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Background <p>One of the most prominent mechanisms for plant cell wall deconstruction in nature, and widely employed in industry, relies on the coordinated action of hydrolytic and oxidative enzymes. However, how redox networks sustain synergistic biomass deconstruction remains incompletely understood, particularly in the industrial workhorse <i>Trichoderma reesei</i>. This fungus lacks a cellobiose dehydrogenase (CDH), a pivotal redox partner for lytic polysaccharide monooxygenases (LPMOs) in many fungal systems. Here, we investigated the oxidative machinery of <i>T. reesei</i> and the contribution of key redox-active enzymes to lignocellulose deconstruction.</p> Results <p>We demonstrate that the oxidative capacity of the <i>T. reesei</i> secretome is largely driven by a single enzyme, <i>Tr</i>LPMO9A, the most abundant oxidoreductase in the secretome. Proteomic analyses also revealed a&#xa0;lower abundance of other redox-active enzymes, including <i>Tr</i>LPMO9B and AA5 oxidase. Although deletion of <i>Tr</i>LPMO9B and <i>Tr</i>AA5 had a less pronounced impact on saccharification efficiency compared with <i>Tr</i>LPMO9A, the secretome remodeling triggered by their deletion, along with the associated decrease in saccharification performance, indicates that these redox enzymes play distinct, non-redundant roles. They likely play a system-level role within a cooperative redox network that fuels oxidative cellulose deconstruction, potentially extending beyond direct catalysis to processes associated with redox balance or protein secretion. Finally, we challenged the CDH-lacking paradigm by heterologously expressing a CDH in <i>T. reesei</i>. In vivo reconstitution of this redox duet increased biomass saccharification by 13–19%, demonstrating a strong synergistic relationship between LPMOs and CDHs even in a native CDH-lacking host.</p> Conclusion <p>These findings define the core oxidative machinery underlying biomass deconstruction in <i>T. reesei</i>, revealing the major cellulose-oxidative role of <i>Tr</i>LPMO9A and the importance of a cooperative redox network for efficient lignocellulose depolymerization. Moreover, successful reconstitution of CDH activity in a naturally CDH-deficient host establishes redox engineering as a promising strategy to enhance industrial biomass conversion.</p>

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TrLPMO9A drives oxidative cellulose depolymerization in Trichoderma reesei and is enhanced by heterologous cellobiose dehydrogenase expression

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月14日

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  • Priscila Thihara Rodrigues,
  • César Rafael Fanchini Terrasan,
  • Nathalia Rodrigues Bulka,
  • Evandro Antonio de Lima,
  • Jose Alberto Diogo,
  • Renan Yuji Miyamoto,
  • Felipe Jun Fuzita,
  • Joaquim Martins Junior,
  • Lucia Daniela Wolf,
  • Adriano Freitas Lima,
  • Douglas Alvaredo Paixão,
  • Meiski Maria Vedovatto,
  • Lara de Oliveira Arinelli,
  • William Godoy,
  • Gabriela Felix Persinoti,
  • Fernanda Mandelli,
  • Mario Tyago Murakami

摘要

Background

One of the most prominent mechanisms for plant cell wall deconstruction in nature, and widely employed in industry, relies on the coordinated action of hydrolytic and oxidative enzymes. However, how redox networks sustain synergistic biomass deconstruction remains incompletely understood, particularly in the industrial workhorse Trichoderma reesei. This fungus lacks a cellobiose dehydrogenase (CDH), a pivotal redox partner for lytic polysaccharide monooxygenases (LPMOs) in many fungal systems. Here, we investigated the oxidative machinery of T. reesei and the contribution of key redox-active enzymes to lignocellulose deconstruction.

Results

We demonstrate that the oxidative capacity of the T. reesei secretome is largely driven by a single enzyme, TrLPMO9A, the most abundant oxidoreductase in the secretome. Proteomic analyses also revealed a lower abundance of other redox-active enzymes, including TrLPMO9B and AA5 oxidase. Although deletion of TrLPMO9B and TrAA5 had a less pronounced impact on saccharification efficiency compared with TrLPMO9A, the secretome remodeling triggered by their deletion, along with the associated decrease in saccharification performance, indicates that these redox enzymes play distinct, non-redundant roles. They likely play a system-level role within a cooperative redox network that fuels oxidative cellulose deconstruction, potentially extending beyond direct catalysis to processes associated with redox balance or protein secretion. Finally, we challenged the CDH-lacking paradigm by heterologously expressing a CDH in T. reesei. In vivo reconstitution of this redox duet increased biomass saccharification by 13–19%, demonstrating a strong synergistic relationship between LPMOs and CDHs even in a native CDH-lacking host.

Conclusion

These findings define the core oxidative machinery underlying biomass deconstruction in T. reesei, revealing the major cellulose-oxidative role of TrLPMO9A and the importance of a cooperative redox network for efficient lignocellulose depolymerization. Moreover, successful reconstitution of CDH activity in a naturally CDH-deficient host establishes redox engineering as a promising strategy to enhance industrial biomass conversion.