A. W. Alfermann

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.

Two new enzymes of rosmarinic acid biosynthesis from cell cultures of Coleus blumei: Hydroxyphenylpyruvate reductase and rosmarinic acid synthase.

Rosmarinic acid biosynthesis can be stimulated in cell cultures of Coleus blumei by culturing the cells in medium with 4% sucrose. In enzyme extracts of these cells two new enzymes of rosmarinic acid biosynthesis were discovered. Hydroxyphenylpyruvate reductase reduces 4-hydroxyphenylpyruvate and 3,4-dihydroxyphenylpyruvate to 4-hydroxyphenyllactate and 3,4-dihydroxyphenyllactate, respectively, using NADH. Rosmarinic acid synthase transfers the caffeoyl moiety of caffeoyl-CoA to the non-phenolic OH-group of 3,4-dihydroxyphenyllactic acid, in course of which rosmarinic acid is formed.

Hinokinin biosynthesis in Linum corymbulosum Reichenb.

SUMMARY Due to their peculiar stereochemistry and numerous biological activities, lignans are of widespread interest. As only a few biosynthetic steps have been clarified to date, we aimed to further resolve the molecular basis of lignan biosynthesis. To this end, we first established that the biologically active lignan (-)-hinokinin could be isolated from in vitro cultures of Linum corymbulosum. Two hypothetical pathways were outlined for the biosynthesis of (-)-hinokinin. In both pathways, (+)-pinoresinol serves as the primary substrate. In the first pathway, pinoresinol is reduced via lariciresinol to secoisolariciresinol by a pinoresinol-lariciresinol reductase, and methylenedioxy bridges are formed later. In the second pathway, pinoresinol itself is the substrate for formation of the methylenedioxy bridges, resulting in consecutive production of piperitol and sesamin. To determine which of the proposed hypothetical pathways acts in vivo, we first isolated several cDNAs encoding one pinoresinol-lariciresinol reductase (PLR-Lc1), two phenylcoumaran benzylic ether reductases (PCBER-Lc1 and PCBER-Lc2), and two PCBER-like proteins from a cDNA library of L. corymbulosum. PLR-Lc1 was found to be enantiospecific for the conversion of (+)-pinoresinol to (-)-secoisolariciresinol, which can be further converted to give (-)-hinokinin. Hairy root lines with significantly reduced expression levels of the plr-Lc1 gene were established using RNAi technology. Hinokinin accumulation was reduced to non-detectable levels in these lines. Our results strongly indicate that PLR-Lc1 participates in (-)-hinokinin biosynthesis in L. corymbulosum by the first of the two hypothetical pathways via (-)-secoisolariciresinol.

High forskolin production in hairy roots of Coleus forskohlii

Abstract Hairy roots of Coleus forskohlii were induced by infection with the Agrobacterium rhizogenes MAFF 03-01724 strain. Growth and forskolin production of two hairy root clones cultured in various liquid media were examined. Hairy root clone B9 grew well in woody plant liquid medium and showed a high forskolin yield (ca. 1.3 mg/ 100 ml flask) after 5 weeks of culture. The time course of growth and forskolin production of the clone B9 cultured in woody plant liquid medium was also examined. Rapid growth started at week 2 and continued until week 5. The highest forskolin yield (ca. 1.6 mg/100 ml flask) was obtained at week 5. Productivity was much higher than that previously reported.

Hydroxyphenylpyruvate Reductase From Cell Suspension Cultures Of Coleus Blumei Benth.

Cell suspension cultures of Coleus blumei Benth. producing high amounts of rosmarinic acid were used to study the biosynthetic pathway of this caffeic acid ester. One of the involved enzymes, the hydroxyphenylpyruvate reductase (HPPR), is characterized in this paper. HPPR catalyzes the NAD (P)H dependent reduction of p-hydroxyphenylpyruvate to p-hydroxyphenyllactate. The enzyme developed maximal activity at an incubation temperature of 37 °C and at a pH of 6.5 to 7.0. The reaction proceeded linearly for an incubation time of 60 min and up to a protein concentration of 0.2 mg per assay. As electron donor HPPR accepted NADH and NADPH with Km-values of 190 µm and 95 µm respectively. The enzyme reduced differently substituted hydroxyphenylpyruvates but not β-phenylpyruvate. The apparent Km-values for the various substrates were at 10 µM for p-hydroxyphenylpyruvate, at 130 µm for 3,4-dihydroxyphenylpyruvate and at 250 µM for 3-methoxy-4-hydroxyphenylpyruvate. HPPR was competitively inhibited by rosmarinic acid and pyruvate with Ki-values of 210 µM and 200 µM respectively. Caffeic acid, p-coumaric acid and cinnamic acid did not affect the enzyme activity but p-coumaroyl-CoA inhibited HPPR

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.

Production of natural products by plant cell biotechnology: results, problems and perspectives

This short review presents some examples, which demonstrate the importance and the potential of plant cell and tissue cultures for a biotechnological production of natural products. On the other hand, it still can not be denied, that despite intensive work for some 30 years all over the world a real breakthrough of this technique has been achieved only very recently (Phyton 2002). The problems we are faced with and some new possibilities to overcome these problems will be discussed (for more detailed and/or special discussion see e.g. Walton et al. 1999, Bourgaud et al. 2001, Stafford 2002.