Chemical Diversification Based on Substrate Promiscuity of a Standalone Adenylation Domain in a Reconstituted NRPS System

A nonribosomal peptide synthetase (NRPS) assembly line (sfa) in Streptomyces thioluteus that directs the formation of the diisonitrile chalkophore SF2768 (1) has been characterized by heterologous expression and directed gene knockouts. Herein, differential metabolic analysis of the heterologous expression strain and the original host led to the isolation of an SF2768 analogue (2, a byproduct of sfa) that possesses N-isovaleryl rather than 3-isocyanobutyryl side chains. The proposed biosynthetic logic of sfa and the structural difference between 1 and 2 suggested substrate promiscuity of the adenylate-forming enzyme SfaB. Further substrate scope investigation of SfaB and a successfully reconstituted NRPS system including a four-enzyme cascade enabled incorporation of diverse carboxylic acid building blocks into peptide scaffolds, and 30 unnatural products were thus generated. This structural diversification strategy based on substrate flexibility of the adenylation domain and in vitro reconstitution can be applied to other adenylation-priming pathways, thus providing a supplementary method for diversity-oriented total synthesis. Additionally, the biocatalytic process of the putative lysine δ-hydroxylase SfaE was validated through the derivatization of two key aldehyde intermediates (2a and 2b), thereby expanding the toolkit of enzymatic C–H bond activation

Biosynthetic Diversification of Fidaxomicin Aglycones by Heterologous Expression and Promoter Refactoring

Fidaxomicin (Dificid) is a commercial macrolide antibiotic for treating Clostridium difficile infection. Total synthesis of fidaxomicin and its aglycone had been achieved through different synthetic schemes. In this study, an alternative biological route to afford the unique 18-membered macrolactone aglycone of fidaxomicin was developed. The promoter refactored fidaxomicin biosynthetic gene cluster from Dactylosporangium aurantiacum was expressed in the commonly used host Streptomyces albus J1074, thereby delivering five structurally diverse fidaxomicin aglycones with the corresponding titers ranging from 4.9 to 15.0 mg L–1. In general, these results validated a biological strategy to construct and diversify fidaxomicin aglycones on the basis of promoter refactoring and heterologous expression

Biosynthesis and Engineered Overproduction of Everninomicins with Promising Activity against Multidrug-Resistant Bacteria

Ribosome-targeting oligosaccharides, everninomicins (EVNs), are promising drug leads with a unique mode of action distinct from that of currently used antibiotics in human therapy. However, the low yields in natural microbial producers hamper an efficient preparation of EVNs for detailed structure–activity relationship analysis. Herein, we enhance the production of EVNs by duplicating the biosynthetic gene cluster (BGC) in Micromonospora sp. SCSIO 07395 and thus obtain multiple EVNs that are sufficient for bioactivity evaluation. EVNs (1–5) are shown to significantly inhibit the growth of multidrug-resistant Gram-positive staphylococcal, enterococcal, and streptococcal strains and Gram-negative pathogens Acinetobacter baumannii and Vibrio cholerae, with micromolar to nanomolar potency, which are comparable or superior to vancomycin, linezolid, and daptomycin. Furthermore, the BGC duplication strategy is proven effective in stepwisely improving titers of the bioactive EVN M (5) from the trace amount to 98.6 mg L–1. Our findings demonstrate the utility of a bioengineering approach for enhanced production and chemical diversification of the medicinally promising EVNs

Biosynthesis and Engineered Overproduction of Everninomicins with Promising Activity against Multidrug-Resistant Bacteria

Ribosome-targeting oligosaccharides, everninomicins (EVNs), are promising drug leads with a unique mode of action distinct from that of currently used antibiotics in human therapy. However, the low yields in natural microbial producers hamper an efficient preparation of EVNs for detailed structure–activity relationship analysis. Herein, we enhance the production of EVNs by duplicating the biosynthetic gene cluster (BGC) in Micromonospora sp. SCSIO 07395 and thus obtain multiple EVNs that are sufficient for bioactivity evaluation. EVNs (1–5) are shown to significantly inhibit the growth of multidrug-resistant Gram-positive staphylococcal, enterococcal, and streptococcal strains and Gram-negative pathogens Acinetobacter baumannii and Vibrio cholerae, with micromolar to nanomolar potency, which are comparable or superior to vancomycin, linezolid, and daptomycin. Furthermore, the BGC duplication strategy is proven effective in stepwisely improving titers of the bioactive EVN M (5) from the trace amount to 98.6 mg L–1. Our findings demonstrate the utility of a bioengineering approach for enhanced production and chemical diversification of the medicinally promising EVNs

Biosynthesis and Engineered Overproduction of Everninomicins with Promising Activity against Multidrug-Resistant Bacteria

Ribosome-targeting oligosaccharides, everninomicins (EVNs), are promising drug leads with a unique mode of action distinct from that of currently used antibiotics in human therapy. However, the low yields in natural microbial producers hamper an efficient preparation of EVNs for detailed structure–activity relationship analysis. Herein, we enhance the production of EVNs by duplicating the biosynthetic gene cluster (BGC) in Micromonospora sp. SCSIO 07395 and thus obtain multiple EVNs that are sufficient for bioactivity evaluation. EVNs (1–5) are shown to significantly inhibit the growth of multidrug-resistant Gram-positive staphylococcal, enterococcal, and streptococcal strains and Gram-negative pathogens Acinetobacter baumannii and Vibrio cholerae, with micromolar to nanomolar potency, which are comparable or superior to vancomycin, linezolid, and daptomycin. Furthermore, the BGC duplication strategy is proven effective in stepwisely improving titers of the bioactive EVN M (5) from the trace amount to 98.6 mg L–1. Our findings demonstrate the utility of a bioengineering approach for enhanced production and chemical diversification of the medicinally promising EVNs

Additional file 1 of Spatiotemporal distribution of migraine in China: analyses based on baidu index

Supplementary Material

Tandem Hydration of Diisonitriles Triggered by Isonitrile Hydratase in Streptomyces thioluteus

The biosynthetic pathway of diisonitrile chalkophore SF2768 was identified in Streptomyces thioluteus through heterologous expression recently. Isolation and structure elucidation of the N-substituted formamides that coexisted with the diisonitriles implied that a hydration event was involved. <i>In vitro</i> enzymatic assays of an endogenous isonitrile hydratase suggested a rare sequential-hydration of the diisonitriles. Additionally, the results of Cu-CAS assays indicate that both partial and complete hydration led to the loss of the copper-chelating ability of SF2768

Discovery of Tetronate-Containing Kongjuemycins from a Coral-Associated Actinomycete and Elucidation of Their Biosynthetic Origin

Tetronate antibiotics make up a growing family of natural products with a wide variety of biological activities. Herein, we report four new tetronates kongjuemycins (KJMs, 5–8) from a coral-associated actinomycete Pseudonocardia kongjuensis SCSIO 11457, and the identification and characterization of the KJM biosynthetic gene cluster (kjm) by heterologous expression, comparative genomic analysis, isotope labeling, and gene knockout studies. The biosynthesis of KJMs is demonstrated to harness diverse precursors from primary metabolism for building secondary metabolites

Additional file 5: of Human-induced pluripotent stem cell-derived macrophages and their immunological function in response to tuberculosis infection

Figure S5. Immunofluorescence images showing the positive expression of Mφ lineage markers MHC-II in iPS-Mφ (A), THP-1-Mφ (B) and ES-Mφ (C). Nuclei are labeled with DAPI. Bar = 100 μm. (TIFF 1462 kb