Hydrophobins are exceptional amphiphilic proteins, yet their industrial adoption is hindered by a dual “production-performance” bottleneck: low natural yields and fixed interfacial properties. Vmh2 from Pleurotus ostreatus is particularly noteworthy for its unique self-assembly into long-range helical fibers, offering distinct advantages over traditional rodlet-forming hydrophobins. In this study, we implemented a synergistic optimization strategy to simultaneously overcome yield limitations and enhance interfacial functionality. By introducing site-directed mutations (N76V and S62I) into the critical Cys3–Cys4 and Cys7–Cys8 loops—which dictate surface polarity—and integrating a high-efficiency endogenous Ygyp promoter, a robust heterologous expression platform was established in Tremella fuciformis yeast-like cells. Our results demonstrate that this coordinated approach significantly elevated transcriptional levels to 11.72-fold higher than the wild type, achieving an average hydrophobin output of 20.60 mg/g DW, representing a substantial increase in hydrophobin yields. Biophysical characterization revealed that strengthening hydrophobic patches precisely modulated surface polarity, with the surface hydrophobicity index (S0) of M12 reaching 11.87 (2.5-fold higher than the wild-type). This structural refinement translated into superior foaming and emulsification activities, proving that targeted polarity modulation can bypass the inherent constraints of natural hydrophobins. This research establishes a scalable platform for industrial protein production and provides a theoretical framework for the rational design of next-generation functional biomaterials. Graphical abstract A synergistic strategy for the high-yield production and functional enhancement of hydrophobin Vmh2 in Tremella fuciformis. a Selection of the high-efficiency endogenous yGYP promoter for optimal transcriptional driving. b Site-directed mutagenesis (N76V/S62I) targeting surface-exposed loops to modulate protein polarity. c Construction of a robust heterologous expression platform in YLCs via Agrobacterium-mediated transformation. d Coordinated optimization resulted in an 11.72-fold increase in mRNA levels, a record-breaking recovery yield of 20.60 mg/g, and a 2.5-fold enhancement in S0 value, significantly improving emulsification and foaming activities.

Heterologous expression and interfacial activity evaluation of Pleurotus ostreatus hydrophobin Vmh2 mutants in Tremella fuciformis

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发表日期：

2026年5月27日

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Yuxin Gou,
Aimin Ma

摘要

Hydrophobins are exceptional amphiphilic proteins, yet their industrial adoption is hindered by a dual “production-performance” bottleneck: low natural yields and fixed interfacial properties. Vmh2 from Pleurotus ostreatus is particularly noteworthy for its unique self-assembly into long-range helical fibers, offering distinct advantages over traditional rodlet-forming hydrophobins. In this study, we implemented a synergistic optimization strategy to simultaneously overcome yield limitations and enhance interfacial functionality. By introducing site-directed mutations (N76V and S62I) into the critical Cys3–Cys4 and Cys7–Cys8 loops—which dictate surface polarity—and integrating a high-efficiency endogenous Ygyp promoter, a robust heterologous expression platform was established in Tremella fuciformis yeast-like cells. Our results demonstrate that this coordinated approach significantly elevated transcriptional levels to 11.72-fold higher than the wild type, achieving an average hydrophobin output of 20.60 mg/g DW, representing a substantial increase in hydrophobin yields. Biophysical characterization revealed that strengthening hydrophobic patches precisely modulated surface polarity, with the surface hydrophobicity index (S₀) of M12 reaching 11.87 (2.5-fold higher than the wild-type). This structural refinement translated into superior foaming and emulsification activities, proving that targeted polarity modulation can bypass the inherent constraints of natural hydrophobins. This research establishes a scalable platform for industrial protein production and provides a theoretical framework for the rational design of next-generation functional biomaterials.

Graphical abstract

A synergistic strategy for the high-yield production and functional enhancement of hydrophobin Vmh2 in Tremella fuciformis. a Selection of the high-efficiency endogenous yGYP promoter for optimal transcriptional driving. b Site-directed mutagenesis (N76V/S62I) targeting surface-exposed loops to modulate protein polarity. c Construction of a robust heterologous expression platform in YLCs via Agrobacterium-mediated transformation. d Coordinated optimization resulted in an 11.72-fold increase in mRNA levels, a record-breaking recovery yield of 20.60 mg/g, and a 2.5-fold enhancement in S₀ value, significantly improving emulsification and foaming activities.