Search Results (32)

This study aims to clarify the dose–response relationship between dietary β-carotene levels and gonadal pigment deposition and regulation mechanisms related to the carotenoid synthesis of Strongylocentrotus intermedius based on a 60-day feeding trial and subsequent transcriptome analysis. Adult sea urchins (initial weight: 9.33 ± 0.21 g) of three cages were given one of the dry feeds with different doses of β-carotene (0 mg/kg, 150 mg/kg, 300 mg/kg) or fresh kelp (Saccharina japonica). The results indicated that the weight gain rate (WGR) of sea urchins increased with the addition of β-carotene, with that of the C300 group being markedly higher than that of the C0 group. The addition of β-carotene significantly improved the redness (a*) and yellowness (b*) values of the gonads, with sea urchins in the C300 group exhibiting closest gonad coloration to those in the kelp-fed group. Meanwhile, β-carotene and echinenone in the gonads of the C300 group showed the highest contents, reaching 1.96 μg/kg and 11.97 μg/kg, respectively. Several differential genes, enriched in the pathways of steroid biosynthesis, oxidative phosphorylation, and ubiquitination, were screened based on transcriptome analysis. Real-time PCR further demonstrated that β-carotene significantly upregulated the expression of cholesterol 25-hydroxylase (CH25H), NADH dehydrogenase subunit 1 (ND1), NADH dehydrogenase subunit 2 (ND2), and NADH dehydrogenase subunit 4 (ND4) while it downregulated the expression of 24-dehydrocholesterol reductase (DHCR24). These results showed that 300 mg/kg β-carotene significantly increased the WGR, redness, and yellowness values, as well as the contents of β-carotene and echinenone in the gonads of S. intermedius. On the one hand, dietary β-carotene increased NADH enzyme activity, which participates in echinenone synthesis by donating electrons for the transformation of β-carotene to echinenone synthesis. On the other hand, the addition of β-carotene inhibited cholesterol synthesis by increasing the expression of CH25H and decreasing the expression of DHCR24, which could in turn increase the fluidity and permeability of the cell membranes and the transport efficiency of β-carotene and echinenone from the digestive tract to the gonads. These results provided fundamental insights into the production of sea urchin gonads with market-favored colors. Full article

Background: Myxol, a monocyclic carotenoid with β- and ψ-end groups, has been identified in only a limited number of bacteria, such as flavobacteria and cyanobacteria. Despite its biological significance, the biosynthetic pathway of myxol is not well understood, and studies on its physiological functions and biological activities are limited because of its rarity. Methods: BLAST homology searches for carotenoid biosynthesis genes in the genome of Nonlabens were performed. The carotenogenesis-related genes in the genome of the marine flavobacteria Nonlabens spongiae were individually cloned and functionally characterized using a heterologous Escherichia coli expression system. Carotenoids from N. spongiae were identified using an LC-MS analysis. Results: We identified a gene cluster involved in carotenoid biosynthesis in the genome of N. spongiae. This cluster includes genes encoding phytoene synthase (CrtB), phytoene desaturase (CrtI), lycopene cyclase (CrtY), carotenoid 1,2-hydratase (CruF), carotenoid 3,4-desaturase (ψ-end group) (CrtD), carotenoid 2-hydroxylase (ψ-end group) (CrtA-OH), and carotene hydro-xylase (CrtZ). Based on the characteristics of these enzymes, the primary products were predicted to be myxol and/or zeaxanthin. A spectroscopic analysis confirmed that myxol was the primary carotenoid. Furthermore, a plasmid containing a reconstructed gene cluster and geranylgeranyl pyrophosphate synthase (CrtE) located outside the cluster was introduced into E. coli. This system predominantly accumulated myxol, indicating that the reconstructed gene cluster enabled efficient myxol production in E. coli. Conclusions: This study highlighted the potential biotechnological applications of the carotenoid biosynthesis gene clusters for myxol production. Full article

Traditional maize possesses low concentrations of provitamin-A and vitamin-E, leading to various health concerns. Mutant alleles of crtRB1 and vte4 that enhance β-carotene (provitamin-A) and α-tocopherol (vitamin-E), respectively, in maize kernels have been explored in several biofortification programs. For genetic improvement of these target nutrients, uniplex-PCR assays are routinely used in marker-assisted selection. However, due to back-to-back breeding seasons, the time required for genotyping individually for each target gene in large backcross populations becomes a constraint for advancing the generations. Additionally, multiple PCR assays for various genes increase the required costs and resources. Here, we aimed to develop a multiplex-PCR assay to simultaneously identify different allelic forms of crtRB1 and vte4 genes and validate them in a backcross-based segregating population. The PCR assay was carried out using newly developed primers for crtRB1 and a gene-specific primer for vte4. The uniplex-PCR assay was standardized for selected primer pairs in the BC₁F₁ population segregating for crtRB1 and vte4 genes. Subsequently, a multiplex-PCR assay for crtRB1 and vte4 genes was developed and employed for genotyping in the BC₁F₁ population. The assay differentiated among four possible genotypic classes, namely crtRB1⁺crtRB1/vte4⁺vte4, crtRB1crtRB1/vte4⁺vte4, crtRB1⁺crtRB1/vte4⁺vte4⁺, and crtRB1crtRB1/vte4⁺vte4⁺. This newly developed multiplex-PCR assay saved 41.7% of the cost and 35.6% of the time compared to two individual uniplex-PCR assays. The developed assay could accelerate maize nutritional quality breeding programs through rapid and cost-effective genotyping for the target genes. This is the first report of a multiplex-PCR assay specific to crtRB1 and vte4 genes for its use in genomics-assisted breeding in maize. Full article

Background: Carotenoids play essential nutritional and physiological roles in aquatic animals. Since aquatic species cannot synthesize carotenoids de novo, they must obtain these compounds from their diet to meet the physiological and adaptive requirements needed in specific aquaculture stages and conditions. Carotenoid supplementation in Artemia represents a promising strategy to enhance pigmentation, health, and growth in aquaculture species, particularly in larvae and other early developmental stages. Methods: In this study, a β-carotene enrichment process was applied to Artemia metanauplii to investigate the biological fate and potential effects of β-carotene. Results: The results indicated significant β-carotene uptake by Artemia, with peak levels observed at 12 h. Alongside β-carotene, two xanthophylls—canthaxanthin and echinenone—were detected in Artemia, each exhibiting distinct patterns during the enrichment and subsequent depletion phases. The transcriptome analysis identified 2705 differentially expressed genes (DEGs), offering valuable insights into gene functions associated with carotenoid absorption, metabolism, and antioxidant mechanisms. The findings suggest that β-carotene enrichment enhances metabolic activity and energy pathways, supporting the physiological functions of Artemia. Notably, unlike other crustaceans, Artemia lack certain enzymes necessary for converting β-carotene into astaxanthin, restricting them to producing keto-carotenoids like canthaxanthin. Furthermore, the study highlights the upregulation of genes encoding lipid transport proteins, such as CD36 and ABC transporters, which may contribute to carotenoid absorption in Artemia. Additional functional insights are provided by the gene BCO2, which regulates pigmentation by preventing excessive carotenoid accumulation, along with ketolase and hydroxylase enzymes in carotenoid metabolic pathways. Conclusions: This research advances our understanding of carotenoid metabolism in crustaceans, with potential implications for aquaculture nutrition and feed formulation. Full article

Physalis pubescens and Physalis alkekengi, members of the Physalis genus, are valued for their delicious and medicinal fruits as well as their different ripened fruit colors—golden for P. pubescens and scarlet for P. alkekengi. This study aimed to elucidate the pigment composition and genetic mechanisms during fruit maturation in these species. Fruit samples were collected at four development stages, analyzed using spectrophotometry and high-performance liquid chromatography (HPLC), and complemented with transcriptome sequencing to assess gene expression related to pigment biosynthesis. β-carotene was identified as the dominant pigment in P. pubescens, contrasting with P. alkekengi, which contained both lycopene and β-carotene. The carotenoid biosynthesis pathway was central to fruit pigmentation in both species. Key genes pf02G043370 and pf06G178980 in P. pubescens, and TRINITY_DN20150_c1_g3, TRINITY_DN10183_c0_g1, and TRINITY_DN23805_c0_g3 in P. alkekengi were associated with carotenoid production. Notably, the MYB-related and bHLH transcription factors (TFs) regulated zeta-carotene isomerase and β-hydroxylase activities in P. pubescens with the MYB-related TF showing dual regulatory roles. In P. alkekengi, six TF families—bHLH, HSF, WRKY, M-type MADS, AP2, and NAC—were implicated in controlling carotenoid synthesis enzymes. Our findings highlight the intricate regulatory network governing pigmentation and provide insights into Physalis germplasm’s genetic improvement and conservation. Full article

Physalis philadelphica, a member of the Solanaceae family, commonly known as Physalis, is a one-year-old herbaceous plant with both medicinal and edible properties, as well as ornamental value. At present, only limited research is available on the flower color of P. philadelphica. This study aimed to elucidate the metabolic characteristics underlying the flower color of P. philadelphica and to identify key genes associated with flower color metabolism. We selected two representative varieties of P. philadelphica with significant differences in flower color, namely, “Tieba” (yellow flower) and “Qingjin” (yellow-purple flower), as the experimental materials. The analysis of related pigment components and the determination of relative content by high-performance liquid chromatography were conducted to investigate the flower color-related metabolic pathways of P. philadelphica. Through next-generation sequencing, these pathways were further investigated for the characteristics and differentially expressed genes (DEGs) associated with flower color formation. The results of the research show that: Anthocyanin is the main component of petal coloring of P. philadelphica var. Qingjin, while malvidin pigment, pelargonidin, delphinidin, and cyanidin are the main components of flower color intensity. Carotenoids are the main components of the petal coloring of P. philadelphica var. Tieba and β-carotene is the main component of flower color intensity. Comparing different developmental stages of these two kinds of Physalis pubescens, we identified two key transcription factors (TFs) (eBP and STAT) that were involved in the inhibition of anthocyanin synthesis and regulate the inhibition of pf05G124640 (dihydroflavonol 4-reductase) and pf09G224140 (anthocyanin synthase) in anthocyanin synthesis. One heat shock transcription factor was found to regulate the flavonoid and flavonol synthesis pathway of pf01G020090 (anthocyanin 3-O-glucosyltransferase); two key TFs (NAC and G2-Like), pf10G255070 (isoricin dehydrogenase) and pf09G237080 (abscisic acid 8′-hydroxylase), played important roles in carotene biosynthesis. This study provides new insights for further exploration of the genetic diversity of petal coloring in P. philadelphica and establishes a foundation for subsequent molecular breeding efforts. Full article

Terpenes are high-value chemicals which can be produced by engineered cyanobacteria from sustainable resources, solar energy, water and CO₂. We previously reported that the euryhaline unicellular cyanobacteria Synechocystis sp. PCC 6803 (S.6803) and Synechococcus sp. PCC 7002 (S.7002) produce farnesene and limonene, respectively, more efficiently than other terpenes. In the present study, we attempted to enhance farnesene production in S.6803 and limonene production in S.7002. Practically, we tested the influence of key cyanobacterial enzymes acting in carbon fixation (RubisCO, PRK, CcmK3 and CcmK4), utilization (CrtE, CrtR and CruF) and storage (PhaA and PhaB) on terpene production in S.6803, and we compared some of the findings with the data obtained in S.7002. We report that the overproduction of RubisCO from S.7002 and PRK from Cyanothece sp. PCC 7425 increased farnesene production in S.6803, but not limonene production in S.7002. The overexpression of the crtE genes (synthesis of terpene precursors) from S.6803 or S.7002 did not increase farnesene production in S.6803. In contrast, the overexpression of the crtE gene from S.6803, but not S.7002, increased farnesene production in S.7002, emphasizing the physiological difference between these two model cyanobacteria. Furthermore, the deletion of the crtR and cruF genes (carotenoid synthesis) and phaAB genes (carbon storage) did not increase the production of farnesene in S.6803. Finally, as a containment strategy of genetically modified strains of S.6803, we report that the deletion of the ccmK3K4 genes (carboxysome for CO₂ fixation) did not affect the production of limonene, but decreased the production of farnesene in S.6803. Full article

Astaxanthin is a carotenoid species with the highest antioxidant capability. Its natural resource is very rare. The biosynthesis of astaxanthin from β-carotene includes a hydroxylation step and a ketolation step, for which the corresponding enzymes have been characterized in a few species. However, the sequence of these two reactions is unclear, and may vary with different organisms. In this study, we aimed to elucidate this sequence in Synechocystis, which is an ideal cyanobacterial synthetic biology chassis. We first silenced the endogenous carotene oxygenase gene SyneCrtO to avoid its possible interference in the carotenoid metabolic network. We then introduced the β-carotene ketolase gene from Haematococcus pluvialis (HpBKT) and the CrtZ-type carotene β-hydroxylase gene from Pantoea agglomerans (PaCrtZ) to this δCrtO strain. Our pigment analysis demonstrated that both the endogenous CrtR-type carotene hydroxylase SyneCrtR and HpBKT have the preference to use β-carotene as their substrate for hydroxylation and ketolation reactions to produce zeaxanthin and canthaxanthin, respectively. However, the endogenous SyneCrtR is not able to further catalyze the 3,3′-hydroxylation of canthaxanthin to generate astaxanthin. From our results, a higher accumulation of canthaxanthin and a much lower level of astaxanthin, as confirmed using liquid chromatography–tandem mass spectrometry analysis, were detected in our transgenic BKT⁺/CrtZ⁺/δCrtO cells. Therefore, we proposed that the bottleneck for the heterologous production of astaxanthin in Synechocystis might exist at the hydroxylation step, which requires a comprehensive screening or genetic engineering for the corresponding carotene hydroxylase to enable the industrial production of astaxanthin. Full article

Given the importance of olive sprouts, it is crucial to explore their potential as an innovative source of bioactive and nutritive compounds through research. Here, we aimed at investigating the potential role of benzylaminopurine (BAP) in improving the tissue chemical composition and bioactivity of olive sprouts. To this end, seeds of two olive varieties (Olea europaea L. vr. Kroniaki and Coratina) were primed with BAP at 25 µM. A substantial enhancement was observed in biomass accumulation by 35% and 30% in Kroniaki and Coratina varieties, respectively. Likewise, the photosynthetic pigments (total chlorophyll, α- and β-carotene, lutein and β-cryptoxanthin) in both varieties were increased, mainly in Coratina. At primary metabolic level, BAP priming improved sprout lipid composition, particularly in Coratina variety. At antioxidant level, BAP priming improved lipid antioxidants (α-, β- and γ-tocopherols) and water-soluble antioxidants (phenols, flavonoids, ascorbate, glutathione and anthocyanins) in both olive varieties. At the anthocyanins level, their precursors (phenylalanine, cinnamic acid, coumaric acid and naringenin) and key biosynthetic enzyme activity (phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), coenzyme A ligase (4CL) and cinnamate 4-hydroxylase (C4H)) were improved in olive varieties, but to a greater extent in Coratina variety. Overall, the sprouts of BAP-primed olive seeds could potentially enhance their nutritional value, suggesting that the sprouts of BAP-primed olive seeds can be used as a food ingredient and additives. Full article

Astaxanthin is a fascinating molecule with powerful antioxidant activity, synthesized exclusively by specific microorganisms and higher plants. To expand astaxanthin production, numerous studies have employed metabolic engineering to introduce and optimize astaxanthin biosynthetic pathways in microorganisms and plant hosts. Here, we report the metabolic engineering of animal cells in vitro to biosynthesize astaxanthin. This was accomplished through a two-step study to introduce the entire astaxanthin pathway into human embryonic kidney cells (HEK293T). First, we introduced the astaxanthin biosynthesis sub-pathway (Ast subp) using several genes encoding β-carotene ketolase and β-carotene hydroxylase enzymes to synthesize astaxanthin directly from β-carotene. Next, we introduced a β-carotene biosynthesis sub-pathway (β-Car subp) with selected genes involved in Ast subp to synthesize astaxanthin from geranylgeranyl diphosphate (GGPP). As a result, we unprecedentedly enabled HEK293T cells to biosynthesize free astaxanthin from GGPP with a concentration of 41.86 µg/g dry weight (DW), which represented 66.19% of the total ketocarotenoids (63.24 µg/g DW). Through optimization steps using critical factors in the astaxanthin biosynthetic process, a remarkable 4.14-fold increase in total ketocarotenoids (262.10 µg/g DW) was achieved, with astaxanthin constituting over 88.82%. This pioneering study holds significant implications for transgenic animals, potentially revolutionizing the global demand for astaxanthin, particularly within the aquaculture sector. Full article

Astaxanthin is a powerful antioxidant and is used extensively as an animal feed additive and nutraceutical product. Here, we report the use of the β-carotene hydroxylase (CBFD1) and the β-carotene ketolase (HBFD1) from Adonis aestivalis, a flowering plant, to produce astaxanthin in E. coli equipped with the P. agglomerans β-carotene pathway and an over-expressed 4-methylerythritol-phosphate (MEP) pathway or the isopentenol utilization pathway (IUP). Introduction of the over-expressed MEP pathway and the IUP resulted in a 3.2-fold higher carotenoid content in LB media at 36 h post-induction compared to the strain containing only the endogenous MEP. However, in M9 minimal media, the IUP pathway dramatically outperformed the over-expressed MEP pathway with an 11-fold increase in total carotenoids produced. The final construct split the large operon into two smaller operons, both with a T7 promoter. This resulted in slightly lower productivity (70.0 ± 8.1 µg/g·h vs. 53.5 ± 3.8 µg/g·h) compared to the original constructs but resulted in the highest proportion of astaxanthin in the extracted carotenoids (73.5 ± 0.2%). Full article

Chlamydomonas reinhardtii is a unicellular green alga that can grow heterotrophically by using acetate as a carbon source. Carotenoids are natural pigments with biological activity and color, which have functions such as antioxidant, anti-inflammatory, vision protection, etc., and have high commercial value and prospects. We transformed Chlamydomonas reinhardtii with the BKT genes from Phaffia rhodozyma (PrBKT) and Chlamydomonas reinhardtii (CrBKT) via plasmid vector, and screened out the stable transformed algal strains C18 and P1. Under the condition that the cell density of growth was not affected, the total carotenoid content of C18 and P1 was 2.13-fold and 2.20-fold higher than that of the WT, respectively. CrBKT increased the levels of β-carotene and astaxanthin by 1.84-fold and 1.21-fold, respectively, while PrBKT increased them by 1.11-fold and 1.27-fold, respectively. Transcriptome and metabolome analysis of C18 and P1 showed that the overexpression of CrBKT only up-regulated the transcription level of BKT and LCYE (the gene of lycopene e-cyclase). However, in P1, overexpression of PrBKT also led to the up-regulation of ZDS (the gene of ζ-carotene desaturase) and CHYB (the gene of β-carotene hydroxylase). Metabolome results showed that the relative content of canthaxanthin, an intermediate metabolite of astaxanthin synthesis in C18 and P1, decreased. The overall results indicate that there is a structural difference between CrBKT and PrBKT, and overexpression of PrBKT in Chlamydomonas reinhardtii seems to cause more genes in carotenoid pathway metabolism to be up-regulated. Full article

A cell suspension culture of saffron (Crocus sativus L.) was previously established from style-derived calli to obtain an in vitro system for crocin, an uncommon and valuable water-soluble apocarotenoid, and carotenoid production suitable for future scaling up. To shed more light on the correlation between apocarotenoid biosynthesis and key-gene expression, in this study, SA was used at 0.5 mM concentration to elicit crocin production and the effects on carotenoid production were analyzed after 6, 12, 24, and 48 h. HPLC-DAD analysis was used for total crocin quantification as well as the other carotenoids zeaxanthin, β-carotene and lutein. Quantitative RT-PCR was used to analyze the transcript levels of saffron apocarotenoid biosynthetic key genes PSY (phytoene synthase), BCH1 (β-carotene hydroxylase), and CCD2 (carotenoid cleavage dioxygenase) after SA elicitation. In saffron suspension-cultured cells elicited by SA, the carotenoid biosynthetic pathway was mostly enhanced toward crocin biosynthesis, known to exert strong biological activity and therapeutic effects, rather than lutein or xanthins. SA increased BCH1 and CCD2 gene expression 15.6 and 3.3 times, respectively, compared to the control at 24 h after elicitation. Although a dynamic change of metabolite contents and gene expression was observed during the 48 h time course in response to SA elicitation, the changes of zeaxanthin and crocin were consistent with the regulation of the corresponding genes BCH and CCD2 during the time course. In conclusion, the effects of SA on regulation of gene expression in the apocarotenoid pathway could be successfully applied for the biotechnological production of crocin. Full article

Carotenoids are important compounds of quality and coloration within sweet potato storage roots, but the mechanisms that govern the accumulation of these carotenoids remain poorly understood. In this study, metabolomic and transcriptomic analyses of carotenoids were performed using young storage roots (S2) and old storage roots (S4) from white-fleshed (variety S19) and yellow-fleshed (variety BS) sweet potato types. S19 storage roots exhibited significantly lower total carotenoid levels relative to BS storage roots, and different numbers of carotenoid types were detected in the BS-S2, BS-S4, S19-S2, and S19-S4 samples. β-cryptoxanthin was identified as a potential key driver of differences in root coloration between the S19 and BS types. Combined transcriptomic and metabolomic analyses revealed significant co-annotation of the carotenoid and abscisic acid (ABA) metabolic pathways, PSY (phytoene synthase), CHYB (β-carotene 3-hydroxylase), ZEP (zeaxanthin epoxidase), NCED3 (9-cis-epoxycarotenoid dioxygenase 3), ABA2 (xanthoxin dehydrogenase), and CYP707A (abscisic acid 8’-hydroxylase) genes were found to be closely associated with carotenoid and ABA content in these sweet potato storage roots. The expression patterns of the transcription factors OFP and FAR1 were associated with the ABA content in these two sweet potato types. Together, these results provide a valuable foundation for understanding the mechanisms governing carotenoid biosynthesis in storage roots, and offer a theoretical basis for sweet potato breeding and management. Full article

Fruit color is one of the most critical characteristics of pepper. In this study, pepper (Capsicum baccatum L.) fruits with four trans-coloring periods were used as experimental materials to explore the color conversion mechanism of pepper fruit. By transcriptome and metabolome analysis, we identified a total of 307 flavonoid metabolites, 68 carotenoid metabolites, 29 DEGs associated with flavonoid biosynthesis, and 30 DEGs related to carotenoid biosynthesis. Through WGCNA (weighted gene co-expression network analysis) analysis, positively correlated modules with flavonoids and carotenoids were identified, and hub genes associated with flavonoid and carotenoid synthesis and transport were anticipated. We identified Pinobanksin, Naringenin Chalcone, and Naringenin as key metabolites in the flavonoid biosynthetic pathway catalyzed by the key genes chalcone synthase (CHS CQW23_29123, CQW23_29380, CQW23_12748), cinnamic acid 4-hydroxylase (C4H CQW23_16085, CQW23_16084), cytochrome P450 (CYP450 CQW23_19845, CQW23_24900). In addition, phytoene synthase (PSY CQW23_09483), phytoene dehydrogenase (PDS CQW23_11317), zeta-carotene desaturase (ZDS CQW23_19986), lycopene beta cyclase (LYC CQW23_09027), zeaxanthin epoxidase (ZEP CQW23_05387), 9-cis-epoxycarotenoid dioxygenase (NCED CQW23_17736), capsanthin/capsorubin synthase (CCS CQW23_30321) are key genes in the carotenoid biosynthetic pathway, catalyzing the synthesis of key metabolites such as Phytoene, Lycopene, β-carotene and ε-carotene. We also found that transcription factor families such as p450 and NBARC could play important roles in the biosynthesis of flavonoids and carotenoids in pepper fruits. These results provide new insights into the interaction mechanisms of genes and metabolites involved in the biosynthesis of flavonoids and carotenoids in pepper fruit leading to color changes in pepper fruit. Full article