Growing Cyclopeptides in Plants

Why Plants Are Ideal Drug Factories

The pharmaceutical industry typically manufactures biological drugs (proteins, peptides) in one of three ways: chemical synthesis, microbial fermentation (bacteria or yeast), or mammalian cell culture. Each has significant limitations:

• Chemical synthesis is expensive for long peptides and produces racemic mixtures requiring resolution
• Fermentation requires expensive fermenters, sterile conditions, and cold chain logistics
• Mammalian cell culture (for antibodies etc.) costs tens of thousands of dollars per gram to produce

Plants offer an alternative: they are solar-powered protein factories. They grow at ambient temperature, require no fermenters, scale through simple agriculture, and can accumulate target proteins at concentrations that make extraction economically viable. For cyclopeptides specifically, plants have an additional advantage: they already naturally produce cyclotides, including the biosynthetic machinery — enzymes, transporters, vacuolar processing — required to fold, cyclise, and store these molecules correctly.

Natural Cyclotide Concentrations

Cyclotides naturally accumulate in plant tissues at concentrations up to 2 mg per gram of dry plant material. For context, many pharmaceutical compounds are manufactured at mg/kg scale in plant extraction processes; 2 mg/g makes cyclotide-producing plants already commercially viable as extraction sources.

Oldenlandia affinis (the original kalata B1 source), Viola species (violets), and Clitoria ternatea (butterfly pea) all naturally accumulate cyclotides at concentrations sufficient for extraction and direct use. Butterfly pea is the basis of Sero-X, the world's first commercial cyclotide product — produced simply by growing the plant and extracting the active compound.

Four Routes to Plant-Based Production

Production Method 1: Native Plant Harvesting

The simplest production approach is growing naturally cyclotide-rich plant species and extracting the compound:

• Butterfly pea (Clitoria ternatea): Source of Sero-X biopesticide; cyclotide content high enough for commercial extraction; grows as a tropical legume in Queensland, India, and Southeast Asia.
• Violet species (Viola): Rich cyclotide content; being explored as extraction sources.
• Oldenlandia affinis: Original kalata B1 source; grown in tropical Africa.

Limitations: the cyclotide mix produced is what the plant naturally makes — you cannot easily change the specific variant or introduce a therapeutic sequence of your choosing.

Production Method 2: Transient Expression in Nicotiana benthamiana

For producing specific, engineered cyclotides (including therapeutic grafts), researchers use transient expression — introducing cyclotide genes into plants temporarily via Agrobacterium-mediated infiltration, without permanently modifying the plant genome.

Nicotiana benthamiana (a relative of tobacco) is the standard platform for this approach — it is highly amenable to infiltration, grows rapidly, and produces large amounts of target protein within 3–7 days of infiltration. Key findings:

• Cyclotide precursor genes can be expressed alongside the butelase-1 gene (the cyclizing enzyme) to produce fully folded, cyclised product
• This approach also works in tobacco, bush bean, lettuce, and canola
• Used primarily for research-scale production and drug development

Production Method 3: Stable Transgenic Crop Plants

For commercial-scale therapeutic production, stable genetic modification offers a scalable route. The target cyclotide gene is permanently integrated into the crop plant genome, so every generation of the plant produces the therapeutic compound.

Professor David Craik's group at UQ has demonstrated stable transgenic production in:

• Potatoes — anti-obesity satiety peptides
• Sunflower — anti-cancer peptides
• Soybean — anti-cancer peptides
• Arabidopsis — pain-relief peptides (research model)

The concept is that a patient could consume a small amount of the engineered crop — a potato chip, a sunflower oil extract, a soy product — and receive a therapeutic dose of the cyclopeptide. Because cyclopeptides survive digestion, the therapeutic compound travels intact through the gut to its target. The food is the drug delivery system.

Production Method 4: Controlled-Environment Indoor Farming

Phyllome (Sydney, Australia) has taken the plant production concept further, combining stable plant expression with controlled-environment indoor vertical farming. Their 1,000 m² robotic facility grows engineered edible plants under precisely controlled light, humidity, and nutrient conditions, optimising cyclotide yield while maintaining consistent, pharmaceutical-grade dosing.

The indoor farming approach offers:

• Year-round production regardless of season or geography
• Precise environmental control for consistent compound concentrations
• Rapid iteration — plant cycles measured in weeks, not seasons
• No dependence on agricultural land or weather

Phyllome's first commercial applications target pain management, cholesterol, and obesity — partnering with Professor Craik's group at UQ and Australian supplements manufacturer Pharmacare.

The Biosynthesis Pathway: How Plants Make Cyclotides

Understanding how plants produce cyclotides has been essential to engineering them. The pathway has five steps:

1. 1

   Gene Transcription

   The cyclotide is encoded in a precursor gene (called an OaAEP1-type gene in O. affinis). The precursor protein includes a signal peptide, N-terminal prodomain, the mature cyclotide sequence, and a C-terminal tail bearing the key Asn/Asp residue that triggers cyclisation.

2. 2

   Vacuolar Processing

   The precursor protein is transported to the plant vacuole via the secretory pathway. There, vacuolar processing enzymes recognise and cleave the prodomains flanking the mature cyclotide sequence, preparing it for cyclisation.

3. 3

   Cyclisation by AEP

   An asparaginyl endopeptidase (AEP) — the class that includes butelase-1, the fastest known peptide ligase — recognises the C-terminal Asn/Asp residue, cleaves it, and simultaneously ligates the newly exposed C-terminus to the N-terminus. This transpeptidation reaction is the key ring-closing step that creates the head-to-tail cyclic backbone.

4. 4

   Oxidative Folding

   Spontaneous or enzyme-assisted oxidation of the six cysteine residues forms the three disulfide bonds of the Cyclic Cystine Knot (CCK). Two pairs of disulfides form a ring; the third threads through the ring, completing the mechanically interlocked knot topology that gives cyclotides their extraordinary stability.

5. 5

   Vacuolar Storage

   Mature cyclotides accumulate in the plant vacuole — the cell's storage compartment — at high concentrations. When plant tissue is damaged by insect feeding or physical injury, cells rupture and release cyclotides as a chemical defence response, which is why they are found predominantly in leaves and stems.

The Sero-X Story: Proof of Commercial Viability

In 2017, Sero-X launched in Australia — proving that cyclotide plant production is commercially viable. Developed by Innovate Ag in partnership with UQ:

• Source: Clitoria ternatea (butterfly pea) extract
• Production: Conventional agricultural growing and aqueous extraction
• Registered: Australian Pesticides and Veterinary Medicines Authority
• Approved with no upper limit of use — exceptional safety record
• Non-toxic to bees, beneficial insects, birds, and mammals at use concentrations
• Active against helicoverpa (cotton bollworm) and other Lepidoptera pests

Sero-X demonstrated that cyclotide plant production can be taken from laboratory curiosity to registered commercial product within a reasonable development timeline — a proof of concept for the therapeutic applications now in development.