Jochen Berlin

Medium- and light-induced formation of serpentine and anthocyanins in cell suspension cultures of Catharanthus roseus

Abstract By irradiation with fluorescent light, medium-induced cell cultures of Catharanthus roseus accumulated anthocyanins and the indole alkaloid serpentine. The formation of both compounds was inhibited by phosphate and nitrogen-containing mineral salts and stimulated by high sucrose concentrations. The accumulation of serpentine was preceded by an increase and subsequent decrease of its biogenetic precursor ajmalicine, which was the predominant alkaloid of medium-induced cultures in the dark. High concentrations of serpentine or anthocyanins were observed only in a small proportion of all cells present in a medium-induced culture. The aglycones of the anthocyanins were identified as petunidin, malvidin and hirsutidin.

Determination and quantification of monoterpenoids secreted into the medium of cell cultures of Thuja occidentalis

Abstract Cell cultures of Thuja occidentalis excrete monoterpenoids into the culture medium. Highly volatile monoterpene hydrocarbons were only detectable in the presence of Miglyol, a water-insoluble non-toxic trap for lipophilic compounds. In the presence of Miglyol the levels of oxygenated monoterpenoids were two-fold higher than in dichloromethane-extracts of culture media. The cells excrete up to 3 mg regular monoterpenoids/g dry wt/day. Continuous extraction of lipophilic compounds by Miglyol also allowed quantification of a Tris—thujaplicinato—(Fe III)-complex.

Increased production of serotonin by suspension and root cultures ofPeganum harmala transformed with a tryptophan decarboxylase cDNA clone fromCatharanthus roseus

Cell suspension and root cultures ofPeganum harmala were established expressing a tryptophan decarboxylase cDNA clone fromCatharanthus roseus under the control of the cauliflower mosaic virus (CaMV) 35S promoter and terminator sequences. The tryptophan decarboxylase activity of some of the transgenic lines was greatly enhanced (25–40 pkat/mg protein) as compared to control cultures (1–5 pkat per mg protein) and remained high during the growth cycle. While the levels of tryptamine, the product of the reaction catalysed by tryptophan decarboxylase, were unchanged in the transgenic lines, their serotonin contents were enhanced up to 10-fold, reaching levels of 1.5 to 2% dry mass. Thus, tryptamine produced by the engineered reaction was apparently immediately used for enhanced serotonin biosynthesis. The yields of serotonin in transgenic lines overexpressing tryptophan decarboxylase activity were further enhanced to 3–5% dry mass by feedingl-tryptophan, while no or only minor effects were seen when control cultures were fed. These data demonstrate that the production of a plant secondary metabolite can be enhanced greatly via genetic manipulation of the level of activity of the rate-limiting enzyme. The amounts of β-carboline alkaloids, the other tryptamine-derived metabolites ofP. harmala, in contrast, were not affected by the overproduction of tryptamine. The information needed for successfully predicting manipulations that enhance production of a secondary metabolite is discussed.

Increased production of cadaverine and anabasine in hairy root cultures of Nicotiana tabacum expressing a bacterial lysine decarboxylase gene

Several hairy root cultures of Nicotiana tabacum varieties, carrying two direct repeats of a bacterial lysine decarboxylase (ldc) gene controlled by the cauliflower mosaic virus (CaMV) 35S promoter expressed LDC activity up to 1 pkat/mg protein. Such activity was, for example, sufficient to increase cadaverine levels of the best line SR3/1-K1,2 from ca. 50 μg (control cultures) to about 700 μg/g dry mass. Some of the overproduced cadaverine of this line was used for the formation of anabasine, as shown by a 3-fold increase of this alkaloid. In transgenic lines with lower LDC activity the changes of cadaverine and anabasine levels were correspondingly lower and sometimes hardly distinguishable from controls. Feeding of lysine to root cultures, even to those with low LDC activity, greatly enhanced cadaverine and anabasine livels, while the amino acid had no or very little effect on controls and LDC-negative lines.

Large scale fermentation and alkaloid production of cell suspension cultures of Catharanthus roseus

Cell cultures of Catharanthus roseus were scaled up to volumes of 50001 using conventional reactors equipped with flat-blade impellers. The behavior of the fermenter grown cells was compared with corresponding shake flask experiments with respect to growth and indole alkaloid inducibility and production. The limits and problems of transferring shake flask experiments of culture systems such as Catharanthus, in which alkaloid production depends greatly upon the physiological state of the cells, to large scale multistage processes is discussed.

Formation of Benzophenanthridine Alkaloids by Suspension Cultures of Eschscholtzia californica

Suspension cultures of Eschscholtzia californica accumulate the dihydro forms of the benzo- phenanthridine alkaloids sanguinarine, chelirubine, macarpine and chelerythrine, all of which are known to be constituents of the Eschscholtzia plant. Under most experimental conditions dihydrochelirubine was found to be the main constituent of the cultured cells. The specific yields of alkaloids varied from zero to 1.7% on a dry weight basis depending on the media conditions. The highest specific yield was 1.5 mg/g dry weight or 13 mg/1 with the growth medium B5. After transfer of the cells into the induction medium IM2 the alkaloid accumulation increased to 17 mg/g dry weight and 146 mg/1. The induction medium contained increased levels of sucrose, decreased levels of phosphate and was devoid of phytohormones. The effect of the various media conditions on the biosynthesis of phenolics was quite different to those found for the alkaloids.

Improved metabolic action of a bacterial lysine decarboxylase gene in tobacco hairy root cultures by its fusion to arbcS transit peptide coding sequence

The gene of a bacterial lysine decarboxylase (ldc) fused to arbcS transit peptide coding sequence (tp), and under the control of the CaMV 35S promoter, was expressed in hairy root cultures ofNicotiana tabacum. The fusion of theldc to the targeting signal sequence improved the performance of the bacterial gene in the plant cells in many respects. Nearly all transgenic hairy root cultures harbouring the35S-tp-ldc gene contained distinctly higher lysine decarboxylase activity (from 1.5 to 30 pkat LDC per mg protein) than those which had been transformed with constructs in which the gene had been directly cloned behind the CaMV 35S promoter. The higher enzyme activity led to the accumulation of up to 0.7% cadaverine on a dry mass basis. In addition, part of the cadaverine pool was used for increased biosynthesis of anabasine, an alkaloid which was hardly detectable in control cultures. The best line contained anabasine levels of 0.5% dry mass, which could further be enhanced by feeding of lysine.

Increased formation of cinnamoyl putrescines by fedbatch fermentation of cell suspension cultures of Nicotiana tabacum.

The tobacco cell line TX1 (Nicotiana tabacum) accumulates up to 10 mg cinnamoyl putrescines/g dry weight. This level was further increased under various growth limiting conditions. The negative effect of accumulated phosphate on the optimal expression of cinnamoyl putrescine biosynthesis was prevented by the fedbatch fermentation technique. A batch fermenter yielded 160 mg cinnamoyl putrescines/1 while a phosphate fedbatch fermentation produced 400 mg/1. This was mainly due to the fact that P in the cells was kept low during the whole growth cycle.

Phosphorus-31 nuclear magnetic resonance investigation of the in vivo regulation of intracellular pH in cell suspension cultures of Nicotiana tabacum: The effects of oxygen supply, nitrogen, and external pH change

31P NMR was used to study the in vivo response of intracellular phosphorus-containing compounds of cell suspension cultures of Nicotiana tabacum to extracellular events. Limitation of the oxygen supply in a static system (sealed tube) caused a strong pH decrease of the cytoplasm and a smaller fall in the vacuolar pH. The rate of this process was independent of the age of the cells, except for those at the end of the growth cycle where either reserve supplies of inorganic phosphate have been depleted or metabolism has ceased. The cells can be returned quickly to their normal pH and metabolic status by reoxygenation after depletion times as long as 4.5 h. Regassing with N2 after anaerobiosis also caused the return of the nearly normal pH status, which indicates that rapid cell acidification is caused almost entirely by CO2 accumulation. In a second type of anaerobiosis, attained by continual passage of N2, cytoplasmic pH fell only slowly to a constant value higher than in the static case. Here acidification appeared to arise, at least in part, from lactate accumulation. Provided aeration occurred, the cytoplasm was maintained at a constant pH of 7.5 +/- 0.1 for changes in the medium pH value between 6.5 and 3. The biochemical and biotechnological implications of these results are discussed.

Site of Action of Growth Inhibitory Tryptophan Analogues in Catharanthus roseus Cell Suspension Cultures

Fourteen tryptophan analogues have been characterized with regard to their growth inhibitory effects on Catharanthus roseus cell cultures, their inhibitory effect on anthranilate synthetase, their incorporation into protein and their substrate affinity to tryptophan decarboxylase. The toxicity of 12 of the analogues was directly correlated with their inhibitory effect on anthranilate synthetase activity. None of the analogues was efficiently incorporated into protein. Only 4 of the analogues (4-methyl-, 4-fluoro-, 5-fluoro- and 5-hydroxytryptophan) were good substrates for tryptophan decarboxylase while all other analogues were rather poor substrates or were not converted at all. Alpha-methyltryptophan was a competitive inhibitor of tryptophan decarboxylase