Introduction 介绍

According to Global Burden of Disease (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].
根据全球疾病负担 （GBD） 分析，全球消费者的游离糖日常消费量正在高速增长，给人类健康带来巨大负担，随之而来的是糖尿病和超重人口的急剧增加。因此，自 2015 年以来，世界卫生组织建议将每日游离糖摄入量减少到总能量摄入量的 10% 以下，以降低患肥胖、糖尿病、高血压和癌症的风险^[1]。

China's annual consumption of free sugar
中国游离糖年消费量

The population has excessive intake of free sugars in China
中国人群游离糖摄入过多

WHO releases document highlighting "sugar reduction"
世卫组织发布强调“减糖”的文件

Since the publication of World Health Organization (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 no calories during the sweetening and digesting process, making them highly sought after and often recommended as a means of controlling blood sugar levels in diabetics.
自从世界卫生组织 （WHO） 关于游离糖摄入量的指南发布以来，人们对探索糖替代品在减少糖摄入量方面的潜在作用的兴趣越来越大。糖替代品与舌头上的甜味受体结合并激活，产生信号，这些信号被传递到大脑以产生甜味^[2]。这些糖替代品在甜味和消化过程中不会产生卡路里，因此备受追捧，并经常被推荐作为控制糖尿病患者血糖水平的一种手段。

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.
从 2015 年到 2022 年，中国代糖的年产量从 170,000 吨增加到 270,000 吨，并保持了较高的增长率^[3]。代糖在食品和饮料行业得到了广泛的应用，代糖饮料的市场份额自 2017 年以来逐年上升，到 2023 年达到 50%。根据 EMIS 数据，全球市场规模已达到 60 亿美元，并将以约 3.75% 的速度增长。这表明代糖已成为世界各地人们必不可少的食物。

Problem 问题

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 one, 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 didn't meet the WHO's recommendation to eat foods. Therefore, there is an urgent need for a safe, reliable and nutritious sweetener^[6].
糖替代品包括各种合成衍生的化学物质和天然提取物，它们通常比糖甜很多倍，因此它们的数量非常少。然而，近年来，人造糖替代品已被证明具有致癌风险。其中，使用最广泛的阿斯巴甜被 WHO 列为风险[^4]。天然提取的糖替代品，如甜菊糖苷和赤藓糖醇，有一些好处，从长远来看，它们不会被人体消化，而是直接通过小肠进入血液，过量食用会导致胃肠道症状和血栓风险增加^[5]。此外，大多数代糖是非营养性的，不符合 WHO 关于吃食物的建议。因此，迫切需要一种安全、可靠和营养的甜味剂^[6]。

The dangers of sugar substitutes
代糖的危害

Thaumatin extraction process
索马甜提取工艺

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 has been approved by the FDA and GRAS for its 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.
目前，一些生物企业正专注于开发一种可溶性天然甜味蛋白，以满足人们的需求：索马甜。这是因为这种甜味蛋白不仅具有现有糖替代品的高甜度特性，而且具有很高的稳定性，并且耐高温和耐酸。更重要的是，它的安全性已获得 FDA 和 GRAS 的批准，可以被人体完全消化成常见的氨基酸^[7]。然而，令人痛心的是，这种甜味蛋白质存在于非洲热带植物 Thaumatococcus danielli （Benth） 的种子中。此类植物数量少，栽培条件恶劣，从种子中提取蛋白质的过程损失大、产量低、成本高，难以满足市场需求。因此，市场迫切需要一种新的生产方法来提高索马甜的产量。

Our method 我们的方法

We designed Sweetein, an efficient production system to produce sweet protein in tomatoes. Based on the "Fruit+Biopower" design, we can obtain nutritious sweeteners from tomato juice without the need to go through the complex purification process.
我们设计了 Sweetein，这是一种在西红柿中生产甜味蛋白的高效生产系统。基于“水果+生物能量”的设计，我们可以从番茄汁中获得营养丰富的甜味剂，而无需经过复杂的纯化过程。

FRUIT: Choosing tomato fruit as a novel production system.
水果：选择番茄果实作为一种新颖的生产系统。

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.
与从生物发酵中提取产品相比，使用水果显然更方便，这有助于我们直接从番茄汁中获得产品。

BIOPOWER: Using synthetic biology to build plant power lines.
BIOPOWER：利用合成生物学构建植物电力线。

Stable Expression 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.
稳定表达系统：植物作为先进的表达系统，在表观遗传学上容易受到外源基因的修饰，导致它们无法正常表达。为了这个生产系统的可持续性，我们必须考虑生产的稳定性，因此，我们选择果实特异性启动子 E8 而不是传统的 35S 启动子，一方面利用内源成分防止外源基因的丢失，另一方面减轻了番茄代谢的负担，大大提高了番茄果实中索马甜表达的稳定性以及遗传稳定性。

Automatic Storage System: As the compartments used for storing nutrients in the fruit cells, the vacuole 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 应用

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 defectiv. 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.
植物合成生物学采用“设计-构建-测试-学习”的研究策略，利用植物的大量基因组、转录组和代谢组，在植物中构建“分子生物学工作室”。在目前的研究中，番茄、烟草、水稻和拟南芥已成为植物合成生物学的模式生物，并已广泛应用于各种重组蛋白和营养物质的生产。此外，植物亚细胞储存和特异性生产是植物合成生物学发展的新方向，可以提高植物生产的产量和稳定性，实现产品的时空控制。

In terms of health concepts:
在健康理念方面：

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!
政策是科学发展和产业创新的总准则。面对当今日益严重的糖尿病和肥胖等全球问题，世界卫生组织 （WHO） 强烈呼吁规律饮食和均衡营养，并减少糖的摄入量。Sweetein 的出现不仅满足了控糖的需要，还从水果和蔬菜中提供了丰富的营养成分，更重要的是，在 iGEM 历史上，我们第一次实现了番茄果实的改性。这是尝试修改 fruits 的第一步。未来，我们希望通过合成生物学的方式，用索马甜代替水果中多余的糖分，让糖尿病和肥胖症患者也能吃到含糖水果！