Synthetic metabolism: metabolic engineering meets enzyme design.

Metabolic engineering aims at modifying the endogenous metabolic network of an organism to harness it for a useful biotechnological task, for example, production of a value-added compound. Several levels of metabolic engineering can be defined and are the topic of this review. Basic 'copy, paste and fine-tuning' approaches are limited to the structure of naturally existing pathways. 'Mix and match' approaches freely recombine the repertoire of existing enzymes to create synthetic metabolic networks that are able to outcompete naturally evolved pathways or redirect flux toward non-natural products. The space of possible metabolic solution can be further increased through approaches including 'new enzyme reactions', which are engineered on the basis of known enzyme mechanisms. Finally, by considering completely 'novel enzyme chemistries' with de novo enzyme design, the limits of nature can be breached to derive the most advanced form of synthetic pathways. We discuss the challenges and promises associated with these different metabolic engineering approaches and illuminate how enzyme engineering is expected to take a prime role in synthetic metabolic engineering for biotechnology, chemical industry and agriculture of the future

Involvement of Noradrenergic Neurotransmission in the Stress- but not Cocaine-Induced Reinstatement of Extinguished Cocaine-Induced Conditioned Place Preference in Mice: Role for β-2 Adrenergic Receptors

The responsiveness of central noradrenergic systems to stressors and cocaine poses norepinephrine as a potential common mechanism through which drug re-exposure and stressful stimuli promote relapse. This study investigated the role of noradrenergic systems in the reinstatement of extinguished cocaine-induced conditioned place preference by cocaine and stress in male C57BL/6 mice. Cocaine- (15 mg/kg, i.p.) induced conditioned place preference was extinguished by repeated exposure to the apparatus in the absence of drug and reestablished by a cocaine challenge (15 mg/kg), exposure to a stressor (6-min forced swim (FS); 20–25°C water), or administration of the α-2 adrenergic receptor (AR) antagonists yohimbine (2 mg/kg, i.p.) or BRL44408 (5, 10 mg/kg, i.p.). To investigate the role of ARs, mice were administered the nonselective β-AR antagonist, propranolol (5, 10 mg/kg, i.p.), the α-1 AR antagonist, prazosin (1, 2 mg/kg, i.p.), or the α-2 AR agonist, clonidine (0.03, 0.3 mg/kg, i.p.) before reinstatement testing. Clonidine, prazosin, and propranolol failed to block cocaine-induced reinstatement. The low (0.03 mg/kg) but not high (0.3 mg/kg) clonidine dose fully blocked FS-induced reinstatement but not reinstatement by yohimbine. Propranolol, but not prazosin, blocked reinstatement by both yohimbine and FS, suggesting the involvement of β-ARs. The β-2 AR antagonist ICI-118551 (1 mg/kg, i.p.), but not the β-1 AR antagonist betaxolol (10 mg/kg, i.p.), also blocked FS-induced reinstatement. These findings suggest that stress-induced reinstatement requires noradrenergic signaling through β-2 ARs and that cocaine-induced reinstatement does not require AR activation, even though stimulation of central noradrenergic neurotransmission is sufficient to reinstate

Malaria pigment crystals as magnetic micro-rotors: Key for high-sensitivity diagnosis

The need to develop new methods for the high-sensitivity diagnosis of malaria has initiated a global activity in medical and interdisciplinary sciences. Most of the diverse variety of emerging techniques are based on research-grade instruments, sophisticated reagent-based assays or rely on expertise. Here, we suggest an alternative optical methodology with an easy-to- use and cost-effective instrumentation based on unique properties of malaria pigment reported previously and determined quantitatively in the present study. Malaria pigment, also called hemozoin, is an insoluble microcrystalline form of heme. These crystallites show remarkable magnetic and optical anisotropy distinctly from any other components of blood. As a consequence, they can simultaneously act as magnetically driven micro-rotors and spinning polarizers in suspensions. These properties can gain importance not only in malaria diagnosis and therapies, where hemozoin is considered as drug target or immune modulator, but also in the magnetic manipulation of cells and tissues on the microscopic scale

Metabolite Profiling Uncovers Plasmid-Induced Cobalt Limitation under Methylotrophic Growth Conditions

BACKGROUND:The introduction and maintenance of plasmids in cells is often associated with a reduction of growth rate. The reason for this growth reduction is unclear in many cases. METHODOLOGY/PRINCIPAL FINDINGS:We observed a surprisingly large reduction in growth rate of about 50% of Methylobacterium extorquens AM1 during methylotrophic growth in the presence of a plasmid, pCM80 expressing the tetA gene, relative to the wild-type. A less pronounced growth delay during growth under non-methylotrophic growth conditions was observed; this suggested an inhibition of one-carbon metabolism rather than a general growth inhibition or metabolic burden. Metabolome analyses revealed an increase in pool sizes of ethylmalonyl-CoA and methylmalonyl-CoA of more than 6- and 35-fold, respectively, relative to wild type, suggesting a strongly reduced conversion of these central intermediates, which are essential for glyoxylate regeneration in this model methylotroph. Similar results were found for M. extorquens AM1 pCM160 which confers kanamycin resistance. These intermediates of the ethylmalonyl-CoA pathway have in common their conversion by coenzyme B(12)-dependent mutases, which have cobalt as a central ligand. The one-carbon metabolism-related growth delay was restored by providing higher cobalt concentrations, by heterologous expression of isocitrate lyase as an alternative path for glyoxylate regeneration, or by identification and overproduction of proteins involved in cobalt import. CONCLUSIONS/SIGNIFICANCE:This study demonstrates that the introduction of the plasmids leads to an apparent inhibition of the cobalt-dependent enzymes of the ethylmalonyl-CoA pathway. Possible explanations are presented and point to a limited cobalt concentration in the cell as a consequence of the antibiotic stress

Identification of Mycobacterium tuberculosis-Specific Th1, Th17 and Th22 Cells Using the Expression of CD40L in Tuberculous Pleurisy

Important advances have been made in the immunodiagnosis of tuberculosis (TB) based on the detection of Mycobacterium tuberculosis (MTB)-specific T cells. However, the sensitivity and specificity of the immunological approach are relatively low because there are no specific markers for antigen-specific Th cells, and some of the Th cells that do not produce cytokines can be overlooked using this approach. In this study, we found that MTB-specific peptides of ESAT-6/CFP-10 can stimulate the expression of CD40L specifically in CD4+ T cells but not other cells from pleural fluid cells (PFCs) in patients with tuberculous pleurisy (TBP). CD4+CD40L+ but not CD4+CD40L− T cells express IFN-γ, IL-2, TNF-α, IL-17 or IL-22 after stimulation with MTB-specific peptides. In addition, CD4+CD40L+ T cells were found to be mostly polyfunctional T cells that simultaneously produce IFN-γ, IL-2 and TNF-α and display an effector or effector memory phenotype (CD45RA−CD45RO+CCR7−CD62L−ICOS−). To determine the specificity of CD4+CD40L+ T cells, we incubated PFCs with ESTA-6/CFP-10 peptides and sorted live CD4+CD40L+ and CD4+CD40L− T cells by flow cytometry. We further demonstrated that sorted CD4+CD40L+, but not CD4+CD40L− fractions, principally produced IFN-γ, IL-2, TNF-α, IL-17 and IL-22 following restimulation with ESTA-6/CFP-10 peptides. Taken together, our data indicate that the expression of CD40L on MTB-specific CD4+ T cells could be a good marker for the evaluation and isolation of MTB-specific Th cells and might also be useful in the diagnosis of TB

Past Achievements and Future Challenges in 3D Photonic Metamaterials

Photonic metamaterials are man-made structures composed of tailored micro- or nanostructured metallo-dielectric sub-wavelength building blocks that are densely packed into an effective material. This deceptively simple, yet powerful, truly revolutionary concept allows for achieving novel, unusual, and sometimes even unheard-of optical properties, such as magnetism at optical frequencies, negative refractive indices, large positive refractive indices, zero reflection via impedance matching, perfect absorption, giant circular dichroism, or enhanced nonlinear optical properties. Possible applications of metamaterials comprise ultrahigh-resolution imaging systems, compact polarization optics, and cloaking devices. This review describes the experimental progress recently made fabricating three-dimensional metamaterial structures and discusses some remaining future challenges

Enhanced antibacterial activity of silver nanoparticles/halloysite nanotubes/graphene nanocomposites with sandwich-like structure

A sandwich-like antibacterial reagent (Ag/HNTs/rGO) was constructed through the direct growth of silver nanoparticles on the surface graphene-based HNTs nanosheets. Herein, various nanomaterials were combined by adhesion effect of DOPA after self-polymerization. Ag/HNTs/rGO posses enhanced antibacterial ability against E. coli and S. aureus compared with individual silver nanoparticles, rGO nanosheets or their nanocomposites

Metabolic and evolutionary insights into the closely-related species Streptomyces coelicolor and Streptomyces lividans deduced from high-resolution comparative genomic hybridization

Whilst being closely related to the model actinomycete Streptomyces coelicolor A3(2), S. lividans 66 differs from it in several significant and phenotypically observable ways, including antibiotic production. Previous comparative gene hybridization studies investigating such differences have used low-density (one probe per gene) PCR-based spotted arrays. Here we use new experimentally optimised 104,000 × 60-mer probe arrays to characterize in detail the genomic differences between wild-type S. lividans 66, a derivative industrial strain, TK24, and S. coelicolor M145

Biocatalytic production of bicyclic β-lactams with three contiguous chiral centres using engineered crotonases

YesThere is a need to develop asymmetric routes to functionalised β-lactams, which remain the most important group of antibacterials. Here we describe biocatalytic and protein engineering studies concerning carbapenem biosynthesis enzymes, aiming to enable stereoselective production of functionalised carbapenams with three contiguous chiral centres. Structurallyguided substitutions of wildtype carboxymethylproline synthases enable tuning of their C-N and C-C bond forming capacity to produce 5-carboxymethylproline derivatives substituted at C-4 and C-6, from amino acid aldehyde and malonyl-CoA derivatives. Use of tandem enzyme incubations comprising an engineered carboxymethylproline synthase and an alkylmalonylCoA forming enzyme (i.e. malonyl-CoA synthetase or crotonyl-CoA carboxylase reductase) can improve stereocontrol and expand the product range. Some of the prepared 4,6-disubstituted-5-carboxymethylproline derivatives are converted to bicyclic β-lactams by carbapenam synthetase catalysis. The results illustrate the utility of tandem enzyme systems involving engineered crotonases for asymmetric bicyclic β-lactam synthesis

“Candidatus Competibacter”-lineage genomes retrieved from metagenomes reveal functional metabolic diversity

The glycogen-accumulating organism (GAO) ‘Candidatus Competibacter’ (Competibacter) uses aerobically stored glycogen to enable anaerobic carbon uptake, which is subsequently stored as polyhydroxyalkanoates (PHAs). This biphasic metabolism is key for the Competibacter to survive under the cyclic anaerobic-‘feast’: aerobic-‘famine’ regime of enhanced biological phosphorus removal (EBPR) wastewater treatment systems. As they do not contribute to phosphorus (P) removal, but compete for resources with the polyphosphate-accumulating organisms (PAO), thought responsible for P removal, their proliferation theoretically reduces the EBPR capacity. In this study, two complete genomes from Competibacter were obtained from laboratory-scale enrichment reactors through metagenomics. Phylogenetic analysis identified the two genomes, ‘Candidatus Competibacter denitrificans’ and ‘Candidatus Contendobacter odensis’, as being affiliated with Competibacter-lineage subgroups 1 and 5, respectively. Both have genes for glycogen and PHA cycling and for the metabolism of volatile fatty acids. Marked differences were found in their potential for the Embden–Meyerhof–Parnas and Entner–Doudoroff glycolytic pathways, as well as for denitrification, nitrogen fixation, fermentation, trehalose synthesis and utilisation of glucose and lactate. Genetic comparison of P metabolism pathways with sequenced PAOs revealed the absence of the Pit phosphate transporter in the Competibacter-lineage genomes—identifying a key metabolic difference with the PAO physiology. These genomes are the first from any GAO organism and provide new insights into the complex interaction and niche competition between PAOs and GAOs in EBPR systems