Construction of an expression system for site-directed mutagenesis of the lantibiotic mersacidin

Abstract

The lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide of 20 amino acids which is produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II. The structural gene of mersacidin (mrsA) and the genes for the enzymes of the biosynthesis pathway, dedicated transporters, producer self-protection proteins, and regulatory factors are organized in a biosynthetic gene cluster. For site-directed mutagenesis of lantibiotics, the engineered genes must be expressed in an expression system that contains all of the factors necessary for biosynthesis, export, and producer self-protection. In order to express engineered mersacidin peptides, a system in which the engineered gene replaces the wild-type gene on the chromosome was constructed. To test the expression system, three mutants were constructed. In S16I mersacidin, the didehydroalanine residue (Dha) at position 16 was replaced with the Ile residue found in the closely related lantibiotic actagardine. S16I mersacidin was produced only in small amounts. The purified peptide had markedly reduced antimicrobial activity, indicating an essential role for Dha16 in biosynthesis and biological activity of mersacidin. Similarly, Glu17, which is thought to be an essential structure in mersacidin, was exchanged for alanine. E17A mersacidin was obtained in good yields but also showed markedly reduced activity, thus confirming the importance of the carboxylic acid function at position 17 in the biological activity of mersacidin. Finally, the exchange of an aromatic for an aliphatic hydrophobic residue at position 3 resulted in the mutant peptide F3L mersacidin; this peptide showed only moderately reduced activity.

FIG. 1.

Construction of clones that are produced by integration and subsequent excision of a plasmid carrying an engineered mrsA gene and the N terminus of mrsR1 (white open reading frames, thick line) into the chromosome (black open reading frames, thin line). The mutation is marked by an asterisk. (A) Wild type. (B and C) The plasmid (thick line, cat resistance gene) has been integrated into the chromosome, and the crossover took place either upstream of the mutation (B) or downstream of the mutation (C). (D) The plasmid has been excised by the second homologous recombination step, which took place either downstream (B) or upstream (C) of the mutation in mrsA, resulting in an exchange of the wild-type mrsA gene for the engineered gene. The primers are shown by arrows: P1, primer 5′; P2, NPTV0; P3, Mut; P4, Umut; P5, PTV01; P6, PTV02.

FIG. 2.

(A) Structure of mersacidin (8) and exchanges that were introduced by site-directed mutagenesis. (B) Structure of the closely related lantibiotic actagardine (34).

FIG. 3.

HPLC-electrospray ionization-MS of S16I mersacidin. (A) Total ion chromatogram obtained after separation of the fraction containing S16I mersacidin. (B) Extracted chromatogram for peaks with masses in the range of 1869.0 to 1872.5 Da. (C) Mass determined for the peak detected at 21.28 min (expected mass for S16I mersacidin, 1,869.32 Da).

FIG. 4.

MS of E17A mersacidin (expected mass, 1767.2 Da) (A) and F3L mersacidin (expected mass, 1791.2 Da) (B).