Dilepton Polarization as a Signature of Plasma Anisotropy.

We propose the angular distribution of lepton pairs produced in ultrarelativistic heavy-ion collisions as a probe of thermalization of the quark-gluon plasma. We focus on dileptons with invariant masses large enough that they are produced through quark-antiquark annihilation in the early stages of the collision. The angular distribution of the lepton in the rest frame of the pair then reflects the angular distribution of quark momenta. At early times, the transverse pressure of the quark-gluon plasma is larger than its longitudinal pressure as a result of the fast longitudinal expansion, which results in an oblate lepton distribution. By contrast, direct (Drell-Yan) production by quarks and antiquarks from incoming nuclei, whose momenta are essentially longitudinal, results in a prolate distribution. As the invariant mass increases, Drell-Yan gradually becomes the dominant source of dilepton production, and the lepton distribution evolves from oblate to prolate. The invariant mass at which the transition occurs is highly sensitive to the equilibration time of the quark-gluon plasma or, equivalently, the shear viscosity over entropy ratio η/s in the early stages of the collision.

Discovery, characterization and engineering of ligases for amide synthesis.

Coronatine and related bacterial phytotoxins are mimics of the hormone jasmonyl-L-isoleucine (JA-Ile), which mediates physiologically important plant signalling pathways1-4. Coronatine-like phytotoxins disrupt these essential pathways and have potential in the development of safer, more selective herbicides. Although the biosynthesis of coronatine has been investigated previously, the nature of the enzyme that catalyses the crucial coupling of coronafacic acid to amino acids remains unknown1,2. Here we characterize a family of enzymes, coronafacic acid ligases (CfaLs), and resolve their structures. We found that CfaL can also produce JA-Ile, despite low similarity with the Jar1 enzyme that is responsible for ligation of JA and L-Ile in plants5. This suggests that Jar1 and CfaL evolved independently to catalyse similar reactions-Jar1 producing a compound essential for plant development4,5, and the bacterial ligases producing analogues toxic to plants. We further demonstrate how CfaL enzymes can be used to synthesize a diverse array of amides, obviating the need for protecting groups. Highly selective kinetic resolutions of racemic donor or acceptor substrates were achieved, affording homochiral products. We also used structure-guided mutagenesis to engineer improved CfaL variants. Together, these results show that CfaLs can deliver a wide range of amides for agrochemical, pharmaceutical and other applications.

Harnessing and engineering amide bond forming ligases for the synthesis of amides.

The amide functional group is ubiquitous in nature and one of the most important motifs in pharmaceuticals, agrochemicals, and other valuable products. While coupling amides and carboxylic acids is a trivial synthetic transformation, it often requires protective group manipulation, along with stoichiometric quantities of expensive and deleterious coupling reagents. Nature has evolved a range of enzymes to construct amide bonds, the vast majority of which utilize adenosine triphosphate to activate the carboxylic acid substrate for amine coupling. Despite the fact that these enzymes operate under mild conditions, as well as possessing chemoselectivity and regioselectivity that obviates the need for protecting groups, their synthetic potential has been largely unexplored. In this review, we discuss recent research into the discovery, characterization, and development of amide bond forming enzymes, with an emphasis on stand-alone ligase enzymes that can generate amides directly from simple carboxylic acid and amine substrates.

De novo Biosynthesis of "Non-Natural" Thaxtomin Phytotoxins.

Thaxtomins are diketopiperazine phytotoxins produced by Streptomyces scabies and other actinobacterial plant pathogens that inhibit cellulose biosynthesis in plants. Due to their potent bioactivity and novel mode of action there has been considerable interest in developing thaxtomins as herbicides for crop protection. To address the need for more stable derivatives, we have developed a new approach for structural diversification of thaxtomins. Genes encoding the thaxtomin NRPS from S. scabies, along with genes encoding a promiscuous tryptophan synthase (TrpS) from Salmonella typhimurium, were assembled in a heterologous host Streptomyces albus. Upon feeding indole derivatives to the engineered S. albus strain, tryptophan intermediates with alternative substituents are biosynthesized and incorporated by the NRPS to deliver a series of thaxtomins with different functionalities in place of the nitro group. The approach described herein, demonstrates how genes from different pathways and different bacterial origins can be combined in a heterologous host to create a de novo biosynthetic pathway to "non-natural" product target compounds.

An Engineered Tryptophan Synthase Opens New Enzymatic Pathways to ß-Methyltryptophan and Derivatives.

ß-Methyltryptophans (ß-mTrp) are precursors in the biosynthesis of bioactive natural products and are used in the synthesis of peptidomimetic-based therapeutics. Currently ß-mTrp is produced by inefficient multistep synthetic methods. Here we demonstrate how an engineered variant of tryptophan synthase from Salmonella (StTrpS) can catalyse the efficient condensation of l-threonine and various indoles to generate ß-mTrp and derivatives in a single step. Although l-serine is the natural substrate for TrpS, targeted mutagenesis of the StTrpS active site provided a variant (ßL166V) that can better accommodate l-Thr as a substrate. The condensation of l-Thr and indole proceeds with retention of configuration at both α- and ß-positions to give (2S,3S)-ß-mTrp. The integration of StTrpS (ßL166V) with l-amino acid oxidase, halogenase enzymes and palladium chemocatalysts provides access to further d-configured and regioselectively halogenated or arylated ß-mTrp derivatives.

Functional Exchangeability of Oxidase and Dehydrogenase Reactions in the Biosynthesis of Hydroxyphenylglycine, a Nonribosomal Peptide Building Block.

A key problem in the engineering of pathways for the production of pharmaceutical compounds is the limited diversity of biosynthetic enzymes, which restricts the attainability of suitable traits such as less harmful byproducts, enhanced expression features, or different cofactor requirements. A promising synthetic biology approach is to redesign the biosynthetic pathway by replacing the native enzymes by heterologous proteins from unrelated pathways. In this study, we applied this method to effectively re-engineer the biosynthesis of hydroxyphenylglycine (HPG), a building block for the calcium-dependent antibiotic of Streptomyces coelicolor, a nonribosomal peptide. A key step in HPG biosynthesis is the conversion of 4-hydroxymandelate to 4-hydroxyphenylglyoxylate, catalyzed by hydroxymandelate oxidase (HmO), with concomitant generation of H2O2. The same reaction can also be catalyzed by O2-independent mandelate dehydrogenase (MdlB), which is a catabolic enzyme involved in bacterial mandelate utilization. In this work, we engineered alternative HPG biosynthetic pathways by replacing the native HmO in S. coelicolor by both heterologous oxidases and MdlB dehydrogenases from various sources and confirmed the restoration of calcium-dependent antibiotic biosynthesis by biological and UHPLC-MS analysis. The alternative enzymes were isolated and kinetically characterized, confirming their divergent substrate specificities and catalytic mechanisms. These results demonstrate that heterologous enzymes with different physiological contexts can be used in a Streptomyces host to provide an expanded library of enzymatic reactions for a synthetic biology approach. This study thus broadens the options for the engineering of antibiotic production by using enzymes with different catalytic and structural features.

Characteristics of Streptomyces griseus biofilms in continuous flow tubular reactors.

The purpose of this study was to investigate the feasibility of cultivating the biotechnologically important bacterium Streptomyces griseus in single-species and mixed-species biofilms using a tubular biofilm reactor (TBR). Streptomyces griseus biofilm development was found to be cyclical, starting with the initial adhesion and subsequent development of a visible biofilm after 24 h growth, followed by the complete detachment of the biofilm as a single mass, and ending with the re-colonisation of the tube. Fluorescence microscopy revealed that the filamentous structure of the biofilm was lost upon treatment with protease, but not DNase or metaperiodate, indicating that the extracellular polymeric substance is predominantly protein. When the biofilm was cultivated in conjunction with Bacillus amyloliquefaciens, no detachment was observed after 96 h, although once subjected to flow detachment. Electron microscopy confirmed the presence of both bacteria in the biofilm and revealed a network of fimbriae-like structures that were much less apparent in single-species biofilm and are likely to increase mechanical stability when developing in a TBR. This study presents the very first attempt in engineering S. griseus biofilms for continuous bioprocess applications.

Microbial biotransformation of aryl sulfanylpentafluorides.

We report, for the first time, the biotransformation of potential pollutants bearing the pentafluorosulfanyl (SF5-) functional group in a fungus and bacteria. Cunninghamella elegans transformed p-methoxy phenyl SF5 via demethylation; Pseudomonas knackmussii and P. pseudoalcaligenes KF707 transformed amino-, hydroxyamino- and diamino- substituted phenyl SF5, forming the N-acetylated derivatives as the main product. Cell-free extract of Streptomyces griseus transformed 4-amino-3-hydroxy-phenyl SF5 to the N-acetylated derivative in the presence of acetyl CoA, confirming that an N-acetyltransferase is responsible for the bacterial biotransformations. Approximately 25% of drugs and 30% of agrochemicals contain fluorine, and the trifluoromethyl group is a prominent feature of many of these since it improves lipophilicity and stability. The pentafluorosulfanyl substituent is seen as an improvement on the trifluoromethyl group and research efforts are underway to develop synthetic methods to incorporate this moiety into biologically active compounds. It is important to determine the potential environmental impact of these compounds, including the potential biotransformation reactions that may occur when they are exposed to microorganisms.

Characterisation of spin coated engineered Escherichia coli biofilms using atomic force microscopy.

The ability of biofilms to withstand chemical and physical extremes gives them the potential to be developed as robust biocatalysts. Critical to this issue is their capacity to withstand the physical environment within a bioreactor; in order to assess this capability knowledge of their surface properties and adhesive strength is required. Novel atomic force microscopy experiments conducted under growth conditions (30°C) were used to characterise Escherichia coli biofilms, which were generated by a recently developed spin-coating method onto a poly-l-lysine coated glass substrate. High-resolution topographical images were obtained throughout the course of biofilm development, quantifying the tip-cell interaction force during the 10 day maturation process. Strikingly, the adhesion force between the Si AFM tip and the biofilm surface increased from 0.8 nN to 40 nN within 3 days. This was most likely due to the production of extracellular polymer substance (EPS), over the maturation period, which was also observed by electron microscopy. At later stages of maturation, multiple retraction events were also identified corresponding to biofilm surface features thought to be EPS components. The spin coated biofilms were shown to have stronger surface adhesion than an equivalent conventionally grown biofilm on the same glass substrate.

Antimicrobial nucleoside antibiotics targeting cell wall assembly: recent advances in structure-function studies and nucleoside biosynthesis.

The quest for new antibiotics, especially those with activity against Gram-negative bacteria, is urgent; however, very few new antibiotics have been marketed in the last 40 years, with this limited number falling into only four new structural classes. Several nucleoside natural product antibiotics target bacterial translocase MraY, involved in the lipid-linked cycle of peptidoglycan biosynthesis, and fungal chitin synthase. Biosynthetic studies on the nikkomycin, caprazamycin and pacidamycin/mureidomycin families are also reviewed.

Development of fluorescent aryltryptophans by Pd mediated cross-coupling of unprotected halotryptophans in water.

A convenient and high yielding procedure for the Suzuki-Miyaura cross-coupling of unprotected bromo- and chlorotryptophans in water provides fluorescent aryltryptophans.

A convenient one-step synthesis of L-aminotryptophans and improved synthesis of 5-fluorotryptophan.

A one-pot biotransformation for the generation of a series of L-aminotryptophans using a readily prepared protein extract containing tryptophan synthase is reported. The extract exhibits remarkable stability upon freeze-drying, and may be stored and used for long periods after its preparation without significant loss of activity.