Selective Photoreceptor Gene Knock‐out Reveals a Regulatory Role for the Growth Behavior of Pseudomonas syringae

The plant pathogen Pseudomonas syringae (Ps) is a well‐established model organism for bacterial infection of plants. The genome sequences of two pathovars, pv. syringae and pv. tomato, revealed one gene encoding a blue and two genes encoding red/far red light‐sensing photoreceptors. Continuing former molecular characterization of the photoreceptor proteins, we here report selective photoreceptor gene disruption for pv. tomato aiming at identification of potentially regulatory functions of these photoreceptors. Transformation of Ps cells with linear DNA constructs yielded interposon mutations of the corresponding genes. Cell growth studies of the generated photoreceptor knock‐out mutants revealed their role in light‐dependent regulation of cell growth and motility. Disruption of the blue‐light (BL) receptor gene caused a growth deregulation, in line with an observed increased virulence of this mutant (Moriconi et al., Plant J., 2013, 76, 322). Bacterial phytochrome‐1 (BphP1) deletion mutant caused unaltered cell growth, but a stronger swarming capacity. Inactivation of its ortholog, BphP2, however, caused reduced growth and remarkably altered dendritic swarming behavior. Combined knock‐out of both bacteriophytochromes reproduced the swarming pattern observed for the BphP2 mutant alone. A triple knock‐out mutant showed a growth rate between that of the BL (deregulation) and the phytochrome‐2 mutant (growth reduction).     

Production of Polyhydroxyalkanoates (PHAs) by Vibrio alginolyticus Strains Isolated from Salt Fields

Vibrio alginolyticus is a halophilic organism usually found in marine environments. It has attracted attention as an opportunistic pathogen of aquatic animals and humans, but there are very few reports on polyhydroxyalkanoate (PHA) production using V. alginolyticus as the host. In this study, two V. alginolyticus strains, LHF01 and LHF02, isolated from water samples collected from salt fields were found to produce poly(3-hydroxybutyrate) (PHB) from a variety of sugars and organic acids. Glycerol was the best carbon source and yielded the highest PHB titer in both strains. Further optimization of the NaCl concentration and culture temperature improved the PHB titer from 1.87 to 5.08 g/L in V. alginolyticus LHF01. In addition, the use of propionate as a secondary carbon source resulted in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). V. alginolyticus LHF01 may be a promising host for PHA production using cheap waste glycerol from biodiesel refining.     

Bisucaberin biosynthesis: an adenylating domain of the BibC multi-enzyme catalyzes cyclodimerization of N-hydroxy-N-succinylcadaverine

The bisucaberin biosynthetic gene cluster has been identified in Vibrio salmonicida and a domain from within the BibC multienzyme encoded by the cluster has been shown to catalyse ATP-dependent dimerisation and macrocyclisation of N-hydroxy-N-succinylcadaverine to form bisucaberin.     

Divalent Transition‐Metal‐Ion Stress Induces Prodigiosin Biosynthesis in Streptomyces coelicolor M145: Formation of Coeligiosins

The bacterium Streptomyces coelicolor M145 reacts to transition‐metal‐ion stress with myriad growth responses, leading to different phenotypes. In particular, in the presence of Co²⁺ ions (0.7 mM ) S. coelicolor consistently produced a red phenotype. This phenotype, when compared to the wild type, differed strongly in its production of volatile compounds as well as high molecular weight secondary metabolites. LC‐MS analysis revealed that in the red phenotype the production of the prodigiosins, undecylprodigiosin and streptorubin B, was strongly induced and, in addition, several intense signals appeared in the LC‐MS chromatogram. Using LC‐MS/MS and NMR spectroscopy, two new prodigiosin derivatives were identified, that is, coeligiosin A and B, which contained an additional undecylpyrrolyl side chain attached to the central carbon of the tripyrrole ring system of undecylprodigiosin or streptorubin B. This example demonstrates that environmental factors such as heavy metal ion stress can not only induce the production of otherwise not observed metabolites from so called sleeping genes but alter the products from well‐studied biosynthetic pathways.     

Pestalotiopens A and B: Stereochemically Challenging Flexible Sesquiterpene‐Cyclopaldic Acid Hybrids from Pestalotiopsis sp.

From the endophytic fungus Pestalotiopsis sp. isolated from the leaves of the Chinese mangrove, Rhizophora mucronata, two novel hybrid sesquiterpene‐cyclopaldic acid metabolites with an unusual carbon skeleton, named pestalotiopens A and B, were obtained, together with the already known phytotoxin altiloxin B. Pestalotiopen B even contains a third, triketide‐derived module. The constitutions and the absolute configurations of the new metabolites and of altiloxin B were unambiguously determined by a combination of spectroscopic methods and quantum‐chemical optical‐rotatory dispersion (ORD) and circular dichroism (CD) calculations. A biosynthetic pathway to pestalotiopens A and B is proposed with altiloxin B as one of the suggested precursors. Pestalotiopen A shows moderate antimicrobial activity against Enterococcus faecalis.     

Functional Genome Mining Reveals a Class V Lanthipeptide Containing a d‐Amino Acid Introduced by an F420H2‐Dependent Reductase

Lantibiotics are a type of ribosomally synthesized and post‐translationally modified peptides (termed lanthipeptides) with often potent antimicrobial activity. Herein, we report the discovery of a new lantibiotic, lexapeptide, using the library expression analysis system (LEXAS) approach. Lexapeptide has rare structural modifications, including N‐terminal (N,N)‐dimethyl phenylalanine, C‐terminal (2‐aminovinyl)‐3‐methyl‐cysteine, and d ‐Ala. The characteristic lanthionine moiety in lexapeptide is formed by three proteins (LxmK, LxmX, and LxmY), which are distinct from enzymes known to be involved in lanthipeptide biosynthesis. Furthermore, a novel F₄₂₀H₂‐dependent reductase (LxmJ) from the lexapeptide biosynthetic gene cluster (BGC) is identified to catalyze the reduction of dehydroalanine to install d ‐Ala. Our findings suggest that lexapeptide is the founding member of a new class of lanthipeptides that we designate as class V. We also identified further class V lanthipeptide BGCs in actinomycetes and cyanobacteria genomes, implying that other class V lantibiotics await discovery.     

Control of the Stereochemical Course of [4+2] Cycloaddition during trans‐Decalin Formation by Fsa2‐Family Enzymes

Enzyme‐catalyzed [4+2] cycloaddition has been proposed to be a key transformation process in various natural product biosynthetic pathways. Recently Fsa2 was found to be involved in stereospecific trans‐decalin formation during the biosynthesis of equisetin, a potent HIV‐1 integrase inhibitor. To understand the mechanisms by which fsa2 determines the stereochemistry of reaction products, we sought an fsa2 homologue that is involved in trans‐decalin formation in the biosynthetic pathway of an enantiomerically opposite analogue, and we found phm7, which is involved in the biosynthesis of phomasetin. A decalin skeleton with an unnatural configuration was successfully constructed by gene replacement of phm7 with fsa2, thus demonstrating enzymatic control of all stereochemistry in the [4+2] cycloaddition. Our findings highlight enzyme‐catalyzed [4+2] cycloaddition as a stereochemically divergent step in natural product biosynthetic pathways and open new avenues for generating derivatives with different stereochemistry.     

Inhibition of Swarming motility of Pseudomonas aeruginosa by Methanol extracts of Alpinia officinarum Hance. and Cinnamomum tamala T. Nees and Eberm

Bacterial drug resistance is a challenge in clinical settings, especially in countries like India. Hence, discovery of novel alternative therapeutics has become a necessity in the fight against drug resistance. Compounds that inhibit bacterial virulence properties form new therapeutic alternatives. Pseudomonas aeruginosa is an opportunistic, nosocomial pathogen that infects immune-compromised patients. Swarming motility is an important virulence property of Pseudomonas which aids it in reaching host cells under nutrient limiting conditions. Here, we report the screening of five plant extracts against swarming motility of P. aeruginosa and show that methanol extracts of Alpinia officinarum and Cinnamomum tamala inhibit swarming motility at 5 μg mL^?1 without inhibiting its growth. These extracts did not inhibit swimming and twitching motilities indicating a mode of action specific to swarming pathway. Preliminary experiments indicated that rhamnolipid production was not affected. This study reveals the potential of the two plants in anti-virulence drug discovery.     

A Unique Tryptophan C‐Prenyltransferase from the Kawaguchipeptin Biosynthetic Pathway

Cyanobactins are a rapidly growing family of linear and cyclic peptides produced by cyanobacteria. Kawaguchipeptins A and B, two macrocyclic undecapeptides reported earlier from Microcystis aeruginosa NIES‐88, are shown to be products of the cyanobactin biosynthetic pathway. The 9 kb kawaguchipeptin (kgp) gene cluster was identified in a 5.26 Mb draft genome of Microcystis aeruginosa NIES‐88. We verified that this gene cluster is responsible for the production of the kawaguchipeptins through heterologous expression of the kgp gene cluster in Escherichia coli. The KgpF prenyltransferase was overexpressed and was shown to prenylate C‐3 of Trp residues in both linear and cyclic peptides in vitro. Our findings serve to further enhance the structural diversity of cyanobactins to include tryptophan‐prenylated cyclic peptides.     

Shimalactone Biosynthesis Involves Spontaneous Double Bicyclo‐Ring Formation with 8π‐6π Electrocyclization

Shimalactones A and B are neuritogenic polyketides possessing characteristic oxabicyclo[2.2.1]heptane and bicyclo[4.2.0]octadiene ring systems that are produced by the marine fungus Emericella variecolor GF10. We identified a candidate biosynthetic gene cluster and conducted heterologous expression analysis. Expression of ShmA polyketide synthase in Aspergillus oryzae resulted in the production of preshimalactone. Aspergillus oryzae and Saccharomyces cerevisiae transformants expressing ShmA and ShmB produced shimalactones A and B, thus suggesting that the double bicyclo‐ring formation reactions proceed non‐enzymatically from preshimalactone epoxide. DFT calculations strongly support the idea that oxabicyclo‐ring formation and 8π‐6π electrocyclization proceed spontaneously after opening of the preshimalactone epoxide ring through protonation. We confirmed the formation of preshimalactone epoxide in vitro, followed by its non‐enzymatic conversion to shimalactones in the dark.     

Boron as a Powerful Reductant: Synthesis of a Stable Boron‐Centered Radical‐Anion Radical‐Cation Pair

Despite the fundamental importance of radical‐anion radical‐cation pairs in single‐electron transfer (SET) reactions, such species are still very rare and transient in nature. Since diborenes have highly electron‐rich B? B double bonds, which makes them strong neutral reductants, we envisaged a possible realization of a boron‐centered radical‐anion radical‐cation pair by SET from a diborene to a borole species, which are known to form stable radical anions upon one‐electron reduction. However, since the reduction potentials of all know diborenes (E_1/2=?1.05/?1.55 V) were not sufficiently negative to reduce MesBC₄Ph₄ (E_1/2=?1.69 V), a suitable diborene, IiPr?(iPr)B?B(iPr)?IiPr, was tailor‐made to comply with these requirements. With a halfwave potential of E_1/2=?1.95 V, this diborene ranks amongst the most powerful neutral organic reductants known and readily reacted with MesBC₄Ph₄ by SET to afford a stable boron‐centered radical‐anion radical‐cation pair.     

RA‐dimer B,a New Dimeric RA‐series Cyclopeptide Incorporating Two Different Types of Cycloisodityrosine Units,from Rubia cordifolia L.

RA‐dimer B, a new cytotoxic RA‐series peptide, was isolated from the roots of Rubia cordifolia L. Its structure was elucidated on the basis of spectroscopic analysis to be a dimeric cyclopeptide composed of deoxybouvardin and allo‐RA‐V. Those two cyclopeptide units are connected by an ether linkage between the phenolic oxygen atom of deoxybouvardin and the ?a carbon atom of Tyr‐6 of allo‐RA‐V. RA‐dimer B was synthesized by the coupling reaction of deoxybouvardin with the boronic acid derivative of allo‐RA‐V, and subsequent deprotection, confirming the relative stereochemistry and establishing the absolute configuration of this peptide. RA‐dimer B showed cytotoxic activity against human promyelocytic leukaemia HL‐60, human colonic carcinoma HCT‐116, and human renal cell carcinoma ACHN cells with IC₅₀ values of 0.59, 0.54, and 0.74 μm , respectively.     

Electrochemical Quartz Crystal Microbalance Studies on the Codeposition of Dextran Sodium Sulfate with the Charge‐Transfer Complexes Generated During Electrooxidation of Benzidine Derivatives

The electrochemical quartz crystal microbalance (EQCM) technique was used to investigate the electrochemistry of three benzidine derivatives, o‐tolidine (o‐TD), 3,3′,5,5′‐tetramethyl‐benzidine (TMB) and o‐dianisidine (o‐DA), in Britton‐Robinson (B‐R) buffer solutions with and without coexisting dextran sodium sulfate (DSS), respectively. During the anodic potential sweep from 0.1 to 0.7 V vs. SCE in pH 5.0 B‐R buffer solution containing o‐TD, the EQCM frequency was decreased during the first‐step oxidation of o‐TD and then increased to some extent during its second‐step oxidation, implying that a poorly soluble charge‐transfer complex (CTC) was produced here as an oxidation intermediate, and its precipitation and then dissolution at the EQCM Au electrode decreased and then increased the frequency. The depth of the V‐shaped time‐dependent frequency response (?Δf_0V) to the redox switching of the CTC/o‐TD couple (0.1–0.37 V vs. SCE) was notably enhanced in the presence of DSS, being due to the formation of a mass‐enhanced CTC‐DSS adduct via electrostatic affinity. Similar phenomena were evident in the TMB system, but the CTC behavior was not observed during o‐DA oxidation in the absence of DSS, namely, the EQCM frequency kept decreasing all the time, due probably to the too high lability of the CTC from o‐DA oxidation, and the coexistence of DSS could well stabilize this CTC and turn on its CTC behavior. The o‐TD system showed the highest sensitivity to DSS and was thus examined in detail. The mechanism for the CTC‐DSS interaction is discussed from EQCM, FT‐IR and UV‐vis data. The CTC‐based EQCM determination of DSS, which is featured by a dynamically renewed surface of the detection electrode, was thus proposed, with a linear range from 0.002 to 1.6 μmol L^?1 and a detection limit down to 0.7 nmol L^?1 (o‐TD system).     

Iterative Assembly of Two Separate Polyketide Chains by the Same Single‐Module Bacterial Polyketide Synthase in the Biosynthesis of HSAF

Antifungal HSAF (heat‐stable antifungal factor, dihydromaltophilin) is a polycyclic tetramate macrolactam from the biocontrol agent Lysobacter enzymogenes. Its biosynthetic gene cluster contains only a single‐module polyketide synthase–nonribosomal peptide synthetase (PKS‐NRPS), although two separate hexaketide chains are required to assemble the skeleton. To address the unusual biosynthetic mechanism, we expressed the biosynthetic genes in two “clean” strains of Streptomyces and showed the production of HSAF analogues and a polyene tetramate intermediate. We then expressed the PKS module in Escherichia coli and purified the enzyme. Upon incubation of the enzyme with acyl‐coenzyme A and reduced nicotinamide adenine dinucleotide phosphate (NADPH), a polyene was detected in the tryptic acyl carrier protein (ACP). Finally, we incubated the polyene–PKS with the NRPS module in the presence of ornithine and adenosine triphosphate (ATP), and we detected the same polyene tetramate as that in Streptomyces transformed with the PKS‐NRPS alone. Together, our results provide evidence for an unusual iterative biosynthetic mechanism for bacterial polyketide–peptide natural products.     

Guanitrypmycin Biosynthetic Pathways Imply Cytochrome P450 Mediated Regio‐ and Stereospecific Guaninyl‐Transfer Reactions

Mining microbial genomes including those of Streptomyces reveals the presence of a large number of biosynthetic gene clusters. Unraveling this genetic potential has proved to be a useful approach for novel compound discovery. Here, we report the heterologous expression of two similar P450‐associated cyclodipeptide synthase‐containing gene clusters in Streptomyces coelicolor and identification of eight rare and novel natural products, the C3‐guaninyl indole alkaloids guanitrypmycins. Expression of different gene combinations proved that the cyclodipeptide synthases assemble cyclo‐l ‐Trp‐l ‐Phe and cyclo‐l ‐Trp‐l ‐Tyr, which are consecutively and regiospecifically modified by cyclodipeptide oxidases, cytochrome P450 enzymes, and N‐methyltransferases. In vivo and in vitro results proved that the P450 enzymes function as key biocatalysts and catalyze the regio‐ and stereospecific 3α‐guaninylation at the indole ring of the tryptophanyl moiety. Isotope‐exchange experiments provided evidence for the non‐enzymatic epimerization of the biosynthetic pathway products via keto–enol tautomerism. This post‐pathway modification during cultivation further increases the structural diversity of guanitrypmycins.     

Enantioselective Syntheses of Rigidiusculamides A and B: Revision of the Relative Stereochemistry of Rigidiusculamide A

The first enantioselective synthesis of cytotoxic natural products rigidiusculamides A (ent‐ 21 ) and B ( 8 ) has been achieved by two synthetic routes. The first one is convergent based on the common intermediate 11 , obtained through a high yielding SmI₂‐mediated Reformatsky‐type reaction. A highly diastereoselective one‐pot Dess–Martin periodinane‐mediated bis‐oxidation allowed the direct conversion of the diastereomeric mixture of 11 into rigidiusculamide B ( 8 ). Isolation of minor diastereomer 21 , in combination with computational work, allowed us to suggest the structure of the natural rigidiusculamide A to be 21 , as synthesized by the second route. Four diastereomers ( 7 , 7 , 22a , and 22b ) and an enantiomer ( 21 ) of rigidiusculamide A ( 21 ) have been synthesized. On the basis of literature precedents and computational work, a biosynthetic pathway for rigidiusculamides A and B was proposed to account for the opposite configuration at C‐5 of those two congeners.     

Heavy Heparin: A Stable Isotope‐Enriched,Chemoenzymatically‐Synthesized,Poly‐Component Drug

Heparin is a highly sulfated, complex polysaccharide and widely used anticoagulant pharmaceutical. In this work, we chemoenzymatically synthesized perdeuteroheparin from biosynthetically enriched heparosan precursor obtained from microbial culture in deuterated medium. Chemical de‐N‐acetylation, chemical N‐sulfation, enzymatic epimerization, and enzymatic sulfation with recombinant heparin biosynthetic enzymes afforded perdeuteroheparin comparable to pharmaceutical heparin. A series of applications for heavy heparin and its heavy biosynthetic intermediates are demonstrated, including generation of stable isotope labeled disaccharide standards, development of a non‐radioactive NMR assay for glucuronosyl‐C5‐epimerase, and background‐free quantification of in vivo half‐life following administration to rabbits. We anticipate that this approach can be extended to produce other isotope‐enriched glycosaminoglycans.     

Biosynthesis of Complex Indole Alkaloids: Elucidation of the Concise Pathway of Okaramines

The okaramines are a class of complex indole alkaloids isolated from Penicillium and Aspergillus species. Their potent insecticidal activity arises from selectively activating glutamate‐gated chloride channels (GluCls) in invertebrates, not affecting human ligand‐gated anion channels. Okaramines B ( 1 ) and D ( 2 ) contain a polycyclic skeleton, including an azocine ring and an unprecedented 2‐dimethyl‐3‐methyl‐azetidine ring. Owing to their complex scaffold, okaramines have inspired many total synthesis efforts, but the enzymology of the okaramine biosynthetic pathway remains unexplored. Here, we identified and characterized the biosynthetic gene cluster (oka ) of 1 and 2 , then elucidated the pathway with target gene inactivation, heterologous reconstitution, and biochemical characterization. Notably, we characterized an α‐ketoglutarate‐dependent non‐heme Fe^II dioxygenase that forged the azetidine ring on the okaramine skeleton.     

Synthesis of a novel DPPA‐containing benzoxazine to flame‐retard epoxy resin with maintained thermal properties

The flame‐retarded epoxy resin with improved thermal properties based on environmentally friendly flame retardants is vital for industrial application. Hereby, a novel reactive‐type halogen‐free flame retardant, 10‐(3‐(4‐hydroxy phenyl)‐3,4‐dihydro‐2H‐benzo[e] [1,3] oxazin‐4‐yl)‐5H‐phenophosphazinine 10‐oxide (DHA‐B) was synthesized via a two‐step reaction route. Its structure was characterized using ¹H, ¹³C, and ³¹P NMR and HRMS spectra. For 4,4′‐diaminodipheny ethane (DDM) and diglycidyl ether of bisphenol A (DGEBA)‐cured systems, the epoxy resin with only 2 wt% loading of DHA‐B passed V‐0 rating of UL‐94 test. Significantly, its glass transition temperature (T_g) and initial decomposition temperature (T_5%) were as high as 169.6°C and 359.6°C, respectively, which were even higher than those of the corresponding original epoxy resin. Besides, DHA‐B decreased the combustion intensity during combustion. The analysis of residues after combustion suggested that DHA‐B played an important role in the condensed phase.