Overview

As the quality of life continues to improve, people have higher requirements for dietary safety and health^[1]. Since the World Health Organization issued dietary guidelines, the market demand for sugar-free and sugar-reduced products has risen sharply^[2]. Sugar substitutes are considered as virtually calorie-free sweeteners and have become a necessity for people to control sugar^[3]. However, scientific research on the safety of sugar substitutes found that the widely used sugar substitutes such as erythritol, sorbitol, may lead to inflammation of the intestines, and increase the risk of blood clots. It is evident that there is a dire need for a healthy and safe sweetener^[4,5].

In order to find a safer sweetener, we focused on Thaumatin, a sweet protein that has gained much attention in recent years^[6]. It comes from the African tropical fruit Thaumatococcus daniellii and is certified by the FDA and GRAS as a safe and reliable sugar substitute^[7]. Meanwhile, in the Integrated Human Practices, we found that the production of Thaumatin is mainly extracted from plant sources. Complexity of the extraction process and the serious depletion of Thaumatin make it difficult to meet the market demand. Therefore, we hope to design an efficient and convenient Thaumatin production system, so as to meet the demand for sugar substitutes, help people achieve the healthy dietary standards of sugar control and sugar reduction, and lower the risk of obesity and diabetes.

In our project, we adopted a "Fruit+Biopower" approach, designing a expression system and a localization-storage system, to develop a production line for Thaumatin in tomato fruits.

---Expression system: We optimized the production chassis and expression parts to ensure efficient and stable Thaumatin expression.

---Localization-storage system: We installed a “GPS” on the sweet protein product to realize the automatical localization and storage of Thaumatin.

Finally, we can obtain the nutritional sweeteners Sweetein directly from tomato juice without going through the complicated process of purification. It can be applied to all kinds of food products through simple juice processing, which greatly improves production efficiency and product utilization, and provides people with a nutritious, safe and cheap sugar substitute.

Expression System

Sweet proteins are sweet compounds found in nature, which are mainly derived from plants in the tropical regions of Africa and Asia. These naturally sourced proteins have gained widespread attention because they are very sweet, sweeter than sucrose, and do not cause health problems or calories compared to artificial sweeteners^[8,9].

Sweet Protein Selection

Currently, a variety of sweet tasting proteins have been identified, such as Thaumatin, Monellin, Brazzein, Curculin, Mabinlin and entadin^[10].

Taking into account the size, sweetness, properties and safety of the proteins, we found that Thaumatin, derived from the aril of the tropical plant Thaumatococcus daniellii (Benth), and Brazzein, from the fruit of P. brazzeana, are not only sweet but also have N-terminal amino acid sequences that translocate them to the Endoplasmic Reticulum(ER), and forming eight disulfide bonds and four disulfide bonds respectively. So, they have the advantages of acid stability and heat stability^[11-13].

Schematic diagram of Thaumatin

Mechanisms by which sugar substitutes produce sweetness

Brazzein fruit and protein structure

And, we learnt that with the development of the industry, natural Thaumatin is approved by FDA and GRAS and is used in chewing gum, chocolate and other foods. Although Brazzein has not been approved, it has been developed by several biological companies because its protein is small enough and its structure is stable, so we can see that the application of Brazzein is very promising, and it may be able to become a leader in the field of sugar substitute in the future.

Therefore, we chose Thaumatin and Brazzein for production.

Chassis Selection

Whether the protein can be successfully expressed is related to the size of the protein, but it still depends on whether the expression in chassis can be properly transcribed, translated and folded^[14].

Prokaryotic expression

Firstly, we tried to express Thaumatin and Brazzein in E. coli by constructing expression vectors and transformed them into E. coli, then induced the expression with IPTG and tested the protein level^[15,16]. Both Thaumatin and Brazzein have the special structure of disulfide bond, and the prokaryotic expression system has natural defects, which is not able to fold proteins correctly to form a complex structure, so the protein expression results were not satisfactory.

Prokaryotic expression plasmid plots of Thaumatin and Brazzein

To ensure the quality of protein expression, we need to select chassis with better expression efficiency.

Expression in plants

Sugar substitutes are used as food additives. We wanted to save purification steps and reduce safety risks in the process of obtaining Thaumatin and Brazzein, so we focused on tomato, a fruit that is often consumed by people in their daily lives.

Therefore, we need to further validate the feasibility of the tomato chassis. In order to accomplish this process quickly, we chose to use the transient expression method. Transient expression consists of two means, one is to soak plant organs with Agrobacterium and the other is to use viral expression vectors. Comparing these two methods, we learned that the Agrobacterium soaking method is difficult to maintain traits. The more fatal problem is that it is difficult to complete the infestation in tomato fruits. While the viral expression vector can maintain the trait for a longer period of time, which is favorable for us to test in tomato fruits. Therefore, we chose to use viral expression vectors to complete this step of verification.

TRV is a double-stranded RNA virus whose genome consists of RNA1 and RNA2. Under the action of reverse transcriptase, the TRV genome is reverse transcribed into cDNA in vitro, which is specifically modified and cloned into non-virulent vectors that are capable of both self-replication and expression of exogenous genes, namely, TRV1 and TRV2^[20]. TRV1 vectors usually contain the RNA-dependent RNA polymerase, RdRp, movement protein (MP) and 16 kD protein. It serves as a helper viral vector that assists in the replication and spread of the virus in plants. TRV2 contains the gene encoding the viral capsid protein, and MCS sequences were artificially inserted into TRV2 for cloning of the target gene. Subsequently, we constructed Thaumatin on the TRV2 vector and injected it into tomato together with the TRV1 vector. When the virus replicates autonomously in tomato cells, it can utilize the material inside tomato cells to express the sweet protein.

At the same time, we made sure that the viral vector could remain in tomato for a period of time by introducing silencing repressor P19 to inhibit the RNA interference system of the tomato plant itself. In addition to this, we introduced mCherry red fluorescent protein partial sequence as a control in order to monitor the effectiveness of the viral vector.

Pathway Design

Tomato became the optimal choice for heterologous expression of Thaumatin and Brazzein in our project. However, as a production system, we need to optimize the pathway for stable, efficient and safe protein expression.

Therefore, we considered using transgenic technology. Transgenic technology is capable of introducing exogenous gene sequences into the genome of a recipient and causing heritable changes in biological traits. Not only has it long been used as a traditional breeding method, but it is also an important aid in the production of products in bioreactors. Therefore, we decided to use transgenic technology to realize the production of sweet proteins.

In the conventional transgenic pathway, expression of Thaumatin and Brazzein initiates expression under the CaMV 35S promoter. However, the CaMV 35S promoter lacks stability.

--On the one hand, a large number of studies have shown that although the CaMV 35S promoter is highly expressed, it affects the expression of neighboring genes, leading to disease in plants.

--On the other hand, the activity of CaMV 35S promoter varies in different tissues and cells of plants, which makes it difficult to ensure the expression level.

Pathway Optimization

In order to ensure the stability of sweet protein expression in transgenic tomato, we need to optimize the promoter. Therefore, we chose the tomato fruit-specific promoter E8^[21,22] instead of the 35S promoter. This tissue-specific promoter can not only accumulate the expression products of target genes in certain organs or tissue sites and increase regional expression, but also avoid unnecessary waste of plant nutrition.

The E8 promoter has the following advantages^[23-25]:

(1) Specific expression

Plants have a typical division of labor in which different organs perform different functions. In the plant body, roots are responsible for drawing water and inorganic salts downward, leaves are responsible for photosynthesis to provide energy, and fruits are responsible for expressing and storing products. The E8 promoter is one of the most widely characteried ripening-specific tomato promoters, and it has shown strong conservation among different tomato varieties. Thus, it enables the specific expression of Thaumatin in tomato fruits without affecting the normal functioning of roots and leaves.

(2) Stable expression

The growth and development of tomato, a respiratory transgressive fruit (ethylene-regulated) among fleshy fruits, is not only regulated by phytohormones and transcription factors, but also requires epigenetic modifications, the most common manifestation of which is the methylation modification of DNA.

Methylation of the DNA itself may physically hinder the binding of transcription factors to genes. At the same time, DNA modified by methylation may be bound by MBD proteins. MBD proteins recruit other modifiers, resulting in the formation of dense, inactive chromatin that leads to failure of DNA unwinding at transcription.

In tomatoes, DNA methylation is widespread as it can occur in both symmetric (CG or CHG) and asymmetric (CHH) environments. On the other hand, there exists a natural SlDML2-mediated active DNA demethylation in tomato, a process that counteracts the effects of methylation modifications. SlDML2 acts as a DNA demethylase that activates the expression of ripening-associated genes such as those related to RIN, ethylene and pigment synthesis, and deregulates the transcriptional repression of ripening-associated gene.

E8, as an ethylene-regulated promoter and being an endogenous promoter in tomato, is particularly important for deregulating transcriptional repression of DNA methylation modifications. The E8 promoter contains two major regions that contribute to its transcriptional regulation:

-- The upstream region of -2181 to -1088 contains ethylene-responsive transcription elements.

-- In the absence of ethylene synthesis, sequences from -409 to -263 in the “downstream” region of -1088 in the transcription start site can be fruit-specific transcribed.

Localization-Storage System

The level of protein depends on how much is synthesized and broken down. If we want to increase protein accumulation, we need to ensure that the amount of expression is high and the amount of catabolism is low. Therefore, in addition to constructing a expression system that utilizes superior chassis and pathway optimization to ensure efficient expression, we also need to pay attention to protein catabolism. Hence, we would like to provide a protected region for Thaumatin within the tomato fruit for Thaumatin storage, reducing the possibility of it being broken down.

Subcellular Circuits

Plant cells consist of multiple membrane-separated organelle compartments, each of which has evolved to optimize and undertake specific biochemical functions. Proteins are carefully choreographed to undergo an assembly line transfer from the nucleus, ribosomes, Endoplasmic Reticulum (ER), and Golgi apparatus, completing the process of transcription, translation, and folding, so that they arrive at the right compartment at the right time through the right route and with the right structure to achieve temporal and spatial specificity, without any artificial or mechanical regulation. It is clear that the subcellular “circuitry” gives the plant production system its automated character, and that the sweet proteins can be automatically localized in a certain compartment by adding a specific “GPS” signal to them.

Storage Compartments

To find the best storage space for sweet proteins, we carefully selected individual subcellular compartments within the cell.

Vacuole, as compartment that occupy most of the space in tomato fruit cells, has the following advantages in storing Thaumatin:

pH environmental aspects:

The liquid environment pH inside the vacuoles is acidic, adapting to the acid stability characteristics of the soluble protein Thaumatin^[26].

Aspects of the contents:

Plants form special vacuoles containing large amounts of specific substances in specific tissues. Plant vacuoles are mainly divided into two distinct chambers: storage chamer and lysis chamber. The typical storage compartment is the protein storage vacuole (PSV) found in seeds, and the typical lysis compartment is the lysing vacuole (LV) found in leaf pulp cells, whereas the PSV contains far fewer protein hydrolyzing enzymes than the LV^[26].

In terms of tissue relationships, tomato fruits and seeds are storage tissues that are more similar in their vacuole properties. As a result, the vacuole within the fruit contain less hydrolytic enzymes and are therefore more suitable for Thaumatin storage.

Fruit development:

Tomato, as a berry, undergoes complex processes such as the transformation of chloroplasts to colored bodies, softening of the cell wall, accumulation of pigments, and changes in hormone levels in the cells during fruit ripening. At this time, the cytoplasmic fluid environment is complex, which is not conducive to the stable existence of Thaumatin. On the contrary, the environment within the vacuole is in a relatively stable state, therefore, the vacuole compartment prevents the effect of the huge environmental changes on Thaumatin during fruit ripening.

Localization Signal

We attached the sequence of glycosporine-N-terminal prepeptide called SPS-NTPP (GenBank accession number: EF638829). It is a vacuolar transport signaling molecule that precisely and efficiently escorts Thaumatin to the vacuole for storage, and after targeting is complete SPS-NTPP is able to self-decompose without any effect, dramatically increasing Thaumatin content in tomato^[27]. Therefore, we constructed the pGD-SPS-NTPP-Thaumatin-EGFP plasmid and transformed it into Agrobacterium GV3101 for transient infestation of Nicotiana benthamiana, thus verifing the targeting function of SPS-NTPP in tobacco leaves.

Sweetein

Natural Thaumatin has a licorice aftertaste, which negatively affects taste in practical applications. In order to meet the sweetness demand in food sensory, Ms. Zhao Liqing, an expert in the direction of food sensory, suggested the screening of bitter taste sites and protein directed evolution for Thaumatin. Considering the continuity of protein directed evolution, we carried out preliminary explorations through visual analysis and molecular docking simulation, hoping to provide theoretical support and useful data for the quality improvement of Thaumatin.

Quality Enhancement

Screening for bittering loci

Bitter taste perception is mediated by type 2 taste receptors (TAS2Rs, also known as T2Rs). Among the members of the TAS2Rs, TAS2R16 is highly expressed in extra-oral tissues and is able to perceive bitterness in food. Thaumatin's bitter taste magnitude depends on the sequence of proteins that can bind to TAS2R16.

The protein sequence of Thaumatin is a peptide chain composed of amino acids, and this specific arrangement of amino acids is known as the primary structure of Thaumatin. There are certain combinations of amino acids within the sequence that determine the folding (alpha helix, beta fold e.g.) within the Thaumatin structure. PPI prediction of Thaumatin's protein sequence and TAS2R16 using a computational approach enables screening of these combinations from the interface of known interacting peptides, thus to determine the bittering site of Thaumatin.

Directed Evolution of Protein

Directed evolution is the most commonly used protein design and modification strategy in protein engineering to rapidly enhance the specific properties of proteins through the establishment of mutant libraries and high-throughput screening methods. In the last decade, with the significant increase in computer computing power and the emergence of advanced algorithms, computer-aided protein design and modification has been greatly emphasized and developed, and has become an important direction newly opened up by protein engineering. The computational design of proteins based on structure simulation and energy calculation has become a tool for us to transform Thaumatin to remove the bitter taste. By random mutation of the screening site, protein structure simulation and combined energy calculation again, the optimal mutation of the bitter taste site is derived, which provides great help for the later experiments.

Learn more in Model.

Process Safety

Sweetein omits the complex steps of purification, allowing direct access to tomato juice which is rich in nutrients and Thaumatin, providing a cost-effective and safe sweetener produced in a synthetic biology factory model.

Thaumatin has good acid stability and thermal stability and can be used as a sweetener in baked foods and various beverages. Automatic production and storage of sugar substitutes using fruits can obtain nutritional sweeteners through simple juicing operations. We can obtain Thaumatin pulp by compression, making it very convenient to be applied in foods such as beverages, candies, pharmaceuticals, and desserts.

The emergence of Sweetein not only meets the need of sugar control, but also provides rich nutrition of fruits and vegetables. More importantly, we have realized the transformation of tomato fruit for the first time in the history of iGEM, which is the first step in attempting to transform fruits. In the future, we expect to use synthetic biology to replace the excess sugar in fruits with Thaumatin, so that most sugar-laden fruits can be consumed by people with diabetes and obesity.