Elisabeth Fuss

Sustainable bioproduction of phytochemicals by plant in vitro cultures: anticancer agents

Due to their complex structure with several chiral centres important anticancer agents are still extracted from plants and not synthesized chemically on a commercial scale. Sustainable bioproduction of the compounds of interest may be achieved by plant in vitro cultures. Undifferentiated callus and suspension cultures, which can be cultivated in large bioreactors easily, very often fail to accumulate the compounds of interest, whereas shoot and root cultures as well hairy roots normally produce the same compounds as in the appropriate organs. The production of anticancer compounds, such as the alkaloids vinblastine, vincristine, paclitaxel (Taxol ® ), camptothecin, or the lignan podophyllotoxin, by plant in vitro cultures is reviewed. Taxanes can be produced in bioreactors using cell suspensions of various Taxus species with good yields; presently paclitaxel is produced on a commercial scale by Phyton Biotech (Germany). Camptothecin has low yields in suspension cultures of Camptotheca acuminata or Nothapodytes foetida (0.0003–0.01%), but a good production (0.1–0.3% dry wt) in root and hairy root cultures of Ophiorrhiza pumila , O. mungos and C. acuminata . Podophyllotoxin can be produced in cell suspension and root as well as hairy root cultures of Podophyllum and various Linum species up to 130 mg/l ( Linum album cell suspensions); its derivative 6-methoxypodophyllotoxin is accumulated in hairy roots of L. persicum up to about 500 mg/l. The in vitro production of dimeric indole alkaloids in Catharanthus roseus has failed so far both in undifferentiated and differentiated in vitro cultures. In cases where in vitro cultures show good yields, they can be employed in biotechnology for the sustainable production of valuable products.

(+)-Pinoresinol/(-)-lariciresinol reductase from Linum perenne Himmelszelt involved in the biosynthesis of justicidin B.

A cDNA encoding a pinoresinol–lariciresinol reductase PLR (PLR‐Lp1) was isolated from a cell culture of Linum perenne Himmelszelt accumulating the arylnaphthalene lignan justicidin B. The recombinant PLR‐Lp1 prefers (+)‐pinoresinol in the first reaction step, but (−)‐lariciresinol in the second step. Therefore, it is the first PLR described with opposite enantiospecificity within the two reaction steps catalysed by PLRs. Hairy root lines transformed with an ihpRNAi construct to suppress plr gene expression show less mRNA accumulation for the plr‐Lp1 gene and PLR enzyme activity. Justicidin B accumulation was reduced down to 24% in comparison to control lines showing the involvement of PLR‐Lp1 in the biosynthesis of justicidin B.

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.

Production of podophyllotoxin in Linum linearifolium in vitro cultures

For the first time, callus and suspension cultures of Linum linearifolium were initiated. Podophyllotoxin (PTOX), a strong antitumor precursor, was isolated from the calli and suspension, as a main lignan besides smaller amount of 6-methoxypodophyllotoxin (6MPTOX). L. linearifolium is now the third Linum species of section Syllinum, with PTOX as the main lignan. The amounts of lignans, especially PTOX, found in L. linearifolium cell cultures are quite high within the studied Linum species until now. The antiproliferative effects of extracts were tested in a panel of human tumor cell lines, using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]-dye reduction assay. The lignan mixtures caused concentration-dependent inhibition of malignant cell proliferation and showed moderate cytotoxic activity. The results clearly demonstrate that the lignan mixture of L. linearifolium exerts inhibitory effects against malignant cells.

Metabolic Profiling of Lignan Variability in Linum species of Section Syllinum native to Bulgaria

Lignans in eighteen samples of Linum species ( L. tauricum ssp. tauricum, serbicum, bulgaricum and linearifolium; L. elegans; L. flavum ssp. sparsiflorum, L. capitatum var. laxiflorum), all members of the section Syllinum occurring in Bulgaria, were analysed by HPLC-ESI/MS and HPLC-UV/DAD. The ESI/MS fragmentation pathways recently established for aryltetralin lignans are now extended to ester and glycoside derivatives. In total, 22 different lignans, mainly of the aryltetralin type, were identified. 6-Methoxypodophyllotoxin and its glucoside were present as major constituents in all samples. Differences between the investigated taxa were observed especially with respect to the accumulation of 6-deoxy-7-hydroxy-aryltetralins such as podophyllotoxin and of 6-hydroxy-7-deoxy-aryltetralin lignans of the peltatin type. The distribution of aryltetralin lignans with different oxygenation patterns in the various samples, and correlations between the chemical data and the molecular phylogeny based on an analysis of ITS sequences of the investigated species are discussed.

Stereochemistry of lignans in Phaleria macrocarpa (Scheff.) Boerl.

Phaleria macrocarpa (Scheff.) Boerl., a member of the Thymelaeaceae, is traditionally used in Indonesia as medicinal plant against cancer. In this context, we isolated the lignans pinoresinol, lariciresinol and matairesinol from different parts of this plant. The enantiomeric composition of these lignans was determined by chiral column analysis. Pinoresinol and lariciresinol were mixtures of both enantiomers with (79 ± 4)% and (55 ± 6)% enantiomeric excess for the (−)-enantiomers, respectively, whereas matairesinol was found as pure (+)-enantiomer.

Isolation of the lignan 6-methoxypodophyllotoxin from Linum boissieri and Biosynthesis of Lignans in this Species

Abstract We were able to establish a suspension culture of Linum boissieri. that produces 6-methoxypodophyllotoxin (6MPT). As a first step to gain insight into the lignan biosynthesis in L. boissieri. cell cultures, we were able to measure phenylalanine ammonia-lyase (PAL) activity in raw protein extracts. PAL is a key enzyme in the early part of the general phenylpropanoid pathway, leading (beside others) to the precursors for lignan biosynthesis.