Introduction

Non-Ribosomal Peptide Synthetases (NRPS) are assembly lines which consist of several modules, each module incorporating one specific amino acid into the growing peptide chain (for a more detailled introduction into NRPS, please refer to our Background Page). As previously shown by both other research groups [1] and our work (Project Peptide-Synthesis), NRPS modules can be rearranged to form a novel assembly line which produces a custom, non-ribosomal peptide. However, the detection of those synhetic peptides using mass spectrometry is still challenging. In order to simplify the detection of peptides created by custom NRPSs, we developed a blue pigment tag for non-ribosomal peptides - the Indigoidine-Tag.

The indigoidine synthetase indC from Photorhabdus luminescens laumondii TT01 consists of an Adenylation domain with an internal Oxidation domain, a Thiolation domain and a ThioEsterase domain. The A-domain adenylates L-glutamine which is then attached to the T-domain via a thioester bond. The TE-domain catalyzes the cyclization of the glutamine and cleaves it from the T-domain. Two cyclic glutamines are oxidized by the Ox-domain, resulting in the blue pigment dimer indigoidine (Fig. 1a)[2]. This leads to a blue phenotype of E. coli cells expressing the indC gene when grown on plates or in liquid cultures (Fig. 1b). The blue pigment can be purified and dissolved in DMSO using a simple protocol.

We fused the indC gene to NRPS modules of the tyrocidine synthesis cluster from Brevibacillus parabrevis to create novel NRPS assembly lines which attach the blue pigment indigoidine to the last amino acid of the synthesized peptide (Fig. 2). After purification, the tagged peptide can be validated using comparative Thin Layer Chromatography (TLC) or Mass-Spectrometry (MassSpec) after purification by High Pressure Liquid Chromatography (HPLC).

The possibility of tagging non-ribosomal peptides makes high-throughput protocols possible. Therefore, we created a standardized procedure for the production of NRPs in our RFC100: i) design of novel NRPSs with our NRPS-Designer software, ii) high-throughput construction of NRPS-libraries, iii) the detection and validation of the synthetic peptides and iv) functional assays with possible upscaling of the peptide production to industrial level.

Results

Showing Inter-Species Module Compatibility by Fusion of Tyrocidine Modules to the Indigoidine Synthetase

Our module-shuffling approach within the tyrocidine cluster was confirmed by MassSpec. 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 asparagine, valine or phenylalanine respectively, all tagged with indigoidine (see Fig. 3).

To assure compatibility, the indigoidine module was always preceded by the C2 module, as the tyc-C2 is specific for glutamine which is required for indigoidine production. SDS-PAGE showed the expected bands for the expression of the NRP synthetases in the transformed BAP1. The E. coli strain BAP1 was used for expression of the NRPS fusions because it carries the required PPTase sfp under the control of a T7 promoter. 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 TLC. The native, purified indigoidine ran further than our purified dipeptides suggesting that the amino acids were indeed fused to the pigment (Fig. 4). The peptides were detected under visible and UV light due to indigoidine’s properties as a dye.

Using Indigoidine as a 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. 5) following the same approach as described above.

Again the constructs pPW06, pPW09, pPW10, pPW11 and pPW12 (Fig. 5) turned E. coli BAP1 colonies blue upon transformation. Even with increasing peptide length, synthesis did not seem to be affected and the dye-properties of the indigoidine were still preserved (Fig. 6).

From this, we deduced that indigoidine possesses characteristics required for a proper peptide tag that we would like to propose for the use within 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 will survive. If the backbone religates or template-backbone is still present in the Gibson-Mix, cells will die due to ccdB expression. The impact of ccdB expression is depicted in Fig. 7

Experimental Validation of Software Predictions

We propose the Indigoidine-Tag as a part of our standards RFC 99 and RFC 100. The crucial tool in this standardized process is the NRPS Designer. The NRPS Designer predicts module boundaries and linker regions based on Hidden Markov Models. We used these predictions for our module shuffling experiments, which all worked successfully in our constructs. However, other tools predict different boarders, hence, we wanted to evaluate the predictions to contribute more data to the NRPS Designer and to provide experimental feedback to our software. 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. 8). 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, we successfully validated the concept of modularity both for intra- and interspecies shuffeling. 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 [3] [4]. How to identify novel NRPSs consisting of compatible modules? Our experimental results (Fig. 4) strongly support the hypothesis that the synthesis of short peptides can be easily monitored when fused to indigoidine. This novel technique that represents an equivalence of the GFP-tag for proteins in NRPs outrules classical detection by Mass-Spec due to the increased efficiency and high-throughput character. Our findings that NRPSs seem to offer a framework going beyond species borders confirm the results shown by Marahiel [1]. Using indigoidine as pigment-module for the fusion results in a fusion NRP which is 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, quantified and even optimized. 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, valin is used, due to less steric hindrance.

Compared to other potential methods for in-vivo tagging of NRPs, as it has been described before [5] and [6], the Indigoidine tag has the apparent advantage that it is relatively small compared to e.g. a 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. Fluorescent proteins (FPs) have the advantage of easy detection but are simply too big to use them. Imagining e.g. GFP synthesized by an NRPS is practically not feasible. In summary, the Indigoidine-Tag, in contrast to all other imaginable tagging methods for NRPs fulfills the required characteristics of being small, inert, universal and easily detectable.

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 [7] [8], is, however in vitro and hence less effective for a high-throughput advance as a functioning in-vivo-method. Besides, the upscaling of such an approach would hardly be feasible due to increasing expenses that would turn down financability. 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 [9]. This approach, however, does not offer any opportunity to evaluate expression in vivo [10].

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. Its properties and effects for peptide synthesis via NRPS compare to the ones of GFP for proteins: it is relatively inert, easily detectable and universal.

Methods

Purification of Indigoidine and Tagged Constructs

1 ml of IPTG-induced, blue culture was spun down at full speed (14,000 rpm) for 20 minutes, washed in 1 ml of methanol and centrifuged once more for 5 minutes at 14,000 rpm. Methanol was discarded and samples were dissolved in 200-400 µl DMSO.

Thin Layer Chromatography

TLC was carried out on silica-gel as immobile phase and Dichloromethane as mobile phase. For the procedure, a 50 ml beaker was filled with ~15 ml Dichloromethane and let stand for about 10 minutes (in order to let the Dichloromethane-vapor fill the beaker). The TLC plate, coated with silica-gel was spotted with sample 0.5 to 1 cm above the lower edge and placed in the beaker. TLC was run until the solvent front was at two thirds of the TLC plate. As indigoidine is light-sensitive, the beaker was covered with aluminium foil in order to prevent direct light irradiation.

Column Chromatography

In order to show that further purification (beyond the regular purification procedure) is feasible and easy with the tagged peptides, we provide a protocol for analysis and purification of the sample via column-chromatography. For this purpose, the purified sample that is dissolved in DMSO is applied to a Pre-Packed column G25, as running buffer, DMSO was used. DMSO is constantly applied to the column to prevent it from drying. All uncolored fractions are discarded, while the blue fraction is kept and collected in a 2 ml Eppendorf tube. This purified extract (with the sample dissolved in DMSO) can then be used for TLC or Mass-Spectrometry if desired.

RFC 100