The Secret World of Non-Canonical Terpene Synthases
In the intricate dance of nature's chemistry, some of the most skilled dancers are masters of disguise.
Imagine a master chef who, instead of following standard recipes, inventively combines ingredients to create entirely new culinary wonders. This is precisely what non-canonical terpene synthases do in nature's kitchen. These molecular mavericks are rewriting the textbook on terpene biosynthesis, challenging long-held assumptions and revealing nature's astonishing ingenuity in creating chemical diversity.
For decades, scientists believed terpene synthesis follows a simple rule: all terpenes are built from five-carbon units. Then came the discovery of irregular terpenes—C11, C16, and even C17 structures that defied this fundamental principle. The discovery of enzymes that create these unusual compounds has unlocked a hidden world of biochemical innovation, with profound implications for medicine, agriculture, and biotechnology 5 .
Terpenoids constitute the largest family of natural products, with over 95,000 known compounds performing essential functions across all living organisms 8 .
Feature conserved DDxxD and NSE/DTE motifs for metal-binding, initiating reactions by eliminating diphosphate 8 .
Contain DxDD motifs that initiate cyclization by protonating double bonds or epoxides 8 .
A comprehensive 2020 review identified twelve distinct families of these unconventional enzymes that perform terpene synthase-like reactions while bearing no resemblance to canonical terpene synthases in sequence or structure 1 .
Perhaps the most striking deviation from tradition is how non-canonical terpene synthases create terpenes with unusual carbon numbers. While traditional terpenes follow the C5 rule (C10, C15, C20, etc.), irregular terpenes with C11, C16, and C17 skeletons are increasingly being discovered 5 .
The secret lies in a newly discovered enzyme family called isoprenyl diphosphate methyltransferases (IDMTs). These enzymes methylate standard terpene precursors to create non-canonical substrates that are then converted into irregular terpenes by specially adapted terpene synthases 5 . This two-step process represents an entirely new biosynthetic route that nature has invented to expand terpene diversity.
The story of how scientists discovered a completely new family of terpene cyclases in Trichoderma fungi illustrates the detective work involved in uncovering nature's biochemical secrets. Trichoderma species are important biocontrol agents in sustainable agriculture, known for producing complex tetracyclic diterpenoids called harzianes and trichodermanins. Despite decades of interest, the enzymes responsible for creating these intricate structures remained elusive 3 .
Researchers faced a significant challenge: none of the predicted terpene synthase genes in Trichoderma genomes produced harziane or trichodermanin diterpenes when expressed in standard laboratory systems. This suggested that the responsible enzymes might be non-canonical terpene synthases that didn't resemble known enzymes 3 .
First, they confirmed that crude protein extracts from T. atroviride B7 could convert GGPP into harzianol I and wickerol A when supplied with magnesium 3 .
They separated the crude protein extract through ammonium sulfate precipitation and ion-exchange chromatography, obtaining three highly active fractions and one weakly active control fraction 3 .
By analyzing these fractions against the T. atroviride B7 genome, they identified 731, 579, 702, and 647 proteins in the respective fractions 3 .
They cultured the fungus in different media—one that produced diterpenoids and one that didn't—then compared gene expression patterns to identify candidates specifically active during diterpenoid production 3 .
They expressed 49 candidate genes in engineered E. coli systems, leading to the identification of a single hypothetical gene (tri4155) that produced trace amounts of harzianol I 3 .
Switching to a different expression vector and co-expressing with a GGPP synthase dramatically boosted production to 3.55 mg/L of harzianol I and 0.20 mg/L of wickerol A 3 .
| Step | Method | Key Outcome |
|---|---|---|
| 1 | Enzyme assay development | Confirmed Mg2+-dependent cyclization activity in crude extracts |
| 2 | Protein fractionation | Isolated three active protein fractions |
| 3 | LC-MS/MS analysis | Identified hundreds of proteins in each active fraction |
| 4 | Transcriptome sequencing | Found 49 differentially expressed candidate genes |
| 5 | Heterologous expression | Discovered tri4155 as the active gene |
| 6 | Expression optimization | Increased yield of harzianol I and wickerol A |
The newly discovered enzyme, named TriDTC (Trichoderma diterpene cyclase), represents a brand new class of terpene cyclases that diverges from all known enzymes 3 . Unlike canonical terpene cyclases, TriDTCs lack the standard motifs and instead likely employ a unique DxxDxxxD aspartate triad for cyclization initiation 3 .
Modulates product specificity
Essential for catalytic activity
Most remarkably, the study revealed that these diterpenoids and their corresponding cyclases play crucial biological roles in regulating the formation of chlamydospores—resistant propagules that enhance Trichoderma's survival and biocontrol efficacy 3 . This finding connects these unusual enzymes directly to the fungus' ecological success and agricultural utility.
| Feature | Canonical Terpene Cyclases | TriDTCs |
|---|---|---|
| Catalytic motifs | DDxxD & NSE/DTE (Class I) or DxDD (Class II) | DxxDxxxD aspartate triad |
| Metal dependence | Mg2+-dependent (Class I) | Mg2+-dependent |
| Product range | Diverse known terpene skeletons | Specific to harziane/trichodermanin skeletons |
| Distribution | Widespread across organisms | Narrow (3 fungal genera) |
| Biological function | Various | Regulating chlamydospore formation |
Studying non-canonical terpene synthases requires specialized experimental approaches and reagents. The following toolkit highlights essential materials and methods that enable discovery in this emerging field.
| Tool/Reagent | Function/Application | Example from Research |
|---|---|---|
| Engineered E. coli expression systems | Heterologous expression and pathway reconstitution | pBbA5c-MevT-MBIS and pCDF-Duet1-crtE plasmids for precursor supply 3 |
| Enzymatic activity-guided fractionation | Identifying enzyme activity without sequence prediction | Ammonium sulfate precipitation and ion-exchange chromatography 3 |
| Isoprenyl diphosphate methyltransferases (IDMTs) | Creating non-canonical substrates | SpSodMT from Serratia plymuthica for C16 building blocks 4 5 |
| Comparative transcriptomics | Linking gene expression to metabolite production | Identifying differentially expressed genes in terpenoid-producing vs. non-producing conditions 3 |
| Structural bioinformatics | Identifying novel catalytic motifs | Predicting DxxDxxxD aspartate triad in TriDTCs 3 |
| Promoter engineering & codon optimization | Activating silent biosynthetic gene clusters | Stepwise activation of the ost gene cluster for ostamycin discovery 8 |
Known Terpenoid Compounds
Distinct Non-Canonical Families
Non-Canonical Terpenes Engineered
The discovery of non-canonical terpene synthases has fundamentally expanded our understanding of nature's chemical innovation strategies. Rather than being limited to two main enzymatic blueprints, nature has repeatedly invented alternative solutions for terpene biosynthesis throughout evolution 1 .
Non-canonical terpene synthases provide new probes for discovering novel terpenoid natural products and the gene clusters responsible for their production 1 .
DiscoveryUnderstanding non-canonical terpene biosynthesis enables metabolic engineers to expand nature's terpene biosynthetic code 4 .
EngineeringThe discovery that Trichoderma diterpene cyclases regulate chlamydospore formation opens new avenues for enhancing fungal biocontrol agents 3 .
AgricultureScientists have successfully engineered yeast to synthesize 10 non-canonical C16 building blocks and identified terpene synthases to convert these into 28 different non-canonical terpenes 4 . Some of these novel compounds display interesting odorant properties, suggesting potential applications in fragrance and flavor industries.
The study of non-canonical terpene synthases reminds us that nature's creativity far exceeds our biochemical imagination. As one review eloquently states, "With every new discovery, the dualistic paradigm of TSs is contradicted and the field of terpene chemistry and enzymology continues to expand" 1 .
These enzymes in disguise challenge us to look beyond established categories and remain open to nature's endless capacity for innovation. They represent not just scientific curiosities but valuable tools for discovering new medicines, developing sustainable biotechnologies, and understanding the intricate chemical conversations that govern life.
As research continues to unveil nature's hidden biochemical repertoire, one thing is certain: the most exciting discoveries often come from where we least expect them, disguised as something else entirely.