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In the post-Green Revolution era, disparities in dietary access, rising obesity rates, demographic shifts, adoption of plant-based diets, and the impact of climate change collectively contribute to a progressive decline in dietary nutritional value, exacerbating B vitamin deficiencies across both low- and high-income countries. While the prevailing focus of biofortification has been on three micronutrients - provitamin A, iron, and zinc - utilizing conventional breeding, it is imperative to diversify biofortification strategies to combat micronutrient malnutrition. Metabolic engineering, facilitated by biotechnological tools, presents a promising avenue, contingent upon advances in fundamental knowledge, technological innovation, regulatory updates, and sustained public funding. Recognizing the intricate metabolic interplay of B vitamins in plants and humans, a comprehensive 'from metabolism to metabolism' approach is crucial for designing effective biofortification strategies that target multiple vitamins. This holistic perspective also extends beyond individual crops to encompass the entire food chain, a complex socioeconomic ecosystem that necessitates a paradigm shift, prioritizing quality over quantity., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Micronutrient deficiencies include shortages of vitamins and minerals. They affect billions of people and are associated with long-range effects on health, learning ability, and huge economic losses. Biofortification of multiple micronutrients can play an important role in combating malnutrition. The challenge, however, is to balance plant growth with nutrient requirements for humans. Here, we summarize the major progress about vitamin biosynthesis and its response to the changing environment. We discuss the interactions among vitamins as well as possible strategies for vitamin biofortification. Finally, we propose to integrate new breeding technologies with metabolic pathway modification to facilitate the biofortification of crops, thereby alleviating the hidden hunger of target populations., (Copyright © 2020 The Author. Published by Elsevier Inc. All rights reserved.)

Ending all forms of hunger by 2030, as set forward in the UN-Sustainable Development Goal 2 (UN-SDG2), is a daunting but essential task, given the limited timeline ahead and the negative global health and socio-economic impact of hunger. Malnutrition or hidden hunger due to micronutrient deficiencies affects about one third of the world population and severely jeopardizes economic development. Staple crop biofortification through gene stacking, using a rational combination of conventional breeding and metabolic engineering strategies, should enable a leap forward within the coming decade. A number of specific actions and policy interventions are proposed to reach this goal.

Folates are essential vitamins in the human diet. Folate deficiency is still very common, provoking disorders such as birth defects and anemia. Biofortification via metabolic engineering is a proven powerful means to alleviate folate malnutrition. A variety of metabolic engineering approaches have been successfully implemented in different crops and tissues. Furthermore, ensuring folate stability is crucial for long-term storage of crop products. However, the current strategies, shown to be successful in rice and tomato, will need to be fine-tuned to enable adequate biofortification of other staples such as potato, wheat and cassava. Thus, there is a need to overcome remaining hurdles in folate biofortification. Overall, biofortification, via breeding or metabolic engineering, will be imperative to effectively combat folate deficiency., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Building upon the growing interest and research on genetically modified (GM) biofortification, its socioeconomic potential has been increasingly examined. We conducted two systematic reviews and meta-analyses to provide comprehensive evidence of consumers' willingness to pay (11 economic valuation studies, 64 estimates) and cost-effectiveness/benefits (five economic evaluation studies, 30 estimates). Worldwide, consumers were willing to pay 23.9% more for GM biofortified food crops. Aside from crop and design-related differences, information provision was deemed crucial. Positive information (nutrition and GM benefits) is associated with the highest consumer willingness to pay, compared with negative, objective, and conflicting GM information, especially when negative information was mentioned last. This health intervention would reduce the aggregated micronutrient deficiency burden in Asia (15.6 million disability-adjusted life years (DALYs)) by 12.5-51.4%, at a low cost of USD 7.9-27.8 per DALY in a pessimistic and optimistic scenario, respectively. Given that GM biofortified crops could tackle hidden hunger in a cost-effective and well-accepted way, its implementation is worth pursuing. A case study on folate biofortification further elaborates on the importance of socioeconomic research and the determinants of their market potential., (© 2016 New York Academy of Sciences.)

Following the growing evidence on biofortification as a cost-effective micronutrient strategy, various researchers have elicited consumers' willingness to pay (WTP) for biofortified crops in an effort to justify and determine their adoption. This review presents a meta-analysis of WTP studies on biofortified foods, either developed through conventional breeding or using genetic modification technology. On the basis of 122 estimates from 23 studies (9507 respondents), consumers are generally willing to pay 21.3% more for biofortified crops. Because WTP estimates are often determined through different valuation methods and procedures, a meta-regression was carried out to examine the role of potential determinants. Aside from contextual factors, such as type of food crop, target nutrient, and region (but not breeding technique), various methodological factors significantly influence premiums, including the type of respondent, nature of the study, study environment, participation fee, and provided information. The findings allow researchers to better anticipate potential methodological biases when examining WTP for (biofortified) foods, while it gives policy makers a broad understanding of the potential demand for different biofortified crops in various settings., (© 2016 New York Academy of Sciences.)

Micronutrient deficiency is a widespread phenomenon, most prevalent in developing countries. Being causally linked to the occurrence of a range of diseases, it affects billions of people worldwide. Enhancing the content of micronutrients in crop products through biotechnology is a promising technique to fight micronutrient malnutrition worldwide. Micronutrient fortification of food products has been implemented in a number of Western countries, but remains inaccessible for poor rural populations in a major part of the developing world. Moreover, evidence of the negative impacts of this practice on human health, at least for some vitamins, is accumulating. Biofortification of crop plants-the enhancement of vitamins and minerals through plant biotechnology-is a promising alternative or complement in the battle against micronutrient deficiencies. Owing to a growing knowledge about vitamin metabolism, as well as mineral uptake and reallocation in plants, it is today possible to enhance micronutrient levels in crop plants, offering a sustainable solution to populations with a suboptimal micronutrient intake., (© 2016 New York Academy of Sciences.)

Background: Understanding thiamin (thiamine; vitamin B1) metabolism in plants is crucial, as it impacts plant nutritional value as well as stress tolerance. Studies aimed at elucidating novel aspects of thiamin in plants rely on adequate assessment of thiamin content. Mass spectrometry-based methods provide reliable quantification of thiamin as well as closely related biomolecules. However, these techniques require expensive equipment and expertise. Microbiological turbidimetric assays can evaluate the presence of thiamin in a given sample, only requiring low-cost, standard lab equipment. Although these microbiological assays do not reach the accuracy provided by mass spectrometry-based methods, the ease with which they can be deployed in an inexpensive and high-throughput manner, makes them a favorable method in many circumstances. However, the thiamin research field could benefit from a detailed step-by-step protocol to perform such assays as well as a further assessment of its potential and limitations. Results: Here, we show that the Saccharomyces cerevisiae thiamin biosynthesis mutant thi6 is an ideal candidate to be implemented in a turbidimetric assay aimed at assessing the content of thiamin and its phosphorylated equivalents (total vitamer B1). An optimized protocol was generated, adapted from a previously established microbiological assay using the thi4 mutant. A step-by-step guidance for this protocol is presented. Furthermore, the applicability of the assay is illustrated by assessment of different samples, including plant as well as non-plant materials. In doing so, our work provides an extension of the applicability of the microbiological assay on top of providing important considerations upon implementing the protocol. Conclusions: An inexpensive, user-friendly protocol, including step-by-step guidance, which allows adequate estimation of vitamer B1 content of samples, is provided. The method is well-suited to screen materials to identify altered vitamer B1 content, such as in metabolic engineering or screening of germplasm. [ABSTRACT FROM AUTHOR]

Summary: Thiamin (or thiamine), known as vitamin B1, represents an indispensable component of human diets, being pivotal in energy metabolism. Thiamin research depends on adequate vitamin quantification in plant tissues. A recently developed quantitative liquid chromatography–tandem mass spectrometry (LC–MS/MS) method is able to assess the level of thiamin, its phosphorylated entities and its biosynthetic intermediates in the model plant Arabidopsis thaliana, as well as in rice. However, their implementation requires expensive equipment and substantial technical expertise. Microbiological assays can be useful in deter‐mining metabolite levels in plant material and provide an affordable alternative to MS‐based analysis.Here, we evaluate, by comparison to the LC–MS/MS reference method, the potential of a carefully chosen panel of yeast assays to estimate levels of total vitamin B1, as well as its biosynthetic intermediates pyrimidine and thiazole in Arabidopsis samples.The examined panel of Saccharomyces cerevisiae mutants was, when implemented in microbiological assays, capable of correctly assigning a series of wild‐type and thiamin biofortified Arabidopsis plant samples.The assays provide a readily applicable method allowing rapid screening of vitamin B1 (and its biosynthetic intermediates) content in plant material, which is particularly useful in metabolic engineering approaches and in germplasm screening across or within species. [ABSTRACT FROM AUTHOR]

Summary: Rice is a major food crop to approximately half of the human population. Unfortunately, the starchy endosperm, which is the remaining portion of the seed after polishing, contains limited amounts of micronutrients. Here, it is shown that this is particularly the case for thiamin (vitamin B1). Therefore, a tissue‐specific metabolic engineering approach was conducted, aimed at enhancing the level of thiamin specifically in the endosperm. To achieve this, three major thiamin biosynthesis genes, THIC, THI1 and TH1, controlled by strong endosperm‐specific promoters, were employed to obtain engineered rice lines. The metabolic engineering approaches included ectopic expression of THIC alone, in combination with THI1 (bigenic) or combined with both THI1 and TH1 (trigenic). Determination of thiamin and thiamin biosynthesis intermediates reveals the impact of the engineering approaches on endosperm thiamin biosynthesis. The results show an increase of thiamin in polished rice up to threefold compared to WT, and stable upon cooking. These findings confirm the potential of metabolic engineering to enhance de novo thiamin biosynthesis in rice endosperm tissue and aid in steering future biofortification endeavours. [ABSTRACT FROM AUTHOR]

'Hidden hunger' involves insufficient intake of micronutrients and is estimated to affect over two billion people on a global scale. Malnutrition of vitamins and minerals is known to cause an alarming number of casualties, even in the developed world. Many staple crops, although serving as the main dietary component for large population groups, deliver inadequate amounts of micronutrients. Biofortification, the augmentation of natural micronutrient levels in crop products through breeding or genetic engineering, is a pivotal tool in the fight against micronutrient malnutrition (MNM). Although these approaches have shown to be successful in several species, a more extensive knowledge of plant metabolism and function of these micronutrients is required to refine and improve biofortification strategies. This review focuses on the relevant B-vitamins (B1, B6, and B9). First, the role of these vitamins in plant physiology is elaborated, as well their biosynthesis. Second, the rationale behind vitamin biofortification is illustrated in view of pathophysiology and epidemiology of the deficiency. Furthermore, advances in biofortification, via metabolic engineering or breeding, are presented. Finally, considerations on B-vitamin multi-biofortified crops are raised, comprising the possible interplay of these vitamins in planta. [ABSTRACT FROM AUTHOR]

Insufficient dietary intake of micronutrients, known as “hidden hunger”, is a devastating global burden, affecting two billion people. Deficiency of folates (vitamin B9), which are known to play a central role in C 1 metabolism, causes birth defects in at least a quarter million people annually. Biofortification to enhance the level of naturally occurring folates in crop plants, proves to be an efficient and cost-effective tool in fighting folate deficiency. Previously, introduction of folate biosynthesis genes GTPCHI and ADCS , proven to be a successful biofortification strategy in rice and tomato, turned out to be insufficient to adequately increase folate levels in potato tubers. Here, we provide a proof of concept that additional introduction of HPPK/DHPS and/or FPGS , downstream genes in mitochondrial folate biosynthesis, enables augmentation of folates to satisfactory levels (12-fold) and ensures folate stability upon long-term storage of tubers. In conclusion, this engineering strategy can serve as a model in the creation of folate-accumulating potato cultivars, readily applicable in potato-consuming populations suffering from folate deficiency. [ABSTRACT FROM AUTHOR]

Genetically modified rice with a high folate content, i.e., folate biofortified rice, was recently developed to tackle folate deficiency. A consumer segmentation study is conducted to explore its market potential in the Chinese Shanxi Province, where the burden of folate deficiency is among the world’s highest. Cluster analysis of 451 rice consumers identified three segments: enthusiasts (14.2%), cautious (41.2%), and opponents (44.6%). Enthusiasts obtain the highest score of GM food knowledge and attitudes and have the highest GM rice acceptance rate, followed by cautious and reluctant consumers. Regarding GM food information, enthusiasts use almost entirely audiovisual channels and rely more on the industry, while other segments depend more on informal channels and anti-GM sources. Trust levels in these channels/sources differ between enthusiasts (high), opponents (neutral), and cautious (low). Based on multinomial regression, a targeted communication approach is recommended with a focus on female, cautious consumers and rural, low-educated opponents. [ABSTRACT FROM AUTHOR]

Improving nutritional health is one of the major socio-economic challenges of the 21st century, especially with the continuously growing and ageing world population. Folate deficiency is an important and underestimated problem of micronutrient malnutrition affecting billions of people worldwide. More and more countries are adapting policies to fight folate deficiency, mostly by fortifying foods with folic acid. However, there is growing concern about this practice, calling for alternative or complementary strategies. In addition, fortification programmes are often inaccessible to remote and poor populations where folate deficiency is most prevalent. Enhancing folate content in staple crops by metabolic engineering is a promising, cost-effective strategy to eradicate folate malnutrition worldwide. Over the last decade, major progress has been made in this field. Nevertheless, engineering strategies have thus far been implemented on a handful of plant species only and need to be transferred to highly consumed staple crops to maximally reach target populations. Moreover, successful engineering strategies appear to be species-dependent, hence the need to adapt them in order to biofortify different staple crops with folate.In this paper, the global occurrence of folate deficiency has been mapped and which staple crops could be targeted for folate biofortification and which engineering strategies could be employed have been investigated. [ABSTRACT FROM PUBLISHER]

Folates are important cofactors in one-carbon metabolism in all living organisms. Since only plants and micro- organisms are capable of biosynthesizing folates, humans depend entirely on their diet as a folate source. Given the low folate content of several staple crop products, folate deficiency affects regions all over the world. Folate biofortification of staple crops through enhancement of pterin and para-aminobenzoate levels, precursors of the folate biosynthesis pathway, was reported to be successful in tomato and rice. This study shows that the same strategy is not sufficient to enhance folate content in potato tubers and Arabidopsis thaliana plants and concludes that other steps in folate biosynthesis and/or metabolism need to be engineered to result in substantial folate accumulation. The findings provide a plausible explanation why, more than half a decade after the proof of concept in rice and tomato, successful folate biofortification of other food crops through enhancement of para-aminobenzoate and pterin content has not been reported thus far. A better understanding of the folate pathway is required in order to determine an engineering strategy that can be generalized to most staple crops. [ABSTRACT FROM AUTHOR]

Folates are of paramount importance in one-carbon metabolism of most organisms. Plants and microorganisms are able to synthesize folates de novo, making them the main dietary source for humans and animals, which are dependent on food or feed supplies for folates. Folate deficiency is an increasing problem in the developing, as well as in the developed regions of the world, affecting millions of people. Different strategies, such as food fortification and folic acid supplementation, remain far from accessible for the poor rural populations in developing countries. Increasing knowledge concerning folate biosynthesis, transport and catabolism does not only deepen our insight on the regulation of folate metabolism but also provides the keys towards folate enhancement through metabolic engineering in bacteria, as well as in plants. Recently, promising results were obtained using such an approach, but further fundamental research is a prerequisite to develop a practicable solution to fight folate deficiency. In parallel, progress in the development and improvement of folate analysis has been made. Here, we provide the state-of-the-art of folate biosynthesis, catabolism, and salvage. Finally, we report on progress in folate biofortification and discuss the agroeconomical aspect of biofortified crop plants. [ABSTRACT FROM AUTHOR]

Biofortification of staple crops could help to alleviate micronutrient deficiencies in humans. We show that folates in stored rice grains are unstable, which reduces the potential benefits of folate biofortification. We obtain folate concentrations that are up to 150 fold higher than those of wild-type rice by complexing folate to folate-binding proteins to improve folate stability, thereby enabling long-term storage of biofortified high-folate rice grains. [ABSTRACT FROM AUTHOR]

In developing countries, people mainly depend on rice as their primary source of calories. However, the thiamine content of rice is below minimal requirements. Biofortification, via genetic engineering, is a cost-effective strategy to increase thiamine content in rice. We report on the optimization of a matrix-specific method, including extensive optimization of the sample preparation to ensure maximal sensitivity and stability. The LC-MS/MS method was fully validated for the simultaneous quantification of thiamine, its precursors 4-methyl-5-(2-hydroxyethyl) thiazole (HET) and 4-amino-2-methyl-5-hydroxymethylpyrimidine (HMP) and its diphosphate derivative (TDP) in both polished and unpolished rice. Bias was below 9% for all analytes and total imprecision (CV%) was within pre-set acceptance criteria (≤15%) for both QCs and real samples. Thiamine monophosphate (TMP), for which no labeled analogue was available at the time of analysis, was determined without internal standard. Although both accuracy and precision criteria were met (bias and CV < 12%), the determination of TMP was considered semi-quantitatively. Moreover, TMP was found to be only a minor thiamine form (<1% of total thiamine in all lines analyzed, both wild-type and genetically engineered), with measurable levels only present in unpolished rice. Finally, the validity and applicability of the procedure were demonstrated via its successful application on rice lines, genetically engineered to enhance thiamine content. Consequently, this method allows to evaluate the success of biofortification strategies in rice. Image 1 • Determination of thiamine, its phosphates and precursors in complex cereal matrix. • Development of essential tool to steer thiamine biofortification in staple crops. • Optimization of sample preparation, ensuring stability labile thiamine vitamers. • Successful application to genetically modified rice, both unpolished and polished. [ABSTRACT FROM AUTHOR]

Micronutrient malnutrition is a persisting problem threatening global human health. Biofortification via metabolic engineering has been proposed as a cost-effective and short-term means to alleviate this burden. There has been a recent rise in the recognition of potential that underutilized, orphan crops can hold in decreasing malnutrition concerns. Here, we illustrate how orphan crops can serve as a medium to provide micronutrients to populations in need, whilst promoting and maintaining dietary diversity. We provide a roadmap, illustrating which aspects to be taken into consideration when evaluating orphan crops. Recent developments have shown successful biofortification via metabolic engineering in staple crops. This review provides guidance in the implementation of these successes to relevant orphan crop species, with a specific focus on the relevant micronutrients iron, zinc, provitamin A and folates. • Micronutrient malnutrition is a persisting global public health problem, particularly affecting poor rural populations. • Promoting a shift towards nutritional quality improvement in highly consumed crops can help mitigating this problem. • Orphan crops hold a prominent role in specific regional diets, often overlapping with several micronutrient deficiencies. • Translation of validated biofortification approaches into orphan crops could significantly aid in the fight against hidden hunger. • Guidance is provided towards orphan crop biofortification, focusing on iron/zinc, provitamin A and folates. [ABSTRACT FROM AUTHOR]

Abstract: High-performance liquid chromatography, coupled to tandem mass spectrometry (HPLC–MS/MS) has been established as the method of choice for the sensitive and simultaneous determination of different folates in a particular matrix, especially when only minute quantities of material are available. Using a previously developed and validated HPLC–MS/MS method as a starting point, we here report on the development and validation of an ultra-performance liquid chromatography (UPLC–MS/MS) method for analysis of folates in rice, which allows higher throughput and better resolution. UPLC was performed under gradient conditions on an Acquity HSS T3 column, followed by tandem mass spectrometry detection. The method was validated based on linearity, sensitivity, precision, accuracy and matrix effects. The limits of detection and the lower limits of quantification varied between 0.06 and 0.45μg/100g and 0.12 and 0.91μg/100g, respectively. Two linear calibration curves were established, one for the low and the other for the high concentration range. Analysis of the distribution and levels of folates in wild-type and folate-biofortified rice showed up to 50-fold enrichment in biofortified rice, with total folate levels of up to 900μg/100g rice. This is the first successful implementation of a UPLC method for the rapid and sensitive quantitative determination of folates in plant material. [Copyright &y& Elsevier]