Expression strategies for the efficient synthesis of antimicrobial peptides in plastids

Abstract

Antimicrobial peptides (AMPs) kill microbes or inhibit their growth and are promising next-generation antibiotics. Harnessing their full potential as antimicrobial agents will require methods for cost-effective large-scale production and purification. Here, we explore the possibility to exploit the high protein synthesis capacity of the chloroplast to produce AMPs in plants. Generating a large series of 29 sets of transplastomic tobacco plants expressing nine different AMPs as fusion proteins, we show that high-level constitutive AMP expression results in deleterious plant phenotypes. However, by utilizing inducible expression and fusions to the cleavable carrier protein SUMO, the cytotoxic effects of AMPs and fused AMPs are alleviated and plants with wild-type-like phenotypes are obtained. Importantly, purified AMP fusion proteins display antimicrobial activity independently of proteolytic removal of the carrier. Our work provides expression strategies for the synthesis of toxic polypeptides in chloroplasts, and establishes transplastomic plants as efficient production platform for antimicrobial peptides.

Appressoria Formation in Phytopathogenic Fungi Suppressed by Antimicrobial Peptides and Hybrid Peptides from Black Soldier Flies

“…Here, CAD-Con was obtained from purified recombination proteins after it was expressed in the E. coli BL21 (DE3) prokaryotic expression system and transformed with a chimeric construct of pET-SUMO-CAD-Con. Hoelecher et al discovered that fusion to SUMO does not appreciably affect the activity of AMPs [58]. Compared with SUMO-C-L, cecropin-like antimicrobial peptide KR12AGPWR6, and hybrid peptide CLP [57,[59][60][61], our hybrid peptide CAD-Con was also expressed normally and exhibited antifungal activities after purification.…”

Efficient in planta production of amidated antimicrobial peptides that are active against drug-resistant ESKAPE pathogens

“…The use of the SUMO domain has further beneficial effects for improved protein accumulation, presumably by increasing protein stability and solubility, thus facilitating AMP purification without adding high concentrations of detergents. A recent publication reported the production of AMPs in plastids using a similar approach involving AMPs fused to an intact SUMO domain 88 . However, compared to our simple expression module for AMP production, which can be applied to many plant species, plastid engineering is sophisticated and suffers from technical hurdles limiting its application.…”

An RNA thermometer in the chloroplast genome of Chlamydomonas facilitates temperature-controlled gene expression

“…systems that are encoded in the plastid genome and would respond to chemical inducers or environmental stimuli), the design of synthetic systems has been pursued. Some progress has been made with the design of synthetic riboswitches ( 35 , 36 , 70 ), but the identification of suitable switches and inducer metabolites has been challenging ( 34 ). This is mainly due to (i) the strong dependence of chloroplast gene expression on post-transcriptional (mainly translational) control, and (ii) the presence of large and diverse metabolite pools inside plastids, which make regulation by external metabolite application difficult and cause substantial leakiness of the switches ( 34 ).…”