Nosiheptide (NOS), belonging to the e series of thiopeptide antibiotics that exhibit potent activity against various bacterial pathogens, bears a unique indole side ring system and regiospecific hydroxyl groups on the characteristic macrocyclic core. Here, cloning, sequencing and characterization of the nos gene cluster from Streptomyces actuosus ATCC 25421 as a model for this series of thiopeptides has unveiled new insights into their biosynthesis. Bioinformatics-based sequence analysis and in vivo investigation into the gene functions show that NOS biosynthesis shares a common strategy with recently characterized b or c series thiopeptides for forming the characteristic macrocyclic core, which features a ribosomally synthesized precursor peptide with conserved posttranslational modifications. However, it apparently proceeds via a different route for tailoring the thiopeptide framework, allowing the final product to exhibit the distinct structural characteristics of e series thiopeptides, such as the indole side ring system. Chemical complementation supports the notion that the S-adenosylmethionine (AdoMet)-dependent protein NosL may play a central role in converting Trp to the key 3-methylindole moiety by an unusual carbon side chain rearrangement, most likely via a radical-initiated mechanism. Characterization of the indole side ring-opened analog of NOS from the nosN mutant strain is consistent with the proposed methyltransferase activity of its encoded protein, shedding light into the timing of the individual steps for indole side ring biosynthesis. These results also suggest the feasibility of engineering novel thiopeptides for drug discovery by manipulating the NOS biosynthetic machinery.Thiopeptides are a growing class of sulfur-rich, highly modified heterocyclic peptides (1). Despite overall structural diversity, they share a characteristic macrocyclic core, consisting of a nitrogen-containing, 6-membered ring central to multiple thiazoles and dehydroamino acids (Figure 1). Nosiheptide-like members, classified as e series thiopeptides according to a central 2,3,5,6-tetrasubstituted pyridine domain, possess an indolic acid ring system that is appended to the side chains of the Ser/Cys and hydroxylated Glu residues of the macrocyclic core via at least two carboxylic ester linkages (e.g. O-and S-linkages for nosiheptide, and two O-linkages * To whom correspondence should be addressed: Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Rd., Shanghai, 200032, China. Wen Liu, wliu@mail.sioc.ac.cn, Tel: 86-21-54925111, Fax: 86-21-64166128. Supporting Information Available: This material is available free of charge via the internet at http://pubs.acs.org.Nucleotide Sequence Accession Number: The sequence reported in this paper has been deposited in GenBank under the accession number FJ438820. Nosiheptide (NOS), produced by Streptomyces actuosus ATCC 25421, is one of the oldest known thiopeptides and has been widely used as a feed additive for animal growth (9,10). The str...

Summary Thiopeptides, with potent activity against various drug-resistant pathogens, contain a characteristic macrocyclic core consisting of multiple thiazoles, dehydroamino acids, and a 6-membered nitrogen heterocycle. Their biosynthetic pathways remain elusive in spite of great efforts by in vivo feeding experiments. Here, cloning, sequencing and characterization of the thiostrepton and siomycin A gene clusters unveiled a new biosynthetic paradigm for the thiopeptide specific core formation, featuring ribosomally synthesized precursor peptides and conserved posttranslational modifications. The paradigm generality for thiopeptide biosynthesis was supported by genome mining and ultimate confirmation of the thiocillin I production in Bacillus cereus ATCC 14579, a strain that was previously unknown as a thiopeptide producer. These findings set the stage to accelerate the discovery of novel thiopeptides by prediction at the genetic level and to generate structural diversity by applying combinatorial biosynthesis methods.

The radical S-adenosylmethionine (S-AdoMet) superfamily contains thousands of proteins that catalyze highly diverse conversions, most of which are poorly understood due to a lack of information regarding chemical products and radical-dependent transformations. We here report that NosL, involved in forming the indole side ring of the thiopeptide nosiheptide (NOS), is a radical S-AdoMet 3-methyl-2-indolic acid (MIA) synthase. NosL catalyzed an unprecedented carbon chain reconstitution of L-Trp to give MIA, showing removal of the Cα-N unit and shift of the carboxylate to the indole ring. Dissection of the enzymatic process upon the identification of products and a putative glycyl intermediate uncovered a radical-mediated, unusual fragmentation-recombination reaction. This finding unveiled a key step in radical S-AdoMet enzyme-catalyzed structural rearrangements during complex biotransformations. Additionally, NosL tolerated fluorinated L-Trps as the substrates, allowing for production of a regiospecifically halogenated thiopeptide that has not been found in over 80 entity-containing, naturally occurring thiopeptide family.

A 45 kb DNA sequencing analysis from Streptomyces hygroscopicus 5008 involved in validamycin A (VAL-A) biosynthesis revealed 16 structural genes, 2 regulatory genes, 5 genes related transport, transposition/integration or tellurium resistance; another 4 genes had no obvious identity. The VAL-A biosynthetic pathway was proposed, with assignment of the required genetic functions confined to the sequenced region. A cluster of eight reassembled genes was found to support VAL-A synthesis in a heterologous host, S. lividans 1326. In vivo inactivation of the putative glycosyltransferase gene (valG) abolished the final attachment of glucose for VAL production and resulted in accumulation of the VAL-A precursor, validoxylamine, while the normal production of VAL-A could be restored by complementation with valG. The role of valG in the glycosylation of validoxylamine to VAL-A was demonstrated in vitro by enzymatic assay.

Lasso peptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) that possess a unique "lariat knot" structural motif. Genome mining-targeted discovery of new natural products from microbes obtained from extreme environments has led to the identification of a gene cluster directing the biosynthesis of a new lasso peptide, designated as chaxapeptin 1, in the genome of Streptomyces leeuwenhoekii strain C58 isolated from the Atacama Desert. Subsequently, 1 was isolated and characterized using high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance methods. The lasso nature of 1 was confirmed by calculating its nuclear Overhauser effect restraint-based solution structure. Chaxapeptin 1 displayed a significant inhibitory activity in a cell invasion assay with human lung cancer cell line A549.

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It is well known that the piezoelectric performance of ferroelectric Pb(Zr,Ti) O 3 (PZT) based ceramics is far inferior to that of ferroelectric single crystals due to ceramics' polycrystalline nature. Herein, it is reported that piezoelectric stress coefficient e 33 = 39.24 C m −2 (induced electric displacement under applied strain) in the relaxor piezoelectric ceramic 0.55Pb(Ni 1/3 Nb 2/3 ) O 3 -0.135PbZrO 3 -0.315PbTiO 3 (PNN-PZT) prepared by the solid state reaction method exhibits the highest value among various reported ferroelectric ceramic and single crystal materials. In addition, its piezoelectric coefficient d 33 * = 1753 pm V −1 is also comparable with that of the commercial Pb(Mg 1/3 Nb 2/3 )O 3 -PbTiO 3 (PMN-PT) piezoelectric single crystal. The PNN-PZT ceramic is then assembled into a cymbal energy harvester. Notably, its maximum output current at the acceleration of 3.5 g is 2.5 mA pp , which is four times of the PMN-PT single crystal due to the large piezoelectric e 33 constants; while the maximum output power is 14.0 mW, which is almost the same as the PMN-PT single crystal harvester. The theoretical analysis on force-induced power output is also presented, which indicates PNN-PZT ceramic has great potential for energy device application.

The fluorinase is an enzyme that catalyses the combination of S-adenosyl-L-methionine (SAM) and a fluoride ion to generate 5'-fluorodeoxy adenosine (FDA) and L-methionine through a nucleophilic substitution reaction with a fluoride ion as the nucleophile. It is the only native fluorination enzyme that has been characterised. The fluorinase was isolated in 2002 from Streptomyces cattleya, and, to date, this has been the only source of the fluorinase enzyme. Herein, we report three new fluorinase isolates that have been identified by genome mining. The novel fluorinases from Streptomyces sp. MA37, Nocardia brasiliensis, and an Actinoplanes sp. have high homology (80-87 % identity) to the original S. cattleya enzyme. They all possess a characteristic 21-residue loop. The three newly identified genes were overexpressed in E. coli and shown to be fluorination enzymes. An X-ray crystallographic study of the Streptomyces sp. MA37 enzyme demonstrated that it is almost identical in structure to the original fluorinase. Culturing of the Streptomyces sp. MA37 strain demonstrated that it not only also elaborates the fluorometabolites, fluoroacetate and 4-fluorothreonine, similar to S. cattleya, but this strain also produces a range of unidentified fluorometabolites. These are the first new fluorinases to be reported since the first isolate, over a decade ago, and their identification extends the range of fluorination genes available for fluorination biotechnology.