Part:BBa_K1954004

Mutacin III biosynthetic device

A decrease in biofilm formation caused by interference with the viability of certain bacterial species presents an approach towards limiting cariogenesis. Our team designed a locus capable of producing and exporting a mature form of an antimicrobial peptide known as mutacin III, first identified in Streptococcus mutans UA787 isolated from a caries-active white female patient in the late 1980s (1). Mutacin III is effective against a wide range of Gram-positive bacteria implicated in dental caries, e.g. other strains of Streptococcus mutans and Actinomyces naeslundii, while Gram-negative bacteria are resistant to inhibition (2). Mutacin III is a ribosomally synthesized 22 amino acid screw-shaped lanthionine-containing peptide. The biosynthesis of mutacin III involves the expression of the structural gene mutA to make a prepropeptide, comprising a C-terminal propeptide and an N-terminal leader peptide from which the former undergoes processing and the latter is cleaved off before export into the medium (1). The specific post-translational modifications make mutacin III distinct from other bacteriocins and are introduced by enzymes coded for by other genes in the locus (mutBCDP). Basing on sequence homologies of genes in the locus with those of other lantibiotic biosynthetic loci it can be inferred that these enzymes catalyze the dehydration (mutB) and cyclization (mutC) of the propeptide serine and threonine residues which can condense with a neighboring cysteine residue (3) leading to the formation of lanthionine or methyllanthionine (thioether) bridges, respectively. The enzyme coded by mutD catalyzes the oxidative decarboxylation of the C-terminal cysteine residue (4) while the product of mutP is a serine protease which cleaves the leader peptide and is likely the last step in the biosynthesis (5). The mature mutacin III is composed of rings connected by flexible linkers (Fig. 1) which may be important in the mechanism of bacteriocidal activity (6). Following export, the peptide is believed to form transmembrane pores as monomer aggregates leading to membrane disruption and efflux of cellular components (7). The content of anionic phospholipids in the membrane has been suggested to be an important factor influencing initial binding – mutacin III has a net positive charge whereas Gram-positive bacteria have a high relative amount of anionic lipids (8).

Fig. 1. The structure of mature mutacin III thought to be identical to mutacin 1140 (8).

The biosynthetic locus of mutacin III was designed by our team in a form allowing for high-yield and fine-tuned expression of the peptide (Fig. 2). We placed a strong T7 promoter upstream of mutA to obtain high levels of the propeptide, a repressible pTet promoter upstream of the mutBCDP co-transcription unit and an inducible araBAD promoter for the mutT gene, coding for the ATP-binding-cassette-like transporter of mutacin III. All illegal restriction sites were removed from the endogenous sequences by silent mutagenesis.

Fig. 2. Simplified diagram of the mutacin III biosynthetic locus designed by UCL iGEM 2016.

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