Proteome remodeling during fungal depolymerization of PBAT microplastics

Proteome remodeling during fungal depolymerization of PBAT microplastics.

Authors: Olga Rusiecka ¹, Rafał Szewczyk ², Przemysław Bernat ¹

1. 1. University of Lodz, Faculty of Biology and Environmental Protection, Department of Industrial Microbiology and Biotechnology, 12/16 Banacha Str., 90-237 Lodz, Poland.
   2. LabExperts sp z o.o., 7A Limbowa Str., 80-175 Gdansk, Poland.

Introduction

Microplastic contamination is an emerging analytical and environmental challenge, particularly in soils where polymer-derived particles alter ecosystem function. Poly(butylene adipate-co-terephthalate) (PBAT), a biodegradable copolyester extensively applied in agriculture, persists as microplastics due to limited degradation under ambient conditions. While microbial plastic biodegradation has been reported, the molecular and enzymatic mechanisms remain insufficiently resolved at the proteome level. Filamentous fungi represent a promising model due to their extracellular enzyme secretion and metabolic plasticity. Here, we investigate PBAT microplastic biodegradation by Trichoderma harzianum KKP534, applying high-resolution LC-MS/MS–based proteomics to characterize extracellular and intracellular protein expression patterns associated with polymer depolymerization and downstream metabolic processing.

Methods

Proteins were extracted separately from extracellular culture filtrates and intracellular fungal biomass, followed by denaturation, reduction, alkylation, and overnight tryptic digestion. Peptides were purified using C18 solid-phase extraction, detergent removal spin columns and analyzed by microflow LC-MS/MS in trap-and-elute configuration. Data-dependent acquisition was performed on a ZenoTOF 7600 mass spectrometer equipped with an OptiFlow ESI source, covering 400–1200 m/z (MS) and 140–1800 m/z (MS/MS). Peptide and protein identification were conducted using PEAKS software with 10 ppm precursor tolerance and 1% FDR against the Trichoderma harzianum (UniProtKB, 37455 entries). Quantitative comparisons were evaluated across biological replicates using appropriate statistical models.

Preliminary results

Comprehensive LC-MS/MS profiling revealed extensive PBAT-induced proteome remodeling. Across all conditions, 192 extracellular protein groups and 3,438 intracellular protein groups were confidently identified, demonstrating deep proteome coverage. The extracellular proteome was dominated by hydrolytic enzymes, including proteases, glycosidases, cellulases, esterases, and cutinases enzymes with known activity toward ester-containing and hydrophobic substrates. Notably, cutinases, α/β-hydrolase family enzymes capable of accommodating high-molecular-weight substrates, were detected and represent strong candidates for PBAT ester bond cleavage.

Proteomic findings were supported by activity assays showing PBAT concentration- and time-dependent increases in proteolytic and lipolytic activity. Chitinases exhibited delayed induction, consistent with secondary degradation or cell wall remodeling processes. Intracellular proteomic data revealed increased abundance of cytochrome P450 monooxygenases and NADPH–cytochrome P450 reductases, indicating activation of oxidative and xenobiotic-processing pathways following uptake of PBAT degradation products.

Additional identification of redox-associated enzymes suggests involvement of oxidative mechanisms, potentially generating reactive oxygen species that further modify polymer fragments. These MS-driven observations support a multistep biodegradation model: extracellular enzymatic depolymerization of PBAT microplastics into low-molecular-weight intermediates, followed by intracellular oxidation and metabolic assimilation. The detection of terephthalic acid (TPA) in post culture extractions confirmed by complementary LC-MS/MS analyses corroborate the proteomics-based interpretation of active polymer degradation.

This research was funded by the National Science Centre, Poland, grant number 2020/39/B/NZ9/00471.

Novel aspect

High-resolution LC-MS/MS reveals coordinated extracellular hydrolase secretion and intracellular P450-mediated metabolism during fungal PBAT microplastic degradation.

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