Carotenoid cleavage oxygenases can cleave carotenoids into a range of biologically important products. Carotenoid isomerooxygenase (NinaB) and β, β-carotene 15, 15\'-monooxygenase (BCO1) are two important oxygenases. In order to understand the roles that both oxygenases exert in crustaceans, we first investigated NinaB-like (EsNinaBl) and BCO1-like (EsBCO1l) within the genome of Chinese mitten crab (Eriocheir sinensis). Their functions were then deciphered through an analysis of their expression patterns, an in vitro β-carotene degradation assay, and RNA interference. The results showed that both EsNinaBl and EsBCO1l contain an RPE65 domain and exhibit high levels of expression in the hepatopancreas. During the molting stage, EsNinaBl exhibited significant upregulation in stage C, whereas EsBCO1l showed significantly higher expression levels at stage AB. Moreover, dietary supplementation with β-carotene resulted in a notable increase in the expression of EsNinaBl and EsBCO1l in the hepatopancreas. Further functional assays showed that the EsNinaBl expressed in E. coli underwent significant changes in its color, from orange to light; in addition, its β-carotene cleavage was higher than that of EsBCO1l. After the knockdown of EsNinaBl or EsBCO1l in juvenile E. sinensis, the expression levels of both genes were significantly decreased in the hepatopancreas, accompanied by a notable increase in the redness (a*) values. Furthermore, a significant increase in the β-carotene content was observed in the hepatopancreas when EsNinaBl-mRNA was suppressed, which suggests that EsNinaBl plays an important role in carotenoid cleavage, specifically β-carotene. In conclusion, our findings suggest that EsNinaBl and EsBCO1l may exhibit functional co-expression and play a crucial role in carotenoid cleavage in crabs.

Carotenoid cleavage oxygenases (CCOs) enzymes play an important role in plant growth and development by producing a wide array of apocarotenoids and their derivatives. These compounds are vital for colouring flowers and fruits and synthesizing plant hormones such as abscisic acid and strigolactones. Despite their importance, the gene family responsible for CCO enzymes in sunflowers has not been identified. In this study, we identify the CCO genes of the sunflower plant to fill this knowledge gap. Phylogenetic and synteny analysis indicated that the Helianthus annnus CCO (HaCCO) genes were conserved in different plant species and they could be divided into three subgroups based on their conserved domains. Analysis using MEME tool and multiple sequence alignment identified conserved motifs in the HaCCO gene sequence. Cis-regulatory elements (CREs) analysis of the HaCCO genes indicated the presence of various responsive elements related to plant hormones, development, and responses to both biotic and abiotic stresses. This implies that these genes may respond to plant hormones, developmental cues, and drought stress, offering potential applications in the development of more resistant crops. Genes belonging to the 9-cis-epoxy carotenoid dioxygenases (NCED) subgroups predominantly exhibited chloroplast localization, whereas the genes found in other groups are primarily localized in the cytoplasm. These 21 identified HaCCOs were regulated by 60 miRNAs, indicating the crucial role of microRNAs in gene regulation in sunflowers. Gene expression analysis under drought stress revealed significant up-regulation of HaNCED16 and HaNCED19, genes that are pivotal in ABA hormone biosynthesis. During organ-specific gene expression analysis, HaCCD12 and HaCCD20 genes exhibit higher activity in leaves, indicating a potential role in leaf pigmentation. This study provides a foundation for future research on the regulation and functions of the CCO gene family in sunflower and beyond. There is potential for developing molecular markers that could be employed in breeding programs to create new sunflower lines resistant to biotic and abiotic stresses.

Durian (Durio zibethinus L.), popularly known as the \"King of fruits,\" holds significant economic importance in Southeast Asia, including Thailand. During its ripening process, the phytohormone abscisic acid (ABA) content has been reported to increase. However, a comprehensive understanding of ABA\'s specific role in durian fruit ripening remains elusive. Furthermore, little is known about the molecular aspects of the carotenoid cleavage pathway in this iconic fruit. Therefore, we performed genome-wide identification of the carotenoid cleavage oxygenase (CCO) family in durian. This family includes the nine-cis-epoxycarotenoid dioxygenases (NCEDs) responsible for ABA production and the carotenoid cleavage dioxygenases exhibiting diverse substrate specificities. Through phylogenetic analysis, we classified 14 CCOs in durian into 8 distinct subfamilies. Notably, each DzCCO subfamily displayed a conserved motif composition. Cis-acting element prediction showed that cis-elements related to plant hormones and environmental stress responses were distributed in the DzCCO promoter. In addition, transcriptome analysis was performed to examine the expression pattern during the fruit development and ripening stages. Interestingly, DzNCED5a, a ripening-associated gene, exhibited the highest expression level at the ripe stage, outperforming other CCOs. Its expression markedly correlated with increased ABA contents during the ripening stages of both the \"Monthong\" variety and other durian cultivars. Transiently expressed DzNCED5a in Nicotiana benthamiana leaves confirmed its function in ABA biosynthesis. These findings highlight the involvement of DzNCED5a in ABA production and its potential importance in durian fruit ripening. Overall, this study provides insights into the significance of CCOs in durian fruit ripening.

Rice constitutes a foundational cereal and plays a vital role in the culinary sector. However, the detriments of abiotic stress on rice quality and productivity are noteworthy. Carotenoid cleavage oxygenases (CCO) hold vital importance as they enable the particular breakdown of carotenoids and significantly contribute towards the growth and response to abiotic stress in rice. Due to the insufficient information regarding rice CCOs and their potential role in abiotic stress, their utilization in stress-resistant genetic breeding remains limited. The current research identified 16 CCO genes within the Oryza sativa japonica group. These OsCCO genes can be bifurcated into three categories based on their conserved sequences: NCEDs (9-Cis-epoxycarotenoid dioxygenases), CCDs (Carotenoid cleavage dioxygenases) and CCD-like (Carotenoid cleavage dioxygenases-like). Conserved motifs were found in the OsCCO gene sequence via MEME analysis and multiple sequence alignment. Stress-related cis-elements were detected in the promoter regions of OsCCOs genes, indicating their involvement in stress response. Additionally, the promoters of these genes had various components related to plant light, development, and hormone responsiveness, suggesting they may be responsive to plant hormones and involved in developmental processes. MicroRNAs play a pivotal role in the regulation of these 16 genes, underscoring their significance in rice gene regulation. Transcriptome data analysis suggests a tissue-specific expression pattern for rice CCOs. Only OsNCED6 and OsNCED10 significantly up-regulated during salt stress, as per RNA seq analyses. CCD7 and CCD8 levels were also higher in the CCD group during the inflorescence growth stage. This provides insight into the function of rice CCOs in abiotic stress response and identifies possible genes that could be beneficial for stress-resistant breeding.

Carotenoids and their cleavage products (norisoprenoids) have excellent functional properties with diverse applications in foods, medicaments, cosmetics, etc. Carotenoids can be oxidatively cleaved through nonspecific reactions or by carotenoid cleavage oxygenases (CCOs), the product of which could further modify food flavor. This review provides comprehensive information on both carotenoid synthesis and cleavage processes with emphasis on enzyme characterization and biosynthetic pathway optimization. The use of interdisciplinary approaches of bioengineering and computer-aided experimental technology for key enzyme modification and systematic pathway design is beneficial to monitor metabolic pathways and assess pathway bottlenecks, which could efficiently lead to accumulation of carotenoids in microorganisms. The identification of CCOs spatial structures isolated from different species has made a significant contribution to the current state of knowledge. Current trends in carotenoid-related flavor modification are also discussed. In particular, we propose the carotenoid-synthesizing yeast Rhodotorula spp. for the production of food bioactive compounds. Understanding the behavior underlying the formation of norisoprenoids from carotenoids using interdisciplinary approaches may point toward other areas of investigation that could lead to better exploiting the potential use of autochthonous yeast in flavor enhancement.

Carotenoid cleavage oxygenases (CCOs) play crucial roles in plant growth and development, as well as in the response to phytohormonal, biotic and abiotic stresses. However, comprehensive and systematic research on the CCO gene family has not yet been conducted in Saccharum. In this study, 47 SsCCO and 14 ShCCO genes were identified and characterized in Saccharum spontaneum and Saccharum spp. R570 cultivar, respectively. The SsCCOs consisted of 38 SsCCDs and 9 SsNCEDs, while ShCCOs contained 11 ShCCDs and 3 ShNCEDs. The SsCCO family could be divided into 7 groups, while ShCCO family into 5 groups. The genes/proteins contained similar compositions within the same group, and the evolutionary mechanisms differed between S. spontaneum and R570. Gene Ontology annotation implied that CCOs were involved in many physiological and biochemical processes. Additionally, 41 SsCCOs were regulated by 19 miRNA families, and 8 ShCCOs by 9 miRNA families. Cis-regulatory elements analysis suggested that CCO genes functioned in the process of growth and development or under the phytohormonal, biotic and abiotic stresses. qRT-PCR analysis indicated that nine CCO genes from different groups exhibited similar expression patterns under abscisic acid treatment, while more divergent profiles were observed in response to Sporisorium scitamineum and cold stresses. Herein, comparative genomics analysis of the CCO gene family between S. spontaneum and R570 was conducted to investigate its evolution and functions. This is the first report on the CCO gene family in S. spontaneum and R570, thus providing valuable information and facilitating further investigation into its function in the future.

NOV1, a stilbene cleavage oxygenase, catalyzes the cleavage of the central double bond of stilbenes to two phenolic aldehydes, using a 4-His Fe(II) center and dioxygen. Herein, we use in-protein quantum mechanical/molecular mechanical (QM/MM) calculations to elucidate the reaction mechanism of the central double bond cleavage of phytoalexin resveratrol by NOV1. Our results showed that the oxygen molecule prefers to bind to the iron center in a side-on fashion, as suggested from the experiment. The quintet Fe-O2 complex with the side-on superoxo antiferromagnetic coupled to the resveratrol radical is identified as the reactive oxygen species. The QM/MM results support the dioxygenase mechanism involving a dioxetane intermediate with a rate-limiting barrier of 10.0 kcal mol-1. The alternative pathway through an epoxide intermediate is ruled out due to a larger rate-limiting barrier (26.8 kcal mol-1). These findings provide important insight into the catalytic mechanism of carotenoid cleavage oxygenases and also the dioxygen activation of non-heme enzymes.

Mucor circinelloides exhibits the complex sexual behaviour that is induced in other Mucoromycotina by a family of apocarotenoids called trisporoids. The genome of M. circinelloides contains four genes encoding putative carotenoid cleavage dioxygenases. The gene products of two of them were sufficient to convert β-carotene into the precursors of three families of apocarotenoids, both in vitro and in the Escherichia coli heterologous in vivo system. The first of these products, CarS, cleaved the C40 β-carotene into the C15 precursor of cyclofarnesoids and a C25 apocarotenal that was converted by the second enzyme, AcaA, into the C18 precursor of trisporoids and the C7 precursor of methylhexanoids. Apocarotenoids were not found in single or mixed cultures of the two strains of opposite sex, whose interaction readily produced zygospores, the sexual fusion cells.