Golden Standard: a complete standard, portable, and interoperative MoClo tool for model and non-model proteobacteria.

Modular cloning has become a benchmark technology in synthetic biology. However, a notable disparity exists between its remarkable development and the need for standardization to facilitate seamless interoperability among systems. The field is thus impeded by an overwhelming proliferation of organism-specific systems that frequently lack compatibility. To overcome these issues, we present Golden Standard (GS), a Type IIS assembly method underpinned by the Standard European Vector Architecture. GS unlocks modular cloning applications for most bacteria, and delivers combinatorial multi-part assembly to create genetic circuits of up to twenty transcription units (TUs). Reliance on MoClo syntax renders GS fully compatible with many existing tools and it sets the path towards efficient reusability of available part libraries and assembled TUs. GS was validated in terms of DNA assembly, portability, interoperability and phenotype engineering in α-, ß-, γ- and δ-proteobacteria. Furthermore, we provide a computational pipeline for parts characterization that was used to assess the performance of GS parts. To promote community-driven development of GS, we provide a dedicated web-portal including a repository of parts, vectors, and Wizard and Setup tools that guide users in designing constructs. Overall, GS establishes an open, standardized framework propelling the progress of synthetic biology as a whole.

Broad-Scope Amination of Aryl Sulfamates Catalyzed by a Palladium Phosphine Complex.

Among phenol-derived electrophiles, aryl sulfamates are attractive substrates since they can be employed as directing groups for C-H functionalization prior to catalysis. However, their use in C-N coupling is limited only to Ni catalysis. Here, we describe a Pd-based catalyst with a broad scope for the amination of aryl sulfamates. We show that the N-methyl-2-aminobiphenyl palladacycle supported by the PCyp2ArXyl2 ligand (Cyp = cyclopentyl; ArXyl2 = 2,6-bis(2,6-dimethylphenyl)phenyl) efficiently catalyzes the C-N coupling of aryl sulfamates with a variety of nitrogen nucleophiles, including anilines, primary and secondary alkyl amines, heteroaryl amines, N-heterocycles, and primary amides. DFT calculations support that the oxidative addition of the aryl sulfamate is the rate-determining step. The C-N coupling takes place through a cationic pathway in the polar protic medium.

Synthetic Control of Metabolic States in Pseudomonas putida by Tuning Polyhydroxyalkanoate Cycle.

Polyhydroxyalkanoates (PHAs) are polyesters produced by numerous microorganisms for energy and carbon storage. Simultaneous synthesis and degradation of PHA drives a dynamic cycle linked to the central carbon metabolism, which modulates numerous and diverse bacterial processes, such as stress endurance, pathogenesis, and persistence. Here, we analyze the role of the PHA cycle in conferring robustness to the model bacterium P. putida KT2440. To assess the effect of this cycle in the cell, we began by constructing a PHA depolymerase (PhaZ) mutant strain that had its PHA cycle blocked. We then restored the flux through the cycle in the context of an engineered library of P. putida strains harboring differential levels of PhaZ. High-throughput phenotyping analyses of this collection of strains revealed significant changes in response to PHA cycle performance impacting cell number and size, PHA accumulation, and production of extracellular (R)-hydroxyalkanoic acids. To understand the metabolic changes at the system level due to PHA turnover, we contextualized these physiological data using the genome-scale metabolic model iJN1411. Model-based predictions suggest successive metabolic steady states during the growth curve and an important carbon flux rerouting driven by the activity of the PHA cycle. Overall, we demonstrate that modulating the activity of the PHA cycle gives us control over the carbon metabolism of P. putida, which in turn will give us the ability to tailor cellular mechanisms driving stress tolerance, e.g., defenses against oxidative stress, and any potential biotechnological applications. IMPORTANCE Despite large research efforts devoted to understanding the flexible metabolism of Pseudomonas beyond the role of key regulatory players, the metabolic basis powering the dynamic control of its biological fitness under disturbance conditions remains largely unknown. Among other metabolic hubs, the so-called PHA cycle, involving simultaneous synthesis and degradation of PHAs, is emerging as a pivotal metabolic trait powering metabolic robustness and resilience in this bacterial group. Here, we provide evidence suggesting that metabolic states in Pseudomonas can be anticipated, controlled, and engineered by tailoring the flux through the PHA cycle. Overall, our study suggests that the PHA cycle is a promising metabolic target toward achieving control over bacterial metabolic robustness. This is likely to open up a broad range of applications in areas as diverse as pathogenesis and biotechnology.

MIXed plastics biodegradation and UPcycling using microbial communities: EU Horizon 2020 project MIX-UP started January 2020.

This article introduces the EU Horizon 2020 research project MIX-UP, "Mixed plastics biodegradation and upcycling using microbial communities". The project focuses on changing the traditional linear value chain of plastics to a sustainable, biodegradable based one. Plastic mixtures contain five of the top six fossil-based recalcitrant plastics [polyethylene (PE), polyurethane (PUR), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS)], along with upcoming bioplastics polyhydroxyalkanoate (PHA) and polylactate (PLA) will be used as feedstock for microbial transformations. Consecutive controlled enzymatic and microbial degradation of mechanically pre-treated plastics wastes combined with subsequent microbial conversion to polymers and value-added chemicals by mixed cultures. Known plastic-degrading enzymes will be optimised by integrated protein engineering to achieve high specific binding capacities, stability, and catalytic efficacy towards a broad spectrum of plastic polymers under high salt and temperature conditions. Another focus lies in the search and isolation of novel enzymes active on recalcitrant polymers. MIX-UP will formulate enzyme cocktails tailored to specific waste streams and strives to enhance enzyme production significantly. In vivo and in vitro application of these cocktails enable stable, self-sustaining microbiomes to convert the released plastic monomers selectively into value-added products, key building blocks, and biomass. Any remaining material recalcitrant to the enzymatic activities will be recirculated into the process by physicochemical treatment. The Chinese-European MIX-UP consortium is multidisciplinary and industry-participating to address the market need for novel sustainable routes to valorise plastic waste streams. The project's new workflow realises a circular (bio)plastic economy and adds value to present poorly recycled plastic wastes where mechanical and chemical plastic recycling show limits.

Ni(II) Precatalysts Enable Thioetherification of (Hetero)Aryl Halides and Tosylates and Tandem C-S/C-N Couplings.

Ni-catalyzed C-S cross-coupling reactions have received less attention compared with other C-heteroatom couplings. Most reported examples comprise the thioetherification of most reactive aryl iodides with aromatic thiols. The use of C-O electrophiles in this context is almost uncharted. Here, we describe that preformed Ni(II) precatalysts of the type NiCl(allyl)(PMe2 Ar') (Ar'=terphenyl group) efficiently couple a wide range of (hetero)aryl halides, including challenging aryl chlorides, with a variety of aromatic and aliphatic thiols. Aryl and alkenyl tosylates are also well tolerated, demonstrating, for the first time, to be competent electrophilic partners in Ni-catalyzed C-S bond formation. The chemoselective functionalization of the C-I bond in the presence of a C-Cl bond allows for designing site-selective tandem C-S/C-N couplings. The formation of the two C-heteroatom bonds takes place in a single operation and represents a rare example of dual electrophile/nucleophile chemoselective process.

Anti-staphylococcal hydrogels based on bacterial cellulose and the antimicrobial biopolyester poly(3-hydroxy-acetylthioalkanoate-co-3-hydroxyalkanoate).

Polymeric hydrogels from bacterial cellulose (BC) have been widely used for the development of wound dressings due to its water holding capacity, its high tensile strength and flexibility, its permeability to gases and liquids, but lacks antibacterial activity. In this work, we have developed novel antimicrobial hydrogels composed of BC and the antimicrobial poly(3-hydroxy-acetylthioalkanoate-co-3-hydroxyalkanoate) (PHACOS). Hydrogels based on different PHACOS contents (20 and 50 wt%) were generated and analysed through different techniques (IR, DSC, TGA, rheology, SEM and EDX) and their bactericidal activity was studied against Staphylococcus aureus. PHACOS20 (BC 80%-PHACOS 20%) hydrogel shows mechanical and thermal properties in the range of human skin and anti-staphylococcal activity (kills 1.8 logs) demonstrating a huge potential for wound healing applications. Furthermore, the cytotoxicity assay using fibroblast cells showed that it keeps cell viability over 85% in all the cases after seven days.

Methane functionalization in water with micellar catalysis.

The functionalization of methane in water as the reaction medium (where it is nearly insoluble) at room temperature using micellar catalysis is described. Aggregates are formed from surfactant molecules and act as methane concentrators, also trapping the catalyst (a silver-based complex) and the diazo reagent (ethyl diazoacetate, EDA), providing yields of ethyl propionate up to 14% (referred to as EDA). This is the first example of methane being functionalized in water at room temperature.

The contribution of microbial biotechnology to sustainable development goals.

The signature and almost unique characteristic of microbial technology is the exceptional diversity of applications it can address, and the exceptional range of human activities and needs to which it is and can be applied. Precisely because sustainability goals have very diverse and complex components and requirements, microbial technology has the ability to contribute substantively on many levels in many arenas to global efforts to achieve sustainability. Indeed, microbial technology could be viewed as a unifying element in our progress towards sustainability.

Determination of the Predatory Capability of Bdellovibrio bacteriovorus HD100.

Bdellovibrio bacteriovorus HD100 is an obligate predator that preys upon a wide variety of Gram negative bacteria. The biphasic growth cycle of Bdellovibrio includes a free-swimming attack phase and an intraperiplasmic growth phase, where the predator replicates its DNA and grows using the prey as a source of nutrients, finally dividing into individual cells (Sockett, 2009). Due to its obligatory predatory lifestyle, manipulation of Bdellovibrio requires two-member culturing techniques using selected prey microorganisms ( Lambert et al., 2003 ). In this protocol, we describe a detailed workflow to grow and quantify B. bacteriovorus HD100 and its predatory ability, to easily carry out these laborious and time-consuming techniques.

To be, or not to be biodegradable that is the question for the bio-based plastics.

Global warming, market and production capacity are being the key drivers for selecting the main players for the next decades in the market of bio-based plastics. The drop-in bio-based polymers such as the bio-based polyethylene terephtalate (PET) or polyethylene (PE), chemically identical to their petrochemical counterparts but having a component of biological origin, are in the top of the list. They are followed by new polymers such as PHA and PLA with a significant market growth rate since 2014 with projections to 2020. Research will provide improved strains designed through synthetic and systems biology approaches; furthermore, the use of low-cost substrates will contribute to the widespread application of these bio- based polymers. The durability of plastics is not considered anymore as a virtue, and interesting bioprospecting strategies to isolate microorganisms for assimilating the recalcitrant plastics will pave the way for in vivo strategies for plastic mineralization. In this context, waste management of bio-based plastic will be one of the most important issues in the near future in terms of the circular economy. There is a clear need for standardized labelling and sorting instructions, which should be regulated in a coordinated way by policymakers and material producers.

A holistic view of polyhydroxyalkanoate metabolism in Pseudomonas putida.

Polyhydroxyalkanoate (PHA) metabolism has been traditionally considered as a futile cycle involved in carbon and energy storage. The use of cutting-edge technologies linked to systems biology has improved our understanding of the interaction between bacterial physiology, PHA metabolism and other cell functions in model bacteria such as Pseudomonas putida KT2440. PHA granules or carbonosomes are supramolecular complexes of biopolyester and proteins that are essential for granule segregation during cell division, and for the functioning of the PHA metabolic route as a continuous cycle. The simultaneous activities of PHA synthase and depolymerase ensure the carbon flow to the transient demand for metabolic intermediates to balance the storage and use of carbon and energy. PHA cycle also determines the number and size of bacterial cells. The importance of PHAs as nutrients for members of the microbial community different to those that produce them is illustrated here via examples of bacterial predators such as Bdellovibrio bacteriovorus that prey on PHA producers and produces specific extra-cellular depolymerases. PHA hydrolysis confers Bdellovibrio ecological advantages in terms of motility and predation efficiency, demonstrating the importance of PHA producers predation in population dynamics. Metabolic modulation strategies for broadening the portfolio of PHAs are summarized and their properties are compiled.

Synthesis, structural characterization, reactivity, and catalytic properties of copper(I) complexes with a series of tetradentate tripodal tris(pyrazolylmethyl)amine ligands.

Novel tris(pyrazolylmethyl)amine ligands Tpa(Me3), Tpa*(,Br), and Tpa(Br3) have been synthesized and structurally characterized. The coordination chemistries of these three new tetradentate tripodal ligands and the already known Tpa and Tpa* have been explored using different copper(I) salts as starting materials. Cationic copper(I) complexes [Tpa(x)Cu]PF6 (1-4) have been isolated from the reaction of [Cu(NCMe)4]PF6 and 1 equiv of the ligand. Complexes 2 (Tpa(x) = Tpa*) and 3 (Tpa(x) = Tpa(Me3)) have been characterized by X-ray studies. The former is a 1D helical coordination polymer, and the latter is a tetranuclear helicate. In both structures, the Tpa(x) ligand adopts a µ(2):κ(2):κ(1)-coordination mode. However, in solution, all of the four complexes form fluxional species. When CuI is used as the copper(I) source, neutral compounds 5-8 have been obtained. Complexes 6-8 exhibit a 1:1 metal-to-ligand ratio, whereas 5 presents 2:1 stoichiometry. Its solid-state structure has been determined by X-ray diffraction, revealing its 3D polymeric nature. The polymer is composed by the assembly of [Tpa2Cu4I4] units, in which Cu4I4 presents a step-stair structure. The Tpa ligands bridge the Cu4I4 clusters, adopting also a µ(2):κ(2):κ(1)-coordination mode. As observed for the cationic derivatives, the NMR spectra of 5-8 show the equivalence of the three pyrazolyl arms of the ligands in these complexes. The reactivities of cationic copper(I) derivatives 1-4 with PPh3 and CO have been explored. In all cases, 1:1 adducts [Tpa(x)CuL]PF6 [L = PPh3 (9-11), CO (12-15)] have been isolated. The crystal structure of [Tpa*Cu(PPh3)]PF6 (9) has been obtained, showing that the coordination geometry around copper(I) is trigonal-pyramidal with the apical position occupied by the tertiary amine N atom. The Tpa* ligand binds the Cu center to three of its four N atoms, with one pyrazolyl arm remaining uncoordinated. In solution, the carbonyl adducts 13-15 exist as a mixture of two isomers; the four- and five-coordinate species can be distinguished by means of their IR νCO stretching bands. Finally, the catalytic activities of complexes 1-4 have been demonstrated in carbene- and nitrene-transfer reactions.

Genome Sequence of Streptomyces exfoliatus DSMZ 41693, a Source of Poly(3-Hydroxyalkanoate)-Degrading Enzymes.

Here we report the draft genome sequence of Streptomyces exfoliatus DSMZ 41693, which includes a gene encoding a poly(3-hydroxyoctanoate) depolymerase, an enzyme which can be used for the industrial synthesis of chiral (R)-3-hydroxyalkanoic acids. In addition, the genome carries numerous genes involved in the biosynthesis of secondary metabolites, including polyketides and terpenes.

Kumada-Tamao-Corriu coupling of heteroaromatic chlorides and aryl ethers catalyzed by (IPr)Ni(allyl)Cl.

The complex (IPr)Ni(allyl)Cl (IPr = 1,3-bis(2,6-diisopropylphenyl)imidazolidene) catalyzes the cross-coupling reactions of heteroaromatic chlorides with aryl Grignard reagents. Catalyst loadings as low as 0.1 mol % have been used to afford the products in excellent yields. This nickel-based catalytic system also promotes the activation of the C(Ar)-O bond of anisoles in the Kumada-Tamao-Corriu reaction under fairly mild conditions.

Non-biaryl atropisomers in organocatalysis.

A new class of 6'-hydroxy cinchona alkaloids, with a non-biaryl atropisomeric functionalisation at position 5' of the quinoline core can be prepared by an easy amination procedure. These are the first derivatives for which the principle of atropisomerism is engrafted in the classical core of the cinchona alkaloids. The aminated cinchona alkaloids are effective organocatalysts for the Michael addition of beta-keto esters to acrolein and methyl vinyl ketone, in up to 93 % ee (ee=enantiomeric excess), as well as for the asymmetric Friedel-Crafts amination of a variety of 2-naphthols, permitting the preparation of the latter in up to 98 % ee. The aminated 8-amino-2-naphthol itself is the first chiral organocatalyst based on non-biaryl atropisomerism. The two enantiomers of this chiral primary amine can be used for the direct alpha-fluorination of alpha-branched aldehydes. The fluorinated compounds can thereby be accessed in up to 90 % ee.

Novel imidazolidine-tetrazole organocatalyst for asymmetric conjugate addition of nitroalkanes.

The Michael addition of nitroalkanes to alpha,beta-unsaturated enones catalyzed by a novel chiral imidazolidine-2-yltetrazole organocatalyst has been investigated. The new more soluble organocatalyst decreases reaction times and improves enantioselectivities compared to other catalysts. The Michael addition adducts were obtained with up to 92% ee. [reaction: see text]

Asymmetric synthesis of succinic semialdehyde derivatives.

The nucleophilic Michael addition of 2-(diphenylmethoxymethyl)-1-methyleneamino pyrrolidine 1Dto prochiral aliphatic and aromatic alkylidene malonates 2 takes place in the presence of MgI(2) to afford the corresponding Michael adducts 3 in excellent yields and good selectivities. In the aromatic series, optically pure (de > 98%) major diastereomers (S,S)-3 were isolated in good yields (77-93%) after chromatographic separation. Direct, racemization-free BF(3).OEt(2)-catalyzed thiolysis of compounds 3 afforded dithioacetals 7. These compounds were transformed into malonates 8 and succinic semialdehyde derivatives 9 by Raney Nickel mediated desulfuration or decarboxylation, respectively.