Bioprocess considerations for production of secondary metabolites by plant cell suspension cultures

Plant cell culture provides a viable alternative over whole plant cultivation for the production of secondary metabolites. In order to successfully cultivate the plant cells at large scale, several engineering parameters such as, cell aggregation, mixing, aeration, and shear sensitivity are taken into account for selection of a suitable bioreactor. The media ingredients, their concentrations and the environmental factors are optimized for maximal synthesis of a desired metabolite. Increased productivity in a bioreactor can be achieved by selection of a proper cultivation strategy (batch, fed-batch, two-stageetc.), feeding of metabolic precursors and extraction of intracellular metabolites. Proper understanding and rigorous analysis of these parameters would pave the way towards the successful commercialization of plant cell bioprocesses.     

Medicinal plant cell suspension cultures: pharmaceutical applications and high-yielding strategies for the desired secondary metabolites

The development of plant tissue (including organ and cell) cultures for the production of secondary metabolites has been underway for more than three decades. Plant cell cultures with the production of high-value secondary metabolites are promising potential alternative sources for the production of pharmaceutical agents of industrial importance. Medicinal plant cell suspension cultures (MPCSC), which are characterized with the feature of fermentation with plant cell totipotency, could be a promising alternative “chemical factory”. However, low productivity becomes an inevitable obstacle limiting further commercialization of MPCSC and the application to large-scale production is still limited to a few processes. This review generalizes and analyzes the recent progress of this bioproduction platform for the provision of medicinal chemicals and outlines a range of trials taken or underway to increase product yields from MPCSC. The scale-up of MPCSC, which could lead to an unlimited supply of pharmaceuticals, including strategies to overcome and solution of the associated challenges, is discussed.     

Exudation: an expanding technique for continuous production and release of secondary metabolites from plant cell suspension and hairy root cultures

This review addresses methods of obtaining secondary metabolites from plant cell suspension and hairy root cultures and their exudates, particularly the physiological mechanisms of secondary metabolites release and trafficking. The efficiency for product recovery of metabolites can be increased by various methods, based on the principle of continuous product release into the cultivation medium. The most common methods for metabolite recovery are elicitation, influencing membrane permeability, and in situ product removal. The biosynthetic pathways can be influenced by cultivation conditions, transformation, or application of elicitors. The membrane permeability can be altered through the application of chemical or physical treatments. Product removal can be greatly increased through a two-phase system and the introduction of absorbents into the cultivation medium. In this review, we describe some improved approaches that have proven useful in these efforts.     

A model that links growth and secondary metabolite production in plant cell suspension cultures

Plant cell suspensions of grape cells (Vitis vinifera L. cv. Gamay Fréaux) were grown in shake flasks operated both in the batch and semicontinuous mode. A mathematical model was developed to describe grape cell growth, sucrose uptake, and secondary metabolite (anthocyanin) production. Parameters were estimated from batch studies data. The model was able to predict results for semicontinuous experiments by only modifying the value of four of these parameters. The modified parameters (maximum specific rate of biomass production, maximum specific rate of substrate consumption for maintenance, maximum specific rate of anthocyanin production, and degradation constant of anthocyanins) were related to the kinetics rather than to the yield of the process. The model introduces the concept of primary and secondary metabolism substrate concentration-dependent competition for precursors. Further, the model was able to predict the evolution of the cell system when substrate is scarce, as the value of the different kinetic constants determines the portion of substrate that is used for biomass production, secondary metabolite production, and cell maintenance. (c) 1995 John Wiley & Sons, Inc.     

Bioreactor engineering for recombinant protein production in plant cell suspension cultures

A review of over 15 years of research, development and commercialization of plant cell suspension culture as a bioproduction platform is presented. Plant cell suspension culture production of recombinant products offers a number of advantages over traditional microbial and/or mammalian host systems such as their intrinsic safety, cost-effective bioprocessing, and the capacity for protein post-translational modifications. Recently significant progress has been made in understanding the bottlenecks in recombinant protein expression using plant cells, including advances in plant genetic engineering for efficient transgene expression and minimizing proteolytic degradation or loss of functionality of the product in cell culture medium. In this review article, the aspects of bioreactor design engineering to enable plant cell growth and production of valuable recombinant proteins is discussed, including unique characteristics and requirements of suspended plant cells, properties of recombinant proteins in a heterologous plant expression environment, bioreactor types, design criteria, and optimization strategies that have been successfully used, and examples of industrial applications.     

A method for the rapid production of fine plant cell suspension cultures

A method has been devised which allows the rapid production of fine suspension cultures of small aggregate size from suspension cultures of large average aggregate size, such as those of Capsicum frutescens. The method, which uses a Waring blender for aseptic homogenisation of cultures, has also been shown to be effective in rapidly producing suspension cultures from callus cultures. The suspension cultures so produced are particularly useful for immobilisation, such as in porous polyurethane foam matrices.     

A new method for the production of fine plant cell suspension cultures

Fine, almost single cell, suspensions were produced from both existing suspension cultures containing large cell clumps and from chopped callus pieces by immobilizing the cells in 4–5 mm diameter calcium alginate beads. The immobilized cells continued to divide inside the beads and at the bead surface, and after 2–3 weeks' culture, fine cell suspensions were formed as a result of loss of the surface cells into the medium. After removal of the cell suspensions by filtration, subsequent culture of the beads in fresh medium resulted in the further production of homogeneous cell suspensions after 1–2 weeks. In this way an almost continuous supply of fine cell suspensions could be obtained from cultures containing large clumps of cells. The cells produced by this method remained in this state for at least one culture period, although in some instances repeated subculture resulted in an increase in the size of cell groups. The technique has been successfully applied to the production of fine cell suspensions ofCatharanthus roseus, Nicotiana tabacum andDaucus carota.     

Trends in the biotechnological production of isoflavonoids in plant cell suspension cultures

Phytochemistry Reviews - Isoflavonoids, a class of flavonoid polyphenolic compounds, are found primarily in leguminous plants. The chemical structure of isoflavonoid is a rearranged 2-phenyl of the...     

Biotechnology for the production of plant secondary metabolites

The production of plant secondary metabolites by means of large-scale culture of plant cells in bioreactors is technically feasible. The economy of such a production is the major bottleneck. For some costly products it is feasible, but unfortunately some of the most interesting products are only in very small amounts or not all produced in plant cell cultures. Screening, selection and medium optimization may lead to 20- to 30-fold increase in case one has producing cultures. In case of phytoalexins, elicitation will lead to high production. But for many of the compounds of interest the production is not inducible by elicitors. The culture of differentiated cells, such as (hairy) root or shoot cultures, is an alternative, but is hampered by problems in scaling up of such cultures. Metabolic engineering offers new perspectives for improving the production of compounds of interest. This approach can be used to improve production in the cell culture, in the plant itself or even production in other plant species or organisms. Studies on the production of terpenoid indole alkaloids have shown that the overexpression of single genes of the pathway may lead for some enzymes to an increased production of the direct product, but not necessarily to an increased alkaloid production. On the other hand feeding of such transgenic cultures with early precursors showed an enormous capacity for producing alkaloids, which is not utilized without feeding precursors. Overexpression of regulatory genes results in the upregulation of a series of enzymes in the alkaloid pathway, but not to an improved flux through the pathway, but feeding loganin does result in increased alkaloid production if compared with wild-type cells. Indole alkaloids could be produced in hairy root cultures of Weigelia by overexpression of tryptophan decarboxylase and strictosidine synthase. Alkaloids could be produced in transgenic yeast overexpressing strictosidine synthase and strictosidine glucosidase growing on medium made out the juice of Symphoricarpus albus berries to which tryptamine is added. Metabolic engineering thus seems a promising approach to improve the production of a cell factory.     

Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures

“Molecular farming” in plants with significant advantages in cost and safety is touted as a promising platform for the production of complex pharmaceutical proteins. While whole-plant produced biopharmaceuticals account for a significant portion of the preclinical and clinical pipeline, plant cell suspension culture, which integrates the merits of whole-plant systems with those of microbial fermentation, is emerging as a more compliant alternative “factory”. However, low protein productivity remains a major obstacle that limits extensive commercialization of plant cell bioproduction platform. This review highlights the advantages and recent progress in plant cell culture technology and outlines viable strategies at both the biological and process engineering levels for advancing the economic feasibility of plant cell-based protein production. Approaches to overcome and solve the associated challenges of this culture system that include non-mammalian glycosylation and genetic instability will also be discussed.     

Rotating drum fermentor for plant cell suspension cultures

A rotating drum fermentor designed for plant cell suspension cultures was constructed and tested. The oxygen transfer coefficient (k(L)a) and power requirements in the fermentor were determined with the water system under various conditions and the relationship between them in the fermentor was clarified. Also, the relationship between k(L)a and the apparent viscosity in the fermentor was investigated in the cell suspension system. The rotating drum fermentor was found to be superior to the mechanically agitated fermentor in the capacity of oxygen supply under high viscosity and low hydrodynamic stress conditions. This finding was also confirmed by the experiments with plant cell suspension cultures.     

刺激剂(elicitor)在植物细胞培养次生代谢物生产中的应用

刺激剂（ｅｌｉｃｉｔｏｒ）在植物细胞培养中被用来作为提高次生代谢物产量的手段。文中概括介绍了微生物、寡聚糖、蛋白质、第二信使及其他物质作为刺激剂在植物细胞培养中的应用及其研究成果。     

Cryopreservation of photosynthetic plant cell suspension cultures

Attempts were made to cryopreserve in liquid nitrogen six different photomixotrophic suspension cultured lines of five different species:Amaranthus powellii Wats.,Datura innoxia Mill.,Glycine max (L.) Merr.,Gossypium hirsutum L. andNicotiana tabacum xNicotiana glutinosa L. fusion hybrid. Only theD. innoxia line, DAT, and theG. max line, SB1, could be successfully recovered as viable, growing, dark green cultures. The successful method utilized a preculture treatment of from 2 to 8 days in a medium containing 3% starch and 3% sorbitol for DAT and 3% sucrose and 3% sorbitol for SB1 cells. The cells survived if frozen with 10% dimethylsulfoxide (DMSO) and 9.1% sorbitol or with 10% DMSO and 8% sucrose. Following a programmed slow-cooling, the cells were thawed in a 40° C bath and could be recovered directly when added to fresh liquid medium. Cryostorage of these lines will save labor and prevent further genetic changes from occurring in these unique suspension cultures.     

A method for growing stationary plant suspension cell cultures

Summary Stationary culture of plant cell suspensions has been achieved. Slurries, produced when small amounts of agar (0.1–0.4%) were added to culture media, were used to suspend plant cells. Growth proceeded more slowly than in standard shake culture, but cells remained viable for months of culture. This method of growing plant cells in stationary culture should be useful for general applications including long-term cell culture, shipment of cultures, and physiological, molecular biological, and pathological studies. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. Editor’s Statement This procedure for growing stationary suspension cultures in an agar slurry should be useful for shipping suspensions and for long-term maintenance of little used or back-up cultures.     

Absolute measurement of cell expansion in plant cell suspension cultures

A method is described for the measurement of intracellular volume (V_i) in cell cultures. In principle, any stable compound that neither penetrates the plasma membrane nor binds to the cells can be used to trace the total extracellular (apoplastic) volume and hence to estimate the intracellular volume. No suitable coloured or UV-absorbing compound could be found among those tested; the main problems were binding to the cell surface and/or instability in the medium. However, [¹⁴C]mannitol was an acceptable apoplastic marker, by use of which we showed that 21–47% of total packed cell volume (PCV) was intracellular, and 14–33% of total settled cell volume (SCV) was intracellular. Therefore, measurements of PCV and SCV misrepresent cell expansion to a variable extent. Cultures of Acer, Rosa, Spinacia and Zea achieved final symplastic volumes of only 9, 14, 6 and 6%, respectively, of the total suspension culture volume.     

Paclitaxel production in suspension cell cultures of Taxus

Five separate cell lines, three of Taxus canadensis Marsh. and two of Taxus cuspidata Sieb. et Zucc., were used to test the effect of carbohydrates and plant growth regulators on the growth of cells and production of paclitaxel in culture. There was no significant correlation between growth of cells and paclitaxel production. While no single medium was developed that was optimal for all cell lines, it was possible to develop a medium for each species that represented a superior combination of growth and paclitaxel production. A combination of NAA and thidiazuron produced the best combination of growth and paclitaxel production in cell lines of T. canadensis, while IAA and BA produced the best results in cell lines of T. cuspidata. A mixture of sucrose and fructose gave the best combination of growth and paclitaxel production. The addition of carbohydrates midway through the growth cycle increased the rate at which paclitaxel accumulated in the culture medium. The highest paclitaxel concentration obtained was 14.78±0.86 mg 1^–1 (n=3).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2ip 6-(,-dimethylamino)-purine - BA 6-benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - NAA -napthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid - thidiazuron 1-phenyl-3 (1,2,3-thiadiazol-5-yl)urea     

Eliciting secondary metabolism in plant cell cultures

Plant cell cultures are potentially rich sources of valuable pharmaceuticals and other biologically active phytochemicals, but relatively few cultures synthesize secondary compounds over extended periods in amounts comparable to those found in the whole plant. Frequently, no secondary metabolites characteristic of the intact plant are produced. So far, the manipulation of culture media, culture conditions and phytohormone levels have, in general, failed to permit commercial production of those phytochemicals useful in medicine and industry. This almost certainly reflects the lack of understanding of basic secondary metabolic regulation in cultured plant cells.

Microbial insult can induce antibiotic phytochemical synthesis in cultured plant cells: the microbial molecules which stimulate synthesis have been called ‘elicitors’. Increased synthesis of secondary products in response to elicitation of various types appear to be the general response of cultured cells. This paper illustrates the immense biotechnological potential of plant cell culture—‘elicitor’ (inducer) interactions to the large scale production of secondary metabolites, and suggests several lines of enquiry that remain to be authoritatively treated.     

Image-based analysis of cell-specific productivity for plant cell suspension cultures

More and more plant cell suspension cultures are regarded as an attractive alternative to mammalian cells as host organism for production of complex recombinant proteins. The most important advantages of the production platform are low costs, easy scalability and enhanced safety by complete lack of animal components in the cultivation media. In order to characterize, understand and control such systems accurately, it is important to determine the cell-specific productivity (Qp) of plant cell-based production platforms. Compared to many microbial and mammalian cells the morphology of plant cells is nonhomogeneous and the cells tend to form aggregates, therefore commercial cell counting systems are too unreliable to determine cell numbers in plant suspension cultures. We addressed this limitation by developing a novel cell counting method based on a combination of cell-staining and automated confocal fluorescence microscopy. This method allowed us, for the first time, to determine the cell-specific productivity of transgenic tobacco (Nicotiana tabacum cv. Bright Yellow-2) cell suspension cultures producing the human antibody M12. In the future this method will be a useful tool in the development of optimized plant cell-based production processes.