Production of a structurally validated cyclotide in rice suspension cells is enabled by a supporting biosynthetic enzyme.

We demonstrate the production of a structurally correct cyclotide in rice suspension cells with co-expression of a ligase-type AEP, which unlocks monocotyledons as production platforms to produce cyclotides. Cyclotides are a class of backbone-cyclic plant peptides that harbor a cystine knot composed of three disulfide bonds. These structural features make cyclotides particularly stable, and thus they have attracted significant attention for their use in biotechnological applications such as drug design. Currently, chemical synthesis is the predominant strategy to produce cyclotides for research purposes. However, synthetic production becomes costly both economically and environmentally at large scale. Plants offer an attractive alternative to chemical synthesis because of their lower cost and environmental footprint. In this study, rice suspension cells were engineered to produce the prototypical cyclotide, kalata B1 (kB1), a cyclotide with insecticidal properties from the African plant Oldenlandia affinis. Engineered rice cells produced structurally validated kB1 at yields of 64.21 µg/g (DW), which was dependent on the co-expression of a peptide ligase-competent asparaginyl endopeptidase OaAEP1 from O. affinis. Without co-expression, kB1 was predominantly produced as linear peptide. Through HPLC-MS co-elution, reduction, alkylation, enzymatic digestion, and proton NMR analysis, kB1 produced in rice was shown to be structurally identical to native kB1. This study reports the first example of an engineered plant suspension cell culture with the required molecular machinery for efficient production and cyclisation of a heterologous cyclotide.