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Nicholas P. Crouch

Bio: Nicholas P. Crouch is an academic researcher. The author has contributed to research in topics: Catharanthus roseus & Secologanin. The author has an hindex of 3, co-authored 3 publications receiving 352 citations.

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TL;DR: The molecular characterization of CYP72A1 from Catharanthus roseus (Madagascar periwinkle) was described nearly a decade ago, but the enzyme function remained unknown, and it is shown by in situ hybridization and immunohistochemistry that the expression in immature leaves is epidermis-specific.
Abstract: Summary The molecular characterization of CYP72A1 from Catharanthus roseus (Madagascar periwinkle) was described nearly a decade ago, but the enzyme function remained unknown. We now show by in situ hybridization and immunohistochemistry that the expression in immature leaves is epidermis-specific. It thus follows the pattern previously established for early enzymes in the pathway to indole alkaloids, suggesting that CYP72A1 may be involved in their biosynthesis. The early reactions in that pathway, i.e. from geraniol to strictosidine, contain several candidates for P450 activities. We investigated in this work two reactions, the conversion of 7-deoxyloganin to loganin (deoxyloganin 7-hydroxylase, DL7H) and the oxidative ring cleavage converting loganin into secologanin (secologanin synthase, SLS). The action of DL7H has not been demonstrated in vitro previously, and SLS has only recently been identified as P450 activity in one other plant. We show for the first time that both enzyme activities are present in microsomes from C. roseus cell cultures. We then tested whether CYP72A1 expressed in E. coli as a translational fusion with the C. roseus P450 reductase (P450Red) has one or both of these activities. The results show that CYP72A1 converts loganin into secologanin.

295 citations

Journal ArticleDOI
TL;DR: The activity was found in microsomes, indicating that tabersonine 6,7-epoxidase was a cytochrome P-450-dependent monooxygenase, and Carbon monoxide, clotrimazole, miconazoles, and cy tochrome C were shown to be strong inhibitors of the enzyme.

60 citations


Cited by
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Shusei Sato, Satoshi Tabata, Hideki Hirakawa, Erika Asamizu  +320 moreInstitutions (51)
31 May 2012-Nature
TL;DR: A high-quality genome sequence of domesticated tomato is presented, a draft sequence of its closest wild relative, Solanum pimpinellifolium, is compared, and the two tomato genomes are compared to each other and to the potato genome.
Abstract: Tomato (Solanum lycopersicum) is a major crop plant and a model system for fruit development. Solanum is one of the largest angiosperm genera1 and includes annual and perennial plants from diverse habitats. Here we present a high-quality genome sequence of domesticated tomato, a draft sequence of its closest wild relative, Solanum pimpinellifolium2, and compare them to each other and to the potato genome (Solanum tuberosum). The two tomato genomes show only 0.6% nucleotide divergence and signs of recent admixture, but show more than 8% divergence from potato, with nine large and several smaller inversions. In contrast to Arabidopsis, but similar to soybean, tomato and potato small RNAs map predominantly to gene-rich chromosomal regions, including gene promoters. The Solanum lineage has experienced two consecutive genome triplications: one that is ancient and shared with rosids, and a more recent one. These triplications set the stage for the neofunctionalization of genes controlling fruit characteristics, such as colour and fleshiness.

2,687 citations

Journal ArticleDOI
TL;DR: The biosynthetic pathways for several representative terpene indole alkaloids are described in detail, showing a diverse array of structures and biological activities.

761 citations

Journal ArticleDOI
TL;DR: The application of genomics to the alkaloid field has accelerated the discovery of cDNAs encoding previously elusive biosynthetic enzymes, and technologies, such as large-scale gene expression analyses and metabolic engineering approaches with transgenic plants, have provided new insights into the regulatory architecture of alkaloids metabolism.
Abstract: Alkaloids represent a highly diverse group of compounds that are related only by the occurrence of a nitrogen atom in a heterocyclic ring. Plants are estimated to produce approximately 12,000 different alkaloids, which can be organized into groups according to their carbon skeletal structures. Alkaloid biosynthesis in plants involves many catalytic steps, catalyzed by enzymes that belong to a wide range of protein families. The characterization of novel alkaloid biosynthetic enzymes in terms of structural biochemistry, molecular and cell biology, and biotechnological applications has been the focus of research over the past several years. The application of genomics to the alkaloid field has accelerated the discovery of cDNAs encoding previously elusive biosynthetic enzymes. Other technologies, such as large-scale gene expression analyses and metabolic engineering approaches with transgenic plants, have provided new insights into the regulatory architecture of alkaloid metabolism.

543 citations

Journal ArticleDOI
TL;DR: Metabolic engineering seems a promising approach to improve the production of a cell factory and can be used in the cell culture, in the plant itself or even production in other plant species or organisms.
Abstract: The production of plant secondary metabolites by means of large-scale culture of plant cells in bioreactors is technically feasible. The economy of such a production is the major bottleneck. For some costly products it is feasible, but unfortunately some of the most interesting products are only in very small amounts or not all produced in plant cell cultures. Screening, selection and medium optimization may lead to 20- to 30-fold increase in case one has producing cultures. In case of phytoalexins, elicitation will lead to high production. But for many of the compounds of interest the production is not inducible by elicitors. The culture of differentiated cells, such as (hairy) root or shoot cultures, is an alternative, but is hampered by problems in scaling up of such cultures. Metabolic engineering offers new perspectives for improving the production of compounds of interest. This approach can be used to improve production in the cell culture, in the plant itself or even production in other plant species or organisms. Studies on the production of terpenoid indole alkaloids have shown that the overexpression of single genes of the pathway may lead for some enzymes to an increased production of the direct product, but not necessarily to an increased alkaloid production. On the other hand feeding of such transgenic cultures with early precursors showed an enormous capacity for producing alkaloids, which is not utilized without feeding precursors. Overexpression of regulatory genes results in the upregulation of a series of enzymes in the alkaloid pathway, but not to an improved flux through the pathway, but feeding loganin does result in increased alkaloid production if compared with wild-type cells. Indole alkaloids could be produced in hairy root cultures of Weigelia by overexpression of tryptophan decarboxylase and strictosidine synthase. Alkaloids could be produced in transgenic yeast overexpressing strictosidine synthase and strictosidine glucosidase growing on medium made out the juice of Symphoricarpus albus berries to which tryptamine is added. Metabolic engineering thus seems a promising approach to improve the production of a cell factory.

507 citations

Journal ArticleDOI
TL;DR: Comparative analysis of the plant CYPomes provides information on the successive steps required for the evolution of land plants, and points to several cases of convergent evolution in plant metabolism.
Abstract: Being by far the largest family of enzymes to support plant metabolism, the cytochrome P450s (CYPs) constitute an excellent reporter of metabolism architecture and evolution. The huge superfamily of CYPs found in angiosperms is built on the successful evolution of 11 ancestral genes, with very different fates and progenies. Essential functions in the production of structural components (membrane sterols), light harvesting (carotenoids) or hormone biosynthesis kept some of them under purifying selection, limiting duplication and sub/neofunctionalization. One group (the CYP71 clan) after an early trigger to diversification, has kept growing, producing bursts of gene duplications at an accelerated rate. The CYP71 clan now represents more than half of all CYPs in higher plants. Such bursts of gene duplication are likely to contribute to adaptation to specific niches and to speciation. They also occur, although with lower frequency, in gene families under purifying selection. The CYP complement (CYPomes) of rice and the model grass weed Brachypodium distachyon have been compared to view evolution in a narrower time window. The results show that evolution of new functions in plant metabolism is a very long-term process. Comparative analysis of the plant CYPomes provides information on the successive steps required for the evolution of land plants, and points to several cases of convergent evolution in plant metabolism. It constitutes a very useful tool for spotting essential functions in plant metabolism and to guide investigations on gene function.

498 citations