M. Petersen

Fungal elicitor preparations and methyl jasmonate enhance rosmarinic acid accumulation in suspension cultures of Coleus blumei

bstract Suspension cultures of Coleus blumei (Lamiaceae) treated with either an elicitor preparation from the culture medium of the phytopathogenic oomycete Pythium aphanidermatum or with methyl jasmonate enhanced accumulation of rosmarinic acid approximately threefold. The specific activities of phenylalanine ammonia lyase and rosmarinic acid synthase were also enhanced after addition of the fungal elicitor. The addition of methyl jasmonate transiently increased activities of phenylalanine ammonia lyase and hydroxyphenylpyruvate reductase, whereas the activity of rosmarinic acid synthase was not stimulated and the activity of tyrosine aminotransferase was slightly and constantly enhanced. Methyl jasmonate stimulated rosmarinic acid accumulation not only when added directly to the culture medium, but also when it could reach the cells only via the gas phase.

Proposed biosynthetic pathway for rosmarinic acid in cell cultures of Coleus blumei Benth

A biosynthetic pathway for rosmarinic acid is proposed. This pathway is deduced from studies of the enzymes detectable in preparations from suspension cells of Coleus blumei. Phenylalanine is transformed to 4-coumaroyl-CoA by the enzymes of the general phenylpropanoid pathway: phenylalanine ammonia-lyase (EC 4.3.1.5), cinnamic acid 4-hydroxylase (EC 1.14.13.11) and hydroxycinnamic acid:CoA ligase (EC 6.2.1.12). Tyrosine is metabolized to 4-hydroxyphenyllactate by tyrosine aminotransferase (EC 2.6.1.5) and hydroxyphenylpyruvate reductase. The ester can be formed from 4-coumaroyl-CoA and 4-hydroxyphenyllactate by the catalytic activity of rosmarinic acid synthase with concomitant release of CoA. Microsomal hydroxylase activities introduce the hydroxyl groups at positions 3 and 3′ of the aromatic rings of the ester 4-coumaroyl-4′-hydroxyphenyllactate giving rise to rosmarinic acid.

Accumulation of podophyllotoxin and related lignans in cell suspension cultures of linum album

Cell suspension cultures of Linum album were established, which were able to synthesize and accumulate lignans. Podophyllotoxin and 5-methoxypodophyllotoxin were the main products and were present as glycosides, together with small amounts of deoxypodophyllotoxin, 5′-demethoxy-5-methoxypodophyllotoxin, lariciresinol, pinoresinol, matairesinol, α- and β-peltatin, as well as the monolignol glucoside, coniferin. In dark and light grown cultures, maximal product yields of lignans of up to 0.2% and 0.5% of the dry weight, respectively, were achieved, mainly consisting of podophyllotoxin.

Cytochrome P450-dependent hydroxylation in the biosynthesis of rosmarinic acid in Coleus

Abstract Three membrane-bound, cytochrome P450-dependent hydroxylases which are involved in the biosynthesis of rosmarinic acid have been characterized in microsomal preparations from cell cultures of Coleus blumei . Cinnamic acid 4-hydroxylase introduces the 4-hydroxyl group into cinnamic acid and forms 4-coumaric acid. This enzyme from Coleus blumei displayed saturation concentrations of 0.5 mM for both cinnamic acid and NADPH. The apparent K m -values were determined to be at 35 and 40 μM, respectively. Hydroxycinnamoyl-hydroxyphenyllactate 3- and 3′-hydroxylases introduce the 3- and 3′-hydroxyl groups into the aromatic rings of rosmarinic acid-like esters like 4-coumaroyl-4′-hydroxyphenyllactate, 4-coumaroyl-3′, 4′-dihydroxyphenyllactate and caffeoyl-4′-hydroxyphenyllactate. 4-Coumaric acid and its CoA-ester as well as 4- hydroxyphenylpyruvate and 4-hydroxyphenyllactate were not accepted as substrates. 3-Hydroxylase was saturated with 250 μM 4-coumaroyl-3′,4′-dihydroxyphenyllac tate and had an apparent K m -value of 12.5 μM for this substrate. The respective values for 3′-hydroxylase and the substrate caffeoyl-4′-hydroxyphenyllactate were 100 and 7 μM. The order of introduction of the 3- and 3′-hydroxyl groups could not be determined. The 3- and 3′-hydroxylations are dependent on O 2 and NADPH; the saturation concentration for both enzymes for NADPH was at 0.5 mM and the apparent K m values at 30 μM. All three hydroxylases were determined to be dependent on cytochrome P450 by inhibition experiments with cytochrome c, ancymidol, metyrapone, miconazole and tetcyclacis as well as by inhibition of the reactions in a gas phase containing CO besides O 2 and the partial reversion of this inhibition after illumination with light at 450 nm wavelength.

Natural product formation by plant cell biotechnology

This short review tries to compile some results, which show the importance and the potential of plant cell and tissue cultures for a biotechnological production of natural products. On the other hand, it can not be denied, that a real breakthrough of this technique has not yet been achieved. The problems involved and possible ways to overcome these will be discussed.

Influence of the carbon source on growth and rosmarinic acid production in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei were characterized with respect to growth and rosmarinic acid formation in media with different sugars and various sugar concentrations. Sucrose is the sugar with the highest stimulating effect on growth and rosmarinic acid accumulation, followed by glucose and fructose. The sugar alcohol mannitol cannot be metabolized by the plant cells. Sucrose is cleaved into glucose and fructose by the Coleus cells. Sucrose concentrations from 1 to 5% have an increasing positive effect on growth and rosmarinic acid synthesis in the cell cultures with a maximum rosmarinic acid content of 12% of the dry weight in medium with 5% sucrose; in medium with 6% sucrose rosmarinic acid accumulation obviously did not reach its highest level in the culture period of 14 days. A very high yield of rosmarinic acid (2 mg ml-1 suspension) could also be achieved by maintaining a sucrose concentration of 2% during the whole culture period. The start of rosmarinic acid synthesis by the cell cultures seems to be regulated by the growth limitation when a nutrient, e.g. phosphate is depleted from the medium. The rate of rosmarinic acid accumulation is related to the amount of carbon left in the medium when growth ceases.

Characterization of rosmarinic acid synthase from cell cultures of coleus blumei

Abstract Rosmarinic acid synthase (RAS) catalyses the transesterification reaction of CoA-activated cinnamic acids, e.g. 4-coumaroyl- or caffeoyl-CoA, and hydroxyphenyllactic acids, e.g. 4-hydroxy- or 3,4-dihydroxyphenyllactic acid, during the biosynthesis of rosmarinic acid. The enzyme isolated from suspension cultures of Coleus blumei is soluble and has an optimal pH of 7.0–7.5. Michaelis-Menten kinetics were observed with respect to the substrates 4-coumaroyl- and caffeoyl-CoA with K m -values of 20 and 33 μM, respectively. Only R (+)-stereoisomers of the hydroxyphenyllactic acid are accepted by RAS. For 4-hydroxy- and 3,4-dihydroxyphenyllactic acid the K m -values were 0.17 and 0.37 mM, respectively. The RAS reaction was inhibited by 4-hydroxymercuribenzoate, the S (−)-stereoisomer of 3,4-dihydroxyphenyllactic acid, hydroxyphenylpyruvates and rosmarinic acid, whereas cinnamic acids were not inhibitory. Coenzyme A showed a non-competitive inhibition. The reverse reaction of RAS, the splitting of rosmarinic acid with help of coenzyme A into caffeoyl-CoA and 3,4-dihydroxyphenyllactate, was readily detected. The kinetic data for this reaction are K m -values of 310 μM for coenzyme A and 15 μM for rosmarinic acid.

The biosynthesis of rosmarinic acid in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei accumulate very high amounts of rosmarinic acid, an ester of caffeic acid and 3,4-dihydroxyphenyllactate, in medium with elevated sucrose concentrations. Since the synthesis of this high level of rosmarinic acid occurs in only five days of the culture period, the activities of the enzymes involved in the biosynthesis are very high. Therefore all the enzymes necessary for the formation of rosmarinic acid from the precursors phenylalanine and tyrosine could be isolated from cell cultures of Coleus blumei: phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, hydroxycinnamoyl:CoA ligase, tyrosine aminotransferase, hydroxyphenylpyruvate reductase, rosmarinic acid synthase and two microsomal 3- and 3′-hydroxylases. The main characteristics of these enzymes of the proposed biosynthetic pathway of rosmarinic acid will be described.

Biosynthesis and accumulation of rosmarinic acid in suspension cultures ofColeus blumei

This communication reviews data on the accumulation and biosynthesis of rosmarinic acid in cell suspension cultures ofColeus blumei. The influence of the medium, mainly the carbohydrate source on growth and rosmarinic acid production in these cell cultures is described. The biosynthetic pathway of rosmarinic acid was elucidated inColeus blumei cell cultures: eight enzymatic activities are involved in the transformation of the precursors phenylalanine and tyrosine to the end product rosmarinic acid.

Isolation of protoplasts and vacuoles from cell suspension cultures of Coleus blumei Benth.

In order to study the accumulation and transport of rosmarinic acid in suspension cells of Coleus blumei we established an efficient method to isolate protoplasts and vacuoles. Protoplasts were disrupted by an osmotic shock in a medium with basic pH containing ethylenediamine tetraacetic acid. The resulting vacuoles were purified on a two-step Ficoll gradient. The comparison of the rosmarinic acid contents of cells, protoplasts and vacuoles showed that the depside is localized in the vacuole. Data concerning the yield and purity of the vacuoles are presented. In addition we show that at the physiological pH of the cytoplasm rosmarinic acid is present almost exclusively as an anion and cannot pass a membrane by simple diffusion. We therefore propose a carrier system for the transport of rosmarinic acid into the vacuole.