New taxane biosynthetic gene encoding a β-phenylalanine-CoA ligase identified in jasmonate-elicited Taxus cells

Abstract

The data-generating power of “omics” technologies is increasingly being harnessed in studies of plant biology to shed light on aspects such as metabolite production patterns, gene sequences and protein expression. Taxol and related taxanes, produced by several Taxus species, are high-value secondary metabolites widely used in cancer therapy due to their antineoplastic activity. To date, several biosynthetic steps of these compounds remain undefined. After carrying out a genome-wide expression analysis of jasmonate-elicited Taxus baccata cell cultures using complementary DNA-amplified fragment length polymorphism (cDNA-AFLP), we found that the increased taxane production in the target cultures was correlated with an extensive elicitor-induced genetic reprogramming. Subsequent in silico analysis allowed us to identify 15 genes with jasmonate-induced differential expression putatively involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene and its predicted 3D structure showed a strong homology with other genes previously reported to encode acyl-CoA ligases, suggesting it was involved in the formation of the taxol lateral chain. In vitro analysis confirmed that the TB768 gene encodes an acyl-CoA ligase, localized in the cytoplasm and able to convert b-phenylalanine into its CoA ester derivative. The latter is then attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. In determining that the TB768 gene encodes a cytosolic CoA ligase with an important role in the taxol biosynthetic pathway, we have corroborated that the combination of a cDNA-AFLP transcriptome profiling approach with bioinformatics analysis constitutes an efficient set of tools for the identification of new genes involved in the metabolism of valuable bioactive compounds. The newly described gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.