Introduction

According to GBD analysis, the daily consumption of free sugars by consumers around the world is increasing at a high rate, placing a huge burden on human health with a consequent sharp increase in diabetes and overweight population. As a result, since 2015, WHO has recommended reducing the daily intake of free sugars to less than 10% of total energy intake to reduce the risk of obesity, diabetes, hypertension and cancer. ^[1]

Since the publication of WHO guidelines on the intake of free sugars, there has been increased interest in exploring the potential role of sugar substitutes in reducing sugar intake. Sugar substitutes bind to and activate sweet taste receptors on the tongue, generating signals that are transmitted to the brain to produce the sweet taste sensation.^[2] These sugar substitutes produce little to no calories during the sweetening and digestion process, making them highly sought after and often recommended as a means of controlling blood sugar levels in diabetics.

From 2015 to 2022, the annual production of sugar substitutes in China increased from 170,000 tons to 270,000 tons and maintained a high growth rate.^[3] Sugar substitutes have been widely used in the food and beverage industry, and the market share of sugar substitute beverages has risen year by year since 2017, reaching 50% by 2023. According to EMIS data, the global market size has reached $6 billion and will grow at a rate of about 3.75%. This shows that sugar substitutes have become an essential food for people all over the world.

Problem

Sugar substitutes include a wide variety of synthetically derived chemicals and natural extracts that are often many times sweeter than sugars, making them desirable in very small quantities. However, in recent years, artificial sugar substitutes have been proven to have carcinogenic risks. Among them, aspartame, the most widely used, is classified as risky by the WHO.^[4] Naturally extracted sugar substitutes such as stevioside and erythritol have some benefits and, in the long run, are not digested by the body and enter the bloodstream directly through the small intestine, where overconsumption can lead to gastrointestinal symptoms and an increased risk of blood clots.^[5] In addition, most sugar substitutes are non-nutritive, which is contrary to the WHO's recommendation to consume foods rich in nutrients that improve body function and health. Therefore, there is an urgent need for a safe, reliable and nutritious sweetener.^[6]

Currently, some biological enterprises are focusing on the development of a soluble natural sweet protein to meet people's needs: thaumatin. This is because this sweet protein not only possesses the characteristics of the existing sugar substitutes with high sweetness but also has high stability and is resistant to high temperatures and acids. More importantly, it is FDA and GRAS approved for safety and can be completely digested by the human body into common amino acids.^[7] However, what is distressing is that this kind of sweet protein exists in the seeds of the African tropical plant Thaumatococcus danielli Benth. The number of such plants is small with harsh cultivation conditions, and the protein extraction process from the seeds has high losses, low yields and high costs, making it difficult to meet the market demand. Therefore, the market is in urgent need of a new production method to increase the yield of thaumatin.

Our method

We designed Sweetein, an efficient production system to produce sweet protein in tomatoes. Based on the "Fruit+Biopower" design, we can obtain nutritional sweeteners from tomato juice without the need to go through the complex purification process.

FRUIT: Choosing tomato fruit as a novel production system.

it is obviously more convenient for us to use the fruit compared to extracting the product from biological fermentation, which facilitates our ability to obtain the product directly from the tomato juice.

BIOPOWER: Using synthetic biology to build plant power lines

Stable Synthesis System: Plants, as advanced expression systems, are epigenetically prone to modification of exogenous genes resulting in their inability to be expressed properly. For the sustainability of this production system, we must consider the stability of production, therefore, we choose the fruit-specific promoter E8 instead of the traditional 35S promoter, which on one hand prevents the loss of exogenous genes by utilizing the endogenous components, and on the other hand, reduces the burden on the metabolism of tomato, and greatly improves the stability of expression of thaumatin in tomato fruits along with the genetic stability.

Automatic Storage System: As the compartments used for storing nutrients in the fruit cells, the vesicles can store excess sugar and other nutrients in the fruit. Considering the natural phenomenon of cell wall fragmentation during fruit ripening, to prevent the effect of this process on the protein, we linked the N-terminal sequence of Thaumatin to the sweet potato sporamine N-terminal propeptide (SPS-NTPP), a vacuolar transport signaling molecule, which can precisely and efficiently escort thaumatin to the vacuole for storage.^[8] After the targeting work, SPS-NTPP could self-decompose without any effect, greatly increasing the content of thaumatin in tomatoes.

Sweetein eliminates the complex purification process and provides direct access to nutrient-rich and thaumatin-rich tomato juice, making Sweetein an undeniably safe nutritive sweetener that solves the current problem of balancing safety with yield and cost in the substitution market.

Sweetein provides a cost-effective and safe synthetic biology factory model, which is expected to provide a viable solution for the future production of medicinal proteins, vaccines, and so on.

Application

Using plant synthetic biology in production system is both cost-effective and sustainable, and has been evaluated as the "major paradigm shift" of cell factories, which has significant advantages over prokaryotic and yeast expression systems:

The microbial expression system is functionally defective, it is difficult to complete the folding of proteins with complex structures, and it is even extremely easy to modify proteins with motifs leading to loss of protein function. The plant expression system, in contrast, is more versatile, and scientists have already successfully utilized plants to produce a wide range of medicines and secondary metabolites that microbes are unable to produce, such as albumin, paclitaxel, and others.

Molecular level

The plant cell is actually an integrated tribe of multiple compartments separated by membranes, each of which has evolved to optimize and assume a specific biochemical function, while any intracellular material is carefully arranged to arrive at the right compartment at the right time via the right route, achieving temporal and spatial specificity without any artificial, mechanical or engineering regulation. A plant cell is a miniature biofermenter. Therefore, we must admit that plant production systems are naturally automated.

Cell level

In the global sugar substitute market, artificial sugar substitutes account for 62% of the market share, and natural plant-derived sugar substitutes account for 30%, while the market share of microbial fermentation is less than 10%. This is because microbial fermentation requires screening and evolution to obtain dominant strains to ensure the stable expression of products, which is much more difficult than plant transgene.

Technical level

Microbial engineering is driven by their rapid reproduction, which can produce large numbers of offspring in a short period of time. However, when microbial engineering enters the market in the form of fermenters, it faces huge engineering and cost problems in purifying the products. More worryingly, the purification process often requires the addition of organic reagents, which makes it difficult to meet safety standards for fermentation products. In contrast, plant-based chassis eliminate the purification step, saving the process, time, and cost of production.

Industrial level

Plant science is moving towards the definition of metabolic pathways and subcellular structure integrated circuits to improve stability and yield to meet the challenges of microbial science that are difficult to break through. Sweetein is a star in the evolution of plant science. in the development of plant science, "fruit + biopower" perfectly utilizes the advantages of plant synthetic biology, which has far-reaching significance for the development of food and nutrition.

In terms of sugar substitute entrepreneurship:

Using fruit for automatic production and storage of sugar, nutritional sweeteners can be obtained by simple juicing operation, and it is very convenient to be used in beverages, sweets, pharmaceuticals, sweets and other foods. Thaumatin is an emerging product in the sugar substitute market, with the global thaumatin market valued at $46 million in 2020 and projected to reach $70 million by 2027, according to Syndicate Research by The Insight Partners. As far as the product is concerned, the creation of Sweetein will significantly reduce the price of thaumatin, and with the help of the global tomato market, the thaumatin industry can promote its outreach, which is a very promising commercialization.

In terms of plant science:

Plant synthetic biology adopts the research strategy of "Design-Build-Test-Learn", which utilizes a large number of genomes, transcriptomes, and metabolomes of plants, to build a "molecular biology studio" in plants. In the current research, tomato, tobacco, rice, and Arabidopsis thaliana have become model organisms for plant synthetic biology, and have been widely used in the production of various recombinant proteins and nutrients. Furthermore, plant subcellular storage and specific production are new directions in the development of plant synthetic biology, which can improve the yield and stability of plant production, and realize the spatial and temporal control of products.

In terms of health concepts:

Policy is the general guideline for scientific development and industrial innovation. In the face of today's increasingly serious global problems of diabetes and obesity, the World Health Organization (WHO) has strongly called for a regular diet and balanced nutrition, and a reduction in the intake of sugar. Sweetein's emergence not only meets the need for sugar control but also provides a wealth of nutrients from fruits and vegetables, and what's more important is that, for the first time in iGEM history, we have achieved the modification of tomato fruit. It is the first step in the attempt to modify fruits. In the future, we hope to replace the excess sugar in fruits with thaumatin by means of synthetic biology, making sugary fruits available to people with diabetes and obesity!