Construction of an Expression System for Site-DirectedMutagenesis of the LantibioticMersacidin

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.

Discovery and in vitro biosynthesis of haloduracin, a two-component lantibiotic

“…1) (17,18,29,30). Importantly, the six-aminoacid-containing MeLan B-ring in Hal␣, which is believed to be important for lipid II binding in mersacidin's C-ring (32), is conserved including the invariant and essential Glu within this ring (33). This MeLan ring is also found in the ␣͞A1-peptides of lacticin 3147 (21), plantaricin W (17), staphylococcin C55 (18), Smb (29), and BHT-A (30), as well as in the A-ring of the lacticin 481 subgroup of single component lantibiotics (3).…”

Posttranslational conversion of l -serines to d -alanines is vital for optimal production and activity of the lantibiotic lacticin 3147

“…This phenomenon has also been associated with a number of other engineered lantibiotics, especially those in which modified amino acids are altered (33). It has been suggested that inhibition of the enzymes of the modification machinery may be the most likely explanation (34). Although nonproduction of LtnA1(7A) and (7V) meant that it was not possible to determine to what extent a D to L conversion would affect the relative activity of LtnA1, it was apparent that significant levels of relative activity were retained upon the replacement of D-alanine with nonchiral amino acids.…”

Fundamental functionality: recent developments in understanding the structure–activity relationships of lantibiotic peptides

“…19 Very surprisingly, this mutation did not abolish activity, unlike with the equivalent mersacidin and haloduracin-a mutants that were both strongly affected by this change. 8,[20][21][22] Other mutants in ring A were found to retain activity, suggesting there is some degree of flexibility in the amino acids of this ring. 23 Mutagenesis has been used to produce weakly active analogs containing the changes S4T, H8P, Q20T and truncation at S27.…”

“…An initial limited study produced the mutants F3L, S16I and E17A, all of which were found to be inactive or very weakly active. 21 A subsequent systematic production of mersacidin analogs via mutagenesis yielded a far larger number of mutants. 22 Three libraries were designed in which amino acids were substituted, inserted or deleted.…”