Biology-Basel
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2025
·Vol. 14
·期 7
·752
·
IF 3.5
Kaixuan Ke,Yufeng Zhang,Xinyi Wang,Zhaoyan Luo,Yangyang Chen,Xianying Fang,Linguo Zhao
Simple Summaryβ-Ionone is a valuable aroma compound widely used in the fragrance, food, and pharmaceutical industries. In this study, we identify and characterize two carotenoid cleavage dioxygenase 1 (CCD1) genes fromOlea europaeaandIpomoea nil. By expressing these genes inE. coli, we evaluate their ability to produce β-ionone and analyze their biochemical properties. Our findings not only provide insights into the functional diversity of CCD1 enzymes across plant species, but also offer new genetic resources for the microbial biosynthesis of natural aroma compounds.The aromatic C13apocarotenoid β-ionone is a high-value natural-flavor and -fragrance compound derived from the oxidative cleavage of carotenoids. Carotenoid cleavage dioxygenases (CCDs) play a pivotal role in the biosynthesis of volatile apocarotenoids, particularly β-ionone. In this study, we report the identification, cloning, and functional characterization of two CCD1 homologs:OeCCD1fromOlea europaeaandInCCD1fromIpomoea nil. These two species, which, respectively, represent a woody perennial and a herbaceous annual, were selected to explore the potential functional divergence of CCD1 enzymes across different plant growth forms. These CCD1 genes were synthesized using codon optimization forEscherichia coliexpression, followed by heterologous expression and purification using a GST-fusion system. In vitro assays confirmed that both enzymes cleave β-carotene at the 9,10 (9′,10′) double bond to yield β-ionone, but onlyOeCCD1exhibits detectable activity on zeaxanthin;InCCD1shows no in vitro cleavage of zeaxanthin. Kinetic characterization using β-apo-8′-carotenal as substrate revealed, forOeCCD1, a Kmof 0.82 mM, Vmaxof 2.30 U/mg (kcat= 3.35 s−1), and kcat/Kmof 4.09 mM−1·s−1, whereasInCCD1displayed Km= 0.69 mM, Vmax= 1.22 U/mg (kcat= 1.82 s−1), and kcat/Km= 2.64 mM−1·s−1. The optimization of expression parameters, as well as the systematic evaluation of temperature, pH, solvent, and metal ion effects, provided further insights into the stability and functional diversity within the plant CCD1 family. Overall, these findings offer promising enzymatic tools for the sustainable production of β-ionone and related apocarotenoids in engineered microbial cell factories.
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Food Chemistry-X
·
2022
·
IF 6.443
Minghui Li,Yanfen Zhang,Xihang Xu,Ying Chen,Jianzhou Chu,Xiaoqin Yao
The rot and deterioration of sprouts are closely related to their physiological state and postharvest storage quality. The study investigated the influences of brassinolide, zeaxanthin, and their combination on physiological metabolism, chlorophyll fluorescence, and nutritional quality of radish sprouts stored at 4 °C. The combined treatments enhanced hypocotyl length, fresh weight, contents of secondary metabolites, nutritional ingredients, glutathione, the photoprotective capacity of PSII, and FRAP level in radish sprouts compared with zeaxanthin alone. The combined treatments enhanced hypocotyl length, fresh weight, glutathione content, F v /F m value, and antioxidant capacity in sprouts compared to brassinolide alone. The combined treatment of zeaxanthin and brassinolide could make radish sprouts keep high biomass and antioxidant capacity by increasing the contents of stress-resistant metabolites and by weakening the photoinhibition of PSII in radish sprouts stored at 4 °C.
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ACS Sustainable Chemistry & Engineering
·
2023
·
IF 8.4
Mengying Shan,Mingdong Yao,Nan Liang,Herong Wang,Nan Wu,Ying Wang,Wenhai Xiao,Ying-jin Yuan
Saffron contains pharmacologic active compounds and a large number of flavor compounds. Both originate from carotenoids by biocatalyzed oxidation. Crocetin in saffron exhibits various attractive bioactivities and could be considered in the treatment of various diseases including cancer, coronary heart disease, and hypertension. However, powerful strategies for the efficient and sustainable source of crocetin are lacking. To address this issue, we demonstrated a one-pot approach to bioconversion of crocetin from zeaxanthin via a dual-enzyme system consisting of carotenoid cleavage dioxygenase 2 (CsCCD2-M1 (R192F/S323A)) and aldehyde dehydrogenase (SynALD) with optimized protein expression and reaction system. Meanwhile, it was observed that the byproduct 3-hydroxy-β-apo-8′-carotenoic acid (3-HACA) severely accumulated due to the imbalance in the activity of the CsCCD2-M1 and SynALD. A suitable molar ratio of 6:1 and the optimized dual-enzyme reaction conditions were developed to eliminate 3-HACA completely. Consequently, the efficient biosynthesis of crocetin (1.48 mg/L/h) and an 80.8% conversion rate of crocetin within 2 h were accomplished after scaling up the optimized system by 10 000 times. Moreover, over 34% of enzyme activity was still retained in the residual enzyme system after three times of recycling. This study not only provides a sustainable means of crocetin production in vitro but also presents a new insight into the rapid construction of one-pot synthesis of high-value compounds with complex structures.
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CHEMISTRY & BIODIVERSITY
·
2021
·Vol. 19
·期 2
·e202100735
·
IF 2.408
Zhipeng Qi,Xianyu Fan,Chunyi Zhu,Dongsheng Chang,Jianjun Pei,Linguo Zhao
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 Fe 2+ increased (0–1 mM). The V max for β-apo-8′-carotenal was 72.5 U/mg, while the K m 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.
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JOURNAL OF PLANT GROWTH REGULATION
·
2023
·
IF 4.8
Islam Md Monirul,Hassan Mahmood Ul,Ishfaq Muhammad,Ripa Farhana Alam,Nadeem Faisal,Ahmad Zeeshan,Xu Jiuliang,Ning Peng,Li Xuexian
Foliar fertilization is an important agricultural practice. The response of crop plants to foliar application of mineral nutrients such as nitrogen (N), phosphorus, and potassium has been well recognized. However, little is known about crop plants’ response to the foliar spray of organic sources of nutrient, in particular, under field conditions. Here, we sprayed glutamine (Gln) at V6, V8, V12, R1 (silk), and R3 (dough) stages of maize hybrid ZD958 to investigate the physiological impacts on grain yield and nutritional quality under sufficient and low-N conditions for two consecutive years. Exogenous application of Gln (1.25 mM) significantly enhanced grain yield approximately by 20.0% under sufficient N and 38.0% under low N, as a combinatorial effect of increases in kernel number and kernel weight. The higher N concentrations in the ear and delaying leaf senescence favor the kernel setting and grain filling, preconditioning higher grain yield at harvest. Further, Gln spray resulted in higher nutritional quality kernels as indicated by higher concentrations of crude proteins, minerals, oil, unsaturated fatty acids (i.e., oleic, and linoleic acids), total carotenoids (i.e., β-carotene, lutein, zeaxanthin, antheraxanthin, and violaxanthin), and α-tocopherol, partially attributing to larger nutrient-rich embryos. Taken together, our findings revealed how Gln promotes maize production and an additional potential to improve grain yield and nutritional quality traits of maize and other closely related cereal crops.
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ISME Journal
·
2021
·Vol. 15
·期 6
·1844-1857
·
IF 10.302
Liu Qing,Li Wei,Liu Di,Li Lingyan,Li Jie,Lv Na,Liu Fei,Zhu Baoli,Zhou Yuguang,Xin Yuhua,Dong Xiuzhu
Bacteria that inhabit glaciers usually produce carotenoids. Here, we report that a group of zeaxanthin-producing glacial Flavobacterium exhibited light-promoted growth. Of the tested 47 strains, 45 showed increased growths but two died under illumination at 50 μmol photon m −2 s −1 . Light stimulation occurred mainly in either anoxic or nutrient-poor cultures, while the same levels of light promotion were found for that grown at 14 and 7 °C. Pigment assays identified overrepresentative zeaxanthin but trace retinal in the light promoted 45 strains, while flexirubin was exclusively in the light-lethal two. Genomic analysis revealed the gene cluster for zeaxanthin synthesis in the 45 strains, in which 37 strains also harbored the proteorhodopsin gene prd . Transcriptomic analysis found that light-induced expressions of both the zeaxanthin synthesis and proteorhodopsin genes. Whereas, deletion of the prd gene in one strain did not diminish light promotion, inhibition of zeaxanthin synthesis did. In comparison, no light promotion was determined in a glacier Cryobacterium luteum that produced a non-zeaxanthin-type carotenoid. Therefore, light stimulation on the glacial Flavobacterium is mostly likely related to zeaxanthin, which could provide better photoprotection and sustain membrane integrity for the organisms living in cold environments.
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Molecular Catalysis
·
2024
·
IF 3.9
Kaixuan Ke,Ling Guo,Zhipeng Qi,Dou Dou,Han Ma,Xianying Fang,Linguo Zhao
Carotenoid cleavage dioxygenases (CCDs) play a pivotal role in the biosynthesis of volatile apocarotenoids, which have significant industrial applications due to their aromatic and bioactive properties. This study focuses on the molecular cloning and biochemical characterization of the LbCCD1 from Lycium barbarum . The LbCCD1 gene was successfully cloned and heterologously expressed in Escherichia coli and several carotenoids were using as substrates for investigate its specificity by in vitro and in vivo experiment. The LbCCD1 protein was able to cleave a variety of carotenoids including β-carotene, zeaxanthin, astaxanthin, and β-apo-8′-carotenal, at the 9, 10 (9′, 10′) double bond to produce β-ionone, 3‑hydroxy-4-oxo-β-ionone, and 3‑hydroxy-β-ionone, respectively in vitro. LbCCD1 could also cleave zeaxanthin and β-carotene at the 9, 10 (9′, 10′) double bond to produce β-ionone, respectively, in E. coli accumulating carotenoids. Interestingly, LbCCD1 did not exhibit cleavage activity on lycopene either in vivo or in vitro unlike other CCD1 family enzymes. In the previous experiment, it was confirmed that LbCCD1 exhibits cleavage activity towards β-apo-8′-carotenal in vitro, so we used β-apo-8′-carotenal as the substrate for characterizing the enzymatic properties. The expression of LbCCD1 was optimized at such conditions (temperature 24 °C, IPTG 0.1 mM, induction time 24 h). The biochemical characterization of LbCCD1 revealed the optimal activities were at pH 9 and 55 °C. The addition of 10 % ethanol could increase enzyme activity to above 15 %. However, the concentration of Fe 2+ has a minimal effect on enzyme activity. The V max for β-apo-8′-carotenal was 8.6 U/mg, while the K m was 0.27 mM. To preliminarily verify the potential of LbCCD1 as a biological component for β-ionone production. By introducing LbCCD1 into the β-carotene-high-producing chassis cell and optimizing the conditions (temperature 30 °C, IPTG 0.01 mM, Fe 2+ concentration 0.05 mM), the β-ionone yield reached 21.45 mg/L. This study focused on one of the CCDs derived from woody plants, which have been relatively underexplored. It lays the groundwork for expanding the CCD enzyme library, identifying suitable CCDs, and investigating the structure-function relationship of CCDs. Furthermore, it sets the stage for engineering novel CCD genes and developing advanced applications of CCDs as biocatalysts and platforms for synthetic biology. These advancements will enable the efficient production of volatile aroma compounds from carotenoids.
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FOOD CHEMISTRY
·
2024
·
IF 8.5
Jiying Zhu,Wei Jia,Jian Peng
Crocus sativus L. is a both medicinal and food bulbous flower whose qualities are geographically characterized. However, identification involving different places of origin of such substances is currently limited to single-omics mediated content analysis. Integrated metabolomics and proteomics, 840 saffron samples from six countries (Spain, Greece, Iran, China, Japan, and India) were analyzed using the QuEChERS extraction method. A total of 77 differential metabolites and 14 differential proteins were identified. The limits of detection of the method were 1.33 to 8.33 μg kg −1 , and the recoveries were 85.56% to 105.18%. Using homology modeling and molecular docking, the Gln84, Lys195, Val182 and Pro184 sites of Crocetin glucosyltransferase 2 were found to be the targets of crocetin binding. By multivariate statistical analysis (PCA and PLS-DA), different saffron samples were clearly distinguished. The results provided the basis for the selection and identification of high quality saffron from different producing areas.
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FOOD HYDROCOLLOIDS
·
2024
·
IF 11
Jipeng Zhang,Xiaojun Zhang,Chongyang Zhu
The self-assembly behavior of proteins and peptides has been widely studied due to their ability to construct various nanostructures, such as nanotubes and nanofibrils. Understanding the self-assembly mechanism is of great significance for developing functional nanomaterials. Herein, we adopt a novel method to construct ovotransferrin (OVT) fibrils by subtilisin A hydrolysis and Ca 2+ ions induced self-assembly with mild reaction conditions. The kinetic process of OVT fibrils formation contains lag, growth, and plateau stage. During the growth stage, OVT peptides self-assemble into precursor structures, which undergo morphological changes of twisted ribbons, helical ribbons, and nanotubes, ultimately closing to form mature fibrils. Ca 2+ ions likely coordinate with the carboxyl groups of OVT peptides, aiding in the self-assembly process. And the secondary conformation is manifested as a β-sheet stacking. The mature OVT fibrils have distinct hollow and left-handed structures. Then OVT fibrils successfully encapsulate zeaxanthin (Zea) and improve its solubility and stability. Besides, OVT fibrils exhibit no in vitro cytotoxicity, and significantly reduce allergenicity to egg allergy patients compared to OVT. OVT fibrils delivery Zea into milk to develop Zea fortified dairy beverage and improve the viscosity of dairy beverage. OVT fibrils show a great potential as nutrient delivery and texture optimization for food application.
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FOOD RESEARCH INTERNATIONAL
·
2025
·
IF 8
Shuo Zhang,Shuang Guo,Jiayu Han,Zhaolian Han,Hailing Yu,Xue Zhang,Ningcan Ma,Zhiqiang Cheng
To achieve the conversion of waste Phellinus igniarius substrate into functional foods, we employed enzymatic hydrolysis-centrifugal fractionation method to extract highly uniform spherical cellulose nanocrystals (CNCs, 14–28 nm) from the waste Phellinus igniarius substrate. These CNCs were used to construct an emulsion delivery system for carotenoids and polyphenols. We utilized biphasic fluorescence staining and Cryo-SEM to visually confirm the adsorption of CNCs at the interface. Furthermore, interfacial shear rheology was applied to perform time-, frequency-, and strain-dependent analyses. These methods provided direct, dynamic, and quantitative evidence that the CNCs rapidly adsorb at the oil-water interface and form a solid-like film dominated by elastic behavior (G′ > G″). This conclusively demonstrates that the emulsification mechanism follows a Pickering-type stabilization. Stabilized emulsions exhibit excellent storage and environmental stability, effectively delaying the degradation of bioactive compounds while preserving their antioxidant activity. In vitro simulated digestion experiments demonstrated that the Pickering mechanism of the CNC-stabilized emulsion offers the advantage of retarding lipid digestion. The bioaccessibility of the four encapsulated bioactive compounds, namely β-carotene, lutein, zeaxanthin, and dihydroquercetin, reached 46.19 ± 1.20 %, 44.66 ± 0.56 %, 40.72 ± 1.17 %, and 38.97 ± 1.16 %, respectively. This study successfully established a bioactive compound delivery system, enabling the conversion of waste Phellinus igniarius substrate into functional food.
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