The colinearity ofcanonical modular polyketide synthases, which creates a direct link betweenmultienzyme structure and the chemical structure of the biosyntheticend-product, has become a cornerstone of knowledge-based genome mining. Here wereport genetic and enzymatic evidence for the remarkable role of anenoylreductase in the polyketide synthase for azalomycin F biosynthesis. This internalenoylreductase domain, previously identified as acting only in the second oftwo chain extension cycles on an initial iterative module, is shown here alsoto catalyse enoylreduction in trans withinthe next module. The mechanism for this rare deviation from colinearity appearsto involve direct cross-modular interaction of the reductase with the longeracyl chain, rather than backtransfer of the substrate into the iterativemodule, suggesting an additional and surprising plasticity in natural PKSassembly-line catalysis.

Streptovaricin C is a naphthalenic ansamycin antibiotic structurally similar to rifamycins with potential anti-MRSA bioactivities. However, the formation mechanism of the most fascinating and bioactivity-related methylenedioxy bridge (MDB) moiety in streptovaricins is unclear. Based on genetic and biochemical evidences, we herein clarify that the P450 enzyme StvP2 catalyzes the MDB formation in streptovaricins, with an atypical substrate inhibition kinetics. Furthermore, X-ray crystal structures in complex with substrate and structure-based mutagenesis reveal the intrinsic details of the enzymatic reaction. The mechanism of MDB formation is proposed to be an intramolecular nucleophilic substitution resulting from the hydroxylation by the heme core and the keto-enol tautomerization via a crucial catalytic triad (Asp89-His92-Arg72) in StvP2. In addition, in vitro reconstitution uncovers that C6-O-methylationand C4-O-acetylation of streptovaricins are necessary prerequisites for the MDB formation. This work provides insight for the MDB formation and adds evidence in support of the functional versatility of P450 enzymes.

Two new spirotetronate natural products, lobophorin L (1) and lobophorin M (2), together with three known lobophorin-like spirotetronate antibiotics (3-5) and two known ansamycins (6-7), were isolated from the marine-derived Streptomyces sp. 4506. The structures of 1 and 2 were established on the basis of HRESIMS as well as 1D and 2D NMR datasets. Antibacterial assay showed that, compounds 1 and 3-5 exhibited strong to moderate antibacterial activities against Micrococcus luteus and Bacillus thuringiensis with MIC values ranging from 0.0625 to 8 μg/mL, while compounds 3 and 6 showed weak antibacterial activities against Staphylococcus aureus and MRSA. The antibacterial activities of the lobophorins in this study indicated that the more substitution number of the sugar moieties at C-9 of the lobophorin, the stronger antimicrobial properties it may deserve, and the higher the oxidation degree of substituent group at C-3_D, the better antibacterial activities of its corresponding compound could be.

The alarming spread of antimicrobial resistance requires the development of novel anti-infective drugs. Despite the recent research focus on the human microbiome and its likely value to understand and exploit inter-bacterial inhibitory phenomena as a source for antimicrobial strategies, the human microbiota has barely been investigated for the purpose of drug development. We performed a large screen analyzing over 3000 human skin isolates to evaluate bacterial competition within the human skin microbiota as a basis for the development of anti-infective therapeutics. We discovered a Staphylococcus hominis strain with strong and broad activity against Gram-positive pathogens that was mediated by the bacteriocin micrococcin P1 (MP1). In "probiotic" approaches, this strain led to reduced Staphylococcus aureus infection and accelerated closure of S. aureus-infected wounds. Furthermore, we used a nanoparticle strategy to overcome the physico-chemical limitations often encountered with natural substances such as MP1 and demonstrate a significant reduction of S. aureus infection by MP1-loaded nanoparticles. Our study gives examples of how analysis of bacterial interactions in the human microbiota can be explored for the development of novel, effective anti-infective strategies.

Hygromycin B is an aminoglycoside antibiotic widely used in industry andbiological research. However, most of its biosynthetic pathway has not been completely identified due to the immense difficulty ingenetic manipulation of the producing strain. To address this problem, wedeveloped an efficient system that combines clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9-associated baseediting and site-specific recombination instead of conventional double-cross-based homologous recombination. This strategywas successfully applied tothe in vivo inactivation of fivecandidate genes involved in the biosynthesis of hygromycin B by generating stop codons or mutating conserved residues within the encoding region. The results revealed that HygJ,HygL and HygD are responsible for successive dehydrogenation, transaminationand transglycosylation of nucleosidediphosphate (NDP)-heptose. Notably, HygY acts as an unusual radical S-adenosylmethionine(SAM)-dependent epimerase for hydroxyl carbons, and HygM serves as a versatilemethyltransferase in multiple parallel metabolic networks. Based on in vivo and in vitro evidence, the biosynthetic pathway for hygromycin B is proposed.