Anaerobic glucose uptake in Pseudomonas putida KT2440 in a bioelectrochemical system.

Providing an anodic potential in a bio-electrochemical system to the obligate aerobe Pseudomonas putida enables anaerobic survival and allows the cells to overcome redox imbalances. In this setup, the bacteria could be exploited to produce chemicals via oxidative pathways at high yield. However, the absence of anaerobic growth and low carbon turnover rates remain as obstacles for the application of such an electro-fermentation technology.

Recursive genome engineering decodes the evolutionary origin of an essential thymidylate kinase activity in KT2440.

Investigating fundamental aspects of metabolism is vital for advancing our understanding of the diverse biochemical capabilities and biotechnological applications of bacteria. The origin of the essential thymidylate kinase function in the model bacterium KT2440, seemingly interrupted due to the presence of a large genomic island that disrupts the cognate gene, eluded a satisfactory explanation thus far. This is a first-case example of an essential metabolic function, likely acquired by horizontal gene transfer, which "landed" in a locus encoding the same activity.

Core and auxiliary functions of one-carbon metabolism in exposed by a systems-level analysis of transcriptional and physiological responses.

The soil bacterium is a robust biomanufacturing host that assimilates a broad range of substrates while efficiently coping with adverse environmental conditions. is equipped with functions related to one-carbon (C1) compounds (e.g.

SEVA 4.0: an update of the Standard European Vector Architecture database for advanced analysis and programming of bacterial phenotypes.

The SEVA platform (https://seva-plasmids.com) was launched one decade ago, both as a database (DB) and as a physical repository of plasmid vectors for genetic analysis and engineering of Gram-negative bacteria with a structure and nomenclature that follows a strict, fixed architecture of functional DNA segments. While the current update keeps the basic features of earlier versions, the platform has been upgraded not only with many more ready-to-use plasmids but also with features that expand the range of target species, harmonize DNA assembly methods and enable new applications.

A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes.

Acetyl-coenzyme A (AcCoA) is a metabolic hub in virtually all living cells, serving as both a key precursor of essential biomass components and a metabolic sink for catabolic pathways for a large variety of substrates. Owing to this dual role, tight growth-production coupling schemes can be implemented around the AcCoA node. Building on this concept, a synthetic C2 auxotrophy was implemented in the platform bacterium Pseudomonas putida through an in silico-informed engineering approach.

Standardization of regulatory nodes for engineering heterologous gene expression: a feasibility study.

The potential of LacI/P , XylS/P , AlkS/P , CprK/P and ChnR/P regulatory nodes, recruited from both Gram-negative and Gram-positive bacteria, as the source of parts for formatting expression cargoes following the Standard European Vector Architecture (SEVA) has been examined. The five expression devices, which cover most known regulatory configurations in bacteria were assembled within exactly the same plasmid backbone and bearing the different functional segments arrayed in an invariable DNA scaffold. Their performance was then analysed in an Escherichia coli strain of reference through the readout of a fluorescence reporter gene that contained strictly identical translation signal elements.

Nutritional and surgical aspects in prostate disorders.

Prostate disorders are commonplace in medicine, especially in older men, with prostatitis, benign prostatic hyperplasia, and prostate cancer being the most abundant pathologies. The complexity of this organ, however, turns treatment into a challenge. In this review, we aim to provide insight into the efficacy of alternative treatments, which are not normally used in conventional medicine, with a particular focus on nutrients.

Combinatorial pathway balancing provides biosynthetic access to 2-fluoro-,-muconate in engineered .

The wealth of bio-based building blocks produced by engineered microorganisms seldom include halogen atoms. Muconate is a platform chemical with a number of industrial applications that could be broadened by introducing fluorine atoms to tune its physicochemical properties. The soil bacterium naturally assimilates benzoate via the -cleavage pathway with ,-muconate as intermediate.

Synthetic metabolism for biohalogenation.

The pressing need for novel bioproduction approaches faces a limitation in the number and type of molecules accessed through synthetic biology. Halogenation is widely used for tuning physicochemical properties of molecules and polymers, but traditional halogenation chemistry often lacks specificity and generates harmful by-products. Here, we pose that deploying synthetic metabolism tailored for biohalogenation represents an unique opportunity towards economically attractive and environmentally friendly organohalide production.

Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways.

Biotechnological production in bacteria enables access to numerous valuable chemical compounds. Nowadays, advanced molecular genetic toolsets, enzyme engineering as well as the combinatorial use of biocatalysts, pathways, and circuits even bring new-to-nature compounds within reach. However, the associated substrates and biosynthetic products often cause severe chemical stress to the bacterial hosts.

Rapid Genome Engineering of Assisted by Fluorescent Markers and Tractable Curing of Plasmids.

Precise genome engineering has become a commonplace technique for metabolic engineering. Also, insertion, deletion and alteration of genes and other functional DNA sequences are essential for understanding and engineering cells. Several techniques have been developed to this end (, CRISPR/Cas-assisted methods, homologous recombination, or λ Red recombineering), yet most of them rely on the use of auxiliary plasmids, which have to be cured after the editing procedure.

Engineering KT2440 for the production of isobutanol.

We engineered for the production of isobutanol from glucose by preventing product and precursor degradation, inactivation of the soluble transhydrogenase SthA, overexpression of the native and genes, and implementation of the feedback-resistant acetolactate synthase AlsS from , ketoacid decarboxylase KivD from , and aldehyde dehydrogenase YqhD from . The resulting strain Iso2 produced isobutanol with a substrate specific product yield ( ) of 22 ± 2 mg per gram of glucose under aerobic conditions. Furthermore, we identified the ketoacid decarboxylase from to be a suitable alternative for isobutanol production, since replacement of from in Iso2 by the variant from yielded an identical Y.

Synthetic control of plasmid replication enables target- and self-curing of vectors and expedites genome engineering of .

Genome engineering of non-conventional microorganisms calls for the development of dedicated synthetic biology tools. is a Gram-negative, non-pathogenic soil bacterium widely used for metabolic engineering owing to its versatile metabolism and high levels of tolerance to different types of stress. Genome editing of .

Accelerated genome engineering of Pseudomonas putida by I-SceI-mediated recombination and CRISPR-Cas9 counterselection.

Pseudomonas species have become reliable platforms for bioproduction due to their capability to tolerate harsh conditions imposed by large-scale bioprocesses and their remarkable resistance to diverse physicochemical stresses. The last few years have brought forth a variety of synthetic biology tools for the genetic manipulation of pseudomonads, but most of them are either applicable only to obtain certain types of mutations, lack efficiency, or are not easily accessible to be used in different Pseudomonas species (e.g.

Cohesin SA2 is a sequence-independent DNA-binding protein that recognizes DNA replication and repair intermediates.

Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids, mediated by the cohesin protein complex, which also plays crucial roles in diverse genome maintenance pathways. Current models attribute DNA binding by cohesin to entrapment of dsDNA by the cohesin ring subunits (SMC1, SMC3, and RAD21 in humans). However, the biophysical properties and activities of the fourth core cohesin subunit SA2 (STAG2) are largely unknown.

Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair.

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different molecules and molecular assemblies in a heterogeneous sample. In the particular context of DNA interacting protein systems, different protein complex forms and their corresponding binding positions on target sites containing DNA fragments can thus be distinguished.

Conservation and Divergence in Nucleotide Excision Repair Lesion Recognition.

Nucleotide excision repair is an important and highly conserved DNA repair mechanism with an exceptionally large range of chemically and structurally unrelated targets. Lesion verification is believed to be achieved by the helicases UvrB and XPD in the prokaryotic and eukaryotic processes, respectively. Using single molecule atomic force microscopy analyses, we demonstrate that UvrB and XPD are able to load onto DNA and pursue lesion verification in the absence of the initial lesion detection proteins.