Peter Brodelius

Immobilized plant cells for the production and transportation of natural products

There has been considerable interest in the past few years in the area of immobilized entities, such as enzymes and microbial cells [ 11, Preparations of this kind have proven to be of value not only for studies of a fundamental nature but also for a number of practical applications including the production and transfo~ation of food stuff additives and drugs. The advantages of using such preparations include, in particular, a lower cost in biotechnolo~cal applications because of the possibility of reusing the ‘biocatalyst’ and there is no need for separation of the product from the catalyst. Furthermore, immobilized preparations are ideally suited for flow-through processes; these advantages of immobilized systems have been amply reviewed [2,3]. In this study we report on an extension of the use of the immob~ization technique to include also, for the first time, plant cells. Isolated growing plant cells have been discussed as a potential source for the production and biotr~sfo~ation of natural products (e.g., [4]). The same advantages mentioned above should also apply to immobilized plant cells. In addition, it appears likely that the chemical potential of normally slow growing plant cells (under batch conditions) could be more efficiently exploited by immobilization. Three examples of the use of immobilized plant cells are given here. First the de novo synthesis of ~thraquinones by entrapped cells of Mo~ndu is demonstrated, second the formation of indole alkaloids from distant precursors by Cat~~Q~th~~ cells and third, the position and stereospecific hydroxyla-

Entrapment of plant cells in different matrices: A comparative study

During recent years the interest for plant tissue cultures as producers of secondary metabolites of commercial value has increased considerably [l-3]. Both callus and suspension cultures might be used in principle, but the latter type is preferred. Low productivity of naturaI products in such tissue cultures is, however, a recognized problem and many attempts are being made to m~ipulate the overah metabolism of the cells in order to increase the total yield of the product in question. We reported the immobilization of whole plant cells by entrapment in calcium alginate gels [4] and believe that this technique is a valuable addition to the general techniques used in plant tissue cultures. The production of secondary products by the immobilized cells was higher than that for freely suspended cells under the same conditions. Thus, immobilized cells of ~u~~r~~ ~~~~f~~~~ produced up to IO-times as much ~thraqu~nones as freely suspended cells [5]. Immobilized plant cells were later studied for biotransformations of natural products [6--S]. The immobilization technique used in [6-g] was also entrapment in calcium alginate gels. Such gels require Ca*+ as stabilizer, which can lead to problems in phosphatecontaining media. Therefore in [7] a special medium lacking phosphate ions was designed. In our initial studies on immobilized plant cells we chose to use entrapment in a&ate because there are also distinct advantages with this immobilization technique> including reversibility of the immobilization which enables the ~nvesiigat~on of the cells in free suspension subsequent to their confinement in the immobilized state. Here, we describe studies on alternative methods

Purification and partial characterization of milk clotting proteases from flowers of Cynara cardunculus

Three proteases (cynarases 1, 2 and 3) with milk-clotting activity have been purified from dried flowers of Cynara cardunculus. The proteases are each composed of one large and one small subunit. The native Mr of the dimeric proteins is 49 000. The three proteases are glycoproteins containing N-linked high mannose type glycans. Cynarase 3 shows the highest proteolytic and milk-clotting activity. All three enzymes express maximum activity at pH 5.1. Inhibitor studies indicate that the cynarases are of the aspartic acid type. Antibodies raised against the large subunit of cynarase 3 cross-reacts with the large subunits of the other two cynarases after destruction of the glycan structure by periodate oxidation.

A launch vector for the production of vaccine antigens in plants

Historically, most vaccines have been based on killed or live‐attenuated infectious agents. Although very successful at immunizing populations against disease, both approaches raise safety concerns and often have limited production capacity. This has resulted in increased emphasis on the development of subunit vaccines. Several recombinant systems have been considered for subunit vaccine manufacture, including plants, which offer advantages both in cost and in scale of production. We have developed a plant expression system utilizing a ‘launch vector’, which combines the advantageous features of standard agrobacterial binary plasmids and plant viral vectors, to achieve high‐level target antigen expression in plants. As an additional feature, to aid in target expression, stability and purification, we have engineered a thermostable carrier molecule to which antigens are fused. We have applied this launch vector/carrier system to engineer and express target antigens from various pathogens, including, influenza A/Vietnam/04 (H5N1) virus.

A general method for the immobilization of cells with preserved viability

SummaryMicrobial, algal, plant and animal cells have been immobilized, with preserved viability, by entrapment in various matrices according to a new bead polymerization technique. The cell polymer/monomer mixture is kept suspended in a hydrophobic phase such as soy, paraffin, or silicon oil, tri-n-butylphosphate, or dibutyphtalate, which is compatible with the cells. The various monomers or polymers tested include agarose, agar, carrageenan, alginate, fibrin, and polyacrylamide. Furthermore, by adjustment of the stirring speed of the suspension, beads of desired diameter can easily be obtained. The entrapped cells are fully viable and biosynthetically active.

Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L

A method based on the laser microdissection pressure catapulting technique has been developed for isolation of whole intact cells. Using a modified tissue preparation method, one outer pair of apical cells and two pairs of sub-apical, chloroplast-containing cells, were isolated from glandular secretory trichomes of Artemisia annua. A. annua is the source of the widely used antimalarial drug artemisinin. The biosynthesis of artemisinin has been proposed to be located to the glandular trichomes. The first committed steps in the conversion of FPP to artemisinin are conducted by amorpha-4,11-diene synthase, amorpha-4,11-diene hydroxylase, a cytochrome P450 monooxygenase (CYP71AV1) and artemisinic aldehyde Delta11(13) reductase. The expression of the three biosynthetic enzymes in the different cell types has been studied. In addition, the expression of farnesyldiphosphate synthase producing the precursor of artemisinin has been investigated. Our experiments showed expression of farnesyldiphosphate synthase in apical and sub-apical cells as well as in mesophyl cells while the three enzymes involved in artemisinin biosynthesis were expressed only in the apical cells. Elongation factor 1alpha was used as control and it was expressed in all cell types. We conclude that artemisinin biosynthesis is taking place in the two outer apical cells while the two pairs of chloroplast-containing cells have other functions in the overall metabolism of glandular trichomes.

Permeabilization of immobilized plant cells, resulting in release of intracellularly stored products with preserved cell viability

SummaryPlant cells, entrapped in agarose or alginate, were permeabilized in a new procedure that retain cell viability, permitting the possibility of reusing biomass after release of intracellular products. Dimethylsulfoxide (DMSO) was used to make the cells permeable. The activity of isocitrate dehydrogenase, an intracellular enzyme, was used as an indicator of plasma membrane permeability. Cells from three plant species require different concentrations of DMSO for complete permeabilization (i.e., for maximal isocitrate dehydrogenase activity). Cells of Catharanthus roseus permeabilized with up to 5% DMSO remained viable, and released 85–90% of the intracellularly stored products (ajmalicine isomers). In model production systems, C. roseus cells entrapped in agarose or alginate beads were intermittently permeabilized for release of products in a cyclic process. An increase in product yield was observed for each cycle because of increase in biomass (cell growth) within the beads.

Immobilized plant cells

Plant cells have been immobilized in alginate, where they have been shown to retain their biological activity. Such systems can be utilized for bioconversions.

Increased secondary product formation in plant cell suspension cultures after treatment with a yeast carbohydrate preparation (elicitor)

A carbohydrate fraction isolated from yeast extract by ethanolic precipitation was used as an elicitor to induce secondary product formation in plant cell suspension cultures. The elicitor preparation is effective in inducing glyceollin isomer synthesis (up to 200 μg glyceollin per g dry wt) in cells of Glycine max and enhancing berberine biosynthesis (up to four-fold) in cells of Thalictrum rugosum. The response of the cell cultures to the elicitor treatment is dependent on the amount of carbohydrate per unit of biomass and on the physiological state of the cells. Cells are optimally induced in late exponential or early stationary growth phases.

Influence of growth regulators and an elicitor on phenylpropanoid metabolism in suspension cultures of Vanilla planifolia.

A cell suspension culture of Vanilla planifolia has been established in MS-medium. 2,4-D suppressed while NAA enhanced the formation of extractable phenolics. Cytokinins appeared to favour lignin biosynthesis. Treatment of the culture with chitosan resulted in the induction of various enzymes of phenylpropanoid metabolism, while the amount of extractable phenolics decreased due to their rapid incorporation into polymeric ligneous material.