Overview
This year, the SZU-China team decided to undertake a project that combines classical molecular biology techniques, foodomics, and innovative approaches based on synthetic biology to successfully express two sweet proteins, Thaumatin and Brazzein, in tomatoes. To achieve this, knowledge from different scientific fields was employed, including molecular biology, biochemistry, food science, and computer science.
On this page, you can find all the results for the production, testing, and localization of our sweet protein product, while also noting that the experimental page provides analytical information on the strategies used to carry out each step.
Protein Expression
E. coli Trial
Escherichia coli, as one of the most common prokaryotes in the research field, has many advantages suitable for protein expression. Therefore, we first chose E. coli as the tool for the trial expression of sweet proteins.
Initially, our team inserted the Thaumatin(~23.02 kD) and Brazzein(~8.67 kD) genes into the pET28a[6] vector to construct a plasmid, 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 E. coli BL21 (DE3), picked single colonies in a Kan (kanamycin)-containing medium for expansion and extracted bacterial proteins for Western Blot (WB) experiments to detect sweet proteins. Since expressing recombinant proteins in E. coli tends to form inclusion bodies[1], we added protein extraction buffer to 4 ml of bacterial culture for lysis, and after centrifugation, we took both the supernatant and the pellet for Western Blot. It was found that both Thaumatin and Brazzein were expressed in E. coli, but the expression levels were low (Fig 1), which may be due to the limitations of the E. coli prokaryotic expression system and the formation of inclusion bodies. This experiment can prove that the sweet proteins expressed by E. coli are not effective, hence we need to optimize the choice of the chassis.
At the same time, in the E. coli experiments, considering the time and difficulty, we chose a crude extraction method. However, the challenges in protein purification operations made us realize that using microorganisms as a chassis not only increases production costs but also raises safety risks[2]. This prompted us to consider using a safer model organism as a sweet protein factory. If an organism is already commonly consumed in people's daily lives, then this chassis would undoubtedly save us the trouble of purification and dispel safety concerns[3]. Thus, it naturally occurred to us to further attempt using the widely loved cherry tomatoes(For more reasons to choose tomatoes, please see the Design page).
TRV Infection Trial
The most common method of genetic modification in plants is Agrobacterium infection, so we only need to select the appropriate plasmid vector and a suitable tomato variety as a chassis to achieve the addition of sweet protein genes in tomatoes. We ultimately chose the Micro-Tom and TRV vectors (for details, see the Outline page).
In order to utilize the TRV vector to introduce the sweet protein gene into Micro-Tom for expression, we first constructed the TRV2_Thaumatin and TRV2_Brazzein plasmids using the TRV2 vector.
Subsequently, we transformed the well-constructed plasmids, along with TRV1, mCherry, and P19 (for details, see the Outline webpage), into Agrobacterium GV3101 as the experimental group. The control group differed only by the absence of the TRV2 plasmid, and Agrobacterium PCR was also performed, yielding positive results (Fig 2), indicating that our plasmids had been successfully transferred into Agrobacterium GV3101.
We selected Micro-Tom among numerous varieties of cherry tomatoes as the chassis system (for details, see the Outline page). After inducing Agrobacterium-mediated expression, we infected tomato leaves with Agrobacterium solution, making small incisions on the abaxial side of the leaves with a syringe needle, taking care to avoid the veins, and then injected the bacterial solution using a needleless syringe. Subsequently, to verify the expression of sweet proteins in the tomato leaves, we harvested the fifth leaf from the bottom of the plant, extracted RNA from a portion of the leaf tissue for RT-PCR, and obtained positive results (Fig 3).
Another part was subjected to Western blot (WB) assay to detect the expression at the protein level, and bands of Thaumatin with a 3x Flag tag (28.10 kD) and Brazzein with a 3x Flag tag (9.48 kD) were successfully detected near 28 kD and 9 kD, respectively (Fig 4). This suggests that our sweet protein genes can be expressed in Micro-Tom plants.
To ensure that our sweet proteins can also be expressed in the fruit of the tomato, we collected red fruits at the appropriate time for protein extraction and performed Western blot analysis, obtaining the same positive results (Fig 5). These experiments indicated that our sweet proteins can not only be expressed in Micro-Tom plants but also successfully expressed in the fruit. This outcome is exhilarating and represents the second milestone in our experimental progress, laying the foundation for our subsequent experiments. It proves that expressing sweet proteins in plant chassis is a feasible approach. The results encouraged us to continue constructing a more stable expression system in tomatoes.
Transgenic tomato 1.0
Genetic modification technology, as a method of introducing target genes into the genome of an organism to cause heritable changes in biological traits, has long been used not only as a traditional breeding method but also plays a significant role in the production of products by bioreactor systems.
In order to make our protein expression system heritable, thereby enabling the production of sweet proteins on a larger scale to meet potential societal demands, we continued to update and iterate, using Agrobacterium transformation to make Micro-Tom a stable sweet protein factory[4].
We initially attempted expression using the eukaryotic promoter 35S, constructing two plasmids, pBWA(V)HS_Thaumatin and pBWA(V)HS_Brazzein. These were transformed into Agrobacterium GV3101 and subjected to PCR screening for the neomycin resistance gene within the bacteria (Fig 6).
Positive bacteria were then used to infect wounded tissue (Fig 7A), and the excised explants were placed on co-cultivation medium and incubated in the dark for 2 days. Once the differentiated seedlings grew to approximately 2-3 cm, the recultured callus was transferred to selection medium and cultivated under the same conditions for 15-30 days. The selected callus was then inoculated onto differentiation medium and cultured under the same conditions for 30-40 days. When the differentiated seedlings reached a height of about 2-3 cm, they were excised from the callus and inoculated onto root induction medium, where they were cultured under the same conditions for an additional 10-15 days to encourage root formation (The process from pre-cultivation of tomato seedlings to post-infection differentiation and rooting is illustrated in Fig 8).
(A) Callus tissue infection. (B) Select positive seedlings with hygromycin.
to post-infection differentiation and rooting
Then we sampled the leaves to extract DNA for PCR, successfully obtaining positive results(Fig 9). Subsequently, we selected some plant leaves to extract proteins for Western Blot (WB) detection and successfully obtained positive results near 28 kDa and 10 kDa(Fig 10), indicating that Thaumatin (28.36 kD) and Brazzein (9.75 kD) were successfully expressed in the transgenic Micro-Tom. This marks the second milestone in our experimental process, suggesting that the transgenic tomato with the ability to stably inherit and produce sweet proteins has been successfully cultivated.
(A) 35S-Thaumatin-3x HA,performing PCR with hygromycin as the
target sequence. Screening 1-33, positive: 1-16, 20, 22-25, 28-33. Positive rate: 27/33 = 82%;
(B) 35S-Brazzein-3x HA,performing PCR with hygromycin as the target sequence. Screening 1-30, positive: 1-14, 16-23, 25, 27-30. Positive rate: 27/30 = 90%.
Although we are well aware that the characteristic of the 35S promoter to express in any part of the plant is not conducive to the production efficiency of sweet proteins. And due to time constraints, we have already begun to conceive and design a fruit-specific expression module. But this does not mean that these 35S transgenic tomatoes have lost their significance. Firstly, the ability to express in leaves does not guarantee successful expression in flowers and fruits. Secondly, these tomatoes not only allow us to become familiar with the operations of a series of experiments on the expression and detection of transgenic plant proteins in advance, but also serve as very suitable control groups, providing a standard for comparison with the results of our subsequent fruit-specific expression experiments. Considering the above factors and time constraints, we still took meticulous care of these plants, closely monitored the plants with positive leaf WB results. At the appropriate time, we collected and flowers and fruits to extract proteins for WB experiments and screen the results (Fig 11), preserving the samples corresponding to the positive bands at -20°C for use as positive controls in subsequent experiments.
Transgenic tomato 2.0
The E8 promoter is one of the most widely characterized and mature fruit-specific tomato promoters, exhibiting strong conservation across different tomato varieties[5][7][9][14]. To achieve efficient and specific expression of sweet proteins in the fruit, we inserted the genes thaumatin and brazzein downstream of the E8 promoter, constructing two plasmids, pBWA(V)HS_Thaumatin and pBWA(V)HS_Brazzein. These were then transformed into Agrobacterium GV3101 and subjected to PCR experiments and obtained positive results(Fig 12) .
We then conducted a tomato transgenic experiment following the same steps as Transgenic Tomato 1.0, and after a period of cultivation, we extracted DNA from the leaves for PCR. The majority of the results were positive, indicating that the target sequence has been successfully integrated into the Micro-Tom plant (Fig 13).
(A) E8-Thaumatin,performing PCR with hygromycin as the target sequence. Screening 1-10, positive: 2, 5-10. Positive rate: 7/10 = 70%.
(B) E8-Brazzein,performing PCR with hygromycin as the target sequence. Screening 1-37, positive: 1-3, 5-7, 9-11, 13, 15-21, 23-34, 36. Positive rate: 30/37 = 81%.
Additionally, we extracted protein from the leaves to verify the fruit-specific expression function of the E8 promoter. We performed Western blot (WB) with E8-Thaumatin (26.20 kD) and E8-Brazzein (7.58 kD) and compared the results with those obtained from the 35S promoter in Transgenic Tomato 1.0 (Fig 16,17). The results showed that the target genes downstream of the E8 promoter were not expressed in the leaves.
To make this difference more pronounced, we specifically tried Western blot with samples of transgenic tomato leaves containing 35S-Thaumatin (28.36 kD) and E8-Thaumatin (26.20 kD) together, and the results showed that the 35S samples had a clear band near 28 kD, while the E8 samples did not show a clear band near 26 kD (Fig 14A,B), again indicating that the E8 promoter does not initiate the expression of the target genes downstream in the leaves .
Flowers are another major important organ of plants. Thus, to ensure our experiment is more comprehensive and the results are convincing and valuable for reference, we also collected tomato flowers at the appropriate time to extract protein for WB experiments(Fig 14C,D), reaching the same conclusion as the leaf experiments .
Since the fruit is our ultimate goal, we naturally collected the red fruits expressing Thaumatin and Brazzein, and similarly extracted protein for WB experiments. Due to the growth cycle of tomatoes, our E8-Thaumatin fruits are still scarce, resulting in a small sample size. However, the results showed that both had distinct bands at around 26-30 kD and 10 kD(Fig 15), respectively, which could already indicate that the E8 promoter can effectively initiate the expression of downstream genes in the fruit.
(A) E8-Thaumatin-1x HA. The target genes downstream
of the E8 promoter were expressed in the fruits.
(B) E8-Brazzein-1x HA. The target genes downstream
of the E8 promoter not expressed in the flowers.
Through comprehensive experiments, after multiple validations and comparisons, we began with the simple recombination expression of sweet proteins, achieved a more natural and safer production environment through the replacement of chassis, optimized the stable and heritable expression of sweet proteins through transgenic methods and improved the utilization rate of sweet proteins by iterating the promoter to achieve fruit-specific expression. Each step has been systematically detected at the molecular level, ultimately achieving the natural, heritable, and specific expression of sweet proteins in Micro-Tom!
Sweetness Detection
To further test the performance of the sweet proteins produced by our natural plant chassis, tomatoes, we conducted the following validation experiments focusing on the concentration, sweetness, and biological functions of the recombinant sweet proteins:
Metabolism test
Macroscopic Phenotypic Metabolism
An indispensable part of producing proteins through heterologous expression is to clarify whether heterologous expression has an impact on the metabolism of the chassis organism and what the impact is[12]. Following the research principle from macro to micro, we first conducted a statistical study on the macroscopic phenotypic metabolism of tomato plants. The figure below (Fig 16) shows a comparison of the phenotypic traits (plant appearance and leaves) of tomato plants in different groups:
Using statistical methods, we collected phenotypic data from each group of tomatoes and analyzed the differences in three of the most representative characteristics: fruit weight, leaf length, and plant height (Fig 16), where the fruit weight was measured for all ripe tomatoes in each group of plants, leaf length was measured on the fifth leaf of similar growth stage and condition tomato plants in the same group, and plant height was recorded for all tomato plants in the same group.
Based on the figure below (Fig 17), it can be seen that there are no significant differences between the three phenotypic traits (fruit weight, leaf length, and plant height) of tomato plants in different groups, indicating that the use of tomato chassis for heterologous expression to produce sweet proteins does not have a significant negative impact on tomato plant metabolism[10].
(A) Data on fruit weight of tomato plants with different expression vectors;(B) Data on leaf length of tomato plants with different expression vectors;(C) Data on plant height of tomato plants with different expression vectors. (ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).
Glucose Colorimetric Assay
In our macroscopic phenotypic metabolism analysis, we have determined that the expression of the sweet protein Thaumatin does not significantly affect the macroscopic phenotypic traits of tomato organs. Following the principle of gradually delving deeper into the molecular level of molecular biology research, we have designed experiments for a more detailed metabolic analysis at the molecular level. Considering the limitations of experimental time and the representativeness of target substances, we chose to first test glucose. For more details, please refer to our Design page. The figure below (Fig 18) shows the results of our detection of glucose content using the Glucose (Glu) Colorimetric Assay Kit (GOD-POD Method)(Elabscience).
TRV-Thau: TRV virus Thaumatin group; TRV-Bra: TRV virus Brazzein group; 35S-Thau: Transgenic tomato 35S promoter Thaumatin group; 35S-Bra: Transgenic tomato 35S promoter Brazzein group; E8-Thau: Transgenic tomato E8 promoter Thaumatin group; E8-Bra: Transgenic tomato E8 promoter Brazzein group
From the figure above, it can be observed that there are no significant differences in glucose content among the various groups of tomato fruits, and the glucose content of all groups falls within the range reported in the literature. However, we have noticed that the glucose content in the experimental groups is slightly but not significantly higher than that in the wild-type and control groups. This could be due to the fact that the growth stages of the control and experimental groups of tomato plants are not entirely aligned, with the experimental group tomatoes being slightly more mature. The conversion of organic acids to glucose may result in a higher content of it.
Expression detection
To further test the capability of our natural plant chassis, tomato, to produce sweet proteins, we conducted the following validation experiments targeting the concentration of recombinant sweet proteins:
Detecting the concentration of sweet proteins produced (ELISA)
Our team used Thaumatin enzyme-linked immunosorbent assay (ELISA) (Shanghai hnybio) to determine the concentration of Thaumatin in tomato fruits. A standard curve was established using Thaumatin standards, and positive plant samples from the Western Blot results were selected as the experimental group. The results are shown in Figure 19.
It can be seen from the above figure that the Thaumatin in the fruits of each experimental group of tomatoes is significantly higher than that in the control group. By comparison, it can be clearly observed that the average expression amount of Thaumatin in the Transgenic tomato E8 promoter group is better than that in the Transgenic tomato 35S promoter group, indicating that the use of the E8 promoter as a promoter will not have a negative impact on the expression content of Thaumatin[7].
Sweetness detection
TAS1R2-sweet protein binding in an innovative assay
We have innovatively put forward a method similar to Enzyme-Linked Immunosorbent Assay (ELISA) for the specific detection of the binding affinity between sweet proteins and human sweet taste receptors[8](Fig 20). (For detailed experimental principles and methods, please refer to the Outline webpage.)
The first column was set as the blank control group. In the first row (except for the first well; the same applies to the second and third rows), it represented the positive control group. The second row corresponded to the experimental group with 35S-Thaumatin fruit protein samples, and the third row was designated for the experimental group with E8-Thaumatin fruit protein samples. As can be seen from the Figure 20, all wells in the experimental groups exhibit color development, indicating that the mature fruits of both transgenic lines contain correctly expressed Thaumatin protein capable of binding to the human sweet receptor protein T1R2.
The absorbance (OD value) was measured at a wavelength of 450 nm using a microplate reader. After obtaining the data, the OD values of the sample wells were subtracted by those of the blank wells. Subsequently, the OD values of the experimental groups were normalized by dividing by the average OD value of the positive control group. The resulting values were plotted as a scatter graph, and the mean values of each dataset were compared(Fig 21).
The chart indicates that the expression levels of 35s-Thaumatin are comparable to those of E8-Thaumatin. Given the limited sample size we utilized, it is not feasible to conclusively determine which promoter confers a superior expression profile. Nevertheless, based on the analysis of the 11 samples, we can deduce that there is no substantial difference in the expression of sweet proteins under the control of the 35s and E8 promoters.
Electronic Tongue Detection
We subjected the control group of tomatoes (non-infected wild-type Micro-Tom tomatoes), transgenic tomatoes with the 35S promoter, and transgenic tomatoes with the E8 promoter to electronic tongue detection. Subsequently, the taste characteristics detected by the electronic tongue were input into the Support Vector Regression (SVR) model for analysis. Based on the model's predictive analysis and calculation of the average values, the wild-type's predicted result was 0.0109, indicating that the control group of tomato samples is equivalent to a 0 ppm standard Thaumatin tomato solution, further confirming the usability of our model. The transgenic tomatoes with the 35S promoter resulted in 21.6013, meaning that the Thaumatin contained within is equivalent to a 21.6013 ppm standard Thaumatin solution. Similarly, it can be concluded that the Thaumatin in tomatoes with the E8 promoter is equivalent to a 21.6040 ppm standard Thaumatin solution.
Following the method established by Grant E. DuBois and D. Eric Walters, we calculated the Thaumatin concentration-response curve. It was determined that the sweetness level of Thaumatin in transgenic tomatoes with the 35S promoter is 8.65, and similarly, the sweetness level of Thaumatin in transgenic tomatoes with the E8 promoter is also 8.65.
Ultimately, based on the sucrose concentration-sweetness level calculation, the Thaumatin in transgenic tomatoes is equivalent to a standard sucrose solution with a concentration of 8.65%.
In summary, from the aforementioned experiments, we have measured the sweetness of Thaumatin in transgenic tomatoes. Thaumatin with a concentration of 11.0591 mg/L in transgenic tomatoes containing the E8 promoter is equivalent in sweetness to a standard sucrose solution with a concentration of 8.65%. This sets a standard for our future product manufacturing, enabling us to provide more precise consumption guidance to users.
Vacuole Localization
After confirming the expression level and functionality of the sweet protein, we aim to enhance the product further. Concerned about the susceptibility of sweet proteins to degradation, we opted to use SPS-NTPP to guide the aggregation of sweet proteins in vacuoles for targeted storage[11][13]. To verify that the addition of SPS-NTPP can direct Thaumatin to vacuoles, we first wanted to replicate the targeting effect of this peptide in our lab, ensuring we can proceed to the next phase of experiments. The most convenient method is to fuse SPS-NTPP with Thaumatin and attach an EGFP protein at the C-terminus; the fluorescence emitted by EGFP will indicate the location of Thaumatin. By comparing the fluorescence distribution of both, we can test whether SPS-NTPP can serve the vacuolar targeting role in this project.
Verification of the intrinsic action of the positioning peptide.
Based on the above concept, we constructed two plasmids, pGD_SPS-NTPP-Thaumatin-EGFP and pGD_Thaumatin-EGFP and introduced them separately into Agrobacterium GV3101 (Fig 23). Considering the time cost and our immediate objectives (to test SPS-NTPP), we used tobacco (Nicotiana benthamiana) for transient infection. Then, confocal observation was used to compare the distribution and aggregation of fluorescence foci.
The results indicate that the green fluorescence, which appears to avoid the red fluorescence (chloroplast autofluorescence), is primarily distributed in the central vacuole of the cell, preliminarily demonstrating that SPS-NTPP can direct the targeted transport of proteins to the vacuole (Fig 24).
Application on Tomatoes
However, we also know that our ultimate goal is to allow the sweet protein in transgenic tomatoes to be localized in the vacuoles of fruit cells for storage to increase the expression of sweet protein. In order to further verify the feasibility of SPS-NTPP localization peptides in tomato fruits, and considering the complexity of transgenic plant culture, after team discussion, we decided to conduct a preliminary leaf infiltration experiment in tomatoes to obtain a general understanding of the SPS-NTPP guidance for sweet protein localization. For more details, please see the Outline webpage.
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