Overview

Iron (Fe) and zinc (Zn) are essential trace elements for human health, yet deficiencies in these nutrients affect approximately 2 billion people worldwide, particularly infants, young children, and pregnant women[1]. In sub-Saharan Africa, diets heavily reliant on maize which provides at least 30% of total caloric intake—pose a high risk of iron and zinc deficiency, as maize endosperm contains very low levels of these elements[2]. While supplementation with iron and zinc compounds can improve nutritional status, economic constraints and low consumer acceptance limit their effectiveness in developing countries[3]. In contrast, biofortification through transgenic crops offers a sustainable and promising approach. Recent advances in genetic engineering have successfully increased edible crop iron content by manipulating genes related to iron uptake and metabolism; for example, co-expression of AtNAS1 (nicotianamine synthase 1), PvFERRITIN, and AfPHYTASE has enhanced iron concentration in rice endosperm[4]. NAS enzymes are key for nicotianamine synthesis, a crucial metal chelator that binds divalent metals like Fe and Zn, thus regulating their uptake, transport, and distribution within plants[5].

However, studies have shown that the C-terminal region of AtNAS1 contains an autoinhibitory signal that affects enzyme activity, which may explain why overexpression of AtNAS1 in transgenic rice does not always significantly increase iron and zinc levels[6]. Based on this, the current study aims to generate site-specific mutations at key C-terminal residues predicted to regulate AtNAS1 activity, purify the mutant proteins via prokaryotic expression in E. coli, and perform enzymatic kinetics analysis compared to wild-type AtNAS1 and a truncated AtNAS1 positive control. The most active mutants will be expressed in Bacillus subtilis for soil-based N. benthamiana cultivation to assess iron and zinc accumulation in leaves and roots. Additionally, transient expression in tobacco combined with qPCR analysis of iron- and zinc-related genes and confocal microscopy of fluorescently tagged proteins will verify mutant AtNAS1 expression, functional impact on downstream genes, and subcellular localization. This work uses tobacco as a model to provide insights and a theoretical basis for alleviating hidden hunger caused by trace element deficiencies in crops.

Cycle1:Autoinhibition Removal and High-Efficiency Enzyme Screening

Design

Based on previous reports of an autoinhibitory signal at the C-terminus of AtNAS1, we performed single- point mutations targeting specific amino acid residues (T287, R288, and G289). We also designed a multi-residue mutation (TRG→AAA) and a C-terminal truncation variant to test their effects on enzyme activity relative to the wild-type protein. All variants were designed and inserted into the pET28a expression vector via homologous recombination for prokaryotic expression in Escherichia coli. Wild-type and truncated AtNAS1 fragments were amplified by high-fidelity PCR, and mutants were constructed using inverse PCR to introduce precise point mutations.

Since nicotinamide synthase activity requires SAM as a substrate and MtnN and AdeD as coenzymes, these coenzyme genes were cloned into the pET28a vector for co-expression or supply during enzymatic assays. This design enabled systematic evaluation of C-terminal mutations and key residues in AtNAS1 while ensuring the availability of the coenzymes and robustly validating all constructs prior to functional analysis.

Fig 1. Schematic diagram of AtNAS1 wild type, C-terminal autoinhibition removal, four point mutations, and two coenzymes connected to the pet28a vector

Build

We amplified the AtNAS1_Wildtype fragments via high-fidelity PCR, resulting in fragment lengths of 963 bp. The right panel of Figure 2 displays the correct bands corresponding to the target sizes of the two genes. Subsequently, we used the restriction endonuclease BamHI to linearize the pET28a vector backbone, as shown in the left panel of Figure 2. After performing agarose gel electrophoresis and recovering the bands, we proceeded with the Infusion assemble kit to obtain the recombinant plasmid.

After the Infusion reaction assay, we transformed the recombinant plasmids into the cloned strain E. coli DH5α. Figure 2A shows the single colonies on the plate. We then selected six colonies from each plate. Next, we used vector universal primers, T7-Forward and T7-Terminal, to detect the insertion of target genes using colony PCR. As shown in Figure 2B, the AtNAS1_Wildtype gene fragment was successfully inserted into the pET28a backbone.

Figure 2. Verification of the transformation results for positive colonies of pET28a-AtNAS1_Wildtype and pET28a-AtNAS1_Truncated plasmids. A: Single colonies on the plate; B: Amplification results from colony PCR.

Finally, we used the universal primer T7-Forward to verify the positive colonies through Sanger sequencing. Figure 3 displays the correct sequences of the recombinant plasmids. We selected the correct colonies for further IPTG induction.

Figure 3. Sanger sequencing results of pET28a-AtNAS1_Wildtype and pET28a-AtNAS1_Truncated positive colony.

To further construct single-base and multi-base mutations, we linearized the pET28A-AtNAS1-Wildtype plasmid using inverse PCR to incorporate specific mutation sites. Subsequently, we constructed the mutated plasmids AtNAS1-T287A, AtNAS1-R288A, AtNAS1-G289A, and AtNAS1-TRGtoAAA via Infusion assay. Following the same construction process as for pET28A-AtNAS1-Wildtype, we also used colony PCR and Sanger sequencing to verify the positive colonies of the mutated plasmids, as shown in Fig. 4.

Fig. 4 The construction process of pET28A-AtNAS1 mutated plasmids. A. Agarose gel electrophoresis of mutated genes; B. Single colonies on the plate; C: Amplification results from colony PCR; D: Sanger sequencing results.

Since the enzymatic activity assay of nicotinamide synthase requires the substrate SAM and two coenzymes, MtnN and AdeD, we cloned the MtnN and AdeD gene fragments from the E. coli XL-1 Blue strain. Using the same construction strategy as for the pET28A-AtNAS1 plasmid, we constructed the expression plasmids pET28a-MtnN and pET28a-AdeD. The construction process is shown in Fig. 5.

Fig. 5 The construction process of pET28A-MtnN and pET28A-AdeD plasmids. A. Agarose gel electrophoresis of MtnN and AdeD genes; B. Single colonies on the plate; C: Amplification results from colony PCR; D: Sanger sequencing results.

We transformed the correct plasmids into the E. coli expression strain BL21 and selected single colonies for culture in preparation for the next step of prokaryotic expression. The plates are shown in Fig. 6.

Fig. 6 Single colonies of recombinant plasmids in E. coli BL21 strain.

Test

Preliminary Experiment for Protein Expression

Based on the lac operator system in the pET28a vector backbone, we used IPTG for protein expression. Initially, we applied a final concentration of 1 mM IPTG for standard protein expression. The expected molecular weights of AtNAS1 (including the mutated proteins), AtNAS1-Truncated, MtnN, and AdeD are 35.5 kDa, 31.7 kDa, 24.4 kDa, and 63.7 kDa, respectively. As shown in Fig. 7, we can clearly observe the protein bands corresponding to the target genes.

Fig. 7 SDS-PAGE analysis of preliminary experiment for target proteins expression.

His-Tag Affinity Purification of Proteins

We utilized the principle of His-tag affinity with Ni²⁺. This allowed us to bind the crude protein extracted from the E. coli expression cells to Ni-NTA beads. After washing away impurities with a low concentration of imidazole, we eluted the protein with a high concentration of imidazole. As shown in Fig. 8, we obtained a high-purity protein with a high expression level. Subsequently, we used dialysis to remove the high concentration of imidazole from the buffer and aliquoted the protein at a concentration of 1 mg/mL for storage, to be used in subsequent functional assays.

Fig. 8 SDS-PAGE analysis of large-scale His-tag protein purification of target proteins. Total: Total Protein; FT: Flow Through.

Enzymatic Reactions Detection of AtNAS1 Mutated Proteins' Catalytic Ability

We assessed the enzymatic activity of nicotinamide synthase (NAS1) mutants in Functional Tests Part 1. The enzymatic activities of the wild-type and various mutants (Truncated, T287A, R288A, G289A, TRG_to_AAA) of NAS1 were evaluated by measuring the initial reaction rates (ΔA/Δt) and calculating the enzyme activities (nkat/mg) based on the parameters of the reaction system (Fig. 9B). Compared to the slope and correlation coefficient of AtNAS1-Wildtype, the enzyme activities of the different mutants varied significantly, with some mutants, such as AtNAS1-T287A and AtNAS1-R288A, exhibiting markedly reduced activities. In contrast, AtNAS1-Truncated and AtNAS1-TRG_to_AAA demonstrated steep slopes and high enzyme catalytic efficiency (Fig. 9A). The AtNAS1-TRG_to_AAA mutant protein, unlike AtNAS1-Truncated, retains the complete C-terminal region of the NAS1 protein, thereby avoiding potential functional redundancies associated with the truncated form. Notably, the TRG_to_AAA mutant also exhibited a relatively higher level of nicotinamide synthesis activity compared to the truncated protein (Fig. 9B). Therefore, the TRG_to_AAA mutant may possess greater potential for application than the truncated variant.

Fig. 9 Kinetic and Thermodynamic Analysis of Engineered Variants of AtNAS1 protein. A. Enzyme Kinetics of AtNAS1 mutated protein; B. Enzyme Activity Analysis.

Molecular Dynamics Fitting Analysis of Catalytic Efficiency in AtNAS1 Mutated Proteins

After testing the enzymatic assays, we further investigated the binding affinities of the AtNAS1-Wildtype and AtNAS1 mutant forms with the substrate S-adenosyl methionine (SAM) using molecular dynamics simulations. As shown in Figure 10, the average binding free energy (dG) between AtNAS1-Wildtype and SAM was -36.83 kcal/mol, while the dG values for AtNAS1-Truncated is -13.63 kcal/mol and the dG values for AtNAS1-TRG_to_AAA is -17.06 kcal/mol. These results indicate that the ligand (SAM) can spontaneously bind to the protein, and the resulting protein-ligand complexes are dynamically stable, suggesting they may possess biological activity. This provides a theoretical energy-based foundation for further studying the biological functions of these ligand-protein complexes. The favorable binding free energies imply that the AtNAS1-Wildtype and AtNAS1-TRG_to_AAA can effectively interact with the SAM substrate, which is a critical step in the enzymatic reactions catalyzed by this protein.

Fig. 10 Molecular dynamics simulation of the binding sffinities between AtNAS1-Wildtype and mutant forms with the substrate S-Adenosyl Methionine (SAM)

Learn

Based on the results of the first round of experiments, we successfully constructed and expressed multiple AtNAS1 variants (wild-type, truncated, and point-mutated) and used them in E. coli. Enzymatic assays showed that the TRG→AAA mutant and the truncated form exhibited the highest catalytic efficiency, while the T287A/R288A mutant exhibited reduced activity. Molecular dynamics simulations confirmed that SAM had strong binding affinity for both the wild-type (ΔG = -36.83 kcal/mol) and the TRG→AAA mutant (ΔG = -17.06 kcal/mol), confirming their excellent substrate interaction abilities.

These results suggest that the C-terminal TRG motif plays a key role in regulating AtNAS1 autoinhibition, and that multi-residue substitutions (TRG→AAA) effectively abolished inhibition while maintaining the full-length integrity of the protein.

For further investigation, we recommend: 1) performing in planta studies using the TRG→AAA mutant in cycle 2 (transient expression in tobacco) and cycle 3 (stable transformation), and 2) implementing ICP-MS to quantify Fe/Zn accumulation in plant tissues.

Cycle2:Gene Expression Levels, and Subcellular Localization

Design

Based on the results from the first round, we selected the TRG→AAA mutant, AtNAS1 truncation, and wild-type for in vitro validation. We chose a tobacco transient expression system because it is rapid and suitable for initial functional screening. This system allows for efficient delivery of constructs via Agrobacterium infiltration, enabling transient expression.

We will clone the coding sequences of the selected mutants (TRG→AAA, truncation, and wild-type) into a plant expression vector (pCambia1305) controlled by the CaMV 35S promoter using homologous recombination. Three plasmids will be constructed for qPCR analysis and subcellular localization. All constructs will be verified by colony PCR sequencing prior to transformation into Agrobacterium GV3101.

Fig. 11 Plasmid maps of wild-type, TRG→AAA, truncation, and pCambia1305-eGFP-6myc vector construction

Build

Our previous results showed that the AtNAS1-Truncated protein and the AtNAS1 triple-mutant AtNAS1-TRG_to_AAA protein have comparable enzyme activity levels. Therefore, in the next step, we would like to validate our hypothesis using a plant model system, like N. benthamiana . First, we constructed the gene sequences of AtNAS1-Wildtype, AtNAS1-Truncated, and AtNAS1-TRG_to_AAA into the dual expression vector pCambia1305, which has a GFP fluorescent protein tag. This will allow us to detect the subcellular localization of the mutants using confocal microscope. As showed in Fig. 9A, we first amplified the gene fragments containing the homologous arms of the pCambia1305 backbone, where the AtNAS1-Wildtype served as the control. We then linearized the pCambia1305 vector using the restriction enzyme BamHI (Fig. 12B). After that, we performed an In-Fusion assembly reaction to construct the recombinant vector (Fig. 12C). Finally, we screened for positive clones using colony PCR (Fig. 12D).

Fig. 12 The construction process of pCambia1305-GFP-AtNAS1-WT, pCambia1305-GFP-AtNAS1-Truncated, and pCambia1305-GFP-AtNAS1-TRG_to_AAA plasmids. A. Agarose gel electrophoresis of genes; B. Linearized pCambia1305 backbone; C. Single colonies on the plate; D: Amplification results from colony PCR.

Test

Detection the Effect of AtNAS1 Mutant Proteins on Subcellular Localization Using Confocal Microscopy

Given that the subcellular localization of a protein can affect its function, we utilized transient expression in N. benthamiana along with microscopy to observe whether the localization patterns of the AtNAS1 mutant proteins differ from the AtNAS1-Wildtype protein. As shown in Fig. 13A, the process of transient expression in tobacco involves Agrobacterium strain in acetosyringone buffer, selecting healthy tobacco leaves, and infiltrating the bacterial suspension into the leaf abaxial surface using a 1 mL syringe. After two days of transient expression, the leaves were analyzed. We first transformed the AtNAS1-Wildtype, AtNAS1-Truncated, and AtNAS1-TRG_to_AAA positive plasmids into the Agrobacterium GV3101 strain and grew them on dual-antibiotic plates containing kanamycin and rifampicin, picking single colonies for further culturing (Fig. 13B). We initially used a handheld fluorescence instrument to detect the protein expression levels, which revealed that the AtNAS1-Truncated and AtNAS1-TRG_to_AAA proteins exhibited higher expression efficiency compared to the AtNAS1-Wildtype protein (Fig. 13C). Further analysis using confocal microscopy showed that the AtNAS1-Truncated and AtNAS1-TRG_to_AAA proteins had stronger fluorescence intensity and were more strongly localized to the endoplasmic reticulum, providing a potential explanation for their higher enzymatic activity (Fig. 13D).

Fig. 13 Subcellular localization of AtNAS1-Wildtype, AtNAS1-Truncated, and AtNAS1-TRG_to_AAA proteins. A. Process of transient transfection in N. benthamiana ; B. Single colonies of recombinant plasmids in Agrobacterium GV3101 strain. The plate contains Kanamycin and Rifampicin dual-antibiotics; C. Detection of GFP protein expression in N. benthamiana leaves using a handheld fluorescence instrument; D. The subcellular localization patterns of AtNAS1-Wildtype, AtNAS1-Truncated, and AtNAS1-TRG_to_AAA in N. benthamiana leaves using confocal microscope.

Detection the Effect of AtNAS1 Mutant Proteins on the Expression of Iron Cycling Related Genes Using Real-time Quantitative PCR In Vivo

To further investigate whether mutations in the AtNAS1 gene could affect the expression of genes related to the absorption and activation of iron and zinc micronutrients, we validated this hypothesis using a N. benthamiana -based culture transient system. First, we used the previously constructed Agrobacterium strains carrying the AtNAS1-Wildtype, AtNAS1-Truncated, and AtNAS1-TRG_to_AAA to infiltrate tobacco leaves. After 3 days of culture, we extracted RNA from the leaves, reverse transcribed it into cDNA, and used quantitative real-time PCR to detect the expression levels of the target genes. The expression levels were normalized using the tobacco NtActin1 gene as an internal reference. As shown in Fig. 14A, compared to the non-transformed tobacco leaves, the tobacco leaves expressing the AtNAS1-Wildtype, AtNAS1-Truncated, and AtNAS1-TRG_to_AAA plasmids significantly enhanced AtNAS1 gene expression, indicating that the transient expression of AtNAS1 in tobacco was successful. Further analysis revealed that the transient overexpression of both the wild-type and mutant AtNAS1 significantly increased the expression of the iron and zinc absorption and transport genes NtIRT1 and NtMTP8 (Fig. 14B-C), while the expression of the iron activation-related gene NtFRO2 remained unchanged (Fig. 14D). Notably, the AtNAS1-Truncated, and AtNAS1-TRG_to_AAA exhibited even higher expression levels of the iron and zinc absorption and transport genes NtIRT1 and NtMTP8, suggesting their greater potential for iron biofortification (Fig. 14B-C).

Fig. 14 Overexpression of wild-type and mutant AtNAS1 affects the expression of genes related to iron and zinc homeostasis. A: Relative expression levels of the AtNAS1 gene in tobacco leaves; B: Relative expression levels of the iron transporter gene NtIRT1 in tobacco leaves; C: Relative expression levels of the zinc transporter gene NtMTP8; D: Relative expression levels of the iron activation gene NtFRO2 in tobacco leaves.

Learn

Based on the combined results from cycles 1 and 2, we demonstrate that TRG→AAA effectively eliminates AtNAS1 autoinhibition while maintaining structural integrity. It also exhibits increased catalytic efficiency (cycle 1 enzymatic analysis) and enhanced in-plant function: confocal microscopy revealed enhanced ER localization, while qPCR confirmed significant upregulation of the metal transporters NtIRT1 and NtMTP8 (14B-C). Molecular dynamics simulations further validated its robust SAM binding affinity (ΔG: wild-type -36.83 kcal/mol vs. TRG→AAA -17.06 kcal/mol), consistent with its enhanced performance. Crucially, the TRG→AAA mutant retains the full-length protein structure while achieving activity comparable to the truncated variant, minimizing potential functional disruption.

These findings provide a mechanistic basis for the observed physiological effects: the ER-localized AtNAS1 mutant enhances nicotinamide synthesis, thereby mobilizing iron/zinc and activating endogenous transport systems, while maintaining metal activation pathways.

In the third cycle, we will:

1) express wild-type and TRG→AAA mutants in Bacillus subtilis for soil cultivation in tobacco;

2) quantify iron and zinc accumulation in stems and leaves using inductively coupled plasma mass spectrometry (ICP-MS) to assess biofortification efficacy under realistic growth conditions;

The transition from controlled models (E. coli, transient tobacco experiments) to soil-based validation addresses environmental relevance and scalability, bridging molecular design with agricultural applications. The TRG→AAA mutant's balanced combination of high activity, intact structure, and minimal ecological risk makes it an ideal candidate for future field-based biofortification solutions.

Cycle3:Biofortification Verification

Design

Based on the results from rounds 1 and 2, the TRG→AAA mutant demonstrated excellent enzyme activity and enhanced upregulation of metal transporters in tobacco. The third round transitioned to soil functional verification under relevant environmental conditions. The wild type, truncated mutant, and TRG→AAA were cloned into the pHT43 vector via homologous recombination to ensure compatibility with Bacillus subtilis. Plasmid integrity was initially verified in Escherichia coli DH5α by colony PCR and Sanger sequencing. Positive clones were transformed into Bacillus subtilis WB800N, screened on resistance plates, and verified by PCR according to established protocols.

Soil Cultivation, Bacillus strains were inoculated at varying concentrations in sterilized soil at a uniform volume. Nicotiana benthamiana seedlings were transplanted into the soil inoculated with Bacillus, and a control group was included. After 2 weeks, stems and leaves were harvested and Fe/Zn concentrations were analyzed by ICP-MS. This design ensured that protein engineering (cycle 1) and gene expression and subcellular localization (cycle 2) were tested under actual agricultural constraints. The expression of Bacillus subtilis reduced the risk of genetic modification while providing a scalable path for future field trials in major crops.

Fig. 15 Plasmid maps of wild-type, TRG→AAA, truncation, and pHT43 vector construction

Build

To further evaluate the application potential of AtNAS1 mutants in plants, we also used N. benthamiana as a model system and used B . subtilis harboring expression plasmids as a bioinoculant for soil-grown tobacco. Using the same construction strategy as for the pET28A-AtNAS1 plasmid, we constructed the B . subtilis expression plasmids pHT43-AtNAS1-Wildtype, pHT43-AtNAS1-Truncated, and pHT43-AtNAS1-TRG_to_AAA. The construction process is shown in Fig. 16.

Fig. 16 The construction process of pHT43-AtNAS1-WT, pHT43-AtNAS1-Truncated, and p pHT43-AtNAS1-TRG_to_AAA plasmids. A. Agarose gel electrophoresis of genes; B. Linearized p pHT43 backbone; C. Single colonies of DH5alpha on the plate; D: Single colonies of B. subtilis on the plate.

Test

Effect of B . subtilis Expressing AtNAS1 Mutated Proteins on N. benthamiana Growth and Biomass

The previous research results have shown that compared to the AtNAS1-Wildtype, both the AtNAS1-Truncated and the AtNAS1-TRG_to_AAA mutants have higher enzyme activity and a high efficiency to activate genes related to iron cycling. Since the AtNAS1-Truncated removes the C-terminal domain of the protein, it may lead to the partial loss of other biological functions, so we are more focused on the application of the AtNAS1-TRG_to_AAA mutant. In our tobacco pot culture experiment, we used B . subtilis as a probiotic. When the tobacco plants grew at the 4-leaf stage, we applied 10 mL of B . subtilis at different concentrations to the root system for root colonization. After two weeks of growth, we measured the plant fresh biomass and then dried and ground the shoot to determine the iron and zinc micronutrient contents using ICP-MS. As shown in Figure 17, we found that after applying B . subtilis , the fresh biomass of the tobacco plants actually showed a decreasing trend, and the leaves exhibited partial curling, which may be related to the applied concentration and still requires further optimization.

Fig. 17 Phenotypic analysis of AtNAS1 transient expressing tobacco plants. A: Images showing the growth of pHT43-AtNAS1-Wildtype and pHT43-AtNAS1-TRG_to_AAA transient expressing tobacco plants under different B . subtilis optical densities (PBS Control, OD=0.5, OD=1.0); B: The fresh weight of tobacco plants.

Effect of B . subtilis Expressing AtNAS1 mutated Proteins on Iron and Zinc Trace Element Contents in Tobacco Leaves Using ICP-MS

After harvesting the tobacco samples with B . subtilis root application, as shown in Figure 18A, we dried the shoot of the tobacco plants at 60°C and ground them into powder. We then performed microwave digestion of the samples using nitric acid and hydrogen peroxide, and used ICP-MS to measure the micronutrient contents. The raw data from the instrument was converted to mg/kg units using standard calibration curves. As shown in Figure 18B, compared to the PBS control, the tobacco plants inoculated with B . subtilis carrying the pHT43-AtNAS1-Wildtype and pHT43-AtNAS1-TRG_to_AAA plasmids showed increased iron (Fe) and zinc (Zn) micronutrient contents in the shoot at different B . subtilis concentrations. However, at the OD=0.5 concentration, there was no significant difference in either Fe or Zn content between the AtNAS1-Wildtype and AtNAS1-TRG_to_AAA treatments. At the OD=1 concentration, the tobacco plants inoculated with the B . subtilis carrying the pHT43-AtNAS1-TRG_to_AAA plasmid had significantly higher Fe content in the shoot compared to the pHT43-AtNAS1-Wildtype, while the Zn content did not differ. This suggests the potential application of the B . subtilis strain carrying the pHT43-AtNAS1-TRG_to_AAA plasmid for iron biofortification. Overall, our experiment has achieved preliminary success, but further optimization is still needed to fully understand the effects of the different B . subtilis treatments on plant biomass and micronutrient accumulation.

Fig. 18 A: Sample processing process, B: Fe element determination results, C: Zn element determination results

Learn

In the third round, we transitioned to soil-based validation, using Bacillus subtilis as a delivery vehicle for AtNAS1 variants in tobacco. The TRG→AAA mutant consistently demonstrated superior performance: ICP-MS analysis revealed a significant increase in iron (Fe) accumulation in tobacco spores at high bacterial density (OD = 1), a 1.5-fold increase compared to the wild type, while zinc (Zn) levels remained stable. This confirmed that the mutant enhanced metal bioavailability without disrupting overall homeostasis. However, the reduction in plant biomass at higher OD concentrations highlighted the need for dose optimization. The successful integration of Bacillus subtilis as a bioinoculant underscores the potential of non-GMO soil amendment strategies for enhancing micronutrient uptake, consistent with real-world agricultural constraints.

Over three cycles, we systematically validated protein engineering solutions for micronutrient biofortification:

• Cycle 1 (in vitro enzymology): TRG→AAA was identified as a highly active mutant (kinetics: ≈ wild-type efficiency; ΔG = -17.06 kcal/mol SAM bound).

• Cycle 2 (In-plant Transient Assays): We confirmed endoplasmic reticulum-localized expression and upregulation of metal transporters (NtIRT1/NtMTP8), linking mutant activity to physiological gene activation.

• Cycle 3 (Soil-Tobacco System): We used ICP-MS to confirm stem iron accumulation and determined functional efficacy under relevant environmental conditions.

The TRG→AAA mutant was the optimal variant, balancing catalytic efficiency, structural integrity, and scalability for agricultural use. Methodologically, we integrated in silico design (molecular dynamics), in vitro validation (enzyme assays), and in-plant testing (transient/soil systems), providing a reproducible workflow for synthetic biology projects.

Contributions to iGEM and Future Teams

We have contributed identified genetic components (e.g., the AtNAS1-TRG→AAA mutant and the pHT43-Bacillus vector) to a registry, enabling future teams to bypass initial screening and directly deploy high-performing variants. Our multi-cycle approach integrating enzyme kinetics, confocal microscopy, qPCR, and ICP-MS provides a template for validating biofortification strategies, highlighting the need to cross-validate molecular data with phenotypic results.

Using Bacillus subtilis (a GRAS organism) for soil delivery mitigates concerns about the release of genetically modified organisms, but further safety considerations are needed, and future efforts, such as the addition of a suicide gene switch, are planned. Our project demonstrates how to integrate protein engineering with agricultural applications, highlighting the steps from rational design to field-ready solutions. Future teams could apply this process to other nutrients/crops or optimize Bacillus-plant synergies to achieve broader sustainability goals.

References

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3. Gibson R S, Hotz C, Temple L, et al. Dietary strategies to combat deficiencies of iron, zinc, and vitamin A in developing countries: development, implementation, monitoring, and evaluation[J]. Food and Nutrition Bulletin, 2000, 21(2): 219-231.

4. Narayanan N, Cueto-Reaño M F, Eroğlu S, et al. Iron biofortification through genetic modification in rice, wheat, and cassava and its potential contribution to nutritional security[J]. CABI Reviews, 2022.

5. Seregin I V, Kozhevnikova A D. Nicotianamine: a key player in metal homeostasis and hyperaccumulation in plants[J]. International journal of molecular sciences, 2023, 24(13): 10822.

6. Seebach H, Radow G, Brunek M, et al. Arabidopsis nicotianamine synthases comprise a common core-NAS domain fused to a variable autoinhibitory C terminus[J]. Journal of Biological Chemistry, 2023, 299(6).