[P005] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P03-395] Vesicle-based platform technology for biopesticides

○Tharathip Hemthanon¹, Florian Bourdeaux¹, Ulrich Schwaneberg¹ (1. Institute of Biotechnology, RWTH Aachen University, 52074 Aachen (Germany))

Keywords:

Vip3A protein,rainfast biopesticide-loaded vesicles,leaf-binding peptides

With the growing demand for sustainable agriculture, effective alternatives to synthetic pesticides are urgently required. Protein-based pesticides represent a promising alternative, as they are typically highly specific, minimizing off-target effects, and can be tailored through protein engineering. A prominent example is the use of Cry proteins from Bacillus thuringiensis (Bt), which are widely applied in biopesticide formulations and transgenic crops. However, the extensive reliance on a limited set of Cry toxins has led to the emergence of resistant pest populations. In contrast to Cry proteins, which form stable crystalline structures, many alternative protein toxins lack sufficient stability for field application. Vip3A toxins are a notable example: they act synergistically with Cry proteins but are soluble and prone to rapid inactivation. Furthermore, they are easily removed from plant surfaces by rainfall. An effective protein-based biopesticide must exhibit environmental stability, rainfastness, and scalable production. To address these challenges, we developed a vesicle-based delivery platform derived from engineered Escherichia coli. These bacteria produce nanoscale vesicles (400–600 nm) via asymmetric division, which are directly loaded with the target biopesticide during biosynthesis. The vesicles are composed of bacterial membrane components, enabling protection of the encapsulated proteins from environmental stress. Importantly, the resulting vesicles are devoid of genomic DNA and therefore do not qualify as genetically modified organisms. Surface functionalization is achieved through cell surface display technologies, allowing the presentation of leaf-binding peptides that promote adhesion to the hydrophobic wax layer of plant surfaces, thereby enhancing rainfastness. The modular design enables straightforward exchange of the vesicle cargo by substituting the expressed protein during production. For proof of concept, vesicle-producing strains were engineered to express GFP, yielding fluorescent vesicles that enabled visualization and tracking on plant leaves. Vesicles displaying material-binding peptides exhibited enhanced surface retention and strong fluorescence signals, indicating high cargo loading. Subsequently, GFP was replaced with an engineered Vip3A toxin, demonstrating the applicability of the system for biopesticide delivery. The platform is broadly compatible with proteins producible in E. coli, underscoring its versatility. Vesicle-producing strains were generated using CRISPR/Cas9 in combination with λ-Red recombination targeting the MinCDE system. Following fermentation, vesicles can be isolated after removal of viable cells and directly applied as biodegradable biopesticides. This platform offers a promising strategy for enhancing the stability and efficacy of protein-based pesticides in sustainable agriculture.