Actinomycetes are well known as an inexhaustible source for antibiotics

The emergence of antibiotic-resistant pathogenic bacteria within the last decades is one reason for the urgent need for new antibacterial agents. A strategy to discover new anti-infective compounds is the evaluation of the genetic capacity of secondary metabolite producers and the activation of cryptic gene clusters (genome mining). One genus known for its potential to synthesize medically important products is Amycolatopsis . However, Amycolatopsis japonicum does not produce an antibiotic under standard laboratory conditions. In contrast to most Amycolatopsis strains, A. japonicum is genetically tractable with different methods. In order to activate a possible silent glycopeptide cluster, we introduced a gene encoding the transcriptional activator of balhimycin biosynthesis, the bbr gene from Amycolatopsis balhimycina ( bbr _Aba ), into A. japonicum . This resulted in the production of an antibiotically active compound. Following whole-genome sequencing of A. japonicum , 29 cryptic gene clusters were identified by genome mining. One of these gene clusters is a putative glycopeptide biosynthesis gene cluster. Using bioinformatic tools, ristomycin (syn. ristocetin), a type III glycopeptide, which has antibacterial activity and which is used for the diagnosis of von Willebrand disease and Bernard-Soulier syndrome, was deduced as a possible product of the gene cluster. Chemical analyses by high-performance liquid chromatography and mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy confirmed the in silico prediction that the recombinant A. japonicum /pRM4- bbr _Aba synthesizes ristomycin A.

Kistamicin is a divergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria. The extensively crosslinked structure of these antibiotics that is essential for their activity makes their chemical synthesis highly challenging and limits their production to bacterial fermentation. Kistamicin contains three crosslinks, including an unusual 15-membered A-O-B ring, despite the presence of only two Cytochrome P450 Oxy enzymes thought to catalyse formation of such crosslinks within the biosynthetic gene cluster. In this study, we characterise the kistamicin cyclisation pathway, showing that the two Oxy enzymes are responsible for these crosslinks within kistamicin and that they function through interactions with the X-domain, unique to glycopeptide antibiotic biosynthesis. We also show that the kistamicin OxyC enzyme is a promiscuous biocatalyst, able to install multiple crosslinks into peptides containing phenolic amino acids.

Halogenation plays a significant role in the activity of the glycopeptide antibiotics (GPAs), although up until now the timing and therefore exact substrate involved was unclear. Here, we present results combined from in vivo and in vitro studies that reveal the substrates for the halogenase enzymes from GPA biosynthesis as amino acid residues bound to peptidyl carrier protein (PCP)-domains from the non-ribosomal peptide synthetase machinery: no activity was detected upon either free amino acids or PCP-bound peptides. Furthermore, we show that the selectivity of GPA halogenase enzymes depends upon both the structure of the bound amino acid and the PCP domain, rather than being driven solely via the PCP domain. These studies provide the first detailed understanding of how halogenation is performed during GPA biosynthesis and highlight the importance and versatility of trans-acting enzymes that operate during peptide assembly by non-ribosomal peptide synthetases.

Covering: up to February 2019 Actinomycetes are Gram positive bacteria of the phylum Actinobacteria. These organisms are one of the most important sources of structurally diverse, clinically used antibiotics and other valuable bioactive products, as well as biotechnologically relevant enzymes. Most strains were discovered by their ability to produce a given molecule and were often poorly characterized, physiologically and genetically. The development of genetic methods for Streptomyces and related filamentous actinomycetes has led to the successful manipulation of antibiotic biosynthesis to attain structural modification of microbial metabolites that would have been inaccessible by chemical means and improved production yields. Moreover, genome mining reveals that actinomycete genomes contain multiple biosynthetic gene clusters (BGCs), however only a few of them are expressed under standard laboratory conditions, leading to the production of the respective compound(s). Thus, to access and activate the so-called “silent” BGCs, to improve their biosynthetic potential and to discover novel natural products methodologies for genetic manipulation are required. Although different methods have been applied for many actinomycete strains, genetic engineering is still remaining very challenging for some “underexplored” and poorly characterized actinomycetes. This review summarizes the strategies developed to overcome the obstacles to genetic manipulation of actinomycetes and allowing thereby rational genetic engineering of this industrially relevant group of microorganisms. At the end of this review we give some tips to researchers with limited or no previous experience in genetic manipulation of actinomycetes. The article covers the most relevant literature published until February 2019.