Biased clique shuffling reveals stabilizing mutations in cellulase Cel7A.

Area of Science:

• Biotechnology
• Enzyme Engineering
• Renewable Energy

Background:

• Renewable fuels from biomass sugars are crucial.
• Fungal lignocellulase enzymes are cost-effective for biomass saccharification but have suboptimal properties like product inhibition and pH sensitivity.
• Enhanced thermostability is desirable to reduce microbial contamination during saccharification.

Purpose of the Study:

• To develop a mutagenesis platform for improving fungal enzyme properties.
• To enhance the operating temperature and thermostability of fungal glycosyl hydrolase Cel7A.
• To create a superior library of active and stable Cel7A variants.

Main Methods:

• Engineered Saccharomyces cerevisiae for high-titer Cel7A secretion.
• Utilized biased clique shuffling (BCS) on 11 Cel7A genes to create a chimera library.
• Screened the library for improved activity and thermostability.

Main Results:

• Achieved Cel7A secretion at 26 mg/L.
• Generated a library of 469 chimeras with 86% activity.
• Identified 51 chimeras with enhanced thermostability, often with mutations near the active site.
• BCS library outperformed an equimolar library.

Conclusions:

• The developed mutagenesis platform effectively improves fungal enzyme properties.
• BCS is a superior method for generating active and thermostable enzyme variants.
• Engineered Cel7A shows promise for efficient biomass saccharification in renewable fuel production.