Engineering a production platform for the potent antipseudomonal antibiotic mureidomycin A by reprogramming the pacidamycin biosynthetic gene cluster

Uridyl peptide antibiotics display potent activity against Pseudomonas aeruginosa, a pathogen causing severe nosocomial infections. The four subclasses-mureidomycins, pacidamycins, napsamycins, and sansanmycins-inhibit the underexplored bacterial target phospho-MurNAc-pentapeptide translocase (MraY). Structurally, these compounds contain a 3'-deoxyuridine linked to a central diaminobutyric acid. The latter is situated within a pseudo tetra- or pentapeptide moiety, assembled by a non-ribosomal peptide synthetase machinery. The subclasses are distinguished by variations in the incorporated amino acids. Native producer strains typically synthesize numerous different uridyl peptide antibiotics in low yields. Since the pacidamycin gene cluster displays higher overall yields compared to the napsamycin cluster in identical expression systems, we aimed to reprogram it into a selective production platform for the highly bioactive mureidomycin A. This approach is intended to streamline isolation processes and support hit-to-lead optimization.

Streptomyces albus J1074 was identified as the most promising host for heterologous expression of the pacidamycin gene cluster. Subsequently, media screening and precursor feeding were conducted, and regulators of closely related gene clusters were introduced into the heterologous host. The overexpression of the NpsM regulator of the napsamycin gene cluster increased production significantly. Next, the pacidamycin gene cluster was engineered to produce the more potent mureidomycins. Variable positions of the peptide moiety were individually targeted by domain or module swaps of the non-ribosomal peptide synthetase machinery. More specifically, these were replaced by homologs of the napsamycin gene cluster, resulting in strains mainly producing sansanmycin A or mureidomycin A. Promoter engineering enhanced mureidomycin A production in the final strain, correlating with increased transcription levels in RNA sequencing experiments on an intermediate strain harboring the same promoters.

Hybrid uridyl peptide antibiotic gene clusters were constructed by genetically engineering the pacidamycin cluster, demonstrating the versatility of these clusters. While the native pacidamycin cluster only produced pacidamycin D and S, the final strain yields the more bioactive mureidomycin A and Met-pacidamycin 5. Additionally, sansanmycin A (syn. Met-pacidamycin 4) can be isolated from an intermediate construct of this study. This expands the access to different analogs and highlights further engineering potential, which will facilitate the future development of these promising antibiotics.