Hybanthus enneaspermus

Doffek, Benjamin, Huang, Yvonne, Huang, Yen-Hua, Chan, Lai Yue, Gilding, Edward K., Jackson, Mark A. & Craik, David J., 2022, Comparative analysis of cyclotide-producing plant cell suspensions presents opportunities for cyclotide plant molecular farming, Phytochemistry (113053) 195, pp. 1-10 : 6

2.5. Scale up of H. enneaspermus cell suspension culture using a rocking motion bioreactor

Rocking motion bioreactors utilizing single-use culture bags provide a convenient and modern approach for scaling up cyclotide production under controlled conditions, without the risks of cross contamination. Given the sustained and relatively high levels of cyO2 accumulation in H. enneaspermus compared to O. affinis in our smaller scale cultures, H. enneaspermus suspension cells were chosen to assess their ability to produce cyO 2 in a rocking motion bioreactor at 5 L final culture volume. A fed batch strategy was employed with 200 mL high density H. enneaspermus culture grown in shake flasks used to first inoculate 800 mL of fresh media in a disposable reactor bag. After 360 h this 1 L culture reached a density of 50% packed cell volume (PCV), at which point a further 4 L of fresh medium was added to achieve the final culture volume of 5 L. Culture conditions (dissolved oxygen %, pH, rocking speed and angle were monitored ( Fig. S4 ). In total 22 g DW of cells were produced at the end of the 480-h culture, which yielded 10.3 mg of pure cyO2 (0.46 mg /g DW). Purified cyO2 from suspension cells was shown via NMR to be identical to plant leaf derived cyO2 ( Fig. 6A ) and was demonstrated to be seven-fold more cytotoxic to insect Sf9 cells than chemosynthetically produced kB1 with a cytotoxic concentration (CC) of 0.27 ± 0.02 μM compared to 1.91 ± 0.02 μM respectively ( Fig. 6B ). Together, the establishment and scale up of H. enneaspermus suspension cells shows the feasibility of producing cyO 2 in a bioreactor, a bracelet-type cyclotide that is recalcitrant to production by chemosynthetic methods.

Figures

Fig. 4. Inducibility of cyclotide accumulation in O. affinis suspension cells. A Cyclotide accumulation profiles in O. affinis suspension cells exposed to biotic (methyl jasmonate, MeJA) and abiotic (NaCl) signals. Replicate (n =3) cultures were assayed 1, 4 and 7 days after addition of inducer. Treatments highlighted by an asterisk are significantly different (P <0.05). Cell morphology of B day 1 cells in 3 mM NaCl, C day 7 cells in 3 mM NaCl and D day 7 cells in 50 mM NaCl.

Fig. 6. A Structural equivalence of cyO2 derived and purified from plant suspension cells or whole plant tissue as determined by NMR spectroscopy. B Cytotoxicity of cyclotides cyO2 and kB1 against Sf9 cells.