Multi-level Integrated Engineering Enhances Neutral β-Glucanase Production in Komagataella phaffii

Area of Science:

• Biotechnology
• Enzyme Engineering
• Industrial Microbiology

Background:

• Neutral β-1,3-glucanase (BgA) is crucial in food processing (brewing, baking, juice clarification).
• Current industrial BgA production faces challenges with low secretion and high costs.

Purpose of the Study:

• To develop an efficient, scalable production system for neutral β-1,3-glucanase (BgA) using Komagataella phaffii.
• To overcome limitations of low secretion titers and high production costs for industrial BgA application.

Main Methods:

• Established a multi-layered engineering framework in K. phaffii.
• Screened promoter and signal peptide libraries for optimal secretion.
• Implemented synergistic transcriptional and translational regulation strategies.
• Validated industrial scalability through fed-batch fermentation.

Main Results:

• Identified P_DAS1 and MFα signal peptide for optimal BgA secretion (5.5 U/mL·OD₆₀₀).
• Co-overexpression of Mxr1 and Mit1 increased BgA titers by 72%.
• Engineering the translation initiation complex yielded a 1.9-fold increase in BgA production.
• Achieved a final BgA titer of 1.1 g/L in a 5-L bioreactor.

Conclusions:

• Demonstrated a robust, integrated engineering strategy for BgA production.
• Successfully established K. phaffii as a competitive host for neutral β-glucanase production.
• Resolved secretory bottlenecks, paving the way for cost-effective industrial enzyme application.