Bioreactor systems for in vitro production of foreign proteins using plant cell cultures

Plant cells have been demonstrated to be an attractive heterologous expression host (using whole plants and in vitro plant cell cultures) for foreign protein production in the past 20years. In recent years in vitro liquid cultures of plant cells in a fully contained bioreactor have become promising alternatives to traditional microbial fermentation and mammalian cell cultures as a foreign protein expression platform, due to the unique features of plant cells as a production host including product safety, cost-effective biomanufacturing, and the capacity for complex protein post-translational modifications. Heterologous proteins such as therapeutics, antibodies, vaccines and enzymes for pharmaceutical and industrial applications have been successfully expressed in plant cell culture-based bioreactor systems including suspended dedifferentiated plant cells, moss, and hairy roots, etc. In this article, the current status and emerging trends of plant cell culture for in vitro production of foreign proteins will be discussed with emphasis on the technological progress that has been made in plant cell culture bioreactor systems.     

Generation of Colors and Flavors in Plant Cell and Tissue Cultures

Plant cell and tissue cultures can be used for the synthesis and production of secondary metabolites like colors, flavors, and sweeteners. Most often, plant cell cultures fail to produce the desired products. In such cases strategies to improve the production of secondary metabolites must be considered.

Plant cell culture technology has now reached the point where a variety of culture types can be critically assessed as potential sources of existing and novel flavors and pigments. This brief review gives examples where progress has been made in the development of plant tissue culture systems.     

Microalgae and Cyanobacteria: How Exploiting These Microbial Resources Can Address the Underlying Challenges Related to Food Sources and Sustainable Agriculture: A Review

For thousands of years, crop production has almost entirely depended on conventional agriculture. However, the reality is changing. The ever-growing population, global climate change, soil degradation and biotic/abiotic stresses are a growing threat to food production and security. Thus, sustainable alternatives to increase crop production for a population projected to reach 9.8 billion by 2050 are a major priority. In addition to vertical and soilless farming, innovative products based on bioresources, including plant growth stimulants, have been a target for sustainable food production. Such solutions have led to the exploitation of microorganisms, including microalgae and cyanobacteria as potential bioresources for food and plant biostimulant products. Microalgae (eukaryotic) and cyanobacteria (prokaryotic) are photosynthetic microorganisms with the capacity to synthesize a vast array of bioactive metabolites from atmospheric CO₂ and inorganic nutrients. The present review outlines the nutritional value of microalgae and cyanobacteria as alternative food resources. The potential aspects of microalgae and cyanobacteria as stabilizers of the net change in soil organic carbon (C) levels for reduced farmland degradation are also highlighted. The applications of microalgae and cyanobacteria as remedies for improved soil structure and fertility, and as enhancers of crop productivity and abiotic stress tolerance in agricultural settings are outlined. This review also discusses the co-cultivation of crops with microalgae or cyanobacteria in hydroponic systems to favor optimum root CO₂/O₂ levels for optimized crop production.

     

曲霉属真菌活性代谢产物及在农业生产中的应用研究进展

曲霉属(Aspergillus)真菌是一类分布广泛的丝状真菌,种类繁多,代谢产物丰富,应用广泛,在农业、医药、生物能源、化妆品、食品发酵等行业均有应用。对近年来曲霉属真菌活性代谢产物在抗菌、抗氧化、抗病毒方面的研究进展进行了综述。结合实验室研究成果,对近年来曲霉属真菌代谢产物在秸秆腐熟菌剂、溶磷生物菌剂、拮抗植物寄生线虫等农业生产领域中的应用进行综述,以期为开发应用曲霉属真菌活性代谢产物的研究提供参考。     

Hairy Root Culture for Mass-Production of High-Value Secondary Metabolites

ABSTRACT

Plant cell cultivations are being considered as an alternative to agricultural processes for producing valuable phytochemicals. Since many of these products (secondary metabolites) are obtained by direct extraction from plants grown in natural habitat, several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of these secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. The main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. Emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. Recent reactors used for mass production of hairy roots can roughly be divided as liquid-phase, gas-phase, or hybrid reactors. The present review highlights the nature, applications, perspectives and scale up of hairy root cultures for the production of valuable secondary metabolites.     

A rational approach for the improvement of biomass production and lipid profile in cacao cell suspensions

Cocoa butter (CB) is produced in the seeds of Theobroma cacao representing 50% of its dry weight. The lipid composition plays an important role in the physicochemical, rheological, and sensory properties of the CB, making this fat a valuable resource for the production of chocolates, cosmetics, and pharmaceuticals. In this paper, are described experimental strategies used for a rational improvement of biomass production and fatty acids in cacao cell suspension cultures. First, the lipid profile in four cacao varieties is characterized, and then, one variety is selected to induce cell suspensions using a direct method without previous establishment of a callus phase. To improve growth and total fat production in cell suspension cultures, modified DKW media and newly designed media culture, based on the mineral concentrations of cacao seeds (cacao biomass production, “CBP”), are analyzed and compared. In addition, the effect of acetate in the lipid profile of cell suspensions is evaluated. Ultrastructural histological analysis of lipid vesicles in cacao seeds and cell suspensions is also performed. The results will show that it is feasible to establish cacao suspensions without the calli step and increase the biomass production by selecting a suitable cacao variety and tissue and also applying a new culture media formulation. In addition, it is possible to synthesize fatty acids in cell cultures and modify the lipid profile adding a precursor of the novo biosynthesis of fatty acids such as the acetate. Transmission electronic microscopy examinations and differential interference contrast microscopy analysis will demonstrate that lipid vesicles are the main reserve substance in both cacao seeds and cell suspensions.     

Structured model and parameter estimation in plant cell cultures of Thevetia peruviana

In this work, a mechanistic model for predicting the dynamic behavior of extracellular and intracellular nutrients, biomass production, and the main metabolites involved in the central carbon metabolism in plant cell cultures of Thevetia peruviana is presented. The proposed model is the first mechanistic model implemented for plant cell cultures of this species, and includes 28 metabolites, 33 metabolic reactions, and 61 parameters. Given the over-parametrization of the model, its nonlinear nature and the strong correlation among the effects of the parameters, a parameter estimation routine based on identifiability analysis was implemented. This routine reduces the parameter’s search space by selecting the most sensitive and linearly independent parameters. Results have shown that only 19 parameters are identifiable. Finally, the model was used for analyzing the fluxes distribution in plant cell cultures of T. peruviana. This analysis shows high uptake of phosphates and parallel uptake of glucose and fructose. Furthermore, it has pointed out the main central carbon metabolism routes for promoting biomass production in this cell culture.

     

Bench to batch: advances in plant cell culture for producing useful products

Despite significant efforts over nearly 30 years, only a few products produced by in vitro plant cultures have been commercialized. Some new advances in culture methods and metabolic biochemistry have improved the useful potential of plant cell cultures. This review will provide references to recent relevant reviews along with a critical analysis of the latest improvements in plant cell culture, co-cultures, and disposable reactors for production of small secondary product molecules, transgenic proteins, and other products. Some case studies for specific products or production systems are used to illustrate principles.     

Bioprocessing of plant cell cultures for mass production of targeted compounds

More than a century has passed since the first attempt to cultivate plant cells in vitro. During this time, plant cell cultures have become increasingly attractive and cost-effective alternatives to classical approaches for the mass production of plant-derived metabolites. Furthermore, plant cell culture is the only economically feasible way of producing some high-value metabolites (e.g., paclitaxel) from rare and/or threatened plants. This review summarizes recent advances in bioprocessing aspects of plant cell cultures, from callus culture to product formation, with particular emphasis on the development of suitable bioreactor configurations (e.g., disposable reactors) for plant cell culture-based processes; the optimization of bioreactor culture environments as a powerful means to improve yields; bioreactor operational modes (fed-batch, continuous, and perfusion); and biomonitoring approaches. Recent trends in downstream processing are also considered. This paper is dedicated to Prof. Dr. Mladenka P. Ilieva on the occasion of her 70th birthday.     

Bacterial rhamnolipids are novel MAMPs conferring resistance to Botrytis cinerea in grapevine

Rhamnolipids produced by the bacteria Pseudomonas aeruginosa are known as very efficient biosurfactant molecules. They are used for a wide range of industrial applications, especially in food, cosmetics and pharmaceutical formulations as well as in bioremediation of pollutants. In this paper, the role of rhamnolipids as novel molecules triggering defence responses and protection against the fungus Botrytis cinerea in grapevine is presented. The effect of rhamnolipids was assessed in grapevine using cell suspension cultures and vitro-plantlets. Ca²⁺ influx, mitogen-activated protein kinase activation and reactive oxygen species production form part of early signalling events leading from perception of rhamnolipids to the induction of plant defences that include expression of a wide range of defence genes and a hypersensitive response (HR)-like response. In addition, rhamnolipids potentiated defence responses induced by the chitosan elicitor and by the culture filtrate of B. cinerea . We also demonstrated that rhamnolipids have direct antifungal properties by inhibiting spore germination and mycelium growth of B. cinerea . Ultimately, rhamnolipids efficiently protected grapevine against the fungus. We propose that rhamnolipids are acting as microbe-associated molecular patterns (MAMPs) in grapevine and that the combination of rhamnolipid effects could participate in grapevine protection against grey mould disease.     

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.     

Biotechnological approaches to the production of shikonins: a critical review with recent updates

Shikonins are commercially important secondary compounds, known for array of biological activities such as antimicrobial, insecticidal, antitumor, antioxidants, etc. These compounds are usually colored and therefore have application in food, textiles and cosmetics. Shikonin and its derivatives, which are commercially most important of the naphthoquinone pigments, are distributed among members of the family Boraginaceae. These include different species of Lithospermum, Arnebia, Alkanna, Anchusa, Echium and Onosma. The growing demand for plant-based natural products has made this group of compounds one of the enthralling targets for their in vitro production. The aim of this review is to highlight the recent progress in production of shikonins by various biotechnological means. Different methods of increasing the levels of shikonins in plant cells such as selection of cell lines, optimization of culture conditions, elicitation, in situ product removal, genetic transformation and metabolic engineering are discussed. The experience of different researchers working worldwide on this aspect is also considered. Further, to meet market demand, the needs for continuous and reliable production systems, as well as future prospects, are included.     

Gas composition strategies for the successful scale-up of Catharanthus roseus cell cultures for the production of ajmalicine

Ajmalicine, serpentine, catharanthine, and vindoline are monoterpenoid indole alkaloids (MIAs) of commercial interest which are produced by the Catharanthus roseus plant. Cultures of C. roseus have been investigated as a potential source of these pharmaceutically important compounds since the early 1960s. In addition, their production from C. roseus cultures has served as a model system for investigating secondary metabolism and for evaluating production-enhancing strategies. Initially, this review will survey (1) the MIAs of interest for large-scale production from plant cell cultures and (2) the volumetric productivities of a specific MIA, ajmalicine, achieved and projected using plant cell cultures. To meet the need for these valuable compounds, the production of these MIAs from plant cell cultures must be successfully reproduced in large-scale aerated and agitated reactors. While the large-scale cultivation of plant cell cultures is currently feasible, initial attempts at scale-up may yield results that differ from that optimized in flasks. To bridge the jump between production in flasks and production in large-scale bioreactors, changes introduced with scale-up such as gas composition must be identified and rationally manipulated to reproduce or even improve growth and secondary metabolite production. Hence, this review will (1) identify the effects of gas composition (i.e., O₂, CO₂, ethylene, or other endogenous volatile compounds) on growth and secondary metabolism and (2) draw operating strategies for optimizing the gas composition for growth of C. roseus cultures and the production of ajmalicine.     

The scale-up of plant cell culture: Engineering considerations

The enormous versatility of plants has continued to provide the impetus for the development of plant tissue culture as a commercial production strategy for secondary metabolites. Unfortunately problems with slow growth rates and low products yields, which are generally non-growth associated and intracellular, have made plant cell culture-based processes, with a few exceptions, economically unrealistic. Recent developments in reactor design and control, elicitor technology, molecular biology, and consumer demand for natural products, are fuelling a renaissance in plant cell culture as a production strategy. In this review we address the engineering consequences of the unique characteristics of plant cells on the scale-up of plant cell culture.Abbreviations a gas-liquid interfacial area per volume - C dissolved oxygen concentration - C^* liquid phase oxygen concentration in equilibrium with the partial pressure of oxygen in the bulk gas phase - K_L overall mass transfer coefficient - k_L liquid film mass transfer coefficient - m_O2 cell maintenance coefficient for oxygen - OTR oxygen transfer rate - OUR oxygen uptake rate - pO₂ partial pressure of oxygen - STR stirred-tank reactor - v.v.m. volume of gas fed per unit operating volume of reactor per minute - X biomass concentration - Y_x/O2 biomass yield coefficient for oxygen - specific growth rate     

Stimulation of ajmalicine production and excretion from Catharanthus roseus: effects of adsorption in situ,elicitors and alginate immobilization

Summary More efficient bioreactors for the production and recovery of secondary metabolites from plant cell cultures are needed. Three factors that have the potential to increase productivity are adsorption in situ, elicitors, and cell immobilization. The effects of these factors on ajmalicine production from Catharanthus roseus are reported in this paper. Elicitation using autoclaved cultures of the mold, Phytophthora cactorum, stimulates a 60% increase in ajmalicine production. The response time to elicitor addition was under 11 h. Adsorption of ajmalicine from the extracellular medium with the neutral resin, Amberlite XAD-7, greatly enhanced the release of ajmalicine (less than 10% extracellular to 40%) with a 40% increase in total productivity. Immobilization in Caalginate beads resulted in a significant increase in the accumulation of ajmalicine in the medium. The effects of elicitation, adsorption and immobilization were synergistic. For a 23-day culture period the amount of ajmalicine in the medium for cells subjected to all three treatments was 90 mg/L compared to 2 mg/L for suspension cultures cultured under otherwise identical conditions. These results suggest that immobilized cell bioreactors may be feasible for continuous production of products normally stored intracellularly in vacuoles in plant cells.     

Betalain production in plant in vitro systems

Betalains have been widely used as natural colorants for many centuries, but their attractiveness for use as colorants of foods (or drugs and cosmetics) has increased recently due to their reportedly high anti-oxidative, free radical scavenging activities and concerns about the use of various synthetic alternatives. The main commercial sources of betalains are powders and concentrates of red beet (Beta vulgaris) or cactus pear (Opuntia ficus-indica) extracts. However, in recent years the technical and commercial feasibility of various in vitro systems to produce them biotechnologically has been explored. These research activities have included assessments of novel approaches for cultivating plant cell or tissue cultures, and diverse bioreactor systems for increasing production levels of secondary metabolites. This paper reviews recent progress in plant in vitro systems for producing betalain pigments. In addition, the factors that could be manipulated, the bioreactor systems that could be used, and the strategies that could be applied to improve betalain production are discussed.     

Improved β-thujaplicin production in Cupressus lusitanica suspension cultures by fungal elicitor and methyl jasmonate

Production of a novel antimicrobial tropolone, beta-thujaplicin, in Cupressus lusitanica suspension cultures was studied by using a variety of chemicals and fungal elicitors. Sodium alginate, chitin, and methyl jasmonate resulted in 2-, 2.5-, and 3-fold higher beta-thujaplicin production, respectively, than in the control. Significantly improved beta-thujaplicin production (187 mg l(-1)) was obtained using a high cell density (180-200 g l(-1)) and fungal elicitor treatment [10 mg (g fresh cells)(-1)] in a production medium with a high ferrous ion concentration (0.3 mM). This improved volumetric productivity was 3- to 4-fold higher than obtained under standard conditions. A synergistic effect of fungal elicitor and ferrous ion on beta-thujaplicin production was also suggested by our study. Plant cell culture technology is a promising alternative for producing a large variety of secondary metabolites that are widely used as food additives, pharmaceuticals, and dairy products (Verpoorte et al. 1999). Thus, beta-thujaplicin production by plant cell cultures was developed with the goal of commercial application (Berlin and Witte 1988; Itose and Sakai 1997; Ono et al. 1998). However, the production of beta-thujaplicin by plant cell cultures is still not competitive for use in industrial applications. In this study, we assessed the effects of methyl jasmonate, alginate, chitin, and fungal elicitor on beta-thujaplicin production; we obtained a significantly elevated beta-thujaplicin production by using an improved culture strategy.     

Potential of plant cells in culture for cosmetic application

Plants and plant derived ingredients are common and of major importance in the fields of pharmacy, food and cosmetics. The cosmetic industry is a fast moving market. Products have short life-cycles and the industry has to come up with innovative products constantly. Most cosmetic products and their applications are defined by active ingredients. These active ingredients may derive from either synthetic sources or from plant sources. Beside this, no other origin like human or animal are accepted or allowed in cosmetics nor are genetically modified plant sources. The whole cosmetic research and development society is therefore desperately seeking for new innovative plant ingredients for cosmetic application. Unfortunately, new plant derived ingredients are limited because several plants of cosmetic interest are not to be used due to following facts: the plants contain toxic metabolites, the plants grow too slow and a seasonal harvesting is not possible, the concentration of plant constituents differ from harvest to harvest or the plant is endangered and not allowed to harvest. With the plant cell culture technology we bring complete new aspects in the development of novel cosmetic plant derived actives. Due to all these findings, we decided to risk the step into plant cell culture derived cosmetic active ingredient production. This article describes the successful establishment of an apple suspension culture producing a high yield of biomass, cultured in disposable, middle-scale bioreactors. The use of a bioactive extract out of these cells for cosmetic application and the efficacy of this extract on mammalian stem cells is also outlined in this article. To obtain a suitable cosmetic product we used the high pressure homogenization technique to decompose the plant cells and release all the beneficial constituents while encapsulating these components at the same time in liquid Nanoparticles. With the plant cell culture technology we bring complete new aspects in the development of novel cosmetic plants derived actives.     

Process optimization of large-scale production of recombinant adeno-associated vectors using dielectric spectroscopy

A well-characterized manufacturing process for the large-scale production of recombinant adeno-associated vectors (rAAV) for gene therapy applications is required to meet current and future demands for pre-clinical and clinical studies and potential commercialization. Economic considerations argue in favor of suspension culture-based production. Currently, the only feasible method for large-scale rAAV production utilizes baculovirus expression vectors and insect cells in suspension cultures. To maximize yields and achieve reproducibility between batches, online monitoring of various metabolic and physical parameters is useful for characterizing early stages of baculovirus-infected insect cells. In this study, rAAVs were produced at 40-l scale yielding ~1 × 10¹⁵ particles. During the process, dielectric spectroscopy was performed by real time scanning in radio frequencies between 300 kHz and 10 MHz. The corresponding permittivity values were correlated with the rAAV production. Both infected and uninfected reached a maximum value; however, only infected cell cultures permittivity profile reached a second maximum value. This effect was correlated with the optimal harvest time for rAAV production. Analysis of rAAV indicated the harvesting time around 48 h post-infection (hpi), and 72 hpi produced similar quantities of biologically active rAAV. Thus, if operated continuously, the 24-h reduction in the production process of rAAV gives sufficient time for additional 18 runs a year corresponding to an extra production of ~2 × 10¹⁶ particles. As part of large-scale optimization studies, this new finding will facilitate the bioprocessing scale-up of rAAV and other bioproducts.