Introduction

Between latitudes 40° and 45° N lies a unique natural habitat for wild ginseng. Known as the “King of Herbs,” ginseng possesses multiple pharmacological activities, largely attributed to its bioactive constituents—ginsenosides. Ginsenosides are glycosylated triterpenoids with multiple functions, including anti-inflammatory, anti-tumor and antioxidant effects^[1], and are widely used in the fields of pharmaceuticals, health products, cosmetics, etc. The market demand for them is increasing year by year. Among them, the rare oleane-type ginsenoside Ro stands out for its unique glucuronic acid fragment, which is related to its enhanced bioavailability and effective pharmacological effects^[2].

At present, Ro is primarily extracted from natural Panax species, a process that is both slow and low-yielding. Traditional ginseng cultivation requires nearly 20 years to reach maturity, and even then, the roots contain less than 0.4% Ro. Thus, plant extraction cannot meet growing market demand^[2]. Furthermore, conventional extraction methods are inefficient, costly, and heavily reliant on organic solvents, which can cause environmental pollution.

In recent years, synthetic biology has been explored as an alternative strategy to produce ginsenosides. However, most biosynthetic approaches rely on glucose as a carbon source^[7], which competes intensely with endogenous yeast metabolism (e.g., ergosterol biosynthesis), resulting in limited precursor availability^[4].

Is it possible to produce the rare ginsenoside Ro using lower-cost raw materials and in a more environmentally friendly way, while maximizing yield?

This is precisely what our project BiOilRo aims to achieve. Through genetic engineering, we designed Saccharomyces cerevisiae strains capable of utilizing glycerol and fatty acids derived from waste oils as carbon sources to biosynthesize rare ginsenoside Ro. This strategy not only provides an innovative solution for the sustainable, efficient, and cost-effective production of ginsenoside Ro, but also addresses urgent global challenges:

Environmental urgency: Reducing reliance on organic solvents and mitigating pollution from waste oils.

Economic and social necessity: Offering a scalable, bio-based alternative to plant extraction, which is constrained by limited natural resources and long growth cycles.

Health relevance: Enabling reliable access to a potent bioactive compound with demonstrated anti-inflammatory and neuroprotective properties, supporting future pharmaceutical and nutraceutical applications.

By turning waste into health, BioOilRo embodies a circular bioeconomy, transforming low-value waste streams into high-value, health-promoting products.

Background

1. Challenges in Ginsenoside Ro Extraction

Ginsenoside Ro is a rare ginsenoside characterized by a unique glucuronic acid structure and has demonstrated significant application value in modern biomedicine. Studies have shown that Ro not only exhibits remarkable anti-inflammatory and antioxidant activities, but also holds great potential in cardiovascular protection as well as immune regulation. It can serve as an important raw material for the development of innovative pharmaceuticals, functional foods, and high-end cosmetics. However, ginsenoside Ro is primarily extracted from Panax species using solvent extraction and purification methods, which yield only limited amounts of product. This approach relies heavily on organic solvents, raising concerns about environmental pollution and safety risks. Furthermore, it is constrained by the long growth cycle of ginseng and the scarcity of natural resources, resulting in high costs and insufficient supply to meet market demand.

2. Waste Oil Pollution

Globally, the issue of waste oil disposal has become increasingly severe. Large volumes of waste oil from diverse sources are often discarded without proper treatment, causing serious ecological damage. This practice not only contributes to elevated carbon emissions but also represents a waste of the abundant carbon resources contained in these oils.

3. Integration of Syn-Bio and S.cerevisiae

Synthetic biology offers an alternative approach. Saccharomyces cerevisiae has been demonstrated to function as a “cell factory” capable of converting glycerol and fatty acids into valuable metabolites^[7]. The yeast possesses endogenous metabolic pathways for glycerol and fatty acid catabolism^[5], generating acetyl-CoA that feeds into the mevalonate (MVA) pathway, which supplies precursors for terpenoid biosynthesis^[9]. Moreover, its native glycosyltransferase (UGT) systems and cytochrome P450 monooxygenases provide a natural platform for modifying sapogenins^[3], making S. cerevisiae an ideal host for engineering the production of complex ginsenosides. Building on this concept, we engineered a Saccharomyces cerevisiae strain capable of efficiently converting glycerol and fatty acids into ginsenoside Ro.

Our Solution

Our approach involves engineering yeast to convert glycerol and fatty acids into ginsenoside Ro. In this project, we aim to integrate heterologous genes responsible for Ro biosynthesis and optimize yeast metabolic pathways to achieve the following objectives:

(1) Increase MVA precursor supply - enhancing the availability of acetyl-CoA-derived intermediates.

(2) Construct the ginsenoside aglycone oleanolic acid (OA) - the attachment of glucuronic acid moieties to Ro.

(3) Express specific UDP-glycosyltransferases - catalyzing the attachment of glucuronic acid moieties to Ro.

(4) Expand the UDP-glucose and UDP-glucuronic acid pools - providing sufficient sugar donors for glycosylation reactions^[6].

Considering that the only reported yeast strain producing Ro reached a titer of ~0.25 mg/L (0.033 mg/g dry weight, about 31% of the content found in ginseng)^[2], even achieving a few milligrams per liter in our system would represent a significant breakthrough.

Innovation

The innovation of our project lies in addressing the critical supply-demand imbalance of ginsenoside Ro through a sustainable biosynthetic platform. As global demand for this bioactive compound continues to rise, we have developed a scalable, efficient, and environmentally friendly production system by engineering Saccharomyces cerevisiae to convert low-value carbon sources into ginsenoside Ro. This approach not only overcomes the limitations of natural extraction but also establishes a viable manufacturing alternative.

Our strategy integrates several pioneering innovations:

For the first time, waste oil-derived glycerol and fatty acids are used as substrates, representing a model of the circular bioeconomy.

A subcellular compartmentalization approach is employed: peroxisomes are engineered as micro-factories to develop a fatty acid-derived acetyl-CoA pool, enhancing Ro biosynthesis — a cutting-edge strategy in synthetic biology^[5].

We successfully achieved the biosynthesis of the rare oleanane-type ginsenoside Ro, a challenging target with significant pharmaceutical value.

BioOilRo provides a sustainable solution to the global shortage of rare ginsenosides by reducing dependence on limited plant resources and aligning with circular economy principles to enable greener, more efficient, and cost-effective production.