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Summary Ketocarotenoids, including astaxanthin, are red lipophilic pigments derived from the oxygenation of β‐carotene ionone rings. These carotenoids have exceptional antioxidant capacity and high commercial value as natural pigments, especially for aquaculture feedstocks to confer red flesh colour to salmon and shrimp. Ketocarotenoid biosynthetic pathways occur only in selected bacterial, algal, fungal and plant species, which provide genetic resources for biotechnological ketocarotenoid production. Toward pathway optimization, we developed a transient platform for ketocarotenoid production using Agrobacterium infiltration of Nicotiana benthamiana leaves with plant (Adonis aestivalis) genes, carotenoid β‐ring 4‐dehydrogenase 2 (CBFD2) and carotenoid 4‐hydroxy‐β‐ring 4‐dehydrogenase (HBFD1), or bacterial (Brevundimonas) genes, β‐carotene ketolase (crtW) and β‐carotene hydroxylase (crtZ). In this test system, heterologous expression of the plant‐derived astaxanthin pathway conferred higher astaxanthin production with fewer ketocarotenoid intermediates than the bacterial pathway. We evaluated the plant‐derived pathway for ketocarotenoid production using the oilseed camelina (Camelina sativa) as a production platform. Genes for CBFD2 and HBFD1 and maize phytoene synthase were introduced under the control of seed‐specific promoters. In contrast to prior research with bacterial pathways, our strategy resulted in nearly complete conversion of β‐carotene to ketocarotenoids, including primarily astaxanthin. Tentative identities of other ketocarotenoids were established by chemical evaluation. Seeds from multi‐season US and UK field sites maximally accumulated ~135 μg/g seed weight of ketocarotenoids, including astaxanthin (~47 μg/g seed weight). Although plants had no observable growth reduction, seed size and oil content were reduced in astaxanthin‐producing lines. Oil extracted from ketocarotenoid‐accumulating seeds showed significantly enhanced oxidative stability and was useful for food oleogel applications.

The Panel on Food Additives and Nutrient Sources added to Food provides a scientific opinion re‐evaluating the safety of mixed carotenes [E 160a (i)] and β‐carotene [E 160a (ii)] when used as food colouring substances. Mixed carotenes [E 160a (i)] and β‐carotene [E 160a (ii)] are authorised as food additives in the EU and have been evaluated previously by the JECFA the latest in 2001 and by the SCF in 1997 and 2000. Both Committees established an Acceptable Daily Intake (ADI) of 0–5 mg/kg bw/day. In this opinion the mixed carotenes are defined according to the Commission Directive 2008/128/EC and consist of two groups of substances: plant carotenes and algal carotenes. β‐Carotene comprises (synthetic) β‐carotene and β‐carotene obtained by fermentation of the fungus Blakeslea trispora. The Panel noted (i) that the specifications of mixed carotenes are inadequate and need to be updated, (ii) that most toxicological studies have been performed with rodents, although rodents, in contrast to humans, very efficiently convert β‐carotene to vitamin A. The Panel concluded that based on the presently available dataset, no ADIs for mixed carotenes and β‐carotene can be established and that the use of (synthetic) β‐carotene and mixed β‐carotenes obtained from palm fruit oil, carrots and algae as food colour is not of safety concern, provided the intake from this use as a food additive and as food supplement, is not more than the amount likely to be ingested from the regular consumption of the foods in which they occur naturally (5–10 mg/day). This would ascertain that the exposure to β‐carotene from these uses would remain below 15 mg/day, the level of supplemental intake of β‐carotene for which epidemiological studies did not reveal any increased cancer risk. Furthermore, the Panel could not conclude on the safety in use of mixed carotenes [E 160a (i)]

Summary Grains of tetraploid wheat (Triticum turgidum L.) mainly accumulate the non‐provitamin A carotenoid lutein—with low natural variation in provitamin A β‐carotene in wheat accessions necessitating alternative strategies for provitamin A biofortification. Lycopene ɛ‐cyclase (LCYe) and β‐carotene hydroxylase (HYD) function in diverting carbons from β‐carotene to lutein biosynthesis and catalyzing the turnover of β‐carotene to xanthophylls, respectively. However, the contribution of LCYe and HYD gene homoeologs to carotenoid metabolism and how they can be manipulated to increase β‐carotene in tetraploid wheat endosperm (flour) is currently unclear. We isolated loss‐of‐function Targeting Induced Local Lesions in Genomes (TILLING) mutants of LCYe and HYD2 homoeologs and generated higher order mutant combinations of lcye‐A, lcye‐B, hyd‐A2, and hyd‐B2. Hyd‐A2 hyd‐B2, lcye‐A hyd‐A2 hyd‐B2, lcye‐B hyd‐A2 hyd‐B2, and lcye‐A lcye‐B hyd‐A2 hyd‐B2 achieved significantly increased β‐carotene in endosperm, with lcye‐A hyd‐A2 hyd‐B2 exhibiting comparable photosynthetic performance and light response to control plants. Comparative analysis of carotenoid profiles suggests that eliminating HYD2 homoeologs is sufficient to prevent β‐carotene conversion to xanthophylls in the endosperm without compromising xanthophyll production in leaves, and that β‐carotene and its derived xanthophylls are likely subject to differential catalysis mechanisms in vegetative tissues and grains. Carotenoid and gene expression analyses also suggest that the very low LCYe‐B expression in endosperm is adequate for lutein production in the absence of LCYe‐A. These results demonstrate the success of provitamin A biofortification using TILLING mutants while also providing a roadmap for guiding a gene editing‐based approach in hexaploid wheat.

The flowers of twenty-three cultivars of Dendranthema grandiflorum Ramat. were investigated to determine anthocyanin and carotenoid levels and to confirm the effects of the pigments on the flower colors using high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI-MS). The cultivars contained the anthocyanins cyanidin 3-glucoside (C3g) and cyanidin 3-(3ʺ-malonoyl) glucoside (C3mg) and the following carotenoids: lutein, zeaxanthin, β-cryptoxanthin, 13-cis-β-carotene, α-carotene, trans-β-carotene, and 9-cis-β-carotene. The cultivar “Magic” showed the greatest accumulation of total and individual anthocyanins, including C3g and C3gm. On the other hand, the highest level of lutein and zeaxanthin was noted in the cultivar “Il Weol”. The cultivar “Anastasia” contained the highest amount of carotenoids such as trans-β-carotene, 9-cis-β-carotene, and 13-cis-β-carotene. The highest accumulation of β-cryptoxanthin and α-carotene was noted in the cultivar “Anastasia” and “Il Weol”. Our results suggested that ‘Magic”, “Angel” and “Relance’ had high amounts of anthocyanins and showed a wide range of red and purple colors in their petals, whereas “Il Weol’, “Popcorn Ball’ and “Anastasia” produced higher carotenoid contents and displayed yellow or green petal colors. Interestingly, “Green Pang Pang”, which contained a high level of anthocyanins and a medium level of carotenoids, showed the deep green colored petals. “Kastelli”, had high level of carotenoids as well as a medium level of anthocyanins and showed orange and red colored petals. It was concluded that each pigment is responsible for the petal’s colors and the compositions of the pigments affect their flower colors and that the cultivars could be a good source for pharmaceutical, floriculture, and pigment industries.

Astaxanthin is one of the strongest natural antioxidants and a red pigment occurring in nature. This C40 carotenoid is used in a broad range of applications such as a colorant in the feed industry, an antioxidant in cosmetics or as a supplement in human nutrition. Natural astaxanthin is on the rise and, hence, alternative production systems are needed. The natural carotenoid producer Corynebacterium glutamicum is a potent host for industrial fermentations, such as million-ton scale amino acid production. In C. glutamicum, astaxanthin production was established through heterologous overproduction of the cytosolic lycopene cyclase CrtY and the membrane-bound β-carotene hydroxylase and ketolase, CrtZ and CrtW, in previous studies. In this work, further metabolic engineering strategies revealed that the potential of this GRAS organism for astaxanthin production is not fully exploited yet. It was shown that the construction of a fusion protein comprising the membrane-bound β-carotene hydroxylase and ketolase (CrtZ~W) significantly increased astaxanthin production under high glucose concentration. An evaluation of used carbon sources indicated that a combination of glucose and acetate facilitated astaxanthin production. Moreover, additional overproduction of cytosolic carotenogenic enzymes increased the production of this high value compound. Taken together, a seven-fold improvement of astaxanthin production was achieved with 3.1 mg/g CDW of astaxanthin.

Astaxanthin is a carotenoid of significant commercial value due to its superior antioxidant potential and wide applications in the aquaculture, food, cosmetic and pharmaceutical industries. A higher ratio of astaxanthin to the total carotenoids is required for efficient astaxanthin production. β-Carotene ketolase and hydroxylase play important roles in astaxanthin production. We first compared the conversion efficiency to astaxanthin in several β-carotene ketolases from Brevundimonas sp. SD212, Sphingomonas sp. DC18, Paracoccus sp. PC1, P. sp. N81106 and Chlamydomonas reinhardtii with the recombinant Escherichia coli cells that synthesize zeaxanthin due to the presence of the Pantoea ananatis crtEBIYZ. The B. sp. SD212 crtW and P. ananatis crtZ genes are the best combination for astaxanthin production. After balancing the activities of β-carotene ketolase and hydroxylase, an E. coli ASTA-1 that carries neither a plasmid nor an antibiotic marker was constructed to produce astaxanthin as the predominant carotenoid (96.6%) with a specific content of 7.4 ± 0.3 mg/g DCW without an addition of inducer.

Summary Biofortification of staple crops is a sustainable strategy to deliver essential micronutrients to impoverished populations in developing countries. Banana is a highly valued crop consumed by over 75% of Ugandans. However, the starchy green cooking bananas have very low levels of pro‐vitamin A (PVA) and heavy dietary reliance on them has been associated with vitamin A deficiency (VAD). Two banana cultivars, hybrid M9 and Nakitembe, were selected for PVA biofortification. A phytoene synthase 2a (MtPsy2a) gene was transformed into the selected cultivars under the control of the constitutive maize polyubiquitin1 promoter or the banana fruit‐preferred ACC oxidase (ACO) promoter. Plants were regenerated on selective media and putatively transgenic plants confirmed by PCR. A total of 356 and 162 transgenic events for M9 and Nakitembe, respectively, were planted in a confined field trial (CFT). Transgenic plants were assessed against non‐transformed controls. Selection was based on phenotype, cycle time, yield, β‐carotene equivalents (β‐CE) and transgene copy number. There were no significant variations in cycle time, but some phenotypic differences were observed between transgenic and non‐transgenic controls. Transgenic fruits had yellow to orange fruit pulps, unlike pulp from non‐transgenic controls that were paler. On average, fruit from transgenic M9 and Nakitembe accumulated fourfold and threefold more β‐CE than non‐transgenic controls, respectively. Five elite lines each of M9 and Nakitembe have been selected for national agronomic performance trials that will aid the selection of lead events to be considered for environmental release.

Plant extract-mediated synthesis is a simple, eco-friendly, and inexpensive method for the preparation of efficient antioxidant and photocatalytic nanoparticles. In this study, lemon peel aqueous extract was used to synthesize CuO nanoparticles (NPs) and then the obtained CuO NPs were modified using polyethylene glycol (PEG). The characteristics, optical properties, antioxidant, and photocatalytic activities of the synthesized nanoparticles were investigated. The CuO NPs and CuO/PEG NPs exhibited sphere-like morphology with an average size of 34 nm and 45 nm and optical bandgap energies of 1.2 eV and 1.5 eV, respectively. The antioxidant activity tests showed that the CuO/PEG NPs exhibited significant scavenging activity with IC50 values of 104.6 μg/mL for the β-carotene scavenging assay and 38.1 μg/mL for the ABTS scavenging assay, while CuO showed lower antioxidant activity of about 150.54 μg/mL for β-Carotene linoleic acid bleaching assay and 59.63 μg/mL for the ABTS scavenging assay. In terms of photocatalytic degradation, CuO/PEG NPs demonstrated higher activity compared to CuO NPs alone. They achieved degradation rates of 99.7% for 4-bromophenol (BP) dye and 99.5% for toluidine blue (TP) dye after 90 min, whereas CuO NPs achieved slightly lower rates. The CuO NPs and CuO/PEG NPs displayed significant photocatalytic degradation activity against amoxicillin (antibiotic), with degradation rates of 91% and 98%, respectively, after 120 min. The reaction kinetics of CuO/PEG NPs and CuO NPs followed a pseudo-first order model, with CuO/PEG NPs exhibiting a higher rate constant than CuO NPs. Overall, modifying the CuO NPs with PEG demonstrated excellent photocatalytic properties for environmental remediation and exhibited antioxidant activity, suggesting their potential use in wastewater treatment and therapeutic applications.

Stressful environmental conditions lead to the production of reactive oxygen species in the chloroplasts, due to limited photosynthesis and enhanced excitation pressure on the photosystems. Among these reactive species, singlet oxygen (¹O₂), which is generated at the level of the PSII reaction center, is very reactive, readily oxidizing macromolecules in its immediate surroundings, and it has been identified as the principal cause of photooxidative damage in plant leaves. The two β-carotene molecules present in the PSII reaction center are prime targets of ¹O₂ oxidation, leading to the formation of various oxidized derivatives. Plants have evolved sensing mechanisms for those PSII-generated metabolites, which regulate gene expression, putting in place defense mechanisms and alleviating the effects of PSII-damaging conditions. A new picture is thus emerging which places PSII as a sensor and transducer in plant stress resilience through its capacity to generate signaling metabolites under excess light energy. This review summarizes new advances in the characterization of the apocarotenoids involved in the PSII-mediated stress response and of the pathways elicited by these molecules, among which is the xenobiotic detoxification.

Micronutrient deficiencies are common in locales where people must rely upon sorghum as their staple diet. Sorghum grain is seriously deficient in provitamin A (β-carotene) and in the bioavailability of iron and zinc. Biofortification is a process to improve crops for one or more micronutrient deficiencies. We have developed sorghum with increased β-carotene accumulation that will alleviate vitamin A deficiency among people who rely on sorghum as their dietary staple. However, subsequent β-carotene instability during storage negatively affects the full utilization of this essential micronutrient. We determined that oxidation is the main factor causing β-carotene degradation under ambient conditions. We further demonstrated that coexpression of homogentisate geranylgeranyl transferase (HGGT), stacked with carotenoid biosynthesis genes, can mitigate β-carotene oxidative degradation, resulting in increased β-carotene accumulation and stability. A kinetic study of β-carotene degradation showed that the half-life of β-carotene is extended from less than 4 wk to 10 wk on average with HGGT coexpression.