Team:DTU-Denmark/Project/Tyrocidine


Tyrocidine

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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. We 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 under [MISSING LINK]. When the promoter can be expressed, the construct becomes a promising tool for the expression of the antibiotic tyrocidine. 
  • A protocol to detect tyrocidine has been established which has been tested by using commercially purchased tyrocidine.

Background

Table 1  Tyrocidine variability
 

Amino acid position

Tyrocidine

3

4

7

A

L-Phe

D-Phe

L-Tyr

B

L-Trp

D-Phe

L-Tyr

C

L-Trp

D-Trp

L-Tyr

D

L-Trp

D-Trp

L-Trp

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 ().

Unfortunately, it has high toxicity towards human blood and reproductive cells and can therefore only be used in topical applications [2]. This makes tyrocidine an interesting target for drug improvement. Oligo mediated recombineering is a promising tool to create new analogues with reduced toxicity. Under MISSING section you can read more about improvement of tyrocidine.

The genes tycA, tycB and tycC encoding the tyrocidine synthetases are located on the tyrocidine operon, which contains three additional open reading frames, labelled tycD, tycE and TycF located downstream of the synthetase genes. The entire operon has a size of 39.5 kb. [3]

 


 

 

 

 

 

 

 

 

 


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 parts of the tyrocidine construct were amplified by PCR reaction and the linearized biobrick plasmid was cut by the adequate restriction enzymes. The parts were run on an agarose gel to verify their length and purified before they were assembled by Gibson assembly. The constructs were transformed into chemocompetent E.coli and grown overnight on an LB agar plate with the specific antibiotics. The reaction was controlled by amplifying the tyrocidine construct in the Gibson assembly mix by PCR or by doing a colony PCR of the transformants and running the amplified fragment on a gel. 

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 MISSING.

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.

References

  1. 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
  2. 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
  3. 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
Technical University of Denmark
Department of Systems Biology
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2800 Kgs. Lyngby
Denmark
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