Puzzling out the role of plant and fungal laccases with auxiliary enzymes during depolymerization and polymerization of lignin in a polysaccharide environment

Context scientific and Societal Challenges

Woody biomass is an important renewable resource, particularly for use in biorefineries. Rich in secondary cell wall components, this biomass (predominant in terrestrial ecosystems) provides a wide range of valuable molecules, including oligosaccharides. However, second-generation biorefineries that rely on hydrolytic enzymes face several challenges, notably the presence of lignin. This complex phenolic polymer is intricately associated with cell wall polysaccharides and restricts the access of fungal hydrolytic enzymes used for saccharification processes, such as bioethanol production. As a result, overcoming this recalcitrance often requires costly and environmentally burdensome pretreatment steps. Interestingly, both lignin polymerization in plant cell walls and lignin depolymerization by microorganisms involve oxidative enzymes known as laccases. Plant cell wall laccases oxidize small phenolic compounds synthesized in the cytosol before their incorporation into the lignin polymer, whereas fungal laccases can break down lignin into smaller oligomers through the action of natural mediators. In both cases, laccase-mediated oxidation generates phenolic oligolignol radicals that are highly reactive and naturally prone to repolymerization. Yet, under natural conditions, lignin oxidation by fungi does not predominantly lead to repolymerization from these small oligomers. Understanding the mechanisms underlying this phenomenon constitutes one of the key scientific questions addressed by the project.

Project Objectives and Expected Outcomes

The DEOX project aimed to develop an in-depth understanding of lignocellulose construction and deconstruction mechanisms in plants through the use of enzymatic cocktails. More specifically, the project sought to compare the activities of plant and fungal laccases, together with associated auxiliary enzymes, in order to elucidate both lignin biosynthesis and lignin depolymerization processes within environments rich in oligo- and polysaccharides. These investigations were conducted using various substrates embedded in matrices enriched in poly- and oligosaccharides, some of which were grafted with hydroxycinnamic acids. During the project, researchers developed an innovative plant material as well as a novel enzymatic cocktail for saccharification. A range of analytical techniques (including nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FTIR), and gas chromatography-mass spectrometry (GC-MS)) were employed to characterize biomass composition and structure and to evaluate the impact of laccases and associated enzymes on polymers present in poplar wood powder and directly in plant tissue sections. In addition, the high-yield production of plant laccases using heterologous expression systems enabled detailed functional characterization of these enzymes, thereby opening new opportunities for structural studies through protein crystallography.

The DEOX project brought together an original consortium of four partners: INRAE-BBF, INRAE-BIOFORA, VIB (Belgium), and INRAE-BIA whose expertise and scientific interests were highly complementary.

Within the consortium, the PVPP team with the kind help of BIBS was primarily responsible for the structural characterization of biomass modified by DEOX enzymes during saccharification. The objective was to better understand the effects of these enzymes on both polysaccharides and lignin polymers. This work led to the successful completion of two doctoral theses by Fanny Vinter and Elina Raad. Several scientific publications under progress from the project, and two patent applications are currently being prepared.

Schematic representation of biomass at different levels

Project synopsis

• Mai 2022 - Oct 2027
• Coordination : Richard Sibout, unité BIA
• Partners/entities involved : INRAE-BBF, INRAE-BIOFORA, VIB (Belgique) et INRAE-BIA Equipes BIA impliquées
• Funding : ANR

Contact :  Richard Sibout