Antimicrobial peptides (AMPs), from both natural and synthetic sources, can be a viable alternative for the treatment of bacterial infections since they are active against a broad range of pathogenic microorganisms (Kamal et al., 2023). AMPs can kill Gram-positive and -negative bacteria, fungi, and parasites, and inactivate enveloped viruses (Zhang, 2025). In addition to anti-infective activities, they can also trigger immune responses (Drayton et al., 2020) and eradicate neoplastic cells (Leite et al., 2018). Thus, AMPs have been developed as therapeutic agents for the treatment of many diseases (Drayton et al., 2020).

AMPs are a class of small polypeptides with a primary structure commonly ranging from 12 to 50 amino acid residues (Luo and Song, 2021). According to their secondary structure, the most-studied AMPs are α-helical, having a positive (+2 to +11) net charge and a significant proportion (∼50 %) of hydrophobic residues (Bin Hafeez et al., 2021), but others are predominantly β-sheet, mixed (α-helical/β-sheet), or non-structured, also known as extended peptides (Koehbach and Craik, 2019). AMPs act on their targets through two distinct mechanisms: (i) disruptive, whereby they destabilize the plasmatic membrane in several possible ways, and (ii) non-disruptive, in which they cross the cell plasmatic membrane by spontaneous translocation and act on intracellular targets (Bellotti and Remelli, 2022, Kumar et al., 2018, Parchebafi et al., 2022, Tian et al., 2022).

AMPs are obtained through three different strategies. The first is direct isolation from natural sources, that is, of the organisms that produce them (Franco et al., 2006, Nesa et al., 2022). However, this method has some limitations, such as the environmental impact of extraction and the low yields, the latter because the source organisms produce them when subjected to specific types of stress (Sarkar et al., 2021). The second strategy is chemical synthesis (Almeida et al., 2020, Cardoso et al., 2018, da Silva et al., 2020, Oliveira et al., 2020, Rodrigues et al., 2018). A common technique used for this is fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS; (Dantas et al., 2019, Fensterseifer et al., 2019). The more classical t-butyloxycarbonyl (Boc) SPPS is generally only used for specific applications (Merrifield, 1963, Rossino et al., 2023). Another method used for peptide synthesis is the SPOT technology, a solid-phase peptide synthesis technique that allows the parallel production of a large number of different peptides on a small scale (Cândido et al., 2019, Frank, 1992).

Although chemical synthesis provides a large amount of highly pure synthetic peptides, it has limitations, such as the difficulty of producing peptides longer than 35 residues with disulfide bonds or post-translational modifications (de Oliveira et al., 2020, Zhang et al., 2021). Chemical residues from synthesis are also an issue: large amounts of solvents such as dichloromethane (DCM), tetrahydrofuran (THF), and dimethylformamide (DMF) are used during the iterations of coupling, washing, deprotection, and washing. These solvents are hazardous, pollute the environment, and increase production costs (Somehsaraie et al., 2022).

Conversely, heterologous expression is a sustainable alternative strategy to obtain AMPs correctly folded in high amounts and low cost (Cunha et al., 2016, de Oliveira et al., 2020, Sousa et al., 2016). The selection of the host organism for this method is carried out according to the target peptide features. Recombinant peptides must retain the properties of their wild-type versions (Sinha and Shukla, 2019). The three-dimensional structure, post-translational modifications, and possible toxicity to the host organism itself are important aspects to be analyzed when choosing a heterologous expression system for an AMP (Zhang et al., 2021).

Currently, several heterologous expression systems are available. Some are based on bacteria, mainly Escherichia coli and Bacillus subtilis (Cui et al., 2018, Deng et al., 2017, Gomes et al., 2018, Parachin et al., 2012, Sinha and Shukla, 2019, Wibowo and Zhao, 2018); others use eukaryotes such as the yeast species Saccharomyces cerevisiae and Komagataella phaffii (formerly Pichia pastoris), the plant species Arabidopsis thaliana and Nicotiana tabacum, Chinese hamster ovary cells (CHO), the insect cell lines Sf9 and Sf21 from Spodoptera frugiperda, and Hi5 from Trichoplusia ni ovarian tissues (Karbalaei et al., 2020; María Eugenia Pachón-Ibáñez, Younes Smani, 2017; Rončević et al., 2019; Sampaio de Oliveira et al., 2020). Among these, yeasts have been considered attractive expression systems for recombinant AMP production because they are easy to manipulate, grow rapidly, perform eukaryotic post-translational modifications, and efficiently secrete the produced proteins. They can also achieve high cell densities and protein yields, making manufacturing processes more economical, while the absence of pyrogens, pathogens, or viral inclusions guarantees the safety of the products (Ergün et al., 2019). Particularly, K. phaffii has additional features that make it an excellent chassis for heterologous AMP production aimed at the medical, food, and cosmetic industries. Herein, we will address the main benefits of this expression system.