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Ketocarotenoids such as astaxanthin and canthaxanthin have important applications in the nutraceutical, cosmetic, food and feed industries. Astaxanthin is derived from beta-carotene by 3-hydroxylation and 4-ketolation at both ionone end groups. These reactions are catalyzed by beta-carotene hydroxylase and beta-carotene ketolase, respectively. The hydroxylation reaction is widespread in higher plants, but ketolation is restricted to a few bacteria, fungi, and some unicellular green algae. The recent cloning and characterization of beta-carotene ketolase genes in conjunction with the development of effective co-transformation strategies permitting facile co-integration of multiple transgenes in target plants provided essential resources and tools to produce ketocarotenoids in planta by genetic engineering. In this review, we discuss ketocarotenoid biosynthesis in general, and characteristics and functional properties of beta-carotene ketolases in particular. We also describe examples of ketocarotenoid engineering in plants and we conclude by discussing strategies to efficiently convert beta-carotene to astaxanthin in transgenic plants.

The nutrients in the human diet ultimately come from plants. However, all our major food crops lack certain essential vitamins and minerals. Although a varied diet provides adequate nutrition, much of the human population, particularly in developing countries, relies on staple crops, such as rice or maize, which does not provide the full complement of essential nutrients. Malnutrition is a significant public health issue in most of the developing world. One way to address this problem is through the enhancement of staple crops to increase their essential nutrient content. Here, we review the current strategies for the biofortification of crops, including mineral fertilization and conventional breeding but focusing on transgenic approaches which offer the most rapid way to develop high-nutrient commercial cultivars.

Nicotiana glauca is a tobacco species that forms flowers with carotenoid-pigmented petals, sepals, pistil, ovary and nectary tissue. The carotenoids produced are lutein, ss-carotene as well as some violaxanthin and antheraxanthin. This tobacco species was genetically modified for ketocarotenoid biosynthesis by transformation with a cyanobacterial crtO ketolase gene under the 35S CaMV promoter. In the transformants, ketocarotenoids were detected in both leaves and flowers. Although astaxanthin was not detected other ketocarotenoids such as 4'-ketolutein, echinenone, 3'-hydroxyechinenone and 4-ketozeaxanthin were present. Accumulation of ketocarotenoids in leaves decreased their photosynthetic efficiency moderately. Under the green house conditions used no impairment of growth and development compared to the wild type was observed. In the crtO-transformants, an unexpected up-regulation of total carotenoid biosynthesis in leaves and especially in flower petals was observed. This led to a total ketocarotenoid concentration in leaves of 136.6 (young) or 156.1 (older) mug/g dry weight and in petals of 165 mug/g dry weight. In our engineered plants, the ketocarotenoid pathway is one step short of astaxanthin. Strategies are discussed to improve N. glauca flowers as a biological system for astaxanthin.

Ketocarotenoids and especially astaxanthin are high-valued pigments used as feed additives. Conventionally, they are provided by chemical synthesis. Their biological production is a promising alternative. For the development of a plant production system, Nicotiana glauca, a species with carotenoid-containing yellow pigmented flower petals, was transformed with a cyanobacterial ketolase gene. The resulting plants accumulated 4-ketozeaxantin (adinoxanthin), which is the first ketocarotenoid synthesized in flower petals by genetic modification. Due to the very late flowering in this tobacco species, N. tabacum was used to optimize the yield and ketocarotenoid product pattern by metabolic engineering of the ketolation steps of carotenogenesis. The highly carotenogenic nectary tissue in the flowers represents a model of a flower chromoplast system. By expression of a ketolase gene, it was possible to engineer the biosynthetic pathway towards the formation of 3'-hydroxyechinenone, 3-hydroxyechinenone, 4-ketozeaxanthin, 4-ketozeaxanthin esters, 4-ketolutein and 4-ketolutein esters. Some of these ketocarotenoids were also formed in the leaves of the trangenic plants. In particular, by co-expression of the ketolase gene in combination with a hydroxylase gene under an ubiquitous promoter, the formation of total carotenoids in nectaries increased by more than 2.5-fold. In the nectaries of this type of transformants, more than 50% of the accumulating carotenoids were keto derivatives. In addition, the levels of ketocarotenoid esters were much lower and a higher percentage of the free ketocarotenoids accumulated. These results open new promising perspectives for a successful metabolic engineering of keto-hydroxy carotenoid production in carotenogenic flowers.

The al-2 cDNA from Neurospora crassa was cloned, expressed and functionally characterized. The enzyme comprised the two catalytic activities of a phytoene synthase and a lycopene cyclase. In contrast to most other lycopene cyclases, single cyclizations were preferentially catalyzed. This N. crassa enzyme is the first CrtYB-type monocyclic-acting lycopene cyclase. Therefore, this cDNA has been evaluated for the heterologous synthesis of monocyclic hydroxy-keto carotenoids by combination with other carotenogenic genes in Escherichia coli. Depending on the degree of desaturation, 4-keto derivatives of gamma-carotene and torulene with additional 2-hydroxy, 3-hydroxy and/or 1'-HO groups were generated and the following asymmetrical carotenoids identified and quantitated: 3-HO-4-keto-gamma-carotene, 2-HO-4-keto-gamma-carotene, 4-keto-1'-HO-gamma-carotene, 3,1'-(HO)(2)-4-keto-gamma-carotene, 3-HO-4-keto-torulene and 2-HO-4-keto-torulene. Among them all the monocyclic gamma-carotene derivatives with 9 conjugated double bonds were not found naturally before. Furthermore, 2-HO-4-keto-torulene with 10 conjugated double bonds is another novel carotenoid.

Summary Two 9- cis- epoxycarotenoid dioxygenase (NCED) cDNAs have been cloned from a petal library of Gentiana lutea . Both cDNAs carry a putative transit sequence for chloroplast import and differ mainly in their length and the 5′-flanking regions. GlNCED1 was evolutionary closely related to Arabidopsis thaliana NCED6 whereas GlNCED2 showed highest homology to tomato NCED1 and A. thaliana NCED3 . The amounts of GlNCED2 transcript were below Northern detection in G. lutea . In contrast, GlNCED1 was specifically expressed at higher levels in developing flowers when petals start appearing. By genetic engineering of tobacco with coding regions of either gene under a constitutive promoter, their function was further analyzed. Although mRNA of both genes was detectable in the corresponding transgenic plants, a physiological effect was only found for GlNCED1 but not for GlNCED2 . In germination experiments of GlNCED1 transgenic lines, delayed radicle formation and cotyledon appearance were observed. However, the transformants exhibited no improved tolerance against desiccation stress. In contrast to other plants with over-expressed NCEDs , prolonged delay of seed germination is the only abscisic-acid-related phenotypic effect in the GlNCED1 transgenic lines.

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.

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.

Cofactor requirement was determined for the heterologous expressed phytoene desaturases from the cyanobacterium Synechococcus and the higher plant Gentiana lutea. The cyanobacterial enzyme is dependent on either NAD(P) or plastoquinone, whereas only quinones such as plastoquinone can function as a cofactor for the phytoene desaturase from G. lutea. Enzyme kinetic studies were carried out to determine a possible competition between the cofactors and the bleaching herbicide norflurazon. For the Synechococcus enzyme, competition between norflurazon and NADP, as well as plastoquinone, could be demonstrated. The K(m) values for these cofactors were 6.6 mM and 0.23 microM, respectively. Inhibition of the phytoene desaturase from G. lutea by norflurazon was also competitive with respect to plastoquinone. The K(m) values of both enzymes for plastoquinone were very close.

A study was undertaken to develop a protoplast regeneration system for pinellia. A yield of 19 29 x 10(5) protoplasts/g F. W. could be obtained from cell suspension cultures incubated in a digestion enzyme solution with 2% cellulase Onzuka R-10, 10% pectinase (Sigma), 0.01% pectolyase Y23. K8P and modified MS media were used to culture protoplasts in: a) liquid, b) liquid-solid double layer, or c) agarose embedded protoplast culture. The former two were conducive to colony formation from protoplast-derived cells. The frequency of cell division was about 8% after 3 days in culture. Gradually adding fresh medium of lower osmotic pressure into the medium for protoplast culture favored cell division. Calli (1-2 mm in diameter) formed after 30-40 days in culture. The calli transferred onto medium supplemented with KT (0.5 mg 1(-1)) and NAA (0.2 mg 1)(-1)) could regenerate plants after 40-50 days. Of 47 plantlets transplanted into plots, 29 flowered and were fertile.

Plant biotechnology journal 19(10), 1901-1920 (2021). doi:10.1111/pbi.13657, Published by Wiley-Blackwell, Oxford

Over the last two decades, many carotenogenic genes have been cloned and used to generate metabolically engineered plants producing higher levels of carotenoids. However, comparatively little is known about the regulation of endogenous carotenogenic genes in higher plants, and this restricts our ability to predict how engineered plants will perform in terms of carotenoid content and composition. During petal development in the Great Yellow Gentian (Gentiana lutea), carotenoid accumulation, the formation of chromoplasts and the upregulation of several carotenogenic genes are temporally coordinated. We investigated the regulatory mechanisms responsible for this coordinated expression by isolating five G. lutea carotenogenic gene (GlPDS, GlZDS, GlLYCB, GlBCH and GlLYCE) promoters by inverse polymerase chain reaction (PCR). Each promoter was sufficient for developmentally regulated expression of the gusA reporter gene following transient expression in tomato (Solanum lycopersicum cv. Micro-Tom). Interestingly, the GlLYCB and GlBCH promoters drove high levels of gusA expression in chromoplast-containing mature green fruits, but low levels in chloroplast-containing immature green fruits, indicating a strict correlation between promoter activity, tomato fruit development and chromoplast differentiation. As well as core promoter elements such as TATA and CAAT boxes, all five promoters together with previously characterized GlZEP promoter contained three common cis-regulatory motifs involved in the response to methyl jasmonate (CGTCA) and ethylene (ATCTA), and required for endosperm expression (Skn-1_motif, GTCAT). These shared common cis-acting elements may represent binding sites for transcription factors responsible for co-regulation. Our data provide insight into the regulatory basis of the coordinated upregulation of carotenogenic gene expression during flower development in G. lutea. This work was supported by the National Natural Science Foundation of China (31270344); Fundamental Research Funds for the Central Universities to Q. Y. (DL13BA06); MICINN, Spain (BIO2011-23324; BIO2011-22525; PIM2010PKB-00746); European Union Framework 7 European Research Council IDEAS Advanced Grant (to P. C.) Program-BIOFORCE.

In this article, we explore the intellectual property (IP) landscape relevant to the production and commercialization of Carolight™, a transgenic multivitamin corn variety created on humanitarian grounds to address micronutrient deficiencies in low-and-middle-income countries. The successful production of this variety requires IP rights risk management because there is a strong protection on inventions and processes via patent portfolios in both developing and industrialized countries. The IP framework is complex, and specialist patent lawyers are usually employed to perform such analysis, but the costs cannot always be met by small, publicly funded projects. We report an alternative strategy, a do-it-yourself patent analysis, to produce a review with limited legal value that can nevertheless lay the foundations for a subsequent more in-depth professional freedom-to-operate opinion. European Union Framework 7 European Research Council IDEAS Advanced Grant Program-BIOFORCE and Proof of Concept grant Multinutrient Maize.

Skin color in poultry is achieved by the addition of natural or synthetic pigments to feed. Crops used routinely in feed formulations offer an alternative cost-effective strategy to replace color additives if they are biofortified with sufficient levels of carotenoids. We tested the hypothesis that high-carotenoid (HC) maize, which was genetically engineered to accumulate high levels of β‐carotene, lutein and zeaxanthin in the endosperm, can replace carotenoid additives in poultry feed by performing two feeding trials using diets incorporating different maize lines with diverse carotenoid compositions: control (wild-type M37W, the parental line), HC, and standard yellow commercial maize supplemented with color additives (marigold flowers and red paprika extracts). The effects of dietary treatments on growth performance, health parameters, color evolution and carotenoid distribution were determined. We found that chickens fed on the HC diet grew normally and developed similar pigmentation to animals fed on a commercial diet supplemented with color additives, although yellowness was significantly higher in the commercial diet due to the high concentration of yellow xanthophylls. Lightness scores in chickens fed on the control, HC and commercial diets were 45.88 ± 1.31, 44.32 ± 1.10 and 44.29 ± 0.99, respectively, in breast muscle, and 51.62 ± 1.33, 49.66 ± 0.96 and 50.10 ± 1.16, respectively, in thigh muscle. Redness scores in chickens fed on the control, HC, and commercial diets were 0.36 ± 0.26, 3.25 ± 0.29 and 3.58 ± 0.29, respectively, in breast muscle, and 1.28 ± 0.37, 4.79 ± 0.39 and 4.85 ± 0.34, respectively, in thigh muscle. Yellowness scores in chickens fed on the control, HC, and commercial diets were 2.45 ± 0.47, 7.61 ± 0.64 and 9.66 ± 0.73, respectively, in breast muscle, and 3.38 ± 0.64, 10.00 ± 1.10 and 12.64 ± 0.97, respectively, in thigh muscle. High-carotenoid maize is therefore a cost-effective alternative to feed supplementation in the poultry industry. This work was supported by La Caixa (Recercaixa project PC084082 VitaMaize: High quality and safe food through antioxidant fortified maize), the Spanish Ministry of Economy and Competitiveness (BIO2014-54426-P; BIO2014-54441-P, FEDER funds) and the Catalan Government (2014 SGR 1296 Agricultural Biotechnology Research Group).

Highlight Metabolomic, proteomic, and transcriptomic analysis of a maize line genetically engineered for enhanced seed carotenoid biosynthesis revealed how the sugar metabolism adapted to meet the additional precursor supply., The aim of this study was to assess whether endosperm-specific carotenoid biosynthesis influenced core metabolic processes in maize embryo and endosperm and how global seed metabolism adapted to this expanded biosynthetic capacity. Although enhancement of carotenoid biosynthesis was targeted to the endosperm of maize kernels, a concurrent up-regulation of sterol and fatty acid biosynthesis in the embryo was measured. Targeted terpenoid analysis, and non-targeted metabolomic, proteomic, and transcriptomic profiling revealed changes especially in carbohydrate metabolism in the transgenic line. In-depth analysis of the data, including changes of metabolite pools and increased enzyme and transcript concentrations, gave a first insight into the metabolic variation precipitated by the higher up-stream metabolite demand by the extended biosynthesis capacities for terpenoids and fatty acids. An integrative model is put forward to explain the metabolic regulation for the increased provision of terpenoid and fatty acid precursors, particularly glyceraldehyde 3-phosphate and pyruvate or acetyl-CoA from imported fructose and glucose. The model was supported by higher activities of fructokinase, glucose 6-phosphate isomerase, and fructose 1,6-bisphosphate aldolase indicating a higher flux through the glycolytic pathway. Although pyruvate and acetyl-CoA utilization was higher in the engineered line, pyruvate kinase activity was lower. A sufficient provision of both metabolites may be supported by a by-pass in a reaction sequence involving phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme.

We generated transgenic rice plants overexpressing Arabidopsis thaliana q-hydroxyphenylpyruvate dioxygenase (HPPD), which catalyzes the first committed step in vitamin E biosynthesis. Transgenic grains accumulated marginally higher levels of total tocochromanols than controls, reflecting a small increase in absolute tocotrienol synthesis (but no change in the relative abundance of the a and c isoforms). In contrast, there was no change in the absolute tocopherol level, but a significant shift from the c to the a isoform. These data confirm HPPD is not rate limiting, and that increasing flux through the early pathway reveals downstream bottlenecks that act as metabolic tipping points. This study was supported by the Ministerio de Ciencia e Innovación, Spain (BFU2007-61413) and European Research Council Advanced Grant (BIOFORCE) to PC. G.F. is the recipient of a Ph.D. fellowship from the Ministerio de Ciencia e Innovación, Spain (BES-2008-003894). The Department of Biotechnology, Government of India provides an Overseas Associateship to D.S.

Designer nucleases allow the creation of new plant genotypes by introducing precisely-targeted double-strand breaks that are resolved by endogenous repair pathways. The major nuclease technologies are meganucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and the CRISPR/Cas9 system. Each comprises a promiscuous endonuclease guided by proteinâDNA or RNAâDNA interactions. A great deal is known about the principles of designer nucleases but much remains to be learned about their detailed behavioral characteristics in different plant species. The outcome of genome engineering reflects the intrinsic properties of each nuclease and target genome, causing variations in efficiency, accuracy, and mutation structure. In this article, we critically discuss the activities of designer nucleases in different cereals representing a broad range of genome characteristics.

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. S.G.-G. is recipient of a fellowship from the Catalan Regional Government (DIUE and ‘‘Fons Social Europeu 2008FIC 00196) Spain. T.C. is supported by the Ramon y Cajal (RyC) program, Spain. PC is grateful for financial support to the Ministry of Science and Innovation, Spain (Grant number BFU2007-61413) and to the European Research Council for advanced grant, BIOFORCE.

Multigene transformation (MGT) is becoming routine in plant biotechnology as researchers seek to generate more complex and ambitious phenotypes in transgenic plants. Every nuclear transgene requires its own promoter, so when coordinated expression is required, the introduction of multiple genes leads inevitably to two opposing strategies: different promoters may be used for each transgene, or the same promoter may be used over and over again. In the former case, there may be a shortage of different promoters with matching activities, but repetitious promoter use may in some cases have a negative impact on transgene stability and expression. Using illustrative case studies, we discuss promoter deployment strategies in transgenic plants that increase the likelihood of successful and stable multiple transgene expression. Work in our laboratory is supported by the Ministry of Science and Innovation, Spain (BFU2007-61413 and BIO2007-30738-E), a European Research Council Advanced Grant (BIOFORCE) to PC, and the CONSOLIDER Agrigenomics program funded by MICINN, Spain

Corolla color in Gentiana lutea L. exhibits a yellow/orange variation. We previously demonstrated that the orange petal color of G. lutea L. var. aurantiaca is predominantly caused by newly synthesized pelargonidin glycosides that confer a reddish hue to the yellow background color, derived from the carotenoids. However, the anthocyanin molecules of these pelargonidin glycosides are not yet fully identified and characterized. Here, we investigated the regulation, content and type of anthocyanins determining the petal coloration of the orange-flowered G. lutea L. var. aurantiaca. Anthocyanins from the petals of G. lutea L. var. aurantiaca were characterized and quantified by HPLC-ESI-MS/MS (High-performance liquid chromatography-electrospray ionization-tandem mass spectrometry) coupled with a diode array detector in flowers at three different stages of development (S1, S3 and S5). Eleven pelargonidin derivatives were identified in the petals of G. lutea L. var. aurantiaca for the first time, but quantitative and qualitative differences were observed at each developmental stage. The highest levels of these pelargonidin derivatives were reached at the fully open flower stage (S5) where all anthocyanins were detected. In contrast, not all the anthocyanins were detected at the budlet stage (S1) and mature bud stage (S3) and those corresponded to more complex pelargonidin derivatives. The major pelargonidin derivatives found at all the stages were pelargonidin 3-O-glucoside, pelargonidin 3,5-O-diglucoside and pelargonidin 3-O-rutinoside. Furthermore, the expression of DFR (dihydroflavonol 4-reductase), ANS (anthocyanidin synthase), 3GT (UDP-glucose:flavonoid 3-O-glucosyltransferase), 5GT (UDP-glucose:flavonoid 5-O-glucosyltransferase) and 5AT (anthocyanin 5- aromatic acyltransferase) genes was analyzed in the petals of three developmental stages, showing that the expression level of DFR, ANS and 3GT parallels the accumulation of the pelargonidin glucosides. Overall, this study enhances the knowledge of the biochemical basis of flower coloration in Gentiana species, and lays a foundation for breeding of flower color and genetic variation studies on Gentiana varieties. This work was supported by the National Natural Science Foundation of China (31270344); MICINN, Spain (BIO2014-54441-P); International Science & Technology Cooperation Project supported by Jilin Provincial Science & Technology Department, China (20190201013JC); PROGRAMA ESTATAL DE INVESTIGACIÓN CIENTÍFICA Y TÉCNICA DE EXCELENCIA, Spain (BIO2015-71703-REDT, BIO2017-90877-REDT); European Cooperation in Science and Technology (EUROCAROTEN, OC-2015-1-19780). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Numerous crops have been bred or engineered to increase carotenoid levels in an effort to develop novel strategies that address vitamin A deficiency in the developing world. The pioneering work in rice (not covered in this review) has been followed up in many additional crops, some of which are staples like rice whereas others are luxury products whose impact on food security is likely to be marginal. This review surveys the progress that has been made in carotenoid breeding and metabolic engineering, focusing on β-carotene enhancement in crops other than rice. We ask if these efforts have the potential to address vitamin A deficiency in developing countries by comparing bioavailable pro-vitamin A levels in wild type and enhanced crops to determine whether nutritional requirements can be met without the consumption of unrealistic amounts of food. The potential impact of carotenoid enhancement should therefore be judged against benchmarks that include the importance of particular crops in terms of global food security, the amount of bioavailable β-carotene, and the amount of food that must be consumed to achieve the reference daily intake of vitamin A. Research in our laboratory is supported by the Ministry of Science and Innovation (MICINN) Grant BFU2007- 61413; European Research Council Advanced Grant BIOFORCE; Center Consolider, MICINN, Spain; COST Action FA0804, Associated Unit CAVA; and the SmartCell, FP7 Integrated Project.