Project description:Nonribosomal peptides represent a large class of metabolites with pharmaceutical relevance. Pteridines, such as pterins, folates, and flavins, are heterocyclic metabolites that often serve as redox-active cofactors. The biosynthetic machineries for construction of these distinct classes of small molecules operate independently in the cell. Here, we discovered an unprecedented nonribosomal peptide synthetase-like-pteridine synthase hybrid biosynthetic gene cluster in Photorhabdus luminescens using genome synteny analysis. P. luminescens is a Gammaproteobacterium that undergoes phenotypic variation and can have both pathogenic and mutualistic roles. Through extensive gene deletion, pathway-targeted molecular networking, quantitative proteomic analysis, and NMR, we show that the genetic locus affects the regulation of quorum sensing and secondary metabolic enzymes and encodes new pteridine metabolites functionalized with cis-amide acyl-side chains, termed pepteridine A (1) and B (2). The pepteridines are produced in the pathogenic phenotypic variant and represent the first reported metabolites to be synthesized by a hybrid NRPS-pteridine pathway. These studies expand our view of the combinatorial biosynthetic potential available in bacteria.
Project description:The disconnect between the genomic prediction of secondary metabolite biosynthetic potential and the observed laboratory production profile of microorganisms is well documented. While heterologous expression of biosynthetic gene clusters (BGCs) is often seen as a potential solution to bridge this gap, it is not immune to many challenges including impaired regulation, the inability to recruit essential building blocks, and transcriptional and/or translational silence of the biosynthetic genes. Here we report the discovery, cloning, refactoring, and heterologous expression of a cryptic hybrid phenazine-type BGC (spz) from the marine actinomycete Streptomyces sp. CNB-091. Overexpression of the engineered spz pathway resulted in increased production and chemical diversity of phenazine natural products belonging to the streptophenazine family, including bioactive members containing an unprecedented N-formylglycine attachment. An atypical discrete adenylation enzyme in the spz cluster is required to introduce the formylglycine moiety and represents a phylogenetically distinct class of adenylation proteins.
Project description:Supporting raw MS data for paper (doi: 10.1002/pmic.202100246) by Nam K.H. et al, titled "Quantitative Proteome Remodeling Characterization of Two Human Reference Pluripotent Stem Cell Lines During Neurogenesis and Cardiomyogenesis".
Index of RAW files uploaded:
- Related to Figure 1 (KN97-120: whole proteome (Unimod: 35; 2016; 4)).
- Related to Figure 2 (KN121-126: phospho proteome (Unimod: 35; 21; 2016; 4))
- Related to Figures 3 and 4 (KN205-228: whole proteome (Unimod: 35; 2016; 4))
Project description:The biosynthetic gene cluster of porothramycin, a sequence-selective DNA alkylating compound, was identified in the genome of producing strain Streptomyces albus subsp. albus (ATCC 39897) and sequentially characterized. A 39.7 kb long DNA region contains 27 putative genes, 18 of them revealing high similarity with homologous genes from biosynthetic gene cluster of closely related pyrrolobenzodiazepine (PBD) compound anthramycin. However, considering the structures of both compounds, the number of differences in the gene composition of compared biosynthetic gene clusters was unexpectedly high, indicating participation of alternative enzymes in biosynthesis of both porothramycin precursors, anthranilate, and branched L-proline derivative. Based on the sequence analysis of putative NRPS modules Por20 and Por21, we suppose that in porothramycin biosynthesis, the methylation of anthranilate unit occurs prior to the condensation reaction, while modifications of branched proline derivative, oxidation, and dimethylation of the side chain occur on already condensed PBD core. Corresponding two specific methyltransferase encoding genes por26 and por25 were identified in the porothramycin gene cluster. Surprisingly, also methyltransferase gene por18 homologous to orf19 from anthramycin biosynthesis was detected in porothramycin gene cluster even though the appropriate biosynthetic step is missing, as suggested by ultra high-performance liquid chromatography-diode array detection-mass spectrometry (UHPLC-DAD-MS) analysis of the product in the S. albus culture broth.
Project description:The GE81112 tetrapeptides (1-3) represent a structurally unique class of antibiotics, acting as specific inhibitors of prokaryotic protein synthesis. Here we report the cloning and sequencing of the GE81112 biosynthetic gene cluster from Streptomyces sp. L-49973 and the development of a genetic manipulation system for Streptomyces sp. L-49973. The biosynthetic gene cluster for the tetrapeptide antibiotic GE81112 (getA-N) was identified within a 61.7-kb region comprising 29 open reading frames (open reading frames), 14 of which were assigned to the biosynthetic gene cluster. Sequence analysis revealed the GE81112 cluster to consist of six nonribosomal peptide synthetase (NRPS) genes encoding incomplete di-domain NRPS modules and a single free standing NRPS domain as well as genes encoding other biosynthetic and modifying proteins. The involvement of the cloned gene cluster in GE81112 biosynthesis was confirmed by inactivating the NRPS gene getE resulting in a GE81112 production abolished mutant. In addition, we characterized the NRPS A-domains from the pathway by expression in Escherichia coli and in vitro enzymatic assays. The previously unknown stereochemistry of most chiral centers in GE81112 was established from a combined chemical and biosynthetic approach. Taken together, these findings have allowed us to propose a rational model for GE81112 biosynthesis. The results further open the door to developing new derivatives of these promising antibiotic compounds by genetic engineering.
Project description:Curacozole is representative of a cyanobactin-like sub-family of ribosomally synthesized and post-translationally modified peptides (RiPPs). The molecule is distinguished by its small macrocyclic structure, a poly-azole sequence that includes a phenyloxazole moiety, and a d-allo-Ile residue. The enzymatic steps required for its formation are not well understood. The predicted biosynthetic gene cluster (BGC) for curacozole in Streptomyces curacoi is cryptic, but is shown to be potently activated upon constitutive expression of the bldA-specified Leu-tRNA(UUA) molecule. Heterologous expression and gene deletion studies have defined the minimum BGC as consisting of seven genes, czlA, D, E, B1, C1, F, and BC. The biosynthetic pathway is highly substrate tolerant, accepting six variants of the precursor peptide CzlA to form new curacozole derivatives. This includes replacing the phenyloxazole moiety of curacozole with indole and p-hydroxyphenyloxazole groups by conversion of the corresponding CzlA Phe18Trp and Phe18Tyr variants. In vitro experiments with purified enzymes demonstrate that CzlD and CzlBC perform cyclodehydration and dehydrogenation reactions, respectively, to form a single oxazole from Ser 22 of CzlA. The curacozole BGC is flanked by czlI, a non-essential but conserved gene of unknown function. In vitro studies demonstrate CzlI to be a non-heme iron(ii) and 2-oxoglutarate-dependent dioxygenase, catalyzing the hydroxylation of Phe18 on CzlA to form the CzlA Phe18Tyr variant, which is then processed to form the p-hydroxyphenyloxazole derivative of curacozole. Overall, this work highlights the amenability of RiPP biosynthesis for engineering the production of new compounds and adds to the repertoire of known RiPP enzymes.
Project description:Members of the diazaquinomycin class of natural products have shown potent and selective activity against Mycobacterium tuberculosis. However, poor aqueous solubility has prevented extensive studies in animal models thus far. Our long-term goal is to harness knowledge regarding diazaquinomycin biosynthesis towards the generation of derivatives for structure-activity relationship studies. We have previously sequenced the genomes of two diazaquinomycin-producing, actinomycete bacteria and identified putative daq biosynthetic gene clusters. Here, we report the heterologous expression of the daq gene cluster from the marine Streptomyces sp. F001 in S. coelicolor M1152. In addition to serving as functional proof for gene cluster assignment, the heterologous expression system reported here is expected to facilitate investigations aimed at elucidating diazaquinomycin biosynthesis.
Project description:Computational analysis of biosynthetic gene clusters (BGCs) has revolutionized natural product discovery by enabling the rapid investigation of secondary metabolic potential within microbial genome sequences. Grouping homologous BGCs into Gene Cluster Families (GCFs) facilitates mapping their architectural and taxonomic diversity and provides insights into the novelty of putative BGCs, through dereplication with BGCs of known function. While multiple databases exist for exploring BGCs from publicly available data, no public resources exist that focus on GCF relationships. Here, we present BiG-FAM, a database of 29,955 GCFs capturing the global diversity of 1,225,071 BGCs predicted from 209,206 publicly available microbial genomes and metagenome-assembled genomes (MAGs). The database offers rich functionalities, such as multi-criterion GCF searches, direct links to BGC databases such as antiSMASH-DB, and rapid GCF annotation of user-supplied BGCs from antiSMASH results. BiG-FAM can be accessed online at https://bigfam.bioinformatics.nl.