Team:Heidelberg/Project/Tyrocidine
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- | <p >Since functionality of isolated modules cannot be shown, as they simply take up single amino acids without linking them to another monomer, we started to design small peptides | + | <p >Since functionality of isolated modules cannot be shown, as they simply take up single amino acids without linking them to another monomer, we started to design small peptides two to four amino acid long. To show the compatibility of the tyrocidine modules with one another, combined different modules via <a href="https://2013.igem.org/Team:Heidelberg/Notebook/Methods" title="wikilink">Gibson Assembly</a>. The resulting constructs led to synthetic NRPSs and the production of five new peptides, i.e. one dipeptide, two tripeptides and two tetrapeptides.<br /><br />We were able to successfully assemble all of our plasmids and continued our work with our synthetic dipeptide (Proline-Leucine) NRPS and tripeptide-I-NRPS (Phenylalanine-Ornithine-Leucine)( |
- | <a class="fancybox fancyFigure" title="<b>Figure 4: Creation of artificial non-ribosomal peptide synthetases from genes encoding for modules of the tyrocidine synthetase via Gibson assembly.</b> | + | <a class="fancybox fancyFigure" title="<b>Figure 4: Creation of artificial non-ribosomal peptide synthetases from genes encoding for modules of the tyrocidine synthetase via Gibson assembly.</b> Corresponding novel module sequences allow synthesis of a synthetic dipeptide (Pro-Leu) and a tripeptide (Phe-Orn-Leu)." href="https://static.igem.org/mediawiki/2013/d/d6/Heidelberg_Shuffling_scheme_di_tri_01.png" rel="gallery1"> |
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- | <a class="fancybox fancyGraphical" title="<b>Figure 4: Creation of artificial non-ribosomal peptide synthetases from genes encoding for modules of the tyrocidine synthetase via Gibson assembly.</b> | + | <a class="fancybox fancyGraphical" title="<b>Figure 4:Creation of artificial non-ribosomal peptide synthetases from genes encoding for modules of the tyrocidine synthetase via Gibson assembly.</b> Corresponding novel module sequences allow synthesis of a synthetic dipeptide (Pro-Leu) and a tripeptide (Phe-Orn-Leu)." href="https://static.igem.org/mediawiki/2013/d/d6/Heidelberg_Shuffling_scheme_di_tri_01.png" style=" width:60%; margin: 10px;" rel="gallery1"> |
<img style="width:40%; padding:1%;border-style:solid;border-width:1px;border-radius: 5px; margin: 10px;" src="https://static.igem.org/mediawiki/2013/d/d6/Heidelberg_Shuffling_scheme_di_tri_01.png"> </img> | <img style="width:40%; padding:1%;border-style:solid;border-width:1px;border-radius: 5px; margin: 10px;" src="https://static.igem.org/mediawiki/2013/d/d6/Heidelberg_Shuffling_scheme_di_tri_01.png"> </img> | ||
- | <figcaption style="width:60%";><b>Figure 4: Creation of artificial non-ribosomal peptide synthetases from genes encoding for modules of the tyrocidine synthetase via Gibson assembly.</b> | + | <figcaption style="width:60%";><b>Figure 4: Creation of artificial non-ribosomal peptide synthetases from genes encoding for modules of the tyrocidine synthetase via Gibson assembly.</b> Corresponding novel module sequences allow synthesis of a synthetic dipeptide (Pro-Leu) and a tripeptide (Phe-Orn-Leu).</figcaption> |
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Revision as of 03:08, 29 October 2013
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Synthetic Peptides. Employing Modularity of NRPS.
![](https://static.igem.org/mediawiki/2013/3/34/Heidelberg_pepsyn_ga.png)
Highlights
- Interchanging of modules from the tyrocidine-cluster and thereby creating novel, synthetic peptides.
- Successful detection of the synthetic peptides produced via Mass-Spectrometry.
- Demonstration that custom NRPSs can be applied for efficient production of synthetic peptides.
Abstract
Non-Ribosomal Peptide Synthetases (NRPSs) are specialized enzymes functioning as peptide assembly lines. NRPSs are composed of distinctive modules which determine the order of amino acids to be incorporated into a peptide and thereby the peptide sequence. Since NRPS have been shown to be highly modular, we performed a semi-rational shuffling approach of NRPS modules to investigate the feasibility of producing artificial peptides by creating custom NRPSs. As starting point we chose the NRPS encoded by the tyrocidine cluster of Brevibacillus Parabrevis and rearranged modules to form novel synthetic peptides of different lengths. Evaluation of recombinant NRPS expression was performed by SDS-PAGE and final peptide products were detected using Mass Spectrometry. We demonstrate the compatibility of different modules when placed in different orders and thereby show that engineered NRPSs can be used for efficient production of synthetic peptides..
Introduction
Tyrocidine is a ten amino acid long non-ribosomal peptide (NRP) produced by the tyrocidine synthetase. This non-ribosomal peptide synthetase (NRPS) is composed of ten modules with specificity for different proteinogenic and non-proteinogenic amino acids [1]. The tyrocidine pathway can be found e.g. in Brevibacillus parabrevis. The gene cluster (
Fig. 1
) encodes three proteins functioning together and constituting the NRPS: tycA, tycB and tycC. Those NRPSs contain different numbers of modules - tycA is a single module NRPS, tycB is composed of three modules and tycC of six modules. Only the complex composed of all three proteins is able to yield the specific NRP product, whereas the single modules are not functional or only produce fragments of the NRP [2].
Every module is again subdivided into domains. Among the most common domains are the Adenylation domain, the Condensation domain and the Thiolation domain. Beside, there are Epimerization domains, Thio-Esterase domains and Communication domains [3] [4] [5].
![](https://static.igem.org/mediawiki/2013/4/44/Heidelberg_TycCluster_Scheme.png)
During the synthesis of non-ribosomal peptides, the growing peptide-chain is transferred from one module to the next. The domains within the modules fulfill distinct functions and determine the modules' amino acid specificity. The C domain catalyzes the condensation of the nascent peptide chain (bound to the T domain) to the amino acid of the next module [6] [7].
The remaining domains vary in their functions. For instance, Com domains are required for mediating protein-protein communication in NRPS composed of multiple proteins and are thus critical for their function [8]. This is, e.g. the case for the communication between the TycA and TycB1 modules present on seperate proteins [9]. Remarkably, all the six domain types described above are present in the tyrocidine synthetase of B. parabrevis [10].
As modules represent almost standalone units, each responsible for the incorporation of a specific amino acid monomer, they can be interchanged to form new non-ribosomal peptide synthetases synthesizing novel peptides [11]. Remarkably, the compatibility among moduoles is not restricted by the substrate specificty of the modules, but requires the general domain order to bew intact. Following this idea, we assembled new synthetic non-ribosomal peptide synthetases by shuffling modules of the tyrocidine NRPS and performed mass spectrometry in order to detect the corresponding synthetic peptide products.
Results
Claims
Here we show that modules of NRPSs can be interchanged resulting in engineered enzymes with novel functionalities. In the following, we have three claims to propose.
- Modules of NRPSs can be interchanged.
- The enzyme modularity allows the synthesis of custom peptides.
- Customized NRPs can be purified and detected by Mass Spectrometry.
The establishment of a standard framework (RFC 100) for in vivo synthesis of customized NRPs by non-ribosomal peptide synthetases required systematic investigation of NRPS modularity and compatibility. As proof of principle, we semi-rationally interchanged NRPS modules, a process we refer to as module shuffling ( Fig. 2 ).
![](https://static.igem.org/mediawiki/2013/6/6c/Heidelberg_SynPep_Overview.png)
Shuffling Modules of a Single NRPS
We tried to interchange modules of the tyrocidine cluster of B. parabrevis in E. coli. As engineering of NRPS by shuffling modules underlies certain constraints, we had to develop certain design principles for NRPS engineering first. These included respecting the domain orders in modules as well as finding optimal linkers for fusing two different modules together.
The condensation domain catalyses the peptide bond formation between the nascent peptide and a new amino acid. We hence concluded, that the module TycA could be introduced at any position within a NRPS when putting it behind a suitable C domain. As the condensation domain takes up the amino acid attached to the preceding module it was removed for modules used as initiation modules. Furthermore, the peptide-peptide interaction is performed by communication domains. To investigate how crucial their functionality actually is the com-domains were replaced by suitable linkers when incorporating corresponding. When respecting these basic design principles for NRPS engineering, the engineering can be conducted in an efficient manner.
Assembling of a Synthetic NRPS
To obtain a basis for shuffling and creation of artificial peptide synthetases, the coding sequences for each individual module were amplified from the tyrocidine cluster by the use of primers optimized for a Gibson assembly strategy. The primers were annealed to the unconserved linker regions between the NRPS domains that were predicted by Pfam. We successfully validated the correct amplification of 12 single DNA fragments comprising different modules as well as corresponding pSB1C3 backbone fragments by electrophoresis ( Fig. 3 ).
![](https://static.igem.org/mediawiki/2013/6/69/Heidelberg_Tyc_All_Fragments.png)
Since functionality of isolated modules cannot be shown, as they simply take up single amino acids without linking them to another monomer, we started to design small peptides two to four amino acid long. To show the compatibility of the tyrocidine modules with one another, combined different modules via Gibson Assembly. The resulting constructs led to synthetic NRPSs and the production of five new peptides, i.e. one dipeptide, two tripeptides and two tetrapeptides.
We were able to successfully assemble all of our plasmids and continued our work with our synthetic dipeptide (Proline-Leucine) NRPS and tripeptide-I-NRPS (Phenylalanine-Ornithine-Leucine)(
Fig. 4
) by transforming them into the E. coli strain BAP1.
![](https://static.igem.org/mediawiki/2013/d/d6/Heidelberg_Shuffling_scheme_di_tri_01.png)
Expression and Detection of the NRPS and its Products
Three hours after induction with IPTG samples expressing the putative synthetases were taken. The expression of the 212 kDa dipeptide synthetase and the 380 kDa tripeptide synthetase was shown by SDS-PAGE (
Fig. 5
).
![](https://static.igem.org/mediawiki/2013/4/47/Heidelberg_SDS_2nd_20130830.png)
Since SDS-PAGE is not sensitive enough to detect small peptides, we wanted to assess the presence of the newly synthesized peptides at different time points by the use of mass spectrometry. Expression of the NRPSs was induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG). Samples were taken at different time points post induction. Since residual salts could potentially disturb the acquisition of small peptide abundances, we washed our LB-Cm culture in M9 minimal medium to minimize this effect and improve the detection of our short NRPs. Supernatant and the bacterial pellet were processed separately with distinct purification protocols to examine the distribution of the synthetic peptides and optimize purity of each sample. Final sample preparation for tandem mass-spectrometry was conducted at the neonate screening facility of the university medical center. There the peptides were hydrolyzed during the acidic butylation reaction.
![](https://static.igem.org/mediawiki/2013/1/15/Heidelberg_Ornithine_tri_retraced.png)
Finally the highly specific m/z profile allowed the identification of different amino acid abundances [12][14]. Since ornithine is a non-proteinogenic amino acid that is incorporated in our tripeptide (Phe-Orn-Leu), we mainly focused on detecting ornithine levels ( Fig. 6 ). Ornithine is a side-product of metabolic routes and has therefore not to be provided in the medium. The abundance of ornithine in the tripeptide samples was strongly elevated with time compared to our dipeptide (Pro-Leu; Fig. 7 ). Due to variation of the general amino acid concentration in both used media and samples, we normalized the ornithine values by the amount of all amino acids present in the respective medium.
![](https://static.igem.org/mediawiki/2013/5/50/Heidelberg_barchart_ornithine.png)
The ornithine level in the supernatant of the tripeptide samples peaked 21 hours upon induction. Samples prepared from bacteria pellets showed minor increases in ornithine levels in comparison to the pure medium and our negative control (untransformed E. coli strain BAP1). To aquire additional data on the existence of our short peptides we sent two samples to analysis via high-resolution electrospray ionization (HR-ESI) mass spectrometry at the mass spectrometry facility of the Institute for Chemistry, based on preliminary results from the ornithine screening. However, we could not obtain conclusive data for our samples, because the background was too high (MS Results: dipeptide and Fig. 8 ).
![](https://static.igem.org/mediawiki/2013/5/5d/Heidelberg_MS_tri_360-540_OCI.png)
In summary, we were able to amplify single modules from the tyrocidine NRPS cluster, and we shuffled them via Gibson Assembly. Two constructs coding for two entirely new NRPSs were successfully transformed. The synthetases are both well expressed on pSB1C3 in E. coli strain BAP1 and their products, short NRPs, can be detected through tandem mass spectrometry finally confirming the functionality of the engineered NRPSs.
Discussion
Interchangeability of Modules
Module compatibility is the vital basis for any standardized work with NRPSs. Hence, the major objective of this project was to investigate flexible interchangeability of modules, which allows for customized synthesis of short peptides via NRPSs, as we propose in our standard (RFC 100). Tyrocidine served as a paradigm for semi-rational rearrangements in the modular structure of NRPSs, as demonstrated by the creation of two different novel peptides through rearrangement of the respective modules that were amplified from B. parabrevis. Comparing the normalized levels of the non-proteinogenic amino acid ornithine in the different samples, we could conclude that the synthetic NRPS is in fact expressed enabling the creation of customized peptides that can be detected via mass spectrometry.
Synthetic Peptides
Before our experiments, there was no evidence whether the synthetic peptides would be released to the medium or remained in the pellet. Showing that the resuspension of lyophilized supernatant in ethanol obtains a higher yield in ornithine content compared to the pellet samples, we can conclude that the small peptides are emitted into the medium. As salt concentrations did not seem to interfere with mass spectrometry measurements, the whole work up process has been successful. The final output of the tandem mass spectrometry demonstrated a highly elevated concentration of synthetic peptides in the medium compared to the cell interior, which leads to an improved protocol for standardized purification of short non-ribosomal peptides (RFC 100).
Furthermore, we found that there was no need to provide additional ornithine to the media since it was incorporated from E. coli as an intermediate product from L-glutamate. Moreover, this assay accounted for the functional incorporation of non-proteinogenic amino acids into artificial non-ribosomal peptides. A high variety of non-proteinogenic amino acids as constituents has already been described in literature [13]. The kinetics of ornithine as a proxy for the production of the tripeptide synthesis implied a more than fifty times higher ornithin content in the samples expressing the respective NRPS providing strong evidence for a functional synthetic NRPS.
In the future, synthesized non-ribosomal peptides could be elongated even further. Therefore, a stepwise-assembly strategy should be considered since the complexity of cloning will increase with the number and size of DNA fragments as well. Of course, additional modules for shuffling will increase the amount of opportunities to create comprehensive constructs. Such an approach is guided by the NRPS Designer, which frames our proposed standard (RFC 100).
Detection Methods
Mass spectrometry often requires several tries and iterations of sample work up procedures to acquire good results. Since the synthetic peptides assembled by our artificial non-ribosomal peptide synthetases were rather small, their properties would not differ significantly from other salts in the solution. As we were able to cut salt concentrations significantly with our purification techniques, no problems of signal interference by the salty background occurred at the tandem mass spectrometry of the neonate screening facility. In combination with a butylation reaction a highly specific compound detection was possible making this method less prone to background noise. This technique could be the reason for the proper detection as it makes the product more distinctive. However, the procedure was not used for the mass spectrometry attempts to detect the whole peptides (as they should be kept intact) which could be the reason for the inconclusive results and also account for a bad or non-ionizability of the synthetic peptides. The generation of different unexpected adducts could be another reason for inconclusive spectra. Hence, for other mass spectrometry measurements an even more improved purification procedure could make the difference and lead to the spectra that were expected. The fragmentation of molecules at the thought target mass did not display significant decay products. Results of the differential approaches were aqcuired at two different facilities minimizing the risk of the mass spectrometre itself as an source of errors.
Increasing masses of the peptides and therefore facilitated purification procedures could be accomplished by means of click chemistry. The higher mass in combination with an easy cleavage would enable a better purification via HPLC with UV and ESI-MS detection [15]. This approach, however, does not offer any opportunity to evaluate expression in vivo’ [16].
Combinatorial Usage
Combinatorial chemistry gained significant recognition over the last decades. Screening of large libraries enables a target-oriented approach of compound research [17]. Exhibiting the manifoldness of non-ribosomal Peptides in combinatorial biology even short NRP sequences result in an impressive number of possible differentially composed products ( Fig. 9 ). As approximately 500 different monomers for NRPS exist even a dipeptide would result in 5002 = 250 000 possible combinations of which, of course only a fraction would obtain a reasonable function. We managed a tripeptide, this is state of the art [Marahiel et al.] and gives us 125 million (5003) potential constructs to screen for, with our (RFC 100). This impressive numbers would increase even more with sequence length and the introduction of possible modifications, as branching or cyclization. In Comparison to cannonical peptide synthesis (223 = 10 648) this is far superior in terms of numbers and high throughput screening possibilities (Tab. 1). Although most of the products won´t inhabit specific features, for instance customized production of toxins, siderophores, pigments, antibiotics, cytostatics, and immunosuppressants would be facilitated. That even small peptides are functional can be seen from the success of peptide therapeutics [18] and from various natural products like the peptide enterobactin, for example. Databases like Norine [19] and Software as the NRPS Designer developed by us will assist these purposes.
Sequence Length | Possible Non-Ribosomal Peptide Combinations* | Possible Ribosomal Peptide Combinations |
2 | 250 000 | 484 |
3 | 125 000 000 | 10 648 |
4 | 62 500 000 000 | 234 256 |
![](https://static.igem.org/mediawiki/2013/8/85/Heidelberg_combinatorial_usage.png)
Conclusion
In Conclusion we were able to prove interchangeability and recombinational potential of modules deriving from a single NRPS. The proper expression of custome dipeptide- and tripeptide-synthetase was shown by SDS-PAGES and an indirect prove of incorporation of non-proteinogenic amino acids accounting for a functional production of the desired target peptide. Thus, we were able to reproduce the work of Marahiel et al. [11] and could open up the engineering diversity of NRPS to the iGEM community. The achievements were enabled through and fed back to the NRPS Designer. This software tool incorporates a database utilizing our experimental on NRPS modules for guiding through user-oriented design of NRPSs.
Methods
Cloning Strategy
The different synthetic non-ribosomal peptide synthetases were assembled on a chloramphenicol resistance backbone (pSB1C3) with an IPTG-inducable lac-promoter and the sequences coding for the desired target modules via Gibson assembly. The resulting constructs of this isothermal pot reaction were transformed into DH10β and tested by restriction digests and sequencing. Positive constructs were isolated and chemically transformed into E. coli BAP1 cells.
SDS-PAGE
To test on the expression of the putative synthetic NRPS SDS-PAGES were performed. Therefore, cell cultures were centrifuged and supernatant discarded. The remaining pellet was resuspended in loading buffer and boiled to rupture the cells. After cooling the mixture down, it was centrifuged and loaded together with the NOVEX pre-stained standard on a 3-8% tris-acetate gradient gel for 1 hour at 180 V tris-acetate running buffer. Finally, the gels were stained with coomassie to visualize the running bands.
Mass Spectrometry
Preparation of Samples
As high salt concentrations interfere with the m/z signal at mass spectrometry, we tried to cut salt concentrations from the very beginning. Therefore, pre-cultures of transformed cells were prepared in LB media containing chloramphenicol and incubated overnight until OD600 ~ 0.6. To reduce the amounts of complex components, the cultures were centrifuged and remaining pellet washed with M9 minimal media. The washed cells were resuspended in M9 minimal media and again incubated until OD600 ~ 0.6 before inducing them with IPTG. After 21 hours pellets and supernatants were centrifuged and processed separated.
Sample Processing
Pellets were resuspended in PBS and lysed via ultra-sonification to release all components in to the buffer. After centrifugation the supernatant was extracted and frozen with liquid nitrogen to avoid peptide hydrolysis.
Supernatants of the samples after separation were frozen with liquid nitrogen and lyophilized to concentrate the solution. The lyophilisate was solubilized in a small amount of methanol, a standard solvent for HPLC and mass spectrometry, and vortexed to mix the phases. The supernatant was then frozen in liquid nitrogen.
Samples of each purification procedure were analysed on a HR-ESI mass spectrometer (Bruker ApexQe hybrid 9.4 T FT-ICR).
Sample Processing for Tandem Mass Spectrometry at Neonate Screening
For evaluation of amino acid levels the frozen pellet and supernatant samples (Sample Processing) were hydrolysed and butylated to achieve a better ionization. Therefore, the samples were pipetted on filter paper sheets and dried chilled overnight. The sheets were extracted with Neo Gen stable isotope standard kit A und B (NSK-AB, EurIsotop) and centrifuged. The resulting supernatant was vaporized and the remaining dried compounds warmed up, before adding butanolic hydrochloric acid. The samples were shaken and heated up to improve the chemical reaction. Liquid residues were vaporated and samples heated up. Finally, an acetonitrile p.A. /water (1:1 vol/vol) solution was added as solvent for tandem mass spectrometry (Micromass Quattro Ultima (ESI-MS/MS)).
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