Molecular optimization of autotransporter-based tyrosinase surface display

Area of Science:

• Microbiology and Biotechnology
• Protein Engineering
• Biocatalysis

Background:

• Cell surface display of recombinant enzymes in Gram-negative bacteria, like Escherichia coli (E. coli), is crucial for applications in whole-cell biocatalysis, environmental pollutant degradation, and affinity screening.
• The autotransporter system is a common method for achieving surface display, but it often suffers from low expression levels and truncated proteins, limiting overall cell-specific activity.

Purpose of the Study:

• To optimize the autotransporter expression system for enhanced display of tyrosinase on the E. coli cell surface.
• To identify key components of the autotransporter vector that influence protein translocation and activity.

Main Methods:

• Systematic evaluation of autotransporter vector variants, including modifications to the promoter region, signal peptide, passenger protein, linker regions, and translocation unit.
• Monitoring the impact of these modifications on tyrosinase translocation via cell-specific activity assays and antibody-based flow cytometry to quantify full-length and degraded passenger proteins.

Main Results:

• Optimization strategies successfully increased the amount of full-length tyrosinase displayed on the E. coli cell surface.
• A five-fold increase in cell-specific activity was achieved compared to the initial autotransporter system.
• Testing seven different heterologous translocation units demonstrated functional expression, with only a 1.6-fold difference in activity among them.

Conclusions:

• The study successfully enhanced tyrosinase surface display and activity on E. coli through autotransporter system optimization.
• These findings provide a foundation for expanding the range of proteins amenable to surface display and developing novel autotransporter-based industrial biocatalysis processes.