Carotenoid Cleavage Dioxygenase Research Articles

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. β-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.

One-pot synthesis of dihydro-β-ionone from carotenoids using carotenoid cleavage dioxygenase and enoate reductase.

Dihydro-β-ionone is a characteristic aroma compound of Osmanthus fragrans and is widely applied in the flavor & fragrance industry. However, the main focus is on chemical synthesis due to the metabolic pathways of dihydro-β-ionone is still unclear. Here, we explored the one-pot synthesis system for dihydro-β-ionone production using carotenoid cleavage dioxygenase (CCD) and enoate reductase. After screening the CCD enzyme, PhCCD1 from the Petunia hybrid was identified as the suitable enzyme for the first step of dihydro-β-ionone synthesis due to the high enzyme activity for carotenoid. The PhCCD1 was expressed in Escherichia coli and further characterized. The optimal activity of PhCCD1 was observed at pH 6.8 and 45°C. The enzyme was stable over the pH range of 6.0-8.0 and had good thermal stability below 40°C. Then, we optimized the coupled reaction conditions for dihydro-β-ionone production by PhCCD1 and enoate reductase AaDBR1 from Artemisia annua. Furthermore, we introduced the NADPH regeneration system with a 1.5-fold enhancement for dihydro-β-ionone production. Collectively, approximately 13.34mg/L dihydro-β-ionone was obtained by the one-pot biosystem with a corresponding molar conversion of 85.8%. For the first time, we successfully designed and constructed a new synthesis pathway for dihydro-β-ionone production in vitro. The coupled catalysis reported herein illustrates the feasibility of producing dihydro-β-ionone from carotenoids and guides further engineering in the food industry.

Accumulation of β-citraurin and expression of carotenoid cleavage dioxygenase4 gene in citrus fruit during maturation

Accumulation of <b>β</b>-citraurin and expression of <i>carotenoid cleavage dioxygenase4</i> gene in citrus fruit during maturation

English

Carotenoids are important substances for the yellow color of peach flesh. In this study, the contents of carotenoids in two yellow flesh peach varieties ('Zhongtao Jinmi' and 'Jinxiang') and one white flesh peach variety ('Bairuyu') were determined by high-performance liquid chromatography. The results showed that the carotenoid content in the two yellow flesh peach varieties increased with fruit development and reached the maximum of 25.80 and 27.63 μg.g-1 at the mature stage, while that of the white flesh peach variety remained at a very low level during the whole fruit development period. Furthermore, transcriptome sequencing was performed to analyze the differentially expressed genes (DEGs) during fruit development among the three peach varieties. Among these DEGs, four candidate genes were identified to be potentially involved in the biosynthesis of carotenoids, including LOC18774744 (encoding UDP-glycosyltransferase 87A1 (UGT87A1)), LOC18792568 (encoding the carotenoid cleavage dioxygenase 4 gene (CCD4)), LOC109949966 (an unannotated gene), and LOC18779468 (encoding xyloglucan endoglucosylase/hydrolase protein 8. qRT-PCR confirmed that the expression of LOC109949966 and LOC18779468 in two yellow flesh varieties was significantly higher than that in the white flesh variety, while it was the opposite case for UGT87A1 and CCD4. Therefore, we speculate that CCD4 is directly involved in regulating carotenoid synthesis, while other three genes may play some indirect roles in the process. Our findings are expected to improve the understanding on the mechanism of carotenoid biosynthesis in peach. © 2022 Friends Science Publishers

Integrative analyses of transcriptome and carotenoids profiling revealed molecular insight into variations in fruits color of Citrus Reticulata Blanco induced by transplantation.

Citrus fruits exhibit vivid color and are favored extensively. However, the biochemical and molecular mechanism of Citrus Reticulata Blanco fruits coloring, especially the effect of transplantation on fruits coloring, is unclear. Herein, RNA-Seq and carotenoids profiling were applied to investigate the effect of transplantation on Orah mandarin fruits coloring. Transplantation induces fruit color shallowing, Ca2+ and ACC level declining and IAA level increasing. Transplantation induced variation in fruit skin and pulp carotenoids, mainly β-citraurin as one of the important pigments of citrus peel. 2253 up-regulated genes, 1103 down-regulated genes in skin and 815 up-regulated genes, 534 down-regulated genes in pulp of transplanted tree fruits were identified by RNA-Seq. The DEGs involved hormone signal, carotenoids biosynthesis and TFs such as MYB and bHLH family TFs. The carotenoid cleavage dioxygenase gene (Ciclev10028113m.g) is positively correlated with β-citraurin and regulated directly and/or indirectly by MYB1R1, PIF4, ACC and IAA. Integrative analyses revealed potential molecular insights into Orah mandarin peel color variation during transplantation.

The Carotenoid Cleavage Dioxygenase Gene CCD7-B, at Large, Is Associated with Tillering in Common Wheat

Wheat, an important cereal crop, is responsible for the livelihoods of many people, and a component of national food security. Tillering, which determines plant architecture and spike number, is a critical agronomic trait of wheat. The carotenoid cleavage dioxygenase 7 (CCD7) has an important effect on the growth of tillers or lateral branches and lateral roots of plants. In order to study the relationship between CCD7 and tillering in wheat, CCD7-B was isolated from 10 Chinese wheat varieties with different tiller numbers. Subsequently, bioinformatics, allelic variation analysis, and field experiments were performed. Wheat CCD7-B belongs to the retinal pigment epithelial membrane receptor (RPE65) superfamily; it displays the greatest homology with monocot CCD7 proteins. Phylogenetic analysis of wheat CCD7-B proteins indicated division into dicotyledonous and monocotyledonous clades. Allelic variation analysis of CCD7-B via SrgAI enzyme digestion (a marker of cleaved amplified polymorphic sequences) suggested that 262 Chinese wheat micro-core collections and 121 Chinese wheat major cultivars from the Yellow and Huai River Valley winter wheat region can be divided into two groups: CCD7-B1 (C/T/T) and CCD7-B2 (G/C/A). CCD7-B1 showed better allelic variation than did CCD7-B2 for increasing the number of effective tillers of wheat varieties in China. This study provides reference data for the application of CCD7-B alleles to wheat breeding and supports further research regarding the mechanism of tillering in common wheat.

Increasing atmospheric CO2 differentially supports arsenite stress mitigating impact of arbuscular mycorrhizal fungi in wheat and soybean plants

Arbuscular mycorrhizal fungi (AMF) are beneficial for the plant growth under heavy metal stress. Such beneficial effect is improved by elevated CO2 (eCO2). However, the mechanisms by which eCO2 improves AMF symbiotic associations under arsenite (AsIII) toxicity are hardly studied. Herein, we compared these regulatory mechanisms in species from two agronomical important plant families – grasses (wheat) and legumes (soybean). AsIII decreased plant growth (i.e., 53.75 and 60.29% of wheat and soybean, respectively) and photosynthesis. It also increased photorespiration and oxidative injury in both species, but soybean was more sensitive to oxidative stress as indicated by higher H2O2 accumulation and oxidation of protein and lipid. eCO2 significantly improved AMF colonization by increasing auxin levels, which induced high carotenoid cleavage dioxygenase (CCDs) activity, particularly in soybean roots. The improved sugar metabolism in plant shoots by co-application of eCO2 and AsIII allocated more sugars to roots sequentially. Sugar accumulation in plant roots is further induced by AMF, resulting in more C skeletons to produce organic acids, which are effectively exudated into the soil to reduce AsIII uptake. Exposure to eCO2 reduced oxidative damage and this mitigation was stronger in soybean. This could be attributed to a greater reduction in photorespiration as well as a stronger antioxidant and detoxification defence systems. The grass/legume-specificity was supported by principal component analysis, which revealed that soybean was more affected by AsIII stress and more responsive to AMF and eCO2. This study provided a mechanistic understanding of the impact of AMF, eCO2 and their interaction on As-stressed grass and legume plants, allowing better practical strategies to mitigate AsIII phytotoxicity.

Identifying Bixa orellana L. New Carotenoid Cleavage Dioxygenases 1 and 4 Potentially Involved in Bixin Biosynthesis.

Carotene cleavage dioxygenases (CCDs) are a large family of Fe2+ dependent enzymes responsible for the production of a wide variety of apocarotenoids, such as bixin. Among the natural apocarotenoids, bixin is second in economic importance. It has a red-orange color and is produced mainly in the seeds of B. orellana. The biosynthesis of bixin aldehyde from the oxidative cleavage of lycopene at 5,6/5′,6′ bonds by a CCD is considered the first step of bixin biosynthesis. Eight BoCCD (BoCCD1-1, BoCCD1-3, BoCCD1-4, CCD4-1, BoCCD4-2, BoCCD4-3 and BoCCD4-4) genes potentially involved in the first step of B. orellana bixin biosynthesis have been identified. However, the cleavage activity upon lycopene to produce bixin aldehyde has only been demonstrated for BoCCD1-1 and BoCCD4-3. Using in vivo (Escherichia coli) and in vitro approaches, we determined that the other identified BoCCDs enzymes (BoCCD1-3, BoCCD1-4, BoCCD4-1, BoCCD4-2, and BoCCD4-4) also participate in the biosynthesis of bixin aldehyde from lycopene. The LC-ESI-QTOF-MS/MS analysis showed a peak corresponding to bixin aldehyde (m/z 349.1) in pACCRT-EIB E. coli cells that express the BoCCD1 and BoCCD4 proteins, which was confirmed by in vitro enzymatic assay. Interestingly, in the in vivo assay of BoCCD1-4, BoCCD4-1, BoCCD4-2, and BoCCD4-4, bixin aldehyde was oxidized to norbixin (m/z 380.2), the second product of the bixin biosynthesis pathway. In silico analysis also showed that BoCCD1 and BoCCD4 proteins encode functional dioxygenases that can use lycopene as substrate. The production of bixin aldehyde and norbixin was corroborated based on their ion fragmentation pattern, as well as by Fourier transform infrared (FTIR) spectroscopy. This work made it possible to clarify at the same time the first and second steps of the bixin biosynthesis pathway that had not been evaluated for a long time.

Structural and Functional Analysis of Nonheme Iron Enzymes BCMO-1 and BCMO-2 from Caenorhabditis elegans.

Carotenoid metabolism is critical for diverse physiological processes. The nematode Caenorhabditis elegans has two genes that are annotated as β-carotene 15,15′-monooxygenase (BCMO) and are 17 centimorgan apart on chromosome II, but the function of BCMO-1 and BCMO-2 remains uncharacterized. Sequence homology indicates that the two enzymes belong to the carotenoid cleavage dioxygenase family that share a seven-bladed β-propeller fold with a nonheme iron center. Here we determined crystal structures of BCMO-1 and BCMO-2 at resolutions of 1.8 and 1.9 Å, respectively. Structural analysis reveals that BCMO-1 and BCMO-2 are strikingly similar to each other. We also characterized their β-carotene cleavage activity, but the results suggest that they may not act as β-carotene 15,15′-oxygenases.

Transgene-free genome editing supports CCD4 role as a negative regulator of β-carotene in banana.

This study aims to understand the regulatory mechanism of the β-carotene homeostasis by establishing transgene-free genome editing in banana. Carotenoid cleavage dioxygenases (CCDs) belong to a miniature gene family having an imperative role in the intricated carotenoid metabolism in plants. Here, the expression pattern of multiple CCDs was correlated with the levels of carotenoid accumulation in two contrasting cultivars, viz., Nendran (high β-carotene) and Rasthali (low β-carotene). The higher expression of the RAS-CCD4 inversely correlated with β-carotene accumulation in fruit-pulp of the Rasthali. The docking analysis followed enzyme assay of purified RAS-CCD4 suggested β-carotene and 10-apo-β-carotenal as its preferred substrates. Bacterial complementation assay affirmed RAS-CCD4 role in β-carotene degradation and then overexpression of the RAS-CCD4 in the Arabidopsis thaliana further validated results in-vivo by the significant reduction in β-carotene. Subsequently, CRISPR/Cas9 mediated editing of CCD4 was demonstrated in the protoplasts and embryogenic cell lines of Rasthali. The carotenoid profiling in stable mutant lines revealed higher fold β-carotene accumulation in non-green tissue (roots) than in green tissue (leaf) compared with the unedited control plants. The differential expression of carotenoid pathway genes was correlated with the metabolites in the edited lines. The study suggests that carotenoid catabolism is regulated by the CCD4 in tissue and cultivar specific manners, and also demonstrated the use of the genome editing tool in developing transgene-free biofortified banana.

Genome-Wide Identification of CCD Gene Family in Six Cucurbitaceae Species and Its Expression Profiles in Melon.

The carotenoid cleavage dioxygenase (CCD) gene family in plants comprises two subfamilies: CCD and 9-cis-epoxycarotenoid dioxygenase (NCED). Genes in the NCED subfamily are mainly involved in plant responses to abiotic stresses such as salt, low temperature, and drought. Members of the NCED subfamily are the most important rate-limiting enzymes in the biosynthesis of abscisic acid (ABA). In the present study, genome-wide analysis was performed to identify CCD gene members in six Cucurbitaceae species, including watermelon (Citrullus lanatus), melon (Cucumis melo), cucumber (C.sativus), pumpkin (Cucurbita moschata), bottle gourd (Lagenaria siceraria), and wax gourd (Benincasa hispida). A total of 10, 9, 9, 13, 8, 8 CCD genes were identified in the six species, respectively, and these genes were unevenly distributed in different chromosomes. Phylogenetic analysis showed that CCD genes of the six species clustered into two subfamilies: CCD and NCED, with five and three independent clades, respectively. The number of exons ranged from 1 to 15, and the number of motifs were set to 15 at most. The cis-acting elements analysis showed that a lot of the cis-acting elements were implicated in stress and hormone response. Melon seedlings were treated with salt, low temperature, drought, and ABA, and then tissue-specific analysis of CCDs expression were performed on the root, stem, upper leaf, middle leaf, female flower, male flower, and tendril of melon. The results showed that genes in CCD family exhibited various expression patterns. Different CCD genes of melon showed different degrees of response to abiotic stress. This study presents a comprehensive analysis of CCD gene family in six species of Cucurbitaceae, providing a strong foundation for future studies on specific genes in this family.

Comprehensive Analysis of Carotenoid Cleavage Dioxygenases Gene Family and Its Expression in Response to Abiotic Stress in Poplar.

Carotenoid cleavage dioxygenases (CCDs) catalyzes the cleavage of various carotenoids into smaller apocarotenoids which are essential for plant growth and development and response to abiotic stresses. CCD family is divided into two subfamilies: 9-cis epoxycarotenoid dioxygenases (NCED) family and CCD family. A better knowledge of carotenoid biosynthesis and degradation could be useful for regulating carotenoid contents. Here, 23 CCD genes were identified from the Populus trichocarpa genome, and their characterizations and expression profiling were validated. The PtCCD members were divided into PtCCD and PtNCED subfamilies. The PtCCD family contained the PtCCD1, 4, 7, and 8 classes. The PtCCDs clustered in the same clade shared similar intron/exon structures and motif compositions and distributions. In addition, the tandem and segmental duplications resulted in the PtCCD gene expansion based on the collinearity analysis. An additional integrated collinearity analysis among poplar, Arabidopsis, rice, and willow revealed the gene pairs between poplar and willow more than that between poplar and rice. Identifying tissue-special expression patterns indicated that PtCCD genes display different expression patterns in leaves, stems, and roots. Abscisic acid (ABA) treatment and abiotic stress suggested that many PtCCD genes are responsive to osmotic stress regarding the comprehensive regulation networks. The genome-wide identification of PtCCD genes may provide the foundation for further exploring the putative regulation mechanism on osmotic stress and benefit poplar molecular breeding.

Genome-wide identification of CsCCD gene family in tea plant (Camellia sinensis) and expression analysis of the oolong tea processing with supplementary LED light

Carotenoid cleavage dioxygenase (CCD) family is important for production of volatile aromatic compounds and synthesis of plant hormones. To explore the biological functions and gene expression patterns of CsCCD gene family in tea plant, genome-wide identification of CsCCD gene family was performed. The gene structures, conserved motifs, chromosome locations, protein physicochemical properties, evolutionary characteristics, interaction network and cis-acting regulatory elements were predicted and analyzed. Real time-quantitative reverse transcription PCR (RT-qPCR) was used to detect the relative expression level of CsCCD gene family members under different leaf positions and light treatments during processing. A total of 11 CsCCD gene family members, each containing exons ranging from 1 to 11 and introns ranging from 0 to 10, were identified. The average number of amino acids and molecular weight were 519 aa and 57 643.35 Da, respectively. Phylogenetic analysis showed the CsCCD gene family was clustered into 5 major groups (CCD1, CCD4, CCD7, CCD8 and NCED). The CsCCD gene family mainly contained stress response elements, hormone response elements, light response elements and multi-factor response elements, and light response elements was the most abundant (142 elements). Expression analysis showed that the expression levels of CsCCD1 and CsCCD4 in elder leaves were higher than those in younger leaves and stems. With the increase of turning over times, the expression levels of CsCCD1 and CsCCD4 decreased, while supplementary LED light strongly promoted their expression levels in the early stage. The expression level of NCED in younger leaves was higher than that in elder leaves and stems on average, and the expression trend varied in the process of turning over. NCED3 first increased and then decreased, with an expression level 15 times higher than that in fresh leaves. In the late stage of turning over, supplementary LED light significantly promoted its gene expression. In conclusion, CsCCD gene family member expressions were regulated by mechanical force and light. These understandings may help to optimize tea processing techniques and improve tea quality.

Evolutionary Origin of the Carotenoid Cleavage Oxygenase Family in Plants and Expression of Pepper Genes in Response to Abiotic Stresses.

Plant carotenoid cleavage oxygenase (CCO) is an enzyme that catalyzes the synthesis of carotenoids and participates in many important physiological functions. The plant CCOs exist in two forms, namely carotenoid cleavage dioxygenase (CCD) and nine-cis epoxide carotenoid dioxygenase (NCED). Although studies have shown that this gene family has been identified in many species, such as Arabidopsis, grape, and tomato, the evolutionary origin of the CCO family and the expression pattern of pepper genes in response to H2O2 and other abiotic stresses are still unclear. In this study, we used the bioinformatics method to identify and analyze the members of the CCO gene family from pepper and other 13 plants from lower to higher plant species based on the whole genome sequence. A total of 158 CCO genes were identified in different plant species and further divided into two groups (e.g., groups I and II). The former was subdivided into CCD7 and CCD8 and have independent evolutionary origins, respectively, while the latter was subdivided into CCD1, CCD4, CCD-like, and NCED, which may have come from a common ancestor. In addition, the results of RNA-seq showed that the expression patterns of pepper CaCCO genes were different in the tissues tested, and only few genes were expressed at high levels such as CaCCD1a, CaCCD4a, CaNCED3, and CaCCD1b. For hydrogen peroxide (H2O2) and other abiotic stresses, such as plant hormones, heat, cold, drought, and NaCl treatments, induction of about half of the CaCCO genes was observed. Moreover, the expression patterns of CaCCOs were further investigated under heat, cold, drought, and NaCl treatments using quantitative real-time PCR (qRT-PCR), and most members were responsive to these stresses, especially some CaCCOs with significant expression changes were identified, such as CaCCD4c, CaCCD-like1, CaCCD8, and CaCCD1b, suggesting the important roles of CaCCOs in abiotic stress responses. All these results will provide a valuable analytical basis for understanding the evolution and functions of the CCO family in plants.

Simultaneous dissection of grain carotenoid levels and kernel color in biparental maize populations with yellow-to-orange grain.

Maize enriched in provitamin A carotenoids could be key in combatting vitamin A deficiency in human populations relying on maize as a food staple. Consumer studies indicate that orange maize may be regarded as novel and preferred. This study identifies genes of relevance for grain carotenoid concentrations and kernel color, through simultaneous dissection of these traits in 10 families of the US maize nested association mapping panel that have yellow to orange grain. Quantitative trait loci were identified via joint-linkage analysis, with phenotypic variation explained for individual kernel color quantitative trait loci ranging from 2.4% to 17.5%. These quantitative trait loci were cross-analyzed with significant marker-trait associations in a genome-wide association study that utilized ∼27 million variants. Nine genes were identified: four encoding activities upstream of the core carotenoid pathway, one at the pathway branchpoint, three within the α- or β-pathway branches, and one encoding a carotenoid cleavage dioxygenase. Of these, three exhibited significant pleiotropy between kernel color and one or more carotenoid traits. Kernel color exhibited moderate positive correlations with β-branch and total carotenoids and negligible correlations with α-branch carotenoids. These findings can be leveraged to simultaneously achieve desirable kernel color phenotypes and increase concentrations of provitamin A and other priority carotenoids.

Enhancement of norisoprenoid and acetoin production for improving the aroma of fermented mango juice by Bacillus subtilis-HNU-B3

Mango juice (MJ) has high nutritional value and decent flavor. However, its aroma could be easily changed by heat treatment during processing, which affects its acceptability and quality. In this study, Bacillus subtilis-HNU-B3 that could transfer carotenoid, was obtained from orchard soil to improve the aroma of fermented mango juice (FMJ). Compared with MJ, the organic acid content of FMJ by B. subtilis-HNU-B3 was significantly changed, and the contents of succinic acid and malic acid were increased to 3.68 mg/mL and 2.04 mg/mL, respectively. Moreover, volatile compounds identified by headspace solid-phase micro-extraction gas-phase mass spectrometry in FMJ were norisoprenoid and acetoin, accounting for 51.52 % of the total volatile substances. In addition, B. subtilis-HNU-B3 fermentation significantly increased the antioxidant capacity and total phenol contents of MJ, and the activity of carotenoid cleavage dioxygenase was 188 U/L. In conclusion, the aroma of FMJ could be improved by B. subtilis-HNU-B3 through increasing the content of norisoprenoid and acetoin. Our results provide theatrical support for Bacillus subtilis to improve the aroma quality of FMJ.

Engineering high levels of saffron apocarotenoids in tomato.

Crocins and picrocrocin are high-value hydrophilic pigments produced in saffron and used commercially in the food and pharmaceutical industries. These apocarotenoids are derived from the oxidative cleavage of zeaxanthin by specific carotenoid cleavage dioxygenases. The pathway for crocins and picrocrocin biosynthesis was introduced into tomato using fruit specific and constitutive promoters and resulted in 14.48 mg/g of crocins and 2.92 mg/g of picrocrocin in the tomato DW, without compromising plant growth. The strategy involved expression of CsCCD2L to produce crocetin dialdehyde and 2,6,6-trimethyl-4-hydroxy-1-carboxaldehyde-1-cyclohexene, and of glycosyltransferases UGT709G1 and CsUGT2 for picrocrocin and crocins production, respectively. Metabolic analyses of the engineered fruits revealed picrocrocin and crocetin-(β-D-gentiobiosyl)-(β-D-glucosyl)-ester, as the predominant crocin molecule, as well as safranal, at the expense of the usual tomato carotenoids. The results showed the highest crocins content ever obtained by metabolic engineering in heterologous systems. In addition, the engineered tomatoes showed higher antioxidant capacity and were able to protect against neurological disorders in a Caenorhabditis elegans model of Alzheimer’s disease. Therefore, these new developed tomatoes could be exploited as a new platform to produce economically competitive saffron apocarotenoids with health-promoting properties.

Elucidation of Phenomena Involving Cyanobacteria in Freshwater Ecosystem by Chemically Ecological Approach

Lakes Sagami and Tsukui are reservoirs constructed by connecting to the Sagami River. Because of eutrophication of the lakes, cyanobacteria have appeared every year. This review deals with phenomena related to occurrence of cyanobacteria that have been observed for 40 years since 1974 at the lakes. These 40 years of observations raised three interesting issues including the retention of cyanobacteria on their surfaces. These phenomena have been attributed to the usual factors, such as illuminance, nutrition and water temperature, but our research results suggested that they cannot be resolved without the introduction of another factor. We have attempted to elucidate various phenomena involving cyanobacteria in lake ecosystems by chemical ecological methods using volatile organic compounds (VOCs) produced by the cyanobacteria as indicators. One of the VOCs, β-cyclocitral, was significantly involved in the above phenomena, which was considered to be produced by the carotenoid cleavage dioxygenase (CCD) of the cyanobacteria. β-Cyclocitral was not produced in the two known CCDs, but two additional CCDs to Microcystis aeruginosa participated to produce the β-cyclocitral. These CCDs did not directly produce β-cyclocitral, but it was accumulated in cells as their precursors. The released β-cyclocitral underwent a Baeyer-Villiger-like oxidation. It was speculated that Microcystis activated the CCD genes through density stress and produced β-cyclocitral, which acted as an allelopathic substance. As a result, the number of cells of cyanobacteria decreased, and the resulting nitrogen and phosphorus were fed to the living cyanobacteria. It is postulated that this "quorum sensing" was functioning in the above-mentioned issues.

Preparation of carotenoid cleavage dioxygenases for X-ray crystallography.

Carotenoid cleavage dioxygenases (CCDs) constitute a superfamily of enzymes that are found in all domains of life where they play key roles in the metabolism of carotenoids and apocarotenoids as well as certain phenylpropanoids such as resveratrol. Interest in these enzymes stems not only from their biological importance but also from their remarkable catalytic properties including their regioselectivity, their ability to accommodate diverse substrates, and the additional activities (e.g., isomerase) that some of these enzyme possess. X-ray crystallography is a key experimental approach that has allowed detailed investigation into the structural basis behind the interesting biochemical features of these enzymes. Here, we describe approaches used by our lab that have proven successful in generating single crystals of these enzymes in resting or ligand-bound states for high-resolution X-ray diffraction analysis.

Overexpression and Characterization of a Novel Plant Carotenoid Cleavage Dioxygenase 1 from Morus notabilis.

Synthesis of β-ionone in microbial cell factories is limited by the efficiency of carotenoid cleavage dioxygenases (CCDs). To obtain genes responsible for specific cleavage of carotenoids generating β-ionone, a novel carotenoid cleavage dioxygenase 1 from Morus notabilis was cloned and overexpressed in Escherichia coli. The MnCCD1 protein was able to cleave a variety of carotenoids at the positions 9, 10 (9', 10') to produce β-ionone, 3-hydroxy-4-oxo-β-ionone, 3-hydroxy-β-ionone, and 3-hydroxy-α-ionone in vitro. MnCCD1 could also cleave lycopene and β-carotene at the 9, 10 (9', 10') bind bond to produce pseudoionone and β-ionone, respectively, in E. coli accumulating carotenoids. The enzyme activity of MnCCD1 was reached 2.98 U/mL at optimized conditions (temperature 28 °C, IPTG 0.1 mM, induction time 24 h). The biochemical characterization of MnCCD1 revealed the optimal activities were at pH 8.4 and 35 °C. The addition of 10 % ethanol could increase enzyme activity at above 15 %. However, an obvious decline was observed on enzyme activity as the concentration of Fe2+ increased (0-1 mM). The Vmax for β-apo-8'-carotenal was 72.5 U/mg, while the Km was 0.83 mM. The results provide a foundation for developing the application of carotenoid cleavage dioxygenases as biocatalysis and synthetic biology platforms to produce volatile aroma components from carotenoids.