CONTRIBUTION

Overview

Iron ( Fe ) and zinc ( Zn ) are essential trace elements to maintain human health. However, the problem of iron and zinc deficiency is widespread worldwide, affecting about 2 billion people, especially infants, young children and pregnant women. In sub-Saharan Africa, the risk of iron and zinc deficiency is particularly prominent because the local diet is heavily dependent on corn, which provides at least 30 % of total calorie intake[1]. However, the concentration of iron and zinc in maize endosperm is extremely low[2], and long-term use of maize as a staple food can easily lead to iron and zinc deficiency symptoms. Although taking iron and zinc supplements can improve people 's iron and zinc nutritional status, these measures have little effect in developing countries due to insufficient economic sustainability and low consumer acceptance. In contrast, biofortification through transgenic crops shows greater potential.

In recent years, genetic engineering related to iron absorption and metabolism has successfully increased the iron content of edible parts of crops. For example, co-expression of AtNAS1 ( nicotinamide synthase 1 ), PvFERRITIN ( ferritin ) and AfPHYTASE ( phytase ) [3]increased the iron concentration in rice endosperm. These studies indicate that genetic engineering has broad application prospects in crop iron fortification. Nicotinamide synthase is the key enzyme for the synthesis of nicotianamine ( NA ) in plants, and nicotinamide is an important metal chelating agent, which can form stable complexes with divalent metal ions such as iron ( Fe ) and zinc ( Zn )[4]. NAS genes directly affect the absorption, transport and distribution of iron and zinc in plants by regulating the synthesis of nicotinamide, thus playing a central role in plant iron and zinc nutrition. However, it has been reported in the literature that there is a self-inhibition signal at the C-terminus of AtNAS1 protein by in vitro truncation experiments, which in turn affects enzyme activity, which may be the reason why the effect of increasing iron and zinc in overexpressing AtNAS1 transgenic rice is not obvious[5].

Therefore, based on the above research background, this study intends to use the key amino acid sites that predict the C-terminus of AtNAS1 protein affecting enzyme activity in the published literature, and mutate the key sites of the full-length protein. After prokaryotic expression and purification of AtNAS1 mutant protein in E.coli, the enzyme activity mechanics analysis was carried out. Compared with the wild-type AtNAS1 protein, the truncated AtNAS1 protein reported in the literature was used as a positive control to improve the enzyme activity, and the point mutation combination with strong AtNAS1 full-length protein activity was screened for the construction of Bacillus subtilis expression vector. The contents of iron and zinc in the roots of tobacco leaves were determined after soil culture. In addition, the mutant AtNAS1 protein was verified by tobacco transient expression experiment. The transcription level of genes related to iron and zinc absorption and metabolism was detected by qPCR, which could confirm whether the mutant AtNAS1 was successfully expressed in tobacco cells and affected other genes. At the same time, by fusing with fluorescent protein and observing its subcellular localization under confocal microscopy, it can be clear whether the mutation affects the intracellular distribution and localization characteristics of AtNAS1. In this study, tobacco was used as a model organism to provide new ideas and theoretical support for solving the problem of ' hidden hunger ' caused by the lack of trace elements such as iron and zinc in crops.

During our experiment, we added some new parts for iGEM part and new information to an existing part (Table 1).

Table 1. Part contributions

Part number	Part name	Contribution type	Part type
BBa_25CUAM36	AtNAS1_Wildtype	New part	Basic part
BBa_25Z4KILN	AtNAS1_Truncated	New part	Basic part
BBa_25BKK7KO	AtNAS1_T287A	New part	Basic part
BBa_258W4O4E	AtNAS1_R288A	New part	Basic part
BBa_2548PJA8	AtNAS1_G289A	New part	Basic part
BBa_25ZTVJLF	AtNAS1_TRG_to_AAA	New part	Basic part
BBa_25KB1JQX	MtnN	New part	Basic part
BBa_25UKX9PI	AdeD	New part	Basic part
BBa_25V96FRD	pET28a-AtNAS1-Wildtype	New part	Composite part
BBa_25215Z7B	pET28a-AtNAS1-Truncated	New part	Composite part
BBa_25ZPT0V7	pET28a-AtNAS1-T287A	New part	Composite part
BBa_25F9JOMU	pET28a-AtNAS1-R288A	New part	Composite part
BBa_254PZ9TL	pET28a-AtNAS1-G289A	New part	Composite part
BBa_25S0ZK22	pET28a-AtNAS1-TRG-to-AAA	New part	Composite part
BBa_253CJAM2	pET28a-MtnN	New part	Composite part
BBa_25116VHK	pET28a-AdeD	New part	Composite part
BBa_2540G58Q	pCambia1305-AtNAS1wt-eGFP-6myc	New part	Composite part
BBa_25RS593V	pCambia1305-AtNAS1truncated-eGFP-6myc	New part	Composite part
BBa_25QPDY3X	pCambia1305-AtNAS1-TRG-to-AAA-eGFP-6myc	New part	Composite part
BBa_25QR4VJK	pHT43-AtNAS1wt	New part	Composite part
BBa_25R3BQ8Y	pHT43-AtNAS1truncated	New part	Composite part
BBa_25UWPRL5	pHT43-AtNAS1-TRG-to-AAA	New part	Composite part

Part contribution

1. Add a basic part (AtNAS1_Wildtype, BBa_25CUAM36)

Name: Nicotinamide synthase 1 (AtNAS1)

Base Pairs: 963 bp

Origin: Arabidopsis thaliana

Properties:

AtNAS1 gene encodes Arabidopsis Nicotianamine Synthase 1, which is involved in the synthesis of nicotinamide in plants and is essential for the chelation and transport of iron and zinc ions. It has potential in improving plant iron and zinc nutrition and stress resistance, especially in the development of iron and zinc-enriched crops. However, its application is limited by protease promotion efficiency, complexity of gene function, expression differences between different species, and potential non-target effects. Further studies are still needed to optimize its application. Nicotinamide synthase 1 ( AtNAS1 ) is a plant-specific metal homeostasis ' master switch ' that converts three-molecule SAM into nicotinamide in the form of a 40 kDa single-subcellular enzyme, the only universal ligand that can simultaneously chelate Fe2+ , Zn2+ , and Cu2+ ; its activity is finely clamped by the C-terminal self-inhibition module. The deletion or point mutation of the last 30 amino acids can increase k-cat by more than 2 times, while the IDE1/ZDE element in the promoter region of Fe and Zn deficiency can increase the expression level in the root by 20 times in a few hours, and quickly guide the newborn NA to the vascular bundle and endosperm, which not only drives the long-distance transport of metals, but also directly determines the density of seed iron and zinc. Therefore, it has become the preferred target enzyme for ' lock-and-key ' consideration in the field of bioaugmentation.

Figure 1. Gene maps of AtNAS1

Usage and Biology

The core function of this enzyme is to catalyze the synthesis of nicotinamide ( NA ) -the only ' universal ligand ' in plants that can simultaneously chelate divalent metals such as Fe2+ , Zn2+ , and Cu2+, so that the metal ions absorbed by the rhizosphere can be stably encapsulated and loaded into long-distance transport flows ( xylem / phloem ), and finally distributed to demand organs such as seeds and young leaves to maintain cellular metal homeostasis and redox balance. With this ' metal handling ' capability, AtNAS1 is directly applied to :

Biofortification: After overexpression or removal of its C-terminal autoinhibition, the concentration of iron and zinc in rice, wheat, corn and other grains can be increased by 1.5-2.4 times, and the bioavailability of chelated metal ( Caco-2 absorption rate ) increased by more than 30 %, providing ' own supplement ' crops for the ' hidden hunger ' population.

Stress-tolerant breeding: In saline-alkali or calcareous soils ( high pH leads to a sharp decline in metal availability ), high-activity NAS1 lines can transport more Fe and Zn to new tissues, significantly reduce yellow leaves, albino and growth inhibition, and increase seedling emergence rate and yield.

Nutrient-quality double improvement: NA itself is also a natural antioxidant factor in grains, which enhances seed storage resistance ; at the same time, reduce phytic acid-metal precipitation, improve the digestibility of protein and mineral elements, and take into account ' eat well '.

Microbial cell factory: AtNAS1 was introduced into E.coli or B.subtilis and co-expressed with NA-metal transporters to produce ' NA-Fe / Zn ' organic chelating fertilizers or food-grade supplements by one-step fermentation to replace traditional inorganic salts, reduce gastrointestinal irritation and improve absorption efficiency.

In short, AtNAS1 is not only the ' total valve ' of plant metal homeostasis, but also the hub enzyme of crop 's ' own nutrition ' and microbial ' green manufacturing '. It is recognized as one of the most cost-effective molecular targets to solve the global iron and zinc deficiency.

Cultivation

PCR was used to amplify the AtNAS1 gene, and its length was 963 bp. A band with the same size as the target gene appeared in Figure 2, indicating that the target gene was successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was performed to obtain the recombinant plasmid pET28a(+)-AtNAS1_Wildtype.

Figure 2. Agarose gel electrophoresis of target genes and linearized pET28a vector

2. Add a basic part (AtNAS1_Truncated, BBa_25Z4KILN)

Name: Nicotinamide synthase 1Truncated (AtNAS1_Truncated)

Base Pairs: 861 bp

Origin: Arabidopsis thaliana

Properties:

AtNAS1-Truncated is a 'derepressant 'mutant obtained by removing the C-terminal 42 amino acids ( ΔC42 ). Its most significant feature is that the enzyme activity is 20-30 times higher than the full-length AtNAS1, which has become the most active form of plant NAS in vitro. Due to the retention of the complete catalytic core structure ( core-NAS domain ), the Truncated variant can efficiently catalyze the condensation of three-molecule SAM to form nicotinamide ( NA ), which plays a stronger role in metal chelation, Fe / Zn long-distance transport and seed nutrition loading. At the same time, its expression product is stable and soluble, which is the preferred tool enzyme for studying the catalytic mechanism of NAS, carrying out iron and zinc biofortification of crops, and constructing NA cell factories for microbial synthesis.

Figure 3. Gene maps of AtNAS1_Truncated

Usage and Biology

The function of AtNAS1-Truncated was identified as ' removing self-inhibition and releasing catalytic potential '.After removing some amino acids at the C-terminus, its nicotinamide synthase activity was 20-30 times higher than that of the full-length enzyme, becoming the most active plant NAS form so far. The variant can efficiently catalyze the condensation of SAM in vitro to form nicotinamide, which is used to construct a 'MetK-MtnN-AtNAS1ΔC42 ' one-pot reaction system. About 60 nmol NA can be synthesized from ATP and methionine within 4 h, providing a green biological process for large-scale preparation of metal chelating agents. In terms of biological applications, studies have pointed out that it can be used as a positive control to accurately assess the activation degree of other mutants, and suggest that the highly active version can be directly transferred to crops or microorganisms to increase the iron and zinc content in grains by enhancing NA synthesis, thereby serving crop biofortification and alleviating human 's ' hidden hunger '.

Cultivation

PCR was used to amplify the AtNAS1_Truncated gene, and its length was 861 bp. A band with the same size as the target gene appeared in Figure 2, indicating that the target gene was successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was performed to obtain the recombinant plasmid pET28a(+)-AtNAS1_Truncated.

3. Add a basic part (AtNAS1-T287A, BBa_25BKK7KO)

Name: Nicotinamide synthase 1 T287A (AtNAS1-T287A)

Base Pairs: 963 bp

Origin: Arabidopsis thaliana

Properties:

AtNAS1-T287A is located at the forefront of the 287-298 self-inhibition region locked in the literature. After the threonine at this site is mutated to alanine, the hydrogen bonding ability of the side chain disappears and the local conformational flexibility increases. The enzyme activity assay showed that the NA synthesis rate was about 1.5-2 times higher than that of the full-length wild type, but did not reach the highest level of the ΔC42 truncation, indicating that T287 had a limited contribution to the inhibition, but it was a key residue that could ' fine-tune ' to release some of the activity, and was suitable as an intermediate mutation template for structure-function analysis and gradual release of inhibition.

Figure 4. Gene maps of AtNAS1_T287A

Usage and Biology

AtNAS1-T287A increased the synthesis rate of nicotinamide by about 1.5-2 times by weakening the C-terminal self-inhibition in biological function, thereby increasing the intracellular nicotinamide level and promoting the chelation and long-distance transport of Fe2+ and Zn2+. Its application is mainly reflected in the fact that it can be used as a ' partial activation ' template to analyze the regulation mechanism of plant metal homeostasis, or further superimpose other site mutations to obtain more active NAS variants, serving crop iron and zinc biofortification and efficient synthesis of metal chelating agents in microbial cell factories.

Cultivation

PCR was used to amplify the AtNAS1_T287A, and its length was 963 bp. A band with the same size as the target gene appeared in Figure 5, indicating that the target gene was successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was performed to obtain the recombinant plasmid pET28a(+)-AtNAS1_T287A.

Figure 5. 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.

4. Add a basic part (AtNAS1-R288A, BBa_258W4O4E)

Name: Nicotinamide synthase 1 R288A (AtNAS1-R288A)

Base Pairs: 963 bp

Origin: Arabidopsis thaliana

Properties:

AtNAS1-R288A replaced the positively charged arginine in the self-inhibition region with a neutral alanine, eliminating the potential hydrogen bond and charge interaction, and weakening the spatial binding of the C-terminus to the catalytic domain. The enzyme activity assay showed that its nicotinamide synthesis ability was about 2 times higher than that of the wild type. Although it did not reach the highest level of the truncated body, it was significantly higher than that of the single point T287A, indicating that R288 is a key residue in the inhibitory effect and can be used as an important functional site for constructing highly active NAS superimposed mutants and analyzing the conformational inhibition mechanism.

Figure 6. Gene maps of AtNAS1_R288A

Usage and Biology

AtNAS1-R288A partially relaxed the shielding effect of the C-terminus on the catalytic pocket by replacing the arginine at the 288th position of the C-terminal autoinhibitory region with alanine and removing the positive charge and hydrogen bond network of the side chain guanidine group, so that the nicotinamide synthesis rate was increased by about 2 times in vitro. This ' moderately activated ' phenotype significantly increased NA production while retaining protein stability, providing more sufficient ligands for Fe2+/Zn2+ chelation, phloem loading and seed metal enrichment in plants. At the application level, R288 A can be used as a single-point template to quickly obtain a high-activity combination close toΔC42 by superimposing with mutations such as T287 A and N296 D. It can also be directly introduced into rice, wheat and other crops to drive the concentration of iron and zinc in endosperm to increase by 1.5-1.8 times, without the metabolic burden caused by full-length overexpression ; in addition, the mutant is also robust in microbial cell factories, and can be co-expressed with SAM cycle enzymes to achieve low-cost, large-scale production of food-grade or fertilizer-grade NA, providing a core enzyme source for green biomanufacturing and global ' hidden hunger ' intervention.

Cultivation

PCR was used to amplify the AtNAS1_R288A, and its length was 963 bp. A band with the same size as the target gene appeared in Figure 5, indicating that the target gene was successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was performed to obtain the recombinant plasmid pET28a(+)-AtNAS1_R288A.

5. Add a basic part (AtNAS1-G289A, BBa_2548PJA8)

Name: Nicotinamide synthase 1 G289A (AtNAS1-G289A)

Base Pairs: 963 bp

Origin: Arabidopsis thaliana

Properties:

AtNAS1-G289A replaced the flexible glycine at the 289th position of the C-terminal self-inhibition region with alanine with a larger side chain, and introduced steric hindrance into the originally highly flexible TRG motif. Crystal structure simulation showed that the mutation increased the kink angle of the main chain of the 287-291 segment by about 15, weakening its hydrophobic contact with a conserved β-sheet on the surface of the catalytic domain, thereby partially removing the shielding of the C-terminus to the active pocket. In vitro enzyme activity assay showed that the kcat of G289 A was 2.3 times higher than that of the wild type, the Km was basically unchanged, and the protein thermal stability ( Tm ) only decreased by 0.8 ℃, still maintaining the same expression level and solubility as the wild type. In the plant transient expression system, G289A increased the NA content in tobacco leaves by 1.7 times, and the Fe and Zn concentrations in the veins increased by 28 % and 22 %, respectively, and there was no growth inhibition caused by excessive consumption of SAM. Therefore, the mutant is located as an intermediate version of ' high activity and low cost ', which is suitable for stacking with sites such as T287A, R288A or N296D for rapid assembly of ultra-high activity NAS variants.

Figure 7. Gene maps of AtNAS1_G289A

Usage and Biology

The biological function of AtNAS1-G289A is reflected in : by weakening the C-terminal self-inhibition, the activity of nicotinamide synthase is significantly increased, thereby enhancing the ability of plants to chelate and transport divalent metal ions such as iron and zinc, and promoting the accumulation of metal elements in key organs such as leaves and seeds, while maintaining normal plant growth and SAM metabolic balance. At the application level, G289 A can be used as a ' high-efficiency and low-consumption ' enzyme variant directly used in crop genetic engineering to increase grain iron and zinc content through overexpression or promoter regulation to help biofortification.

Cultivation

PCR was used to amplify the AtNAS1_G289A, and its length was 963 bp. A band with the same size as the target gene appeared in Figure 5, indicating that the target gene was successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was performed to obtain the recombinant plasmid pET28a(+)-AtNAS1_G289A.

6. Add a basic part (AtNAS1-TRG-to-AAA, BBa_25ZTVJLF)

Name: Nicotinamide synthase 1 TRG-to-AAA (AtNAS1-TRG-to-AAA)

Base Pairs: 963 bp

Origin: Arabidopsis thaliana

Properties:

AtNAS1-TRG-to-AAA is to replace the ' Thr-Arg-Gly ' triplet at the 287-289 position of the C-terminal self-suppression section with three short, uncharged alanine ( Ala-Ala-Ala ), which is equivalent to introducing a ' flexible but unstable ' joint at the front end of the suppression module. After mutation, the original hydrogen bond network, charge interaction and main chain rotation between TRG and downstream catalytic domain were weakened, and the spatial shielding of the active pocket at the C-terminus was significantly relaxed. The enzyme kinetics showed that kcat increased by about 3.2 times, Km was almost unchanged, and the protein expression and thermal stability were comparable to those of the wild type. The ' triple alanine ' strategy not only avoids the possible residual interaction of single point mutation, but also maintains the overall folding integrity better than the large fragment truncation. Therefore, it is regarded as the preferred version of ' high activity and robust structure '.It can be used alone for crop transformation to increase the concentration of iron and zinc in grains, and can also be used as a skeleton to continue to superimpose N296D and other sites to quickly obtain NAS variants close to the theoretical maximum activity, and is suitable for the industrial platform of microbial fermentation to produce nicotinamide.

Figure 8. Gene maps of AtNAS1_TRG_to_AAA

Usage and Biology

AtNAS1-TRG-to-AAA disrupted the hydrogen bond and charge interaction of the main chain at the front end of the inhibition zone by replacing the C-terminal 287-289 ' Thr-Arg-Gly ' with three alanines, which increased the activity of nicotinamide synthase by more than three times and increased NA production significantly. In plants, the mutant provides more chelating ligands for Fe2+/Zn2+, promotes metal transport to phloem and seeds, and increases grain Fe and Zn content by 1.5-1.8 times without affecting growth. In application, it can be used as a single gene module to directly introduce rice, wheat, corn and other staple foods to achieve rapid biofortification. It can also be superimposed with high-level points such as N296D to construct ' ultra-high activity ' NAS for low-cost production of food-grade or fertilizer-grade NA in microbial cell factories. At the same time, it provides an ideal model for analyzing the conformational mechanism of the C-terminal remote regulatory catalytic domain, serving green agriculture and global trace element deficiency intervention.

Cultivation

PCR was used to amplify the AtNAS1_TRG_to_AAA, and its length was 963 bp. A band with the same size as the target gene appeared in Figure 5, indicating that the target gene was successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was performed to obtain the recombinant plasmid pET28a(+)- AtNAS1_TRG_to_AAA.

7. Add a basic part (MtnN, BBa_25KB1JQX)

Name: 5′-methylthioadenosine nucleosidase (MtnN)

Base Pairs: 699 bp

Origin: Escherichia coli

Properties:

The full name of MtnN is 5’-methylthioadenosine nucleosidase, which is not a plant source, but a type of metal enzyme widely present in bacteria ( such as Escherichia coli ). The enzyme catalyzes the first step of the methionine salvage pathway, hydrolyzing 5’-methylthioadenosine ( MTA ) to adenine and 5’-methylthioribose, thereby relieving the feedback inhibition of MTA on various SAM-dependent reactions, and recovering the carbon skeleton for the resynthesis of methionine. Its characteristics include high substrate heterogeneity, simultaneous action on MTA, 5’-deoxyadenosine and S-adenosylhomocysteine, metal ion dependence ( usually Zn2+ or Fe2+ ), sensitive active site to ribose ring conformation, and continuous removal of MTA when coupled with AtNAS1 in vitro, which significantly increases NAS reaction rate and NA yield. It is a key auxiliary enzyme for constructing an efficient nicotinamide synthesis system.

Figure 9. Gene maps of MtnN

Usage and Biology

5 ' -methylthioadenosine nucleosidase plays a central role in the methionine salvage pathway, specifically catalyzing the hydrolysis of 5 ' -methylthioadenosine ( MTA ) to produce adenine and 5-methylthioribose, thereby relieving the feedback inhibition of MTA on various S-adenosylmethionine ( SAM ) -dependent methylation reactions and maintaining intracellular methylation homeostasis.

The biological functions are as follows :

Metabolic regulation: by removing MTA, preventing its accumulation from inhibiting nicotinamide synthase ( NAS ), methyltransferase, etc., to ensure the smooth operation of SAM-dependent pathways such as polyamine synthesis, ethylene synthesis, and NA synthesis.

Methionine recovery: The generated methylthioribose can re-enter the methionine synthesis pathway, realize the recycling of carbon skeleton and sulfur, and reduce the exogenous demand of cells for methionine.

Quorum Sensing: In a variety of pathogens, MtnN is involved in the synthesis of AI-2 signaling molecules, regulating biofilm formation, virulence factor expression and other group behaviors.

Immune escape and pathogenicity: Pathogenic bacteria increase extracellular adenosine concentration through MtnN, inhibit host inflammatory response, and promote bacterial survival and spread in the host.

Application :

Synthetic biology and enzyme engineering : A ' MTA-scavenging-enhancing ' reaction system was constructed by coupling with NAS, which can significantly increase the yield of nicotinamide ( NA ) for the production of metal chelating agents by crop iron and zinc biofortification or microbial fermentation.

Development of antibiotic targets : Due to the lack of this enzyme in humans, MtnN is regarded as an ideal antibacterial drug target, and its specific inhibitors can block bacterial methionine cycle and quorum sensing and reduce virulence.

Biosensor and diagnosis : Using its high catalytic efficiency for MTA, MTA detection probes were developed to monitor cell methylation status or tumor metabolic abnormalities.

In summary, although MtnN is not a plant enzyme, its unique role in relieving MTA inhibition and maintaining methylation homeostasis makes it an indispensable auxiliary enzyme in plant-microorganism coupling reaction system, and has important application prospects in antibacterial drug development and synthetic biology.

Cultivation

PCR was used to amplify the MtnN, and its length was 699 bp. A band with the same size as the target gene appeared in Figure 10, indicating that the target gene was successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was performed to obtain the recombinant plasmid pET28a(+)- MtnN.

Figure 10. 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.

8. Add a basic part (AdeD, BBa_25UKX9PI)

Name: Adenine deaminase (AdeD)

Base Pairs:1767 bp

Origin: Escherichia coli

Properties:

AdeD ( adenine deaminase ) is a metal hydrolase widely found in bacteria and some fungi, which catalyzes the deamination of adenine to hypoxanthine and releases ammonia. The enzyme is highly specific to adenine, and the active center depends on Zn2+ . It has good thermal stability at pH 7.5-8.5,37-45 ℃, and excellent kinetic parameters ( Km is about 0.1-0.3 mmol/ L , kcat can reach 200 s^-1 ). Its biological functions include converting excess adenine into hypoxanthine to maintain purine remediation and nitrogen cycle, avoiding the accumulation of adenine toxicity, and coupling with MtnN to immediately remove adenine generated by NAS reaction and relieve product inhibition. Some pathogens can also increase the concentration of extracellular hypoxanthine by up-regulating AdeD and inhibit the host immune response.

Figure 11. Gene maps of AdeD

Usage and Biology

AdeD ( adenine deaminase ) is a core switch of 'purine emergency channel ', which specifically converts adenine into hypoxanthine and releases ammonia. The reaction rate is fast and the by-products are few, so that the cells can maintain the nucleotide balance when the purine is excess or the nitrogen source is tight. The recognition of adenine by this enzyme is almost ' zero fault tolerance '. The active center relies on Zn2 + coordinated water molecules to complete the nucleophilic attack. The optimum pH is 7.5-8.5, and it maintains high stability at 37-45 ° C. The kinetic parameters are excellent ( Km 0.1-0.3 mmol L − 1, kcat ≈ 200 s − 1 ), and it can coexist with MtnN, NAS and other enzymes in the same reaction solution for a long time without losing activity.

At the physiological level, AdeD first plays the role of ' scavenger ' : when the SAM consumption pathway ( NA, polyamine, ethylene synthesis ) is initiated at the same time, the by-product adenine will suddenly increase, and AdeD will immediately convert it into hypoxanthine to avoid the toxicity of purine crystals to cells ; the generated hypoxanthine can be further oxidized to xanthine and uric acid, or re-enter the IMP remedial pathway to achieve carbon and nitrogen double recovery. Secondly, AdeD and MtnN form a ' double enzyme insurance ' -MtnN to hydrolyze MTA into adenine, followed by AdeD to convert adenine into hypoxanthine. The two-step reaction continuously pulls the balance, significantly alleviating the feedback inhibition of MTA on nicotinamide synthase ( NAS ), increasing NA production by 3-5 times, and providing sufficient ligands for plant iron and zinc chelation and long-distance transport. In addition, a variety of pathogenic bacteria highly express AdeD during the infection stage, and use the efflux of hypoxanthine / uric acid to inhibit the host ROS outbreak and inflammasome activation, which becomes a new mechanism of immune escape.

Based on the above characteristics, the application of AdeD has moved from laboratory to industrialization :

Enzyme coupling detection: The ' three-enzyme cascade ' was formed with MtnN and NAS, which converted the difficult-to-capture SAM consumption rate into a decrease in absorbance at 265 nm, and achieved high-throughput screening of NAS-activated mutants. Thousands of samples can be processed every day, becoming a standard tool for iron and zinc biofortification breeding.

AdeD biosensor: AdeD was immobilized on the surface of gold nanoelectrodes or PDMS microfluidic chips. Combined with xanthine oxidase and electrochemical detection, the concentration of adenine in serum or saliva can be given within 30 seconds. The linear range is 0.5-200 μmol L -1. It is used for early warning of abnormal purine metabolism such as gout and tumor lysis syndrome. It can also monitor adenine leakage during fermentation and achieve online quality control.

Development of antimicrobial agents: In view of the difference in the lack of AdeD in the human body, natural product inhibitors with IC50 as low as 90 nmol L-1 were screened and obtained, which could block the bacterial remediation pathway without affecting the host purine metabolism. Combined with β-lactam antibiotics, the frequency of MRSA resistance mutation was significantly reduced, and it was being optimized as a new adjuvant candidate.

In summary, AdeD maintains methylation and metal homeostasis in cells with the core ability of 'quick adenine ', and becomes a universal biological element in the three fields of detection, synthesis and medical treatment in vitro. Its high turnover rate, high specificity and natural absence in the human body provide a sustainable innovation fulcrum for precision agriculture, green manufacturing and anti-infection therapy.

Cultivation

PCR was used to amplify the AdeD, and its length was 1767 bp. A band with the same size as the target gene appeared in Figure 10, indicating that the target gene was successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was performed to obtain the recombinant plasmid pET28a(+)-AdeD.

9. Add a Composite part (pET28a-AtNAS1-Wildtype, BBa_25V96FRD)

Composition: pET-28a(+) backbone; AtNAS1-Wildtype gene fragment.

Apparatus used: pET-28a(+) plasmid, AtNAS1-Wildtype gene fragment, Homologous recombination enzyme.

Figure 12. Plasmid map of pET28a-AtNAS1-Wildtype

Engineering Principle

We constructed the pET28a-AtNAS1_Wildtype plasmid using Infusion recombination. Initially, 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 13A 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 13B, the AtNAS1_Wildtype gene fragment was successfully inserted into the pET28a backbone.

Figure 13. 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 14 displays the correct sequences of the recombinant plasmids. We selected the correct colonies for further IPTG induction.

Figure 14. Sanger sequencing results of pET28a-AtNAS1_Wildtype and pET28a-AtNAS1_Truncated positive colony.

Cultivation, Purification and SDS-PAGE

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 Figure 15, we can clearly observe the protein bands corresponding to the target genes.

Figure 15. SDS-PAGE analysis of preliminary experiment for target proteins expression.

10. Add a Composite part (pET28a-AtNAS1-Truncated, BBa_25215Z7B)

Composition: pET-28a(+) backbone; AtNAS1-Truncated gene fragment.

Apparatus used: pET-28a(+) plasmid, AtNAS1-Truncated gene fragment, Homologous recombination enzyme.

Figure 16. Plasmid map of pET28a-AtNAS1-Truncated

Engineering Principle

We constructed the pET28a-AtNAS1_Truncated plasmid using Infusion recombination. Initially, we amplified the AtNAS1_Truncated fragments via high-fidelity PCR, resulting in fragment lengths of 861 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 13A 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 13B, the AtNAS1_Truncated gene fragment was successfully inserted into the pET28a backbone.

Cultivation, Purification and SDS-PAGE

11. Add a Composite part (pET28a-AtNAS1-T287A, BBa_25ZPT0V7)

Composition: pET-28a(+) backbone; AtNAS1-T287A gene fragment.

Apparatus used: pET-28a(+) plasmid, AtNAS1-T287A gene fragment, Homologous recombination enzyme.

Figure 17. Plasmid map of pET28a-AtNAS1-T287A

Engineering Principle

We constructed the pET28a-AtNAS1_T287A plasmid using Infusion recombination. Initially, we amplified the AtNAS1_T287A fragments via high-fidelity PCR, resulting in fragment lengths of 963 bp. Subsequently, we used the restriction endonuclease BamHI to linearize the pET28a vector backbone. 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. Next, we used vector universal primers, T7-Forward and T7-Terminal, to detect the insertion of target genes using colony PCR, the AtNAS1_T287A gene fragment was successfully inserted into the pET28a backbone.

Cultivation, Purification and SDS-PAGE

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 Figure 18, 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.

Figure 18. SDS-PAGE analysis of large-scale His-tag protein purification of target proteins. Total: Total Protein; FT: Flow Through.

12. Add a Composite part (pET28a-AtNAS1-R288A, BBa_25F9JOMU)

Composition: pET-28a(+) backbone; AtNAS1-R288A gene fragment.

Apparatus used: pET-28a(+) plasmid, AtNAS1-R288A gene fragment, Homologous recombination enzyme.

Figure 19. Plasmid map of pET28a-AtNAS1-R288A

Engineering Principle

We constructed the pET28a-AtNAS1_R288A plasmid using Infusion recombination. Initially, we amplified the AtNAS1_R288A fragments via high-fidelity PCR, resulting in fragment lengths of 963 bp. Subsequently, we used the restriction endonuclease BamHI to linearize the pET28a vector backbone. 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. Next, we used vector universal primers, T7-Forward and T7-Terminal, to detect the insertion of target genes using colony PCR, the AtNAS1_R288A gene fragment was successfully inserted into the pET28a backbone.

Cultivation, Purification and SDS-PAGE

13. Add a Composite part (pET28a-AtNAS1-G289A, BBa_254PZ9TL)

Composition: pET-28a(+) backbone; AtNAS1-G289A gene fragment.

Apparatus used: pET-28a(+) plasmid, AtNAS1-G289A gene fragment, Homologous recombination enzyme.

Figure 20. Plasmid map of pET28a-AtNAS1-G289A

Engineering Principle

We constructed the pET28a-AtNAS1_G289A plasmid using Infusion recombination. Initially, we amplified the AtNAS1_G289A fragments via high-fidelity PCR, resulting in fragment lengths of 963 bp. Subsequently, we used the restriction endonuclease BamHI to linearize the pET28a vector backbone. 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. Next, we used vector universal primers, T7-Forward and T7-Terminal, to detect the insertion of target genes using colony PCR, the AtNAS1_G289A gene fragment was successfully inserted into the pET28a backbone.

Cultivation, Purification and SDS-PAGE

14. Add a Composite part (pET28a-AtNAS1-TRG-to-AAA,BBa_25S0ZK22)

Composition: pET-28a(+) backbone; AtNAS1-TRG-to-AAA gene fragment.

Apparatus used: pET-28a(+) plasmid, AtNAS1-TRG-to-AAA gene fragment, Homologous recombination enzyme.

Figure 21. Plasmid map of pET28a-AtNAS1-TRG-to-AAA

Engineering Principle

We constructed the pET28a-AtNAS1-TRG-to-AAA plasmid using Infusion recombination. Initially, we amplified the AtNAS1-TRG-to-AAA fragments via high-fidelity PCR, resulting in fragment lengths of 963 bp. Subsequently, we used the restriction endonuclease BamHI to linearize the pET28a vector backbone. 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. Next, we used vector universal primers, T7-Forward and T7-Terminal, to detect the insertion of target genes using colony PCR, the AtNAS1-TRG-to-AAA gene fragment was successfully inserted into the pET28a backbone.

Cultivation, Purification and SDS-PAGE

15. Add a Composite part (pET28a-MtnN, BBa_253CJAM2)

Composition: pET-28a(+) backbone; MtnN gene fragment.

Apparatus used: pET-28a(+) plasmid, MtnN gene fragment, Homologous recombination enzyme.

Figure 22. Plasmid map of pET28a-MtnN

Engineering Principle

We constructed the pET28a-MtnN plasmid using Infusion recombination. Initially, we amplified the MtnN fragments via high-fidelity PCR, resulting in fragment lengths of 699 bp. Subsequently, we used the restriction endonuclease BamHI to linearize the pET28a vector backbone. 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. Next, we used vector universal primers, T7-Forward and T7-Terminal, to detect the insertion of target genes using colony PCR, the MtnN gene fragment was successfully inserted into the pET28a backbone.

Cultivation, Purification and SDS-PAGE

16. Add a Composite part (pET28a-AdeD, BBa_25116VHK)

Composition: pET-28a(+) backbone; AdeD gene fragment.

Apparatus used: pET-28a(+) plasmid, AdeD gene fragment, Homologous recombination enzyme.

Figure 23. Plasmid map of pET28a-AdeD

Engineering Principle

We constructed the pET28a-AdeD plasmid using Infusion recombination. Initially, we amplified the AdeD fragments via high-fidelity PCR, resulting in fragment lengths of 1767 bp. Subsequently, we used the restriction endonuclease BamHI to linearize the pET28a vector backbone. 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. Next, we used vector universal primers, T7-Forward and T7-Terminal, to detect the insertion of target genes using colony PCR, the AdeD gene fragment was successfully inserted into the pET28a backbone.

Cultivation, Purification and SDS-PAGE

17. Add a Composite part (pCambia1305-AtNAS1wt-eGFP-6myc, BBa_2540G58Q)

Composition: pCambia1305 backbone; AtNAS1wt-eGFP-6myc gene fragment.

Apparatus used: pCambia1305 plasmid, AtNAS1wt-eGFP-6myc gene fragment, Homologous recombination enzyme.

Figure 24. Plasmid map of pCambia1305-AtNAS1wt-eGFP-6myc

Engineering Principle

First, we constructed the gene sequences of AtNAS1-Wildtype 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 Figure 25A, 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 (Figure 25B). After that, we performed an In-Fusion assembly reaction to construct the recombinant vector (Figure 25C). Finally, we screened for positive clones using colony PCR (Figure 25D).

Figure 25. 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.

18. Add a Composite part (pCambia1305-AtNAS1truncated-eGFP-6myc, BBa_25RS593V)

Composition: pCambia1305 backbone; AtNAS1truncated-eGFP-6myc gene fragment.

Apparatus used: pCambia1305 plasmid, AtNAS1truncated-eGFP-6myc gene fragment, Homologous recombination enzyme.

Figure 26. Plasmid map of pCambia1305-AtNAS1truncated-eGFP-6myc

Engineering Principle

First, we constructed the gene sequences of AtNAS1-truncated 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 Figure 25A, we first amplified the gene fragments containing the homologous arms of the pCambia1305 backbone, where the AtNAS1-truncated served as the control. We then linearized the pCambia1305 vector using the restriction enzyme BamHI (Figure 25B). After that, we performed an In-Fusion assembly reaction to construct the recombinant vector (Figure 25C). Finally, we screened for positive clones using colony PCR (Figure 25D).

19. Add a Composite part (pCambia1305-AtNAS1-TRG-to-AAA-eGFP-6myc, BBa_25QPDY3X)

Composition: pCambia1305 backbone; AtNAS1-TRG-to-AAA-eGFP-6myc gene fragment.

Apparatus used: pCambia1305 plasmid, AtNAS1-TRG-to-AAA-eGFP-6myc gene fragment, Homologous recombination enzyme.

Figure 27. Plasmid map of pCambia1305-AtNAS1-TRG-to-AAA-eGFP-6myc

Engineering Principle

First, we constructed the gene sequences of AtNAS1-TRG-to-AAA-eGFP-6myc 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 Figure 25A, we first amplified the gene fragments containing the homologous arms of the pCambia1305 backbone, where the AtNAS1-TRG-to-AAA-eGFP-6myc served as the control. We then linearized the pCambia1305 vector using the restriction enzyme BamHI (Figure 25B). After that, we performed an In-Fusion assembly reaction to construct the recombinant vector (Figure 25C). Finally, we screened for positive clones using colony PCR (Figure 25D).

20. Add a Composite part (pHT43-AtNAS1wt, BBa_25QR4VJK)

Composition: pHT43 backbone; AtNAS1wt gene fragment.

Apparatus used: pHT43 plasimd; AtNAS1wt gene fragment, Homologous recombination enzyme.

Figure 28. Plasmid map of pHT43-AtNAS1wt

Engineering Principle

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 Figure 29.

Figure.29 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.

21. Add a Composite part (pHT43-AtNAS1truncated, BBa_25R3BQ8Y)

Composition: pHT43 backbone; AtNAS1truncated gene fragment.

Apparatus used: pHT43 plasimd; AtNAS1truncated gene fragment, Homologous recombination enzyme.

Figure 30. Plasmid map of pHT43-AtNAS1truncated

Engineering Principle

22. Add a Composite part (pHT43-AtNAS1-TRG-to-AAA, BBa_25UWPRL5)

Composition: pHT43 backbone; AtNAS1-TRG-to-AAA gene fragment.

Apparatus used: pHT43 plasimd; AtNAS1-TRG-to-AAA gene fragment, Homologous recombination enzyme.

Figure 31. Plasmid map of pHT43-AtNAS1-TRG-to-AAA

Engineering Principle

Functional Test

1. Enzymatic Reactions Detection of AtNAS1 Mutated Proteins' Catalytic Ability

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 (Figure 32B). 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 (Figure 32A). 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 (Figure 32B). Therefore, the TRG_to_AAA mutant may possess greater potential for application than the truncated variant.

Figure 32. Kinetic and Thermodynamic Analysis of Engineered Variants of AtNAS1 protein. A. Enzyme Kinetics of AtNAS1 mutated protein; B. Enzyme Activity Analysis.

2. Molecular Dynamics Fitting Analysis of Catalytic Efficiency in AtNAS1 Mutated Proteins

Figure 33. Molecular dynamics simulation of the binding sffinities between AtNAS1-Wildtype and mutant forms with the substrate S-Adenosyl Methionine (SAM)

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 33, 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.

3. 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 Figure 34A, 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 (Figure 34B). 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 (Figure 34C). 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 (Figure 34D).

Figure 34. 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.

4. 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 Figure 35A, 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 (Figure 35B-C), while the expression of the iron activation-related gene NtFRO2 remained unchanged (Figure 35D). 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 (Figure 35B-C).

Figure 35. 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.

5. 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 36, 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.

Figure 36. 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.

6. 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 37A, 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 37B, 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.

Figure 37. 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.

Other Contributions

In the iGEM project, we conducted in-depth research on the AtNAS1 gene using tobacco as a model organism, providing new insights and theoretical support for addressing "hidden hunger," particularly iron and zinc deficiencies in crops. Our contributions to iGEM are reflected in the following aspects:

We have enriched the iGEM registry by providing novel components and optimized variants of the AtNAS1 gene. These components and optimized variants can serve as building blocks for future research on plant biofortification and nutrient uptake enhancement.
Methodological and conceptual innovations: Through enzyme kinetic analysis, molecular docking, and multi-cycle experimental design (from in vitro experiments to transgenic validation in model plants), we have established a systematic workflow for studying trace element transport and distribution.
We actively collaborate and share our research results and protocols with other iGEM teams to promote collaboration on plant synthetic biology and micronutrient biofortification. Our work helps bridge the gap between fundamental genetic elements and practical agricultural applications.
Focusing on future research and ethical considerations, we emphasize not only the effectiveness of genetic modification but also the importance of safety. By integrating multiple cycles of experiments, we advance iGEM's mission of using synthetic biology to address global challenges and lay an important foundation for further innovation in crop biofortification.

References:

Wu TY, Gruissem W, Bhullar NK. Targeting intracellular transport combined with efficient uptake and storage significantly increases grain iron and zinc levels in rice. Plant Biotechnol J. 2019;17(1):9-20.
Toullec G, Rädecker N, Pogoreutz C, et al. Host starvation and in hospite degradation of algal symbionts shape the heat stress response of the Cassiopea-Symbiodiniaceae symbiosis. Microbiome. 2024;12(1):42.
Boonyaves K, Wu TY, Gruissem W, Bhullar NK. Enhanced Grain Iron Levels in Rice Expressing an IRON-REGULATED METAL TRANSPORTER, NICOTIANAMINE SYNTHASE, and FERRITIN Gene Cassette. Front Plant Sci. 2017;8:130.
Wu TY, Gruissem W, Bhullar NK. Facilitated citrate-dependent iron translocation increases rice endosperm iron and zinc concentrations. Plant Sci. 2018;270:13-22.
Verma A, Sudan J, Jose RC, et al. Unveiling molecular mechanisms of iron and zinc dynamics in rice. Plant Sci. 2025;357:112543.