Class II two-peptide lanthipeptide proteases: exploring LicTP for biotechnological applications

Area of Science:

• Biochemistry
• Molecular Biology
• Microbial Biotechnology

Background:

• Lantibiotic biosynthesis involves specific proteases encoded by contiguous genes.
• The enzymatic machinery for lantibiotic production is a source of proteases with unique specificities.
• Lichenicidin A2 (LicA2) trimming proteases (LicP and LicT) play a role in lantibiotic maturation.

Purpose of the Study:

• To investigate the in vivo activity of LicA2 trimming proteases (LicP and LicT).
• To evaluate the impact of specific residues and peptide size on protease activity.
• To assess the potential of the LicA2 leader peptide as a tool for producing and secreting heterologous peptides.

Main Methods:

• In vivo investigation of LicP and LicT protease activity.
• Site-directed mutagenesis of lichenicidin hexapeptide residues and motifs.
• Fusion of heterologous peptides (insulin A, amylin, epidermin) to the LicA2 leader peptide for secretion studies.

Main Results:

• A negatively charged residue, preferably Glutamic acid (Glu), at the cleavage site is essential for LicP protease efficacy.
• Specific mutations in the lichenicidin hexapeptide (e.g., Val-4Ala, Asp-5Ala, Asn-6Ser) and GG-motif to GA alteration increased processing rates.
• Heterologous peptides, including insulin A, amylin, and epidermin, were successfully produced and secreted into the supernatant of Escherichia coli when fused to the LicA2 leader peptide.

Conclusions:

• The study clarifies the activity of lantibiotic-related proteases and transporters.
• Mutations enhancing processing rates offer potential for improved lichenicidin production in E. coli.
• The LicA2 leader peptide can be utilized as a secretion tag for various peptides, demonstrating its industrial application potential in microbial biofactories.