Analysis of aggregate size as a process variable affecting paclitaxel accumulation in Taxus suspension cultures

Abstract

Plant cell aggregates have long been implicated in affecting cellular metabolism in suspension culture, yet the rigorous characterization of aggregate size as a process variable and its effect on bioprocess performance has not been demonstrated. Aggregate fractionation and analysis of biomass-associated product is commonly used to assess the effect of aggregation, but we establish that this method is flawed under certain conditions and does not necessarily agree with comprehensive studies of total culture performance. Leveraging recent advances to routinely measure aggregate size distributions, we developed a simple method to manipulate aggregate size and evaluate its effect on the culture as a whole, and found that Taxus suspension cultures with smaller aggregates produced significantly more paclitaxel than cultures with larger aggregates in two cell lines over a range of aggregate sizes, and where biomass accumulation was equivalent prior to elicitation with methyl jasmonate. T. cuspidata P93AF cultures with mean aggregate sizes of 690 μm and 1100 μm produced 22 mg/L and 11 mg/L paclitaxel, respectively, a 2-fold increase for smaller aggregates, and T. cuspidata P991 cultures with mean aggregate sizes of 400 μm and 840 μm produced 6 mg/L and 0.3 mg/L paclitaxel, respectively, an increase of 20-fold for smaller aggregates. These results demonstrate the importance of validating experiments aimed at a specific phenomenon with total process studies, and provide a basis for treating aggregate size as a targeted process variable for rational control strategies.

Contribution of taxane biosynthetic pathway gene expression to observed variability in paclitaxel accumulation in Taxus suspension cultures

“…All chemicals were obtained from Sigma-Aldrich Co. (St. Louis, MO), unless otherwise noted. Cell cultures were maintained, as described previously [33]. For elicitation, 200 μM methyl jasmonate (MeJA) was added on day 7 post-transfer during the mid-exponential phase of growth, as described previously [34].…”

“…This culture was aseptically separated into two cultures (one “small aggregate culture” and one “large aggregate culture”) using a filter corresponding to its mean aggregate size, based on correlations developed in previous work [30]. Three biological replicates each of small aggregate culture and large aggregate culture were initiated and maintained as 200 mL cultures in 500 mL shake flasks, as described previously [33]. Measurements for biomass were taken on the day of inoculation (day zero) and on the day of elicitation with MeJA (day seven).…”

“…Cells within these aggregates are subject to different microenvironments, leading to cell-cell differences in morphology [31] and metabolism [32]. Recently, we have developed an approach to assess the effect of aggregate size as a process variable on paclitaxel accumulation, and have demonstrated that cultures with smaller aggregates accumulate significantly more paclitaxel upon elicitation with MeJA than cultures with larger aggregates [33]. Using this approach, it is possible to predictably and rapidly obtain cultures with different biosynthetic capabilities upon MeJA elicitation.…”

“…Using qRT-PCR, we first compared expression profiles in cultures elicited with MeJA that produce detectable levels of paclitaxel and unelicited cultures that do not produce detectable levels of paclitaxel, to elucidate the on/off state of paclitaxel accumulation. Next, we established cultures with small and large aggregate distributions [33] that exhibit variable paclitaxel accumulation patterns upon elicitation with MeJA, to enable comparison of gene expression between cultures in which taxane accumulation is positive but significantly different. Using this method we were able to examine cultures that had 15-fold and 2-fold differences in paclitaxel accumulation after MeJA elicitation, respectively.…”

Aggregate cell suspension cultures of Psoralea corylifolia improved phytoestrogens production

“…Influence of cell aggregation on secondary metabolites was demonstrated in suspension culture of Catharanthus roseus and Taxus cuspidate [16,27]. Kolewe et al [29] reported that size of cell aggregates represented as active parameter to influence the paclitaxel levels in Taxus cuspidata cell suspension cultures. Comparing the product capability by cell aggregates of different sizes, present results concluded that the daidzein produced by cell aggregate of 1.2 mm in suspension cultures was ~11-fold much higher than that of roots of two-month-old naturally grown plants.…”