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

Abstract

The enzymatic machinery involved in the biosynthesis of lantibiotic is an untapped source of proteases with different specificities. Lanthipeptide biosynthesis requires proteolysis of specific target sequences by known proteases, which are encoded by contiguous genes. Herein, the activity of lichenicidin A2 (LicA2) trimming proteases (LicP and LicT) was investigated in vivo. Firstly, the impact of some residues and the size of the peptide were evaluated. Then followed trials in which LicA2 leader was evaluated as a tag to direct production and secretion of other relevant peptides. Our results show that a negatively charged residue (preferably Glu) at cleavage site is important for LicP efficacy. Some mutations of the lichenicidin hexapeptide such as Val-4Ala, Asp-5Ala, Asn-6Ser, and the alteration of GG-motif to GA resulted in higher processing rates, indicating the possibility of improved lichenicidin production in Escherichia coli. More importantly, insulin A, amylin (non-lanthipeptides), and epidermin were produced and secreted to E. coli supernatant, when fused to the LicA2 leader peptide. This work aids in clarifying the activity of lantibiotic-related transporters and proteases and to evaluate their possible application in industrial processes of relevant compounds, taking advantage of the potential of microorganisms as biofactories. KEY POINTS: • LicM2 correct activity implies a negatively charged residue at position -1. • Hexapeptide mutations can increase the amount of fully processed Bliβ. • LicA2 leader peptide directs LicTP cleavage and secretion of other peptides.

Keywords: Chimeric genes; Lanthipeptides; Lantibiotics; Proteases; Recombinant peptides; Site-directed mutagenesis.

Fig. 1

Lichenicidin gene cluster containing the genes essential for the production of Bliα (green) and for the production of Bliβ (blue); LicT is responsible for cleaving the precursor peptides C-terminally of the double-Gly motif (underlined) and for the transport of both peptides out of the cell. LicP cleaves the remaining hexapeptide attached to Bliβ in the extracellular space; in gray are other genes encoding putative regulatory enzymes and self-immunity genes, not essential for lichenicidin production in E. coli. Adapted from Caetano et al. (2011)

Fig. 2

Schematic representation of the mutations inserted into Bliβ’s hexapeptide. The native sequence is shown on top: hexapeptide in yellow, core peptide in pink, and the remaining leader sequence in blue. The mutations inserted are represented in green when multiple amino acids were replaced or in red for single amino acid mutations. Deletion of an amino acid is represented by a circle containing a “–.” The cleavage sites of LicT and LicP enzymes are also represented

Fig. 3

Quantification of peptide (top) and bioactivity against Kocuria rhizophila (bottom) of Bliβ hexapeptide mutants. The ratio between the native Bliβ concentration/activity (LicA2) is represented in black, green diamonds represent increased quantity/activity, and red diamonds decreased. * indicates statistically significant differences in bioactivity and quantification compared to the control (p < 0.05). ^a) Fully modified Bliβ not detected

Fig. 4

Alignment of LicA2 with hexapeptides from other closely related lantibiotics: haloduracin A2, plantaricin W A2, cytolysins, and cerecidins. White circles represent amino acids conserved among all the sequences, while the decrease in the conservation level is represented by red circles: light red for higher conservation and dark red for lower. At the bottom, representation of the relative frequency of hexapeptide amino acids

Fig. 5

Representation of constructions made with chimeric genes. In blue, LicA2 leader sequence with double Gly motif highlighted; in yellow, the hexapeptide with a Glu residue in position -1; in pink, the core peptide is indicated, which is replaced by various core sequences; position 1 was mutated to Thr if required to maintain the cleavage site. Conditions tested: control, LicP cleavage, LicT cleavage, and transport; LicTP combined activity