Making plants into cost-effective bioreactors for highly active antimicrobial peptides

Abstract

As antibiotic-resistant bacterial pathogens become an ever-increasing concern, antimicrobial peptides (AMPs) have grown increasingly attractive as alternatives. Potentially, plants could be used as cost-effective AMP bioreactors; however, reported heterologous AMP expression is much lower in plants compared to E. coli expression systems and often results in plant cytotoxicity, even for AMPs fused to carrier proteins. We wondered if there were a physical factor that made heterologous AMPs difficult to express in plants. Using a meta-analysis of protein databases, we determined that native plant AMPs were significantly less cationic than AMPs native to other taxa.To apply this finding to plant expression, we tested the transient expression of 10 different heterologous AMPs, ranging in charge from +7 to -5, in the the tobacco, Nicotiana benthamiana. We first tested several carrier proteins and were able to express AMPs only with elastin-like polypeptide (ELP). Conveniently, ELP fusion allows for a simple, cost-effective temperature shift purification. Using the ELP system, all five anionic AMPs expressed well, with two at unusually high levels (375 and 563 µg/gfw). Furthermore, antimicrobial activity against Staphylococcus epidermidis was an order of magnitude stronger (average MIC = 0.26 µM) than that typically seen for AMPs expressed in E. coli expression systems. Unexpectedly, this high level of antimicrobial activity was associated with the uncleaved fusion peptide. In contrast, all previous reports of AMPs expressed in both plant and E. coli expression systems show cleavage from the fusion partner to be required before activity is seen. In summary, we describe a means of expressing AMP fusions in plants in high yield, purified with a simple temperature-shift protocol, resulting in a fusion peptide with high antimicrobial activity, without the need for a peptide cleavage step.

Expression strategies for the efficient synthesis of antimicrobial peptides in plastids

“…Production of AMPs on the surface of a recombinant plant virus that was used to infect plants yielded up to 25 µg/g fresh weight of AMP (for SP1-1 27 , 29 ). By contrast, anionic AMPs fused to the elastin protein and expressed transiently in plants appear to accumulate to higher levels than cationic AMPs, reaching yields of up to 563 µg/g FW, corresponding to 113 µg/g FW of AMP (for ADP2 27 , 32 ). However, cationic AMPs are more versatile antimicrobial agents than anionic AMPs, because they have a stronger binding affinity to microbial membranes, due to the abundant presence of anionic compounds in the membranes of bacteria and fungi (including lipopolysaccharides in gram-negative bacteria, lipoteichoic acid in gram-positive bacteria and mannans in fungi 78 ).…”

“…Expression from the plastid (chloroplast) genome is a particularly attractive method of heterologous protein production in plants, because chloroplasts can accumulate very large amounts of foreign protein 22 – 24 , and offer the additional benefits of transgene exclusion from pollen transmission and the absence of epigenetic transgene silencing 25 . Production of AMPs or antimicrobial proteins in plants has been attempted by transient expression, stable nuclear transformation and plastid transformation 26 – 32 .…”

Smart tools for antimicrobial peptides expression and application: The elastic perspective

“…All the anionic AMPs described as ELP fusion partners are characterized by the presence of intra-chain disulfide bonds, showing a cysteine knot, which is typical for defensin-like structures. Only one was expressed as an ELP fusion in a bacterial recombinant system (Table 1, #5), whereas the other negatively charged AMPs were successfully expressed as ELP fusions in a plant system, (Table 1, #14 to #18) according to the finding that, in general, AMPs of plant origin are significantly less cationic than the others (Ghidey et al, 2020).…”

Advances in the use of plants as potential biofactories in the production of antimicrobial peptides

“…Using algorithms to explore the genome of living organisms available in different databases can reveal thousands of biomolecules of economic interest. [ 1 ] Among the biomolecules with high added value are antimicrobial peptides (AMPs).…”

“…In this context, to explore the diversity of action in in vitro and/or in vivo studies and to obtain a commercial product, large‐scale production may present too many challenges. [ 1,10,11 ]…”