Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Kamil Żmijewski, Luba Prout
Date created: 2016-10-16 11:00:00
Date modified: 2016-10-19 02:13:09
Mutacin III biosynthetic device
| Types | DnaRegion |
| Roles | Device engineered_region |
| Sequences | BBa_K1954004_sequence (Version 1) |
Description
The biosynthetic locus was engineered by our team in a form allowing for high-yield and fine-tuned expression of mutacin III (Fig. 1). 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. 1. Simplified diagram of the mutacin III biosynthetic locus designed by UCL iGEM 2016.
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 (4). 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 (6) 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 (7) while the product of mutP is a serine protease which cleaves the leader peptide and is likely the last step in the biosynthesis (8). The mature mutacin III is composed of rings connected by flexible linkers (Fig. 2) which may be important in the mechanism of bacteriocidal activity (9). Following export, the peptide is believed to form transmembrane pores as monomer aggregates leading to membrane disruption and efflux of cellular components (10). 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 (11).
Fig. 2. The structure of mature mutacin III (10).
In one investigation of the activity of mutacin-related lantibiotic gallidermin it became clear that lantibiotics are more effective in preventing biofilm formation rather than in exterminating microorganisms already embedded in biofilms (12). To reflect this, our device could be used to transform E. coli cells and employed as an anti-cariogenic strategy in replacement therapy (Fig. 3). Such a novel bacterial strain would demonstrate features of a successful effector strain as it would not cause disease by itself and because it could displace the host pathogenic bacteria. Importantly, there are very few existing examples of lantibiotic resistance compared with antibiotics and only one mechanism of resistance to mutacin III, known as CprRK in Clostridium difficile, has been established (13). Moreover, the fact that a closely related lantibiotic nisin has been shown to exhibit low in vivo toxicity levels (14) and has been widely used as food preservative from as early as mid 1940s (15) further encourages the prospect of considering the employment of mutacin III as an anti-cariogenic agent.
Notes
All restriction sites were removed by silent mutagenesis.Source
Our device was based on the sequence of AF154675.1| Sequence Annotation | Location | Component / Role(s) |
| BBa_I14033 -35 -10 BBa_B0034 mutR TetR 1 -35 TetR 2 -10 BBa_I0500 AraC rrnB T1 rrnB T2 T7 promoter Pbad BBa_B0034 mutA T7 terminator BBa_B0034 muB mutC mutD mutP rrnB T1 BBa_I0500 BBa_B0034 mutT BBa_B1002 | 1,38 4,9 27,32 47,58 59,913 67,85 86,91 92,110 109,114 129,1338 129,1207 958,1029 1121,1148 1179,1197 1208,1338 1220,1231 1238,1429 1507,1554 1638,1649 1656,4626 4615,5889 5913,6479 6484,7827 7872,7943 8022,8187 8205,8216 8223,9848 9864,9897 | promoter feature/promoter promoter feature/promoter feature/promoter promoter feature/BioBrick engineered_region feature/cds CDS feature/binding non_covalent_binding_site promoter feature/promoter non_covalent_binding_site feature/binding promoter feature/promoter engineered_region feature/BioBrick feature/misc sequence_feature feature/stop stop_codon feature/stop stop_codon feature/promoter promoter promoter feature/promoter feature/rbs ribosome_entry_site CDS feature/cds stop_codon feature/stop feature/rbs ribosome_entry_site feature/cds CDS feature/cds CDS feature/cds CDS CDS feature/cds feature/stop stop_codon feature/promoter promoter feature/rbs ribosome_entry_site feature/cds CDS sequence_feature feature/misc |