Team:DTU-Denmark/Project/Tyrocidine
Introduction to Tyrocidine
Tyrocidine is a mixture of non-ribosomal antibiotics, naturally expressed by Brevibacillus parabrevis. Due to its toxicity, tyrocidine is only suited for topical use. The project has focused on expressing the non-ribosomal peptide synthetase in Bacillus subtilis, an established model organism, and thereby making it accessible for improvement by oligo mediated recombineering. By using recombineering to alter the active site determining the substrate specificity, new variants of the antibiotic can be created which could be screened for reduced toxicity.
Achievements
- All parts except for the repressor have been assembled. Read more in the lab notebook.
- A protocol to detect tyrocidine has been established, which has been tested by using commercially purchased tyrocidine as a standard.
Background
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Tyrocidine is a mixture of non-ribosomal, cyclic antibiotics produced by Brevibacillus parabrevis and has a unique mode of action wherein it disrupts the function of the cell membrane. Tyrocidine consists of four decapeptides varying at three amino acids which are produced by an NRPS system which is capable of incorporating structurally similar amino acids at position 3, 4, and 7 [1]. The three enzymes Tyrocidine Synthetase A, B, and C contain 1, 3, and 6 modules, respectively, and assemble Tyrocidine in an assembly line manner.
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Amino acid position |
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Tyrocidine |
3 |
4 |
7 |
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A |
L-Phe |
D-Phe |
L-Tyr |
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B |
L-Trp |
D-Phe |
L-Tyr |
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C |
L-Trp |
D-Trp |
L-Tyr |
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D |
L-Trp |
D-Trp |
L-Trp |
Table 2. Tyrocidine amino acid variablility in tyrocidine A-D for position 3, 4, and 7.
Unfortunately, Tyrocidine has high toxicity towards human blood and reproductive cells and can therefore only be used in topical applications [2]. Under lab-on-a-disc you can read more about the cytotoxic effects of this as well and other NRPs. The approach is to use the compound's hemolytic properties for developing a screening platform which could be used for screening a big number of samples simultaneously.
This limitation and its mode of operation as antibiotic makes tyrocidine an interesting target for drug improvement, see Figure 1. Oligo mediated recombineering is a promising tool to create new analogues with reduced toxicity.
The genes tycA, tycB and tycC encoding the tyrocidine synthetases are located on the tyrocidine operon, which contains three additional open reading frames, labeled tycD, tycE and TycF located downstream of the synthetase genes. The entire operon has a size of 39.5 kb[3].
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Figure 1: Structure of tyrocidine A.
Experimental Design
Due to the big size of the biosynthetic cluster, an amplification with standard PCR is not feasible. We designed a construct for the expression of tyrocidine which can integrate into the lacA site of Bacillus subtilis, an established model organism. By counter-selection with a toxic gene cassette, the tyrocidine operon can insert into the construct by homologous recombination. The toxic gene is placed under control of the Pxyl promoter, which is repressed by a repressor in the absence of xylose. If xylose is present, the repressor leaves the operon and the toxic gene is transcribed. The construct is flanked by two sequences homologous to the lacA sequence, which allow integration into the genome of Bacillus subtilis.
Since our efforts to express tyrocidine have been hampered by difficulties in assembling all parts, we have tried to tackle the problem with a number of different experimental designs.
- Cloning all of the parts described above into a single plasmid.
- Splitting the construct up into two fragments which could subsequently be cloned together by simple restriction enzyme cloning. Since the efficiency of the used method for assembling the fragments, the Gibson assembly, decreases with increasing length, this approach rules out assembly problems due to the length of the construct.
- Assembling all parts in one plasmid while cloning the XylR repressor in a second plasmid, which can be cloned together by using restriction enzymes cloning.
Discussion
Our efforts to express tyrocidine have been hampered by difficulties in assembling all parts. All parts could be assembled except for the xylose repressor xylR, which we have not been able to assemble with any of our various experimental designs described here.
The repressor has been amplified from the BioBrick BBa K733002. The part has been used by the iGEM team of Hong Kong University of Science and Technology 2012, which indicates that the biobrick is functional.
If given more time, the tyrocidine construct could be amplified with primers containing restriction sites and cloned into a plasmid which already contains XylR by classic cloning techniques, like the BioBrick BBa_K733002 or BBa_K733018. This plasmid could then be further amplified in E.coli and transformed into a B. subtilis strain which already has the xylose repressor integrated into its genome.
Furthermore it has to be taken into consideration, that the BioBrick used for the construct which was distributed might be defect. A problem to which our attention has been drawn during our participation in the Giant Jamboree in Boston.
References
- Lipmann, F., Roskoski, R., Gevers, W., & Kleinkauf, H. (1970). Tyrocidine biosynthesis by three complementary fractions from Bacillus brevis (ATCC 8185). Biochemistry, 9(25), 4839–4845. doi:10.1021/bi00827a002
- Joo, S.-H. (2012). Cyclic Peptides as Therapeutic Agents and Biochemical Tools. Biomolecules and Therapeutics, 20(1), 19–26. doi:10.4062/biomolther.2012.20.1.019
- Mootz, H.D., & Marahiel, M.A. (1997). The tyrocidine biosynthesis operon of Bacillus brevis: Complete nucleotide sequence and biochemical characterization of functional internal adenylation domains. Journal of Bacteriology, 179(21):6843-50
