A solution of 10 g/L of sodium alginate (Satialgine types used [Sanofi trademark]: SG800 and S1100 with manuronic/guluronic ratio of 0.5 and 1.2, respectively) containing invertase (0.08 g of protein/L) was dropped into 0.1 M CaCl2 solution buffered at pH 4.0, 7.0, or 8.0. The beads were left to harden in CaCl2 solution for 24 h. The high immobilization yield of 60% occurred with SG800 at pH 8.0. The activity of soluble and insoluble invertase was measured against pH (2.8-8.0), sucrose concentration (4.5-45 mM), and temperature (30-60 degrees C). Both forms presented an optimum pH of 4.6. However, the soluble invertase was stable at the overall pH interval studied, whereas insoluble invertase lost 30% of its original activity at pH > 5.0. At temperatures above 40 degrees C, the insoluble form was more stable than the soluble one. The kinetic constants and activation energies (Ea) for free invertase were KM = 41.2 mM, Vmax = 0.10 mg of TRS/(min.mL), and Ea 28 kJ/mol for entrapped invertase they were (KM)ap = 7.2 mM, (Vmax)ap = 0.060 mg of TRS/(min.mL), and (Ea)ap = 24 kJ/mol.
Regulation of gene expression in bacteria results from the interplay between hundreds of transcriptional factors (TFs) at target promoters. However, how the arrangement of binding sites for TFs generates the regulatory logic of promoters is not well-known. Here, we generated and fully characterized a library of synthetic complex promoters for the global regulators, CRP and IHF, in Escherichia coli, which are formed by a weak -35/-10 consensus sequence preceded by four combinatorial binding sites for these two TFs. Using this approach, we found that while cis-elements for CRP preferentially activate promoters when located immediately upstream of the promoter consensus, binding sites for IHF mainly function as "UP" elements and stimulate transcription in several different architectures in the absence of this protein. However, the combination of CRP- and IHF-binding sites resulted in emergent properties in these complex promoters, where the activity of combinatorial promoters cannot be predicted from the individual behavior of its components. Taken together, the results presented here add to the information on architecture-logic of complex promoters in bacteria.
Filamentous fungi are remarkable organisms naturally specialized in deconstructing plant biomass and this feature has a tremendous potential for biofuel production from renewable sources. The past decades have been marked by a remarkable progress in the genetic engineering of fungi to generate industry-compatible strains needed for some biotech applications. In this sense, progress in this field has been marked by the utilization of high-throughput techniques to gain deep understanding of the molecular machinery controlling the physiology of these organisms, starting thus the Systems Biology era of fungi. Additionally, genetic engineering has been extensively applied to modify wellcharacterized promoters in order to construct new expression systems with enhanced performance under the conditions of interest. In this review, we discuss some aspects related to significant progress in the understating and engineering of fungi for biotechnological applications, with special focus on the construction of synthetic promoters and circuits in organisms relevant for industry. Different engineering approaches are shown, and their potential and limitations for the construction of complex synthetic circuits in these organisms are examined. Finally, we discuss the impact of engineered promoter architecture in the single-cell behavior of the system, an often-neglected relationship with a tremendous impact in the final performance of the process of interest. We expect to provide here some new directions to drive future research directed to the construction of high-performance, engineered fungal strains working as microbial cell factories.
Neuropathic pain is one of the most important clinical consequences of injury to the somatosensory system. Nevertheless, the critical pathophysiological mechanisms involved in neuropathic pain development are poorly understood. In this study, we found that neuropathic pain is abrogated when the kynurenine metabolic pathway initiated by the enzyme indoleamine 2,3-dioxygenase (IDO1) is ablated pharmacologically or genetically. Mechanistically, it was found that IDO1-expressing dendritic cells (DCs) accumulated in the dorsal root leptomeninges and led to an increase in kynurenine levels in the spinal cord. In the spinal cord, kynurenine was metabolized by kynurenine-3-monooxygenase-expressing astrocytes into a pronociceptive metabolite 3-hydroxykynurenine. Ultimately, 3-hydroxyanthranilate 3,4dioxygenase-derived quinolinic acid formed in the final step of the canonical KYNPATH was also involved in neuropathic pain development through the activation of the glutamatergic N-methyl-D-aspartate (NMDA) receptor. In conclusion, these data revealed a novel role for DCs driving neuropathic pain development through elevation of the kynurenine metabolic pathway. This novel paradigm offers potential new targets for drug development against this type of chronic pain.
Environmental bacteria are endowed with several regulatory systems that have potential applications in biotechnology. In this report, we characterize the arsenic biosensing features of the ars response system from Chromobacterium violaceum in the heterologous host Escherichia coli. We show that the native Pars/arsR system of C. violaceum outperforms the chromosomal ars copy of E. coli when exposed to micromolar concentrations of arsenite. To understand the molecular basis of this phenomenon, we analyzed the interaction between ArsR regulators and their promoter target sites as well as induction of the system at saturating concentrations of the regulators. In vivo titration experiments indicate that ArsR from C. violaceum has stronger binding affinity for its target promoter than the regulator from E. coli does. Additionally, arsenite induction experiments at saturating regulator concentration demonstrates that although the Pars/arsR system from E. coli displays a gradual response to increasing concentration of the inducer, the system from C. violaceum has a steeper response with a stronger promoter induction after a given arsenite threshold. Taken together, these data demonstrate the characterization of a novel arsenic response element from an environmental bacterium with potentially enhanced performance that could be further explored for the construction of an arsenic biosensor.
SummaryRegulation of gene expression in bacteria results from the interplay between transcriptional factors (TFs) at target promoters, and how the arrangement of binding sites determines the regulatory logic of promoters is not well known. Here, we generated and fully characterized a library of synthetic complex promoters for the global regulators, CRP and IHF, in Escherichia coli, formed by a weak -35/-10 consensus sequence preceded by four combinatorial binding sites for these TFs. We found that while cis-elements for CRP preferentially activate promoters when located immediately upstream of the promoter consensus, binding sites for IHF mainly function as "UP" elements and stimulate transcription in several different architectures in the absence of this protein.However, the combination of CRP-and IHF-binding sites resulted in emergent properties in these complex promoters, where the activity of combinatorial promoters cannot be predicted from the individual behavior of its components. Taken together, the results presented here add to the information on architecture-logic of complex promoters in bacteria.__________________________________________________________________________________________
Bacterial transcriptional factors (TFs) and their target promoters are key devices for engineering of complex circuits in many biotechnological applications. Yet, there is a dearth of well characterized inducer-responsive TFs that could be used in the context of an animal or human host. In this work we have deciphered the inducer recognition mechanism of two AraC/XylS regulators from Pseudomonas putida (BenR and XylS) for creating a novel expression system responsive to acetyl salicylate (i.e. Aspirin). Using protein homology modeling and molecular docking with the cognate inducer benzoate and a suite of chemical analogues, we identified the conserved binding pocket of these two proteins. Using site directed mutagenesis, we identified a single amino acid position required for efficient inducer recognition and transcriptional activation. While modification of this position in BenR abolishes protein activity, its modification in XylS increases the response to several aromatic compounds, including acetyl salicylic acid to levels close to those achieved by the canonical inducer. Moreover, by constructing chimeric proteins with swapped N-terminal domains, we created novel regulators with mixed promoter and inducer recognition profiles. As a result, a collection of engineered TFs was generated with enhanced response to a well characterized and largely innocuous molecule with a potential for eliciting heterologous expression of bacterial genes in animal carriers.
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