Engineering of Streptoalloteichus tenebrarius 2444 for Sustainable Production of Tobramycin.

Tobramycin is a broad-spectrum aminoglycoside antibiotic agent. The compound is obtained from the base-catalyzed hydrolysis of carbamoyltobramycin (CTB), which is naturally produced by the actinomycete Streptoalloteichus tenebrarius. However, the strain uses the same precursors to synthesize several structurally related aminoglycosides. Consequently, the production yields of tobramycin are low, and the compound's purification is very challenging, costly, and time-consuming. In this study, the production of the main undesired product, apramycin, in the industrial isolate Streptoalloteichus tenebrarius 2444 was decreased by applying the fermentation media M10 and M11, which contained high concentrations of starch and dextrin. Furthermore, the strain was genetically engineered by the inactivation of the aprK gene (∆aprK), resulting in the abolishment of apramycin biosynthesis. In the next step of strain development, an additional copy of the tobramycin biosynthetic gene cluster (BGC) was introduced into the ∆aprK mutant. Fermentation by the engineered strain (∆aprK_1-17L) in M11 medium resulted in a 3- to 4-fold higher production than fermentation by the precursor strain (∆aprK). The phenotypic stability of the mutant without selection pressure was validated. The use of the engineered S. tenebrarius 2444 facilitates a step-saving, efficient, and, thus, more sustainable production of the valuable compound tobramycin on an industrial scale.

The small MbtH-like protein encoded by an internal gene of the balhimycin biosynthetic gene cluster is not required for glycopeptide production.

The balhimycin biosynthetic gene cluster of the glycopeptide producer Amycolatopsis balhimycina includes a gene (orf1) with unknown function. orf1 shows high similarity to the mbtH gene from Mycobacterium tuberculosis. In almost all nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters, we could identify a small mbtH-like gene whose function in peptide biosynthesis is not known. The mbtH-like gene is always colocalized with the NRPS genes; however, it does not have a specific position in the gene cluster. In all glycopeptide biosynthetic gene clusters the orf1-like gene is always located downstream of the gene encoding the last module of the NRPS. We inactivated the orf1 gene in A. balhimycina by generating a deletion mutant. The balhimycin production is not affected in the orf1-deletion mutant and is indistinguishable from that of the wild type. For the first time, we show that the inactivation of an mbtH-like gene does not impair the biosynthesis of a nonribosomal peptide.

Genetic analysis of the balhimycin (vancomycin-type) oxygenase genes.

In the balhimycin biosynthesis three oxygenases OxyA, OxyB and OxyC are responsible for the oxidative phenol coupling reactions, which lead to the ring-closures between the aromatic amino acid side chains in the heptapeptide aglycone. These ring-closures constrain the peptide backbone into the cup-shaped conformation that is required for binding to the Lys-D-Ala-D-Ala-terminus of the cell wall precursor peptide and represent one of the essential features of glycopeptide antibiotics. In the balhimycin biosynthetic gene cluster the oxygenase genes oxyA, oxyB and oxyC have been identified downstream of the peptide synthetase genes. Reverse transcription (RT)-PCR analyses revealed that these oxygenase genes in Amycolatopsis balhimycina are co-transcribed. Non-polar mutants (NPoxyA, DeltaoxyB and DeltaoxyC) were constructed, cultivated in production medium and assayed for the presence of glycopeptides and glycopeptide precursors by HPLC-ESI-MS. The mutant NPoxyA produces mainly monocyclic, the mutant DeltaoxyB linear and the mutant DeltaoxyC bicyclic peptides. These results definitely confirm the sequence of the three oxidative ring-closing steps (OxyB-OxyA-OxyC). The heterologous complementation of the mutant strains with the corresponding oxygenase genes from the vancomycin producer A. orientalis restored the production of balhimycin, which proves the functional equivalence of the oxygenases from the balhimycin and vancomycin producer. For the first time it is now possible to combine the genetic data obtained from the balhimycin producer with the biochemical and structural data obtained from the vancomycin producer.