Part:BBa_K5160004
Brazzein
Overview
When selecting sweet proteins, we noticed Brazzein, a sweet protein that is comparable to Thaumatin. However, due to its lack of safety certification, it has not been able to enter the market for public consumption. Nevertheless, we have characterized it during the experimental stage and filled out the relevant part of the form, hoping that it will be widely used in the future. Brazzein is derived from the Pentadiplandra brazzeana Baillon (P. brazzeana) plant, which is found in the tropical rainforests of Africa. It is a sweet-tasting protein with strong thermal stability and acid resistance. It can bind to the T1R2 and T1R3 sweet taste receptors on the human tongue, triggering a sweet sensation. Furthermore, it can be completely digested by the human body into common amino acids for absorption, and this process generates almost no calories. It is evident that Brazzein is a promising sweet protein. We have provided information about Brazzein here and hope to inspire and offer suggestions for future iGEM projects.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 133
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 133
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 133
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 133
- 1000COMPATIBLE WITH RFC[1000]
Biology
Brazzein is derived from the pulp of Pentadiplandra brazzeana Baillon (P. brazzeana), which grows in the tropical rainforests of Africa. Each fruit features a red, shell-like epicarp, beneath which are three to five kidney-shaped seeds surrounded by a thick layer of soft, red pulp that contains Brazzein. Brazzein is the smallest sweet-tasting protein, with its peptide chain consisting of 54 amino acids and a molecular weight of only 6.5 kDa. In mature fruits, the content of Brazzein ranges from 0.05% to 0.2% by weight. It exists in three forms: Type I Brazzein, Type II Brazzein, and Type III Brazzein. Type I Brazzein accounts for 80% of the total Brazzein and contains pyroglutamic acid at the N-terminus; Type II Brazzein has glutamine at the N-terminus, which subsequently converts to pyroglutamine; and Type III Brazzein lacks the N-terminal glutamine (or pyroglutamic acid).
Brazzein triggers the human sense of sweetness by binding to the T1R2 and T1R3 sweet taste receptors on the tongue. Meanwhile, Brazzein's flavor profile is similar to sucrose, but its sweetness intensity is 500-2000 times that of sucrose. This significant sweetness is attributed to the multi-site binding of Brazzein to the sweet taste receptors. The primary interaction sites between Brazzein and the sweet receptors are Loop43 (site 1) and the N-terminal and C-terminal regions (site 2). Additionally, the nearby Loop33 and Loop9-19 (site 3) make significant contributions to the sweetness of Brazzein.
Besides, Brazzein is a protein with excellent thermal stability and acid resistance. Due to its simple structure and four stable disulfide bonds, Brazzein exhibits high tolerance to high temperatures and extreme pH levels. It can maintain its protein properties after incubation at 98°C for 2 hours, at 80°C for 4.5 hours, and within a pH range of 2.5-8 at 80°C for 4 hours. Meanwhile, it also demonstrates outstanding thermal stability across a wide range of pH values. Furthermore, it is non-toxic and non-allergenic, and has been consumed by indigenous people in Africa for thousands of years.
In conclusion, Brazzein is an excellent sugar substitute.
Design
Gene sequence
In the literature, we found the gene sequence for heterologous expression of Brazzein. Subsequently, we performed a BLAST search on NCBI and found that it retrieved only one result. Therefore, we obtained the complete sequence for heterologous expression of Brazzein.
Pathway Design
Prokaryotic Expression
In order to effectively characterize Brazzein, we made a series of attempts and efforts. Firstly, we conducted initial trials in Escherichia coli. We chose the most common strain, E. coli BL21 (DE3). The target sequence we designed was induced by the T7 promoter and ligated with the plasmid pET-28(+) to construct our expression vector. However, since E. coli is a prokaryote, it cannot correctly express eukaryotic products such as Brazzein. Additionally, heterologous expression of proteins in E. coli often results in the formation of inclusion bodies. Therefore, our initial attempt was not ideal, and we were unable to correctly express properly folded Brazzein.
Plant Expression
Subsequently, we shifted our focus to plant production systems and made corresponding modifications to both the genetic pathway and the host plant. Among various options, we noticed tomatoes, a crop with high nutritional value, significant market economic value, and ease of cultivation. In our exploration process, we first used a modified TRV virus designed for cloning to attempt transient expression. Transient expression is an effective method for verifying the usability of plant expression vectors and can shorten the verification time. Therefore, we cloned the target sequence of Brazzein onto the TRV2 gene of the TRV virus and induced its expression under the 35S promoter. We then injected the TRV vector into Agrobacterium for amplification and subsequently into tomato leaves for validation. After completing this series of work, we found that the tomato plant host was indeed capable of correctly folding and expressing the sweet protein Brazzein, indicating that the tomato plant host is a good choice.
Afterwards, we achieved GMO (genetically modified organism) whole-gene expression in tomatoes. Transgenic technology can introduce exogenous gene sequences into the host's genome and cause heritable changes in biological traits. We utilized the integration capability of the Agrobacterium Ti plasmid to introduce the target gene into the callus tissue of tomatoes, achieving heterologous whole-plant expression. Brazzein was also correctly expressed in the transgenic tomatoes. However, we subsequently found that even under the induction of the strong 35S promoter, the yield of Brazzein in transgenic tomatoes was far from meeting production demands. Therefore, in order to increase the expression level of Brazzein in tomatoes and reduce its degradation, we conducted a new round of exploration and designed a new synthesis system and storage system.
During discussions with plant biology experts, we noted that the non-fruit parts of tomatoes, such as leaves and stems, are not popular for consumption. Therefore, to reduce the metabolic burden on these non-fruit parts and avoid expression inhibition caused by DNA methylation in tomatoes, we used the tomato endogenous fruit-specific expression promoter E8. We inserted the Brazzein gene downstream of the E8 promoter and transformed it into Agrobacterium, which was then injected into tomatoes for validation.
In summary, we designed the expression of Brazzein in tomatoes through transgenic technology. Under the induction of the E8 promoter, Brazzein was successfully expressed in high quantities of tomatoes.
Construction
Prokaryotic Expression
We selected the most commonly used strain, Escherichia coli BL21 (DE3), for expression verification. Our target gene sequence is induced by the T7 promoter and ligated to the plasmid pET-28a (+) to construct the expression vector. Subsequently, we transformed the vector into E. coli BL21 (DE3) and picked monoclonal colonies for expanded culture in Kan-containing medium.
Plant Expression
We constructed the TRV2-35s-Brazzein plasmid using the Tobacco rattle virus (TRV). After amplification in Agrobacterium tumefaciens GV3101, the plasmid was used to infect tomato plants for verification of expression.
Whole-plant Expression of GMO (Genetically Modified Organism)
Subsequently, we constructed the 35S_Brazzein plasmid on the binary vector pBWA(V)HS and transformed it into Agrobacterium tumefaciens GV3101 for infection and verification of expression in tomato plants.
Fruit-specific Expression
By utilizing the E8 promoter, a fruit ripening-specific promoter, we constructed the pCAMBIA1301_Brazzein plasmid. This plasmid was then transformed into Agrobacterium tumefaciens GV3101 for subsequent infection and verification of expression in tomato plants.
Results
1. Prokaryotic Expression
Protein Verification
We constructed the Brazzein:pET-28(+) vector and placed the strong promoter 35S in front of the open reading frame. The hope was to enhance the expression level of the plasmid. Subsequently, we used whole-genome synthesis technology to transform the aforementioned expression vector into Escherichia coli BL21 (DE3), picked single colonies from kanamycin (Kan) containing medium for amplification, and extracted bacterial protein for Western Blot (WB) assay to detect the sweet protein. We found (Figure 10), after IPTG induction, the ability of E. coli to express Brazzein was not very satisfactory.
2. Plant Expression
DNA Verification
Colony PCR Identification
We constructed the TRV2_Brazzein plasmid using the TRV2 vector, and then transformed the constructed plasmid along with TRV1 into Agrobacterium GV3101 for induced expression. Afterwards, we selected single colonies of Agrobacterium and performed colony PCR identification. From the result figure (Figure 11), it can be seen that Brazzein (176bp) has a distinct band near 100bp, thereby verifying that our transformed Agrobacterium has carried the target sequence and has the capability to express RNA, which plays a very important role in our further experiments that follow.
RNA Verification
Subsequently, we selected the micro-Tom variety of cherry tomatoes as our chassis. We then infected tomato leaves with Agrobacterium, and as described above, we first checked the RNA in the tomato leaves and performed RT-PCR. From the result figure (Figure 12), it can be seen that Brazzein (176bp) has a distinct band near 100bp. This indicates that the virus-infected tomato plants have begun to express the sweet protein. This experimental result is of milestone significance for the expression of sweet proteins in plant chassis and has laid the foundation for our further validation experiments at the protein level.
Protein Expression
We collected leaves and fruits from tomato plants infected with the TRV virus using the 35S promoter, and extracted proteins for Western blot (WB) detection. The results (as shown in the figure below) indicated that Brazzein was correctly expressed in tomatoes.
GMO Whole-Plant Expression
Colony PCR
We attempted expression using the eukaryotic 35S promoter, constructed the pBWA(V)HS_Brazzein plasmid, transformed it into Agrobacterium GV3101, and performed PCR screening on the bacteria. Then, we used the positive bacteria to infect callus tissue. Afterwards, we selected single colonies of Agrobacterium and carried out colony PCR identification. From the result figure (Figure 15), it can be seen that Brazzein (176bp) has a distinct band near 100bp, thereby verifying that our transformed Agrobacterium has carried the target sequence and has the capability to express RNA, indicating that the tomato chassis is usable.
Seedling DNA
We then sampled the leaves for DNA extraction and performed PCR, successfully obtaining positive results (Figure 16). This marks the first milestone in our experimental process, indicating that the genetically modified tomatoes with the stable inheritance capability to produce sweet proteins have been successfully cultivated.
Protein Verification
We attempted expression using the eukaryotic 35S promoter, constructed the pBWA(V)HS_Brazzein plasmid, transformed it into Agrobacterium GV3101, and then used the positive bacteria to infect callus tissue. We then collected samples of leaves, flowers, and fruits, extracted protein, and performed Western Blot (WB) detection. As shown in the results (Figures 17, 18, 19), we obtained positive results. This marks the first milestone in our experimental process, indicating that the genetically modified tomatoes with the ability to stably inherit and produce sweet proteins have been successfully cultivated.
Fruit-specific Expression
We inserted the gene brazzein downstream of the E8 promoter to construct the pBWA(V)HS_Brazzein plasmid, which was then transformed into Agrobacterium GV3101. Positive bacteria were then used to infect callus tissue. We then collected samples of leaves, flowers, and fruits, extracted protein, and performed Western Blot (WB) detection. As shown in the results (Figures 20, 21, 22), we obtained positive results. This proves that the E8 promoter can correctly express Brazzein in tomatoes.
Application Prospects
Considering that Brazzein has not yet obtained FDA safety certification and cannot be provided to the public as a food product, we have merely considered the potential applications of Brazzein in the future. We hope that this information can provide inspiration and assistance to interested iGEM teams, facilitating the formation of their projects.
Firstly, given the favorable properties of Brazzein, we hope to accelerate its safety approval processes, such as nutritional evaluations and pathological assessments, thereby promoting its faster entry into the market. Secondly, we anticipate that Brazzein will find widespread and innovative applications in the food industry. In our project, we have provided a plan for an innovative application of Brazzein. We innovatively proposed a tomato-based sugar substitute production system. By utilizing the E8 promoter, a fruit ripening-specific promoter, we aim to achieve mass production of Brazzein in tomatoes, addressing the issue of low yield due to its plant origin and meeting market demands. In our design, Brazzein can be directly extracted from tomato juice without the need for complex extraction processes. This not only avoids industrial pollution but also ensures that Brazzein is natural and harmless.
We believe that as a new type of sweetener, Brazzein has a bright future and can create a richer world of sweetness, benefiting more people.
Reference
[1] Assadi-Porter FM, Maillet EL, Radek JT, Quijada J, Markley JL, Max M. Key amino acid residues involved in multi-point binding interactions between brazzein, a sweet protein, and the T1R2-T1R3 human sweet receptor. J Mol Biol. 2010 May 14;398(4):584-99. doi: 10.1016/j.jmb.2010.03.017. Epub 2010 Mar 17. PMID: 20302879; PMCID: PMC2879441.
[2] Markova EV, Leonova EI, Sopova JV. [Sweet protein brazzein as a promising sweetener]. Vopr Pitan. 2024;93(1):61-71. Russian. doi: 10.33029/0042-8833-2024-93-1-61-71. Epub 2024 Jan 19. PMID: 38555610.
[3] Saraiva A, Carrascosa C, Ramos F, Raheem D, Pedreiro S, Vega A, Raposo A. Brazzein and Monellin: Chemical Analysis, Food Industry Applications, Safety and Quality Control, Nutritional Profile and Health Impacts. Foods. 2023 May 10;12(10):1943. doi: 10.3390/foods12101943. PMID: 37238762; PMCID: PMC10217172.
[4] 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.
[5] Yoo SY, Bomblies K, Yoo SK, Yang JW, Choi MS, Lee JS, Weigel D, Ahn JH. The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene. Planta. 2005 Jun;221(4):523-30. doi: 10.1007/s00425-004-1466-4. Epub 2005 Jan 29. PMID: 15682278.
[6] Singh AK, Ghosh D, Chakraborty S. Optimization of Tobacco Rattle Virus (TRV)-Based Virus-Induced Gene Silencing (VIGS) in Tomato. Methods Mol Biol. 2022;2408:133-145. doi: 10.1007/978-1-0716-1875-2_9. PMID: 35325421.
| None |