Functional dissection of transport processes during glycopeptide antibiotic production

Glycopeptide antibiotics (GPAs) - e.g., vancomycin - are natural products that have been used for many years as a last resort and are an important group of agents to fight bacterial infections by inhibiting cell wall biosynthesis. GPAs are modified heptapeptides that are synthesized by non-ribosomal peptide synthetases (NRPS) from proteinogenic and non-proteinogenic amino acids. However, their modification results in a variety of different structures. In addition to all the biosynthetic proteins, we noticed that a transport-related protein was always co-encoded in the associated biosynthetic gene cluster (BGC). Interestingly, we found that in all 89 BGCs we analyzed, this was an ABC transporter. In A. balhimycina, the transporter Tba is essential for the export of the GPA balhimycin. However, a detailed functional analysis of its specificity and interactions in the cellular context is not yet available, which is the primary goal of our work.

We performed a phylogenetic analysis of GPA ABC transporters in the context of other transporters encoded by producer strains and found that they form a distinct clade. We have established a balhimycin export assay that allows the investigation of the specificity of different GPA transporters in vivo by HPLC-MS and bioassays. This indicates substrate specificity of different GPA transporters, despite high similarity in sequence and structure. Furthermore, MD simulation and mutagenesis studies led us to suggest, which parts of the GPA are important for the interaction with the transporter. To confirm this in vitro, we are trying to purify Tba. Additionally, our data indicate a regulatory mechanism of the balhimycin production in absence of the native transporter. We have therefore used different omics approaches to investigate it further. So far, we have ruled out another regulation at the transcriptional level.
Finally, we are trying to understand the role of GPA production and export in a cellular context by using MS-based methods to identify protein-protein interactions after proximity biotinylation and immunoprecipitation.