Biosensor-Based Optimization of Cutinase Secretion by Corynebacterium glutamicum

Area of Science:

• Biotechnology
• Molecular Biology
• Industrial Microbiology

Background:

• Corynebacterium glutamicum is a key industrial host for recombinant protein secretion.
• Optimizing heterologous protein secretion is crucial for biotechnological applications.
• Developing efficient reporter systems aids in strain engineering for enhanced protein production.

Purpose of the Study:

• To optimize the secretion of Fusarium solani pisi cutinase using a fluorescence-based reporter system in C. glutamicum.
• To identify improved signal peptides and protein variants that enhance cutinase secretion and activity.
• To demonstrate the utility of a biosensor for directed evolution of secretory strains.

Main Methods:

• Development and application of a fluorescence-based (eYFP) C. glutamicum reporter strain.
• Utilized split green fluorescent protein (GFP) assay for cutinase detection and quantification.
• Employed error-prone PCR for mutant library generation and fluorescence-activated cell sorting (FACS) for screening.
• Applied site-directed mutagenesis to validate identified mutations.

Main Results:

• Identified five PelSP variants with 4- to 6-fold increased cutinase secretion (up to 4,100 U/L).
• Discovered two NprESP variants improving secretion by ~35% (achieving ~5,500 U/L).
• Found mutations in the signal peptide's h-region increased hydrophobicity, boosting secretion.
• Isolated a cutinase variant with increased specific activity due to an A85V mutation.

Conclusions:

• The biosensor-based approach significantly improved cutinase secretion by C. glutamicum.
• Engineered signal peptides, particularly PelSP variants, enhanced secretion efficiency.
• The developed reporter system is a valuable tool for optimizing secretion of any target protein.