Rational domestication of a plant-based recombinant expression system expands its biosynthetic range

Abstract

Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead, and an orally active analgesic is demonstrated.

Keywords: Asparaginyl endopeptidase (AEP); CRISPR/Cas9; cyclotide; gene editing; insecticide; peptide; plant molecular farming; protease; recombinant; therapeutic.

Fig. 1.

Therapeutic peptide expression in N. benthamiana. (A) SFTI-1, SFTI-1_N, and therapeutic peptide candidates were prepared for plant-based expression by insertion of the peptide-coding sequence into the Oak1 gene, replacing the sequence domain for the cyclotide kB1. The processing of the engineered SFTI-1 precursors is predicted to be controlled by a papain-like cysteine protease (PLCP) and an asparaginyl endopeptidase (AEP) at the N- and C-terminus, respectively. The Oak1 signal peptide (SP), N-terminal propeptide (NTPP), and N-terminal repeat (NTR) sequence ensure that the precursor enters the endomembrane system with targeting towards the vacuole. (B) Co-expression of engineered SFTI-1 precursors with the AEP ligase OaAEP1b in N. benthamiana resulted in MALDI-MS detection of either degraded peptides (shaded in peach) or cyclic peptides (shaded in purple).

Fig. 2.

Comparison of recombinant peptide endogenous processing in wild-type versus ΔAEP N. benthamiana. (A) The Oak1 precursor is predicted to be processed initially at the N-terminus by a papain-like cysteine protease (PLCP) (blue triangle), followed by asparaginyl endopeptidase (AEP) processing (green triangle) at the C-terminus. Without the co-expression of an AEP ligase, endogenous AEPs that prefer hydrolysis over ligation will compete for the expressed substrate with amino- and carboxypeptidases (red triangles). (B) MALDI-TOF-MS analysis (representative) of Oak1_HIIAA expression in wild-type N. benthamiana (NB) alongside the ΔAEP gene-edited accession. Only the signals highlighted in green can be attributed to endogenous AEP processing, with the remainder representing carboxypeptidase C-terminal processing events. (C) Mean and SD (n=3) of the individual kB1 identified masses as a percentage of the total kB1 peptide MS signal detected by MALDI-TOF-MS.

Fig. 3.

Comparative expression of cyclic peptides in wild-type versus ΔAEP N. benthamiana. Representative MALDI-TOF-MS of cyclic peptides accumulated in (A) wild-type N. benthamiana (NB) and (B) the ΔAEP accession in transient co-expression with OaAEP1b. Test cyclotides are encoded in the Oak1 precursor that natively contains kB1, or with kB1 swapped for kB2, a cyclotide that ends with an Asp residue. MS signals for cyclic peptides are highlighted to match the colours in (C) and (D). An arrow indicates the MS signal for the internally spiked peptide control that served to normalize MS signals for relative quantification. (C) Mean and SD (n=3) of relative kB1 and kB2 MS signals detected in crude peptide extracts of infiltrated N. benthamiana (NB) and the ΔAEP accession. (D) Mean and SD (n=3) of relative SFTI-1_KLK4_N, SFTI-1_KLK4_D, and SFTI-1_KLK5_N MS signals detected in crude peptide extracts of infiltrated N. benthamiana (NB) and the ΔAEP accession.

Fig. 4.

Rescue of AEP function in ΔAEP N. benthamiana. Representative MALDI-TOF-MS of (A) cyclic SFTI-1_KLK4_N and (B) cyclic kB1 accumulation in the ΔAEP accession upon co-expression of peptide precursor, AEP ligase, and NbAEP genes. (C) Mean and SD (n=3) of the percentage of MS signal representing cyclic kB1 upon co-expression.

Fig. 5.

Expression of two recombinant peptides negatively affected by endogenous AEP activity. (A) The insecticidal peptide Pa1b is encoded by the pea albumin 1 gene (PA1 gene) that additionally codes for the larger PA1a domain of unknown function. The Pa1b N-terminus is predicted to be released via a signal peptide (SP) cleavage event during co-translation into the endoplasmic reticulum. The protease responsible for the release of the Pa1b C-terminus is unknown. Two internal Asn sites are present (red triangles) that may represent putative AEP processing sites. (B) Representative MALDI-TOF-MS of crude peptide extracts prepared from infiltrated leaves of N. benthamiana (NB) and ΔAEP. Indicated by an arrow is the MS signal for the internally spiked peptide control that served to normalize MS signals for relative quantification. (C) Mean and SD (n=3) of the relative MS signal representing Pa1b-G and Pa1b-G(Mox) peptides. (D) The conotoxin Vc1.1 and its analogue Vc1.1[N9W] were prepared for plant-based expression by insertion of peptide-coding sequence into the Oak1 gene, replacing both the cyclotide kB1 domain and the C-terminal tail. Processing was predicted to be controlled by a papain-like cysteine protease (PLCP) (brown arrow). One internal Asn site (indicated by a red arrow) is present in Vc1.1, substituted for a Trp in Vc1.1[N9W]. (E) Representative MALDI-TOF-MS of crude peptide extracts prepared from infiltrated leaves of N. benthamiana (NB) and ΔAEP. (F) Mean and SD (n=3) of the relative MS signal representing Vc1.1 and Vc1.1[N9W].