Bacteria use small diffusible molecules to exchange information in a process called quorum sensing. An important class of autoinducers used by Gram-negative bacteria is the family of N-acylhomoserine lactones. Here, we report the discovery of a previously undescribed nonenzymatically formed product from N-(3-oxododecanoyl)-L-homoserine lactone; both the N-acylhomoserine and its novel tetramic acid degradation product, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, are potent antibacterial agents. Bactericidal activity was observed against all tested Gram-positive bacterial strains, whereas no toxicity was seen against Gram-negative bacteria. We propose that Pseudomonas aeruginosa utilizes this tetramic acid as an interference strategy to preclude encroachment by competing bacteria. Additionally, we have discovered that this tetramic acid binds iron with comparable affinity to known bacterial siderophores, possibly providing an unrecognized mechanism for iron solubilization. These findings merit new attention such that other previously identified autoinducers be reevaluated for additional biological functions.tetramic acid ͉ bactericidal agents ͉ evolution

Cell density-dependent coordination of gene expression in bacteria has been termed "quorum sensing" (QS). 1 N-acyl L-homoserine lactones (AHLs) are produced by over 70 species of Gram-negative bacteria, and structural differences within AHLs occur in the length and oxidation state of the acyl side chain (Figure 1). Upon reaching a critical threshold concentration, AHLs bind to their cognate receptor proteins, triggering the expression of target genes. AHLs have been shown to play an important role in the establishment and course of bacterial infections. One example of a Gram-negative pathogen that employs AHL-based QS to regulate the expression of its pathogenicity factors is Pseudomonas aeruginosa. This common environmental microorganism has acquired the ability to take advantage of weaknesses in the host defenses to become an opportunistic pathogen in humans. Most prominent is the role of P. aeruginosa in patients suffering from cystic fibrosis (CF). Over the last 15 years, much progress has been made in elucidating the molecular mechanisms underlying P. aeruginosa pathogenicity. 2 Two different AHLs, N-(3-oxododecanoyl) homoserine lactone (3-oxo-C 12 -AHL) and N-butyryl homoserine lactone (C 4 -AHL), have been identified as QS signaling molecules in P. aeruginosa. Genes regulated by this QS mechanism encode enzymes such as elastases A and B, catalase, and superoxide dismutase as well as exotoxins. 2Interference with QS signaling has been suggested as a new approach for anti-infective therapy. 3 In fact, this strategy has yielded interesting results using AHL analogs as QS antagonists in P. aeruginosa. 4 Alternatively, we have embarked on a program utilizing antibodies to inhibit AHL-mediated quorum sensing signaling in P. aeruginosa. AHL-based QS systems represent an ideal target for antibody-based anti-infective therapy given the highly conserved molecular scaffold and extracellular distribution of AHLs.Our initial hapten design for the elicitation of anti-AHL antibodies focused on synthesizing a set of molecules highly congruent in structure to AHLs while also possessing a pendant carboxylic acid functionality that would enable carrier proteins BSA or KLH conjugates to be easily accessed. However, we were aware that such molecules might also be prone to hydrolysis (ring opened products) under conditions required for chemical coupling and also immunization. Thus, to guide our hapten design the stability of several AHLs under physiological conditions was investigated; we synthesized a number of AHL analogs and their corresponding ring opened hydrolysis products, both of which contained a 4-methoxyphenyl amide group that allowed for a detailed investigation into the rate of hydrolysis of the AHL analogues using HPLC with UV detection (Figure 2 Each compound was assayed for its hydrolysis rate in phosphate buffer saline (PBS), pH 7.2, at a concentration of 200 μM at 37 °C. The half lives of each of the lactones varied from 13.7 to 18.1 hours. Interestingly, the oxidation state and chain length did not influence...

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Microbial strains are considered promising hosts for production of flavonoids because of their rapid growth rate and suitability for large-scale manufacturing. However, productivity and titer of current recombinant strains still do not meet the requirements of industrial processes. Genetically encoded biosensors have been applied for high-throughput screening or dynamic regulation of biosynthetic pathways for enhancing the performance of microbial strains that produce valuable chemicals. Currently, few protein sensor-regulators for flavonoids exist. Unlike the protein-based trans-regulating controllers, riboswitches can respond to their ligands faster and eliminate off-target effects. Here, we developed artificial riboswitches that activate gene expression in response to naringenin, an important flavonoid. RNA aptamers for naringenin were developed using SELEX and cloned upstream of a dual selectable marker gene to construct a riboswitch library. Two in vivo selection routes were applied separately to the library by supplementing naringenin at two different concentrations during enrichments to modulate the operational ranges of the riboswitches. The selected riboswitches were responsive to naringenin and activated gene expression up to 2.91-fold. Operational ranges of the riboswitches were distinguished on the basis of their selection route. Additionally, the specificity of the riboswitches was assessed, and their applicability as dynamic regulators was confirmed. Collectively, the naringenin riboswitches reported in this work will be valuable tools in metabolic engineering of microorganisms for the production of flavonoids.

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Racemic aromatic β-amino acids have been kinetically resolved into (R)-β-amino acids with high enantiomeric excess (>99%) by a novel ω-TA with ca. 50% conversion.

Here, we have utilized the incorporation of non‐canonical amino acids as a tool kit to improve enzyme properties for organic synthesis applications. The global incorporation of 3‐fluorotyrosine (FY) into ω‐transaminase (ω‐TA) to give ω‐TA[FY] enhanced the thermostability and organic solvent tolerance without altering substrate specificity and enantioselectivity. Moreover, ω‐TA[FY] was able to completely convert 25 mM of acetophenone into (S)‐1‐phenylethylamine (ee>99%) in the presence of 20% DMSO (v/v) which is ∼2‐fold higher when compared to wild‐type ω‐TA.magnified image

Primary ureas have been used as substrates in rhodium-catalyzed N-H insertion reactions with an array of diazocarbonyls. The insertion reaction is efficient and gives excellent selectivity and yields. The products from the insertion reaction with diazoketones cyclize readily in the presence of acid to yield the corresponding imidazolones that can be further derivatized by N-alkylation with alkyl, allyl, and benzyl halides. Alternatively, the imidazolones were treated with phosphorus oxybromide to form the corresponding 2-bromoimidazoles that were further functionalized using a Suzuki coupling reaction.