Ursula Mocek

Genetic transformation of mature Taxus: an approach to genetically control the in vitro production of the anticancer drug, taxol

Abstract This report demonstrates genetic transformation of two Taxus species. Taxus brevifolia and Taxus baccata, and expression of bacterial genes transferred into the plant genome by Agrobacterium tumefaciens. We used two strains of Agrobacterium tumefaciens (Bo542 and C58) to inoculate shoot segments of mature yew trees. The highest gall formation frequency (28.3%) was achieved with Taxus baccata using the Bo542 strain. Agrobacterium tumefaciens strain Bo542 induced significantly more galls (24%) than strin C58 (4%). Although we were able to induce on both Taxus species, Taxus baccata showed significantly higher susceptibility (14%) than Taxus brevifolia (7%). In contrast to untransformed callus cultures, the gall cell lines proliferated on phytormone-free medium and produced agropine as the result of T-DNA transfer. Southern blot analysis showed the presence of T-DNA sequence in the genome of these cell lines. Taxol and related taxane produced by the transgenic callus cultures were identified by mass spectrometry and immunoassay with monoclonal antibodies specific for taxol.

An antifungal compound from Solanum nigrescens

The antifungal activity of Solanum nigrescens extracts has been traced to the presence of a spirostanol glycoside, cantalasaponin-3.

Biosynthetic studies on taxol

The biosynthesis of the plant antitumor agent, taxol, was studied by feeding radioactive or stable isotope-labeled precursors to cut stems or to inner bark tissue of Taxus brevifolia. The labeled taxol was purified to radiochemical purity and subjected to chemical degradation or was analyzed by electrospray tandem mass spectrometry. It was demonstrated that in the plant taxol is synthesized from baccatin-111, containing the fully functionalized diterpene moiety, and a precursor of the phenylpropanoid side chain. The latter arises from phenylalanine not via cinnamic acid, but via B-phenylalanine and phenylisoserine. Benzoylation of the side chain occurs only after its attachment to the diterpene moiety. The benzoate moiety is also formed from phenylalanine via B-phenylalanine and phenylisoserine, not via cinnamic acid. Our studies focused initially on the origin of the phenylpropanoid side chain and its mode of attachment to the diterpene moiety. Feeding experiments with radiolabeled precursors were carried out with cut twigs of Taxus brevifolia to which the radioactive material was administered through the cut stem in a small volume of water. Extensive purification of the resulting taxol, first by repeated HPLC and then by co-crystallization with non-labeled carrier material was found necessary to achieve constant specific radioactivity or, in other cases, to remove traces of radioactive impurities. Under these conditions incorporation of (7-14C)benzoic acid, as its N-acetylcysteamine thioester (0.09%), and ( 13-3Hlbaccatin-III (0.12%) was obtained. Degradation of the radioactive taxol from the benzoate feeding experiment showed that 88% of the radioactivity was recovered in the side chain fragment and only 1 1 % in the baccatin-111, suggesting that under the conditions of the experiment relatively little of the diterpene moiety was synthesized de novo. Upon degradation of the taxol from the baccatin-I11 feeding experiments, all the radioactivity was recovered in the baccatin-111 moiety, none in the side chain fragment.

Diversions of the Shikimate Pathway — The Biosynthesis of Cyclohexanecarboxylic Acid

The shikimic acid pathway1 has evolved in plants and microorganisms to provide for the synthesis of the aromatic amino acids, phenylalanine, tyrosine and tryptophan, as well as a number of other essential aromatic compounds, e.g., jo-aminobenzoic acid, the precursor of folic acid, or]D-hydroxybenzoic acid, the precursor of ubiquinone. A vast number of secondary metabolites, e.g., alkaloids or phenylpropanoids, are derived from these aromatic end products of the pathway. In addition, nature has invented a variety of diversions along the pathway which lead to a range of additional natural products. While the majority of secondary metabolites are derived from late segments of the shikimate pathway, at or beyond the stage of the branch point intermediate, chorismate, a few diversions also occur in the prechorismate part of the pathway. One of these, the reduction of shikimic acid to cyclohexanecarboxylic acid, forms the topic of this chapter.