Considerations for the process development of insect‐derived antimicrobial peptide production

Abstract

Antimicrobial peptides (AMPs) could evolve into new therapeutic lead molecules against multi-resistant bacteria. As insects are a rich source of AMP, the identification and characterization of insect-derived AMPs is particularly emphasized. One challenge of bringing these molecules into market, e.g., as a drug, is to develop a cost-efficient large-scale production process. Due to the fact that a direct AMP isolation from insects is not economical and that chemical synthesis is recommended for peptide sizes below 40 amino acids, a viable option is heterologous AMP production. Therefore, previous knowledge concerning the expression of larger proteins can be adapted, but due to the AMP nature (e.g., small size, bactericide) additional challenges have to be faced during up and downstream processing. Nonetheless the bottleneck for large-scale AMP production is the same as for proteins; mainly the downstream process. This review introduces opportunities for insect-derived AMP production, like the choice of the expression system (based on previously derived data), depending on the AMP nature, as well as new purification strategies like elastin-like peptide/intein based purification strategies. All of these aspects are discussed with regard to large-scale processes and costs.

Reprogramming biological peptides to combat infectious diseases

“…95 Synthetic biology approaches of recombining DNA offer a more sustainable, scalable, and cost-effective production of AMPs than the chemical-synthesis route because synthetic biology offers an unusually high degree of flexibility for genetically engineering microorganisms such as bacteria and yeasts. 96 The heterologous expression of AMPs is generally performed by expressing a fusion protein to facilitate microbial production as well as simplified purification, decreasing toxicity to the producer cell and increasing resistance to enzymatic degradation. Usually, the carrier proteins are then cleaved and separated from AMPs.…”

Recent achievements and perspectives for large-scale recombinant production of antimicrobial peptides

“…Pichia pastoris (P. pastoris) has recently gained popularity as an eukaryotic expression host for the production of AMPs. The advantages of P. pastoris as compared to E. coli are that it facilitates extracellular expression of AMPs and most posttranslational modifications, including disulfide bond formation, O-and N-glycosylation and processing of signal sequences correctly (Müller et al 2015). Similar to E. coli, codon optimization in P. pastoris is needed to avoid translational errors (Meng et al 2017).…”

“…A few fusion tags has made non-chromatography based purification of fusion protein possible, that is, by using a combined elastin-like polypeptide (ELP) and intein (Müller et al 2015;Sousa et al 2016), and four-helix bundle protein DAMP4 (Sun et al 2018a;Sun et al 2018b;Wibowo et al 2017;Wibowo et al 2015;Zhao et al 2015).…”

“…Recombinant DNA technology offers more sustainable, scalable and cost-effective production of AMPs than the chemical-synthesis route due to its flexibility in genetically engineering microorganisms such as bacteria and yeasts (Müller et al 2015). In this system, AMPs are generally expressed as fusion proteins to facilitate microbial production as well as simplified purification.…”

“…Deng et al reviewed different types of cell factories used to produce AMPs, including Escherichia coli, Bacillus subtilis, Pichia pastoris and Saccharomyces cerevisae (Deng et al 2017). Müller et al also reviewed various host strains with added methods for the isolation and purification of AMPs (Müller et al 2015). However, high-cell-density fermentation of bacteria and yeasts in bioreactor to achieve large-scale production of AMPs has not been reviewed.…”