Abstract

Long COVID has led to an increase in myocarditis cases, a condition with limited treatment options. Traditional Chinese medicine (Salvia miltiorrhiza, Danshen) contains tanshinones and carnosic acid, which are known for their anti-inflammatory and cardioprotective effects. However, the conventional extraction of these compounds is costly, time-consuming, and unsustainable. To address this, we are utilizing Saccharomyces cerevisiae to construct a synthetic pathway that efficiently produces tanshinones and carnosic acid by leveraging its inherent mevalonate (MVA) pathway. This method offers a scalable, sustainable solution for producing cardiovascular drugs and may have broader implications for treating cardiovascular and cerebrovascular diseases.

Inspiration

Our project was inspired by the growing concern over the long-term health impacts of COVID-19, particularly cardiovascular complications such as myocarditis. With rising numbers of patients experiencing persistent symptoms post-infection, including inflammation of the heart muscle, there is a pressing need for new therapeutic approaches. Additionally, we were inspired by the work of Nobel laureate Prof. Tu Youyou, whose discovery of artemisinin in Chinese medicine revolutionized malaria treatment. Drawing from this, we explored Salvia miltiorrhiza as a source of cardioprotective compounds and aimed to employ modern synthetic biology to produce these beneficial molecules in yeast.

1 What issue do we focus on?

1.1 Long COVID and Myocarditis

Since the outbreak of COVID-19 in 2019, long-term complications like myocarditis have emerged as a significant public health concern. A study in JAMA found that 6.2% of patients experience persistent symptoms three months post-infection, and 0.9% continue to live with long COVID even after one year.

Myocarditis, the inflammation of heart muscle tissue, is one of the most serious cardiovascular complications associated with COVID-19. In severe cases, it can progress to chronic inflammatory cardiomyopathy, impacting heart function long-term. Despite the high incidence, current treatment options for myocarditis are limited and focus primarily on managing symptoms. This emphasizes the need for new therapeutic approaches targeting the underlying inflammation and myocardial damage.

Figure 1 Schematic diagram of the cardiovascular effects of the virus after entering the human body

Figure 2 Clinical data questionnaire on patients with myocarditis during the COVID-19 pandemic

2 Salvia miltiorrhiza (Danshen)

Salvia miltiorrhiza (S. Miltiorrhiza, Danshen), a prevalent Chinese herbal medicine, is characterized by its fleshy roots, which are reddish-brown on the outside and white inside (Fig 5). Danshen has been used in traditional Chinese medicine for thousands of years, mainly to treat "heartburn" (formerly called cardiovascular disease). Its roots serve as a medicinal component, containing various lipid-soluble and water-soluble constituents with anti-inflammatory, antitumor, antibacterial, and neuroprotective properties, including carnosic acid, tanshinone, tanshinone IIA, and cryptotanshinone.

These constituents are extensively applied in managing inflammation-related diseases by, for instance, ameliorating inflammation in cardiovascular and cerebrovascular ailments, diminishing inflammatory responses by hindering pro-inflammatory factor release, and inhibiting apoptosis(Fig 5).

Figure 5 Salvia miltiorrhiza (Danshen)

3 Tanshinones and Carnosic acid

3.1 Tanshinones

Tanshinones are rosin diterpenes with a common neighboring or para-naphthoquinone chromophore, found mainly in the rhizomes of the Chinese medicinal plant Salvia miltiorrhiza (Danshen). Tanshinone is considered to be the main bioactive component of the Salvia miltiorrhiza herb, which helps to treat cardiovascular diseases. Among them, Tanshinone IIA has been developed and utilized especially intensively in China, and has been used as one of the main clinical drugs for cardiovascular diseases.

It exhibits vasodilating properties, which enhance blood circulation and lower blood pressure, playing a crucial role in the management of cardiovascular diseases. Its ability to address coronary heart disease by improving coronary circulation and inhibiting thrombotic disorders is noteworthy. Additionally, it has the potential to reduce myocardial damage induced by hypoxia (Fig 7).

Figure 7 Pharmacological activity and effects of Tan IIA

3.2 Carnosic acid (CA)

Carnosic acid (CA), a natural phenolic diterpene, is naturally occurring in genus Salvia of the Labiatae family such as Danshen, Salvia officinalis. Recently, a higher concentration of CA was found in the leaves of Salvia miltiorrhiza of the genus Labiatae family, reaching up to 3.05%. Despite the extraction rate now being at a maximum of 98%, the extracted content remains relatively low. CA possesses multiple health benefits and medicinal values, functioning as antioxidants, anti-inflammatories, and anti-proliferatives.

Figure 8 The chemical structures of CA

4 Plant-based production and its shortcomings

Currently, these two compounds are mainly extracted from plants, such as Salvia miltiorrhiza. As these plant-based materials are typically obtained through two sources, wild harvesting or farm cultivation, they show several limitations and drawbacks.

• High cost: Plants’ cultivation requires extensive space (arable land or greenhouses), fertilizers, and labor-intensive field management, and the extraction from plants is costly.
• Unstable supply of raw materials: Plant growth is susceptible to weather, pests, diseases, and variations in sowing area, causing fluctuations in raw material quantities, leading to imbalances in market supply and demand.
• Poor purity and stability of the products: Due to the less-understood mechanism of solvent extraction, the purity and stability of the final products produced from plant extracts may be affected.
• Environmental and ecological damage: The extraction of tanshinones from plants generates a significant amount of production waste and chemical residues, leading to environmental pollution. Wild harvesting of plants poses risks of environmental and ecological damage due to the long growth cycles and limited resources.

Thus, it is urgent to develop a more sustainable, cost-effective, and scalable approach.

5 Our Solution: Yeast-based Biosynthesis of Tanshinones and Carnosic Acid

In plants, tanshinones are primarily synthesized through the methylerythritol phosphate (MEP) pathway, while carnosic acid is produced via a similar terpenoid synthesis process. In yeast, however, we rely on the MVA pathway to produce isoprene pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), the building blocks for terpenoid synthesis. By introducing key enzymes from the Salvia miltiorrhiza pathway into yeast, we aim to efficiently convert these intermediates into tanshinones and carnosic acid. This method offers a sustainable alternative to traditional plant extraction, with the potential to scale up production while minimizing environmental impact.

Figure 9 Biosynthetic pathway of tanshinone IIA and CA

6 Advantages of Yeast-based Production

Our yeast-based platform for producing tanshinones and carnosic acid offers several key advantages over traditional plant extraction:

• Sustainability: By using yeast, we reduce the environmental impact associated with large-scale plant cultivation and extraction, such as deforestation, water use, and pollution.
• Cost-effectiveness: Yeast fermentation is a scalable process, potentially lowering the costs associated with producing these bioactive compounds.
• Consistency and purity: Our system provides a controlled environment for biosynthesis, ensuring higher purity and consistency compared to plant extraction, which can be influenced by factors such as weather and soil conditions.

Our research concentrates on the biosynthesis of tanshinones and critical bioactive compounds from Salvia miltiorrhiza using yeast as a means to combat myocarditis. Through the efficient generation of these compounds, such as tanshinones and carnosic acid, we seek to provide an affordable and sustainable therapy for cardiovascular diseases. This groundbreaking biosynthesis platform marks a significant advancement in the production of natural medicinal compounds, enhancing broader healthcare availability.

Conclusions

By harnessing the power of synthetic biology, our project aims to revolutionize the production of tanshinones and carnosic acid, providing a sustainable and scalable solution to treat long COVID-related myocarditis. Our method not only offers a novel approach to drug production but also addresses a critical need in global healthcare.

We believe this project will have broad applications in drug development, with potential benefits extending to the treatment of various cardiovascular and inflammatory diseases.

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