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

Abstract

Summary 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.

Changes in gene transcription and taxane production in elicited cell cultures of Taxus×media and Taxus globosa

“…In a recent transcriptomic study of T. chinensis, Li et al (2012) found that 16 h after MeJA elicitation the most expressed genes were TXS and T7bOH, whereas levels of DBAT, DBTNBT and BAPT were low. The CoA ligase gene could also be considered as another limiting gene of taxol biosynthesis, since its response to elicitation in T. media cell cultures was not very remarkable, similar to our previous observations in MeJA-treated T. baccata cell cultures (Ramirez-Estrada et al, 2015).…”

“…When cDNA-AFLP analysis was combined with in silico studies, several candidate genes were identified for the different uncharacterized enzymes, including a CoA transferase. In recent functional studies, we have confirmed that this enzyme is a new b-phenylalanine CoA ligase involved in the formation of the taxol side chain (Ramirez-Estrada et al, 2015).…”

“…As previously indicated, the CoA ligase gene encodes the enzyme that activates b-phenylalanine to b-phenylalanine CoA, before its attachment to B (Ramirez-Estrada et al, 2015). Its expression peaked at 4 h of CD + CORO elicitation, when transcript levels were 3.2-, 8.7-and 6-fold higher than in the cell cultures treated with CORO or CD separately, or without elicitation, respectively.…”

“…6 and 7). Most of these genes have been previously studied in different Taxus systems under the action of diverse elicitors, but the expression level of the CoA ligase gene has never been determined under the treatments described here, since its involvement in the activation of b-phenylanine to b-phenylanine CoA to form the C13 side chain of taxol has only been reported very recently (Ramirez-Estrada et al, 2015).…”

Improving 10-deacetylbaccatin III-10-β-O-acetyltransferase catalytic fitness for Taxol production

“…Other functional groups include two hydroxyl groups (at C1 and C7) and two acetyl groups (at C4 and C10). It has been postulated that 20 enzymes are involved in 19 steps of the biosynthetic pathway from the universal diterpenoid precursor geranylgeranyl diphosphate (GGPP) to Taxol, of which six corresponding genes remains to be identified, i.e., C1β-hydroxylase, C4,C20-epoxydase, C4b,C20-oxomutase, C9α-hydroxylase, C9-oxidase and C2′ side-chain hydroxylase454647. The first committed step is the cyclization of GGPP to taxa-4(5), 11(12)-diene.…”