The Impact of Heterologous Regulatory Genes from Lipodepsipeptide Biosynthetic Gene Clusters on the Production of Teicoplanin and A40926.

StrR-like pathway-specific transcriptional regulators (PSRs) function as activators in the biosynthesis of various antibiotics, including glycopeptides (GPAs), aminoglycosides, aminocoumarins, and ramoplanin-like lipodepsipeptides (LDPs). In particular, the roles of StrR-like PSRs have been previously investigated in the biosynthesis of streptomycin, novobiocin, GPAs like balhimycin, teicoplanin, and A40926, as well as LDP enduracidin. In the current study, we focused on StrR-like PSRs from the ramoplanin biosynthetic gene cluster (BGC) in Actinoplanes ramoplaninifer ATCC 33076 (Ramo5) and the chersinamycin BGC in Micromonospora chersina DSM 44151 (Chers28). Through the analysis of the amino acid sequences of Ramo5 and Chers28, we discovered that these proteins are phylogenetically distant from other experimentally investigated StrR PSRs, although all StrR-like PSRs found in BGCs for different antibiotics share a conserved secondary structure. To investigate whether Ramo5 and Chers28, given their phylogenetic positions, might influence the biosynthesis of other antibiotic pathways governed by StrR-like PSRs, the corresponding genes (ramo5 and chers28) were heterologously expressed in Actinoplanes teichomyceticus NRRL B-16726 and Nonomuraea gerenzanensis ATCC 39727, which produce the clinically-relevant GPAs teicoplanin and A40926, respectively. Recombinant strains of NRRL B-16726 and ATCC 39727 expressing chers28 exhibited improved antibiotic production, although the expression of ramo5 did not yield the same effect. These results demonstrate that some StrR-like PSRs can "cross-talk" between distant biosynthetic pathways and might be utilized as tools for the activation of silent BGCs regulated by StrR-like PSRs.

Heterologous Expression Reveals Ancient Properties of Tei3­A VanS Ortholog from the Teicoplanin Producer Actinoplanes teichomyceticus

Glycopeptide antibiotics (GPAs) are among the most clinically successful antimicrobials. GPAs inhibit cell-wall biosynthesis in Gram-positive bacteria via binding to lipid II. Natural GPAs are produced by various actinobacteria. Being themselves Gram-positives, the GPA producers evolved sophisticated mechanisms of self-resistance to avoid suicide during antibiotic production. These self-resistance genes are considered the primary source of GPA resistance genes actually spreading among pathogenic enterococci and staphylococci. The GPA-resistance mechanism in Actinoplanes teichomyceticus­the producer of the last-resort-drug teicoplanin­has been intensively studied in recent years, posing relevant questions about the role of Tei3 sensor histidine kinase. In the current work, the molecular properties of Tei3 were investigated. The setup of a GPA-responsive assay system in the model Streptomyces coelicolor allowed us to demonstrate that Tei3 functions as a non-inducible kinase, conferring high levels of GPA resistance in A. teichomyceticus. The expression of different truncated versions of tei3 in S. coelicolor indicated that both the transmembrane helices of Tei3 are crucial for proper functioning. Finally, a hybrid gene was constructed, coding for a chimera protein combining the Tei3 sensor domain with the kinase domain of VanS, with the latter being the inducible Tei3 ortholog from S. coelicolor. Surprisingly, such a chimera did not respond to teicoplanin, but indeed to the related GPA A40926. Coupling these experimental results with a further in silico analysis, a novel scenario on GPA-resistance and biosynthetic genes co-evolution in A. teichomyceticus was hereby proposed.

Genetics Behind the Glycosylation Patterns in the Biosynthesis of Dalbaheptides.

Glycopeptide antibiotics are valuable natural metabolites endowed with different pharmacological properties, among them are dalbaheptides used to treat different infections caused by multidrug-resistant Gram-positive pathogens. Dalbaheptides are produced by soil-dwelling high G-C Gram-positive actinobacteria. Their biosynthetic pathways are encoded within large biosynthetic gene clusters. A non-ribosomally synthesized heptapeptide aglycone is the common scaffold for all dalbaheptides. Different enzymatic tailoring steps, including glycosylation, are further involved in decorating it. Glycosylation of dalbaheptides is a crucial step, conferring them specific biological activities. It is achieved by a plethora of glycosyltransferases, encoded within the corresponding biosynthetic gene clusters, able to install different sugar residues. These sugars might originate from the primary metabolism, or, alternatively, their biosynthesis might be encoded within the biosynthetic gene clusters. Already installed monosaccharides might be further enzymatically modified or work as substrates for additional glycosylation. In the current minireview, we cover recent updates concerning the genetics and enzymology behind the glycosylation of dalbaheptides, building a detailed and consecutive picture of this process and of its biological evolution. A thorough understanding of how glycosyltransferases function in dalbaheptide biosynthesis might open new ways to use them in chemo-enzymatic synthesis and/or in combinatorial biosynthesis for building novel glycosylated antibiotics.