Terpenoids are a kind of natural products which exist in plants, fungi, insects and many other organisms.
These compounds not only shine in the pharmaceutical field, such as artemisinin and paclitaxel, but are also widely used in industries such as fragrances and materials.
However, traditional research methods face the bottleneck of high repetition rate, low yield and low screening efficiency, and the mining progress of novel terpenoids is slow, which is difficult to meet the medical and industrial needs.
An article co-published by Professor Liu Tiangang and Academician Deng Zixin details innovative methods for the discovery of terpenoid natural products.
Titled “Innovative approaches in the discovery of terpenoid natural products,” it appears in the journal Current Opinion in Microbiology.
There are three main strategies for the exploration of terpenoids: direct isolation of natural terpenoids, activation of terpenoids (clusters) through homologous recombination, and heterologous expression, so as to isolate and identify a large number of terpenoids with different structures.
At the same time as the discovery of new terpenes has proliferate, repeat discoveries have also increased, and the yield of discovered terpenes products is usually low.
In light of these issues, this review examines advances in research on efficient, high-value terpenes and discusses future prospects for terpene extraction strategies.
In this paper, the application of high-yield terpene-producing microbial chassis in the efficient synthesis of terpenes and the discovery of novel terpenes is reviewed. How the automated high-throughput platform can accelerate the screening and characterization of terpene compounds is discussed.
Finally, the research team also looks forward to the broad prospects and potential challenges of these innovative technologies in promoting the mining and application of natural products and realizing customized synthesis.
High yield chassis applications
The optimized microbial chassis with high yield terpenes not only supports the efficient synthesis of existing terpenes, but also provides a platform for the discovery of new terpenes.
Using a “targeted anabolic” strategy, the researchers optimized the microbial terpene synthesis pathway by precisely regulating the proportion of key enzymes through in vitro titration.
This technology has greatly improved the yield and stability of terpenes, laying a solid foundation for industrial production.
For example, in Escherichia coli, 35 terpene compounds, including two sesquiterpenes and five new diterpenes, were detected from the heteroexpression of terpene synthase (TS) derived from two fungi.
By introducing 31 genes, 1088 terpene biosynthesis pathways constructed, and 228 novel diterpene compounds detected, greatly enriching the chemical diversity of terpenes.
Saccharomyces cerevisiae has become an important base for terpene research because of its unique metabolic characteristics and industrialization potential.
By optimizing the metabolic pathway, the researchers achieved efficient production of sesquiterpenes and discovered several novel skeleton compounds.
Using gene editing technology, the yield of key compounds significantly increased, which opened up A new way for the production of anticancer active compound englerin A.
Based on the yeast platform, the scientists discovered 75 non-classical terpenes through directed mutation and combined biosynthesis strategies, and used the Golden Gate assembly technique to assemble 10 diterpene synthases and four CYPs from different plant sources, and detected more than 200 diterpenes on the Saccharopyces cerevisiae platform. Of these, 162 were new compounds.
This platform not only used for the discovery of high-value terpenes of plant origin, but also revolutionized the synthesis process of vitamin E and successfully synthesized (-)-eremophilene with high efficiency, demonstrating its potential as a spice and biological insecticide.
These results demonstrate the key role of metabolic engineering in increasing the production and diversity of terpenes.
A terpene library constructed on a high-throughput platform
The efficiency of traditional low-flux terpene mining methods is low, but the application of automated high-flux platform greatly improves the research efficiency.
Using the heterologous expression platform, the researchers expressed 41 fungically-derived gene clusters in Saccharomyces cerevisiae with high throughput, successfully detected 22 compounds, including 5 terpenes and 17 polyketones, and finally identified 14 novel compound structures.
By combining the automated platform with the cerevisiae chassis, the researchers characterized 34 novel chimeric enzymes PTTS and identified 24 new compounds, including two entirely new sesquiterpenes.
The conversion of type I ganoderic acid to type II Ganoderic acid successfully achieved by using 158 kinds of P450 oxidase from Ganoderma lucidum.
The high-throughput screening based on the saccharomyces cerevisiae platform realized the sequential production of geranyl, nocaol and nocadione.
Aspergillus oryzae has inherent advantages in that its endogenous post-modification system can modify the terpene skeleton produced by exogenous genes, thereby diversifying the output of terpene and significantly improving the diversity of terpene skeleton.
From 39 fungal gene clusters, the researchers constructed 208 mutant strains and detected 185 terpene compounds, including 62 new skeletons and 123 post-modifications.
Among them, sesquiterpene mangicol J showed significant inhibitory activity against interleukin-6, and its yield increased 111 times after metabolic engineering.
This platform provides a new strategy for rapid mining and functional exploration of high-value terpenes.
Artificial intelligence facilitates the efficient discovery of new terpenes
In terpene mining, repeated discovery is one of the main problems.
The researchers were able to reduce the repetition of the same terpenes by screening for TSs with low similarity to known functional sequences.
Artificial intelligence technology provides powerful modeling tools. Based on the known TS crystal structure, the researchers analyzed the non-conserved amino acid sites of FgMS through homologous modeling.
Six new sesquiterpenes were synthesized by the construction of its mutants F65L and F153G.
Reasonable protein modification based on TS crystal structure helps to expand the structural diversity of sesquiterpenes and diterpenes.
Type I PTTS, TvTS and MpMS with novel triterpene synthesis mechanisms screened for the first time on the Saccharomyces cerevicae platform, and their ability to synthesize macrocyclic triterpenes talaropentaene and macroprohomene confirmed.
Isotopic labeling and crystal structure analysis revealed that the synthesis of these triterpenes begins with the polymerization of IPP and DMAPP and the removal of HexPP pyrophosphate groups, thus generating new triterpene scaffolders through a novel cyclization mechanism.
Using AlphaFold2 to predict the crystal structure of TvTS and MpMS and substrate docking analysis, the researchers found that chimeric enzyme CgCS could catalyze the formation of non-squalene triterpenol.
This discovery challenges the traditional concept, reveals the wide applicability of type I PTTS in triterpene skeleton synthesis, and opens up a new direction for terpene research.
Remarkable progress has made in terpene research, from high-yield chassis microbes to automated high-throughput workstations to AI-assisted mining techniques, which provide strong support for solving bottlenecks in terpene research.
The high-yield chassis technology optimizes the synthesis capacity of microorganisms through metabolic engineering, enabling the efficient production of terpenes and the mining of novel molecules.
The automated high-throughput platform significantly improves screening efficiency and provides a reliable tool for rapid discovery and accurate characterization of natural products.
The introduction of artificial intelligence technology has allowed researchers to explore never-before-seen terpene structures through rational design and functional prediction, greatly expanding the research boundaries in this field.
The collaborative application of these technologies not only promotes the basic research of terpene chemistry, but also provides new ideas for drug development, green chemical production, and the development of high value-added materials.
In the future, with the further integration of biotechnology and information science, terpene research expected to move towards a more intelligent, large-scale and customized era, and make greater contributions to solving humanity’s major problems in the fields of health, environment and energy.
Reference link:
1.Cheng S, Wang X, Deng Z, et al. Innovative approaches in the discovery of terpenoid natural products[J]. Current Opinion in Microbiology, 2025, 83: 102575.
