R. van der Heijden

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

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.

High-performance liquid chromatography with on-line coupled UV, mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products.

A high-performance liquid chromatography (HPLC) method with on-line coupled ultraviolet (UV), mass spectrometry (MS) and biochemical detection for acetylcholinesterase (AChE) inhibitory activity has been developed. By combining the separation power of HPLC, the high selectivity of biochemical detection, and the ability to provide molecular mass and structural information of MS, AChE inhibitors can be rapidly identified. The biochemical detection was based on a colorimetric method using Ellmans reagent. The detection limit of galanthamine, an AChE inhibitor, in the HPLC-biochemical detection is 0.3 nmol. The three detector lines used, i.e., UV, MS and Vis for the biochemical detection were recorded simultaneously and the delay times of the peaks obtained were found to be consistent. This on-line post-column detection technique can be used for the identification of AChE inhibitors in plant extracts and other complex mixtures such as combinatorial libraries.

Engineering the plant cell factory for secondary metabolite production

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.

RPLC-ion-trap-FTMS method for lipid profiling of plasma : method validation and application to p53 mutant mouse model

A reversed-phase liquid chromatography-linear ion trap-Fourier transform ion cyclotron resonance-mass spectrometric method was developed for the profiling of lipids in human and mouse plasma. With the use of a fused-core C 8 column and a binary gradient, more than 160 lipids belonging to eight different classes were detected in a single LC-MS run. The method was fully validated and the analytical characteristics such as linearity ( R (2), 0.994-1.000), limit of detection (0.08-1.28 microg/mL plasma), repeatability (RSD, 2.7-7.9%) and intermediate precision (RSD, 2.7-15.6%) were satisfactory. The method was successfully applied to p53 mutant mice plasma for studying some phenotypic effects of p53 expression.

Effect of terpenoid precursor feeding and elicitation on formation of indole alkaloids in cell suspension cultures of Catharanthus roseus

The effects of terpenoid precursor feeding and elicitation by a biotic elicitor on alkaloid production of Catharanthus roseus suspension cultures were studied. After addition of secologanin, loganin or loganic acid an increase in the accumulation of ajmalicine and strictosidine and a decrease of tryptamine level was observed in non-elicited cells. Elicitation increased tryptamine accumulation in non-fed cells but it did not further increase alkaloid accumulation in precursor-fed cells. A decrease of tryptamine level was also observed, despite the induction of the tryptamine pathway after elicitation. Feeding mevalonic acid did not increase alkaloid accumulation in any studied case.

High-performance liquid chromatographic determination of indole alkaloids in a suspension culture of Tabernaemontana div aricata

Abstract A method is reported for the determination of alkaloids in cell cultures of Tabernaemontana species. The alkaloids were isolated by means of C 8 pre-concentration columns. After recovery they were analysed on a Phenyl type of column using phosphate buffer (pH 3.9)-acetonitrile-2-methoxyethanol (80:15:5) as the mobile phase. Detection was effected at 275 and 313 nm and peak-height ratios were used for identification.

Plant cell biotechnology for the production of secondary metabolites

Plant cells, which in general are shear-stress tolerant, can be cultured on a large scale in stirred bioreactors. The costs of a natural product, at a production of 3gA, would be about 430 US

Distribution of ginkgolides and terpenoid biosynthetic activity in Ginkgo biloba

/kg. To increase yields metabolic engineering seems to be a promising approach, but requires the understanding of the regulation of secondary metabolism at all its levels: genes, enzymes, products, transport and compartmentation. From Carharanthus roseus already several genes have been cloned and successfully expressed in among others tobacco and C. roseus.

Induction of triterpene biosynthesis by elicitors in suspension cultures of Tabernaemontana species.

Abstract The terpene trilactone content (bilobalide and ginkgolides) of extracts prepared from leaves of terminal buds, rosettes and side branches, from stem and root bark, and from root and root meristems of three-year-old Ginkgo biloba plants was determined. The aerial parts were relatively rich in bilobalide while ginkgolides were the major constituents of the underground parts. The formation of farnesyl and geranylgeranyl pyrophosphate was monitored by incubating cell-free extracts prepared from the corresponding plant parts with [1-14C]isopentenyl pyrophosphate. Extracts prepared from leaves of the terminal buds displayed terpenoid biosynthetic activity, suggesting that terpene trilactone synthesis might occur in actively growing tissues.

Isopentenyl diphosphate isomerase and prenyltransferase activities in rubiaceous and apocynaceous cultures

Treatment of suspension cultures of some Tabernaemontana species (Apocynaceae) with elicitors (e.g. cellulase, Candida albicans) result in a rapid de novo production of antimicrobial active triterpenes. The triterpenes are identified as ursene carboxylic acid derivatives. These triterpenes are not produced by an elicited cell suspension culture of Catharanthus roseus, another Apocynaceae.