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.
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.
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.
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.
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.