Identification and characterization of methoxy- and dimethoxyhydroquinone 1,2-dioxygenase from Phanerochaete chrysosporium.

White-rot fungi, such as Phanerochaete chrysosporium, are the most efficient degraders of lignin, a major component of plant biomass. Enzymes produced by these fungi, such as lignin peroxidases and manganese peroxidases, break down lignin polymers into various aromatic compounds based on guaiacyl, syringyl, and hydroxyphenyl units. These intermediates are further degraded, and the aromatic ring is cleaved by 1,2,4-trihydroxybenzene dioxygenases. This study aimed to characterize homogentisate dioxygenase (HGD)-like proteins from P. chrysosporium that are strongly induced by the G-unit fragment of vanillin. We overexpressed two homologous recombinant HGDs, PcHGD1 and PcHGD2, in Escherichia coli. Both PcHGD1 and PcHGD2 catalyzed the ring cleavage in methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ). The two enzymes had the highest catalytic efficiency (kcat/Km) for MHQ, and therefore, we named PcHGD1 and PcHGD2 as MHQ dioxygenases 1 and 2 (PcMHQD1 and PcMHQD2), respectively, from P. chrysosporium. This is the first study to identify and characterize MHQ and DMHQ dioxygenase activities in members of the HGD superfamily. These findings highlight the unique and broad substrate spectra of PcHGDs, rendering them attractive candidates for biotechnological applications.IMPORTANCEThis study aimed to elucidate the properties of enzymes responsible for degrading lignin, a dominant natural polymer in terrestrial lignocellulosic biomass. We focused on two homogentisate dioxygenase (HGD) homologs from the white-rot fungus, P. chrysosporium, and investigated their roles in the degradation of lignin-derived aromatic compounds. In the P. chrysosporium genome database, PcMHQD1 and PcMHQD2 were annotated as HGDs that could cleave the aromatic rings of methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ) with a preference for MHQ. These findings suggest that MHQD1 and/or MHQD2 play important roles in the degradation of lignin-derived aromatic compounds by P. chrysosporium. The preference of PcMHQDs for MHQ and DMHQ not only highlights their potential for biotechnological applications but also underscores their critical role in understanding lignin degradation by a representative of white-rot fungus, P. chrysosporium.

Three-Dimensional Printer-Assisted Electrospinning for Fabricating Intricate Biological Tissue Mimics.

Although regenerative medicine necessitates advanced three-dimensional (3D) scaffolds for organ and tissue applications, creating intricate structures across scales, from nano- to meso-like biological tissues, remains a challenge. Electrospinning of nanofibers offers promise due to its capacity to craft not only the dimensions and surfaces of individual fibers but also intricate attributes, such as anisotropy and porosity, across various materials. In this study, we used a 3D printer to design a mold with polylactic acid for gel modeling. This gel template, which was mounted on a metal wire, facilitated microfiber electrospinning. After spinning, these structures were treated with EDTA to remove the template and were then cleansed and dried, resulting in 3D microfibrous (3DMF) structures, with average fiber diameters of approximately 1 µm on the outer and inner surfaces. Notably, these structures matched their intended design dimensions without distortion or shrinkage, demonstrating the adaptability of this method for various template sizes. The cylindrical structures showed high elasticity and stretchability with an elastic modulus of 6.23 MPa. Furthermore, our method successfully mimicked complex biological tissue structures, such as the inner architecture of the voice box and the hollow partitioned structure of the heart's tricuspid valve. Achieving specific intricate shapes required multiple spinning sessions and subsequent assemblies. In essence, our approach holds potential for crafting artificial organs and forming the foundational materials for cell culture scaffolds, addressing the challenges of crafting intricate multiscale structures.

In Vitro Synthesis and Design of Kinesin Biomolecular Motors by Cell-Free Protein Synthesis.

Kinesin is a biomolecular motor that generates force and motility along microtubule cytoskeletons in cells. Owing to their ability to manipulate cellular nanoscale components, microtubule/kinesin systems show great promise as actuators of nanodevices. However, classical in vivo protein production has some limitations for the design and production of kinesins. Designing and producing kinesins is laborious, and conventional protein production requires specific facilities to create and contain recombinant organisms. Here, we demonstrated the in vitro synthesis and editing of functional kinesins using a wheat germ cell-free protein synthesis system. The synthesized kinesins propelled microtubules on a kinesin-coated substrate and showed a higher binding affinity with microtubules than E. coli-produced kinesins. We also successfully incorporated affinity tags into the kinesins by extending the original sequence of the DNA template by PCR. Our method will accelerate the study of biomolecular motor systems and encourage their wider use in various nanotechnology applications.

Mannose- and Mannobiose-Specific Responses of the Insect-Associated Cellulolytic Bacterium Streptomyces sp. Strain SirexAA-E.

The cellulolytic insect symbiont bacterium Streptomyces sp. strain SirexAA-E secretes a suite of carbohydrate-active enzymes (CAZymes), which are involved in the degradation of various polysaccharides in the plant cell wall, in response to the available carbon sources. Here, we examined a poorly understood response of this bacterium to mannan, one of the major plant cell wall components. SirexAA-E grew well on mannose, carboxymethyl cellulose (CMC), and locust bean gum (LBG) as sole carbon sources in the culture medium. The secreted proteins from each culture supernatant were tested for their polysaccharide-degrading ability, and the composition of secreted CAZymes in each sample was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that mannose, LBG, and CMC induced the secretion of mannan and cellulose-degrading enzymes. Interestingly, two α-1,2-mannosidases were abundantly secreted during growth on mannose and LBG. Using genomic analysis, we found a unique 12-bp palindromic sequence motif at 4 locations in the SirexAA-E genome, two of which were found upstream of the above-mentioned α-1,2-mannosidase genes, along with a newly identified mannose and mannobiose-responsive transcriptional regulator, SsManR. Furthermore, the previously reported cellobiose-responsive repressor, SsCebR, was determined to also use mannobiose as an effector ligand. To test whether mannobiose induces the sets of genes under the control of the two regulators, SirexAA-E was grown on mannobiose, and the secretome composition was analyzed. As hypothesized, the composition of the mannobiose secretome combined sets of CAZymes found in both LBG and CMC secretomes, and thus they are likely under the regulation of both SsManR and SsCebR. IMPORTANCEStreptomyces sp. SirexAA-E, a microbial symbiont of biomass-harvesting insects, secretes a suite of polysaccharide-degrading enzymes dependent on the available carbon sources. However, the response of this bacterium to mannan has not been documented. In this study, we investigated the response of this bacterium to mannose, mannobiose, and galactomannan (LBG). By combining biochemical, proteomic, and genomic approaches, we discovered a novel mannose and mannobiose responsive transcriptional regulator, SsManR, which selectively regulates three α-1,2-mannosidase-coding genes. We also demonstrated that the previously described cellobiose responsive regulator, SsCebR, could use mannobiose as an effector ligand. Overall, our findings suggest that the Streptomyces sp. SirexAA-E responds to mannose and mannooligosaccharides through two different transcriptional repressors that regulate the secretion of the plant cell wall-degrading enzymes to extract carbon sources in the host environment.

AtTrxh3, a Thioredoxin, Is Identified as an Abscisic AcidBinding Protein in Arabidopsis thaliana.

Abscisic acid (ABA, 1) is a plant hormone that regulates various plant physiological processes such as seed developing and stress responses. The ABA signaling system has been elucidated; binding of ABA with PYL proteins triggers ABA signaling. We have previously reported a new method to isolate a protein targeted with a bioactive small molecule using a biotin linker with alkyne and amino groups, a protein cross-linker, and a bioactive small molecule with an azido group (azido probe). This method was used to identify the unknown ABA binding protein of Arabidopsis thaliana. As a result, AtTrxh3, a thioredoxin, was isolated as an ABA binding protein. Our developed method can be applied to the identification of binding proteins of bioactive compounds.

Identification of recombinant AtPYL2, an abscisic acid receptor, in E. coli using a substrate-derived bioactive small molecule, a biotin linker with alkyne and amino groups, and a protein cross-linker.

Target protein identification of bioactive small molecules is one of the most important research in forward chemical genetics. The affinity chromatography technique to use a resin bound with a small molecule is often used for identification of a target protein of a bioactive small molecule. Here we report a new method to isolate a protein targeted with a bioactive small molecule using a biotin linker with alkyne and amino groups, protein cross-linker containing disulfide bond, and a bioactive small molecule with an azido group (azido probe). After an azido probe is associated with a target protein, the complex of a target protein and azido probe is covalently bound through the biotin linker by azide-alkyne Huisgen cycloaddition and protein cross-linker containing disulfide bond. This ternary complex is immobilized on an affinity matrix with streptavidin, and then the target protein is selectively eluted with a buffer containing a reducing agent for cleavage of disulfide bonds. This method uses a probe having an azido group, which a small functional group, and has the possibility to be a solution strategy to overcome the hindrance of a functional group introduced into the probe that reduces association a target protein. The effectiveness of the method in this study was shown using linker 1, 3'-azidoabscisic acid 3, and protein cross-linker containing a disulfide bond (DTSSP 5).

Antarctic heterotrophic bacterium Hymenobacter nivis P3T displays light-enhanced growth and expresses putative photoactive proteins.

Hymenobacter nivis P3T is a heterotrophic bacterium isolated from Antarctic red snow generated by algal blooms. Despite being non-photosynthetic, H. nivis was dominantly found in the red snow environment that is exposed to high light and UV irradiation, suggesting that this species can flourish under such harsh conditions. In order to further understand the adaptive strategies on the snow surface environment of Antarctica, the genome of H. nivis P3T was sequenced and analyzed, which identified genes putatively encoding for light-reactive proteins such as proteorhodopsin, phytochrome, photolyase and several copies of cryptochromes. Culture-based experiments revealed that H. nivis P3T growth was significantly enhanced under light conditions, while dark conditions had increased extracellular polymeric substances. Furthermore, the expression of several putative light-reactive proteins was determined by proteomic analysis. These results indicate that H. nivis P3T is able to potentially utilize light, which may explain its dominance on the red snow surface environment of Antarctica. ORIGINALITY-SIGNIFICANCE STATEMENT: The role of proteorhodopsin in heterotrophic bacteria is not well-characterized, as only a handful of proteorhodopsin-harbouring isolates were shown to have a light-enhanced phenotype through culture-based experiments to date. This is the first study that demonstrates light-stimulated growth and protein expression evidence of photoactive proteins for a non-marine psychrophile and for a member of the genus Hymenobacter. It is also the first study that provides comprehensive proteome information for this genus. This study presents significant results in understanding the adaptive mechanism of a heterotrophic non-photosynthetic bacterium thriving on the snow surface environment of Antarctica as well as demonstrating the role of light-utilization in promoting growth, possibly through proteorhodopsin.

Smooth muscle-like Ca2+-regulation of actin-myosin interaction in adult jellyfish striated muscle.

Cnidaria is an animal phylum, whose members probably have the most ancestral musculature. We prepared and characterized, for the first time to our knowledge, native actomyosin from the striated myoepithelium of the adult moon jelly Aurelia sp. The actomyosin contained myosin, paramyosin-like protein, Ser/Thr-kinase, actin, and two isoforms of tropomyosin, but not troponin, which is known to activate contraction dependent on intracellular Ca2+ signaling in almost all striated muscles of bilaterians. Notably, the myosin comprised striated muscle-type heavy chain and smooth muscle-type regulatory light chains. In the presence of Ca2+, the Mg-ATPase activity of actomyosin was stimulated and Ser21 of the regulatory light chain was concomitantly phosphorylated by the addition of calmodulin and myosin light chain kinase prepared from chicken smooth muscle. Collectively, these results suggest that, similar to smooth muscle, the contraction of jellyfish striated muscle is regulated by Ca2+-dependent phosphorylation of the myosin light chain.

Universal reconnection of non-Abelian cosmic strings.

We show that local and semilocal strings in Abelian and non-Abelian gauge theories with critical couplings always reconnect classically in collision, by using moduli space approximation. The moduli matrix formalism explicitly identifies a well-defined set of the vortex moduli parameters. Our analysis of generic geodesic motion in terms of those shows right-angle scattering in head-on collision of two vortices, which is known to give the reconnection of the strings.

Moduli space of non-abelian vortices.

We completely determine the moduli space M(N,k) of k vortices in U(N) gauge theory with N Higgs fields in the fundamental representation. Its open subset for separated vortices is found as the symmetric product (CxCP(N-1))k/(see text)k. Orbifold singularities of this space correspond to coincident vortices and are resolved resulting in a smooth moduli manifold. The relation to Kähler quotient construction is discussed.

Skyrmions from instantons inside domain walls.

Some years ago, Atiyah and Manton described a method to construct approximate Skyrmion solutions from Yang-Mills instantons. Here we present a dynamical realization of this construction using domain walls in a five-dimensional gauge theory. The non-Abelian gauge symmetry is broken in each vacuum but restored in the core of the domain wall, allowing instantons to nestle inside the wall. We show that the world volume dynamics of the wall is given by the Skyrme model, including the four-derivative term, and the instantons appear as domain wall Skyrmions.

Construction of non-Abelian walls and their complete moduli space.

We present a systematic method to construct exactly all Bogomol'nyi-Prasad-Sommerfield multiwall solutions in supersymmetric U(N(C)) gauge theories in five dimensions with N(F) hypermultiplets in the fundamental representation for infinite gauge coupling. The moduli space of these non-Abelian walls is found to be the complex Grassmann manifold SU(N(F)) divided by SU(N(C))xSU(N(F)-N(C))xU(1) endowed with a deformed metric.