Transcript profiling of jasmonate-elicited Taxus cells reveals a β-phenylalanine-CoA ligase.

Karla Ramírez‐Estrada,Cedric Notredame,Miriam Onrubia,Teresa Altabella,Alain Goossens,Lidia Osuna,Robin Vanden Bossche,Javier Palazon,Rosa M Cusido,Elisabeth Moyano

doi:10.1111/pbi.12359

Abstract

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.

Highlights

Taxol is the registered trade name of Bristol-Myers Squibb for the generic drug paclitaxel, which is currently considered as one of the most effective anticancer drugs ever developed
We report on the characterization, sequencing and cloning of a gene selected through cDNA-amplified fragment length polymorphism (AFLP) and in silico analysis, which encodes b-phenylalanineCoA ligase, an enzyme involved in the formation of the taxol side chain, one of the unresolved steps of the taxol biosynthetic pathway
Transcript profiling reveals potential candidates for five unknown steps of taxane biosynthesis In this work, cDNA-AFLP was applied for genomewide transcript profiling of T. baccata cells cultured in a two-stage system (Palazon et al, 2003)

Summary

Introduction

Taxol is the registered trade name of Bristol-Myers Squibb for the generic drug paclitaxel, which is currently considered as one of the most effective anticancer drugs ever developed. The production of taxol in Taxus spp. cell cultures has become a major challenge for plant biotechnology due to the growing worldwide demand and its scarcity in nature (Onrubia et al, 2013a). The natural source of taxol is the inner bark of several Taxus spp., where it accumulates at a very low concentration. For these reasons, several research laboratories and biotechnological companies are involved in designing new strategies based on elicited Taxus cell cultures (ETCCs) to optimize the biotechnological production of this anticancer compound. In the highly complex taxol biosynthetic pathway, the first product bearing the taxane ring is taxa-4(5),11(12)-diene, catalysed from (E,E,E)-geranylgeranyl diphosphate (GGPP) by the plastidic enzyme taxadiene synthase (TXS) (Hezari et al, 1997; Koepp et al, 1995). A succession of steps, involving hydroxylases, acyl transferases and other enzymes, leads to the production of

Results

Discussion

Conclusion