Metabolic engineering of Pseudomonas fluorescens for the production of vanillin from ferulic acid

Vanillin is one of the most important flavors in the food industry and there is great interest in its production through biotechnological processes starting from natural substrates such as ferulic acid. Among bacteria, recombinant Escherichia coli strains are the most efficient vanillin producers, whereas Pseudomonas spp. strains, although possessing a broader metabolic versatility, rapidly metabolize various phenolic compounds including vanillin. In order to develop a robust Pseudomonas strain that can produce vanillin in high yields and at high productivity, the vanillin dehydrogenase (vdh)-encoding gene of Pseudomonas fluorescens BF13 strain was inactivated via targeted mutagenesis. The results demonstrated that engineered derivatives of strain BF13 accumulate vanillin if inactivation of vdh is associated with concurrent expression of structural genes for feruloyl-CoA synthetase (fcs) and hydratase/aldolase (ech) from a low-copy plasmid. The conversion of ferulic acid to vanillin was enhanced by optimization of growth conditions, growth phase and parameters of the bioconversion process. The developed strain produced up to 8.41 mM vanillin, which is the highest final titer of vanillin produced by a Pseudomonas strain to date and opens new perspectives in the use of bacterial biocatalysts for biotechnological production of vanillin from agro-industrial wastes which contain ferulic acid.     

Hydroxylamine as a thermal destabiliser of bacteriorhodopsin

The light-catalysed reaction of hydroxylamine (HA) with retinal is one of the basic features of bacteriorhodopsin (BR). Surprisingly, according to recent results, neither the photocycle and proton pumping of BR, nor the trans–cis isomerisation of retinal is prerequisite for photobleaching of BR in the presence of HA. How, then, is the accessibility of retinal to HA enhanced on illumination? We studied whether local thermal denaturation of BR, proposed recently, could provide an explanation for HA-promoted bleaching. According to our results, HA does not alter the absorption spectrum and the photocycle kinetics of BR substantially at room temperature, even at molar concentrations, but grossly affects the temperature of thermal denaturation. At pH 7, the presence of 0.5 M HA reduces the denaturation temperature from 100°C to as low as 72°C. The decrease is proportional to the logarithm of the HA concentration over more than three orders of magnitude, and even 0.5 mM HA has a significant effect. In addition, photobleaching becomes considerably faster with increasing temperature in the presence of HA, it takes a few seconds at 50–60°C. Our results suggest that photobleaching of BR in the presence of HA can be explained by overall destabilisation of the structure of the protein and local thermal denaturation that has already accounted for the photobleaching of the HA-free BR at elevated temperatures. These results further support the importance of thermooptic effects in protein photoreactions and identify HA as a thermal destabiliser of BR.     

Characterization of the novel antifungal chitosanase PgChP and the encoding gene from Penicillium chrysogenum

The protein PgChP is a new chitosanase produced by Penicillium chrysogenum AS51D that showed antifungal activity against toxigenic molds. Two isoforms were found by SDS-PAGE in the purified extract of PgChP. After enzymatic deglycosylation, only the smaller isoform was observed by SDS-PAGE. Identical amino acid sequences were obtained from the two isoforms. Analysis of the molecular mass by electrospray ionization-mass spectrometry revealed six major peaks from 30 to 31 kDa that are related to different levels of glycosylation. The pgchp gene has 1,146 bp including four introns and an open reading frame encoding a protein of 304 amino acids. The translated open reading frame has a predicted mass of 32 kDa, with the first 21 amino acids comprising a signal peptide. Two N glycosylation consensus sequences are present in the protein sequence. The deduced sequence showed high identity with fungal chitosanases. A high level of catalytic activity on chitosan was observed. PgChP is the first chitosanase described from P. chrysogenum. Given that enzymes produced by this mold species are granted generally recognized as safe status, PgChP could be used as a food preservative against toxigenic molds and to obtain chitosan oligomers for food additives and nutraceuticals.     

Interactions Between Sabre‐Tooth Blennies and Their Reef Fish Victims: Effects Of Enforced Repeated Game Structure and Local Abundance on Victim Aggression

The conditions under which humans benefit from contributing to a public good have attracted great interest; in particular the potential role of punishment of cheaters is hotly debated. In contrast, similar studies on other animals are lacking. In this study, we describe for the first time how the course of interactions between parasitic sabre‐tooth blennies (the cheaters) and their reef fish victims can be used to study both punishment and the emergence of public goods. Sabre tooth blennies (Plagiotremus sp.) sneak up from behind to bite off small pieces of scales and/or mucus from other fish. Victims regularly show spontaneous aggression as well as aggressive responses to blenny attacks. In a between species comparison, we tested how the probability of chasing a blenny is affected by (1) the option of avoiding interactions with a blenny by avoiding its small territory, and (2) variation in local abundance of conspecifics. We found that resident victim species are more aggressive towards blennies than visiting species. This difference persisted when we controlled for victim size and territoriality, suggesting that it is the enforced repeated game structure that causes residents to chase blennies. In residents, we also found a negative correlation between aggression towards blennies and local abundance, which suggests that the benefits of chasing are diluted with increasing local abundance. We discuss the implication of these results for future studies.     

Costs and Benefits of Sociality Differ Between Female Guanacos Living in Contrasting Ecological Conditions

According to current theory, anti‐predator benefits promote group formation in open‐dwelling ungulates. An inverse relationship between vigilance effort and group size has been documented frequently and thought to reflect the consequent decrease in perceived predation risk as group size increases. In contrast, competition costs are supposed to set the upper limit to the number of individuals that can forage together. As anti‐predator behavior is no longer functional in the absence of predation and competition costs might be affected by resource distribution, the net benefit of aggregation will depend on the particular combination of predation risk and habitat structure experienced by the individual. To test this hypothesis, group‐size effects on female time allocation and within‐group aggression rate were compared between two guanaco populations exposed to contrasting levels of puma predation. Habitat structure within both sites consisted of mosaics of shrublands and grasslands, and group‐size effects were also compared between these habitat types. Females under predation risk showed a strong reduction in vigilance as the number of adults in the group increased, whereas females from the predator‐free population showed overall low levels of vigilance, regardless of group size. These results emphasize the anti‐predator significance of the group‐size effect on female vigilance, as well as guanaco plasticity to adjust time allocation to local conditions. On the other hand, within‐group aggression rate increased with the number of adults in the group. Aggression rate was almost null within groups located in grasslands but was significantly higher in shrublands, regardless of predation risk, suggesting that the more heterogeneous distribution of shrubs increases the interference competition level. These results strengthen the notion of predation pressure and habitat structure as major determinants of the balance between costs and benefits of group living, and highlight the potential of individual behavioral patterns to make qualitative predictions about group‐size variation within territorial ungulates.     

The norepinephrine transporter (NET) radioligand (S,S)-[18F]FMeNER-D2 shows significant decreases in NET density in the human brain in Alzheimer's disease: A post-mortem autoradiographic study

Earlier post-mortem histological and autoradiographic studies have indicated a reduction of cell numbers in the locus coeruleus (LC) and a corresponding decrease in norepinephrine transporter (NET) in brains obtained from Alzheimer's disease (AD) patients as compared to age-matched healthy controls. In order to test the hypothesis that the regional decrease of NET is a disease specific biomarker in AD and as such, it can be used in PET imaging studies for diagnostic considerations, regional differences in the density of NET in various anatomical structures were measured in whole hemisphere human brain slices obtained from AD patients and age-matched control subjects in a series of autoradiographic experiments using the novel selective PET radioligand for NET (S,S)-[¹⁸F]FMeNER-D₂. (S,S)-[¹⁸F]FMeNER-D₂ appears to be a useful imaging biomarker for quantifying the density of NET in various brain structures, including the LC and the thalamus wherein the highest densities are found in physiological conditions. In AD significant decreases of NET densities can be demonstrated with the radioligand in both structures as compared to age-matched controls. The decreases in AD correlate with the progress of the disease as indicated by Braak grades. As the size of the LC is below the spatial resolution of the PET scanners, but the size of the thalamus can be detected with appropriate spatial accuracy in advanced scanners, the present findings confirm our earlier observations with PET that the in vivo imaging of NET with (S,S)-[¹⁸F]FMeNER-D₂ in the thalamus is viable. Nevertheless, further studies are warranted to assess the usefulness of such an imaging approach for the early detection of changes in thalamic NET densities as a disease-specific biomarker and the possible use of (S,S)-[¹⁸F]FMeNER-D₂ as a molecular imaging biomarker in AD.     

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol‐enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.