Targeted control of supporting pathways in paclitaxel biosynthesis with CRISPR-guided methylation.

Introduction: Plant cell culture biomanufacturing is rapidly becoming an effective strategy for production of high-value plant natural products, such as therapeutic proteins and small molecules, vaccine adjuvants, and nutraceuticals. Many of these plant natural products are synthesized from diverse molecular building blocks sourced from different metabolic pathways. Even so, engineering approaches for increasing plant natural product biosynthesis have typically focused on the core biosynthetic pathway rather than the supporting pathways. Methods: Here, we use both CRISPR-guided DNA methylation and chemical inhibitors to control flux through the phenylpropanoid pathway in Taxus chinensis, which contributes a phenylalanine derivative to the biosynthesis of paclitaxel (Taxol), a potent anticancer drug. To inhibit PAL, the first committed step in phenylpropanoid biosynthesis, we knocked down expression of PAL in Taxus chinensis plant cell cultures using a CRISPR-guided plant DNA methyltransferase (NtDRM). For chemical inhibition of downstream steps in the pathway, we treated Taxus chinensis plant cell cultures with piperonylic acid and caffeic acid, which inhibit the second and third committed steps in phenylpropanoid biosynthesis: cinnamate 4-hydroxylase (C4H) and 4-coumaroyl-CoA ligase (4CL), respectively. Results: Knockdown of PAL through CRISPR-guided DNA methylation resulted in a profound 25-fold increase in paclitaxel accumulation. Further, through the synergistic action of both chemical inhibitors and precursor feeding of exogenous phenylalanine, we achieve a 3.5-fold increase in paclitaxel biosynthesis and a similar reduction in production of total flavonoids and phenolics. We also observed perturbations to both activity and expression of PAL, illustrating the complex transcriptional co-regulation of these first three pathway steps. Discussion: These results highlight the importance of controlling the metabolic flux of supporting pathways in natural product biosynthesis and pioneers CRISPR-guided methylation as an effective method for metabolic engineering in plant cell cultures. Ultimately, this work demonstrates a powerful method for rewiring plant cell culture systems into next-generation chassis for production of societally valuable compounds.

The Interface amongst Conserved and Specialized Pathways in Non-Paclitaxel and Paclitaxel Accumulating Taxus Cultures.

Plant cell cultures derived from Taxus are used to produce valuable metabolites like paclitaxel, a chemotherapeutic drug. Methyl jasmonate elicitation enhances paclitaxel accumulation, but also inhibits culture growth and increases phenylpropanoid biosynthesis, two side effects that detract from taxane accumulation. To understand the connection between all of these processes, a systems approach is applied to investigate cell-wide metabolism in Taxus. Non-paclitaxel and paclitaxel accumulating cultures were elicited over single and multi-generational periods, and subsequent changes in conserved and specialized metabolism were quantified. Methyl jasmonate typically resulted in decreased growth and increased metabolite content in paclitaxel accumulating cultures. Conversely, elicitation typically resulted in either no change or decrease in accumulation of metabolites in the non-paclitaxel accumulating cultures. In both sets of cultures, variability was seen in the response to methyl jasmonate across generations of cell growth. Consolidation of these data determined that paclitaxel accumulation and basal levels of phenolic and flavonoid compounds are indirectly correlated with aggregate size. These approaches assess alternative metabolic pathways that are linked to paclitaxel biosynthesis and provide a comprehensive strategy to both understand the relationship between conserved and specialized metabolism in plants and in the design of strategies to increase natural product yields in plant cell culture.

A population balance model to modulate shear for the control of aggregation in Taxus suspension cultures.

Cellular aggregation in plant suspension cultures directly affects the accumulation of high value products, such as paclitaxel from Taxus. Through application of mechanical shear by repeated, manual pipetting through a 10 ml pipet with a 1.6 mm aperture, the mean aggregate size of a Taxus culture can be reduced without affecting culture growth. When a constant level of mechanical shear was applied over eight generations, the sheared population was maintained at a mean aggregate diameter 194 µm lower than the unsheared control, but the mean aggregate size fluctuated by over 600 µm, indicating unpredictable culture variability. A population balance model was developed to interpret and predict disaggregation dynamics under mechanical shear. Adjustable parameters involved in the breakage frequency function of the population balance model were estimated by nonlinear optimization from experimentally measured size distributions. The optimized model predictions were in strong agreement with measured size distributions. The model was then used to determine the shear requirements to successfully reach a target aggregate size distribution. This model will be utilized in the future to maintain a culture with a constant size distribution with the goal of decreasing culture variability and increasing paclitaxel yields.

Jasmonate-responsive expression of paclitaxel biosynthesis genes in Taxus cuspidata cultured cells is negatively regulated by the bHLH transcription factors TcJAMYC1, TcJAMYC2, and TcJAMYC4.

Taxus cell suspension culture is a sustainable technology for the industrial production of paclitaxel (Taxol®), a highly modified diterpene anti-cancer agent. The methyl jasmonate (MJ)-mediated paclitaxel biosynthetic pathway is not fully characterized, making metabolic engineering efforts difficult. Here, promoters of seven genes (TASY, T5αH, DBAT, DBBT, PAM, BAPT, and DBTNBT), encoding enzymes of the paclitaxel biosynthetic pathway were isolated and used to drive MJ-inducible expression of a GUS reporter construct in transiently transformed Taxus cells, showing that elicitation of paclitaxel production by MJ is regulated at least in part at the level of transcription. The paclitaxel biosynthetic pathway promoters contained a large number of E-box sites (CANNTG), similar to the binding sites for the key MJ-inducible transcription factor AtMYC2 from Arabidopsis thaliana. Three MJ-inducible MYC transcription factors similar to AtMYC2 (TcJAMYC1, TcJAMYC2, and TcJAMYC4) were identified in Taxus. Transcriptional regulation of paclitaxel biosynthetic pathway promoters by transient over expression of TcJAMYC transcription factors indicated a negative rather than positive regulatory role of TcJAMYCs on paclitaxel biosynthetic gene expression.

Multi-scale engineering of plant cell cultures for promotion of specialized metabolism.

To establish plant culture systems for product synthesis, a multi-scale engineering approach is necessary. At the intracellular level, the influx of 'omics' data has necessitated development of new methods to properly annotate and establish useful metabolic models that can be applied to elucidate unknown steps in specialized metabolite biosynthesis, define effective metabolic engineering strategies and increase enzyme diversity available for synthetic biology platforms. On an intercellular level, the presence of aggregates in culture leads to distinct metabolic sub-populations. Recent advances in flow cytometric analyses and mass spectrometry imaging allow for resolution of metabolites on the single cell level, providing an increased understanding of culture heterogeneity. Finally, extracellular engineering can be used to enhance culture performance through media manipulation, co-culture with bacteria, the use of exogenous elicitors or modulation of shear stress.

Methyl jasmonate represses growth and affects cell cycle progression in cultured Taxus cells.

KEY MESSAGE: Methyl jasmonate elicitation of Taxus cultures enhances paclitaxel accumulation, but represses growth by inhibition of cell cycle progression. Growth repression is evident both at the culture level and transcriptional level. Methyl jasmonate (MeJA) elicitation is an effective strategy to induce and enhance synthesis of the anticancer agent paclitaxel (Taxol(®)) in Taxus cell suspension cultures; however, concurrent decreases in growth are often observed, which is problematic for large-scale bioprocessing. Here, increased accumulation of paclitaxel in Taxus cuspidata suspension cultures with MeJA elicitation was accompanied by a concomitant decrease in cell growth, evident within the first 3 days post-elicitation. Both MeJA-elicited and mock-elicited cultures exhibited similar viability with no apoptosis up to day 16 and day 24 of the cell culture period, respectively, suggesting that growth repression is not attributable to cell death. Flow cytometric analyses demonstrated that MeJA perturbed cell cycle progression of asynchronously dividing Taxus cells. MeJA slowed down cell cycle progression, impaired the G1/S transition as observed by an increase in G0/G1 phase cells, and decreased the number of actively dividing cells. Through a combination of deep sequencing and gene expression analyses, the expression status of Taxus cell cycle-associated genes correlated with observations at the culture level. Results from this study provide valuable insight into the mechanisms governing MeJA perception and subsequent events leading to repression of Taxus cell growth.

Metabolic engineering approaches for production of biochemicals in food and medicinal plants.

Historically, plants are a vital source of nutrients and pharmaceuticals. Recent advances in metabolic engineering have made it possible to not only increase the concentration of desired compounds, but also introduce novel biosynthetic pathways to a variety of species, allowing for enhanced nutritional or commercial value. To improve metabolic engineering capabilities, new transformation techniques have been developed to allow for gene specific silencing strategies or stacking of multiple genes within the same region of the chromosome. The 'omics' era has provided a new resource for elucidation of uncharacterized biosynthetic pathways, enabling novel metabolic engineering approaches. These resources are now allowing for advanced metabolic engineering of plant production systems, as well as the synthesis of increasingly complex products in engineered microbial hosts. The status of current metabolic engineering efforts is highlighted for the in vitro production of paclitaxel and the in vivo production of ß-carotene in Golden Rice and other food crops.

Terminal sterilization of alginate hydrogels: efficacy and impact on mechanical properties.

Terminal, or postprocessing, sterilization of composite biomaterials is crucial for their use in wound healing and tissue-engineered devices. Recent research has focused on optimizing traditional biomaterial formulations to create better products for commercial and academic use which incorporate hydrophobic compounds or secondary gel networks. To use a hydrogel in a clinical setting, terminal sterilization is necessary to ensure patient safety. Lyophilization, gamma-irradiation, and ethylene oxide treatment all have negative consequences when applied to alginate scaffolds for clinical use. Here, we aim to find alternative terminal sterilization methods for alginate and alginate-based composite hydrogels which maintain the structure of composite alginate networks for use in biomedical applications. A thorough investigation of the effect of common sterilization methods on swollen alginate-based hydrogels has not been reported and therefore, this work examines autoclaving, ethanol washing, and ultraviolet light as sterilization techniques for alginate and alginate/Pluronic® F68 composite hydrogels. Preservation of structural integrity is evaluated using shear rheology and analysis of water retention, and efficacy of sterilization is determined via bacterial persistence within the hydrogel. Results indicate that ethanol sterilization is the best method of those investigated because ethanol washing results in minimal effects on mechanical properties and water retention and eliminates bacterial persistence. Furthermore, this study suggests that ethanol treatment is an efficacious method for terminally sterilizing interpenetrating networks or other composite hydrogel systems.

Cellular aggregation is a key parameter associated with long term variability in paclitaxel accumulation in Taxus suspension cultures.

Plant cell cultures provide a renewable source for synthesis and supply of commercially valuable plant-derived products, particularly for secondary metabolites. However, instability in product yields over multiple passages has hampered the efficient and sustainable use of this technology. Paclitaxel accumulation in Taxus cell suspension culture was quantified over multiple passages and correlated to mean aggregate size, extracellular sugar level, ploidy, and cell cycle distribution. Paclitaxel levels varied approximately 6.9-fold over the six-month timeframe investigated. Of all of the parameters examined, only mean aggregate size correlated with paclitaxel accumulation, where a significant negative correlation (r = - 0.75, p < 0.01) was observed. These results demonstrate the relevance of measuring, and potentially controlling, aggregate size during long term culture passages, particularly for plant suspensions where industrially relevant secondary metabolites are not pigmented to enable rapid culture selection.

Addition of perfluorocarbons to alginate hydrogels significantly impacts molecular transport and fracture stress.

Perfluorocarbons (PFCs) are used in biomaterial formulations to increase oxygen (O(2) ) tension and create a homogeneous O(2) environment in three-dimensional tissue constructs. It is unclear how PFCs affect mechanical and transport properties of the scaffold, which are critical for robustness, intracellular signaling, protein transport, and overall device efficacy. In this study, we investigate composite alginate hydrogels containing a perfluorooctyl bromide (PFOB) emulsion stabilized with Pluronic(®) F68 (F68). We demonstrate that PFC addition significantly affects biomaterial properties and performance. Solution and hydrogel mechanical properties and transport of representative hydrophilic (riboflavin), hydrophobic (methyl and ethyl paraben), and protein (bovine serum albumin, BSA) solutes were compared in alginate/F68 composite hydrogels with or without PFOB. Our results indicate that mechanical properties of the alginate/F68/PFOB hydrogels are not significantly affected under small strains, but a significant decrease fracture stress is observed. The effective diffusivity D(eff) of hydrophobic small molecules decreases with PFOB emulsion addition, yet the D(eff) of hydrophilic small molecules remained unaffected. For BSA, the D(eff) increased and the loading capacity decreased with PFOB emulsion addition. Thus, a trade-off between the desired increased O(2) supply provided by PFCs and the mechanical weakening and change in transport of cellular signals must be carefully considered in the design of biomaterials containing PFCs.

Paclitaxel uptake and transport in Taxus cell suspension cultures.

The transport of paclitaxel in Taxus canadensis suspension cultures was studied with a fluorescence analogue of paclitaxel (Flutax-2(®)) in combination with flow cytometry detection. Experiments were carried out using both isolated protoplasts and aggregated suspension cell cultures. Flutax-2(®) was shown to be greater than 90% stable in Taxus suspension cultures over the required incubation time (24 hours). Unlabeled paclitaxel was shown to inhibit the cellular uptake of Flutax-2(®), although structurally similar taxanes such as cephalomannine, baccatin III, and 10-deacetylbaccatin III did not inhibit Flutax-2(®) uptake. Saturation kinetics of Flutax-2(®) uptake was demonstrated. These results indicate the presence of a specific transport system for paclitaxel. Suspension cells elicited with methyl jasmonate accumulated 60% more Flutax-2(®) than unelicited cells, possibly due to an increased cellular storage capacity following methyl jasmonate elicitation. The presence of a specific mechanism for paclitaxel transport is an important first result that will provide the basis of more detailed studies as well as the development of targeted strategies for increased paclitaxel secretion to the extracellular medium.

Liquid-liquid extraction for recovery of paclitaxel from plant cell culture: solvent evaluation and use of extractants for partitioning and selectivity.

A major challenge in the production of metabolites by plant cells is the separation and purification of a desired product from a number of impurities. An important application of plant cell culture is the biosynthesis of the anticancer agent paclitaxel. Liquid-liquid extraction plays a critical role in the recovery of paclitaxel and other valuable plant-derived products from culture broth. In this study, the extraction of paclitaxel and a major unwanted by-product, cephalomannine, from plant cell culture broth into organic solvents is quantified. Potential solvent mixtures show varying affinity and selectivity for paclitaxel over cephalomannine. The partition coefficient of paclitaxel is highest in ethyl acetate and dichloromethane, with measured values of 28 and 25, respectively; however, selectivity coefficients are less than 1 for paclitaxel over cephalomannine for both solvents. Selectivity coefficient increases to 1.7 with extraction in n-hexane, but the partition coefficient decreases to 1.9. Altering the pH of the aqueous phase results in an increase in both recovery and selectivity using n-hexane but does not change the results for other solvents significantly. The addition of extractants trioctylamine (TOA) or tributylphosphate (TBP) to n-hexane gives significantly higher partition coefficients for paclitaxel (8.6 and 23.7, respectively) but no selectivity. Interestingly, when 20% hexafluorobenzene (HFB) is added to n-hexane, the partition coefficient remains approximately constant, but the selectivity coefficient for paclitaxel over cephalomannine improves to 4.5. This significant increase in selectivity early in the purification process has the potential to simplify downstream processing steps and significantly reduce overall purification costs.

Identification and expression analysis of methyl jasmonate responsive ESTs in paclitaxel producing Taxus cuspidata suspension culture cells.

BACKGROUND: Taxol(®) (paclitaxel) promotes microtubule assembly and stabilization and therefore is a potent chemotherapeutic agent against wide range of cancers. Methyl jasmonate (MJ) elicited Taxus cell cultures provide a sustainable option to meet the growing market demand for paclitaxel. Despite its increasing pharmaceutical importance, the molecular genetics of paclitaxel biosynthesis is not fully elucidated. This study focuses on identification of MJ responsive transcripts in cultured Taxus cells using PCR-based suppression subtractive hybridization (SSH) to identify genes involved in global pathway control. RESULTS: Six separate SSH cDNA libraries of paclitaxel-accumulating Taxus cuspidata P991 cell lines were constructed at three different post-elicitation time points (6h, 18h and 5 day) to identify genes that are either induced or suppressed in response to MJ. Sequencing of 576 differentially screened clones from the SSH libraries resulted in 331 unigenes. Functional annotation and Gene Ontology (GO) analysis of up-regulated EST libraries showed enrichment of several known paclitaxel biosynthetic genes and novel transcripts that may be involved in MJ-signaling, taxane transport, or taxane degradation. Macroarray analysis of these identified genes unravelled global regulatory expression of these transcripts. Semi-quantitative RT-PCR analysis of a set of 12 candidate genes further confirmed the MJ-induced gene expression in a high paclitaxel accumulating Taxus cuspidata P93AF cell line. CONCLUSIONS: This study elucidates the global temporal expression kinetics of MJ responsive genes in Taxus suspension cell culture. Functional characterization of the novel genes identified in this study will further enhance the understanding of paclitaxel biosynthesis, taxane transport and degradation.

A population balance equation model of aggregation dynamics in Taxus suspension cell cultures.

The nature of plant cells to grow as multicellular aggregates in suspension culture has profound effects on bioprocess performance. Recent advances in the measurement of plant cell aggregate size allow for routine process monitoring of this property. We have exploited this capability to develop a conceptual model to describe changes in the aggregate size distribution that are observed over the course of a Taxus cell suspension batch culture. We utilized the population balance equation framework to describe plant cell aggregates as a particulate system, accounting for the relevant phenomenological processes underlying aggregation, such as growth and breakage. We compared model predictions to experimental data to select appropriate kernel functions, and found that larger aggregates had a higher breakage rate, biomass was partitioned asymmetrically following a breakage event, and aggregates grew exponentially. Our model was then validated against several datasets with different initial aggregate size distributions and was able to quantitatively predict changes in total biomass and mean aggregate size, as well as actual size distributions. We proposed a breakage mechanism where a fraction of biomass was lost upon each breakage event, and demonstrated that even though smaller aggregates have been shown to produce more paclitaxel, an optimum breakage rate was predicted for maximum paclitaxel accumulation. We believe this is the first model to use a segregated, corpuscular approach to describe changes in the size distribution of plant cell aggregates, and represents an important first step in the design of rational strategies to control aggregation and optimize process performance.

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

Variability in product accumulation is one of the major obstacles limiting the widespread commercialization of plant cell culture technology to supply natural product pharmaceuticals. Despite extensive process engineering efforts, which have led to increased yields, plant cells exhibit variability in productivity that is poorly understood. Elicitation of Taxus cultures with methyl jasmonate (MeJA) induces paclitaxel accumulation, but to varying extents in different cultures. In the current study, cultures with different aggregation profiles were established to create predictable differences in paclitaxel accumulation upon MeJA elicitation. Expression of known paclitaxel biosynthetic genes in MeJA-elicited cultures exhibiting both substantial (15-fold) and moderate (2-fold) differences in paclitaxel accumulation was analyzed using quantitative reverse transcriptase PCR. Each population exhibited the characteristic large increase in paclitaxel pathway gene expression following MeJA elicitation; however, differences in expression between populations were minor, and only observed for the cultures with the 15-fold variation in paclitaxel content. These data suggest that although upregulation of biosynthetic pathway gene expression contributes to observed increases in paclitaxel synthesis upon elicitation with MeJA, there are additional factors that need to be uncovered before paclitaxel productivity can be fully optimized.

Recent advances towards development and commercialization of plant cell culture processes for the synthesis of biomolecules.

Plant cell culture systems were initially explored for use in commercial synthesis of several high-value secondary metabolites, allowing for sustainable production that was not limited by the low yields associated with natural harvest or the high cost associated with complex chemical synthesis. Although there have been some commercial successes, most notably paclitaxel production from Taxus sp., process limitations exist with regards to low product yields and inherent production variability. A variety of strategies are being developed to overcome these limitations including elicitation, in situ product removal and metabolic engineering with single genes and transcription factors. Recently, the plant cell culture production platform has been extended to pharmaceutically active heterologous proteins. Plant systems are beneficial because they are able to produce complex proteins that are properly glycosylated, folded and assembled without the risk of contamination by toxins that are associated with mammalian or microbial production systems. Additionally, plant cell culture isolates transgenic material from the environment, allows for more controllable conditions over field-grown crops and promotes secretion of proteins to the medium, reducing downstream purification costs. Despite these benefits, the increase in cost of heterologous protein synthesis in plant cell culture as opposed to field-grown crops is significant and therefore processes must be optimized with regard to maximizing secretion and enhancing protein stability in the cell culture media. This review discusses recent advancements in plant cell culture processing technology, focusing on progress towards overcoming the problems associated with commercialization of these production systems and highlighting recent commercial successes.

Transport of biological molecules in surfactant-alginate composite hydrogels.

Obstructed transport of biological molecules can result in improper release of pharmaceuticals or biologics from biomedical devices. Recent studies have shown that nonionic surfactants, such as Pluronic® F68 (F68), positively alter biomaterial properties such as mesh size and microcapsule diameter. To further understand the effect of F68 (incorporated at concentrations well above the critical micelle concentration (CMC)) in traditional biomaterials, the transport properties of BSA and riboflavin were investigated in F68-alginate composite hydrogels, formed by both internal and external cross-linking with divalent cations. Results indicate that small molecule transport (represented by riboflavin) was not significantly hindered by F68 in homogeneously (internally) cross-linked hydrogels (up to an 11% decrease in loading capacity and 14% increase in effective diffusion coefficient, D(eff)), while protein transport in homogeneously cross-linked hydrogels (represented by BSA) was significantly affected (up to a 43% decrease in loading capacity and 40% increase in D(eff)). For inhomogeneously cross-linked hydrogels (externally cross-linked by CaCl(2) or BaCl(2)), the D(eff) increased up to 50 and 83% for small molecules and proteins, respectively. Variation in the alginate gelation method was shown to affect transport through measurable changes in swelling ratio (30% decrease) and observable changes in cross-linking structure as well as up to a 3.6- and 11.8-fold difference in D(eff) for riboflavin and BSA, respectively. Aside from the expected significant changes due to the cross-linking method utilized, protein transport properties were altered due to mesh size restrictions (10-25 nm estimated by mechanical properties) and BSA-F68 interaction (DLS). Taken as a whole, these results show that incorporation of a nonionic surfactant at concentrations above the CMC can affect device functionality by impeding the transport of large biological molecules.

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

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 before elicitation with methyl jasmonate. Taxus cuspidata (T. cuspidata) P93AF cultures with mean aggregate sizes of 690 and 1,100 µm produced 22 and 11 mg/L paclitaxel, respectively, a twofold increase for smaller aggregates, and T. cuspidata P991 cultures with mean aggregate sizes of 400 and 840 µm produced 6 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.

Statistical optimization of single-cell production from Taxus cuspidata plant cell aggregates.

Flow-cytometric characterization of plant cell culture growth and metabolism at the single-cell level is a method superior to traditional culture average measurements for collecting population information. Investigation of culture heterogeneity and production variability by obtaining information about different culture subpopulations is crucial for optimizing bio-processes for enhanced productivity. Obtaining high yields of intact and viable single cells from aggregated plant cell cultures is an enabling criterion for their analysis and isolation using high-throughput flow cytometric methods. The critical parameters affecting the enzymatic isolation of single cells from aggregated Taxus cuspidata plant cell suspensions were optimized using response-surface methodology and factorial central composite design. Using a design of experiments approach, the output response single-cell yield (SCY, percentage of cell clusters containing only a single cell) was optimized. Optimal conditions were defined for the independent parameters cellulase concentration, pectolyase Y-23 concentration, and centrifugation speed to be 0.045% (w/v), 0.7% (w/v), and 1200 × g, respectively. At these optimal conditions, the model predicted a maximum SCY of 48%. The experimental data exhibited a 72% increase over previously attained values and additionally validated the model predictions. More than 99% of the isolated cells were viable and suitable for rapid analysis through flow cytometry, thus enabling the collection of population information from cells that accurately represent aggregated suspensions. These isolated cells can be further studied to gain insight into both growth and secondary metabolite production, which can be used for bio-process optimization.

Flow cytometric methods to investigate culture heterogeneities for plant metabolic engineering.

Plant cell cultures provide an important method for production and supply of a variety of natural products, where conditions can be easily controlled, manipulated, and optimized. Development and optimization of plant cell culture processes require both bioprocess engineering and metabolic engineering approaches. Cultures are generally highly heterogeneous, with significant variability amongst cells in terms of growth, metabolism, and productivity of key metabolites. Taxus cultures produce the important anti-cancer agent Taxol((R)) (i.e., paclitaxel) and have demonstrated significant variability amongst cell populations in culture with regard to paclitaxel accumulation, cell cycle participation, and protein synthesis. To fully understand the link between cellular metabolism and culture behavior and to enable targeted metabolic engineering approaches, cultures need to be studied at a single cell level. This chapter describes the application of plant cell flow cytometric techniques to investigate culture heterogeneity at the single cell level, in order to optimize culture performance through targeted metabolic engineering. Flow cytometric analytical methods are described to study Taxus single cells, protoplasts, and nuclei suspensions with respect to secondary metabolite accumulation, DNA content, cell size, and complexity. Reproducible methods to isolate these single particle suspensions from aggregated Taxus cultures are discussed. Methods to stain both fixed and live cells for a variety of biological markers are provided to enable characterization of cell phenotypes. Fluorescence-activated cell sorting (FACS) methods are also presented to facilitate isolation of certain plant cell culture populations for both analysis and propagation of superior cell lines for use in bioprocesses.