S04 - Session O6 - Tamarillo cell suspension-based bioreactors – optimization of a novel plant-based system for elicited enzymatic production

Authors: Bruno Casimiro *, Inês Lopes, Mariana Correia, Jorge Canhoto, Luisa F Rojas, Paula Veríssimo, Sandra Correia

Plant molecular farming describes the production of important metabolites in plants, with several advantages over other systems when considering their safety and potential for large-scale and low-cost productions. Plant cell suspension (PCS) cultures are regarded as one of the most suitable molecular farming platforms for plant-based production of valuable materials. In this context, PlantREACT was projected with the main goal of developing a novel plant-based bioreactor system to produce bioactive compounds with applicability in the agri-food industry. Based on effective protocols for tamarillo ( Solanum betaceum Cav.) micropropagation and PCS cultures establishment from induced calli lines, we first started to optimize the cells' growth and to characterize their stress-related intracellular/extracellular protein profiles under in vitro controlled conditions. These assays showed that tamarillo PCS secrete a wide range of enzymes, namely proteases and glycoside hydrolases, commonly associated with inhibiting pathogen-derived cell wall degrading enzymes. By using different biotic elicitors (e.g., chitosan, yeast extract), enzyme production was enhanced, particularly for chitosan (0.1g/L) elicited extracts showing higher glycosidase, alkaline phosphatase and protease activities. Further proteomic analysis is being conducted to identify differentially expressed proteins between treatments, and the effectiveness of the obtained extracts assayed on bioactivity tests. Also, a protocol for Agrobacterium -mediated transformation of the induced cell lines is being optimized for recombinant protein production. In parallel, selected PCS cultures are being scaled up for operation in a 5L-stirred-tank bioreactor equipped with an aeration system. To obtain consistently sized aggregates, PCS were sieved (200-300μm) and operating conditions were determined by estimating the transfer rate and oxygen consumption and the resistance of plant cells to hydrodynamic stress at the flask level (by cell viability determination). The results so far obtained are good indicators of the potential of tamarillo cell suspension cultures as a novel system for a safe and low-cost production of high demanded metabolites.