the rational design of strains to tune coupling with redox partners can improve P450 activity in yeast. Along with improving redox dynamics, P450 optimization could be enhanced via augmentation of the ER anchoring regions to improve the localization and expression of plant derived P450s in yeast; or the inclusion of non-enzymatic ER scaffold proteins engineered to bind the P450s for the formation of pseudo-metabolons . Taxol biosynthesis in the native host T. brevifolia is a complex pathway requiring nineteen enzymatic conversions, with eight of these enzymes yet to be identified/characterized. This includes eleven ER anchored enzymes with the remaining predicted as soluble cytosolic enzymes.