Rice callus as a high-throughput platform for synthetic biology and metabolic engineering of carotenoids.

Carotenoids are a large class of important lipid-soluble phytonutrients that are widely used as nutritional supplements due to their health-promoting activities. For example, ß-carotene is the precursor for vitamin A synthesis, and astaxanthin is a powerful antioxidant. However, these carotenoids cannot be synthesized de novo by humans. These properties of ß-carotene and astaxanthin make them attractive targets for metabolic engineering in rice (Oryza sativa) endosperm because rice is an important staple food in developing countries, and rice endosperm is devoid of carotenoids. In this chapter, we introduce an assay based on rice embryogenic callus for the rapid functional characterization of genes involved in carotenoid biosynthesis and accumulation. The system is also an ideal platform to characterize cereal endosperm specific promoters. Four diverse cereal endosperm specific promoters were demonstrated to be active in rice callus despite their restricted activity in mature plants. The use of endosperm specific promoters that are expressed in rice callus, but remain silent in regenerated vegetative tissue, directs accumulation of carotenoids in the endosperm without interfering with plant growth. Rice callus is a useful platform for improving gene editing methods and for further optimizing pathway engineering. Thus, the rice callus platform provides a unique opportunity to test strategies for metabolic engineering of synthetic carotenoid pathways, leading to novel carotenoid-biofortified crops.

Metabolic Engineering of Crocin Biosynthesis in Nicotiana Species.

Crocins are high-value soluble pigments that are used as colorants and supplements, their presence in nature is extremely limited and, consequently, the high cost of these metabolites hinders their use by other sectors, such as the pharmaceutical and cosmetic industries. The carotenoid cleavage dioxygenase 2L (CsCCD2L) is the key enzyme in the biosynthetic pathway of crocins in Crocus sativus. In this study, CsCCD2L was introduced into Nicotiana tabacum and Nicotiana glauca for the production of crocins. In addition, a chimeric construct containing the Brevundimonas sp. ß-carotene hydroxylase (BrCrtZ), the Arabidopsis thaliana ORANGE mutant gene (AtOrMut), and CsCCD2L was also introduced into N. tabacum. Quantitative and qualitative studies on carotenoids and apocarotenoids in the transgenic plants expressing CsCCD2L alone showed higher crocin level accumulation in N. glauca transgenic plants, reaching almost 400 µg/g DW in leaves, while in N. tabacum 36 µg/g DW was obtained. In contrast, N. tabacum plants coexpressing CsCCD2L, BrCrtZ, and AtOrMut accumulated, 3.5-fold compared to N. tabacum plants only expressing CsCCD2L. Crocins with three and four sugar molecules were the main molecular species in both host systems. Our results demonstrate that the production of saffron apocarotenoids is feasible in engineered Nicotiana species and establishes a basis for the development of strategies that may ultimately lead to the commercial exploitation of these valuable pigments for multiple applications.

A global perspective on carotenoids: Metabolism, biotechnology, and benefits for nutrition and health.

Carotenoids are lipophilic isoprenoid compounds synthesized by all photosynthetic organisms and some non-photosynthetic prokaryotes and fungi. With some notable exceptions, animals (including humans) do not produce carotenoids de novo but take them in their diets. In photosynthetic systems carotenoids are essential for photoprotection against excess light and contribute to light harvesting, but perhaps they are best known for their properties as natural pigments in the yellow to red range. Carotenoids can be associated to fatty acids, sugars, proteins, or other compounds that can change their physical and chemical properties and influence their biological roles. Furthermore, oxidative cleavage of carotenoids produces smaller molecules such as apocarotenoids, some of which are important pigments and volatile (aroma) compounds. Enzymatic breakage of carotenoids can also produce biologically active molecules in both plants (hormones, retrograde signals) and animals (retinoids). Both carotenoids and their enzymatic cleavage products are associated with other processes positively impacting human health. Carotenoids are widely used in the industry as food ingredients, feed additives, and supplements. This review, contributed by scientists of complementary disciplines related to carotenoid research, covers recent advances and provides a perspective on future directions on the subjects of carotenoid metabolism, biotechnology, and nutritional and health benefits.

Biofortification of crops with nutrients: factors affecting utilization and storage.

Biofortification is an effective and economical method to improve the micronutrient content of crops, particularly staples that sustain human populations in developing countries. Whereas conventional fortification requires artificial additives, biofortification involves the synthesis or accumulation of nutrients by plants at source. Little is known about the relative merits of biofortification and artificial fortification in terms of nutrient bioaccessibility and bioavailability, and much depends on the biochemical nature of the nutrient, which can promote or delay uptake, and determine how efficiently different nutrients are transported through the blood, stored, and utilized. Data from the first plants biofortified with minerals and vitamins provide evidence that the way in which nutrients are presented can affect how they are processed and utilized in the human body. The latest studies on the effects of the food matrix, processing and storage on nutrient transfer from biofortified crops are reviewed, as well as current knowledge about nutrient absorption and utilization.

The distribution of carotenoids in hens fed on biofortified maize is influenced by feed composition, absorption, resource allocation and storage.

Carotenoids are important dietary nutrients with health-promoting effects. The biofortification of staple foods with carotenoids provides an efficient delivery strategy but little is known about the fate and distribution of carotenoids supplied in this manner. The chicken provides a good model of human carotenoid metabolism so we supplemented the diets of laying hens using two biofortified maize varieties with distinct carotenoid profiles and compared the fate of the different carotenoids in terms of distribution in the feed, the hen's livers and the eggs. We found that after a period of depletion, pro-vitamin A (PVA) carotenoids were preferentially diverted to the liver and relatively depleted in the eggs, whereas other carotenoids were transported to the eggs even when the liver remained depleted. When retinol was included in the diet, it accumulated more in the eggs than the livers, whereas PVA carotenoids showed the opposite profile. Our data suggest that a transport nexus from the intestinal lumen to the eggs introduces bottlenecks that cause chemically-distinct classes of carotenoids to be partitioned in different ways. This nexus model will allow us to optimize animal feed and human diets to ensure that the health benefits of carotenoids are delivered in the most effective manner.

Engineered maize as a source of astaxanthin: processing and application as fish feed.

Astaxanthin from a transgenic maize line was evaluated as feed supplement source conferring effective pigmentation of rainbow trout flesh. An extraction procedure using ethanol together with the addition of vegetal oil was established. This resulted in an oily astaxanthin preparation which was not sufficiently concentrated for direct application to the feed. Therefore, a concentration process involving multiple phase partitioning steps was implemented to remove 90 % of the oil. The resulting astaxanthin raw material contained non-esterified astaxanthin with 12 % 4-keto zeaxanthin and 2 % zeaxanthin as additional carotenoids. Isomeric analysis confirmed the exclusive presence of the 3S, 3'S astaxanthin enantiomer. The geometrical isomers were 89 % all-E, 8 % 13-Z and 3 % 9-Z. The incorporation of the oily astaxanthin preparation into trout feed was performed to deliver 7 mg/kg astaxanthin in the final feed formulation for the first 3.5 weeks and 72 mg/kg for the final 3.5 weeks of the feeding trial. The resulting pigmentation of the trout fillets was determined by hue values with a colour meter and further confirmed by astaxanthin quantification. Pigmentation properties of the maize-produced natural astaxanthin incorporated to 3.5 µg/g dw in the trout fillet resembles that of chemically synthesized astaxanthin. By comparing the relative carotenoid compositions in feed, flesh and feces, a preferential uptake of zeaxanthin and 4-keto zeaxanthin over astaxanthin was observed.

Carotenoid-enriched transgenic corn delivers bioavailable carotenoids to poultry and protects them against coccidiosis.

Carotenoids are health-promoting organic molecules that act as antioxidants and essential nutrients. We show that chickens raised on a diet enriched with an engineered corn variety containing very high levels of four key carotenoids (ß-carotene, lycopene, zeaxanthin and lutein) are healthy and accumulate more bioavailable carotenoids in peripheral tissues, muscle, skin and fat, and more retinol in the liver, than birds fed on standard corn diets (including commercial corn supplemented with colour additives). Birds were challenged with the protozoan parasite Eimeria tenella and those on the high-carotenoid diet grew normally, suffered only mild disease symptoms (diarrhoea, footpad dermatitis and digital ulcers) and had lower faecal oocyst counts than birds on the control diet. Our results demonstrate that carotenoid-rich corn maintains poultry health and increases the nutritional value of poultry products without the use of feed additives.

Cloning and Functional Characterization of the Maize (Zea mays L.) Carotenoid Epsilon Hydroxylase Gene.

The assignment of functions to genes in the carotenoid biosynthesis pathway is necessary to understand how the pathway is regulated and to obtain the basic information required for metabolic engineering. Few carotenoid ε-hydroxylases have been functionally characterized in plants although this would provide insight into the hydroxylation steps in the pathway. We therefore isolated mRNA from the endosperm of maize (Zea mays L., inbred line B73) and cloned a full-length cDNA encoding CYP97C19, a putative heme-containing carotenoid ε hydroxylase and member of the cytochrome P450 family. The corresponding CYP97C19 genomic locus on chromosome 1 was found to comprise a single-copy gene with nine introns. We expressed CYP97C19 cDNA under the control of the constitutive CaMV 35S promoter in the Arabidopsis thaliana lut1 knockout mutant, which lacks a functional CYP97C1 (LUT1) gene. The analysis of carotenoid levels and composition showed that lutein accumulated to high levels in the rosette leaves of the transgenic lines but not in the untransformed lut1 mutants. These results allowed the unambiguous functional annotation of maize CYP97C19 as an enzyme with strong zeinoxanthin ε-ring hydroxylation activity.

A question of balance: achieving appropriate nutrient levels in biofortified staple crops.

The biofortification of staple crops with vitamins is an attractive strategy to increase the nutritional quality of human food, particularly in areas where the population subsists on a cereal-based diet. Unlike other approaches, biofortification is sustainable and does not require anything more than a standard food-distribution infrastructure. The health-promoting effects of vitamins depend on overall intake and bioavailability, the latter influenced by food processing, absorption efficiency and the utilisation or retention of the vitamin in the body. The bioavailability of vitamins in nutritionally enriched foods should ideally be adjusted to achieve the dietary reference intake in a reasonable portion. Current vitamin biofortification programmes focus on the fat-soluble vitamins A and E, and the water-soluble vitamins C and B9 (folate), but the control of dosage and bioavailability has been largely overlooked. In the present review, we discuss the vitamin content of nutritionally enhanced foods developed by conventional breeding and genetic engineering, focusing on dosage and bioavailability. Although the biofortification of staple crops could potentially address micronutrient deficiency on a global scale, further research is required to develop effective strategies that match the bioavailability of vitamins to the requirements of the human diet.

Can the world afford to ignore biotechnology solutions that address food insecurity?

Genetically engineered (GE) crops can be used as part of a combined strategy to address food insecurity, which is defined as a lack of sustainable access to safe and nutritious food. In this article, we discuss the causes and consequences of food insecurity in the developing world, and the indirect economic impact on industrialized countries. We dissect the healthcare costs and lost productivity caused by food insecurity, and evaluate the relative merits of different intervention programs including supplementation, fortification and the deployment of GE crops with higher yields and enhanced nutritional properties. We provide clear evidence for the numerous potential benefits of GE crops, particularly for small-scale and subsistence farmers. GE crops with enhanced yields and nutritional properties constitute a vital component of any comprehensive strategy to tackle poverty, hunger and malnutrition in developing countries and thus reduce the global negative economic effects of food insecurity.

Biofortification of plants with altered antioxidant content and composition: genetic engineering strategies.

Antioxidants are protective molecules that neutralize reactive oxygen species and prevent oxidative damage to cellular components such as membranes, proteins and nucleic acids, therefore reducing the rate of cell death and hence the effects of ageing and ageing-related diseases. The fortification of food with antioxidants represents an overlap between two diverse environments, namely fortification of staple foods with essential nutrients that happen to have antioxidant properties (e.g. vitamins C and E) and the fortification of luxury foods with health-promoting but non-essential antioxidants such as flavonoids as part of the nutraceuticals/functional foods industry. Although processed foods can be artificially fortified with vitamins, minerals and nutraceuticals, a more sustainable approach is to introduce the traits for such health-promoting compounds at source, an approach known as biofortification. Regardless of the target compound, the same challenges arise when considering the biofortification of plants with antioxidants, that is the need to modulate endogenous metabolic pathways to increase the production of specific antioxidants without affecting plant growth and development and without collateral effects on other metabolic pathways. These challenges become even more intricate as we move from the engineering of individual pathways to several pathways simultaneously. In this review, we consider the state of the art in antioxidant biofortification and discuss the challenges that remain to be overcome in the development of nutritionally complete and health-promoting functional foods.

Nutritious crops producing multiple carotenoids--a metabolic balancing act.

Plants and microbes produce multiple carotenoid pigments with important nutritional roles in animals. By unraveling the basis of carotenoid biosynthesis it has become possible to modulate the key metabolic steps in plants and thus increase the nutritional value of staple crops, such as rice (Oryza sativa), maize (Zea mays) and potato (Solanum tuberosum). Multigene engineering has been used to modify three different metabolic pathways simultaneously, producing maize seeds with higher levels of carotenoids, folate and ascorbate. This strategy may allow the development of nutritionally enhanced staples providing adequate amounts of several unrelated nutrients. By focusing on different steps in the carotenoid biosynthesis pathway, it is also possible to generate plants with enhanced levels of several nutritionally-beneficial carotenoid molecules simultaneously.

Simultaneous expression of Arabidopsis ρ-hydroxyphenylpyruvate dioxygenase and MPBQ methyltransferase in transgenic corn kernels triples the tocopherol content.

The quantity and composition of tocopherols (compounds with vitamin E activity) vary widely among different plant species reflecting the expression, activity and substrate specificity of enzymes in the corresponding metabolic pathway. Two Arabidopsis cDNA clones corresponding to ρ-hydroxyphenylpyruvate dioxygenase (HPPD) and 2-methyl-6-phytylplastoquinol methyltransferase (MPBQ MT) were constitutively expressed in corn to further characterize the pathway and increase the kernel tocopherol content. Transgenic kernels contained up to 3 times as much γ-tocopherol as their wild type counterparts whereas other tocopherol isomers remained undetectable. Biofortification by metabolic engineering offers a sustainable alternative to vitamin E supplementation for the improvement of human health.

Critical evaluation of strategies for mineral fortification of staple food crops.

Staple food crops, in particular cereal grains, are poor sources of key mineral nutrients. As a result, the world's poorest people, generally those subsisting on a monotonous cereal diet, are also those most vulnerable to mineral deficiency diseases. Various strategies have been proposed to deal with micronutrient deficiencies including the provision of mineral supplements, the fortification of processed food, the biofortification of crop plants at source with mineral-rich fertilizers and the implementation of breeding programs and genetic engineering approaches to generate mineral-rich varieties of staple crops. This review provides a critical comparison of the strategies that have been developed to address deficiencies in five key mineral nutrients-iodine, iron, zinc, calcium and selenium-and discusses the most recent advances in genetic engineering to increase mineral levels and bioavailability in our most important staple food crops.

Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways.

Vitamin deficiency affects up to 50% of the world's population, disproportionately impacting on developing countries where populations endure monotonous, cereal-rich diets. Transgenic plants offer an effective way to increase the vitamin content of staple crops, but thus far it has only been possible to enhance individual vitamins. We created elite inbred South African transgenic corn plants in which the levels of 3 vitamins were increased specifically in the endosperm through the simultaneous modification of 3 separate metabolic pathways. The transgenic kernels contained 169-fold the normal amount of beta-carotene, 6-fold the normal amount of ascorbate, and double the normal amount of folate. Levels of engineered vitamins remained stable at least through to the T3 homozygous generation. This achievement, which vastly exceeds any realized thus far by conventional breeding alone, opens the way for the development of nutritionally complete cereals to benefit the world's poorest people.

[Determination and fingerprint analysis of tetramethylpyrazine and ferulic acid in Ligusticum chuanxiong].

OBJECTIVE: To determine the contents of tetramethylpyrazine (TMP) and ferulic acid in Ligusticum chuanxiong from different producing areas and seasons. METHODS: The contents of TMP and ferulic acid were determined by HPLC, and then analyzed by Chromatographic Fingerprints. RESULTS: The contents of TMP and ferulic acid from different seasons were obviously different from each other. It was much higher in "laoxiong" than that in "naixiong". The similarity of fingerprints was high if the samples were collected from the same season, or the same areas, but not different seasons. CONCLUSIONS: The contents of TMP and ferulic acid were different from different producing areas. The evident variety of Ligusticum chuanxiong's fingerprints from different collecting seasons, Laoxiong and Naixiong, was not relevant for clinical use as the same medicine.

Maize cDNAs expressed in endosperm encode functional farnesyl diphosphate synthase with geranylgeranyl diphosphate synthase activity.

Isoprenoids are the most diverse and abundant group of natural products. In plants, farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are precursors to many isoprenoids having essential functions. Terpenoids and sterols are derived from FPP, whereas gibberellins, carotenoids, casbenes, taxenes, and others originate from GGPP. The corresponding synthases (FPP synthase [FPPS] and GGPP synthase [GGPPS]) catalyze, respectively, the addition of two and three isopentenyl diphosphate molecules to dimethylallyl diphosphate. Maize (Zea mays L. cv B73) endosperm cDNAs encoding isoprenoid synthases were isolated by functional complementation of Escherichia coli cells carrying a bacterial gene cluster encoding all pathway enzymes needed for carotenoid biosynthesis, except for GGPPS. This approach indicated that the maize gene products were functional GGPPS enzymes. Yet, the predicted enzyme sequences revealed FPPS motifs and homology with FPPS enzymes. In vitro assays demonstrated that indeed these maize enzymes produced both FPP and GGPP and that the N-terminal sequence affected the ratio of FPP to GGPP. Their functionality in E. coli demonstrated that these maize enzymes can be coupled with a metabolon to provide isoprenoid substrates for pathway use, and suggests that enzyme bifunctionality can be harnessed. The maize cDNAs are encoded by a small gene family whose transcripts are prevalent in endosperm beginning mid development. These maize cDNAs will be valuable tools for assessing the critical structural properties determining prenyl transferase specificity and in metabolic engineering of isoprenoid pathways, especially in cereal crops.

cDNAs for the synthesis of cyclic carotenoids in petals of Gentiana lutea and their regulation during flower development.

cDNAs encoding lycopene epsilon -cyclase, lycopene beta-cyclase, beta-carotene hydroxylase and zeaxanthin epoxidase were isolated from a Gentiana lutea petal cDNA library. The function of all cDNAs was analyzed by complementation in Escherichia coli. Transcript levels during different stages of flower development of G. lutea were determined and compared to the carotenoid composition. Expression of all genes increased by a factor of up to 2, with the exception of the lycopene epsilon -cyclase gene. The transcript amount of the latter was strongly decreased. These results indicate that during flower development, carotenoid formation is enhanced. Moreover, metabolites are shifted away from the biosynthetic branch to lutein and are channeled into beta-carotene and derivatives.

cDNA cloning and expression of carotenogenic genes during flower development in Gentiana lutea.

All cDNAs involved in carotenoid biosynthesis leading to lycopene in yellow petals of Gentiana lutea have been cloned from a cDNA library. They encode a geranylgeranyl pyrophosphate synthase, a phytoene synthase, a phytoene desaturase and a zeta-carotene desaturase. The indicated function of all cDNAs was established by heterologous complementation in Escherichia coli. The amino acid sequences deduced from the cDNAs were between 47.5% and 78.9% identical to those reported for the corresponding enzymes from other higher plants. Southern analysis suggested that the genes for each enzyme probably represent a small multi-gene family. Tissue-specific expression of the genes and expression during flower development was investigated. The expression of the phytoene synthase gene, psy, was enhanced in flowers but transcripts were not detected in stems and leaves by northern blotting. Transcripts of the genes for geranylgeranyl pyrophosphate (ggpps), phytoene desaturase (pds) and zeta-carotene desaturase (zds) were detected in flowers and leaves but not in stems. Analysis of the expression of psy and zds in petals revealed that levels of the transcripts were lowest in young buds and highest in fully open flowers, in parallel with the formation of carotenoids. Obviously, the transcription of these genes control the accumulation of carotenoids during flower development in G. lutea. For pds only a very slight increase of mRNA was found whereas the transcripts of ggpps decreased during flower development.