H. J. G. ten Hoopen

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.

Effects of hydrodynamic stress on cultured plant cells: A literature survey

Abstract In this paper a literature survey is presented on the effects of hydrodynamic stress (“shear stress”) on cultured plant cells. Hydrodynamic stress has mostly negative effects on cells, these effects can be designated as “damage.” Various symptoms of cell damage as damage indicator are discussed: alteration of morphology, release of intracellular compounds, alteration of metabolism and productivity, and loss of viability. A compilation is made of the experimental techniques that have been used to investigate the shear sensitivity of plant cells. From the literature reviewed it is clear that huge differences in hydrodynamic stress sensitivity exist among various plant cell lines. The opinion that plant cells are by all means sensitive to hydrodynamic stress has to be revised. It is concluded that for the development of a productiond system based on plant cells, hydrodynamic stress sensitivity should be determined preferably in a down-scaled version of the production system.

Growth of a Catharanthus roseus cell suspension culture in a modified chemostat under glucose-limiting conditions

SummaryA system for the continuous cultivation of plant cells has been developed, based on a commercially available 3–1 turbine-stirred fermentor. A special device was constructed to provide for homogeneous effluent from the culture at low dilution rates. Two steady states with Catharanthus roseus cells growing under glucose limitation are described with respect to biomass yield on the carbon and energy source glucose, specific oxygen consumption, specific carbon dioxide production and (by)product formation. From a carbon balance for each steady state it is shown that the flow of carbon to the culture (as glucose) practically equalled the flow of carbon from the culture (as biomass, carbon dioxide and (by)product). Biomass yields on glucose were 0.31 g/g and 0.35 g/g at dilution rates of 0.0060 l/h and 0.0081 l/h respectively. The striking difference between the obtained yield coefficients and biomass yield commonly found for batch-cultured plant cells is discussed.

Dissimilation curves as a simple method for the characterization of growth of plant cell suspension cultures.

A simple non-invasive method for the characterization of growth of a plant cell suspension in a single culture flask is given. The dissimilation of sugars by a cell-culture causes a loss of weight of the contents of the culture flask, and can therefore be used to follow the growth in that single culture flask. Because a correction for water evaporation is necessary, accurate results can only be obtained when a stable closure is used (e.g. Silicosen T-type plugs). The dissimilation curves obtained in this way were correlated to the concentration of sugars in the medium, the dry weight and the fresh weight. From these correlations the amount of intracellularly stored carbohydrates could be estimated. Rate constants for CO2-diffusion were determined for different types of closure. These values allowed the estimation of CO2 levels inside the culture flasks from the dissimilation curves (CO2 release curves). The dissimilation curves obtained using this method can easily be related to other types of growth curves. Different growth-phases can be clearly distinguished, e.g. lag-phase, exponential growth-phase and stationary-phase.

Effects of hydrodynamic stress on the growth of plant cells in batch and continuous culture

Abstract Plant cell suspensions of different species were subjected to various levels of hydrodynamic stress generated by a Rushton impeller in a fermenter. Experiments were carried out in both batch and continuous culture. To assess the effects of hydrodynamic stress, various culture parameters were followed. Comparisons were made with growth characteristics obtained at low levels of hydrodynamic stress. The cell lines Catharanthus roseus and Nicotiana tabacum were capable of growth under conditions of high hydrodynamic stress which were similar to those in a 25-m 3 stirred-tank production fermenter. Cinchona robusta and Tabernaemontana divaricata were found to be more sensitive to hydrodynamic stress. The variation in the hydrodynamic stress sensitivity of the cells depended strongly on the cell line used and could not be explained from the experimental data. The dependence of the hydrodynamic stress sensitivity of a cell line might be governed by a combination of effects: the species investigated, the subcultivation regime, and the growth conditions.

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

The negligible role of carbon dioxide and ethylene in ajmalicine production by Catharanthus roseus cell suspensions

/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.

Influence of temperature on growth and ajmalicine production by Catharantus roseus suspension cultures

SummaryRemoval of gaseous metabolites in an aerated fermenter affects ajmalicine production by Catharanthus roseus negatively. Therefore, the role of CO2 and ethylene in ajmalicine production by C. roseus was investigated in 3 l fermenters (working volume 1.8 l) with recirculation of a large part of the exhaust air. Removal of CO2, ethylene or both from the recirculation stream did not have an effect on ajmalicine production. Inhibition of ethylene biosynthesis in shake flasks with Co2+, Ni2+ or aminooxyacetic acid did not affect ajmalicine production. However, the removal of CO2 did enhance the amount of extracellular ajmalicine.

The application of continuous culture for plant cell suspensions

Industrial production of valuable secondary metabolites by plant cell cultures is generally hampered by low productivity. This productivity is controlled by several factors, one of these is temperature. Secondary metabolites are in most cases produced in a two-stage process: biomass growth, followed by secondary metabolite production. In part I of this study the optimal temperature for biomass growth was investigated aiming at: maximal formation of biosynthetic active biomass, minimal formation of useless by-products. These processes each had their characteristic temperature dependence. The growth of Catharanthus roseus biomass for the production of ajmalicine was found to be optimal at 27.5°C. The effects of oxygen limitation are discussed. In part II the temperature effect on ajmalicine production was investigated. The productivity was governed by two processes: induction and production. Induction and production were both optimal at 27.5°C. The length of the induction period was easily estimated from a rapid concentration decrease of the precursor tryptamine.

Gaseous metabolites and the ajmalicine production rate in high density cell cultures of Catharanthus roseus

Continuous culture of plant cell suspensions has been developed during the last 35 years. Starting from rather imperfect set-ups, nowadays much better equipment is used for studies on growth and production kinetics or cell physiology. In this review the development of equipment and theory, as well as the applications are discussed.