Institute for Molecular Bioscience, Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland, Brisbane, QLD, 4072, Australia.
Planta. 2020 Nov 5;252(6):97. doi: 10.1007/s00425-020-03505-z.
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.
我们展示了在水稻悬浮细胞中通过共表达连接酶型 AEP 来生产结构正确的环肽，这为生产环肽解锁了单子叶植物这一生产平台。环肽是一类具有三对二硫键组成的半胱氨酸结的植物肽。这些结构特征使环肽特别稳定，因此它们在生物技术应用中引起了广泛关注，如药物设计。目前，化学合成是生产环肽用于研究目的的主要策略。然而，大规模的化学合成在经济和环境方面都变得非常昂贵。由于成本较低且对环境的影响较小，植物为化学合成提供了一种有吸引力的替代方案。在这项研究中，我们通过工程改造水稻悬浮细胞来生产原型环肽 kalata B1（kB1），这是一种来自非洲植物 Oldenlandia affinis 的具有杀虫特性的环肽。工程化的水稻细胞以 64.21 µg/g（DW）的产率生产出结构验证的 kB1，这依赖于从 O. affinis 共表达具有肽连接酶活性的天冬酰胺内肽酶 OaAEP1。没有共表达时，kB1主要以线性肽的形式产生。通过 HPLC-MS 共洗脱、还原、烷基化、酶消化和质子 NMR 分析，表明在水稻中生产的 kB1 在结构上与天然 kB1 相同。这项研究报告了首例具有生产和环化异源环肽所需分子机制的工程植物悬浮细胞培养的例子。
