Producing Medicinal Phytochemicals from Phyllanthus acuminatus in Plant - Cell Suspension Cultures

Pharmaceutical use is not feasible for important medicinal compounds derived from certain plant materials, including Phyllanthus acuminatus roots, due to their low natural abundance. New technologies in non-traditional biomass generation are needed to produce these remarkable natural compounds. Therefore, this article describes a methodology for establishing Phyllanthus acuminatus plant-cell suspensions from callus cultures: An evaluation on inoculum concentration and agitation speed displayed significant changes in plant cell growth kinetics. It was determined that treatment with 2 g of inoculum in 25 mL of medium and 100 rpm agitation creates the best conditions for generating thick cell suspensions. Likewise, treatment with 2 g of inoculum and 120 rpm agitation produces the best conditions for establishing fine cell suspensions. Phytochemical comparison through high-resolution mass spectrometry of P. acuminatus roots and plant cell suspension extracts confirmed presence in the plant cell culture of multiple phyllantostatins of pharmaceutical interest. Here, we demonstrate that Phyllanthus acuminatus can be cultured in plant cell suspensions to produce secondary metabolites of medical interest – technology that could be scaled up for implementation in industrial bioprocesses.

Engineering Considerations to Produce Bioactive Compounds from Plant Cell Suspension Culture in Bioreactors

The large-scale production of plant-derived secondary metabolites (PDSM) in bioreactors to meet the increasing demand for bioactive compounds for the treatment and prevention of degenerative diseases is nowadays considered an engineering challenge due to the large number of operational factors that need to be considered during their design and scale-up. The plant cell suspension culture (CSC) has presented numerous benefits over other technologies, such as the conventional whole-plant extraction, not only for avoiding the overexploitation of plant species, but also for achieving better yields and having excellent scaling-up attributes. The selection of the bioreactor configuration depends on intrinsic cell culture properties and engineering considerations related to the effect of operating conditions on thermodynamics, kinetics, and transport phenomena, which together are essential for accomplishing the large-scale production of PDSM. To this end, this review, firstly, provides a comprehensive appraisement of PDSM, essentially those with demonstrated importance and utilization in pharmaceutical industries. Then, special attention is given to PDSM obtained out of CSC. Finally, engineering aspects related to the bioreactor configuration for CSC stating the effect of the operating conditions on kinetics and transport phenomena and, hence, on the cell viability and production of PDSM are presented accordingly. The engineering analysis of the reviewed bioreactor configurations for CSC will pave the way for future research focused on their scaling up, to produce high value-added PDSM.

LEDitSHAKE: a lighting system to optimize the secondary metabolite content of plant cell suspension cultures

Plant secondary metabolites are widely used in the food, cosmetic and pharmaceutical industries. They can be extracted from sterile grown plant cell suspension cultures, but yields and quality strongly depend on the cultivation environment, including optimal illumination. Current shaking incubators do not allow different light wavelengths, intensities and photoperiods to be tested in parallel. We therefore developed LEDitSHAKE, a system for multiplexed customized illumination within a single shaking incubator. We used 3D printing to integrate light-emitting diode assemblies into flask housings, allowing 12 different lighting conditions (spectrum, intensity and photoperiod) to be tested simultaneously. We did a proof of principle of LEDitSHAKE using the system to optimize anthocyanin production in grapevine cell suspension cultures. The effect of 24 different light compositions on the total anthocyanin content of grapevine cell suspension cultures was determined using a Design of Experiments approach. We predicted the optimal lighting conditions for the upregulation and downregulation of 30 anthocyanins and found that short-wavelength light (blue, UV) maximized the concentration of most anthocyanins, whereas long-wavelength light (red) had the opposite effect. Therefore our results demonstrate proof of principle that the LEDitSHAKE system is suitable for the optimization of processes based on plant cell suspension cultures.

Evidence of starch accumulation in TBY-2 cells in the presence of auxin

Tobacco cell suspension (TBY-2) is known to produce starch when cultured in medium supplemented with cytokinin or in hormone-free medium. Unexpectedly, TBY-2 cells, continuously cultivated on auxin alone, were also able to accumulate starch at the beginning of stationary growth phase with a yield of 9.22 ± 0.68 percent. This starch production was strongly correlated with a 25-fold increase in starch synthase activity. Moreover, this TBY-2 line was able to produce an amylopectin-rich starch with a ratio amylopectin over amylose of 2.7 which also linked to typical small granules (size around 1.4 µm). According to our preliminary results, this plant cell suspension could produce a low-cost amylopectin rich starch needed in the food industry for production of edible film or bioplastic without impacts from climate or season changes.

Plant Extracellular Vesicles and Nanovesicles: Focus on Secondary Metabolites, Proteins and Lipids with Perspectives on Their Potential and Sources

While human extracellular vesicles (EVs) have attracted a big deal of interest and have been extensively characterized over the last years, plant-derived EVs and nanovesicles have earned less attention and have remained poorly investigated. Although a series of investigations already revealed promising beneficial health effects and drug delivery properties, adequate (pre)clinical studies are rare. This fact might be caused by a lack of sources with appropriate qualities. Our study introduces plant cell suspension culture as a new and well controllable source for plant EVs. Plant cells, cultured in vitro, release EVs into the growth medium which could be harvested for pharmaceutical applications. In this investigation we characterized EVs and nanovesicles from distinct sources. Our findings regarding secondary metabolites indicate that these might not be packaged into EVs in an active manner but enriched in the membrane when lipophilic enough, since apparently lipophilic compounds were associated with nanovesicles while more hydrophilic structures were not consistently found. In addition, protein identification revealed a possible explanation for the mechanism of EV cell wall passage in plants, since cell wall hydrolases like 1,3-β-glucosidases, pectinesterases, polygalacturonases, β-galactosidases and β-xylosidase/α-L-arabinofuranosidase 2-like are present in plant EVs and nanovesicles which might facilitate cell wall transition. Further on, the identified proteins indicate that plant cells secrete EVs using similar mechanisms as animal cells to release exosomes and microvesicles.

LEDitSHAKE – A Lighting System to Optimize the Secondary Metabolite Content of Plant Cell Suspension Cultures

1.1 Background Plant secondary metabolites are widely used in the food, cosmetic and pharmaceutical industries. They can be extracted from naturally grown plants or plant cell suspension cultures grown under sterile conditions. In the latter case, yields and quality strongly depend on the cultivation environment, including optimal illumination. Current shaking incubators do not allow different light wavelengths, intensities and photoperiods to be tested in parallel. We therefore developed a system for multiplexed customized illumination within a single shaking incubator, and used it to optimize anthocyanin production in grapevine cell suspension cultures. 1.2 Results We used 3D printing to integrate light-emitting diode assemblies into flask housings to develop the LEDitSHAKE system, allowing 12 different lighting conditions (spectrum, intensity and photoperiod) to be tested simultaneously in a single shaking incubator. We used a Design of Experiments approach to determine the effect of 24 different light compositions on the total anthocyanin content of replicate grapevine cell suspension cultures. All tested conditions achieved higher yields than standard illumination or dark cultivation, and the optimal spectrum (8.3 µmol m− 2 s− 1 red, 8.3 µmol m− 2 s− 1 green, 33.3 µmol m− 2 s− 1 blue, and UV turned on) increased the total anthocyanin concentration by 2.42-fold after 4 weeks. Based on the resulting model, we predicted the optimal lighting conditions for the upregulation and downregulation of 30 anthocyanins, and found that short-wavelength light (blue, UV) maximized the concentration of most anthocyanins, whereas long-wavelength light (red) had the opposite effect. For example, the cyanidin glucoside concentration was predicted to increase 2.99-fold compared to the source culture with optimized illumination (12 µmol m− 2 s− 1 green, 38 µmol m− 2 s− 1 blue, and UV turned on for 1 h/day). 1.3 Conclusions The LEDitSHAKE system enables the screening of up to 12 different lighting conditions in terms of spectrum, intensity and photoperiod within a single shaking incubator using a Design of Experiments approach, as exemplified by the optimization of the anthocyanin content and composition in grapevine cell suspension cultures. Our results demonstrate proof of principle that the LEDitSHAKE system is suitable for the optimization of processes based on plant cell suspension cultures.

Root Cultures for Secondary Products

Plants are source of many high-value secondary compounds used as drugs, food additives, flavors, pigments and pesticides. The production of these compounds in nature faces to many difficulties because of the dependence on weather, soil … Furthermore, these compounds are usually limited by species, periods of growth or stress. The utilization of plant cells in vitro for the secondary compounds has gained increasing attention over past decades. However, the yield is still low, probably due to the degree of cell differentiation. Therefore, root culture is focused on research as an alternative to cell cultures to produce secondary compounds because of high rate proliferation, great potential in the production with high and stable yields. Hairy roots and adventitious roots have a high ability to biosynthesize secondary compounds in vitro with high and fairly stable in yield in comparison with plant cell suspension cultures. Nowadays, it is feasible to expand the scale of root cultures in bioreactors, which makes it possible to produce secondary compounds on an industrial scale.