Optimization of induction of eGFP secretion in Brassica rapa rapa hairy roots
Factors to be assessed for secretion induction in B. rapa rapahairy roots were selected according to earlier studies, which have shown to improve recombinant protein secretion in hairy root platforms, including KNO3, NAA and PVP as a stabilizer (Drake et al., 2003; Drake et al., 2009; Häkkinen et al., 2014; Wongsamuth and Doran, 1997). In addition, we wanted to see whether methyl jasmonate (MeJA), a known elicitor for a broad range of plant metabolites, could play a role in recombinant protein secretion. Some studies have reported the use of MeJA to increase the expression rate of recombinant proteins (Fraissinet-Tachet et al., 1998; Karimzadegan et al., 2019). The idea behind using this organic compound as reagent for recombinant protein optimization is that when plants experience herbivore attacks and wounding, plant defense mechanisms get triggered through jasmonate signaling pathways, resulting in re-allocation of plant resources to defense mechanisms instead of developmental processes. Hence, energy and resources like amino acids can be shifted to synthesize other proteins, and the heterologous proteins can be one of them.
Preliminary secretion induction assays in B. rapa rapa hairy roots had shown that addition of 2,4-D in the culture media was able to increase the extracellular levels of recombinant proteins such as lipase or eGFP (Ele Ekouna et al., 2017b). Thus, the starting 2,4-D level was initially set to 1 mg/l. In addition, PVP level was also set at 1.5 g/l according to Häkkinen et al., 2014. The studied factors included to the design of experiment (DoE) consisted of KNO3, NAA and MeJA. The optimization iteration was performed in three experimental rounds (Table 1 ).
The outcome of secretion induction optimization as model descriptors and equations are shown in Table 2 . The 1stsecretion induction optimization round indicated that KNO3 and MeJA had significant effect on the eGFP secretion whereas NAA had no influence. The highest eGFP secretion, 22 mg/g DW B. rapa rapa hairy root biomass, was obtained in the studied range with low MeJA and high KNO3 levels (Fig. 2A ).
The 2nd secretion optimization experiment was performed so that the optimal secretion point was placed in the center of the experimental range (Table 1 ). Again, the highest eGFP secretion was gained with high KNO3 induction. Noteworthy is that the role of MeJA was observed to be minimal. At highest the eGFP secretion was reaching 16 mg /g DW B. rapa rapahairy root biomass (Fig. 2B ). Indeed, the insignificant role of MeJA was confirmed in the 3rd secretion induction experiment were only KNO3 had a significant effect on eGFP secretion. It was shown that the optimal concentration based on the model would be 20.4 g/l KNO3 which would yield 16.2 mg/g DW eGFP (~ 260 mg/l) (Fig. 2C ). In the study of Häkkinen et al. , 2014, the addition of 14 g/l KNO3 and 19 mg/l NAA to the B5mod culture medium increased the amount of recombinant M12 antibody recovered 30 fold.
as described above, for B. rapa rapa hairy root expressing eGFP, NAA and MeJA did not give additional benefit in secretion induction. Earlier, Drake et al. (2009) showed that the addition of growth regulators (NAA, indolebutyric acid (IBA), 6-benzylaminopurine (BAP) and kinetin (KIN)) to hydroponic plants all cased an increase in the root biomass, induced the secretion of recombinant proteins and also increased their stability in the hydroponic culture medium. NAA had the greatest effect on rhizosecretion with improving the antibody yields into the liquid media ca. 50-fold when compared to non-supplemented hydroponic media. In the current study, auxin was added in the form of 2,4-D and it is likely that additional auxin supplementation with NAA did not extend the auxin-induced effect in hairy roots.
In order to verify the identified optimal secretion induction conditions for B. rapa rapa hairy roots expressing eGFP, an empirical set up shown in Fig. 3A was designed. The biomass accumulation was statistically significantly lower when secretion induction, PVP+2,4-D+KNO3, was applied when compared to all other treatment and non-induced samples (Fig. 3B ). Nevertheless, the PVP+2,4-D+KNO3 secretion induction resulted in highest eGFP secretion of 342 mg/l being 3.4-fold higher than in the non-induced sample that reached level of 101 mg/l. The highest secretion value corresponded to 42 mg/g DW hairy roots.