1. Background & Inspiration

Globally, deficiencies in the trace elements iron (Fe) and zinc (Zn) constitute the most prevalent form of malnutrition, known as "hidden hunger." [1]. The World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO) estimate that this affects over 2 billion people, particularly in developing countries [2]. Iron deficiency is the leading cause of anemia worldwide, severely impacting both pregnancy and childhood; zinc deficiency, on the other hand, impairs immune function and increases children's risk of infectious diseases such as diarrhea and pneumonia [3-5].

This problem is particularly acute in regions such as sub-Saharan Africa that rely on single staple crops (such as corn, rice, and wheat)[7]. The endosperm of these crops is naturally low in Fe/Zn and contains high concentrations of phytates. Consequently, even adequate dietary intake may not meet the body's micronutrient needs[8]. The knock-on effects of this malnutrition not only lead to significant medical expenses but also reduce labor productivity, posing a long-term obstacle to global economic development[9].

Traditional interventions to address hidden hunger primarily include dietary supplements, which are effective for acute deficiencies but difficult to scale up, facing compliance and economic sustainability challenges[11-12]. Fortification involves adding trace nutrients to processed foods. This approach relies on processing facilities and well-established distribution systems, resulting in low coverage for populations living in remote areas and relying primarily on subsistence farming. While dietary diversification may be the most ideal long-term strategy, it is difficult to achieve in the short term due to economic constraints, climate change, and limited agricultural infrastructure[13].

Given the limitations of traditional strategies, genetic engineering-driven crop biofortification has emerged as a sustainable and cost-effective alternative[15]. Biofortification aims to increase the trace element concentration in edible parts of crops by modifying their genetic characteristics[16]. Its core advantage is that once highly nutritious crop varieties are developed, farmers can cultivate them continuously, with near-zero efficiency costs, and their nutritional coverage is widespread, reaching the poorest populations most in need[17].

Fig 4. Overview of three major biofortification strategies to enhance nutritional contents in various crops (Created in BioRender.com)[18].

The nicotianamine synthase (NAS) gene family occupies a central position in the plant trace element metabolism regulatory network. NAS catalyzes the synthesis of nicotinamide (NA), a non-proteinogenic amino acid with strong metal chelating capacity[19]. Therefore, increasing NAS expression or activity is widely considered a universal and effective genetic strategy for increasing iron and zinc content in crops. However, previous studies have shown that simply overexpressing NAS often results in less than expected iron and zinc enrichment in crop endosperm, suggesting that the protein may have inherent activity-limiting mechanisms[20].

This project was initiated based on an in-depth analysis of existing bottlenecks in biofortification. Literature reports suggest that the C-terminus of the AtNAS1 protein may function as an autoinhibitory domain, limiting the catalytic efficiency of the full-length enzyme through association with the active site or conformational changes. This discovery directly inspired the design of this project. If the C-terminal autoinhibitory signal can be precisely disrupted or removed through site-directed mutagenesis, the catalytic efficiency of AtNAS1 could theoretically be improved, leading to higher concentrations of nicotinamide in plants and more efficient transport and distribution of iron and zinc from vegetative to edible organs. This provides novel insights and technical support for addressing global "hidden hunger."

2. Design

This project focuses on optimizing the activity of natural proteins and enhancing biofortification effects, with experimental design divided into three levels.

1. Protein Engineering: Autoinhibition Removal and High-Efficiency Enzyme Screening

Based on tertiary structure prediction and conservation analysis of AtNAS1, key amino acid residues at the C-terminus that may affect substrate binding or active site conformation were precisely identified. Multiple point mutations (such as alanine scanning) were designed to disrupt autoinhibitory signals. Prokaryotic Expression and Purification: The WT and all mutants were cloned into the pET28a vector and expressed in E. coli. High-purity protein purification was achieved using nickel affinity chromatography and gel filtration chromatography to ensure the accuracy of subsequent enzymatic analysis. Using S-Adenosyl Methionine (SAM) as a substrate, the catalytic efficiency of the wild-type, truncated form (positive control), and all point mutants was accurately determined.

2. Molecular Mechanism Analysis, Gene Expression Levels, and Subcellular Localization

A tobacco transient expression system was used to rapidly verify the effects of mutants on the endogenous gene. qPCR was used to analyze changes in the transcriptional levels of target genes, including key metal transporters. Subcellular localization and functional zonation: An AtNAS1-GFP fusion expression vector was constructed and localized in tobacco epidermal cells using confocal microscopy, with particular attention paid to whether its distribution in the cytoplasm and endoplasmic reticulum was altered. Changes in localization may indicate that the mutant affects transmembrane transport of NA or metal chelation efficiency in specific organelles.

3. Biofortification Verification

Mutants with higher activity were constructed on new vectors and transformed into Bacillus subtilis. Resistance-positive plants were screened and grown under standardized soil culture conditions to observe tobacco growth. Trace element quantification: Tobacco leaf samples were collected and, after dry ashing or wet digestion, iron and zinc content was accurately quantified using highly sensitive inductively coupled plasma mass spectrometry (ICP-MS) to quantify the biofortification effect.

3. Goal

Identify and validate AtNAS1 mutants with significantly higher catalytic efficiency than the wild-type, achieving significant and stable increases in Fe and Zn content in tobacco plants. This provides direct and robust proof-of-concept for the subsequent application of this technology in the biofortification of major crops such as maize, rice, and wheat. The ultimate goal is to provide an economically sustainable and environmentally friendly genetic engineering solution to address global "hidden hunger" and improve the health of vulnerable populations in developing regions.

4. References

1. Kumar A, Kerketta A, Dewali S, et al. Tackling hidden hunger: understanding micronutrient deficiency and effective mitigation strategies[M]//Emerging solutions in sustainable food and nutrition security. Cham: Springer International Publishing, 2023: 305-319.

2. Canton H. Food and agriculture organization of the United Nations—FAO[M]//The Europa directory of international organizations 2021. Routledge, 2021: 297-305.

3. Means R T. Iron deficiency and iron deficiency anemia: implications and impact in pregnancy, fetal development, and early childhood parameters[J]. Nutrients, 2020, 12(2): 447.

4. Abu-Ouf N M, Jan M M. The impact of maternal iron deficiency and iron deficiency anemia on child’s health[J]. Saudi medical journal, 2015, 36(2): 146.

5. Durrani A M, Parveen H. Zinc deficiency and its consequences during pregnancy[J]. Microbial biofertilizers and micronutrient availability: The role of zinc in agriculture and human health, 2021: 69-82.

6. Huertas R, Karpinska B, Ngala S, et al. Biofortification of common bean (Phaseolus vulgaris L.) with iron and zinc: Achievements and challenges[J]. Food and Energy Security, 2023, 12(2): e406.

7. Frossard E, Bucher M, Mächler F, et al. Potential for increasing the content and bioavailability of Fe, Zn and Ca in plants for human nutrition[J]. Journal of the Science of Food and Agriculture, 2000, 80(7): 861-879.

8. Singh D, Prasanna R. Potential of microbes in the biofortification of Zn and Fe in dietary food grains. A review[J]. Agronomy for Sustainable Development, 2020, 40(2): 15.

9. Knoetze C. Feasting on foreign aid: a political economic examination of foreign aid's potential role in perpetuating chronic hunger and starvation in Malawi[J]. 2015.

10. Benjamin J, Idowu O, Babalola O K, et al. Cereal production in Africa: the threat of certain pests and weeds in a changing climate—a review[J]. Agriculture & Food Security, 2024, 13(1): 18.

11. Lowe N M. The global challenge of hidden hunger: perspectives from the field[J]. Proceedings of the Nutrition Society, 2021, 80(3): 283-289.

12. Hodge J. Hidden hunger: approaches to tackling micronutrient deficiencies[J]. 2016.

13. Mustafa M A, Mayes S, Massawe F. Crop diversification through a wider use of underutilised crops: A strategy to ensure food and nutrition security in the face of climate change[M]//Sustainable solutions for food security: combating climate change by adaptation. Cham: Springer International Publishing, 2019: 125-149.

14. Kumar A, Kerketta A, Dewali S, et al. Tackling hidden hunger: understanding micronutrient deficiency and effective mitigation strategies[M]//Emerging solutions in sustainable food and nutrition security. Cham: Springer International Publishing, 2023: 305-319.

15. Oguamanam C. Toward a Constructive Engagement: Agricultural Biotechnology as a Public Health Incentive in Less-Developed Countries[J]. J. Food L. & Pol'y, 2011, 7: 257.

16. Ofori K F, Antoniello S, English M M, et al. Improving nutrition through biofortification–A systematic review[J]. Frontiers in nutrition, 2022, 9: 1043655.

17. Malik K A, Maqbool A. Transgenic crops for biofortification[J]. Frontiers in Sustainable Food Systems, 2020, 4: 571402.

18. Shohael A M, Kelly J, Venkataraman S, et al. Unlocking opportunities and overcoming challenges in genetically engineered biofortification[J]. Nutrients, 2025, 17(3): 518.

19. Zhou X, Li S, Zhao Q, et al. Genome-wide identification, classification and expression profiling of nicotianamine synthase (NAS) gene family in maize[J]. BMC genomics, 2013, 14(1): 238.

20. 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.

21. Zhou X, Li S, Zhao Q, et al. Genome-wide identification, classification and expression profiling of nicotianamine synthase (NAS) gene family in maize[J]. BMC genomics, 2013, 14(1): 238.