Biosynthesis and Regulation of Grisemycin, a New Member of the Linaridin Family of Ribosomally Synthesized Peptides Produced by Streptomyces griseus IFO 13350

Abstract

Our recent identification and genetic analysis of the biosynthetic gene cluster for production of the ribosomally synthesized and posttranslationally modified peptide cypemycin revealed a new class of peptide natural products, the linaridins. Here we describe the identification and characterization of grisemycin, a linaridin produced by a previously unidentified gene cluster in Streptomyces griseus IFO 13350. Mass spectrometric analysis revealed that grisemycin possesses at least three of the modifications found in cypemycin, as well as an analogous leader peptidase cleavage site. Expression of putative grisemycin biosynthetic genes in a Streptomyces coelicolor A3(2) derivative, combined with deletion of the gene encoding the grisemycin precursor peptide, confirmed the identity of the grisemycin gene cluster. Both grisemycin and cypemycin depend on the transcriptional activator AdpA for wild-type levels of production.Although the ribosomal translational machinery generally uses only the 20 genetically encoded amino acids for protein and peptide synthesis, nature has invented several mechanisms to expand on this structural repertoire by posttranslational modification of the constituent amino acids. Several new classes of posttranslationally modified peptides have been identified based on recent advances in microbial genome sequencing, biochemistry, and genetics. Examples include the thiopeptides (9,15,17,26), patellamides (24), linear heterocyclized toxins (13), and linaridins (3). Interest in these compounds stems from the identification of previously undescribed peptides with potentially interesting biological properties and the discovery and characterization of new types of modification enzymes. Such enzymes could well play a role in rational peptide engineering (22). A ribosomally synthesized and modified peptide is typically encoded as a prepropeptide that consists of a propeptide that is subject to posttranslational modifications and a leader sequence that is thought to serve as a recognition signal for biosynthetic and/or transporter proteins and as a means of sequestering the compound in an inactivate form while it is inside the cell (21).Our work on the posttranslationally modified peptide cypemycin revealed a new family of linear, dehydrated peptides, the linaridins (3). Several previously unrecognized linaridin gene clusters were identified bioinformatically in different bacterial phyla and even in the Archaea. Here we describe the identification of grisemycin as the product of a previously unidentified linaridin gene cluster found in Streptomyces griseus IFO 13350. We show that grisemycin contains at least three of the posttranslational modifications found in cypemycin, and we identify the grisemycin biosynthetic gene cluster. We also show that the production of both grisemycin and cypemycin is regulated by the transcriptional activator AdpA, a master regulatory protein that links ␥-butyrolactone signaling to the expression of natural product gene clusters-and morphological differentiation-in many str...

Nucleophilic 1,4-Additions for Natural Product Discovery

“…griseus is a known producer of grisemycin, of which the mass of the labeled natural product did correlate (Figure 4a–b). MS/MS fragmentation analysis yielded a seven amino acid sequence tag confirming the identity of the compound as grisemycin (Figure 4c) (15). The labeling and identification of grisemycin, a member of the linaridin class of natural products, further validated our reactivity-based screen while also highlighting the usefulness of bioinformatic integration to rapidly dereplicate known compounds.…”

Pleiotropic regulatory genes bldA , adpA and absB are implicated in production of phosphoglycolipid antibiotic moenomycin

“…No CSR genes are found in the moe cluster; the presence of essential moenomycin-specific regulatory gene(s) elsewhere in the S. ghanaensis genome is unlikely given that we were able to recreate moenomycin production in several heterologous hosts [10]. Although CSR-free SM gene clusters in actinomycetes have been considered the exception rather than the rule [11,12], the number has increased steadily as numerous whole genomes have been sequenced and analysed [13–16]. These gene clusters represent a poorly understood archetype of regulation of actinomycete SM, where CSRs are not involved.…”

Structural and functional insight into an unexpectedly selective N -methyltransferase involved in plantazolicin biosynthesis

“…The N-terminal methylation of ribosomal peptides from bacteria is exceptionally rare. With respect to natural products, the only compounds that we are aware of that undergo N-terminal dimethylation besides PZN are the linaridin antibiotics (e.g., cypemycin, grisemycin), the best studied of which contain N α ,N α -dimethylAla (12)(13)(14). Through genetic manipulation and alteration of cultivation conditions, PZN biosynthetic intermediates have been intercepted, permitting investigation into the details of downstream tailoring reactions and the bioactivity of partially processed substrates (3).…”