Co-expression of a cyclizing asparaginyl endopeptidase enables efficient production of cyclic peptides in planta

Abstract

Cyclotides are ultra-stable, backbone-cyclized plant defence peptides that have attracted considerable interest in the pharmaceutical industry. This is due to their range of native bioactivities as well as their ability to stabilize other bioactive peptides within their framework. However, a hindrance to their widespread application is the lack of scalable, cost-effective production strategies. Plant-based production is an attractive, benign option since all biosynthetic steps are performed in planta. Nonetheless, cyclization in non-cyclotide-producing plants is poor. Here, we show that cyclic peptides can be produced efficiently in Nicotiana benthamiana, one of the leading plant-based protein production platforms, by co-expressing cyclotide precursors with asparaginyl endopeptidases that catalyse peptide backbone cyclization. This approach was successful in a range of other plants (tobacco, bush bean, lettuce, and canola), either transiently or stably expressed, and was applicable to both native and engineered cyclic peptides. We also describe the use of the transgenic system to rapidly identify new asparaginyl endopeptidase cyclases and interrogate their substrate sequence requirements. Our results pave the way for exploiting cyclotides for pest protection in transgenic crops as well as large-scale production of cyclic peptide pharmaceuticals in plants.

Keywords: Asparaginyl endopeptidase; Nicotiana benthamiana; SFTI; cyclic peptide; cyclotide; kalata B1; plant-made pharmaceutical; transient expression.

Fig. 1.

Cyclotide structure and production in N. benthamiana. (A) The prototypic cyclotide, kalata B1, is encoded by Oak1, which is composed of an ER signal peptide (ER SP), an N-terminal propeptide (NTPP), the mature cyclotide domain and a C-terminal propeptide (CTPP). The precursor protein is proteolytically processed to mature kalata B1 with the final steps involving cleavage at the NTPP (grey arrow) followed by cleavage at the CTPP (black arrow) and subsequent cyclization by peptide bond formation between the first glycine residue and the last asparagine residue of the mature cyclotide domain. The cyclic protein backbone and the knotted arrangement of three disulfide bonds (yellow) impart exceptional stability whilst the loop regions contribute native bioactivities or can be replaced with other bioactive peptides. The ribbon structure is based on PDB code 1NB1. (B) Representative MALDI-TOF mass spectra of peptides produced by transient expression of Oak1 alone or co-expressed with OaAEP1_b in N. benthamiana. The production of predominantly linear peptides when Oak1 is expressed alone in N. benthamiana has been reported previously (Gillon et al., 2008; Conlan et al., 2012). The position of cyclic kB1 is indicated with a dotted line; cyc, cyclic product; lin, linear product.

Fig. 2.

Production of kalata B1, kalata B2 and kalata B3 by transient co-expression of the cyclotide precursors with a cyclizing AEP in N. benthamiana. (A) Oak2(kB2-kB3) and Oak4 are multidomain cyclotide sequences that present different residues from Oak1 at the P1-P1′ positions of the C-terminal AEP processing site (Oak1: NG; Oak2: DG; Oak4: DS). See Fig. 1 for a description of domains and processing steps. (B) Mean percentage of cyclic kB1 (blue bars), cyclic kB2 (dark blue bars), and cyclic kB3 (light blue bars) relative to all assigned peptides±SEM based on mass spectra peak areas for Oak1, Oak2(kB2-kB3), and Oak4 expressed alone or co-expressed with OaAEP1_b, OaAEP2, or OaAEP3. (C) Schematic representation of substrates co-infiltrated with AEPs from Clitoria ternatea. In Oak1-HV the native CTPP sequence of Oak1 (GLPSLAA) was replaced with HV. Cter M is a native cyclotide precursor from C. ternatea comprising the cyclotide domain embedded within an albumin precursor protein (Poth et al., 2011). In Cter M-kB1, the native cyclotide domain was replaced with kB1 within the Cter M precursor. (D) Mean relative percentage of cyclic kB1 relative to all assigned peptides±SEM based on mass spectra peak areas for Oak1-HV and Cter M-kB1 expressed alone or co-expressed with CtAEP1, CtAEP2, or CtAEP6. All data were derived from a minimum of three independent replicates. Different letters indicate significant differences found by Tukey’s ANOVA (P<0.05). Representative MALDI-TOF mass spectra, number of replicates, observed monoisotopic masses and mean relative percentages of all assigned peptides are shown in Supplementary Fig. S2.

Fig. 3.

Production of grafted cyclic peptides in N. benthamiana. Mean relative percentage of cyclic peptide relative to all assigned peptides±SEM based on mass spectra peak areas for Oak1-KNK-kB1 (orange bars), Oak1-DK-kB1 (aqua bars), Oak1-MOG3 (purple bars), and Oak1-kB1(T20K) (green bars) expressed alone or co-expressed with OaAEP1_b or OaAEP3. All data were derived from a minimum of three independent replicates. Different letters indicate significant differences found by Tukey’s ANOVA (P<0.05). Representative MALDI-TOF mass spectra, number of replicates, observed monoisotopic masses and mean relative percentages of all assigned peptides are shown in Fig. S10.