taxane and jasmonic-acid

Plant cell cultures constitute eco-friendly biotechnological platforms for the production of plant secondary metabolites with pharmacological activities, as well as a suitable system for extending our knowledge of secondary metabolism. Despite the high added value of taxol and the importance of taxanes as anticancer compounds, several aspects of their biosynthesis remain unknown. In this work, a genomewide expression analysis of jasmonate-elicited Taxus baccata cell cultures by complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) indicated a correlation between an extensive elicitor-induced genetic reprogramming and increased taxane production in the targeted cultures. Subsequent in silico analysis allowed us to identify 15 genes with a jasmonate-induced differential expression as putative candidates for genes encoding enzymes involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene showed a strong homology, including a very similar predicted 3D structure, with other genes previously reported to encode acyl-CoA ligases, thus suggesting a role in the formation of the taxol lateral chain. Functional analysis confirmed that the TB768 gene encodes an acyl-CoA ligase that localizes to the cytoplasm and is able to convert β-phenylalanine, as well as coumaric acid, into their respective derivative CoA esters. β-phenylalanyl-CoA is attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. The identification of this gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.

Topics: Amino Acid Sequence; Amplified Fragment Length Polymorphism Analysis; Bridged-Ring Compounds; Chromatography, High Pressure Liquid; Computer Simulation; Cyclopentanes; Cytosol; DNA, Complementary; Gene Expression Profiling; Gene Expression Regulation, Plant; Genes, Plant; Genetic Association Studies; Ligases; Models, Molecular; Oxylipins; Paclitaxel; Phenylalanine; Plant Proteins; RNA, Messenger; Sequence Alignment; Tandem Mass Spectrometry; Taxoids; Taxus

Cell lines of Taxus x media Rehd. were established via embryo and callus culture and grown on B5 medium containing 10 microM 2,4-D and 1 microM kinetin. Jasmonic acid (JA) was used to elicit taxane synthesis in a selected yew cell line. 100 microM JA was added 7 days after subculture. JA strongly increased taxane content in the cells and in the medium. At the time of the highest amount in elicited cultures, the enhancement of paclitaxel production was 19-fold and 4-fold in the cells and medium, respectively, compared to controls (non-elicited cultures). The time course of taxane accumulation in the cells and in the medium and the release of taxanes into the medium were not changed after JA elicitation.

Topics: 2,4-Dichlorophenoxyacetic Acid; Adenine; Bridged-Ring Compounds; Cells, Cultured; Culture Techniques; Cyclopentanes; Kinetin; Oxylipins; Seeds; Taxoids; Taxus