The actinomycete Nonomuraea sp. strain ATCC 39727 produces the
glycopeptide A40926, the precursor of
dalbavancin. Biosynthesis of
A40926 is encoded by the dbv gene cluster, which contains 37
protein-coding sequences that participate in
antibiotic biosynthesis, regulation, immunity, and export. In addition to the positive regulatory
protein Dbv4, the A40926-biosynthetic gene cluster encodes two additional putative regulators, Dbv3 and Dbv6. Independent mutations in these genes, combined with bioassays and liquid chromatography-mass spectrometry (LC-MS) analyses, demonstrated that Dbv3 and Dbv4 are both required for
antibiotic production, while inactivation of dbv6 had no effect. In addition, overexpression of dbv3 led to higher levels of
A40926 production. Transcriptional and quantitative reverse transcription (RT)-PCR analyses showed that Dbv4 is essential for the transcription of two operons, dbv14-dbv8 and dbv30-dbv35, while Dbv3 positively controls the expression of four monocistronic transcription units (dbv4, dbv29, dbv36, and dbv37) and of six operons (dbv2-dbv1, dbv14-dbv8, dbv17-dbv15, dbv21-dbv20, dbv24-dbv28, and dbv30-dbv35). We propose a complex and coordinated model of regulation in which Dbv3 directly or indirectly activates transcription of dbv4 and controls biosynthesis of
4-hydroxyphenylglycine and the heptapeptide backbone,
A40926 export, and some tailoring reactions (mannosylation and
hexose oxidation), while Dbv4 directly regulates biosynthesis of
3,5-dihydroxyphenylglycine and other tailoring reactions, including the four cross-links, halogenation, glycosylation, and acylation.
IMPORTANCE: This report expands knowledge of the regulatory mechanisms used to control the biosynthesis of the
glycopeptide antibiotic A40926 in the actinomycete Nonomuraea sp. strain ATCC 39727.
A40926 is the precursor of
dalbavancin, approved for treatment of skin
infections by Gram-positive bacteria. Therefore, understanding the regulation of its biosynthesis is also of industrial importance. So far, the regulatory mechanisms used to control two other similar
glycopeptides (
balhimycin and
teicoplanin) have been elucidated, and beyond a common step, different clusters seem to have devised different strategies to control
glycopeptide production. Thus, our work provides one more example of the pitfalls of deducing regulatory roles from bioinformatic analyses only, even when analyzing gene clusters directing the synthesis of structurally related compounds.