Part:BBa_K5160003
Thaumatin Ⅱ
Overview
In order to provide people with a safe sweetener, our project hopes to propose a method for the efficient production of Thaumatin. Thaumatin comes from a tropical fruit in Africa and has been certified by the FDA and GRAS as a safe and reliable sugar substitute. Thaumatin binds to receptors on the human tongue to induce a sweet taste. This process produces almost no calories. At the same time, It can be fully digested by the human body and produce no side effects. This shows that Thaumatin is a very safe and reliable sweetener, which makes it an excellent choice for sugar reduction and control. About the engineering cycle and iteration of Thaumatin. Please go to the engineering section to find out more about the engineering cycle and the iteration of Thaumatin production.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 109
Illegal NotI site found at 146
Illegal NotI site found at 248 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 142
Illegal NgoMIV site found at 308 - 1000COMPATIBLE WITH RFC[1000]
Usage and biology
Thaumatin is derived from the aril of the tropical plant Thaumatococcus daniellii (Benth). Bamboo yam contains two types of Thaumatin: Thaumatin I and Thaumatin II. One of them, Thaumatin II, has a higher sweetness. It consists of 207 amino acids, and the amino acids are coiled and folded to form eight disulfide bonds. As a result, the protein structure of Thaumatin is stable, thermally and acid-stable.
Thaumatin belongs to the family of five pathogenesis-related proteins (PR5), which are characterized by significant antifungal as well as phytopathogenic activity. Thus, in bamboo yam, the main function of Thaumatin is to protect seeds and fruits against abiotic stresses. After discovering the sweet flavor characteristics of Thaumatin, people have developed it and used it in various foods.
Thaumatin binds to sweetness receptors T1R2 and T1R3 on the human tongue, activating G-protein-coupled receptors on the membrane surface, generating an electrical signal that is transmitted to the cerebral cortex, causing sweetness perception. As a protein, Thaumatin consists of common amino acids and can be fully digested and absorbed by the body without generating calories.
Design
Sequence identification
We found the original record of heterologous expression of Thaumatin from the literature. However, the recorded fragment was incomplete, so we blasted the incomplete sequence in NCBI to obtain the complete cds sequence. Finally, we obtained the complete sequence of Thaumatin II on the whole genome.
Chassis selection
Because of the complex structure of Thaumatin, we hoped to find a suitable chassis to express it efficiently.
The successful expression of the protein is related to its size, but it mainly depends on whether the protein expressed in the chassis can be properly transcribed, translated and folded.
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. 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.
Considering the practical application of sugar substitutes as a food additive, and the fact that fermentation production requires the complex process of protein purification, which may introduce safety concerns, we aim to simplify the Thaumatin production process by eliminating the need for purification steps. Thus, we have turned our focus to a fruit commonly consumed in daily life—tomatoes.
Pathway design
To validate the feasibility of the tomato platform, we need a quick testing method. Therefore, we aim to achieve this goal through transient expression. Many studies have shown that transient infection can be accomplished by Agrobacterium dipping or by viral vector leaf injection. Comparing the two methods, we found that Agrobacterium dipping struggles to maintain traits. A more critical issue is that it is difficult for it to complete infection in tomato fruits. In contrast, viral expression vectors can maintain traits for a longer period, which is advantageous for testing in tomato fruits. Therefore, we chose to use viral expression vectors for this step of validation.
TRV is a double-stranded RNA virus with a genome consisting of RNA1 and RNA2. With the reverse transcriptase, the TRV genome is reverse transcribed into cDNA in vitro. Specific modifications and cloning of the cDNA transform it into a non-toxic vector that retains both self-replication ability and the ability to express foreign genes, known as TRV1 and TRV2. The TRV1 vector usually contains genes encoding RNA-dependent RNA polymerase (RdRp), movement protein (MP), and a 16 kDa protein. It serves as an auxiliary viral vector to assist in the replication and spread of the virus within the plant. TRV2 contains the gene encoding the viral coat protein, and an artificially inserted MCS sequence is used for cloning the target gene on TRV2. We then inserted Thaumatin on the TRV2 vector and co-injected it with the TRV1 vector into tomatoes. As the virus replicates autonomously within the tomato cells, it can utilize intracellular substances to express Thaumatin.
At the same time, we inhibited the RNA interference system of tomato plants themselves by introducing silencing repressor P19 to ensure that the viral vectors could remain in tomato for a period of time. In addition, in order to monitor the effectiveness of the viral vector, we introduced mCherry red fluorescent protein partial sequence as a control.
(A) The mechanism of the introduction of the target fragment into tomato cell through the TRV vector.
(B) The expression pathway of Thaumatin.
(C) The expression pathway of P19.
(D) The expression pathway of the partial sequence of mCherry.
Pathway optimization
We verified that Thaumatin is correctly expressed in tomato. In order to make the production of Thaumatin sustainable, we have changed the methods of production. Transgenic technology enables the introduction of exogenous gene sequences into the host genome, resulting in heritable changes in biological traits. Not only has it been used for a long time as a traditional breeding method, but it has also been instrumental in the production of products in bioreactors.
Transgenic technology is based on a sequence of T-DNA on the plasmid of Agrobacterium. After Agrobacterium invades plant cells through wounds, it inserts the T-DNA into the genome of the target plant.
Agrobacterium Ti plasmid is a double-stranded covalently closed circular DNA molecule. It contains four functional regions, which are T-DNA region, Vir region, Con region and Ori region. The T-DNA region is the fragment that can be transferred and integrated into the plant genome; the Vir region encodes proteins that are involved in the processing and transfer of T-DNA; the Con region is related to the binding and transfer of the plasmid; and the Ori region is the plasmid replication initiation site.
When the plant is wounded, the damage cells will secrete a large number of phenolic compounds. These phenolics can induce the expression of Vir genes in Agrobacterium. Through chemotaxis, Agrobacterium recognizes and attaches to the wounded area of the plant. The expression product of the Vir gene cuts off a single strand of T-DNA from the Ti plasmid to form a single-stranded T-DNA. Subsequently, the single-stranded T-DNA binds to the expression products of the Vir gene and the Con gene to form a complex. The complex transfers the T-DNA to the plant cell through contact between Agrobacterium and the plant cell. Upon entering the plant cell, the T-DNA is able to randomly integrate into the plant chromosome. This process can be single-copy or multi-copy, but is usually preferred near the coding or regulatory regions of a gene.
In the conventional transgenic pathway, synthesis 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, making it difficult to ensure the expression level.
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 instead of the 35S promoter. This tissue-specific promoter can not only accumulate the expression products of target genes in certain organs or tissues and increase regional expression, but also avoid unnecessary waste of plant nutrition.
The E8 promoter has a highly specific expression pattern in tomato fruit. It is mainly activated at specific stages of tomato fruit development, driving preferential expression of downstream genes in fruit tissues. The E8 promoter is significantly more active in fruit compared to other tissues such as roots, stems and leaves. It can be utilized to achieve precise regulation of specific genes in fruits. By linking the target gene to the E8 promoter, the target gene can be expressed only in tomato fruits, avoiding unwanted effects in other tissues. This is important for improving the quality and characteristics of tomato fruits.
During fruit ripening, the E8 promoter can initiate the expression of a series of genes that are involved in physiological processes such as fruit color change, texture softening, and flavor substance synthesis. These changes are the result of the mutual regulation of the E8 promoter and ethylene.
There are at least two major regions in the E8 promoter sequence that contribute to its transcriptional regulation. One is the upstream region from -2181 to -1088, which contains ethylene-responsive transcriptional elements. When ethylene concentration is elevated, the expression of the E8 promoter and ethylene synthesis genes increases simultaneously, forming a positive feedback regulatory mechanism that further promotes fruit ripening. And in the absence of ethylene synthesis, another region comes into play. The sequence from -409 to -263 of the transcription start site is sufficient for ripening-specific transcription.
Storage location
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.
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.
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.
We attached the sequence of glycosporine-N-terminal prepeptide 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. 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.
Plasmid construction
In prokaryotes
We chose one of the most common strains E. coli BL21(DE3) for fermentation production. The target sequence we designed was controlled by the T7 promoter on the plasmid pET-28a(+) to obtain our expression vector. Subsequently, we transformed the vector into E. coli BL21(DE3) and picked the colonies for amplification on the medium containing kanamycin.
In plants
We constructed the TRV2-35S-Thaumatin plasmid using the virus TRV (Tobacco rattle virus) and infested tomato plants. We used this transient infestation to verify the viability of tomato chassis.
Next, we constructed CaMV 35S-Thaumatin and E8-Thaumatin plasmids using the binary vectors pBWA(V)HS and pCAMBIA1301 as backbones for tomato transgenic expression respectively.
To validate that the incorporation of SPS-NTPP can direct Thaumatin to be localized in the vacuole, we initially aimed to reproduce the localization effect of this targeting peptide in our laboratory, which was a guarantee indicating that we could proceed to the next stage of experiments. The most expedient approach was to link SPS-NTPP with Thaumatin and attach an EGFP protein at the rear end. The fluorescence emitted by it could thereby indicate the position of Thaumatin. Hence, we constructed the pGD_SPS-NTPP-Thaumatin-EGFP plasmid.
Characterization
Synthetic validation
Ⅰ. Expression prokaryotic
We extracted bacterial proteins for Western Blot to detect whether Thaumatin is expressed or not. We found that the expression of Thaumatin in E. coli was not satisfactory after induction by IPTG (Fig 12). During the experiment, we found that E. coli was prone to forming inclusion bodies which made it extremely difficult to extract the protein, so we improved the experimental method. We disrupted the cells, centrifuged them to get the supernatant for Western Blot, but the results still showed that the expression of Thaumatin was extremely low. This indicates that E. coli is not suitable as a chassis for Thaumatin expression and we need to change the expression system.
PC1: BSA (1 μg); PC2: BSA (2 μg); NC: Cell lysate without induction;
Lane 1: Cell lysate with induction for 16 h at 15℃; Lane 2: Cell lysate with induction for 4 h at 37℃;
Lane NC1: Supernatant of cell lysate without induction; Lane 3: Supernatant of cell lysate with induction for 16 h at 15℃;
Lane 4: Supernatant of cell lysate with induction for 4 h at 37℃; Lane NC2: Pellet of cell lysate without induction;
Lane 5: Pellet of cell lysate with induction for 16 h at 15℃; Lane 6: Pellet of cell lysate with induction for 4 h at 37℃.
Ⅱ. Validation of tomato chassis
Agarose gel electrophoresis:
The colonies were amplified by PCR with specific primers and the products were obtained and then subjected to agarose gel electrophoresis. From the results, it can be seen that the band of Thaumatin (719 bp) has a band appearing near 700 bp (Fig 13). This can verify that our transformed Agrobacterium has carried on the target sequence. This is an important milestone and lays the foundation for further experiments.
RNA expression test:
After ensuring that the plasmid was successfully transferred into Agrobacterium GV3101, we amplified Agrobacterium into culture and assembled the virus by inducing it with MMA. Next, we injected the virus into the leaves of four-leaf-old Micro-Tom. After the plants grew up, the leaves were collected for RNA extraction and RT-PCR experiments were performed to verify the transcription of Thaumatin gene in the leaves. According to the results, it can be seen that the RNA band of Thaumatin (719 bp) has a band near 700 bp, which indicates that Thaumatin has begun to be expressed in tomato plants, and we can proceed to the next step of verification (Fig 14).
Protein expression:
We collected leaves, flowers and fruits of TRV virus-infected tomato plants and extracted proteins for WB assay. According to the results, it was clear that tomato was able to express Thaumatin. Based on the immunoenzymatic ELISA for Thaumatin, we concluded that the average level of transient expression of Thaumatin was 11.2873 mg/L in the virus-infested tomato experiment (Fig 15).
(B)Western Blot results for Thaumatin after TRV infection to fruits of tomato.
(C) Detection of Thaumatin in tomato fruits infected with TRV.
Ⅲ. Whole-plant expression
Agarose gel electrophoresis:
To confirm the plasmid was transformed into Agrobacterium, a fragement on hygromycin B resistance gene HygR was amplified by PCR with specific primers and the products were obtained and then subjected to agarose gel electrophoresis. From the results, it can be seen that the amplification result of the fragment (719 bp) has a band appearing near 700bp (Fig 16). This can be verified that our transformed Agrobacterium has carried the target sequence.
(C) Detection of Thaumatin in tomato fruits infected with TRV.
DNA test:
We chose positive bacteria to infect the callus tissue, and when the callus tissue grew to a certain extent, it was inoculated and cultivated in rooting medium (Fig 17), and when the plant grew leaves, we picked the leaves to test whether the plasmid was inseted into the genome of tomato. The results of the analysis showed that HygR DNA could be detected (Fig 18).
Protein expression:
We collected leaf, flower and fruit samples, extracted proteins for Western Blot assay and successfully obtained positive results around 28 kD, indicating successful expression of Thaumatin (28.36 kD) in transgenic Micro-Tom (Fig 19A,B,C). This is the first milestone in our experimental process, indicating that transgenic tomatoes with stable genetic ability to produce sweet proteins have been successfully bred.
Based on the ELISA for Thaumatin, we concluded that the average level of transient expression of Thaumatin in the virus-infested tomato experiment was 11.1598 mg/L (Fig 19D).
plants expressing Thaumatin under the 35S promoter.(D)Thuamatin expression of tomato fruit controlled by 35S promoter.
Ⅳ. Specific expression
Transformation success:
We used specific primers for PCR and proved the success of our expression vector into Agrobacterium GV3101 by agarose gel electrophoresis (Fig 20).
Expression at DNA level:
After the plants grew leaves, we picked the leaves to test whether our genes were present in the genome of tomato. The results of the analysis showed that all of Thaumatin's DNA could be detected (Fig 21).
Expression at protein level:
We harvested fruit samples and extracted proteins for WB assay. Eventually, we only succeeded in detecting the presence of a positive band around 28 kD in the fruit, but not in the leaves and flowers (Fig 22). This result suggests that Thaumatin (28.36 kD) was successfully expressed in our transgenic tomato under the initiation of the E8 promoter.
(A) Protein was examined in transgenic tomato leaf with the E8 promoter.
(B) Protein was examined in transgenic tomato flower with the E8 promoter.
(C) Protein was examined in transgenic tomato fruits with the E8 promoter.
We analyzed the efficacy of the E8 promoter to express proteins. We determined the concentration of Thaumatin expressed with the CaMV 35S promoter and the E8 promoter in transgenic tomatoes by ELISA. By comparison, we obtained that the average level of Thaumatin expressed under the induction of the constitutive promoter 35S was 11.1598 mg/L; while the expression level using the fruit ripening-specific promoter was 11.0591 mg/L (Fig 23). There was no statistically significant difference, indicating that the expression level of the fruit-specific promoter E8 had reached the expression level of the strong promoter CaMV 35S. Compared with 35S, the E8 promoter not only ensures fruit-specific expression and avoids burdening other parts of the tomato plant, but also ensures the high expression level of Thaumatin.
Sweetness test
After verifying the viability of the tomato chassis, we needed to ensure that the protein was structurally and functionally correct. We utilized the human-derived receptor and the electronic tongue to test the sweetness of Thaumatin.
Ⅰ. Improved ELISA for sweetness measurement
In order to use this principle to determine whether the sweetness protein in tomato can bind to the human receptor protein T1R2, we innovatively proposed a “double sandwich” method. Firstly, T1R2 is immobilized on a solid-phase carrier carrying an antibody against T1R2, and then a protein sample is added to allow Thaumatin to bind to T1R2, which is the key to our innovation. Next, we use an HRP enzyme-labeled secondary antibody to bind specifically to the already bound Thaumatin. Finally, we added the enzyme substrate TMB, which catalyzes a chromogenic reaction of the substrate, as a means of determining whether the sweet protein produced by tomato can bind to T1R2. As a control, we incubate a Thaumatin standard carrying the HRP enzyme as a secondary antibody on a solid-phase carrier and then use TMB for color development.
Based on this principle, we can give a judgment criterion: if the wells turn yellow with the addition of the substrate TMB, the Thaumatin in the sample can bind to T1R2, indicating that the protein has a sweet taste.
Our result allows us to determine whether the Thaumatin protein can bind to each other with the human-derived receptor protein T1R2 (Fig 24). However, our result from this method cannot reflect the sweetness index of the sample, this is because we cannot get the connection between absorbance and sweetness index. Finally, we were also unable to estimate the sweetness index of the tomato samples. Therefore, we further explored the sweetness detection method.
Ⅱ. Electronic tongue detects sweetness
Model construction
Before the test, we used the electronic tongue to establish our own sweetness model. Since the sweet protein Thaumatin is a protein substance rather than a common sugar, we referred to the concentration-response curve of sweeteners, conducted a large number of preliminary experiments, and set up Thaumatin tomato standard solutions with different concentration gradients (0%, 0.5%, 1%, 1.5%, 2%) to establish SVM sweetness models (parameters all set to 1, 2, 3, 4, 5).
SVM Model Analysis
We conducted electronic tongue detection on the control group tomatoes (i.e., uninfected wild Micro-Tom tomatoes), transgenic tomatoes with the 35S promoter, and transgenic tomatoes with the E8 promoter. Subsequently, the taste characteristics detected by the electronic tongue were input into the SVR model for analysis. According to the model prediction analysis and calculating the average value of the data, the prediction result of the wild type is 0.0109, indicating that the tomato sample of the control group is equivalent to the standard Thaumatin tomato solution with a concentration of 0 ppm, further demonstrating the usability of our model. The result of transgenic tomatoes with the 35S promoter is 21.6013, that is, the Thaumatin contained therein is equivalent to the standard Thaumatin solution with a concentration of 21.6013 ppm. By the same reasoning, it can be concluded that the Thaumatin in tomatoes with the E8 promoter is equivalent to the standard Thaumatin solution with a concentration of 21.6040 ppm.
Subsequently, according to the concentration-response relationship curve of Thaumatin established by Grant E. DuBois and D. Eric Walters, it can be calculated that the sweetness level of Thaumatin in transgenic plants containing the 35S promoter is 8.65, and the sweetness level of Thaumatin in transgenic tomatoes containing the E8 promoter is also 8.65.
Finally, according to the calculation based on the concentration-sweetness level of sucrose, it can be known that Thaumatin in transgenic tomatoes is equivalent to the standard sucrose solution with a concentration of 8.65%.
Metabolic detection
Heterologously expressed proteins may have an impact on the metabolism of chassis organisms. Considering the final product and safety of our project, we hope to determine whether the expression of Thaumatin affects the normal physiological metabolism of tomatoes through detection related to tomato metabolism. Following the research principle from macroscopic to microscopic, we carried out apparent detection, glucose concentration analysis and mass spectrometry analysis respectively.
Apparent detection
We compared the plant appearance and leaf appearance of tomato plants in different groups, and measured their plant height, leaf length and fruit weight (Fig 27). Through statistical analysis, we concluded that there was no significant difference among different groups of tomato plants in the three apparent traits (Fig 28), indicating that the heterologous expression of producing sweet protein in the tomato chassis has no significant negative impact on the plant height, leaf and fruit growth of tomato plants.
(a) Data on fruit weight of tomato plants with different expression vectors in each group;
(b) Data on leaf length of tomato plants with different expression vectors in each group;
(c) Data on plant height of tomato plants with different expression vectors in each group.
(ns P > 0.05; * P < 0.05;** P < 0.01;P < 0.001;P < 0.0001).
Glucose concentration analysis
Glucose is an important indicator of plant physiological metabolism and participates in the metabolic processes within and between plant cells. In our project, the content of glucose affects the sweetness of tomatoes and has an impact on the production of the final product. Therefore, we need to compare the content of glucose between experimentally treated tomato plants and wild plants to test whether the heterologously expressed Thaumatin has an impact on tomato glucose metabolism. We selected wild-type tomatoes and experimentally treated tomatoes with the same growth time and similar growth states for comparison and conducted experiments using a glucose concentration detection kit. According to the result analysis, there is no significant difference in glucose content between the experimental group and the control group(Fig 29).
Storage location verification
We used tobacco leaves as rapid verification experimental materials. The Thaumatin with EGFP guided by the vacuole localization peptide SPS-NTPP was infected into tobacco leaves by using an Agrobacterium vector. The observation results of confocal microscopy imaging showed that there were clustered green spots at the vacuole positions of tobacco leaf cells, proving that our SPS-NTPP protein can be normally expressed and function in tobacco cells (Fig 30).
In order to further verify the feasibility of the SPS-NTPP localization peptide in tomato fruits, and considering the complexity of transgenic plant cultivation, after discussion by the team, we decided to first conduct a pre-experiment of infecting leaves in tomatoes. We used Agrobacterium to infect tomato leaves with Thaumatin carrying the SPS-NTPP localization peptide and green fluorescent protein, and then continued to observe the feasibility of the localization peptide under confocal microscopy. From the figure, it can be analyzed that green fluorescent spots are clustered in the cells, and red spots are widely distributed in various positions of the cells. The green spots are caused by green fluorescent protein, while the red spots are caused by chloroplasts. Because when we designed the plasmid, we co-expressed and combined green fluorescent protein with Thaumatin, so the green fluorescent spots we see now represent the aggregation of Thaumatin.(Fig 31) Secondly, we see that the chloroplasts of red spots are clustered around the green spots without mixing to produce yellow light, which proves that they are stored separately. Because chloroplasts exist in the cell matrix, and most of the remaining part is vacuoles, we can preliminarily infer that the heterologously expressed SPS-NTPP localization peptide in tomatoes is effective. This verifies the rationality of expressing and localizing Thaumatin in tomato fruits.
Proof of concept: quality enhancement
Natural Thaumatin has a licorice aftertaste, which negatively affects the taste in practical applications. To meet the sensory sweetness demands in food, we optimized the structure of Thaumatin. Through visualization analysis and molecular docking simulations, we conducted a preliminary exploration, aiming to provide theoretical support and useful data for improving the quality of Thaumatin.
Structure simulation
We used AlphaFold to predict the structures of the human receptors T1R2 and T1R3, as well as Thaumatin, obtaining protein structure models.
PPI Prediction
Sweetness
We predicted the binding of Thaumatin to the sweetness receptors T1R2 and T1R3 by alphafold to identify the sites where they bind. This work helps us detect the sweetness of Thaumatin.
Bitterness
In our research, we found that Thaumatin can bind to the bitter taste receptor T2R16, so we need to perform PPI simulations of Thaumatin and T2R16 to identify the bitter taste site. This will help us to modify Thaumatin, thus improving its taste and quality.
Considering that there is no decomposition of Thaumatin in the oral cavity, we used alphafold to predict the docking of intact Thaumatin and T2R16 to obtain three bitter sites that exhibit the periphery of Thaumatin.
The protein-protein docking score between Thaumatin and T2R16 is -287.
The docking score was calculated using the knowledge-based iterative scoring functions ITScorePP or ITScorePR. A more negative docking score indicates a more likely binding model, with typical docking scores for protein-protein/DNA/RNA complexes around -200.
The residues on Thaumatin involved in hydrogen bond formation are LYS-49, GLU-89, CYS-158, and THR-190. Among them, the hydrogen bonds formed by LYS-49, GLU-89, and CYS-158 are relatively strong. However, considering that LYS-49 is located on the bitter peptide, we have decided to exclude it from mutation.
Directed Evolution
After excluding the site on the bitter peptide, LYS-49, we identified three mutation sites: GLU-89, CYS-158, and THR-190. We mutated these sites to alanine (Ala), a small, non-polar amino acid with a minimal side chain (-CH₃). Alanine is commonly used in protein mutagenesis because its simple structure allows for easier evaluation and analysis when designing mutants. In certain cases, mutating to alanine does not significantly affect the protein's three-dimensional structure.
Through calculation and comparison, the protein-protein docking score between Thaumatin and T2R16 is -284.9. So, the CYS-158 site of Thaumatin was mutated to reduce the bitter taste and optimize the taste of Thaumatin.
However, since mutated proteins do not comply with laws and regulations and cannot be put into production use without testing. In order to put Sweetein into production use as soon as possible, the protein-directed evolution part of the protein is currently only for proof-of-concept and has not been put into use for the time being in our project, and we look forward to putting the optimized Thaumatin into the market in the future after a reasonable evaluation to gain popular acceptance.
Application Prospects
The automatic production and storage of sugar substitutes from fruits, followed by a simple juicing operation to obtain nutritive sweeteners, can be easily applied to beverages, confectionery, pharmaceuticals, sweets, and other foodstuffs.
Our product 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 are expected to replace the excess sugar in fruits with Thaumatin by means of synthetic biology, so that most of the fruits that are overloaded with sugar can be consumed by people with diabetes and obesity.
Reference
[1]Saraiva, A.; Carrascosa, C.; Raheem, D.; Ramos, F.; Raposo, A. Natural Sweeteners: The Relevance of Food Naturalness for Consumers, Food Security Aspects, Sustainability and Health Impacts. Int. J. Environ. Res. Public Health 2020.
[2]The artificial sweetener erythritol and cardiovascular event risk, February 2023.
[3]Inglett, G.E., May, J.F. Tropical plants with unusual taste properties. Econ Bot 22, 326-331.1968.
[4]Differential subcellular targeting of recombinant human a1-proteinase inhibitor influences yield, biological activity and in planta stability of the protein in transgenic tomato plants,Plant Science,Volume 196, November 2012.
[5]Transgenic Plants as Producers of Supersweet Protein Thaumatin II, Living reference work entry, 19 August 2016.
[6]TMasuda T, Okubo K, Baba S, Suzuki M, Tani F, Yamasaki M, Mikami B. Structure of thaumatin under acidic conditions: Structural insight into the conformations in lysine residues responsible for maintaining the sweetness after heat-treatment. Food Chem. 2022 Sep 30;389:132996. doi: 10.1016/j.foodchem.2022.132996. Epub 2022 Apr 18.
[7]de Jesús-Pires C, Ferreira-Neto JRC, Pacifico Bezerra-Neto J, Kido EA, de Oliveira Silva RL, Pandolfi V, Wanderley-Nogueira AC, Binneck E, da Costa AF, Pio-Ribeiro G, Pereira-Andrade G, Sittolin IM, Freire-Filho F, Benko-Iseppon AM. Plant Thaumatin-like Proteins: Function, Evolution and Biotechnological Applications. Curr Protein Pept Sci. 2020;21(1):36-51. doi: 10.2174/1389203720666190318164905. PMID: 30887921.
[8]Ohta K, Masuda T, Tani F, Kitabatake N. The cysteine-rich domain of human T1R3 is necessary for the interaction between human T1R2-T1R3 sweet receptors and a sweet-tasting protein, thaumatin. Biochem Biophys Res Commun. 2011 Mar 18;406(3):435-8. doi: 10.1016/j.bbrc.2011.02.063. Epub 2011 Feb 15. PMID: 21329673.
[9]Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113-30. doi: 10.1016/0022-2836(86)90385-2. PMID: 3537305.
[10]Hirai T, Kim YW, Kato K, Hiwasa-Tanase K, Ezura H. Uniform accumulation of recombinant miraculin protein in transgenic tomato fruit using a fruit-ripening-specific E8 promoter. Transgenic Res. 2011 Dec;20(6):1285-92. doi: 10.1007/s11248-011-9495-9. Epub 2011 Feb 27. PMID: 21359850.
[11]Differential subcellular targeting of recombinant human a1-proteinase inhibitor influences yield, biological activity and in planta stability of the protein in transgenic tomato plants,Plant Science,Volume 196, November 2012.
[12]Joseph JA, Akkermans S, Nimmegeers P, Van Impe JFM. Bioproduction of the Recombinant Sweet Protein Thaumatin: Current State of the Art and Perspectives. Front Microbiol. 2019 Apr 8;10:695. doi: 10.3389/fmicb.2019.00695. PMID: 31024485; PMCID: PMC6463758.
[13]Witkowski, M., Nemet, I., Alamri, H. et al. The artificial sweetener erythritol and cardiovascular event risk. Nat Med (2023).
[14]X. Li, L. Staszewski, H. Xu, K. Durick, M. Zoller, E. Adler, Human receptors for sweet and umami taste,PNAS, 99 (2002).
[15]A.N. Pronin, H. Tang, J. Connor, W. Keung, Identification of ligands for two bitter T2R receptors, Chem. Senses, 29 (2004).
[16]Key Amino Acid Residues Involved in Multi-Point Binding Interactions between Brazzein, a Sweet Protein, and the T1R2-T1R3 Human Sweet Receptor. Author links open overlay pane, Fariba M. Assadi-Porter,Journal of Molecular Biology Volume 398, Issue 4, 14 May 2010.
[17]Refolding the sweet-tasting protein thaumatin II from insoluble inclusion bodies synthesised in Escherichia coli.Food Chemistry Volume 71, Issue 1, October 2000.
[18]Maat, M.Y. Toonen, C. Visser, C.T. Verrips Synthesis and processing of the plant protein thaumatin in yeastCell, 37 (1984).
[19]L. Edens, H. Van der Wel, Microbial synthesis of the sweet-tasting plant protein thaumatin Trends in Biotechnology, 3 (1985).
[20]Matsuoka K, Nakamura K. Propeptide of a precursor to a plant vacuolar protein required for vacuolar targeting. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):834-8. doi: 10.1073/pnas.88.3.834. PMID: 1992474; PMCID: PMC50908.
| None |