Production Challenges of AMPs

Antimicrobial peptides

Antimicrobial peptides (AMPs) are small molecules capable of acting against various pathogenic microorganisms, such as bacteria and fungi. In nature, they are produced by different organisms, including invertebrates, vertebrates, and even plants^[1].

Their mechanism of action is generally associated with disruption of the plasma membrane through pore formation, leading to cytoplasmic leakage and cell death. In addition, some AMPs can interfere with vital metabolic processes, including protein synthesis, cell wall biosynthesis, and enzymatic activity, thereby inhibiting pathogen growth and survival^[1].

In agriculture, the application of AMPs to control diseases caused by pathogenic microorganisms has proven to be a promising strategy. Conventional phytopathology management techniques rely mainly on the use of pesticides, antibiotics, and other non-sustainable chemical compounds. To address these limitations, our team investigated the main challenges associated with these traditional practices, aiming to understand how AMPs can improve this scenario in global agriculture, particularly in citrus production.

Pesticides

Pesticides are chemical compounds widely used to prevent and control pests, aiming to increase agricultural productivity. Among the commercially available pesticides are herbicides, insecticides, fungicides, and bactericides, which are used to manage different types of plant diseases.

According to data from the Food and Agriculture Organization of the United Nations (FAO), global agriculture applied 3.5 million tons of these compounds in 2021 - twice the amount recorded in 1991, indicating that the consumption and application of agrochemicals are likely to increase over the years^[2]. In Brazil, the scenario is alarming; the same FAO 2021 survey indicated that 719,500 tons of agrochemicals were used in Brazilian crops, making it the largest consumer of pesticides in the world (Figure 1)^[2].

Although the use of pesticides is a type of pest management that contributes to maintaining agricultural productivity, the consequences of using these compounds for human health and the environment are negative and concerning.

From an environmental perspective, the lack of specificity of pesticides leads to the intoxication of non-target organisms, such as birds, beneficial microorganisms, and pollinators. As a result, the biological processes of these living beings, including reproduction, growth, and metabolism are compromised, ultimately disrupting the ecological interactions in which these organisms play essential roles^[3].

In terms of human health, estimates from the United Nations Environment Programme (UNEP) indicate that 3 million people are poisoned by pesticides annually, with approximately 200,000 deaths worldwide^[4]. This high toxicity is largely associated with the ability of agrochemicals to elevate levels of reactive oxygen species in the body, reducing antioxidant defenses and leaving cells vulnerable to oxidative stress.

Consequently, vital components such as proteins, lipids, and nucleic acids become unbalanced, making the human body susceptible to diseases, including poisoning and, in severe cases, cancer^[5].

In citrus production, according to the Pesticide Action Network (PAN), oranges are among the 12 most contaminated foods with agrochemicals, with substances classified as Highly Hazardous Pesticides (HHPs) by the UN (Figure 2). In many cases, residues accumulate in the peel, where fungicides are applied to extend shelf life. However systemic pesticides can penetrate the pulp, making superficial washing insufficient to remove them^[6].

Antibiotics

The use of antibiotics in agriculture began in the mid-1950s, with the application of streptomycin to combat fire blight, caused by the bacterium Erwinia amylovora, in apple and pear orchards. This initial application of antibiotics was highly successful, reducing the incidence of fire blight by 98%. However, by 1962, reports of streptomycin-resistant bacterial strains emerged, and other types of antibiotics were subsequently applied over the years to control this plant disease^[7]. This pattern, based on the introduction of a new antibiotic and the emergence of resistant pathogenic strains, has been observed over the years for different diseases (Figure 3). For instance, bacterial spot and crown gall were initially controlled with a single type of antibiotic, but within a few years, new resistant strains appeared, once again driving disease incidence in agricultural production^[7].

The United States Environmental Protection Agency (EPA) authorized, in 2016, the use of the antibiotics streptomycin and oxytetracycline by Florida citrus growers to combat Greening on an emergency basis. As the disease progressed, the use of these antibiotics was expanded to citrus production in other U.S. states.

However, both the Food and Drug Administration (FDA) and the Centers for Disease Control and Prevention (CDC) opposed this measure, citing concerns about the potential emergence of resistant strains of the pathogenic bacterium Candidatus Liberibacter sp.^[8]

Other crop protection products

In citrus farming, copper is a mineral widely applied to control fungal and bacterial diseases. This compound is sprayed onto the leaf surface of orange trees, acting against pathogens by inhibiting the activity of their proteins and enzymes, which causes several cellular disturbances such as degradation of the lipid membrane and DNA^[9].

Frequent applications of copper result in the accumulation of this compound in the soil, which is then absorbed by the roots of orange trees. High levels of copper directly affect citrus physiology, leading to increased oxidative stress and, consequently, various metabolic dysfunctions. The cupric ion (Cu²⁺) can irreversibly bind to the active sites of key metabolic enzymes, inactivating their functions. Moreover, studies indicate that high concentrations of copper also prevent the optimal absorption of manganese and zinc by the roots, rendering plants deficient in these components^[9].

Copper applications in citrus production have significant environmental impacts. Due to soil leaching processes, high concentrations of copper reach various ecosystems, including aquatic ecosystems^[10]. In these systems, studies indicate that the sensory systems of fish, particularly the olfactory system, are highly affected by exposure to high copper levels, due to alterations in gene transcription pathways. Additionally, dysfunction in gill osmoregulation has been reported, caused by the inactivation of enzymes essential for maintaining ionic balance (Figure 4)^[10].

Benefits of Using AMPs in Agriculture

As previously discussed, the management strategies adopted in agriculture, especially in citrus production, present challenges that negatively affect both the environment and human health. Within this scenario, the use of AMPs in combating the main phytopathologies that reduce orange production emerges as a very promising alternative. Naturally produced by a wide range of organisms, AMPs stand out for their targeted mode of action and their reduced environmental footprint compared to conventional pesticides^[11].

The main advantage of using AMPs lies in reducing environmental impacts. Unlike broad-spectrum pesticides, which can cause imbalances in the agricultural ecosystem, AMPs are biodegradable and more specific to target pathogens. This favors more sustainable citrus production, in line with market demands for safe fruits and environmentally responsible farming practices^[12,13].

Nevertheless, it is important to acknowledge a limitation of AMP use: the need for recurrent applications. Since these compounds can be rapidly degraded in the environment, their effectiveness tends to be limited in the short term, requiring continuous management to ensure plant protection. In this context, our project seeks to identify these challenges related to AMP application and to develop solutions that enable their use as a tool for integrated management in orange orchards.

Identification of AMPs for Application in Agriculture

Many antimicrobial peptides have been identified and described in scientific articles, however, not all of them show potential for direct application in agriculture. The biochemical characteristics and modes of action of these molecules must be carefully considered before conducting experiments on infected plants. In this context, our team conducted an extensive literature review to understand which AMPs have been primarily explored for agricultural applications, especially in citrus production, and to identify their target pathogens and associated phytopathologies.

Tachyplesin I is a natural cationic antimicrobial peptide with a β-hairpin structure, isolated from the horseshoe crab. Studies report its activity against fungi and both Gram-positive and Gram-negative bacteria. However, the application of this AMP presents challenges due to its hemolytic activity on human erythrocytes^[14]. In the context of combating Greening, there are reports of the application of Tachyplesin I in cultures of Agrobacterium tumefaciens and Sinorhizobium meliloti, used as models for Candidatus Liberibacter sp. These experiments confirmed the effectiveness of this AMP at low minimum inhibitory concentrations^[15].

SMAP-29 is a semi-synthetic antimicrobial peptide of 29 amino acids, derived from sheep cathelicidins and obtained by chemical synthesis in its amidated C-terminal form, which confers a helical structure essential for antimicrobial activity. In the literature, there are records of this peptide’s activity against medically relevant bacteria and fungi, such as Pseudomonas aeruginosa and Cryptococcus neoformans^[16]. In the context of Greening, under the same experimental conditions as Tachyplesin I, the application of SMAP-29 is described as highly effective, with low minimum inhibitory concentrations^[15].

D4E1 is a synthetic antimicrobial peptide active against a broad range of fungi and bacteria. Among the fungal species most susceptible to D4E1 are Thielaviopsis basicola, Verticillium dahliae, Fusarium moniliforme, Phytophthora cinnamomi, and Phytophthora parasitica. Meanwhile, Pseudomonas syringae and Xanthomonas campestris are the most susceptible bacterial species^[17]. In Greening models, under the same experimental conditions as Tachyplesin I and SMAP-29, the application of D4E1 is also described as highly effective, with low inhibitory concentrations and low hemolytic activity^[15].

Melittin is a natural linear cationic antimicrobial peptide identified in the venom of the honeybee Apis mellifera. Several studies have explored the medical applications of this AMP, including against certain types of cancer. However, the main challenge of its application lies in its high hemolytic activity on human erythrocytes^[18]. In the context of Greening, under the same experimental conditions as Tachyplesin I, SMAP-29, and D4E1, the application of Melittin is described as highly effective, with low minimum inhibitory concentrations^[15].

Cecropin B is a natural peptide with lytic activity identified in insects such as Hyalophora cecropia. It is effective against Gram-negative and some Gram-positive bacteria at low concentrations without harming eukaryotic cells^[19]. Moreover, in the context of Greening, studies indicate that the expression of the Cecropin B gene in infected plants can confer greater resistance to the disease^[20].

Attacin A is a natural antimicrobial peptide secreted by insects in response to bacterial infections. This AMP acts mainly against Gram-negative bacteria, and its use has been explored synergistically with cecropins and lysozymes. In the literature, it is also reported that transgenic orange trees expressing the AttA peptide showed increased resistance to Xanthomonas axonopodis, the causal agent of Citrus Canker^[21]. In the context of Greening, there are no reports of the application of this AMP.

MaSAMP is a natural antimicrobial peptide isolated from the plant Microcitrus australasica, known as the Australian finger lime. It functions both by lysing pathogenic bacteria and by inducing plant resistance genes that activate systemic defense responses, showing potential against bacterial phytopathologies, including Greening^[22,23].

Ctx

From the literature review of AMPs, we were able to recognize the need to quantify important parameters for the application of these molecules, such as hemolytic activity and the minimum inhibitory concentration (MICs) against target pathogens. In addition, we understood that the AMPs listed are important molecules that could be used in the future by adapting our production and biotechnological strategies, as well for the hardware development.

However, the Pepcitrus project was based on the application of an AMP that is little explored in the literature and had not been previously mentioned. This antimicrobial peptide is called Ctx, and it was introduced to us by Prof. Dr. Eduardo Vicente from São Paulo State University (UNESP), who contributed to the characterization of this molecule and conducted experiments to assess its effectiveness against a range of microorganisms. In addition, the professor also evaluated the hemolytic activity of Ctx and found, through in vitro tests, that the AMP loses its hemolytic potential once ingested and processed by the digestive system. This feature is essential for our project, although our application does not involve the ingestion of the AMP by animals or humans, it is important that our molecule is not harmful to health, considering possible accidental contaminations.

A key factor that motivated our interest in Ctx is its origin. Ctx is a natural antimicrobial peptide isolated from the frog Hypsiboas albopunctatus, a species native to the Brazilian Cerrado (Figure 5)^[24]. By using this AMP, our project not only explores an underutilized molecule but also highlights and values Brazil’s biodiversity.

Production challenges of AMPs

Another important aspect to be considered in our project is related to the production of AMPs. To this end, we sought to understand the commercialization of antimicrobial peptides in the current context, as well as the challenges behind the synthesis of these molecules.

The antimicrobial peptide market has shown steady growth in recent years, driven mainly by concerns about resistance to conventional antibiotics and the need for novel therapeutic solutions across various sectors, such as healthcare, agriculture, and livestock. According to a report by Mordor Intelligence, the global antimicrobial peptide market is projected to grow at a compound annual growth rate (CAGR) of 5.1% between 2024 and 2029, reaching a value of approximately USD 7.85 billion by 2029 (Figure 6)^[25].

Although the AMP market is of great global interest, the production of these molecules faces several obstacles. Currently, peptide synthesis is predominantly carried out through chemical pathways, notably by solid-phase peptide synthesis (SPPS). This technique allows precise and highly pure sequence synthesis; however, it is costly, time-consuming, has low yield, and requires highly toxic chemical solvents^[26].

Given this scenario, the production of antimicrobial peptides through fermentation represents a promising solution to address these challenges. This process enables the production of peptides using genetically modified microorganisms capable of converting carbon sources into AMPs, offering significant advantages: reduced production costs, improved scalability, and a sustainable, environmentally friendly process. Consequently, fermentation enhances the long-term accessibility of these peptides in the market^[27].

Key Aspects of AMP Production in Our Project

During the design of our project, we carefully considered structural and mechanistic aspects of AMPs that pose challenges for their biotechnological production.

From the conversations we had with our stakeholders, especially with Peptidus, we were able to understand the real challenges associated with AMP synthesis by microorganisms, which often result in low production yields. Based on these insights, we discussed several strategies used in the industrial sector to improve our project. Understanding mechanisms such as in tandem production was essential for the design of our expression cassette, facilitating complex downstream processes like peptide purification.

Another important aspect considered in the design of our project refers to the mechanism of action of AMPs. Since antimicrobial peptides cause the disruption of the plasma membrane of pathogenic microorganisms, they can also harm the microbial production chassis, making the biological synthesis of these peptides unfeasible. To overcome this, we designed genetic constructs based on coupling our AMP to larger proteins, such as GFP and yeast cell wall-associated proteins, in order to temporarily inactivate the antimicrobial activity of the peptide. In addition, we designed strategies to ensure the separation of our molecule of interest from the larger fused proteins through cleavage and purification techniques. Altogether, this set of strategies enabled AMP production by our chassis without degrading them.

It is important to note that our strategy of coupling our peptide to larger proteins was inspired by natural AMP biosynthesis in animals, especially frogs. Buforin I is an AMP isolated from the toad Bufo gargarizans, which synthesizes this peptide naturally fused to the histone H2A. Upon microbial challenge, part of histone H2A is cleaved, releasing active AMP Buforin I. This mechanism ensures that the AMP is activated only when needed, preventing damage to the host’s metabolism^[29]. Using the same logic, our peptide remains fused to another protein during its synthesis and is later cleaved to exert its antimicrobial activity exclusively against pathogenic microorganisms.

Acceptance of AMPs and the International Market

The European market, the primary destination for Brazilian orange juice, values products with high food safety standards and low environmental impact. Implementing AMPs as an alternative or complement to traditional fungicides can not only improve the reputation of the Brazilian citrus production chain among European consumers, but also meet the strict regulatory requirements of the European Union, especially regarding the reduction of pesticide residues and sustainable agricultural practices.

Given the risks associated with the prolonged use of agrochemicals—such as toxicity, food residues, and pathogen resistance—the adoption of AMPs offers a sustainable, effective, and socially responsible solution for pre- and post-harvest disease management. Substituting or combining AMPs with lower doses of chemical products can markedly decrease negative impacts on human health and the environment, while also increasing market acceptance and consumer confidence internationally.

Thus, investing in orange production represents not only a focus on a high-value agricultural commodity but also a contribution to global health and food security. In light of current phytosanitary challenges, its preservation and valorization are both urgent and essential.