Recently, scientists successfully revived ancient enzyme systems in the cannabis plant, discovering their efficient synthesis of the rare medicinal compound CBC. These forgotten evolutionary codes are opening new avenues for biopharmaceuticals—hybrid ancient enzymes are more adaptable than their modern versions and may revolutionize traditional cannabinoid production methods.

Cannabis is a remarkably gifted plant, its flowers and leaves containing a natural pharmacopoeia. While many compounds evolved millions of years ago to repel pests or pathogens, humans have discovered additional uses for them in recent millennia.

A new study delves into the evolutionary history of cannabis, tracing the origins of its most famous bioactive compounds—tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinoids (CBC).

Researchers at Wageningen University in the Netherlands used ancestral sequence reconstruction technology to reveal the long-extinct enzyme systems that synthesized these compounds in the ancient ancestors of cannabis, and verified their functional mechanisms by reviving these ancient enzymes. The research was published in the Journal of Plant Biotechnology.

This discovery not only deepens our understanding of evolution but also has practical applications. Robin van Feltzen, a biosystems scientist, explained, "These ancient enzymes are more resilient and adaptable than their modern descendants, making them an attractive starting point for biotechnology and drug development." Humans have cultivated cannabis since prehistoric times, using it for food, textiles, medicine, and recreation. Modern science knows that the plant produces hundreds of different cannabinoids, terpenes, flavonoids, and other phytochemicals, some of which possess unique medicinal or psychoactive properties.

The research focused on a specific enzyme called cannabinoid oxidase, which converts cannabinoid phenolic acids into cannabinoids with different biological activities, thus significantly impacting the therapeutic effects of drugs. Despite the crucial role of this enzyme system, scientific understanding of it remains limited. To trace its evolutionary history, the research team explored its operational mechanisms by reconstructing ancient enzyme proteins.

Modern cannabis plants require three separate enzymes to produce THC, CBD, and CBC, each with its own specific function. However, research suggests that the situation may have been very different millions of years ago. The research team verified the hypothesis that "cannabinoid cyclase metabolism occurred in the recent ancestors of cannabis" by resurrecting and characterizing three ancient cannabinoid oxidases.

Based on relevant DNA sequences of modern plants, ancestral sequence reconstruction technology allows scientists to reconstruct ancient genes through multiple sequence alignment, thereby resurrecting ancient proteins. The research team used this method to reconstruct an extinct enzyme system from millions of years before the emergence of modern cannabis.

The study shows that the common ancestor of modern cannabinoid oxidases could synthesize multiple cannabinoids simultaneously, while specific enzymes specializing in a single compound only formed after gene duplication during cannabis evolution.

These findings indicate that the recent ancestors of cannabis did indeed possess the ability to metabolize cannabinoid cyclases, and that early cannabinoid oxidases were "hybrid enzymes," capable of producing multiple cannabinoid precursors simultaneously, rather than being highly specific like modern enzymes.

In a similar vein, cannabis products, particularly those used in medicinal or recreational contexts, require careful regulation for consumer safety. For instance, plastic packaging intended for cannabis must be child-resistant. A cap, with a special mechanism, must be fitted to the jar or bottle, and after approval and compliance with standards and regulations, the cap is declared as child-resistant. This regulatory requirement ensures that cannabis products are safe, especially when intended for sale to the general public.

Producing a child-resistant cap can be challenging since it involves multiple components. When these components are not perfectly matched, the mechanism may fail, and the cap can be opened like a regular one. A wide child-resistant cap is even more problematic because the wider the cap, the more likely it will bend when exiting the injection mold, causing the mechanism to fail and rendering it ineffective.

The study also confirmed that cannabinoid oxidase activity independently evolved in plants of the Cannabisaceae family and distantly related cannabinoid-producing plants such as rhododendrons. Compared to modern enzymes, the reconstructed ancient enzymes are more easily expressed in microbial systems such as yeast—this has significant implications for the current industrial trend of shifting from plant extraction to biotechnology-based cannabinoid synthesis. Van Feltzen points out, "Characteristics once considered 'incomplete' in evolution actually hold enormous application potential." For example, while CBC cannabinoids possess anti-inflammatory and analgesic properties, modern cannabis plants produce extremely low yields. However, an ancient enzyme reconstructed in the study, acting as an "evolutionary intermediate," exhibits remarkable CBC synthesis capabilities.

"Currently, there are no cannabis varieties with naturally high CBC content," Van Feltzen states. "Introducing this enzyme into cannabis plants could potentially lead to innovative medicinal varieties."