By Ashley Stender | September 2, 2025

Natural products are produced by living organisms to help them adapt and survive in their environments. Many natural products are celebrated for their potential to repel pests, fight fungi, and trigger beneficial plant responses. But discovery alone isn’t enough. To be used in agriculture, these molecules must be made in quantity, sustainably, and affordably, which remains an enduring challenge in plant science and synthetic biology. C-SPIRIT’s Aim 4: Biological Synthesis of Bioactive Compounds tackles this problem directly by developing ways to produce natural products, or bioactive compounds, in plant and microbial hosts.

“In our lab we’re interested in making insect sex pheromones and insect antifeedants for pest control,” says Nicola Patron, a plant synthetic biologist at the University of Cambridge and the C-SPIRIT Aim 4 lead. 

The goal of C-SPIRIT’s Aim 4 is to translate biosynthetic discoveries into usable outputs: molecules that can be manufactured for testing and eventually scaled for use in agricultural systems. From pheromones to antifungal terpenoids, the focus is on compounds with real-world potential.

A Modular Approach to Biosynthesis

Patron’s lab specializes in metabolism and genetic engineering. Her team has spent years working to make genetic engineering easier and more accessible, a philosophy that aligns naturally with C-SPIRIT’s mission of building globally relevant tools for sustainable agriculture.

Patron was invited to C-SPIRIT due to her participation in the Plant Cell Atlas, with both groups being under the direction of Seung Yon (Sue) Rhee. “Sue contacted members of the Plant Cell Atlas project to see who might be involved in this area of research,” Patron says. “I had a good conversation with her about what they were hoping to do. And it sounded like a really interesting opportunity.”

C-SPIRIT’s international structure enables a collaborative approach to distributed biosynthesis challenges. Republic of Korea-based teams led by Sang-Ho Kang and Tae-Jin Oh are engineering the biosynthesis of coumarin and anthraquinone, compounds useful in plant defense and against pathogens and herbivores. Currently, they are assessing new candidates’ genes through comparative genome analysis. Their findings are crucial for identifying viable gene targets for downstream engineering, which they will perform in microbes.

In the UK, the Patron, Lichman and Graham labs are focused on the production of terpenoids in plants and yeast. These include monoterpenes as well as antifeedant diterpenes and triterpenes, all with promising roles in pest and disease control.

From Sequence to Synthesis

The work of Aim 4 builds directly on discoveries from the previous Aims within C-SPIRIT’s pipeline. Once a bioactive compound is identified (Aim 1: Metabolite Discovery) and annotated (Aim 2: Metabolite Annotation & Database Development), and the genes responsible are mapped (Aim 3: Gene & Pathway Discovery), researchers attempt to reconstruct the pathway in a production host such as Nicotiana benthamiana or microbial systems.

Euphorbia diterpenoids with antifeedant potential are among the compounds under evaluation. Additionally, researchers are looking into irregular monoterpenoids, compounds that form the basis of coccidian sex pheromones, and terpenoids with known antifungal activity. All of these require careful reconstitution and testing to determine their functional effects.

“We might not know exactly what those molecules are doing, but if we can make them, then we can test them. And if they show interesting activity, we can continue to explore their potential,” says Patron.

“Pheromones that are currently on the market are all made by chemical synthesis, and that’s because they’re chemically quite simple. But there’s a lot of really important crop pests for which that’s not the case. Some plants make very similar and sometimes identical molecules to insect sex pheromones. We don’t really know why, but we can use this to increase our toolkit,” Patron explains.

To support these experiments, Benjamin Lichman’s lab at the University of York manufactured a custom device that enables extraction and assay work at the plate scale, improving throughput. In tandem, Ian Graham’s lab is coordinating enzyme screening and host evaluation to increase yields of antifungal terpenoids.

Patron emphasized that cross-lab integration is one of the most rewarding aspects of C-SPIRIT’s model. “We have people who are experts in phytochemistry, people who are experts in genetics and genomics, and people who are experts in engineering biosynthesis,” she says. “You need all those people to actually make this work.”

Looking Beyond Nature

C-SPIRIT’s long-term strategy goes beyond rebuilding natural pathways. With modular constructs and diverse biosynthetic tools, researchers hope to produce families of novel molecules that are more effective or more stable than their natural counterparts.

“We could produce a panel of molecules that don’t exist in nature and might be more powerful antifeedants,” Patron says. “We can do this by combining enzymes from different species to produce new variations on chemical structures.”

To support this vision, the Graham lab is conducting comparative structural analyses of isoprenoid synthases and assembling a curated set of biosynthetic parts for combinatorial design. All available, relevant terpenoid plasmids are being collated and shared across the C-SPIRIT network. These resources are already being used to design experiments that test new combinations of genes and pathways.

“The genes for producing the coccidian sex pheromones are currently unknown and they are very difficult to chemically synthesize,” Patron explains. “We are trying to make them by engineering plant enzymes that produce similar molecules.”

This is where biosynthesis offers an alternative, and where Aim 4 can go beyond simple reproduction. It’s a chance to pursue a new chemical space, guided by biology.

Preparing for Scale

Reconstructing biosynthetic pathways is only the beginning. Getting measurable amounts of product from engineered hosts remains one of the biggest bottlenecks in the field. Patron’s lab is optimizing GC-MS protocols for monoterpenoid detection, while researchers in the Republic of Korea are running parallel expression and activity tests. The Lichman lab continues to refine their approach to increasing testing throughput in plants, contributing to the development of scalable workflows.

“It’s a known challenge in synthetic biology… scale-up is difficult,” Patron says. “But if you have a molecule and there isn’t any other way to access it, then it’s worth devoting resources to scale-up.”

Scalability isn’t just about volume, but also about making reliable, transferable workflows. One of C-SPIRIT’s defining values is open science: ensuring that constructs, protocols, and results are available for others to test and improve.

“It’s very difficult to do this as a single lab, because there’s so many steps,” Patron says. “I think that’s one of the things that C-SPIRIT is doing that’s so nice. It’s making that process reproducible.”

As these workflows mature, researchers will be able to test compounds across different geographies and systems. This is critical for understanding how bioactive compounds behave in real agricultural environments and for building the case for their sustainable use.

Connecting the Pipeline and Beyond

Each of C-SPIRIT’s aims feeds into the next. By the time a compound or natural product reaches Aim 4, researchers have already moved from wild species to identified metabolites to annotated gene pathways. What remains is to make those pathways work, and to turn what’s known into what’s usable.

“There’s a few different ways that you can use pheromones for pest control if you have an affordable source,” Patron says. This is what Aim 4 is ultimately about: enabling real-world deployment of molecules that would otherwise remain locked inside living organisms. Researchers turn biosynthetic insight into functional, scalable solutions that support the broader C-SPIRIT vision of sustainable agriculture.

“I think that C-SPIRIT is really exciting,” Patron says. “It’s so international and so collaborative. I really like the model.”

With the help of Aim 4, C-SPIRIT is designing systems that can produce meaningful quantities of bioactive compounds for testing in diverse agricultural settings, transforming discovery into  testable products.