Plant cell suspension cultures are widely used for the production of recombinant proteins and secondary metabolites. One of the most important steps during process development is the optimization of yields by testing different cultivation parameters, including the components of the growth medium. However, we have shown that the biomass yield of a cell suspension culture derived from the pear cultivar Pyrus communis cv. Champagner Bratbirne can be significantly improved solely by varying the temperature, inoculum density, illumination, and incubation time. In contrast to medium optimization, these simple physical factors are easily controlled and varied, thereby reducing the effort required. Using an experimental design approach, we improved the biomass yield from 146 g fresh weight (FW)/L to 407 g FW/L in only 5 weeks, simultaneously reducing the costs of goods sold per kg biomass from €125 to €45. Our simple approach therefore offers a rapid, efficient and economical process for the optimization 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.

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.

En este trabajo se presentan los resultados finales obtenidos en el marco de un Proyecto de Investigación en curso que tiene por objetivo general estudiar la aplicación del bloque de legalidad vigente en la región del Mercosur sobre Arbitraje Comercial Internacional, a la luz de la jurisprudencia del periodo 2002-