Introduction

The Non-Ribosomal Peptide Synthetases (NRPS) offer a highly modular system as those multi-enzyme complexes are composed of several subunits, called modules. Every module is responsible for adding one amino acid to the nascent peptide, however it is not the smallest entity in a NRPS, as it 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 [1] [2] [3].

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. An amino acid is first adenylated by the A domain and then bound to the T domain (also called Peptidyl-Carrier-Protein domain) via a thioester bond for subsequent reactions in the nascent NRP. The C domain then catalyzes the condensation of the already synthesized peptide chain (bound to the T domain) with the amino acid of the next module [4] [5]. The T domain itself does not exhibit any substrate specificity. Instead, it is merely a carrier domain to keep the peptide attached to the NRPS complex [2]. The core of every T domain is a conserved 4’-phosphopanthetheinylated (4’-PPT) Serine. The 4’-PPT residue is added by a 4’-PhosphoPanthetheinyl-Transferase (PPTase)activating the NRPS as a prosthetic group (Fig. 1) [6].

The remaining domains vary in their functions. Every NRPS is terminated by a TE domain that cleaves the thioester bond between the synthesized NRP and the last T domain [7]. E domains perform an epimerization reaction from the L- to the D-conformation or vice versa [8] [9]. Com domains are required for protein-protein interactions between subsequent modules that are not encoded on the same gene [10]. This is the case for the communication between the TycA and TycB1 module [11].

We realized the idea of tagging short Non-Ribosomal Peptides (NRPs) by using the Indigoidine synthetase of Photorhabdus luminescens [12]. As this special NRPS is only composed of one module, it does not contain any C domain, comparably with any other initiation module. Furthermore, the A domain is carrying an insertion of several hundreds of basepairs, changing its character to being an A-Ox domain. In addition to the adenylation, this particular domain also oxidizes the amino acid [12] (Fig. 2). Hence, the domain removes two hydrogen atoms such that a C-C double bond is formed. The oxidation leads in the case of glutamine, which is the substrate of the Indigoidine synthetase, to a cyclization.

Results

Showing inter-species module compatibility by fusion of Tyrocidine modules to the Indigoidine synthetase

Our module-shuffling approach was confirmed by mass spectrometry. Access to such special technical devices and the referring expertise is demanded for detection of small peptides but often limited. That is why we were thinking of a potential alternative to test for the synthesis of NRPs. We wanted to establish an assay accessible to the majority of the community for validation of the presence of custom peptides. Since the synthetase for indigoidine consists of only a single module, it could serve as a paradigm for the fusion to modules of other NRPSs. The pigment Indigoidine could potentially ease detection of peptides when they are fused to the dye that is visible by eye.

Thus we wanted to investigate whether it is possible to fuse Indigoidine as a tag to NRPs of other pathways even originating from other species. In the following, we will showcase the extent of NRPSs’ modular compatibility by creating inter-species module-fusions between the Indigoidine synthetase from P. luminescens and modules of the Tyrocidine synthetase from B. parabrevis. In those fusion NRPs, Indigoidine serves as a tag that eases identification of E. coli clones synthesizing the customized peptide.

Fusing single amino acids to Indigoidine

First, we combined single modules of the Tyrocidine synthetase with the indC synthetase resulting in three distinct NRPSs producing Asparagin, Valine or Phenylalanine respectively, all tagged with Indigoidine (see Fig. 4).

To assure compatibility the constructs were designed in such a way that the C domain of the C2 module was always used, given its specificity for Glutamine required for the Indigoidine production. SDS-PAGE showed the expected bands for the expression of the NRP synthetases in the transformed BAPI. The E. coli strain BAPI was used for expression of the NRPS fusions because it carries the required PPTase endogenously. All three of the fusion variants turned the colonies blue even before expression induction with IPTG. The blue pigment thus served a first indicator that peptide synthesis was successful. To further verify the existence of the fusion peptide, we ran comparative thin-layer chromatography (TLC). The native, purified Indigoidine ran further than our purified dipeptides suggesting that the amino acids were indeed fused to the pigment (Fig. 5). The peptides were detected under visible and UV light due to Indigoidine’s properties as a dye.

We sent a sample of our purified Val-Ind NRP and purified Indigoidine to the mass spectrometry facility at the Institute for Chemistry handling these samples the same way as the samples sent from the Module Shuffling experiments. (Val-Ind MS and Negative Control Ind)

Using Indigoidine as tag for non-ribosomal peptides

To gather additional evidence for our functional Indigoidine tag, we assembled seven variants with up to three modules in front of the Indigoidine synthetase (Fig. 6) following the same approach as described above.

Again the constructs pPW06, pPW09, pPW10, pPW11 and pPW12 turned BAPI colonies blue upon transformation. We tested the fusion of those peptides by comparative TLC with native Indigoidine. Even with increasing peptide length, synthesis did not seem to be affected and the dye-properties of the Indigoidine were still preserved (Fig. 7).

From this, we deduced that Indigoidine possesses characteristics required for a proper peptide tag that we would like to propose for use to the community (RFC 100). For this purpose we have created a ccdB-dependent vector to ease the tagging of NRPs, accessible through the parts registry. Design of such constructs is enabled with our software, the NRPS Designer. The ccdB-helper-construct (BBa_K1152007) is designed to lower the background of negative transformants: If the insert is cloned in the vecor correctly, there is no active ccdB present and the cell survives. If the backbone religates or template-backbone is still present in the Gibson-Mix, cells will die due to ccdB. The clearly visible impact of ccdB is visible in Fig. 8

Experimental validation of software predictions

The NRPS Designer predicts module boundaries and linker regions based on Pfam. We used these predictions for our module shuffling experiments, which all worked well in our constructs. However we feel that the borders and linker regions predicted are rather vague and want to evaluate the predictions to contribute more data to the NRPS Designer. Therefore we systematically varied the module boundaries of the A domain and C domain of C2 within the Val-Ind-construct in relation to the Pfam prediction. Based on this altered domain positioning, eight constructs were designed combining narrow, broad and original Pfam module regions (Fig. 9). Their functionality was always compared to the original construct based on the boundaries obtained from Pfam. We could induce the production of Val-Ind in two of our constructs: pLV03 (narrow and Pfam border) and pLV08 (broad and narrow border)

To summarize, with our inner- and interspecies module shuffling, we successfully validated the concept of modularity. We have integrated our experimental data and fuelled it into our software to improve its prediction accuracy. To make NRPSs and their custom design more accessible to the community, we have submitted standardized versions of the modules we used for our shuffling experiments, to the parts registry (see the parts registry entries).

Discussion

The bottleneck to test libraries of combinatorial NRPSs with an array of different modules is in fact the screening for functional enzymes [13] [14]. How to identify novel NRPSs consisting of compatible modules? Our experimental results (Fig. 5) strongly support the hypothesis that the synthesis of short peptides can be easily monitored when fused to Indigoidine. here obtained by module shuffling within the Tyrocidine cluster, could not be detected in a high-throughput manner yielded in the design and synthesis of fusion peptides that are composed of. The approach of combining one or more modules from the Tyrocidine cluster and the Indigoidine module, encoded by indC from P. luminescens represents an entirely new finding. NRPSs seem to offer a framework that does not only go beyond species borders, as already shown by Marahiel [15], but the resulting fusion NRP is synthesized and even detectable by eye.

With this, we offer a novel and very efficient way of tagging NRPs with Indigoidine. The dye can be easily measured and quantified as it has been shown in the work of the Indigoidine-group. This aspect furthermore appends a valuable feature of the NRPS Designer. Users now have the opportunity to easily add an Indigoidine tag when they design their constructs, which is also described in our proposed RFC (RFC 100). Noteworthy, our experiments we could show that the Indigoidine tag works fine when put behind modules for different amino acids, but the tagging clearly works best, when an apolar, small spacer amino-acid such as Glycine, Alanine or, as in our experiments, Valine is used. Since these amino acids adjacent to the pi electron system of the Indigoidine interfere least with the delocalized electron cloud of the mesomeric benzene ring systeme, electron excitation by electromagnetic radiation can occur more easily. Since the difference of energy betweeen HOMO and LUMO states is minimized a longer wavelength is observed (Planck’s constant).

Compared with other potential methods for in-vivo tagging of NRPs, as it has been described before [16] and [17], the Indigoidine tag has the apparent advantage that it is relatively small compared to e.g. a Haemagglutinine tag. Similarly, using fluorescent proteins (FPs) as tag is hardly feasible as the peptides that should be tagged are often smaller than the chromophore of those FPs. Imagining e.g. GFP synthesized by an NRPS is practically not feasible. Something similar accounts for other tags, such as the His tag, for which four to nine Histidines are required. Synthesizing a short peptide with several modules for Histidine is imaginable, but would double or triple the size of the required NRPS, and hence of the vector, which is highly ineffective. In this context, the Indigoidine tag would be especially suited for analytical sample labeling.

As far as in-vitro approaches are concerned, there are, in principle, two ways. I) Either one could add a tagging agent to the cells or the medium before purification – which would be the case for e.g. Click-Chemistry. II) Or one could add a certain tag such as a His tag to the NRPS and perform the entire synthesis of the NRP in vitro. The latter approach has been widely used [18] [19], is, however in vitro and hence less effective for a high-throughput advance as a functioning in-vivo-method. We ourselves thought of using methods such as Click-Chemistry for the purification of the short, synthetic peptides. Increased masses and easy cleavage enable high purities and can be determined via HPLC with UV and ESI-MS detection [20]. This approach, however, does not offer any opportunity to evaluate expression in vivo’ [21].

Hence, using and Indigoidine-tag, which can be added to the nascent NRP in the process of its formation by adding a 4 kbp Indigoidine-module to the NRPS is a novel and effective approach for labeling NRPs for quantitative expression analysis.