Oldenlandia affinis subsp. suspension

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 : 5

2.4. Loss of cyclotides in O. affinis suspension cells is due to rapid downregulation of transcript

Considering the reduced diversity and changes observed for cyclotide accumulation in O. affinis suspension cells, we probed the expression level of various peptide precursor genes and relevant ligase-type and proteolytic-type AEPs. Ligase-type AEPs, in this case OaAEP1 and OaAEP3, are essential for performing the head-to-tail cyclization in vitro and in vivo heterologously ( Harris et al., 2015, 2019; Poon et al., 2018). We found that transcript levels for both ligase-type AEPs OaAEP1 and OaAEP3, and protease-type AEP OaAEP2, are highest in suspension cells assayed at one week after initiation before gradually declining but remain stable thereafter ( Fig. 5A ). Likewise, the cyclotide precursor genes Oak1 and Oak4 encoding kB1 and kB2, respectively, exhibit minimal to zero expression in suspension cells, whereas transcripts encoding OaCyO2 increase in callus and suspension cell cultures but are not detected in leaf ( Fig. 5B ). Overall, the patterns of cyclotide precursor transcript abundance reasonably match what was observed for relative amounts of accumulated cyclotide ( Fig. 5C ). Importantly the sustained expression of ligase-type AEP in O. affinis suspension cells suggest that they would be viable hosts of custom cyclotide precursors should they be introduced as transgenes.

The observed loss of cyclotide diversity in cell suspensions has both positive and negative implications for the use of cell suspensions as cyclotide production platforms. The reduced diversity may restrict the types of cyclotides that are amenable for expression in suspensions, yet the reduced diversity is coincident with reduced complexity in purifying cyclotides from cultures. Serendipitously, production of two biofunctional cyclotides, cyO2 and cyO13, in H. enneaspermus suspension cells and cyO 2 in O. affinis was observed. Both cyO2 and cyO13 are antifungal agents against agricultural pathogens, with cyO2 possessing the strongest activity against six plant-infecting fungi and the highest anti-cancer cytotoxicity of Violaceae cyclotides tested thus far ( Slazak et al., 2018). CyO2 and cyO13 are both bracelet-type cyclotides, which have proven difficult to fold during chemosynthesis thus leading to poor synthetic yields ( Leta Aboye et al., 2008). The development of suspension cells for their efficient production and purification would be advantageous.

Figures

Fig. 5. Assessment of transcript or peptide levels present in O. affinis leaf, calli, and suspension cells at days 1, 14, 21 and 28. A Relative transcript expression determined by qPCR (n = 4) of asparaginyl endopeptidases OaAEP1, OaAEP2 and OaAEP3. B Relative transcript expression determined by qPCR (n = 4) of select cyclotide precursors Oak1 encoding kB1, Oak4 encoding kB2, and OaCyO2 encoding cyO2. C Relative peptide levels determined by MALDI-TOF- MS (n = 4) of O. affinis cyclotides.