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Shin Hasegawa - One of the best experts on this subject based on the ideXlab platform.
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modification of Limonoid metabolism in suspension cell culture of citrus
Plant Biotechnology, 2002Co-Authors: Tomoko Endo, Shin Hasegawa, Masayuki Kita, Takehiko Shimada, Takaya Moriguchi, Tetsushi Hidaka, Ryoji Matsumoto, Mitsuo OmuraAbstract:For the metabolic engineering of bio-functional substances in Citrus, metabolic changes of Limonoids were investigated in embryogenic cell cultures of Citrus and Fortunella. When embryogenic cells of Citrus and Fortunella were cultured for four weeks in the liquid subculture medium containing nomilin, most species converted nomilin to obacunone and limonin. In addition, calamin group Limonoids were produced in Fortunella and Calamondin cells. Deacetylnomilin was produced from nomilin in the cells of C. ichangensis and its related species. Exogenously added limonin into medium was not metabolized. The embryoid formation in C.aurantium did not affect the Limonoid metabolism. All cells cultured without the presence of nomilin or limonin did not generate any Limonoids. Furthermore, transgenic C. sinensis cell harboring a chimeric Limonoid UDP-glucosyltransferase (Limonoid GTase) gene was generated. The conversion of limonin into limonin 17-β-D-glucopyranoside (LG) demonstrated an efficient assay system for the transgene.
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analysis of Limonoid glucosides from citrus by electrospray ionization liquid chromatography mass spectrometry
Journal of Agricultural and Food Chemistry, 2001Co-Authors: Thomas K Schoch, Gary D Manners, Shin HasegawaAbstract:An electrospray ionization liquid chromatography−mass spectrometry (ESI-LC-MS) method for the detection and quantitation of Limonoid glucosides has been developed. Negative ions [M − H+]- characteristic of six Limonoid glucosides can be detected and quantified from selected ion monitoring chromatograms using carminic acid as an internal standard. The described method has been applied to the analysis of Limonoid glucoside content in various liquid and solid Citrus spp. samples as well as complex mixtures of partially purified Limonoid glucosides. Rapid and sensitive qualitative screening of samples for Limonoid glucosides can also be accomplished with slight modifications of the method. Keywords: Limonoid glucoside; limonin 17β-d-glucopyranoside; electrospray ionization; liquid chromatography−mass spectrometry; Citrus
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molecular cloning and characterization of a novel gene encoding Limonoid udp glucosyltransferase in citrus
FEBS Letters, 2000Co-Authors: Masayuki Kita, Shin Hasegawa, Takaya Moriguchi, Ryoji Matsumoto, Yutaka Hirata, Tomoko Endoinagaki, Charles G Suhayda, Mitsuo OmuraAbstract:We isolated a cDNA clone encoding Limonoid UDP-glucosyltransferase (Limonoid GTase) from the albedo of Satsuma mandarin (Citrus unshiu Marc.) and investigated the contribution to Limonoid glucoside accumulation in fruit. The isolated cDNA clone (CitLGT) was 1732 bp in length encoding 511 deduced amino acids with a predicted molecular mass of 57.5 kDa. The products of in vitro translation from an expression vector had the Limonoid GTase activity. Southern blot analysis of genomic DNA indicated that CitLGT was present as a single copy gene in the Citrus genome. The amount of transcript corresponding to CitLGT mRNA changed the same way as the fluctuation of limonin glucoside content during fruit development of navel orange (Citrus sinensis Osb.). This indicates that the transcription of CitLGT regulates the conversion of Limonoid aglycones to glucosides in citrus fruit.
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Limonoid glucosides in fruit juice and processing by products of satsuma mandarin citrus unshiu marcov
Journal of Food Science, 1995Co-Authors: Yoshihiko Ozaki, Shigeru Ayano, Nobuya Inaba, Masaki Miyake, Mark A. Berhow, Shin HasegawaAbstract:In a study on improving utilization of waste from processing of Satsuma mandarin (Citrus unshiu Marcov.) the Limonoid glucosides of fruit, juice, and by-products were measured using HPLC and TLC. All materials had the 17-β-D-glucopyranoside derivatives of Limonoids reported in other commercial citrus fruit. Citrus molasses was a good source for industrial scale extraction of Limonoid glucosides. An extraction system using polystyrene divinylbenzene resins, was developed which could be expanded to industrial scale.
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Limonoids in pummelos [Citrus grandis (L.) Osbeck]
Journal of Food Science, 1995Co-Authors: Hideaki Ohta, Shin HasegawaAbstract:Juice and seeds from 16 cultivars of mature pummelo fruits were analyzed for Limonoid content. Pummelo juice contained an average of 18 ppm limonin and 29 ppm total Limonoid glucosides. Compared to other juices, ptmnnelo contained very high concentrations of limonin and very low concentrations of Limonoid glucosides. Limonin, nomilin, obacunone and trace amounts of deacetyhromilin were found in pummelo seeds. The 17–β-D-glucopyranoside derivatives (glucosides) of nomilin, nomilinic acid and obacunone were also present. Total Limonoid aglycone concentration in the seeds ranged from 773 ppm to 9,900 ppm and total Limonoid glucosides ranged from 130 ppm to 1,912 ppm.
Li Shen - One of the best experts on this subject based on the ideXlab platform.
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xylomolones a d from the thai mangrove xylocarpus moluccensis assignment of absolute stereostructures and unveiling a convergent strategy for Limonoid biosynthesis
Journal of Organic Chemistry, 2019Co-Authors: Attila Mandi, Junjun Liu, Li Shen, Tibor KurtanAbstract:Two new 9,10- seco Limonoids with a central 3,4-dihydro-2 H-pyran motif, named xylomolones A and B (1 and 2, respectively), possessing identical absolute configurations but reversed alignments of ring A, compared to the tricyclic core of rings B-D, were obtained from seeds of the Thai mangrove Xylocarpus moluccensis, together with a highly modified 1,2- seco Limonoid, named xylomolone C (3), containing a novel 3-oxabicyclo[3.2.1]octane-2,7-dione motif, and a new C11-terpenic acid methyl ester (4). The relative and absolute configurations of 1-4 were evidenced by extensive nuclear magnetic resonance (NMR) investigations combined with density functional theory and time-dependent density functional theory calculations of electronic circular dichroism, specific optical rotation, and 13C NMR data. The co-isolation of 1-4 allowed us to propose a novel convergent strategy for Limonoid biosynthesis for the first time. This study demonstrates that mangroves of the genus Xylocarpus harbor new Limonoid biosynthetic routes.
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Xylomolones A–D from the Thai Mangrove Xylocarpus moluccensis: Assignment of Absolute Stereostructures and Unveiling a Convergent Strategy for Limonoid Biosynthesis
2019Co-Authors: Attila Mándi, Junjun Liu, Li Shen, Tibor KurtánAbstract:Two new 9,10-seco Limonoids with a central 3,4-dihydro-2H-pyran motif, named xylomolones A and B (1 and 2, respectively), possessing identical absolute configurations but reversed alignments of ring A, compared to the tricyclic core of rings B–D, were obtained from seeds of the Thai mangrove Xylocarpus moluccensis, together with a highly modified 1,2-seco Limonoid, named xylomolone C (3), containing a novel 3-oxabicyclo[3.2.1]octane-2,7-dione motif, and a new C11-terpenic acid methyl ester (4). The relative and absolute configurations of 1–4 were evidenced by extensive nuclear magnetic resonance (NMR) investigations combined with density functional theory and time-dependent density functional theory calculations of electronic circular dichroism, specific optical rotation, and 13C NMR data. The co-isolation of 1–4 allowed us to propose a novel convergent strategy for Limonoid biosynthesis for the first time. This study demonstrates that mangroves of the genus Xylocarpus harbor new Limonoid biosynthetic routes
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scaffold diversity oriented synthesis of Limonoid dimers discovery of an axially chiral agent with in vivo anti breast cancer activity
Organic chemistry frontiers, 2018Co-Authors: Yang Yang, Junjun Liu, Li Shen, Zhi ShiAbstract:Starting from the natural monomeric Limonoids 1 and 1′, we designed and synthesized eight new Limonoid dimers of four skeletons (two symmetric and two non-symmetric), containing a rotationally hindered C15C15′ central axis (2, 2′), an embedded C15-spiro-fused 2H-pyran motif (3, 3′), and an axially chiral C15–C15′ central bond (4, 4′, and 5a, 5b), respectively, by oxidative carbon–carbon radical coupling. The dimeric architectures of these compounds, particularly absolute configurations of C15C15′ and C15–C15′ central axes, were unequivocally determined by extensive NMR investigations, single-crystal X-ray diffraction analyses, and electronic circular dichroism spectral comparison. The C2-symmetric dimer 5b with a (M)-configured C15–C15′ central axis exhibited selective and potent cytotoxicities against human triple-negative breast cancer (TNBC) MD-MBA-231 and MD-MBA-453 cells with IC50 values of 5.57 ± 1.48 and 3.93 ± 0.75 μM, respectively. This induces cell-cycle arrest in the G2/M phase and apoptosis, and enhances the accumulation of reactive oxygen species (ROS) in cells. Importantly, 5b suppressed the growth of MDA-MB-453 tumor xenografts with no significant side effects in Balb/c nude mice. After intravenous injection with 5b at a dose of 30 mg kg−1 every two days for four weeks, the inhibitory rate for TNBC MDA-MB-453 tumor xenografts was 63.83%. It was concluded that not only the presence of 6R-OH/6′R-OH groups, but also the stable (M)-configuration of the C15–C15′ central axis is pivotal for the in vivo anti-TNBC activity of 5b. This is the first report on an axially chiral Limonoid dimer with in vivo anticancer activity.
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Twenty-Nine New Limonoids with Skeletal Diversity from the Mangrove Plant, Xylocarpus moluccensis
MDPI AG, 2018Co-Authors: Jianzhi Zhang, Yiguo Dai, Li ShenAbstract:Twenty-nine new Limonoids—named xylomolins A1–A7, B1–B2, C1–C2, D–F, G1–G5, H–I, J1–J2, K1–K2, L1–L2, and M–N, were isolated from the seeds of the mangrove plant, Xylocarpus moluccensis. Compounds 1–13 are mexicanolides with one double bond or two conjugated double bonds, while 14 belongs to a small group of mexicanolides with an oxygen bridge between C1 and C8. Compounds 15–19 are khayanolides containing a Δ8,14 double bond, whereas 20 and 21 are rare khayanolides containing a Δ14,15 double bond and Δ8,9, Δ14,15 conjugated double bonds, respectively. Compounds 22 and 23 are unusual Limonoids possessing a (Z)-bicyclo[5.2.1]dec-3-en-8-one motif, while 24 and 25 are 30-ketophragmalins with Δ8,9, Δ14,15 conjugated double bonds. Compounds 26 and 27 are phragmalin 8,9,30-ortho esters, whereas 28 and 29 are azadirone and andirobin derivatives, respectively. The structures of these compounds, including absolute configurations of 15–19, 21–23, and 26, were established by HRESIMS, extensive 1D and 2D NMR investigations, and the comparison of experimental electronic circular dichroism (ECD) spectra. The absolute configuration of 1 was unequivocally established by single-crystal X-ray diffraction analysis, obtained with Cu Kα radiation. The diverse cyclization patterns of 1–29 reveal the strong flexibility of skeletal plasticity in the Limonoid biosynthesis of X. moluccensis. Compound 23 exhibited weak antitumor activity against human triple-negative breast MD-MBA-231 cancer cells with an IC50 value of 37.7 μM. Anti-HIV activities of 1, 3, 8, 10, 11, 14, 20, 23–25, and 27 were tested in vitro. However, no compounds showed potent inhibitory activity
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trangmolins a f with an unprecedented structural plasticity of the rings a and b new insight into Limonoid biosynthesis
Chemistry: A European Journal, 2016Co-Authors: Li Shen, Torsten Bruhn, Patchara Pedpradab, Gerhard BringmannAbstract:The absolute stereostructures of trangmolins A–F (1–6), Limonoids with three new and one known topologies of the rings A and B, were unambiguously determined by NMR spectroscopic investigations, single-crystal XRD analysis, and quantum-chemical electronic circular dichroism calculations. Compounds 1–3 contain a hexahydro-1H-inden-4-one motif, compound 4 comprises a hexahydro-2,6-methanobenzofuran-7-one cage, and compound 5 consists of a hexahydro-2H-2,8-epoxychromene scaffold. The C1−C30 linkage in 1–3 and the C3−C30 connection in 4 form two unprecedented types of ring A/B-fused carbobicyclic cores: viii and ix. The oxidative cleavage of the C2−C3 bond in 5 and heterocyclization in 4 and 5 constitute the unprecedented tricyclic 6/6/5 ring A/B1/B2- and 6/5/6 ring A1A2/B-fused topologies, respectively, which are uncovered, for the first time, in the construction of Limonoid architectures. The diverse cyclization patterns of 1–6 reveal an unparalleled structural plasticity of rings A and B in Limonoid biosynthesis.
Michel Lagarde - One of the best experts on this subject based on the ideXlab platform.
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Limonoid compounds inhibit sphingomyelin biosynthesis by preventing cert protein dependent extraction of ceramides from the endoplasmic reticulum
Journal of Biological Chemistry, 2012Co-Authors: Francoise Hullinmatsuda, Nario Tomishige, Shota Sakai, Reiko Ishitsuka, Kumiko Ishii, Asami Makino, Peter Greimel, Mitsuhiro Abe, Elad L Laviad, Michel LagardeAbstract:To identify novel inhibitors of sphingomyelin (SM) metabolism, a new and selective high throughput microscopy-based screening based on the toxicity of the SM-specific toxin, lysenin, was developed. Out of a library of 2011 natural compounds, the Limonoid, 3-chloro-8β-hydroxycarapin-3,8-hemiacetal (CHC), rendered cells resistant to lysenin by decreasing cell surface SM. CHC treatment selectively inhibited the de novo biosynthesis of SM without affecting glycolipid and glycerophospholipid biosynthesis. Pretreatment with brefeldin A abolished the Limonoid-induced inhibition of SM synthesis suggesting that the transport of ceramide (Cer) from the endoplasmic reticulum to the Golgi apparatus is affected. Unlike the Cer transporter (CERT) inhibitor HPA-12, CHC did not change the transport of a fluorescent short chain Cer analog to the Golgi apparatus or the formation of fluorescent and short chain SM from the corresponding Cer. Nevertheless, CHC inhibited the conversion of de novo synthesized Cer to SM. We show that CHC specifically inhibited the CERT-mediated extraction of Cer from the endoplasmic reticulum membranes in vitro. Subsequent biochemical screening of 21 Limonoids revealed that some of them, such as 8β-hydroxycarapin-3,8-hemiacetal and gedunin, which exhibits anti-cancer activity, inhibited SM biosynthesis and CERT-mediated extraction of Cer from membranes. Model membrane studies suggest that 8β-hydroxycarapin-3,8-hemiacetal reduced the miscibility of Cer with membrane lipids and thus induced the formation of Cer-rich membrane domains. Our study shows that certain Limonoids are novel inhibitors of SM biosynthesis and suggests that some biological activities of these Limonoids are related to their effect on the ceramide metabolism. Background: Pharmacological inhibitors of sphingolipid metabolism and transport are useful for both biological and therapeutic research. Results: High throughput microscopy-based screening identified Limonoids as inhibitors of sphingomyelin biosynthesis by preventing the membrane extraction of ceramide. Conclusion: Some therapeutic properties of Limonoids might be related to their effects on ceramide metabolism. Significance: This study provides insights into the role of the ceramide domains in sphingolipid metabolism.
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Limonoid compounds inhibit sphingomyelin biosynthesis by preventing cert protein dependent extraction of ceramides from the endoplasmic reticulum
Journal of Biological Chemistry, 2012Co-Authors: Francoise Hullinmatsuda, Nario Tomishige, Shota Sakai, Reiko Ishitsuka, Kumiko Ishii, Asami Makino, Peter Greimel, Mitsuhiro Abe, Elad L Laviad, Michel LagardeAbstract:Abstract To identify novel inhibitors of sphingomyelin (SM) metabolism, a new and selective high throughput microscopy-based screening based on the toxicity of the SM-specific toxin, lysenin, was developed. Out of a library of 2011 natural compounds, the Limonoid, 3-chloro-8β-hydroxycarapin-3,8-hemiacetal (CHC), rendered cells resistant to lysenin by decreasing cell surface SM. CHC treatment selectively inhibited the de novo biosynthesis of SM without affecting glycolipid and glycerophospholipid biosynthesis. Pretreatment with brefeldin A abolished the Limonoid-induced inhibition of SM synthesis suggesting that the transport of ceramide (Cer) from the endoplasmic reticulum to the Golgi apparatus is affected. Unlike the Cer transporter (CERT) inhibitor HPA-12, CHC did not change the transport of a fluorescent short chain Cer analog to the Golgi apparatus or the formation of fluorescent and short chain SM from the corresponding Cer. Nevertheless, CHC inhibited the conversion of de novo synthesized Cer to SM. We show that CHC specifically inhibited the CERT-mediated extraction of Cer from the endoplasmic reticulum membranes in vitro. Subsequent biochemical screening of 21 Limonoids revealed that some of them, such as 8β-hydroxycarapin-3,8-hemiacetal and gedunin, which exhibits anti-cancer activity, inhibited SM biosynthesis and CERT-mediated extraction of Cer from membranes. Model membrane studies suggest that 8β-hydroxycarapin-3,8-hemiacetal reduced the miscibility of Cer with membrane lipids and thus induced the formation of Cer-rich membrane domains. Our study shows that certain Limonoids are novel inhibitors of SM biosynthesis and suggests that some biological activities of these Limonoids are related to their effect on the ceramide metabolism.
Bhimanagouda S Patil - One of the best experts on this subject based on the ideXlab platform.
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apigenin and naringenin suppress colon carcinogenesis through the aberrant crypt stage in azoxymethane treated rats
Experimental Biology and Medicine, 2010Co-Authors: Tety Leonardi, Bhimanagouda S Patil, Jairam K P Vanamala, Stella S Taddeo, Laurie A Davidson, Mary E Murphy, Naisyin Wang, Raymond J Carroll, Robert S Chapkin, Joanne R LuptonAbstract:Epidemiological evidence suggests that a diet abundant in fruits and vegetables may protect against colon cancer. Bioactive compounds, including flavonoids and Limonoids, have been shown to possess antiproliferative and antitumorigenic effects in various cancer models. This experiment investigated the effects of four citrus flavonoids and one Limonoid mixture at the promotion stage of chemically induced colon cancer in rats. Male Sprague-Dawley rats (n = 10 rats/group) were randomly allocated to one of six diets formulated to contain 0.1% apigenin, 0.02% naringenin, 0.1% hesperidin, 0.01% nobiletin, 0.035% limonin glucoside/obacunone glucoside mixture or a control diet (0% flavonoid/Limonoid). Rats received experimental diets for 10 weeks and were injected with azoxymethane (15 mg/kg) at weeks 3 and 4. Excised colons were evaluated for aberrant crypt foci (ACF) formation, colonocyte proliferation (proliferating cell nuclear antigen assay), apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling assay) and expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) (immunoblotting). When compared with the control diet, apigenin lowered the number of high multiplicity ACF (HMACF >4 aberrant crypts/focus) by 57% (P < 0.05), while naringenin lowered both the number of HMACF by 51% (P < 0.05) and the proliferative index by 32% (P < 0.05). Both apigenin and naringenin increased apoptosis of luminal surface colonocytes (78% and 97%, respectively; P < 0.05) when compared with the control diet. Hesperidin, nobiletin and the limonin glucoside/obacunone glucoside mixture did not affect these variables. The colonic mucosal protein levels of iNOS or COX-2 were not different among the six diet groups. The ability of dietary apigenin and naringenin to reduce HMACF, lower proliferation (naringenin only) and increase apoptosis may contribute toward colon cancer prevention. However, these effects were not due to mitigation of iNOS and COX-2 protein levels at the ACF stage of colon cancer.
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apigenin and naringenin suppress colon carcinogenesis through the aberrant crypt stage in azoxymethane treated rats
Experimental Biology and Medicine, 2010Co-Authors: Tety Leonardi, Bhimanagouda S Patil, Jairam K P Vanamala, Stella S Taddeo, Laurie A Davidson, Mary E Murphy, Naisyin Wang, Raymond J Carroll, Robert S Chapkin, Joanne R LuptonAbstract:Epidemiological evidence suggests that a diet abundant in fruits and vegetables may protect against colon cancer. Bioactive compounds, including flavonoids and Limonoids, have been shown to possess anti-proliferative and anti-tumorigenic effects in various cancer models. This experiment investigated the effects of four citrus flavonoids and one Limonoid mixture at the promotion stage of chemically induced colon cancer in rats. Male Sprague Dawley rats (n = 10 rats/group) were randomly allocated to one of six diets formulated to contain 0.1% apigenin, 0.02% naringenin, 0.1% hesperidin, 0.01% nobiletin, 0.035% limonin glucoside/obacunone glucoside mixture, or a control diet (0% flavonoid/Limonoid). Rats received experimental diets for 10 wk and were injected with azoxymethane (15 mg/kg) at wk 3 and 4. Excised colons were evaluated for aberrant crypt foci (ACF) formation, colonocyte proliferation (PCNA assay), apoptosis (TUNEL assay), and expression of iNOS and COX-2 (immunoblotting). When compared to the control diet, apigenin lowered the number of high multiplicity ACF (HMACF > 4 AC/focus) by 57% (P < 0.05), while naringenin lowered both the number of HMACF by 51% (P < 0.05) and the proliferative index by 32% (P < 0.05). Both apigenin and naringenin increased apoptosis of luminal surface colonocytes (78% and 97%, respectively; P < 0.05) when compared to the control diet. Hesperidin, nobiletin, and the limonin glucoside/obacunone glucoside mixture did not affect these variables. The colonic mucosal protein levels of iNOS or COX-2 were not different among the six diet groups. The ability of dietary apigenin and naringenin to reduce HMACF, lower proliferation (naringenin only), and increase apoptosis may contribute toward colon cancer prevention. However, these effects were not due to mitigation of iNOS and COX-2 protein levels at the ACF stage of colon cancer.
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Hydrotropic extraction of bioactive limonin from sour orange (Citrus aurantium L.) seeds
Food Chemistry, 2008Co-Authors: Deepak V. Dandekar, Guddadarangavvanahally K. Jayaprakasha, Bhimanagouda S PatilAbstract:Limonoids are potential bioactive compounds present only in citrus among fruits and vegetables. A new process for extraction of Limonoid aglycones from sour orange (Citrus aurantium L.) seeds was investigated using aqueous hydrotropic solutions. The extraction efficiency was dependent on hydrotrope concentration, extraction temperature and percent of raw material loaded. Two hydrotropes such as sodium salicylate (Na-Sal) and sodium cumene sulphonate (Na-CuS) were studied using Box-Behnken experiment design. Response surface analysis (RSA) of data was performed to study the effect of parameters on extraction efficiency. Prominent Limonoid aglycone such as limonin was extracted and quantified for process optimization. Both hydrotropes gave maximum limonin yield at 2 M concentration, extraction temperature of 45 °C and 10% solid loading. A maximum limonin yield of 0.65 mg/g seeds was obtained using Na-CuS whereas only 0.46 mg/g seed was obtained using Na-Sal. Using this process, the use of organic solvents can be reduced dramatically to keep the process environmental friendly for the extraction of bioactive compounds.
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simultaneous determination of citrus Limonoid aglycones and glucosides by high performance liquid chromatography
Analytica Chimica Acta, 2007Co-Authors: Amit Vikram, G K Jayaprakasha, Bhimanagouda S PatilAbstract:High performance liquid chromatography (HPLC) method has been developed for simultaneous quantification of Limonoid aglycones and glucosides on a reversed phase C18 column using a binary solvent system, coupled with diode array detector. Seven Limonoids such as limonin, nomilin, isolimonic acid, ichangin, isoobacunoic acid, limonin 17-beta-D glucopyranoside and deacetyl nomilinic acid 17-beta-D glucopyranoside were separated and detected at 210 nm. Furthermore, Limonoids were separated, identified and quantified in four varieties of citrus fruits and seeds using developed method. Limonin and limonin glucoside were found to be the predominant Limonoid aglycone and glucoside, respectively, in all tested samples. The sensitivity of the method was found to be 0.25-0.50 microg for tested Limonoids.
Mitsuo Omura - One of the best experts on this subject based on the ideXlab platform.
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modification of Limonoid metabolism in suspension cell culture of citrus
Plant Biotechnology, 2002Co-Authors: Tomoko Endo, Shin Hasegawa, Masayuki Kita, Takehiko Shimada, Takaya Moriguchi, Tetsushi Hidaka, Ryoji Matsumoto, Mitsuo OmuraAbstract:For the metabolic engineering of bio-functional substances in Citrus, metabolic changes of Limonoids were investigated in embryogenic cell cultures of Citrus and Fortunella. When embryogenic cells of Citrus and Fortunella were cultured for four weeks in the liquid subculture medium containing nomilin, most species converted nomilin to obacunone and limonin. In addition, calamin group Limonoids were produced in Fortunella and Calamondin cells. Deacetylnomilin was produced from nomilin in the cells of C. ichangensis and its related species. Exogenously added limonin into medium was not metabolized. The embryoid formation in C.aurantium did not affect the Limonoid metabolism. All cells cultured without the presence of nomilin or limonin did not generate any Limonoids. Furthermore, transgenic C. sinensis cell harboring a chimeric Limonoid UDP-glucosyltransferase (Limonoid GTase) gene was generated. The conversion of limonin into limonin 17-β-D-glucopyranoside (LG) demonstrated an efficient assay system for the transgene.
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molecular cloning and characterization of a novel gene encoding Limonoid udp glucosyltransferase in citrus
FEBS Letters, 2000Co-Authors: Masayuki Kita, Shin Hasegawa, Takaya Moriguchi, Ryoji Matsumoto, Yutaka Hirata, Tomoko Endoinagaki, Charles G Suhayda, Mitsuo OmuraAbstract:We isolated a cDNA clone encoding Limonoid UDP-glucosyltransferase (Limonoid GTase) from the albedo of Satsuma mandarin (Citrus unshiu Marc.) and investigated the contribution to Limonoid glucoside accumulation in fruit. The isolated cDNA clone (CitLGT) was 1732 bp in length encoding 511 deduced amino acids with a predicted molecular mass of 57.5 kDa. The products of in vitro translation from an expression vector had the Limonoid GTase activity. Southern blot analysis of genomic DNA indicated that CitLGT was present as a single copy gene in the Citrus genome. The amount of transcript corresponding to CitLGT mRNA changed the same way as the fluctuation of limonin glucoside content during fruit development of navel orange (Citrus sinensis Osb.). This indicates that the transcription of CitLGT regulates the conversion of Limonoid aglycones to glucosides in citrus fruit.