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Pseudomonas species have become promising cell factories for the production of natural products due to their inherent robustness. Although these bacteria have naturally evolved strategies to cope with different kinds of stress, many biotechnological applications benefit from engineering of optimised chassis strains with specially adapted tolerance traits. Here, we explored the formation of outer membrane vesicles (OMV) of Pseudomonas putida KT2440. We found OMV production to correlate with the recombinant production of a natural compound with versatile beneficial properties, the tripyrrole prodigiosin. Further, several P. putida genes were identified, whose up‐ or down‐regulated expression allowed controlling OMV formation. Finally, genetically triggering vesiculation in production strains of the different alkaloids prodigiosin, violacein, and phenazine‐1‐carboxylic acid, as well as the carotenoid zeaxanthin, resulted in up to three‐fold increased product yields. Consequently, our findings suggest that the construction of robust strains by genetic manipulation of OMV formation might be developed into a useful tool which may contribute to improving limited biotechnological applications. [ABSTRACT FROM AUTHOR]

The primary photochemistry is similar among the flavin‐bound sensory domains of light–oxygen–voltage (LOV) photoreceptors, where upon blue‐light illumination a covalent adduct is formed on the microseconds time scale between the flavin chromophore and a strictly conserved cysteine residue. In contrast, the adduct‐state decay kinetics vary from seconds to days or longer. The molecular basis for this variation among structurally conserved LOV domains is not fully understood. Here, we selected PpSB2‐LOV, a fast‐cycling (τrec 3.5 min, 20 °C) short LOV protein from Pseudomonas putida that shares 67% sequence identity with a slow‐cycling (τrec 2467 min, 20 °C) homologous protein PpSB1‐LOV. Based on the crystal structure of the PpSB2‐LOV in the dark state reported here, we used a comparative approach, in which we combined structure and sequence information with molecular dynamic (MD) simulations to address the mechanistic basis for the vastly different adduct‐state lifetimes in the two homologous proteins. MD simulations pointed toward dynamically distinct structural region, which were subsequently targeted by site‐directed mutagenesis of PpSB2‐LOV, where we introduced single‐ and multisite substitutions exchanging them with the corresponding residues from PpSB1‐LOV. Collectively, the data presented identify key amino acids on the Aβ‐Bβ, Eα‐Fα loops, and the Fα helix, such as E27 and I66, that play a decisive role in determining the adduct lifetime. Our results additionally suggest a correlation between the solvent accessibility of the chromophore pocket and adduct‐state lifetime. The presented results add to our understanding of LOV signaling and will have important implications in tuning the signaling behavior (on/off kinetics) of LOV‐based optogenetic tools. [ABSTRACT FROM AUTHOR]

Human guanylate‐binding protein 1 (hGBP1) belongs to the family of dynamin‐like proteins and is activated by addition of nucleotides, leading to protein oligomerization and stimulated GTPase activity. In vivo, hGBP1 is post‐translationally modified by attachment of a farnesyl group yielding farn‐hGBP1. In this study, hydrodynamic differences in farn‐hGBP1 and unmodified hGBP1 were investigated using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and analytical size‐exclusion chromatography (SEC). In addition, we performed small‐angle X‐ray scattering (SAXS) experiments coupled with a SEC setup (SEC‐SAXS) to investigate structural properties of nonmodified hGBP1 and farn‐hGBP1 in solution. SEC‐SAXS measurements revealed that farnesylation keeps hGBP1 in its inactive monomeric and crystal‐like conformation in nucleotide‐free solution, whereas unmodified hGBP1 forms a monomer–dimer equilibrium both in the inactive ground state in nucleotide‐free solution as well as in the activated state that is trapped by addition of the nonhydrolysable GTP analogue GppNHp. Nonmodified hGBP1 is structurally perturbed as compared to farn‐hGBP. In particular, GppNHp binding leads to large structural rearrangements and higher conformational flexibility of the monomer and the dimer. Structural changes observed in the nonmodified protein are prerequisites for further oligomer assemblies of farn‐hGBP1 that occur in the presence of nucleotides. Database: All SEC‐SAXS data, corresponding fits to the data and structural models are deposited in the Small Angle Scattering Biological Data Bank [SASBDB (Nucleic Acids Res, 43, 2015, D357)] with project IDs: SASDEE8, SASDEF8, SASDEG8, SASDEH8, SASDEJ8, SASDEK8, SASDEL8 and SASDEM8. [ABSTRACT FROM AUTHOR]

The human membrane‐bound α/β‐hydrolase domain 6 (ABHD6) protein modulates endocannabinoid signaling, which controls appetite, pain and learning, as well as being linked to Alzheimer's and Parkinson's diseases, through the degradation of the key lipid messenger 2‐arachidonylglycerol (2‐AG). This makes ABHD6 an attractive therapeutic target that lacks structural information. In order to better understand the molecular mechanism of 2‐AG‐hydrolyzing enzymes, the PA2949 protein from Pseudomonas aeruginosa, which has 49% sequence similarity to the ABHD6 protein, was cloned, overexpressed, purified and crystallized. Overexpression of PA2949 in the homologous host yielded the membrane‐bound enzyme, which was purified in milligram amounts. Besides their sequence similarity, the enzymes both show specificity for the hydrolysis of 2‐AG and esters of medium‐length fatty acids. PA2949 in the presence of n‐octyl β‐d‐glucoside showed a higher activity and stability at room temperature than those previously reported for PA2949 overexpressed and purified from Escherichia coli. A suitable expression host and stabilizing detergent were crucial for obtaining crystals, which belonged to the tetragonal space group I4122 and diffracted to a resolution of 2.54 Å. This study provides hints on the functional similarity of ABHD6‐like proteins in prokaryotes and eukaryotes, and might guide the structural study of these difficult‐to‐crystallize proteins. Homologous expression of the membrane‐bound esterase PA2949 from Pseudomonas aeruginosa PA01 and the purification of detergent‐solubilized enzyme resulted in stable PA2949 protein that crystallized. The crystals obtained were used for X‐ray analysis and diffracted to a resolution of 2.74 Å. [ABSTRACT FROM AUTHOR]