iGEM 2024 — Team thailand-ris

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% set header\_image\_url = 'https://static.igem.wiki/teams/5120/header-photos/project-notebook.webp' %}
{% block page\_content %}{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one
iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{%
endblock %}
{% set header\_image\_url = 'https://static.igem.wiki/teams/5120/header-photos/project-engineering.webp' %}
{% block page\_content %}

* [Dry Lab](#DryLab)
* [Kill Switch](#KillSwitch)
* [Wet Lab Design](#Wetlab)
* [Conclusion](#Conclusion)
* [References](#References)

## ![sidebar_icon](https://static.igem.wiki/teams/5120/generalimgs/sidebaropen.png)

# **Dry Lab**

---

### Transient (co)expression of isoflavone biosynthetic genes in *Nicotiana benthamiana*

    Puerarin, daidzein, and genistein are isoflavonoids, a class of natural products that act as phytoestrogens in mammals and that are synthesised in an isoflavone pathway. These compounds are commonly found in household plants like soybeans, whereas puerarin is only found in the *Pueraria* genus. *Nicotiana benthamiana* has been used as an effective and easy plant for the functional characterisation of various plant proteins and enzymes (Sainsbury & Lomonossoff, 2014). Specifically, *N. benthamiana* is “amenable to protein overexpression through well-established *Agrobacterium infiltration* methods for DNA delivery... [due to] its native capacity to secrete heme peroxidases” (Goodin et al. 2008). However, because it lacks isoflavone synthase (IFS), *N. benthamiana* is unable to produce isoflavones. Thus, we performed transient co-expression of *N. benthamiana* leaves with a set of six isoflavone biosynthetic genes from *P. mirifica*: *chalcone synthase (PmCHS), chalcone reductase (PmCHR), chalcone isomerase (PmCHI), isoflavone synthase (PmIFS), 2-hydroxyisoflavanone (PmHID)*, and *C-glycosyltransferase (PmC-UGT)* genes. This set of genes would allow *N. benthamiana* to produce puerarin, daidzein, and genistin. To augment isoflavone production, this collection of isoflavone biosynthetic genes was co-expressed with the *Arabidopsis* R2R3 MYB12 transcription factor (*AtMYB12*), thus amplifying the expression of flavonoid biosynthetic genes. Along with the six isoflavone biosynthetic genes from *P. mirifica* and *AtMYB12*, we also used *Aequorea victoria* green fluorescent protein (*GFP*) as a control group in our experiment.

|  |  |
| --- | --- |
| ![resultsfigure1.png](https://static.igem.wiki/teams/5120/engineering/original-fig-1.png) | ![resultsfigure2.png](https://static.igem.wiki/teams/5120/engineering/original-fig-2.png) |

*Fig. 1 (the right hand image is a zoomed in version to the left pathway of the left image): Isoflavonoid and proposed miroestrol biosynthetic pathways in P. mirifica. Modified from Suntichaikamolkul et al. (2022, p. 63). Enzyme abbreviations: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, coumarate-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; IFS, isoflavone synthase; HID, 2- hydroxyisoflavanone dehydratase; C-UGT, C-glycosyltransferase; O- UGT, O-glycosyltransferase; CYP, cytochrome P450; IFR, isoflavone reductase; PT, prenyltransferase*

    In Fig. 1, daidzein and puerarin follow the same ‘left’ pathway. Genistein, on the other hand, follows the ‘right’ pathway. However, both pathways utilised the same sequence of enzymes to catalyse each substrate. Our experiment will not only be able to showcase *N. benthamiana*’s ability to produce isoflavones, but also to identify its preferred metabolic pathway: puerarin or genistin.

### *Agrobacterium* Vector-Mediated Infiltration and Vector Construction

    To allow for transient (co)expression of isoflavone biosynthetic genes in *N. benthamiana*, we utilised *Agrobacterium* vector-mediated infiltration (commonly referred to as agro-infiltration) as a method to perform transient transformation. This method was chosen as it leverages the natural ability of *Agrobacterium tumefaciens* to transfer DNA (T-DNA) into plant cells, facilitating rapid analysis of gene function and protein interactions without the need for stable transformation that allows for genetic modification to be present in future generations, but risks cross contamination with wildtypes and takes a longer experimental period.

### Vector Design

    We first needed to design the plasmids required for transient expression of the isoflavonoid biosynthesis pathway. Since we knew we were going to achieve our results from *N. benthamiana* through agroinfiltration, the team decided that it would be best to use an existing plasmid backbone as a template and build on it.

    We selected the pEAQ-HT-DEST1 vector because it was specifically designed for transient expression in plants as opposed to other vectors such as pCAMBIA which are designed for stable transformation.

There were also other features in the pEAQ DEST1 plasmid that appealed to us:

1. It contains a strong CaMV 35S promoter for high-level expression of our compound.
2. It is equipped with elements such as the LB and RB T-DNA repeats, which are necessary for T-DNA transfer during *Agrobacterium*-mediated plant transformation.
3. It suppresses RNA silencing of the transfer complex via the P19 suppressor gene4.
4. It has kanamycin resistance (KanR) for selection of transformed bacteria and plants.

    Then, we cloned six pEAQ-HT-DEST1 vectors using In-Fusion Cloning to contain the six aforementioned transient expression factors obtained from *P. mirifica*. We chose to do this in six different vectors rather than one because it would be easier for us to identify problems, if there are any later in the engineering cycle, if we had six vectors to alternate infiltration of rather than one giant vector.

    We also cloned 2 additional pEAQ-HT-DEST1 vectors to with GFP to use as a control for determining if agroinfiltration was successful and AtMYB12 to bolster production of substrates required for the isoflavonoid biosynthetic pathway so our modifications wouldn’t have any effects on other processes in the plant and that our target compounds would have increased production.

|  |  |
| --- | --- |
| ![resultsfigure1.png](https://static.igem.wiki/teams/5120/engineering/1-peaq-atmyb12.png) | ![resultsfigure2.png](https://static.igem.wiki/teams/5120/engineering/2-peaq-pmchi.png) |
| ![resultsfigure3.png](https://static.igem.wiki/teams/5120/engineering/3-peaq-pmifs.png) | ![resultsfigure4.png](https://static.igem.wiki/teams/5120/engineering/4-peaq-pmchs.png) |
| ![resultsfigure5.png](https://static.igem.wiki/teams/5120/engineering/5-peaq-pmchr.png) | ![resultsfigure6.png](https://static.igem.wiki/teams/5120/engineering/6-peaq-pmcgt.png) |
| ![resultsfigure7.png](https://static.igem.wiki/teams/5120/engineering/7-peaq-pmhid.png) | ![resultsfigure8.png](https://static.igem.wiki/teams/5120/engineering/8-peaq-gfp.png) |

*Fig. 2: Vector diagram for each of the eight transformed Agrobacterium; AtMYB12, PmCHI, PmIFS, PmCHS, PmCHR, PmCGT, PmHID, GFP*

# **Kill Switch**

---

    Our team also wanted to create safety measures to prevent the spread of our modified plant outside of controlled environments. After some consideration we chose to create a kill switch that would activate depending on specific conditions.

We first wanted to choose a condition based on which our kill switch would activate. We originally thought about making the plant dependent on specific nutrients, which would only be available in controlled environments. The kill switch would activate and kill the plant without these nutrients. Further consideration showed that this would not be cost-effective for producing our supplements in the long run. Thus we decided to go with the atPfr-promoter, which is light-inducible and responds to red wavelengths of light, so our plant is dependent on a red light filtered growing environment. Furthermore, using our method to produce the isoflavonoids would only require a one-time investment in a red light filter, and if the plant went outside, the kill switch would activate almost immediately with red light being everywhere.

    Then we had to choose a terminator sequence. We chose Barnase, which is a ribonuclease that causes degradation of single stranded RNAs. We chose this for our killing mechanism because the expression of this protein without its inhibitor Barstar would result in cell death and cause the target plant to die.

    We then selected the pEAQ-HT-DEST1 vector as a base for the kill switch because its parameters fit the most with what we needed. The vector diagram in Figure 3 shows the structure of the kill switch.

![killsiwtch.png](https://static.igem.wiki/teams/5120/engineering/peaq-kill-switch.png)

*Fig.3: Vector diagram for the kill switch*

# **Wet Lab Design**

---

### Building and Testing Round 1

1. **1. Cloning of our synthesised plasmid**

       Our project's wet lab began with cloning our bacteria via In-Fusion Cloning method. Unlike the traditional cloning method of using ligation enzymes that depends on specific restriction sites, limiting the choice of vectors and inserts, In-Fusion Cloning allows for a directional cloning of multiple fragments into any vector. First, we used restriction enzymes to linearise pHEAQ-HT-DEST1. Then, we combined the linearised vector with the PCR-amplified insert (referring to the T-DNA section on each of the eight diagrams as shown in Fig. 2)5 and the In-Fusion Cloning Master mixed to perform the cloning reaction (as per the kit’s instructions). From then on, the resulting solution (from the combination phase of In-Fusion Cloning) was transformed into *E. coli* strain DH5-α via the heat shock transformation method.

       Out of eight transient expression factors, six from *P. mirifica*-*PmCHR, PmCHI, PmIFS, PmHID, PmCHS*, and *PmCGT*—were cloned by us. However, AtMYB12 and GFP were already available in the lab and were thus not cloned.

   5Before transformation, we performed PCR and RNA gel electrophoresis to confirm that the length of our recombinant vector is relatively the same as the theoretical length.
2. **2. Growing bacteria**

       We grew the transformed *E. coli* strain DH5-α on a petri dish filled with Luria-Bertani (LB) agar, and kanamycin.6 The base LB agar includes four grams of peptone, two grams of yeast extract, four grams of table salt (NaCl), and three grams of agar. *E. coli* colonies verified by colony PCR were then sent for DNA sequencing. The plasmids identified as positive clones were then transformed into *A. tumefaciens*, which was left to grow in a new petri dish with LB agar (same base formulae) supplemented with not only kanamycin but also rifamycin and gentamicin.

   6KanR is added as a safety step. Regular *E. coli* are susceptible to kanamycin. However, the insert that we cloned is specifically designed to include the kanamycin-resistant gene. Therefore, the bacteria's survival in this medium confirms the successful transformation of our recombinant plasmid into *E. coli*.

## 3. Infiltration of *N. benthamiana*

After two days, we again verified the *Agrobacterium* using colony PCR and agarose gel electrophoresis. We then selected positive clones for overnight inoculation. Subsequently, we washed the overnight culture three times with sterilised distilled water. The pallets were then resuspended in a resuspension solution of 10 mM MgCl2, 10 mM MES-K, and 100 μM acetosy-ringone, pH 5.6, before being mixed into 3 different solutions:

1. Control: *Agrobacterium* with >pEAQ-GFP vector.
2. *Agrobacterium* harbouring the *AtMYB12*, along with an upstream array of isoflavone biosynthetic genes, including *PmCHR, PmCHI, PmIFS, PmHID*, and *PmCHS* which culminate the pathway at daidzein or genistein.
3. *Agrobacterium* harbouring the *AtMYB12*, along with an upstream array of isoflavone biosynthetic genes including *PmCHR, PmCHI, PmIFS, PmHID, PmCHS*, and *PmCGT* which culminate the pathway for puerarin or daidzein.

We mixed *Agrobacterium* in equal amounts for solutions 2 and 3 (adjusting the OD to 0.5).

After each of the solutions has rested at room temperature for two-three hours, we infiltrate each solution into the top four leaves of roughly 4-week-old *N. benthamiana* trees. We then leave these trees to grow for an additional five days.

## 4. Extraction and analysis

Before extracting the isoflavones, we freeze-dried the collected leaves of the modified *N. benthamiana*. Then we perform extraction using our sample and 500 ul of 50% methanol and 20 ug/ml of isovitexin as an internal standard. After that, using only the extraction's supernatant, we performed HPLC analysis. We used a reversed-phase C18 column (50 x 4.6 mm) filled with 5 M core-shell silica. The linear gradient of the mobile phase was set from 95:5 to 85:15 of 1% acetic acid (v/v) in water and 1% acetic acid (v/v) in acetonitrile, with a flow rate of 1 ml/min for 45 min. The column temperature during the test was set at 45 degrees Celsius. Using UV absorbance (254 nm and 280 nm), we monitored the eluate. Calibration curves of chromatogram peak areas for the standard compounds were produced.

## Building and Testing Round 2

After our first round of engineering, the peak of expected isoflavones did not obviously show based on HPLC results. Thus, we repeated the aforementioned process for Step 2: Growing Bacteria and Step 3: Infiltration again, though with some variation in our methodology for extraction and analysis. These variations include the following:

1. ### Shifts from using dry weight samples to fresh weight samples in step 4 - extraction and analysis.

   1. The fresh weight sample was done by grinding the sample into fine powder directly after collection instead of freeze-drying the sample first before grinding.
   2. Justification: Following the utilisation of dry weight as the sample, HPLC analysis revealed that the calibration curves of the chromatogram exhibit peaks corresponding to the standard compounds daidzein, genistin, daidzein, and genistein. However, the aforementioned peaks are insufficient. Thus, we switched from dry weight to fresh weight samples.
2. ### Change in HPLC gradient

   1. The mobile phase was set to 100% solvent A and 0% solvent B (at 0 mins) to 55% solvent A and 45% solvent B (at 45 mins). Solvent A, 0.1% of acetic acid in distilled water, and solent B, 0.1% acetic acid in acetonitrile with a flow rate of 1 ml/min while maintaining the column temperature at 45°C and the sample injection at 20µl. However, the parameters used in the first round of HPLC were similar, but there was a change in the gradient: 95% solvent A and 5% solvent B (at 5 min) and 15% solvent B and 85% solvent A (at 40 min). In addition, the sample injected was increased from 10µl to 20µl.
   2. Justification: Altering the gradient rate enables the attenuation of noise surrounding the peak of each compound while concurrently enhancing the prominence of other peaks.

![final 2.png](https://static.igem.wiki/teams/5120/engineering/hplc-gradient-for-analysis-1.webp)

###### *Fig. 4: Graph showing the flow rate of each solvent (solvent A and solvent B) for the first HPLC analysis; 95% solvent A and 5% solvent B (at 5 min) and 15% solvent B and 85% solvent A (at 40 min).*

![final 2.png](https://static.igem.wiki/teams/5120/engineering/hplc-gradient-for-analysis-2.webp)

###### *Fig. 5: Graph showing the flow rate of each solvent (solvent A and solvent B) for the second analysis; 100% solvent A and 0% solvent B (at 0 mins) and 55% solvent A and 45% solvent B (at 45 mins).*

# **Conclusion**

---

    The results of the agro-infiltrated *N. benthamiana* leaves reflect the effectiveness of modifying and agro-infiltration of the pEAQ-HT-DEST 1 vector with the isoflavonoid genes, resulting in the production of isoflavone compounds. The data shows that the expression of these genes, along with an overflow of the metabolites in the isoflavonoid pathway, allowed the plant to produce isoflavones such as puerarin and daidzein. The infiltration of our plasmid with the isoflavonoid genes enables a new, synthesised isoflavonoid pathway for *N. benthamiana* in support of a treatment for neurodegenerative diseases such as Alzheimer’s.

# References

---

1. Sainsbury, F., & Lomonossoff, G. P. (2014). *Transient expressions of synthetic biology in plants*. Current Opinion in Plant Biology, 19, 1–7. <https://doi.org/10.1016/j.pbi.2014.02.003>

2. Goodin, M. M., Zaitlin, D., Naidu, R. A., & Lommel, S. A. (2008). *Nicotiana benthamiana: Its History and Future as a Model for Plant–Pathogen Interactions*. *Molecular Plant-Microbe Interactions*, 21(8), 1015–1026. <https://doi.org/10.1094/mpmi-21-8-1015>

3. Suntichaikamolkul, N., Akashi, T., Mahalapbutr, P., Sanachai, K., Rungrotmongkol, T., Bassard, J., Schaller, H., De-Eknamkul, W., Vimolmangkang, S., Yamazaki, M., & Sirikantaramas, S. (2022). *Daidzein Hydroxylation by CYP81E63 Is Involved in the Biosynthesis of Miroestrol in Pueraria mirifica*. Plant and Cell Physiology, 64(1), 64–79. <https://doi.org/10.1093/pcp/pcac140>. Diagram is obtained from figure 1 of the journal which is titled: Isoflavonoid and proposed miroestrol biosynthetic pathways in *P. mirifica*.

4. Debler, J. W., Henares, B. M., & Lee, R. C. (2021). *Agroinfiltration for transient gene expression and characterisation of fungal pathogen effectors in cool-season grain legume hosts*. Plant Cell Reports, 40(5), 805–818. <https://doi.org/10.1007/s00299-021-02671-y>

5. Before transformation, we performed PCR and RNA gel electrophoresis to confirm that the length of our recombinant vector is relatively the same as the theoretical length.

6. KanR is added as a safety step. Regular *E. coli* are susceptible to kanamycin. However, the insert that we cloned is specifically designed to include the kanamycin-resistant gene. Therefore, the bacteria's survival in this medium confirms the successful transformation of our recombinant plasmid into *E. coli*.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% set header\_image\_url = 'https://static.igem.wiki/teams/5120/header-photos/project-experiments.webp' %}
{% block page\_content %}{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% set header\_image\_url = 'https://static.igem.wiki/teams/5120/header-photos/project-results.webp' %}
{% block page\_content %}

* [Results](#results)
* [Proposed Implementation](#ProposedImplementation)

## ![sidebar_icon](https://static.igem.wiki/teams/5120/generalimgs/sidebaropen.png)

# **Results**

---

    Isoflavonoids, particularly genistein and puerarin, play crucial roles in the medicinal and biological functions of *Pueraria lobata v. mirifica*. The bioengineering of metabolic pathways to optimize the production of these compounds has become a vital area of research, especially for enhancing yields of valuable phytoestrogens such as puerarin. This study aimed to engineer the biosynthetic pathway of *P. mirifica* to produce genistein, puerarin, and daidzein in non-leguminous plants.

    To begin the process, *Nicotiana benthamiana* was transformed using the vector (pHEAQ-HT-DEST1), which contains key genes involved in isoflavonoid synthesis, including PmCHS, PmCHR, PmCHI, PmIFS, PmHID, and PmCGT, as well as AtMYB12. These genes were introduced to enhance the pathway from phenylalanine to daidzein and genistein, aiming to boost their production.

    PCR amplification and gel electrophoresis were then performed to confirm the successful insertion and functionality of the genistein within the host plant. The presence of the expected bands verified the genetic transformation and ensured the integrity of the engineered constructs, a cortical step before proceeding with activating the desired metabolic pathway.

![resultsfigure1.png](https://static.igem.wiki/teams/5120/results/resultsfigure1.png)

*Figure 1. Gel electrophoresis result following PCR amplification, showing detection of the genes PmCHS, PmCHR, PmCHI, PmIFS, PmHID, PmCGT*

    Based on Figure 1, showing a gel electrophoresis result following PCR amplification, likely performed to confirm the presence of the inserted genes in *Nicotiana benthamiana*. The gel has distinct bands, which indicate the successful amplification of specific gene fragments from the vector (pHEAQ-HT-DEST1) that were used to introduce the genes PmCHS, PmCHR, PmCHI, PmIFS, PmHID, PmCGT, and AtMYB12 into the host plant. The presence of bright, distinct bands in certain lanes suggests the correct insertion of the target genes. These results confirm that the transformation was partially successful, allowing the next step in activating the metabolic pathway to enhance daidzein and genistein production. This electrophoresis result is critical for verifying the integrity and functionality of the genetic constructs before proceeding with further analysis of experiments.

    In Figure 2, the red boxes in the HPLC chromatogram highlight the peaks of various compounds: puerarin, isovitexin, daidzein, and genistein. Each red box represents the expected retention time range for these compounds. The puerarin peak appears at around 12–15 minutes, which suggests the detection of puerarin in the samples represented by certain coloured lines (i.e., purple, black). However, the peak is not as prominent as it should be, suggesting a low concentration. Isovitexin is marked around 22–23 minutes, with a much more prominent peak observed, indicating its presence in the sample (notably in the green line). For daidzein and genistein, located between 36-50 minutes, the red boxes highlight these areas, but the absence of significant peaks (flat or minimal lines) indicates that these compounds were not detected in the samples.

![resultsfigure2.png](https://static.igem.wiki/teams/5120/results/resultsfigure2.png)

*Figure 2. HPLC chromatogram showing the detection of 4 compounds: puerarin (with a small peak), isovitexin (with a prominent peak) and minimal to no detection of daidzein and genistein*

    The experiment followed a systematic approach to pathway engineering *N. benthamiana* plants were transformed using the pHEAQ-HT-DEST1 vector, which allowed for efficient agrobacterium-mediated transformation. The vector contained LB and RB elements necessary for T-DNA insertion, along with NeoR/KanR resistance markers for selecting successfully transformed plants. After transformation, the plants were tested for isoflavonoid production using High-Performance Liquid Chromatography (HPLC). In the first analysis, daidzein was detected in significant amounts, while puerarin was not.

    Because of the lack of puerarin detected, we increased sample size and improved HPLC detection parameters (i.e., increasing sample injection from 10µl to 20µl, adjusting gradient solvent concentrations, and raising column oven temperature to 45°C) provided more reliable data, but puerarin was still only present at very low concentrations.

![resultsfigure3.png](https://static.igem.wiki/teams/5120/results/resultsfigure3.png)

*Figure 3. HPLC chromatogram showing the detection of puerarin, daidzein, genistin, iso-vitexin, daidzein and genistein in transformed Nicotiana benthamiana samples*

    The chromatogram in the above figure highlights the detection of key isoflavonoid compounds—*puerarin, daidzein, genistin, iso-vitexin, daidzein,* and *genistein*—in *Nicotiana benthamiana* samples following transformation. Each red box in the figure corresponds to one of these compounds, showing the specific peaks generated during HPLC analysis. The gre line serves as the control sample, providing a baseline for comparison, while the coloured lines (purple, pink, blue) represent chromatograms from different experimental samples, each reflecting the presence and concentration of these target compounds. The peaks in these coloured lines indicate the success of the transformation, confirming that the engineered pathway effectively produced the desired isoflavonoids, such as *daidzein* and *genistein*. This chromatogram provides crucial evidence that the genetic constructs were functional and led to the production of key metabolites in the engineered plants.

    The chromatogram obtained from High-Performance Liquid Chromatography (HPLC) of our samples from our second trial reveals several key peaks corresponding to distinct compounds, as shown in Figure 2. The first peak, observed at around 16.0 minutes, represents puerarin, followed by a peak at approximately 17.0 minutes, which corresponds to daidzein. Further along, a peak at 22.0 minutes is attributed to genistin, while the peak at 23.0 minutes represents isovitexin. The peak at 27.0 minutes signifies daidzein, and finally, the peak at 31.0 minutes corresponds to genistein. These compounds are clearly labelled in the chromatography, highlighting their respective retention times.

![resultsfigure4.png](https://static.igem.wiki/teams/5120/results/resultsfigure4.png)

*Figure 4, concentration levels of three compounds—daidzein, genistein, and genistin—in Nicotiana benthamiana (in collaboration with K. Tantisuwanichkul, 2024)*

    Moreover, the bar graph in Figure 4 shows that genestin was detected in higher quantities, showing that some genistein was glucosylated into genestin as there are elevated genistin levels relative to daidzein and genistein.

    In our second round of testing, we also added PmCGT to the glucosylation of daidzein into puerarin. However, the chromatogram (Figure 1) showed that puerarin was nearly undetectable. This suggests that the glycosylation step may have been inefficient or that upstream bottlenecks prevented adequate precursor supply for puerarin synthesis.

    As shown in Figure 3 and Figure 4, the concentrations of three main compounds were quantified after the two engineering rounds. Daidzein was detected in both rounds, with the second round showing a slight improvement. Similarly, genistin levels reached approximately 350 µg/g and ~300 µg/g, respectively, for each round of testing. These findings, represented in Figure 2, underscore the disparity in compound accumulation and the failure to boost puerarin production despite pathway modifications. The second stage was crucial in highlighting the inefficiency of the conversion of daidzein to puerarin, despite the addition of a new enzyme intended to enhance this pathway step. This result from this stage highlights the need for further investigation into enzyme activity for PmCGT and possible competition between pathways for conversion of daidzein to puerarin.

    A key issue appears to be the inadequate conversion of genistein into puerarin. While genistin accumulated, puerarin was not detected, indicating a bottleneck at the C-UGT step. This inefficient activity of PmCGT may have hindered puerarin synthesis, or the system may have favoured the production of genistin. Additionally, the upstream conversion of naringenin to genistein was less efficient, limiting the availability of genistein for further conversion. The introduction of multiple enzymes such as CHR and CHI without balancing the expression of CHS could lead to alternative pathway activation, reducing the overall production of daidzein and puerarin. This imbalance may explain why genistin accumulated but not puerarin. The isoflavonoid pathway involves the conversion of phenylalanine into p-coumaric acid, which is then used to produce isoliquiritigenin and naringenin. IFS and HID are responsible for converting naringenin into daidzein and genistein. Despite the introduction of HID, genistein levels remained low. It is likely that the pathway favoured the conversion of daidzein over genistein, limiting the availability of genistein for subsequent puerarin synthesis. The failure to detect puerarin in significant amounts suggests that PmCGT activity was suboptimal. There is a possibility that puerarin or daidzein was present in the samples, but in quantities too small for detection. Additionally, competition between UGT enzymes may have favoured the production of genistin over puerarin.

    These results highlight the complexity of engineering the isoflavonoid pathway in *P. mirifica*. While some success was achieved in increasing genistein and daidzein levels, the lack of puerarin points to key areas for improvement. Future efforts should focus on balancing enzyme expression levels, optimizing the conversion of genistein to puerarin, and improving detection methods for trace amounts of puerarin. Additionally, we could further investigate alternative hosts or pathway engineering strategies to enhance the production of valuable isoflavonoids.

# **Proposed Implementation**

---

    The Thailand-RIS team focused on providing a proof of concept demonstrating the possibility and viability of relocating the isoflavonoid pathway for the production of puerarin from *Pueraria lobata v. mirifica* to *Nicotiana benthamiana*; however, there are many elements we would like to further implement in our project, as an expansion of the work we’ve already done.

    The immediate next step would be to permanently engineer *N. benthamiana* through stable transformation instead of agroinfiltration. As ensuring the sustainability of the production of puerarin is one of the main goals of the project, it would be essential to develop a transgenic *N. benthamiana* that does not require agroinfiltration for every individual leaf and plant. This is not only time consuming but also requires special expertise and adds unnecessary costs that would once again make puerarin less accessible. Performing stable transformation on *N. benthamiana* would allow it and its future generations to autonomously produce puerarin, daidzein, and genistein. This would better establish the sustainability and accessibility of our solution, as the plant would not necessarily be constrained to special facilities but ideally be available for farmers to grow.

    Another implementation we would like to propose is testing whether *Glycine max* is viable for the relocation of the enzymes required for the isoflavonoid pathway. This is something to highly consider because *G. max* is a leguminous plant much like *P. mirifica*, and thus already contains more enzymes needed for the pathway than *N. benthamiana*. As a result, it would simplify the relocation of the pathway and limit errors to a certain extent, and possibly be more successful because of it. Moreover, *G. max* is a plant widely grown in Thailand. Especially considering the stigmatization of GMOs that is still very much prevalent in the country, transforming a well-known plant may decrease the resistance to the idea of farming and consuming (puerarin produced by) a genetically modified plant. On top of that, *G. max* is better suited for the tropical climate of Thailand as opposed to *N. benthamiana*, which makes it a more feasible candidate for commercialization and production outside of controlled conditions. Factoring in the 3-month growing time of *G. max*, the plant still remains a viable option as a chassis for the production of puerarin, which underscores the importance of a future investigation of transforming *G. max* for our project.

    Future work would need to consider measures for biocontainment of our transgenic plant. Performing stable transformation on *N. benthamiana* and *G. max* if possible increases the sustainability of the production of puerarin, but comes with a new set of challenges once the plants are released beyond containment. These issues include the risk of propagation and spread of the plant outside of the farm area, which could lead to the consequences of cross contamination and competition for resources with wild species in the environment. This could be prevented with the integration of a “kill switch,” which could be done by further modifying the plant’s genes so that it becomes dependent on a certain nutrient not found in the environment. The *N. benthamiana* (or *G. max*) would not be able to survive outside of farms–including seeds that carry the modified genes–and thus it would stay contained in the area where that nutrient would be artificially provided to it. Even if we were to choose the biomanufacturing route of producing puerarin supplements by performing constant agroinfiltration, a similar “kill switch” can be implemented for the *Agrobacterium tumefaciens* used for infiltration. These kill switches, or auxotrophic modifications, could trigger bacterial death outside of controlled conditions to prevent their spread. In both cases, strategic measures would be utilized to ensure that the engineered organisms remain contained within the environmental settings of the production, minimizing the risk of unintended ecological impacts. Furthermore, to follow through with the use and biocontainment of modified plants, the Thailand-RIS team would seek approval from institutional biosafety committees and compliance with national regulations governing the use of genetically modified organisms in agriculture. Thailand-RIS has proposed some models for biocontainment outlined in our parts registry and safety protocols.

     On a final note, we recognize the potential interests seeking to monopolize puerarin production through genetically modified plants that may arise if our project was expanded and commercialized, and how counterproductive that would be in making puerarin an accessible treatment method for everyone. To address this, we would need to further discuss with policymakers and seek the aid of the government to implement a more inclusive policy. The Thailand-RIS team is aware that this will be a difficult endeavor and may not be realistic, but if we were to follow through with our future work as outlined above, this would be one of the priorities to protect local farmers and the general public as a whole.

{% endblock %}