Description

Introduction

Plant natural metabolites, or plant secondary metabolites, are bioactive compounds produced by plants that play critical roles in their survival, such as defence against pests and diseases.

Many plants produce metabolites with properties that are of interest to health and industry. The global market for plant-derived natural products is expanding rapidly due to their wide-ranging applications in pharmaceuticals, cosmetics, and food industries. By 2027, the global plant extract market is projected to grow to $61.5 billion, largely driven by consumer demand for natural and chemical-free products[1]. In the pharmaceutical and dietary supplement sectors, botanical and plant-derived drugs are expected to generate an additional $20.93 billion between 2023 and 2028[2]. This growth reflects the increasing preference for natural products in various industries, emphasizing the importance of plant metabolites in meeting consumer and industrial needs.

Challenges and Alternatives in Plant-Based Natural Metabolite Production

However, these plant natural metabolites sometimes occur in complex mixtures or within a limited number of cell types reducing accessibility. Additionally, their intricate stereochemistry often complicates chemical synthesis. As a result, high demand for these natural products can place immense strain on the organisms that produce them. A notable example is the Pacific yew (Taxus brevifolia), whose population suffered a 30% decline due to the extraction of paclitaxel, leading to its classification as 'near threatened'[3].

Reconstructing plant metabolic pathways in microbial hosts offers a viable alternative for producing these compounds. This method can yield high outputs and allows the combination of enzymes from various species to create novel biosynthetic pathways. Nevertheless, the commercial viability of this emerging industry remains uncertain, and concerns have been raised about the sustainability of the feedstocks used in microbial cultivation[4].

Photosynthetic organisms like plants provide an appealing alternative chassis. These systems benefit from easy access to metabolic precursors from photosynthesis, multiple subcellular compartments, and the compatibility of heterologously expressed enzymes[5]. However, genetic modification in plants is often labor-intensive. The selection of plants for synthetic biology modifications to enable the production of natural metabolites is a key question we are addressing.

The Need for Transformation and Upgrading in the Tobacco Industry

The Traditional Tobacco Industry is Facing Multifaceted Challenges.

As global anti-smoking policies become increasingly stringent, the traditional tobacco industry is encountering unprecedented challenges. Various factors are contributing to a yearly decline in tobacco consumption, resulting in a gradual shrinkage in the market demand for traditional tobacco products.

On the one hand, governments around the world are imposing stricter regulations on tobacco, enhancing oversight of tobacco products through measures such as increasing tobacco taxes, banning smoking in public places, and prohibiting advertisements, according to the "WHO Global Tobacco Epidemic Report 2023," the number of countries implementing comprehensive tobacco control policies increased from 42 in 2007 to 106 in 2020(Fig.1).

On the other hand, rising public health awareness has led to a decrease in the number of smokers, further promoting the implementation of smoking bans. According to the "WHO Global Report on Trends in Prevalence of Tobacco Use 2000-2025"in 2000, around a third (32.7%) of the global population (both sexes combined) and aged 15 years and older were current users of some form of tobacco. By 2020, this rate has declined to under a quarter (22.3%) of the global population. Assuming that current efforts in tobacco control are maintained in all countries, the rate is projected to decline further to around a fifth (20.4%) of the global population by 2025(Fig.2).

In the face of potential market contraction and policy pressures, the tobacco industry must seek new directions for development. Therefore, transformation and upgrading have become inevitable choices for the industry. To achieve sustainable development, exploring the applications of tobacco in high-value-added fields such as medicine, agriculture, and biotechnology has become a key avenue for driving industry growth.

New Applications of Tobacco: Nicotiana benthamiana

Nicotiana benthamiana, an Australian native herb known for its secondary growth characteristic of dicotyledonous plants, is an important model organism in plant biology research. It offers several advantages over other plants, including low cultivation costs, a short growth cycle, high efficiency in genetic transformation and transient expression, ease of recombinant protein isolation and purification, and high biomass yield. A prized feature of N. benthamiana is its amenability to rapid, transient expression using a technique known as agroinfiltration, in which strains of Agrobacterium tumefaciens carrying genes or pathways of interest are injected into leaves(Fig.3). These benefits make N. benthamiana an ideal chassis for synthetic biology research.

To date, several classes of molecules have been successfully produced in N. benthamiana:

• Alkaloids: strychnine[6], diosgenin[7], piperine[8],...
• Phenolic compounds: montbretin A[9], melitidin[10],...
• Diterpenoid: the anticancer paclitaxel[11], the allelopathic momilactone B[12],...

Limitations of N. benthamiana

While N.benthamiana is a promising chassis for biomanufacturing, it still has inevitable drawbacks.

Like other Nicotiana species, N.benthamiana accumulates high amounts of secondary metabolites, like chlorogenic acid and its derivatives. Its own metabolites will divert too many metabolic streams, reducing the efficiency of heterologous synthesis.

In addition, the current chassis modification logic for N.benthamiana is mostly bottom-up, with chassis designed based on known target products, which is target product specific and not universally applicable. We wondered whether it would be possible to develop a N.benthamiana chassis that could increase the heterologous synthesis yield of different target products.

Our Solution: VersaTobacco

As traditional tobacco products decline, the industry is looking for ways to utilize tobacco crops in a more sustainable and health-conscious manner. Leveraging tobacco plants like Nicotiana benthamiana as biofactories for plant-derived metabolites offers a dual benefit: revitalizing the tobacco industry while addressing the inefficiencies in natural product synthesis. Repurposing tobacco through synthetic biology could provide a scalable, efficient alternative for producing high-demand plant metabolites.

In this context, VersaTobacco emerges as a solution that addresses both the inefficiencies in metabolite production and the need for tobacco industry transformation.

VersaTobacco is a combination of "Versatile" and "Tobacco," symbolizing the multipurpose potential of the Nicotiana benthamiana chassis. In the VersaTobacco project, by optimizing the metabolic network of Nicotiana benthamiana, we aim to develop a universal plant chassis capable of heterologously producing specific plant natural products within a short period.

VersaTobacco is mainly composed of two parts:

Construction of a Low Chlorogenic Acid Chassis: We first built a genome-scale metabolic model (GSMM) of Nicotiana benthamiana and used the COBRA algorithm to analyze and devise an optimal metabolic flux distribution strategy. Subsequently, we employed CRISPR/Cas9 technology to perform a combinatorial knockout of critical genes in chlorogenic acid synthesis (NbHQTs), successfully constructing a low-chlorogenic-acid Nicotiana benthamiana chassis.

Chassis Function Validation: To validate the efficiency of heterologous synthesis in the chassis, we introduced the metabolic pathways for the heterologous synthesis of caffeoylmalate, resveratrol, and crocin into the chassis. By comparing the production yields of these three metabolites in VersaTobacco with those in the wild type, we evaluated the efficiency of the chassis. During this process, we employed semi-rational enzyme design and directed evolution to modify key enzymes within the metabolic pathways, enhancing the synthesis capacity for the target products.

For more information, please refer to the modeling section and the design section.

We have also conducted an initial exploration of the industrial-scale production model for VersaTobacco. By cultivating VersaTobacco in large quantities in a greenhouse, we can perform efficient agroinfiltration using a vacuum infiltration device when the plants reach the optimal leaf stage . After 24 hours of dark incubation followed by 3 to 5 days of greenhouse cultivation, the leaves can be harvested for compound extraction and purification(Fig.4). This process allows for the rapid, large-scale production of target compounds within 2 to 3 weeks.

For more information, please refer to the integrated human practices section.