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Norman G Lewis - One of the best experts on this subject based on the ideXlab platform.
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Pinoresinol lariciresinol reductase substrate versatility enantiospecificity and kinetic properties
Chirality, 2020Co-Authors: Julianne K Hwang, Laurence B Davin, Syed G A Moinuddin, Norman G LewisAbstract:Two western red cedar Pinoresinol-lariciresinol reductase (PLR) homologues were studied to determine their enantioselective, substrate versatility, and kinetic properties. PLRs are downstream of dirigent protein engendered, coniferyl alcohol derived, stereoselective coupling to afford entry into the 8- and 8'-linked furofuran lignan, Pinoresinol. Our investigations showed that each PLR homolog can enantiospecifically metabolize different furofuran lignans with modified aromatic ring substituents, but where phenolic groups at both C4/C4' are essential for catalysis. These results are consistent with quinone methide intermediate formation in the PLR active site. Site-directed mutagenesis and kinetic measurements provided additional insight into factors affecting enantioselectivity and kinetic properties. From these data, PLRs can be envisaged to allow for the biotechnological potential of generation of various lignan skeleta, that could be differentially "decorated" on their aromatic ring substituents, via the action of upstream dirigent proteins.
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dirigent protein mediated lignan and cyanogenic glucoside formation in flax seed integrated omics and maldi mass spectrometry imaging
Journal of Natural Products, 2015Co-Authors: Doralyn S Dalisay, Laurence B Davin, Kyewon Kim, Hong Yang, Choonseok Lee, Oliver Rubel, Benjamin P Bowen, Norman G LewisAbstract:An integrated omics approach using genomics, transcriptomics, metabolomics (MALDI mass spectrometry imaging, MSI), and bioinformatics was employed to study spatiotemporal formation and deposition of health-protecting polymeric lignans and plant defense cyanogenic glucosides. Intact flax (Linum usitatissimum) capsules and seed tissues at different development stages were analyzed. Transcriptome analyses indicated distinct expression patterns of dirigent protein (DP) gene family members encoding (-)- and (+)-Pinoresinol-forming DPs and their associated downstream metabolic processes, respectively, with the former expressed at early seed coat development stages. Genes encoding (+)-Pinoresinol-forming DPs were, in contrast, expressed at later development stages. Recombinant DP expression and DP assays also unequivocally established their distinct stereoselective biochemical functions. Using MALDI MSI and ion mobility separation analyses, the Pinoresinol downstream derivatives, secoisolariciresinol diglucoside (SDG) and SDG hydroxymethylglutaryl ester, were localized and detectable only in early seed coat development stages. SDG derivatives were then converted into higher molecular weight phenolics during seed coat maturation. By contrast, the plant defense cyanogenic glucosides, the monoglucosides linamarin/lotaustralin, were detected throughout the flax capsule, whereas diglucosides linustatin/neolinustatin only accumulated in endosperm and embryo tissues. A putative biosynthetic pathway to the cyanogens is proposed on the basis of transcriptome coexpression data. Localization of all metabolites was at ca. 20 μm resolution, with the web based tool OpenMSI enabling not only resolution enhancement but also an interactive system for real-time searching for any ion in the tissue under analysis.
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non host disease resistance response in pea pisum sativum pods biochemical function of drr206 and phytoalexin pathway localization
Phytochemistry, 2015Co-Authors: Herana Kamal Seneviratne, Laurence B Davin, Doralyn S Dalisay, Kyewon Kim, Syed G A Moinuddin, Hong Yang, Christopher M Hartshorn, Norman G LewisAbstract:Continually exposed to potential pathogens, vascular plants have evolved intricate defense mechanisms to recognize encroaching threats and defend themselves. They do so by inducing a set of defense responses that can help defeat and/or limit effects of invading pathogens, of which the non-host disease resistance response is the most common. In this regard, pea (Pisum sativum) pod tissue, when exposed to Fusarium solani f. sp. phaseoli spores, undergoes an inducible transcriptional activation of pathogenesis-related genes, and also produces (+)-pisatin, its major phytoalexin. One of the inducible pathogenesis-related genes is Disease Resistance Response-206 (DRR206), whose role in vivo was unknown. DRR206 is, however, related to the dirigent protein (DP) family. In this study, its biochemical function was investigated in planta, with the metabolite associated with its gene induction being Pinoresinol monoglucoside. Interestingly, both Pinoresinol monoglucoside and (+)-pisatin were co-localized in pea pod endocarp epidermal cells, as demonstrated using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging. In addition, endocarp epidermal cells are also the site for both chalcone synthase and DRR206 gene expression. Taken together, these data indicate that both (+)-pisatin and Pinoresinol monoglucoside function in the overall phytoalexin responses.
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an historical perspective on lignan biosynthesis monolignol allylphenol and hydroxycinnamic acid coupling and downstream metabolism
Phytochemistry Reviews, 2003Co-Authors: Laurence B Davin, Norman G LewisAbstract:This review describes discoveries from this laboratory on monolignol, allylphenol and hydroxycinnamic acid coupling, and downstream metabolic conversions, affording various lignan skeleta. Stereoselective 8-8′ coupling (dirigent protein-mediated) of coniferyl alcohol to afford (+)-Pinoresinol is comprehensively discussed, as is our current mechanistic/kinetic understanding of the protein’s radical-radical binding, orientation and coupling properties, and insights gained for other coupling modes, e.g. affording (−)-Pinoresinol. In a species dependent manner, (+)- or (−)-Pinoresinols can also undergo enantiospecific reductions, catalyzed by various bifunctional Pinoresinol-lariciresinol reductases (PLR), to afford lariciresinol and then secoisolariciresinol. With X-ray structures giving a molecular basis for differing PLR enantiospecificities, comparisons are made herein to the X-ray structure of the related enzyme, phenylcoumaran benzylic ether reductase, capable of 8-5′ linked lignan regiospecific reductions. Properties of the enantiospecific secoisolariciresinol dehydrogenase (also discovered in our laboratory and generating 8-8′ linked matairesinol) are summarized, as are both in situ hybridization and immunolocalization of lignan pathway mRNA/proteins in vascular tissues. This entire 8-8′ pathway thus overall affords secoisolariciresinol and matairesinol, viewed as cancer preventative agent precursors, as well as intermediates to cancer treating substances, such as podophyllotoxin derivatives. Another emphasis is placed on allylphenol/hydroxycinnamic acid coupling and associated downstream metabolism, e.g. affording the antiviral creosote bush lignan, nordihydroguaiaretic acid (NDGA), and the fern lignans, blechnic/brainic acids. Regiospecific 8-8′ allylphenol coupling is described, as is characterization of the first enantiospecific membrane-bound polyphenol oxidase, (+)-larreatricin hydroxylase, involved in NDGA formation. Specific [13C]-labeling also indicated that Blechnum lignans arise from stereoselective 8-2′ hydroxycinnamic acid coupling.
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secondary and quaternary structures of the Pinoresinol forming dirigent protein
Biochemistry, 2002Co-Authors: Steven C Halls, Norman G LewisAbstract:The (+)-Pinoresinol-forming dirigent protein is the first protein capable of stereoselectively coupling two coniferyl alcohol derived radical species, in this case to give the 8−8‘ linked (+)-Pinoresinol. Only dimeric cross-linked dirigent protein structures were isolated when 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide was used as cross-linking agent, whereas the associated oxidase, presumed to generate the corresponding free radical substrate, was not detected. Native Forsythia intermedia dirigent protein isoforms were additionally subjected to MALDI-TOF and ESI-MS analyses, which established the presence of both monomeric masses of 23−25 kDa and dimeric dirigent protein species ranging from 46 to 49 kDa. Analytical ultracentrifugation, sedimentation velocity, and sedimentation equilibrium analyses of the native dirigent protein in open solution confirmed further its dimeric nature as well as a propensity to aggregate, with the latter being dependent upon both temperature and solution ionic strengt...
Madhura Shettigar - One of the best experts on this subject based on the ideXlab platform.
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oxidative catabolism of Pinoresinol is initiated by an unusual flavocytochrome encoded by translationally coupled genes within a cluster of Pinoresinol coinduced genes in pseudomonas sp strain sg ms2
Applied and Environmental Microbiology, 2020Co-Authors: Madhura Shettigar, Sahil Balotra, Annette B Kasprzak, Stephen L Pearce, Michael J Lacey, Matthew C Taylor, Jianwei Liu, David M CahillAbstract:Burkholderia sp. SG-MS1 and Pseudomonas sp. SG-MS2 have previously been found to mineralize (+)-Pinoresinol through a common catabolic pathway. Here we use comparative genomics, proteomics, protein semi-purification and heterologous expression to identify a flavoprotein from the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family in SG-MS2 which carries out the initial hydroxylation of (+)-Pinoresinol at the benzylic carbon. The cognate gene is translationally coupled with a downstream cytochrome gene and the cytochrome is required for activity. The flavoprotein has a unique combination of cofactor binding and cytochrome requirements for the VAO/PCMH family. The heterologously expressed enzyme has a Km of 1.17 μM for (+)-Pinoresinol. The enzyme is over-expressed in the strain SG-MS2 upon exposure to (+)-Pinoresinol, along with 45 other proteins, 22 of which were found to be encoded by genes in an approximately 35.1 kb cluster also containing the flavoprotein and cytochrome genes. Homologs of 18 of these 22 genes, plus the flavoprotein and cytochrome genes, were also found in a 38.7 kb cluster in SG-MS1. The amino acid identities of four of the other proteins encoded within the SG-MS2 cluster suggest they could catalyze conversion of hydroxylated Pinoresinol to protocatechuate and 2-methoxyhydroquinone. Nine other proteins upregulated in SG-MS2 on exposure to (+)-Pinoresinol appear to be homologs of proteins known to comprise the protocatechuate and 2-methoxyhydroquinone catabolic pathways but only three of the cognate genes lie within the cluster containing the flavoprotein and cytochrome. IMPORTANCE (+)-Pinoresinol is an important plant defense compound, a major food lignan for humans and some other animals, and the model compound used to study degradation of the β-β′ linkages in lignin. We report a gene cluster in a Pseudomonas and a Burkholderia strain which is involved in oxidative catabolism of (+)-Pinoresinol. The flavoprotein component of the α-hydroxylase which heads the pathway belongs to the 4-phenol oxidizing (4PO) subgroup of the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family but constitutes a novel combination of cofactor and electron acceptor properties for the family. It is translationally coupled with a cytochrome gene whose product is also required for activity. The work throws new light on the biology of (+)-Pinoresinol and its transformation to other bioactive molecules. Potential applications of the findings include new options for deconstructing lignin into useful chemicals and the generation of new phytoestrogenic enterolactones from lignans.
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isolation of the Pinoresinol mineralizing pseudomonas sp strain sg ms2 and elucidation of its catabolic pathway
Applied and Environmental Microbiology, 2017Co-Authors: Madhura Shettigar, Sahil Balotra, Michael J Lacey, David M Cahill, Andrew C Warden, Hanspeter E Kohler, Daniel Rentsch, John G Oakeshott, Gunjan PandeyAbstract:Pinoresinol is a dimer of two β-β'-linked coniferyl alcohol molecules. It is both a plant defense molecule synthesized through the shikimic acid pathway and a representative of several β-β-linked dimers produced during the microbial degradation of lignin in dead plant material. Until now, little has been known about the bacterial catabolism of such dimers. Here we report the isolation of the efficient (+)-Pinoresinol-mineralizing Pseudomonas sp. strain SG-MS2 and its catabolic pathway. Degradation of Pinoresinol in this strain is inducible and proceeds via a novel oxidative route, which is in contrast to the previously reported reductive transformation by other bacteria. Based on enzyme assays and bacterial growth, cell suspension, and resting cell studies, we provide conclusive evidence that Pinoresinol degradation in strain SG-MS2 is initiated by benzylic hydroxylation, generating a hemiketal via a quinone methide intermediate, which is then hydrated at the benzylic carbon by water. The hemiketal, which stays in equilibrium with the corresponding keto alcohol, undergoes an aryl-alkyl cleavage to generate a lactone and 2-methoxyhydroquinone. While the fate of 2-methoxyhydroquinone is not investigated further, it is assumed to be assimilated by ring cleavage. The lactone is further metabolized via two routes, namely, lactone ring cleavage and benzylic hydroxylation via a quinone methide intermediate, as described above. The resulting hemiketal again exists in equilibrium with a keto alcohol. Our evidence suggests that both routes of lactone metabolism lead to vanillin and vanillic acid, which we show can then be mineralized by strain SG-MS2.IMPORTANCE The oxidative catabolism of (+)-Pinoresinol degradation elucidated here is fundamentally different from the reductive cometabolism reported for two previously characterized bacteria. Our findings open up new opportunities to use lignin for the biosynthesis of vanillin, a key flavoring agent in foods, beverages, and pharmaceuticals, as well as various new lactones. Our work also has implications for the study of new Pinoresinol metabolites in human health. The enterodiol and enterolactone produced through reductive transformation of Pinoresinol by gut microbes have already been associated with decreased risks of cancer and cardiovascular diseases. The metabolites from oxidative metabolism we find here also deserve attention in this respect.
Laurence B Davin - One of the best experts on this subject based on the ideXlab platform.
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Pinoresinol lariciresinol reductase substrate versatility enantiospecificity and kinetic properties
Chirality, 2020Co-Authors: Julianne K Hwang, Laurence B Davin, Syed G A Moinuddin, Norman G LewisAbstract:Two western red cedar Pinoresinol-lariciresinol reductase (PLR) homologues were studied to determine their enantioselective, substrate versatility, and kinetic properties. PLRs are downstream of dirigent protein engendered, coniferyl alcohol derived, stereoselective coupling to afford entry into the 8- and 8'-linked furofuran lignan, Pinoresinol. Our investigations showed that each PLR homolog can enantiospecifically metabolize different furofuran lignans with modified aromatic ring substituents, but where phenolic groups at both C4/C4' are essential for catalysis. These results are consistent with quinone methide intermediate formation in the PLR active site. Site-directed mutagenesis and kinetic measurements provided additional insight into factors affecting enantioselectivity and kinetic properties. From these data, PLRs can be envisaged to allow for the biotechnological potential of generation of various lignan skeleta, that could be differentially "decorated" on their aromatic ring substituents, via the action of upstream dirigent proteins.
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dirigent protein mediated lignan and cyanogenic glucoside formation in flax seed integrated omics and maldi mass spectrometry imaging
Journal of Natural Products, 2015Co-Authors: Doralyn S Dalisay, Laurence B Davin, Kyewon Kim, Hong Yang, Choonseok Lee, Oliver Rubel, Benjamin P Bowen, Norman G LewisAbstract:An integrated omics approach using genomics, transcriptomics, metabolomics (MALDI mass spectrometry imaging, MSI), and bioinformatics was employed to study spatiotemporal formation and deposition of health-protecting polymeric lignans and plant defense cyanogenic glucosides. Intact flax (Linum usitatissimum) capsules and seed tissues at different development stages were analyzed. Transcriptome analyses indicated distinct expression patterns of dirigent protein (DP) gene family members encoding (-)- and (+)-Pinoresinol-forming DPs and their associated downstream metabolic processes, respectively, with the former expressed at early seed coat development stages. Genes encoding (+)-Pinoresinol-forming DPs were, in contrast, expressed at later development stages. Recombinant DP expression and DP assays also unequivocally established their distinct stereoselective biochemical functions. Using MALDI MSI and ion mobility separation analyses, the Pinoresinol downstream derivatives, secoisolariciresinol diglucoside (SDG) and SDG hydroxymethylglutaryl ester, were localized and detectable only in early seed coat development stages. SDG derivatives were then converted into higher molecular weight phenolics during seed coat maturation. By contrast, the plant defense cyanogenic glucosides, the monoglucosides linamarin/lotaustralin, were detected throughout the flax capsule, whereas diglucosides linustatin/neolinustatin only accumulated in endosperm and embryo tissues. A putative biosynthetic pathway to the cyanogens is proposed on the basis of transcriptome coexpression data. Localization of all metabolites was at ca. 20 μm resolution, with the web based tool OpenMSI enabling not only resolution enhancement but also an interactive system for real-time searching for any ion in the tissue under analysis.
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non host disease resistance response in pea pisum sativum pods biochemical function of drr206 and phytoalexin pathway localization
Phytochemistry, 2015Co-Authors: Herana Kamal Seneviratne, Laurence B Davin, Doralyn S Dalisay, Kyewon Kim, Syed G A Moinuddin, Hong Yang, Christopher M Hartshorn, Norman G LewisAbstract:Continually exposed to potential pathogens, vascular plants have evolved intricate defense mechanisms to recognize encroaching threats and defend themselves. They do so by inducing a set of defense responses that can help defeat and/or limit effects of invading pathogens, of which the non-host disease resistance response is the most common. In this regard, pea (Pisum sativum) pod tissue, when exposed to Fusarium solani f. sp. phaseoli spores, undergoes an inducible transcriptional activation of pathogenesis-related genes, and also produces (+)-pisatin, its major phytoalexin. One of the inducible pathogenesis-related genes is Disease Resistance Response-206 (DRR206), whose role in vivo was unknown. DRR206 is, however, related to the dirigent protein (DP) family. In this study, its biochemical function was investigated in planta, with the metabolite associated with its gene induction being Pinoresinol monoglucoside. Interestingly, both Pinoresinol monoglucoside and (+)-pisatin were co-localized in pea pod endocarp epidermal cells, as demonstrated using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging. In addition, endocarp epidermal cells are also the site for both chalcone synthase and DRR206 gene expression. Taken together, these data indicate that both (+)-pisatin and Pinoresinol monoglucoside function in the overall phytoalexin responses.
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an historical perspective on lignan biosynthesis monolignol allylphenol and hydroxycinnamic acid coupling and downstream metabolism
Phytochemistry Reviews, 2003Co-Authors: Laurence B Davin, Norman G LewisAbstract:This review describes discoveries from this laboratory on monolignol, allylphenol and hydroxycinnamic acid coupling, and downstream metabolic conversions, affording various lignan skeleta. Stereoselective 8-8′ coupling (dirigent protein-mediated) of coniferyl alcohol to afford (+)-Pinoresinol is comprehensively discussed, as is our current mechanistic/kinetic understanding of the protein’s radical-radical binding, orientation and coupling properties, and insights gained for other coupling modes, e.g. affording (−)-Pinoresinol. In a species dependent manner, (+)- or (−)-Pinoresinols can also undergo enantiospecific reductions, catalyzed by various bifunctional Pinoresinol-lariciresinol reductases (PLR), to afford lariciresinol and then secoisolariciresinol. With X-ray structures giving a molecular basis for differing PLR enantiospecificities, comparisons are made herein to the X-ray structure of the related enzyme, phenylcoumaran benzylic ether reductase, capable of 8-5′ linked lignan regiospecific reductions. Properties of the enantiospecific secoisolariciresinol dehydrogenase (also discovered in our laboratory and generating 8-8′ linked matairesinol) are summarized, as are both in situ hybridization and immunolocalization of lignan pathway mRNA/proteins in vascular tissues. This entire 8-8′ pathway thus overall affords secoisolariciresinol and matairesinol, viewed as cancer preventative agent precursors, as well as intermediates to cancer treating substances, such as podophyllotoxin derivatives. Another emphasis is placed on allylphenol/hydroxycinnamic acid coupling and associated downstream metabolism, e.g. affording the antiviral creosote bush lignan, nordihydroguaiaretic acid (NDGA), and the fern lignans, blechnic/brainic acids. Regiospecific 8-8′ allylphenol coupling is described, as is characterization of the first enantiospecific membrane-bound polyphenol oxidase, (+)-larreatricin hydroxylase, involved in NDGA formation. Specific [13C]-labeling also indicated that Blechnum lignans arise from stereoselective 8-2′ hydroxycinnamic acid coupling.
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dirigent mediated podophyllotoxin biosynthesis in linum flavum and podophyllum peltatum
Phytochemistry, 2000Co-Authors: Zhiqiang Xia, Laurence B Davin, Michael A Costa, John Proctor, Norman G LewisAbstract:Given the importance of the antitumor/antiviral lignans, podophyllotoxin and 5-methoxypodophyllotoxin, as biotechnological targets, their biosynthetic pathways were investigated in Podophyllum peltatum and Linum flavum. Entry into their pathways was established to occur via dirigent mediated coupling of E-coniferyl alcohol to afford (+)-Pinoresinol; the encoding gene was cloned and the recombinant protein subsequently obtained. Radiolabeled substrate studies using partially purified enzyme preparations next revealed (+)-Pinoresinol was enantiospecifically converted sequentially into (+)-lariciresinol and (-)-secoisolariciresinol via the action of an NADPH-dependent bifunctional Pinoresinol/lariciresinol reductase. The resulting (-)-secoisolariciresinol was enantiospecifically dehydrogenated into (-)-matairesinol, as evidenced through the conversion of both radio- and stable isotopically labeled secoisolariciresinol into matairesinol, this being catalyzed by the NAD-dependent secoisolariciresinol dehydrogenase. (-)-Matairesinol was further hydroxylated to afford 7'-hydroxymatairesinol, this being efficiently metabolized into 5-methoxypodophyllotoxin. Thus much of the overall biosynthetic pathway to podophyllotoxin has been established, that is, from the dirigent mediated coupling of E-coniferyl alcohol to the subsequent conversions leading to 7'-hydroxymatairesinol.
Yan Zhang - One of the best experts on this subject based on the ideXlab platform.
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tracing the mass flow from glucose and phenylalanine to Pinoresinol and its glycosides in phomopsis sp xp 8 using stable isotope assisted tof ms
Scientific Reports, 2019Co-Authors: Yan Zhang, Junling Shi, Zhixia Zhao, Yanlin Liu, Xixi Zhao, Zhenhong GaoAbstract:Phomopsis sp. XP-8, an endophytic fungus from the bark of Tu-Chung (Eucommia ulmoides Oliv) showed capability to biosynthesize Pinoresinol (Pin) and Pinoresinol diglucoside (PDG) from glucose (glu) and phenylalanine (Phe). To verify the mass flow in the biosynthesis pathway, [13C6]-labeled glu and [13C6]-labeled Phe were separately fed to the strain as sole substrates and [13C6]-labeled products were detected by ultra-high-performance liquid chromatography-quadrupole time of flight mass spectrometry. As results, [13C6]-labeled Phe was incorporated into [13C6]-cinnamylic acid (Ca) and p-coumaric acid (p-Co), and [13C12]-labeled Pin, which revealed that the Pin benzene ring came from Phe via the phenylpropane pathway. [13C6]-Labeled Ca and p-Co, [13C12]-labeled Pin, [13C18]-labeled Pinoresinol monoglucoside (PMG), and [13C18]-labeled PDG products were found when [13C6]-labeled glu was used, demonstrating that the benzene ring and glucoside of PDG originated from glu. It was also determined that PMG was not the direct precursor of PDG in the biosynthetic pathway. The study identified the occurrence of phenylalanine- lignan biosynthesis pathway in fungi at the level of mass flow.
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verification of the phenylpropanoid Pinoresinol biosynthetic pathway and its glycosides in phomopsis sp xp 8 using 13c stable isotope labeling and liquid chromatography coupled with time of flight mass spectrometry
bioRxiv, 2018Co-Authors: Yan Zhang, Junling Shi, Yao Liu, Zhixia Zhao, Q Zhao, Zhenhong GaoAbstract:Phomopsis sp. XP-8, an endophytic fungus from the bark of Tu-Chung (Eucommia ulmoidesOliv), revealed the Pinoresinol diglucoside (PDG) biosynthetic pathway after precursor feeding measurements and genomic annotation. To verify the pathway more accurately, [13C6]-labeled glucose and [13C6]-labeled phenylalanine were separately fed to the strain as sole substrates and [13C6]-labeled products were detected by ultra-high performance liquid chromatography-quantitative time of flight mass spectrometry. As results, [13C6]-labeled phenylalanine was found as [13C6]-cinnamylic acid and p-coumaric acid, and [13C12]-labeled Pinoresinol revealed that the Pinoresinol benzene ring came from phenylalanine via the phenylpropane pathway. [13C6]-Labeled cinnamylic acid and p-coumaric acid, [13C12]-labeled Pinoresinol, [13C18]-labeled Pinoresinol monoglucoside (PMG), and [13C18]-labeled PDG products were found when [13C6]-labeled glucose was used, demonstrating that the benzene ring and glucoside of PDG originated from glucose. It was also determined that PMG was not the direct precursor of PDG in the biosynthetic pathway. The study verified the occurrence of the plant-like phenylalanine and lignan biosynthetic pathway in fungi.
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genomic analysis reveals the biosynthesis pathways of diverse secondary metabolites and Pinoresinol and its glycoside derivatives in phomopsis sp xp 8
2018Co-Authors: Zhenhong Gao, Yan Zhang, Zhiwei Zhang, Jinxin Che, Yanlin Liu, Junling ShiAbstract:ObjectiveSequencing and analysis of Phomopsis sp. XP-8 genome are beneficial to reveal the potential metabolic pathways of this strain and the key genes related to the biosynthesis of Pinoresinol and its glycoside derivatives and other secondary metabolites. MethodsWe sequenced Phomopsis sp. XP-8 genome by the Illumina HiSeq 2500 high throughput sequencing platform. Then gene prediction and functional annotation were analyzed using different softwares. ResultsThe final assembled genome size was approximately 55.2 Mb with an overall GC content of 53.5%. Further annotation analyses predicted 17094 protein-coding genes and 310 non-coding RNA genes. A large set of candidate genes involved in the production of Pinoresinol, its glycoside derivatives and other secondary metabolites were identified. Orthology and phylogenetic analysis revealed that Phomopsis sp. XP-8 and 5 Ascomycota share 12635 orthologous genes and 5626 gene families. ConclusionPhomopsis sp. XP-8 possessed genomic basis for production of diverse secondary metabolites, including Pinoresinol and its glycoside derivatives. This study provides basis for the further metabolic engineering of Pinoresinol and its glucoside production.
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strategies to enhance the production of Pinoresinol and its glucosides by endophytic fungus phomopsis sp xp 8 isolated from tu chung bark
AMB Express, 2018Co-Authors: Jing Zhu, Yan Zhang, Junling Shi, Lu Yan, Chunmei Jiang, Dongyan ShaoAbstract:To improve the production yield of (+)-Pinoresinol (Pin), (+)-Pinoresinol monoglucoside (PMG), and (+)-Pinoresinol diglucoside (PDG), different methods were conducted, including co-culture with resveratrol-producing Alternaria sp. MG1 spores and addition of Tu-chung in a medium at the start of cultivation, ultrasound treatment (40 kHZ, 10 min) on 5-day culture, and addition of ethanol and sodium butyrate on Day 3, followed by cultivation for an additional period of 2 days. At the end of the cultivation period (5 days), the liquid phase was collected for product analysis. Cells were collected for the determination of gene expression levels and then used in bioconversion using resting cells for another period of 2 days. The liquid phase was measured to determine the output of the target products and the expression levels of the key genes related to the biosynthesis of these compounds. Consequently, co-culture with Alternaria MG1 and addition of Tu-chung bark in the medium efficiently increased Pin, PMG, and PDG production yield in the biosynthesis systems using potato dextrose broth medium and resting cells of Phomopsis sp. XP-8. The key genes related to the biosynthesis of these compounds were significantly upregulated. However, in the majority of cases, the addition of ethanol and sodium butyrate, and ultrasound treatment decreased the production yield of Pin, PMG, and PDG. The change in production yield was not consistently accompanied by a change in gene expression.
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comparison of Pinoresinol diglucoside production by phomopsis sp xp 8 in different media and the characterisation and product profiles of the cultivation in mung bean
Journal of the Science of Food and Agriculture, 2016Co-Authors: Yan Zhang, Junling Shi, Zhenhong Gao, Jinxin Che, Dongyan Shao, Yanlin LiuAbstract:Background Phomopsis sp. XP-8 is an endophytic fungus with the ability to produce Pinoresinol diglucoside (PDG) in vitro and thus has potential application in biosynthesis of PDG independent of plants. In order to enhance the production of PDG, 18 different natural materials were tested in solid-state cultivation of Phomopsis sp. XP-8. Results Most of the tested natural materials promoted the production of PDG. A supplement derived from mung beans produced the highest PDG yield and better fungal growth than the other materials. Also, Pinoresinol monoglucoside, Pinoresinol and other substrates (phenylalanine, p-coumaric acid, cinnamic acid, caffeic acid, and ferulic acid) were obtained after fermentation on mung beans. Furthermore, PDG production was much higher when mung beans were incorporated into solid state agar versus a liquid medium. The highest Pinoresinol diglucoside production (72.1 mg kg(-1) in fresh culture) was obtained in 9 days using a solid state culture of Phomopsis sp. XP-8 on a mung bean grain medium containing 100 g kg(-1) glucose. Mung bean water-soluble polysaccharide was identified as a major promoter of PDG production by Phomopsis sp. XP-8. Conclusion Mung bean, especially its water-soluble polysaccharide fraction, was an efficient natural material to promote PDG production by Phomopsis sp. XP-8. © 2015 Society of Chemical Industry.
Yanlin Liu - One of the best experts on this subject based on the ideXlab platform.
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tracing the mass flow from glucose and phenylalanine to Pinoresinol and its glycosides in phomopsis sp xp 8 using stable isotope assisted tof ms
Scientific Reports, 2019Co-Authors: Yan Zhang, Junling Shi, Zhixia Zhao, Yanlin Liu, Xixi Zhao, Zhenhong GaoAbstract:Phomopsis sp. XP-8, an endophytic fungus from the bark of Tu-Chung (Eucommia ulmoides Oliv) showed capability to biosynthesize Pinoresinol (Pin) and Pinoresinol diglucoside (PDG) from glucose (glu) and phenylalanine (Phe). To verify the mass flow in the biosynthesis pathway, [13C6]-labeled glu and [13C6]-labeled Phe were separately fed to the strain as sole substrates and [13C6]-labeled products were detected by ultra-high-performance liquid chromatography-quadrupole time of flight mass spectrometry. As results, [13C6]-labeled Phe was incorporated into [13C6]-cinnamylic acid (Ca) and p-coumaric acid (p-Co), and [13C12]-labeled Pin, which revealed that the Pin benzene ring came from Phe via the phenylpropane pathway. [13C6]-Labeled Ca and p-Co, [13C12]-labeled Pin, [13C18]-labeled Pinoresinol monoglucoside (PMG), and [13C18]-labeled PDG products were found when [13C6]-labeled glu was used, demonstrating that the benzene ring and glucoside of PDG originated from glu. It was also determined that PMG was not the direct precursor of PDG in the biosynthetic pathway. The study identified the occurrence of phenylalanine- lignan biosynthesis pathway in fungi at the level of mass flow.
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genomic analysis reveals the biosynthesis pathways of diverse secondary metabolites and Pinoresinol and its glycoside derivatives in phomopsis sp xp 8
2018Co-Authors: Zhenhong Gao, Yan Zhang, Zhiwei Zhang, Jinxin Che, Yanlin Liu, Junling ShiAbstract:ObjectiveSequencing and analysis of Phomopsis sp. XP-8 genome are beneficial to reveal the potential metabolic pathways of this strain and the key genes related to the biosynthesis of Pinoresinol and its glycoside derivatives and other secondary metabolites. MethodsWe sequenced Phomopsis sp. XP-8 genome by the Illumina HiSeq 2500 high throughput sequencing platform. Then gene prediction and functional annotation were analyzed using different softwares. ResultsThe final assembled genome size was approximately 55.2 Mb with an overall GC content of 53.5%. Further annotation analyses predicted 17094 protein-coding genes and 310 non-coding RNA genes. A large set of candidate genes involved in the production of Pinoresinol, its glycoside derivatives and other secondary metabolites were identified. Orthology and phylogenetic analysis revealed that Phomopsis sp. XP-8 and 5 Ascomycota share 12635 orthologous genes and 5626 gene families. ConclusionPhomopsis sp. XP-8 possessed genomic basis for production of diverse secondary metabolites, including Pinoresinol and its glycoside derivatives. This study provides basis for the further metabolic engineering of Pinoresinol and its glucoside production.
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comparison of Pinoresinol diglucoside production by phomopsis sp xp 8 in different media and the characterisation and product profiles of the cultivation in mung bean
Journal of the Science of Food and Agriculture, 2016Co-Authors: Yan Zhang, Junling Shi, Zhenhong Gao, Jinxin Che, Dongyan Shao, Yanlin LiuAbstract:Background Phomopsis sp. XP-8 is an endophytic fungus with the ability to produce Pinoresinol diglucoside (PDG) in vitro and thus has potential application in biosynthesis of PDG independent of plants. In order to enhance the production of PDG, 18 different natural materials were tested in solid-state cultivation of Phomopsis sp. XP-8. Results Most of the tested natural materials promoted the production of PDG. A supplement derived from mung beans produced the highest PDG yield and better fungal growth than the other materials. Also, Pinoresinol monoglucoside, Pinoresinol and other substrates (phenylalanine, p-coumaric acid, cinnamic acid, caffeic acid, and ferulic acid) were obtained after fermentation on mung beans. Furthermore, PDG production was much higher when mung beans were incorporated into solid state agar versus a liquid medium. The highest Pinoresinol diglucoside production (72.1 mg kg(-1) in fresh culture) was obtained in 9 days using a solid state culture of Phomopsis sp. XP-8 on a mung bean grain medium containing 100 g kg(-1) glucose. Mung bean water-soluble polysaccharide was identified as a major promoter of PDG production by Phomopsis sp. XP-8. Conclusion Mung bean, especially its water-soluble polysaccharide fraction, was an efficient natural material to promote PDG production by Phomopsis sp. XP-8. © 2015 Society of Chemical Industry.
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production of Pinoresinol diglucoside Pinoresinol monoglucoside and Pinoresinol by phomopsis sp xp 8 using mung bean and its major components
Applied Microbiology and Biotechnology, 2015Co-Authors: Yan Zhang, Junling Shi, Zhenhong Gao, Jinxin Che, Ruiming Yangwu, Huanshi Jiang, Yanlin LiuAbstract:Phomopsis sp. XP-8 is an endophytic fungus that has the ability to produce Pinoresinol diglucoside (PDG) in vitro and thus has potential application for the biosynthesis of PDG independent of plants. When cultivated in mung bean medium, PDG production was significantly improved and Pinoresinol monoglucoside (PMG) and Pinoresinol (Pin) were also found in the culture medium. In this experiment, starch, protein, and polysaccharides were isolated from mung beans and separately used as the sole substrate in order to explore the mechanism of fermentation and identify the major substrates that attributed to the biotransformation of PDG, PMG, and Pin. The production of PDG, PMG, and Pin was monitored using high-performance liquid chromatography (HPLC) and confirmed using HPLC-MS. Activities of related enzymes, including phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL) were analyzed and tracked during the cultivation. The reaction system contained the compounds isolated from mung bean in the designed amount. Accumulation of phenylalanine, cinnamic acid, p-coumaric acid, PDG, PMG, and Pin and the activities of PAL, C4H, and 4CL were measured during the bioconversion. PMG was found only when mung bean polysaccharide was analyzed, while production of PDG and Pin were found when both polysaccharide and starch were analyzed. After examining the monosaccharide composition of the mung bean polysaccharide and the effect of the different monosaccharides had on the production of PMG, PDG, and Pin, galactose in mung bean polysaccharide proved to be the major factor that stimulates the production of PMG.