Institute for Molecular Bioscience, Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland, Brisbane, Queensland, 4072, Australia.
Institute for Molecular Bioscience, Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland, Brisbane, Queensland, 4072, Australia.
Phytochemistry. 2022 Mar;195:113053. doi: 10.1016/j.phytochem.2021.113053. Epub 2021 Dec 16.
Cyclotides are a class of ribosomally-synthesized plant peptides that function in plants as a defense against insects and fungal pathogens. Their unique structure comprises a cyclized peptide backbone threaded by three disulfide bonds, that imparts structural stability, a desirable quality for peptide-based therapeutics or insecticides. Producing these peptides synthetically is challenging due to the amount of chemical waste produced and inefficiency of folding certain cyclotides. Thus, it is desirable to develop a means to access cyclotide biosynthesis in their native hosts, cultured in defined conditions, at both laboratory and commercial scale. Here we developed suspension cell cultures from two species previously unexplored for cyclotide production in suspension cells, Clitoria ternatea L., Hybanthus enneaspermus F. Muell., as well as with Oldenlandia affinis (Roem. & Schult.) DC., a species reported previously to accumulate cyclotides in cell suspensions. We assessed the growth rate, cyclotide production and gene expression for the various species. We found that while many cyclotides had reduced expression in Oldenlandia affinis suspension cells when compared to plant organs, those in Clitoria ternatea and Hybanthus enneaspermus maintained or increased expression levels. The cyclotides that continued to be expressed in suspension cultures shared similar sequence and biophysical properties as a group, regardless of phylogenetic origin of the host. Of particular interest was the discovery of inducibility by NaCl of cyclotide expression in O. affinis, cycloviolacin O2 expression in O. affinis, and the scale up of cycloviolacin O2 production in H. enneaspermus. Together the results presented here highlight the utility of plant cell suspensions as modalities to produce macrocyclic peptides.
环肽是一类核糖体合成的植物肽，在植物中作为抵御昆虫和真菌病原体的防御物发挥作用。它们独特的结构由一个环化的肽骨架组成，由三个二硫键贯穿，赋予了结构稳定性，这是肽类治疗药物或杀虫剂的理想特性。由于产生这些肽的化学废物产生量和某些环肽折叠的效率低下，因此合成这些肽具有挑战性。因此，人们希望开发一种方法，在其天然宿主中获得环肽生物合成，在定义的条件下，在实验室和商业规模上进行培养。在这里，我们从两个以前在悬浮细胞中环肽生产中未被探索的物种中开发了悬浮细胞培养物，即三叶蝴蝶藤（Clitoria ternatea L.）和密序山蚂蝗（Hybanthus enneaspermus F. Muell.），以及老鹳草（Oldenlandia affinis (Roem. & Schult.) DC.），该物种以前被报道在细胞悬浮液中积累环肽。我们评估了各种物种的生长速度、环肽产量和基因表达。我们发现，虽然与植物器官相比，许多环肽在老鹳草悬浮细胞中的表达减少，但三叶蝴蝶藤和密序山蚂蝗中的环肽维持或增加了表达水平。在悬浮培养物中继续表达的环肽共享相似的序列和物理化学性质，无论宿主的系统发育起源如何。特别有趣的是发现 NaCl 可诱导老鹳草中环肽的表达、O. affinis 中环维奥啉 O2 的表达，以及 H. enneaspermus 中环维奥啉 O2 产量的扩大。这里呈现的结果共同强调了植物细胞悬浮液作为生产大环肽的一种手段的实用性。
