Work

Engineering a native signal peptide and N-glycosylation sites enables active production of a mushroom Hericium erinaceus laccase in the Pichia pastoris expression system

Year: 2025

Type: article

Abstract: Recombinant fungal laccase production in a yeast expression system is one of way to enhance its yield for biotechnological applications. However, fungal laccase genes are sometime not expressed in heterologous hosts. In this study, the previously non-expressed laccase Lac1b from the mushroom Hericium erinaceus was engineered to produce an active form in the Pichia pastoris expression system. Both the introduction of a native signal peptide and the reduction of the degree for N-linked glycosylation sequons were applied to the recombinant laccase Lac1b (rLac1b) produced in this methylotrophic yeast. These two strategies successfully enabled extracellular production of rLac1b with five N-glycosylation sequons (rLac1b_G5), instead of the inactive form with the α-mating factor signal peptide and ten glycosylation sites. Biochemical analysis of purified rLac1b_G5 showed a broad substrate spectrum and strong activity toward sinapinic acid and ferulic acid, which contain a propanoid chain and serve as natural lignin precursors. The highly substrate-specific rLac1b_G5 revealed catalytic efficiencies of 219.8 s −1 mM −1 , 73.6 s −1 mM −1 , 6.7 s −1 mM −1 , and 6.1 s −1 mM −1 toward 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), 2,6-dimethylphenol, guaiacol, and, hydroquinone, respectively. In addition, rLac1b_G5 displayed enhanced oxidation activity in the presence of polyoxyethylene sorbitol ester detergents and stability in miscible organic solvents, benefiting its biocatalytic use by improving substrate solubility in aqueous reactions. Overall, these results suggest that signal peptide choice and glycosylation levels are key factors in obtaining active recombinant laccase in yeast. The biocatalytic properties of rLac1b could support lignin valorization via bioconversion of aromatic precursor compounds.

Source: International Journal of Biological Macromolecules

DOI: 10.1016/j.ijbiomac.2025.147814

Authors: Quang Anh Nguyen, Thuat Van La, Ji Young Kang, Bong Hyun Sung, Seonghun Kim

Institutions: Korea Research Institute of Bioscience and Biotechnology, Korea University of Science and Technology

Language: English

Topic: Fungal and yeast genetics research

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Responsible consumption and production

Open Access status: hybrid

Estimated APC paid: $4,390