Identification of rate-limiting enzymes involved in paclitaxel biosynthesis pathway affected by coronatine and methyl-β-cyclodextrin in Taxus baccata L. cell suspension cultures

Abstract

Background: Paclitaxel is a potent antitumor alkaloid widely used for the treatment of several cancer types. This valuable secondary metabolite naturally exists in the inner bark of Taxus species in very low amounts. The small-scale production of paclitaxel in Taxus cell cultures requires utilization of several elicitors.

Objective: The main objective of this work was to identify key genes that encode rate-limiting enzymes in paclitaxel biosynthesis pathway by investigating the possible relationship between paclitaxel production and a set of 13 involved genes' relative expression in Taxus baccata L. cell suspension cultures affected by coronatine and methyl-β-cyclodextrin.

Methods: In the present research, the most important key genes were identified using gene expression profiling evaluation and paclitaxel production assessment in Taxus baccata L. cell cultures affected by mentioned elicitors.

Results and conclusion: Gene expression levels were variably increased using methyl-β-cyclodextrin, and in some cases, a synergistic effect on transcript accumulation was observed when culture medium was supplemented with both elicitors. It was revealed that DBAT, BAPT, and DBTNBT are the most important rate-limiting enzymes in paclitaxel biosynthesis pathway in Taxus baccata L. cell suspension cultures under coronatine and methyl-β-cyclodextrin elicitation condition. Moreover, PAM was identified as one of the important key genes especially in the absence of β-phenylalanine. In cell cultures affected by these elicitors, paclitaxel was found largely in the culture media (more than 90%). The secretion of this secondary metabolite suggests a limited feedback inhibition and reduced paclitaxel toxicity for producer cells. It is the result of the ABC gene relative expression level increment under methyl-β-cyclodextrin elicitation and highly depends on methyl-β-cyclodextrin's special property (complex formation with hydrophobic compounds). Paclitaxel biosynthesis was obviously increased due to the effect of coronatine and methyl-β-cyclodextrin elicitation, leading to the production level of 5.62 times higher than that of the untreated cultures. Graphical abstract Rate Limiting Enzymes in Paclitaxel Biosynthesis Pathway: DBAT, BAPT, DBTNBT and PAM.

Keywords: Cell suspension culture; Coronatine; Methyl-β-cyclodextrin; Paclitaxel; Taxus baccata L.; Transcription profiling.

Graphical abstract

Rate Limiting Enzymes in Paclitaxel Biosynthesis Pathway: DBAT, BAPT, DBTNBT and PAM.

Fig. 1

A summarized paclitaxel biosynthesis pathway. IPPI; isopentenyl diphosphate isomerase, GGPP; geranylgeranyl diphosphate synthase, TXS; taxadiene synthase, T1βH; taxane 1β-hydroxylase, T2αH; taxane 2α-hydroxylase, T5αH; taxane 5α-hydroxylase, T7βH; taxane 7β-hydroxylase, T9αH; taxane 9α-hydroxylase, T10βH; taxane 10β-hydroxylase, T13αH; taxane 13α-hydroxylase, T14βOH; taxane 14β-hydroxylase, TDAT; taxa-4(20),11(12)-diene-5α-ol-O-acetyltransferase, TBT; taxane-2α-O-benzoyl transferase, DBAT; 10-deacetyl baccatin III-10-O-acetyltransferase, BAPT; baccatin III-3-amino 13-phenylpropanoyl- CoA transferase, T2´H; taxane 2´-hydroxylase, DBTNBT; de-benzoyltaxol N-benzoyl transferase, PAM; phenylalanine amino mutase and CoA Ligase. Adopted from [25] and [30]. Colored names point out to the genes that have been investigated in this research

Fig. 2

Biomass production evaluated by measuring T. baccata cells’ dry mass (g/L) in optimized two-step medium over the period of 22 days. Each value is the average of three biological × three technical replicates ± SE

Fig. 3

Cell-associated and extracellular paclitaxel amounts in CD + Cor treated and untreated samples. Each value is the average of two replicates ± SE. Y-axis: paclitaxel amounts (μg/g); X-axis: days after cell culture initiation in untreated and treated cultures. Uppercase and lowercase letters show the results of mean comparative test accomplished on cell-associated and extracellular paclitaxel contents, respectively. Dissimilar letters showsignificant difference between compared treatments. # shows non-detectable paclitaxel amounts

Fig. 4

Relative expression level of genes with highly increased expression (T13αH and TBT) in Taxus baccata L. cell suspension cultures during 5 days in production medium (from day 4 to 8) supplemented with CD (50 mM) individually or in combination with Cor (1 μM). Y-axis: gene expression relative to that of the expression level in cell suspension cultures maintained for 13 days in the growth medium, prior to 4 days in the production medium; X-axis: equivalent time or time after Cor elicitation in cell suspension cultures affected by CD and CD + Cor, respectively. Each value is the average of two biological × two technical replicates ± SE. **; significant difference between compared treatments at p-value < 0.01, CD; Methyl-β-Cyclodextrin and CD+Cor; Methyl-β-Cyclodextrin and Coronatine

Fig. 5

Relative expression level of genes with moderately increased expression (GGPP, TXS, T5αH, T10βH, T14βH, T7βH and DBAT) in Taxus baccata L. cell suspension cultures during 5 days in production medium (from day 4 to 8) supplemented with CD (50 mM) individually or in combination with Cor (1 μM). Y-axis: gene expression relative to that of the expression level in cell suspension cultures maintained for 13 days in the growth medium, prior to 4 days in the production medium; X-axis: equivalent time or time after Cor elicitation in cell suspension cultures affected by CD and CD + Cor, respectively. Each value is the average of two biological × two technical replicates ± SE. **; significant difference between compared treatments at p-value < 0.01, CD; Methyl-β-Cyclodextrin and CD+Cor; Methyl-β-Cyclodextrin and Coronatine

Fig. 6

Relative expression level of genes with slightly increased expression (PAM, BAPT, DBTNBT, and ABC) in Taxus baccata L. cell suspension cultures during 5 days in production medium (from day 4 to 8) supplemented with CD (50 mM) individually or in combination with Cor (1 μM). Y-axis: gene expression relative to that of the expression level in cell suspension cultures maintained for 13 days in the growth medium, prior to 4 days in the production medium; X-axis: equivalent time or time after Cor elicitation in cell suspension cultures affected by CD and CD + Cor, respectively. Each value is the average of two biological × two technical replicates ± SE. * and **;significant difference between compared treatments at p-value < 0.05 and p-value < 0.01, respectively, CD; Methyl-β-Cyclodextrin and CD+Cor; Methyl-β-Cyclodextrin and Coronatine.