Production of Dehydroamino Acid-Containing Peptides by Lactococcus lactis

Abstract

Nisin is a pentacyclic peptide antibiotic produced by some Lactococcus lactis strains. Nisin contains dehydroresidues and thioether rings that are posttranslationally introduced by a membrane-associated enzyme complex, composed of a serine and threonine dehydratase NisB and the cyclase NisC. In addition, the transporter NisT is necessary for export of the modified peptide. We studied the potential of L. lactis expressing NisB and NisT to produce peptides whose serines and threonines are dehydrated. L. lactis containing the nisBT genes and a plasmid coding for a specific leader peptide fusion construct efficiently produced peptides with a series of non-naturally occurring multiple flanking dehydrobutyrines. We demonstrated NisB-mediated dehydration of serines and threonines in a C-terminal nisin(1-14) extension of nisin, which implies that also residues more distant from the leader peptide than those occurring in prenisin or any other lantibiotic can be modified. Furthermore, the feasibility and efficiency of generating a library of peptides containing dehydroresidues were demonstrated. In view of the particular shape and reactivity of dehydroamino acids, such a library provides a novel source for screening for peptides with desired biological and physicochemical properties.Lantibiotics are (methyl)lanthionine-containing antibiotics (4). Lantibiotics contain (methyl)lanthionines, i.e., thioethercontaining amino acids, which are posttranslationally introduced by enzymes coded for by genes of the lantibiotic's operon. These enzymes dehydrate serine and threonine residues, thereby converting them into dehydroalanine and dehydrobutyrine residues, respectively. Subsequently, (methyl)lanthionines are formed by enzymatic coupling of the dehydroresidues to cysteines. Functional reconstitution of in vitro activity of one bifunctional enzyme, lacticin M (27), and of the cyclase NisC (14) has been achieved. The structure of NisC has also been determined (14), providing detailed information about its catalytic mechanism.The pentacyclic lantibiotic nisin is produced by some Lactococcus lactis strains. Nisin is ubiquitously applied as a food preservative that inhibits the growth of mainly gram-positive spoilage and pathogenic bacteria. By binding to the pyrophosphate of lipid II with its N-terminal region, which encompasses the A and B rings, nisin exerts two different antimicrobial activities (1, 7). First, a transmembrane pore composed of both nisin and lipid II causes dissipation of the transmembrane ion gradients. Second, binding of nisin to lipid II causes cell killing by inhibition of cell wall synthesis. Nisin is composed of four methyllanthionines, one lanthionine, two dehydroalanines, one dehydrobutyrine, and twenty-six unmodified amino acids (6, 12). The dehydration reactions are carried out by the nisin dehydratase NisB. Replacement of the dehydroalanine at positions 5 of nisin (2) and subtilin (15) very much reduces the capacity to inhibit the outgrowth of spores. NisB can also dehydrate serine and threonine residu...

Biosynthesis and Transport of the Lantibiotic Mutacin 1140 Produced by Streptococcus mutans

“…The absence of any inhibitory activity of these products also demonstrates that the truncated leader peptide keeps mutacin 1140 in an inactive structural state. It has been shown that the biosynthesis of nisin in the absence of LanP produces partially cleaved leader peptide products (41,42), with the full-length leader peptide being the main product (43). Unlike nisin, only a single truncated leader peptide product is observed for the mutacin 1140 leader peptide.…”

Site-Directed Mutations in the Lanthipeptide Mutacin 1140

“…There have been over 80 different directed mutations reported on the nisin core peptide (23,24). In addition, a large bank of randomly mutated nisin derivatives was screened, which resulted in the identification of three mutants that significantly improved the bioactivity of nisin (23).…”

In Vitro Reconstitution and Substrate Specificity of a Lantibiotic Protease

“…It may act in a protective role for the producing organism, as its removal is required for bioactivity (10)(11)(12)(13)(14)(15). The leader peptide could also serve as a recognition motif for the transport machinery because non-lantibiotic peptides attached to leader peptides are secreted (16,17), or as a scaffold for the lantibiotic synthetases (11,(18)(19)(20). Recently, it was shown that the leader sequence of LctA, while important for processing by LctM, is not absolutely required for activity (21).…”