We describe for the first time the full reaction coordinate regarding the photoisomerization of red-absorbing norbornadienes (NBDs) to quadricyclanes (QCs). Our studies go beyond steady-state investigations by using an arsenal of time-resolved techniques. Importantly, the red absorption of NBDs is made possible by a different charge-transfer character; adjusting its strength enables control over the photoreversibility of the rearrangement. In the case of strong charge-transfer character (a weakly electron-withdrawing ester and a strongly electron-donating dimethylaniline), photoirradiation with visible light into the delocalized charge-transfer absorption of NBD affords QC reversibly. In stark contrast, UV photoirradiation into the NBD localized excited state leads to a photoinduced degradation and cannot be back-isomerized to NBD under any circumstances. If the charge-transfer character is weak (a weakly electron-withdrawing ester and a weakly electron-donating phenyl), reversibility is seen independently of the photoirradiation light.

Cobalt catalysts are immobilized on the surface of iron oxide nanoparticles for the preparation of highly active quasi-homogeneous catalysts toward an efficient release of photochemically stored energy in norbornadiene-based photoswitches. The facile separation of the iron oxide nanoparticles through exploitation of the intrinsic magnetic properties of this material enables efficient cyclization of energy storage and release. Through the transition from cobalt (II) salphen to cobalt porphyrins, a 22.6-fold increase in the catalytic efficiency of the QC-NBD back-conversion is achieved, with an initial TOF of up to 3.64 s(-1) and excellent TON of over 3305. In addition, a series of novel "push-pull" functionalized norbornadiene derivatives is prepared, featuring excellent absorption properties with maxima up to 366 nm, quantum yields around 70 %, high energy storage capacities of up to 98.0 kJ mol(-1), and outstanding thermal stability with t(1/2) (25 degrees C) over 100 days. Finally, the energy storage potential of these molecular solar thermal (MOST) systems is harnessed in a heat release experiment. This demonstrates the potential of norbornadiene-based photoswitches in combination with efficient magnetic catalysts for the generation of environmentally benign process heat.

With respect to molecular switches, initializing the quadricyclane (QC) to norbornadiene (NBD) back-reaction by light is highly desirable. Our previous publication provided a unique solution for this purpose by utilizing covalently bound C60 . In this work, the fundamental processes within these hybrids has been investigated. Variation of the linker unit connecting the NBD/QC moiety with the fullerene core is used as a tool to tune the properties of the resulting hybrids. Utilizing the Prato reaction, two unprecedented NBD/QC - fullerene hybrids having a long-rigid and a short-rigid linker were synthesized. Molecular dynamics simulations revealed that this results in an average QC-C60 distance of up to 14.2 Å. By comparing the NBD-QC switching of these derivatives with the already established one having a flexible linker, valuable mechanistic insights were gained. Most importantly, spatial convergence of the QC moiety and the fullerene core is inevitable for an efficient back-reaction.

The synthesis and properties of various norbornadiene/quadricyclane (NBD/QC) fullerene hybrids are reported. By cyclopropanation of C60 with malonates carrying the NBD scaffold a small library of NBD-fullerene monoadducts and NBD-fullerene hexakisadducts was established. The substitution pattern of the NBD scaffold, as well as the electron affinity of the fullerene core within these hybrid systems, has a pronounced impact on the properties of the corresponding energy rich QC derivatives. Based on this, the first direct photoisomerization of NBD-fullerene hybrids to their QC derivatives was achieved. Furthermore, it was possible to use the redox-active fullerene core of a QC-fullerene monoadduct to enable the back reaction to form the corresponding NBD-fullerene monoadduct. Combining these two processes enables switching between NBD and QC simply by changing the irradiation wavelength between 310 and 400 nm. Therefore, turning this usually photo/thermal switch into a pure photoswitch. This not only simplifies the investigation of the underlying processes of the NBD-QC interconversion within the system, but also renders such hybrids interesting for applications as molecular switches.

The Cover Feature illustrates an energy-harvesting battery based on the photochemical rearrangement of norbornadiene (NBD) into the highly strained metastable isomer quadricyclane (QC). The chemically stored energy can easily be accessed via treatment with a magnetic cobalt(II) catalyst resulting in an energy release of approximately 88.3 kJ/mol. After magnetic recovery of the catalyst, NBD can undergo repeated cycles of emission free energy storage and release. More information can be found in the Article by T. Luchs et al. on page 52 in Issue 1, 2020 (DOI: 10.1002/cptc.201900194).

The valence isomerization of norbornadiene (NBD) and quadricyclane (QC) has been widely studied as a potential molecular solar thermal (MOST) energy storage and release system. By combining this interconversion couple with C60 fullerene, a hybrid system with novel properties was created. Within this system, light-induced switching between NBD and QC in both directions is possible, thereby transforming the former photo/thermal system into a pure photoswitch. More information can be found in the Full Paper by P. Lorenz and A. H. Hirsch on page 5220.

We report a novel, inexpensive and effective process for the repeatable photoisomerization of norbornadiene (NBD) to its metastable isomer quadricyclane (QC), followed by catalytically induced strain energy release via back-conversion of QC to NBD. By utilization of a quasi-homogeneous catalyst based on magnetic core-shell nanoparticles, tedious purification steps are avoided. The core of this material is comprised of Fe3O4 and a catalytically active cobalt(II) complex is anchored on the particle surface as a self-assembled monolayer (SAM). These core-shell nanoparticles [Fe3O4-CoSalphen] combine a high surface area of catalytically active molecules with straightforward separation by the action of an external magnetic field. In combination with the promising interconversion couple NBD1-QC1, which features outstanding stability (t(1/2)=450 days at room temperature) and a high energy storage potential (88.34 kJ/mol), the nanoparticle catalyst [Fe3O4-CoSalphen] shows great potential for technical applications in molecular solar thermal (MOST) energy-storage systems.

To date, commercial laccase preparations are used in the food, textile, and paper and pulp industries (mild pH). Laccases are attractive in the synthesis of dye molecules or oxidative lignin treatment, which take place at high pH (>= 8.0). So far, one fungal laccase has been reported to be active at alkaline pH. Herein, engineering of the fungal laccase from Melanocarpus albomyces (MaL) for increased activity toward the substrate 2,6-dimethoxyphenol at pH (>= 9.0) is reported. Through a knowledge-gaining directed evolution (KnowVolution) campaign, the key positions Leu365 and Leu513 were identified to increase alkaline tolerance. Both positions are located in close proximity of the T1Cu site. Molecular docking and simulations studies reveal that both substitutions act in a synergic way to stabilize and improve laccase activity at higher pH. Kinetic characterization of the final variant MaL-M1 (L365E/L513M) revealed at pH 9.8 a threefold improved k(cat) (k(cat)=(6.0 +/- 0.2) s(-1)) compared with that of wild-type M. albomyces laccase (k(cat)=(2.11 +/- 0.07) s(-1)).

AbstractA novel Dye-decolorizing peroxidase from the basidiomycete Pleurotus sapidus was screened for dyedecolorizing peroxidase activity with 2,2‘-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid), Remazol Brilliant Blue R and Guaiacol. Additionally, the catalytic efficiency on degrading β-carotene into volatile products, and the catalyst storage stability with three different additives were also studied. The apparent inhibition constant (KS) was 51.7 μM. Optimal reaction rates (Vmax) and affinity constants (Km) towards the reducing substrates were obtained using Michaelis-Menten kinetic theory. The trend in the calculated Km’s was found to be 7.0 mM > 0.524 mM > 0.051 mM for Guaiacol, 2,2‘-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and Remazol Brilliant Blue R. The storage stability of the catalyst was evaluated with 7.0% w/v PEG400, 7.0% w/v PEG1450 and 0.1% w/v Tween®80 at 5°C over a period of 45 days. The study revealed the longest activity conservation with PEG1450, where rDyP had lost 30% of initial activity. The enzyme solution presented similar pH and temperature dependence to known fungal dye-decolorizing peroxidases with most prolific enzymatic activities registered at pH 4.0 and temperatures below 30°C. An interesting property of the catalyst was oxidation observed in the absence of hydrogen peroxide.

The coding sequence of a peroxidase from the secretome of Pleurotus sapidus was cloned from a cDNA library. Bioinformatic analyses revealed an open reading frame of 1551 bp corresponding to a primary translation product of 516 amino acids. The DyP-type peroxidase was heterologously produced in Trichoderma reesei with an activity of 55,000 U L−1. The enzyme was purified from the culture supernatant, biochemically characterized and the kinetic parameters were determined. The enzyme has an N-terminal signal peptide composed of 62 amino acids. Analysis by Blue Native PAGE and activity staining with ABTS, as well as gel filtration chromatography showed the native dimeric state of the enzyme (115 kDa). Analysis of the substrate range revealed that the recombinant enzyme catalyzes, in addition to the conversion of some classic peroxidase substrates such as 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) and substituted phenols like 2,6–dimethoxyphenol, also the decolorization of the anthraquinonic dye Reactive Blue 5. The enzyme also catalyzes bleaching of natural colorants such as β-carotene and annatto. Surprisingly, β-carotene was transformed in the presence and absence of H2O2 by rPsaDyP, however enzyme activity was increased by the addition of H2O2. This indicates that the rPsaDyP has an oxidase function in addition to a peroxidase activity. As a consequence of the high affinity to the characteristic substrate Reactive Blue 5 the rPsaDyP belongs functionally to the dyp-type peroxidase family.

BACKGROUND:Bacillus subtilis is an important cell factory for the biotechnological industry due to its ability to secrete commercially relevant proteins in large amounts directly into the growth medium. However, hyper-secretion of proteins, such as α-amylases, leads to induction of the secretion stress-responsive CssR-CssS regulatory system, resulting in up-regulation of the HtrA and HtrB proteases. These proteases degrade misfolded proteins secreted via the Sec pathway, resulting in a loss of product. The aim of this study was to investigate the secretion stress response in B. subtilis 168 cells overproducing the industrially relevant α-amylase AmyM from Geobacillus stearothermophilus, which was expressed from the strong promoter P(amyQ)-M.RESULTS:Here we show that activity of the htrB promoter as induced by overproduction of AmyM was "noisy", which is indicative for heterogeneous activation of the secretion stress pathway. Plasmids were constructed to allow real-time analysis of P(amyQ)-M promoter activity and AmyM production by, respectively, transcriptional and out-of-frame translationally coupled fusions with gfpmut3. Our results show the emergence of distinct sub-populations of high- and low-level AmyM-producing cells, reflecting heterogeneity in the activity of P(amyQ)-M. This most likely explains the heterogeneous secretion stress response. Importantly, more homogenous cell populations with regard to P(amyQ)-M activity were observed for the B. subtilis mutant strain 168degUhy32, and the wild-type strain 168 under optimized growth conditions.CONCLUSION:Expression heterogeneity of secretory proteins in B. subtilis can be suppressed by degU mutation and optimized growth conditions. Further, the out-of-frame translational fusion of a gene for a secreted target protein and gfp represents a versatile tool for real-time monitoring of protein production and opens novel avenues for Bacillus production strain improvement.

BACKGROUND:Filamentous fungi are frequently used as production platforms in industrial biotechnology. Most of the strains involved were known as reproducing exclusively asexually thereby preventing the application of conventional strain breeding techniques. In the last decade, evidence was obtained that a number of these imperfect fungi possess a sexual life cycle, too. Trichoderma reesei, an industrial producer of enzymes for food, feed and biorefinery purposes, is heterothallic and takes a special position among industrially utilized species as all industrial strains are derived from the single MAT1-2 isolate QM6a. Consequently, strain improvement by crossing is not feasible within this strain line as this necessitates a MAT1-1 mating partner. Simply switching the mating type in one of the mating partners to MAT1-1, however, is not sufficient to produce a genotype capable of sexual reproduction with QM6a MAT1-2.RESULTS:We have used a systems biology approach to identify genes restoring sexual reproduction in the QM6a strain line. To this end, T. reesei QM6a was crossed with the MAT1-1 wild-type strain CBS999.97. The descendants were backcrossed 8-times in two lineages with QM6a to obtain mating competent MAT1-1 strains with a minimal set of CBS999.97 specific genes. Comparative genome analysis identified a total of 73 genes of which two-encoding an unknown C2H2/ankyrin protein and a homolog of the WD-protein HAM5-were identified to be essential for fruiting body formation. The introduction of a functional ham5 allele in a mating type switched T. reesei QM6a allowed sexual crossing with the parental strain QM6a.CONCLUSION:The finding that Trichoderma reesei is generally capable of undergoing sexual reproduction even under laboratory conditions raised hope for the applicability of classical breeding techniques with this fungus as known for plants and certain yeasts. The discovery that the wild-type isolate QM6a was female sterile, however, precluded any progress along that line. With the discovery of the genetic cause of female sterility and the creation of an engineered fertile strain we now provide the basis to establish sexual crossing in this fungus and herald a new era of strain improvement in T. reesei.

The conidium plays a critical role in the life cycle of many filamentous fungi, being the primary means for survival under unfavorable conditions. To investigate the transcriptional changes taking place during the transition from growing hyphae to conidia in Trichoderma reesei, microarray experiments were performed. A total of 900 distinct genes were classified as differentially expressed, relative to their expression at time zero of conidiation, at least at one of the time points analyzed. The main functional categories (FunCat) overrepresented among the upregulated genes were those involving solute transport, metabolism, transcriptional regulation, secondary metabolite synthesis, lipases, proteases, and, particularly, cellulases and hemicellulases. Categories overrepresented among the downregulated genes were especially those associated with ribosomal and mitochondrial functions. The upregulation of cellulase and hemicellulase genes was dependent on the function of the positive transcriptional regulator XYR1, but XYR1 exerted no influence on conidiation itself. At least 20% of the significantly regulated genes were nonrandomly distributed within the T. reesei genome, suggesting an epigenetic component in the regulation of conidiation. The significant upregulation of cellulases and hemicellulases during this process, and thus cellulase and hemicellulase content in the spores of T. reesei, contributes to the hypothesis that the ability to hydrolyze plant biomass is a major trait of this fungus enabling it to break dormancy and reinitiate vegetative growth after a period of facing unfavorable conditions.

Metagenomics – the application of the genomics suit of technologies to uncultivated microorganisms – is coming of age. Sophisticated technologies are being developed and adapted to this promising genetic resource to make increasing use of the seemingly boundless molecular and functional diversity. Particular progress has been made in the areas of randomly proliferating limited‐source DNA, massively parallel sequencing without cloning, isolating specific target sequences from highly complex template mixtures, high‐throughput assay systems targeting metabolic pathways, artificial transcriptional regulators activating reporter genes to indicate enzymatic substrate conversion and cDNA cloning from extracted mRNA to directly clone actively expressed genes from a microbial consortium. However, challenges still lie ahead. Most prominently, the efficient heterologous expression of a plethora of potentially interesting enzymes from unknown source organisms is not readily achieved.