Evolved α-factor prepro-leaders for directed laccase evolution in Saccharomyces cerevisiae

Area of Science:

• Biotechnology
• Molecular Biology
• Enzyme Engineering

Background:

• Functional expression of fungal laccases in Saccharomyces cerevisiae is challenging.
• Signal peptide engineering is a promising strategy to improve enzyme secretion.
• Directed evolution requires efficient methods for assessing enzyme activity and secretion.

Purpose of the Study:

• To investigate the efficacy of native and evolved α-factor prepro-leaders for enhancing the secretion of diverse fungal laccases in S. cerevisiae.
• To evaluate the impact of different prepro-leaders on laccase activity across varying redox potentials.
• To optimize fungal laccase expression for directed evolution applications.

Main Methods:

• Construction of twelve expression vectors fusing S. cerevisiae α-factor prepro-leaders (native and evolved) to four fungal laccases (PM1L, PcL, TspC30L, MtL).
• Cultivation of engineered yeast strains in selective medium with galactose for expression induction.
• High-throughput screening of laccase secretion and activity using low- and high-redox potential mediators in microcultures.
• Flask-based production in rich medium to assess secretion efficiency.

Main Results:

• Evolved prepro-leaders, particularly αPcL, enhanced secretion and activity of medium- and high-redox potential laccases (PcL, PM1L, TspC30L) up to sevenfold compared to the native α-factor prepro-leader.
• The low-redox potential laccase MtL showed a different pattern, with the native prepro-leader yielding the highest expression.
• In flask cultures, evolved prepro-leaders consistently outperformed the native signal peptide, achieving over 50-fold secretion enhancement.
• A clear correlation was observed between prepro-leader type and laccase redox potential in determining secretion efficiency.

Conclusions:

• Evolved α-factor prepro-leaders are crucial for optimizing the functional expression and secretion of fungal laccases in S. cerevisiae.
• The choice of prepro-leader should be tailored to the specific laccase and its redox potential for maximal secretion.
• This strategy is highly effective for improving fungal laccase yields in directed evolution campaigns.