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.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2000.00922.x

Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase.

The technology of large-scale plant cell culture is feasible for the industrial production of plant-derived fine chemicals. Due to low or no productivity of the desired compounds the economy is only in a few cases favorable. Various approaches are studied to increase yields, these encompass screening and selection of high producing cell lines, media optimization, elicitation, culturing of differentiated cells (organ cultures), immobilization. In recent years metabolic engineering has opened a new promising perspectives for improved production in a plant or plant cell culture.

Metabolic engineering of plant secondary metabolite pathways for the production of fine chemicals

In the past hundred years, synthetic drugs had gradually replaced plant-derived drugs, except for antibiotics and antitumor drugs. Due ; ; to the development of experimentation with molecules, new openings and perspectives appear for natural products. Progress in bio\ i technology, biochemistry and research on metabolism are also the cause of renewed interest in natural resources. j » This interest is based on growing awareness that a large number of secondary metabolites playing a significant role for the prevention ; [ of diseases are also present in our food. The food-processing industry is heavily investing in this branch, which results in an improve,' ment of the overall quality of our diet, including the development of functional food and neutraceuticals. ,

Pharmacognosy in the new millennium : Leadfinding and biotechnology

Plant secondary metabolism is very important for traits such as flower color, flavor of food, and resistance against pests and diseases. Moreover, it is the source of many fine chemicals such as drugs, dyes, flavors, and fragrances. It is thus of interest to be able to engineer the secondary metabolite production of the plant cell factory, e.g. to produce more of a fine chemical, to produce less of a toxic compound, or even to make new compounds, Engineering of plant secondary metabolism is feasible nowadays, but it requires knowledge of the biosynthetic pathways involved. To increase secondary metabolite production different strategies can be followed, such as overcoming rate limiting steps, reducing flux through competitive pathways, reducing catabolism and overexpression of regulatory genes. For this purpose genes of plant origin can be overexpressed, but also microbial genes have been used successfully. Overexpression of plant genes in microorganisms is another approach, which might be of interest for bioconversion of readily available precursors into valuable fine chemicals. Several examples will be given to illustrate these various approaches. The constraints of metabolic engineering of the plant cell factory will also be discussed. Our limited knowledge of secondary metabolite pathways and the genes involved is one of the main bottlenecks.

Engineering the plant cell factory for secondary metabolite production.

Methods described in this paper are confined to in vitro dedifferentiated plant cell suspension cultures, which are convenient for the large-scale production of fine chemicals in bioreactors and for the study of cellular and molecular processes, as they offer the advantages of a simplified model system for the study of plants when compared with plants themselves or differentiated plant tissue cultures. The commonly used methods of initiation of a callus from a plant and subsequent steps from callus to cell suspension culture are presented in the protocol. This is followed by three different techniques for subculturing (by weighing cells, pipetting and pouring cell suspension) and four methods for growth measurement (fresh- and dry-weight cells, dissimilation curve and cell volume after sedimentation). The advantages and disadvantages of the methods are discussed. Finally, we provide a two-step (controlled rate) freezing technique also known as the slow (equilibrium) freezing method for long-term storage, which has been applied successfully to a wide range of plant cell suspension cultures.

Initiation, growth and cryopreservation of plant cell suspension cultures

AUSTKACT.-A capillary gas chromatographic analysis is described by which non-dcrlvatizrd indolr alkaloids and indole-related compounds can br sepvrnted and identified whclr thr sysrem is coupled ro a mass spectrometer. By the USC of this analysis some phrnolics and stcrols could also be separuzd. The phenolics coniferyl alcohol and srnapyl alcohol and the stcrols campestrrol and stigmastem were identified in ‘~~&rr~~tvucln~rrrru Jwuril-utd cell suspcnsion cultures.

Capillary gas chromatographic analysis of indole alkaloids : investigation of the indole alkaloids present in Tabernaemontana divaricata cell suspension culture

Terpenoid indole alkaloid biosynthesis and enzyme activities in two cell lines of Tabernaemontana divaricata

From the aerial parts of Pentas lanceolata, belonging to the family Rubiaceae, a series of iridoid glucosides was isolated by preparative HPLC. Seven iridoid glucosides were identified. Besides asperuloside and asperulosidic acid, characteristic iridoids for Rubiaceae, five new iridoids were isolated, namely, tudoside (1), 13R-epi-gaertneroside (2), 13R-epi-epoxygaertneroside (3), and a mixture of E-uenfoside (4) and Z-uenfoside (5). Further, it was shown that the compound reported as citrifolinin B (6) is in fact the same as tudoside and should be revised. Also, the configuration of the previously reported iridoids gaertneroside and epoxygaertneroside has been elucidated.

Iridoids from Pentas lanceolata.

Revision of the structures of citrifolinin A, citrifolinoside, yopaaoside A, yopaaoside B, and morindacin, iridoids from Morinda citrifolia L. and Morinda coreia Ham.

An isocratic HPLC system is described which allows the separation of the iridoid and indole precursors of terpenoid indole alkaloids, which are present in a single crude extract. The system consists of a column of LiChrospher 60 RP select B 5 μm, 250 x 4 mm (Merck) with an eluent of 1 % formic acid-acetonitrile-trichloroacetic acid (100 :10 :0.25, v :v :w) at a flow of 1.2 ml/min. In the suspension cultures of Catharanthus roseus secologanin and tryptophan were detected. In the cultures of Tabernaemontana divaricata loganin, tryptophan, and tryptamine accumulated.

Denise Dagnino

Papers

Capillary gas chromatographic analysis of indole alkaloids : investigation of the indole alkaloids present in Tabernaemontana divaricata cell suspension culture

Terpenoid indole alkaloid biosynthesis and enzyme activities in two cell lines of Tabernaemontana divaricata

Iridoids from Pentas lanceolata.

Revision of the structures of citrifolinin A, citrifolinoside, yopaaoside A, yopaaoside B, and morindacin, iridoids from Morinda citrifolia L. and Morinda coreia Ham.

Analysis of several iridoid and indole precursors of terpenoid indole alkaloids with a single HPLC run.