Accumulation of docosapentaenoic acid (n-3 DPA) in a novel isolate of the marine ichthyosporean Sphaeroforma arctica.

OBJECTIVE: Currently, there is lack of a consistent and highly enriched source for docosapentaenoic acid (n-3 DPA, C22:5), and this work report the isolation of microorganism that naturally produces n-3 DPA. RESULTS: In this work, we screened microorganisms in our culture collections with the goal to isolate a strain with high levels of n-3 DPA. We isolated a strain of Sphaeroforma arctica that produces up to 11% n-3 DPA in total fatty acid and has a high n-3 DPA to DHA/EPA ratio. The cell growth of the isolated strain was characterized using microscopy imaging and flow cytometer technologies to confirm the coenocytic pattern of cell divisions previously described in S. arctica. Our novel isolate of S. arctica grew more robustly and produced significantly more n-3 DPA compared to previously isolated and described strains indicating the uniqueness of the discovered strain. CONCLUSION: Overall, this work reports a first isolate n-3 DPA producing microorganism and establishes the foundation for future strain improvement and elucidation of the physiological function of this LC-PUFA for human nutrition and health.

Highly selective whole-cell 25-hydroxyvitamin D3 synthesis using molybdenum-dependent C25-steroid dehydrogenase and cyclodextrin recycling.

BACKGROUND: The global prevalence of vitamin D (VitD) deficiency associated with numerous acute and chronic diseases has led to strategies to improve the VitD status through dietary intake of VitD-fortified foods and VitD supplementation. In this context, the circulating form of VitD3 (cholecalciferol) in the human body, 25-hydroxy-VitD3 (calcifediol, 25OHVitD3), has a much higher efficacy in improving the VitD status, which has motivated researchers to develop methods for its effective and sustainable synthesis. Conventional monooxygenase-/peroxygenase-based biocatalytic platforms for the conversion of VitD3 to value-added 25OHVitD3 are generally limited by a low selectivity and yield, costly reliance on cyclodextrins and electron donor systems, or by the use of toxic co-substrates. RESULTS: In this study, we used a whole-cell approach for biocatalytic 25OHVitD3 synthesis, in which a molybdenum-dependent steroid C25 dehydrogenase was produced in the denitrifying bacterium Thauera aromatica under semi-aerobic conditions, where the activity of the enzyme remained stable. This enzyme uses water as a highly selective VitD3 hydroxylating agent and is independent of an electron donor system. High density suspensions of resting cells producing steroid C25 dehydrogenase catalysed the conversion of VitD3 to 25OHVitD3 using either O2 via the endogenous respiratory chain or externally added ferricyanide as low cost electron acceptor. The maximum 25OHVitD3 titer achieved was 1.85 g L-1 within 50 h with a yield of 99%, which is 2.2 times higher than the highest reported value obtained with previous biocatalytic systems. In addition, we developed a simple method for the recycling of the costly VitD3 solubiliser cyclodextrin, which could be reused for 10 reaction cycles without a significant loss of quality or quantity. CONCLUSIONS: The established steroid C25 dehydrogenase-based whole-cell system for the value-adding conversion of VitD3 to 25OHVitD3 offers a number of advantages in comparison to conventional oxygenase-/peroxygenase-based systems including its high selectivity, independence from an electron donor system, and the higher product titer and yield. Together with the established cyclodextrin recycling procedure, the established system provides an attractive platform for large-scale 25OHVitD3 synthesis.

In Vivo Efficacy and Safety Evaluations of Therapeutic Splicing Correction Using U1 snRNA in the Mouse Retina.

Efficacy and safety considerations constitute essential steps during development of in vivo gene therapies. Herein, we evaluated efficacy and safety of splice factor-based treatments to correct mutation-induced splice defects in an Opa1 mutant mouse line. We applied adeno-associated viruses to the retina. The viruses transduced retinal cells with an engineered U1 snRNA splice factor designed to correct the Opa1 splice defect. We found the treatment to be efficient in increasing wild-type Opa1 transcripts. Correspondingly, Opa1 protein levels increased significantly in treated eyes. Measurements of retinal morphology and function did not reveal therapy-related side-effects supporting the short-term safety of the treatment. Alterations of potential off-target genes were not detected. Our data suggest that treatments of splice defects applying engineered U1 snRNAs represent a promising in vivo therapeutic approach. The therapy increased wild-type Opa1 transcripts and protein levels without detectable morphological, functional or genetic side-effects in the mouse eye. The U1 snRNA-based therapy can be tailored to specific disease gene mutations, hence, raising the possibility of a wider applicability of this promising technology towards treatment of different inherited retinal diseases.

From Sugars to Nutritional Products - Active Ingredients.

A modern trend to carbon dioxide neutral production processes is based on renewable raw materials derived from sugars. Herein, an overview on modern approaches to fine chemicals for the nutritional industry is presented. In comparison to the traditional fossil-fuel-based processes the development of sustainable alternative transformations is necessary to enable the full potential of the new sustainable feedstocks.

Photoredox-Catalyzed Dehydrogenative Csp3-Csp2 Cross-Coupling of Alkylarenes to Aldehydes in Flow.

Executing photoredox reactions in flow offers solutions to frequently encountered issues regarding reproducibility, reaction time, and scale-up. Here, we report the transfer of a photoredox-catalyzed benzylic coupling of alkylarenes to aldehydes to a flow chemistry setting leading to improvements in terms of higher concentration, shorter residence times, better yields, ease of catalyst preparation, and enhanced substrate scope. Its applicability has been demonstrated by a multi-gram-scale reaction using high-power light-emitting diodes (LEDs), late-stage functionalization of selected active pharmaceutical ingredients (APIs), and also a photocatalyst recycling method.

Photoredox Generation of Sulfonyl Radicals and Coupling with Electron Deficient Olefins.

Various sulfone tetrazoles were activated via iridium photoredox catalysis in the presence of DMAP to give dialkyl sulfones. The presumed sulfone radical intermediates were trapped by a range of electron-deficient olefins in generally good to excellent yields.

A Practical Method for Continuous Production of sp3-Rich Compounds from (Hetero)Aryl Halides and Redox-Active Esters.

A practically useful coupling reaction between aromatic halides and redox-active esters was realized by nickel catalysis through the use of a packed zinc bed column in continuous flow. Multiple reuse of the column showed a negligible decrease in efficiency, affording high space/time yields. A wide range of substrates, including a number of heteroaryl halides and polyfunctional materials were coupled in generally good yields. Longer-time and larger-scale experiments further demonstrates the robustness of the system.

A New World for Chemical Synthesis?

This perspective seeks to provide an overarching vision of the current state of chemical synthesis methodology using machinery as enabling tools. It highlights current capabilities and limitations in this highly digitallyconnected world and suggests areas where new opportunities may arise in the future by going well beyond our present levels of innovation and automation. There is a new need for improved downstream processing tools, advanced reactor design, computational predictive algorithms and integration of robotic systems to maximise the human resource to facilitate a new era in the assembly of our functional materials.

A Photoredox Coupling Reaction of Benzylboronic Esters and Carbonyl Compounds in Batch and Flow.

Mild cross-coupling reaction between benzylboronic esters with carbonyl compounds and some imines was achieved under visible-light-induced iridium-catalyzed photoredox conditions. Functional group tolerance was demonstrated by 51 examples, including 13 heterocyclic compounds. Gram-scale reaction was realized through the use of computer-controlled continuous flow photoreactors.

A Novel C-Terminal Mutation in Gsdma3 (C+/H-) Leads to Alopecia and Corneal Inflammatory Response in Mice.

Purpose: Mutations in the gene encoding Gasdermin A3 (Gsdma3) have been described to cause severe skin phenotypes, including loss of sebaceous glands and alopecia, in mice. We discovered a novel C-terminal mutation in Gsdma3 in a new mouse line and characterized a less frequently reported corneal phenotype, likely caused by degeneration of Meibomian glands of the inner eyelid. Methods: We used histologic methods to evaluate the effects of the C+/H- mutation on sebaceous gland and skin morphology as well as Meibomian glands of the inner eyelid and corneal tissue. Chromosomal aberrations were excluded by karyogram analyses. The mutation was identified by Sanger sequencing of candidate genes. Results: Analyses of skin samples from affected mice confirmed the frequently reported phenotypes associated with mutations in Gsdma3: Degeneration of sebaceous glands and complete loss of pelage. Immunologic staining of corneal samples suggested an inflammatory response with signs of neovascularization in half of the affected older mice. While the corneal phenotype was observed at irregular time points, mainly after 6 months, its appearance coincided with a degeneration of Meibomian glands in the eyelids of affected animals. Conclusions: The mutation described herein is associated with inflammation and neovascularization of corneal tissue. Simultaneous degeneration of Meibomian glands in affected animals suggested a change in tear-film composition as the underlying cause for the corneal phenotype. Our data further support that different pathogenic mechanisms underlie some of the reported mutations in Gsdma3.

Short-term regulation of murine colonic NBCe1-B (electrogenic Na+/HCO3(-) cotransporter) membrane expression and activity by protein kinase C.

The colonic mucosa actively secretes HCO3(-), and several lines of evidence point to an important role of Na+/HCO3(-) cotransport (NBC) as a basolateral HCO3(-) import pathway. We could recently demonstrate that the predominant NBC isoform in murine colonic crypts is electrogenic NBCe1-B, and that secretagogues cause NBCe1 exocytosis, which likely represents a component of NBC activation. Since protein kinase C (PKC) plays a key role in the regulation of ion transport by trafficking events, we asked whether it is also involved in the observed NBC activity increase. Crypts were isolated from murine proximal colon to assess PKC activation as well as NBC function and membrane abundance using fluorometric pHi measurements and cell surface biotinylation, respectively. PKC isoform translocation and phosphorylation occurred in response to PMA-, as well as secretagogue stimulation. The conventional and novel PKC inhibitors Gö6976 or Gö6850 did not alter NBC function or surface expression by themselves, but stimulation with forskolin (10(-5) M) or carbachol (10(-4) M) in their presence led to a significant decrease in NBC-mediated proton flux, and biotinylated NBCe1. Our data thus indicate that secretagogues lead to PKC translocation and phosphorylation in murine colonic crypts, and that PKC is necessary for the increase in NBC transport rate and membrane abundance caused by cholinergic and cAMP-dependent stimuli.

Increased epithelial permeability is the primary cause for bicarbonate loss in inflamed murine colon.

BACKGROUND: Bicarbonate loss into the lumen occurs during intestinal inflammation in different species. However, candidate pathways like CFTR or DRA are inhibited in the inflamed gut. This study addressed the question whether and how inflammation-associated increased intestinal permeability may result in epithelial HCO(3)(-) loss. METHODS: Murine proximal colon was studied because it does not express functional DRA but is inflamed in the tumor necrosis factor α overexpressing mouse model (TNF(ΔARE)). Luminal alkalization, (3)H-mannitol fluxes, impedance spectroscopy, and dilution potentials were measured in Ussing chambers, whereas expression and localization of tight junction-associated proteins were analyzed by Western blots and immunohistochemistry. RESULTS: Luminal alkalization rates and (3)H-mannitol fluxes were increased in TNF(+/ΔARE) proximal colon, whereas forskolin-stimulated I(sc) was not altered. Epithelial resistance was reduced, but subepithelial resistance increased. The epithelial lining was intact, and enterocyte apoptosis rate was not increased despite massively increased Th1 cytokine levels and lymphoplasmacellular infiltration. Measurement of dilution potentials suggested a loss of cation selectivity with increased anion permeability. Western analysis revealed a downregulation of occludin expression and an upregulation of both claudin-2 and claudin-5, with no change in ZO-1, E-cadherin, claudin-4, and claudin-8. Immunohistochemistry suggested correct occludin localization but reduced tight junction density in TNF(+/ΔARE) surface epithelium. CONCLUSIONS: Inflammation during TNF-α overexpression leads to increased epithelial permeability in murine proximal colon, decreased tight junctional cation selectivity, and increased HCO(3)(-) loss into the lumen. Inflammation-associated colonic HCO(3)(-) loss may occur through leaky tight junctions rather than through HCO(3)(-) secreting ion transporters.

Biocatalytic reductions: from lab curiosity to "first choice".

Enzyme-catalyzed reductions have been studied for decades and have been introduced in more than 10 industrial processes for production of various chiral alcohols, alpha-hydroxy acids and alpha-amino acids. The earlier hurdle of expensive cofactors was taken by the development of highly efficient cofactor regeneration methods. In addition, the accessible number of suitable dehydrogenases and therefore the versatility of this technology is constantly increasing and currently expanding beyond asymmetric production of alcohols and amino acids. Access to a large set of enzymes for highly selective C=C reductions and reductive amination of ketones for production of chiral secondary amines and the development of improved D-selective amino acid dehydrogenases will fuel the next wave of industrial bioreduction processes.

The structure of L-hydantoinase from Arthobacter aurescens leads to an understanding of dihydropyrimidinase substrate and enantio specificity.

L-Hydantoinase from Arthrobacter aurescens (L-Hyd) is a member of the dihydropyrimidinases which in turn belong to the cyclic amidases. Dihydropyrimidinases catalyze the reversible hydrolytic ring opening of dihydropyrimidines as the second step in the catabolism of pyrimidines. In biotechnology, their hydroloytic activity on five-membered cyclic diamides (hydantoins) is used in the enantio-specific production of amino acids from racemic hydantoins. L-Hyd differs from most of the other dihydropyrimidinases by an L-enantio specificity and by lacking activity on possible natural substrates such as dihydropyrimidines. In this paper, we describe the three-dimensional structure of L-Hyd which was solved by molecular replacement using a homology model and subsequently refined to 2.6 A resolution. Each subunit of the tetrameric L-Hyd consists of an elliptically distorted (alpha/beta)(8)-barrel domain, which hosts the active site, and a beta-sheet domain. In the active site, a binuclear zinc center activates a water molecule for nucleophilic attack on the substrates' amide bond. L-Hyd shows a strong homology both in fold and in metal coordination in the active site to another dihydropyrimidinase from Thermus sp. (D-hydantoinase) and to a slightly lesser degree to ureases, dihydroorotase and phosphotriesterase. Using the homology to ureases, a model for the transition state was modeled in the active site of L-Hyd and D-hydantoinase. This model could provide an explanation for the different substrate and enantio selectivities of both dihydropyrimidinases.