Advancing Biomimetic Functions of Synthetic Cells through Compartmentalized Cell-Free Protein Synthesis.

Synthetic cells are artificial constructs that mimic the structures and functions of living cells. They are attractive for studying diverse biochemical processes and elucidating the origins of life. While creating a living synthetic cell remains a grand challenge, researchers have successfully synthesized hundreds of unique synthetic cell platforms. One promising approach to developing more sophisticated synthetic cells is to integrate cell-free protein synthesis (CFPS) mechanisms into vesicle platforms. This makes it possible to create synthetic cells with complex biomimetic functions such as genetic circuits, autonomous membrane modifications, sensing and communication, and artificial organelles. This Review explores recent advances in the use of CFPS to impart advanced biomimetic structures and functions to bottom-up synthetic cell platforms. We also discuss the potential applications of synthetic cells in biomedicine as well as the future directions of synthetic cell research.

Temperature-responsive membrane permeability of recombinant fusion protein vesicles.

In this study, we investigate the changes in the permeability of the recombinant fusion protein vesicles with different membrane structures as a function of solution temperature. The protein vesicles are self-assembled from recombinant fusion protein complexes composed of an mCherry fused with a glutamic acid-rich leucine zipper and a counter arginine-rich leucine zipper fused with an elastin-like polypeptide (ELP). We have found that the molecular weight cut-off (MWCO) of the protein vesicle membranes varies inversely with solution temperature by monitoring the transport of fluorescent-tagged dextran dyes with different molecular weights. The temperature-responsiveness of the protein vesicle membranes is obtained from the lower critical solution temperature behavior of ELP in the protein building blocks. Consequently, the unique vesicle membrane structures with different single-layered and double-layered ELP organizations impact the sensitivity of the permeability responses of the protein vesicles. Single-layered protein vesicles with the ELP domains facing the interior show more drastic permeability changes as a function of temperature than double-layered protein vesicles in which ELP blocks are buried inside the membranes. This work about the temperature-responsive membrane permeability of unique protein vesicles will provide design guidelines for new biomaterials and their applications, such as drug delivery and synthetic protocell development.

Heterogeneous Synthetic Vesicles toward Artificial Cells: Engineering Structure and Composition of Membranes for Multimodal Functionalities.

The desire to develop artificial cells to imitate living cells in synthetic vesicle platforms has continuously increased over the past few decades. In particular, heterogeneous synthetic vesicles made from two or more building blocks have attracted attention for artificial cell applications based on their multifunctional modules with asymmetric structures. In addition to the traditional liposomes or polymersomes, polypeptides and proteins have recently been highlighted as potential building blocks to construct artificial cells owing to their specific biological functionalities. Incorporating one or more functionally folded, globular protein into synthetic vesicles enables more cell-like functions mediated by proteins. This Review highlights the recent research about synthetic vesicles toward artificial cell models, from traditional synthetic vesicles to protein-assembled vesicles with asymmetric structures. We aim to provide fundamental and practical insights into applying knowledge on molecular self-assembly to the bottom-up construction of artificial cell platforms with heterogeneous building blocks.

Tuning the Structural Integrity and Mechanical Properties of Globular Protein Vesicles by Blending Crosslinkable and NonCrosslinkable Building Blocks.

Vesicles made from functionally folded, globular proteins that perform specific biological activities, such as catalysis, sensing, or therapeutics, show potential applications as artificial cells, microbioreactors, or protein drug delivery vehicles. The mechanical properties of vesicle membranes, including the elastic modulus and hardness, play a critical role in dictating the stability and shape transformation of the vesicles under external stimuli triggers. Herein, we have developed a strategy to tune the mechanical properties and integrity of globular protein vesicle (GPV) membranes of which building molecules are recombinant fusion protein complexes: a mCherry fused with an acidic leucine zipper (mCherry-ZE) and a basic leucine zipper fused with an elastin-like polypeptide (ZR-ELP). To control the mechanical properties of GPVs, we introduced a nonstandard amino acid (para-azidophenylalanine (pAzF)) into the ELP domains (ELP-X), which enabled the creation of crosslinked vesicles under ultraviolet (UV) irradiation. Crosslinked GPVs made from mCherry-ZE/ZR-ELP-X complexes presented higher stability than noncrosslinked GPVs under hypotonic osmotic stress. The degree of swelling of GPVs increased as less crosslinking was achieved in the vesicle membranes, which resulted in the disassembly of GPVs into membraneless coacervates. Nanoindentation by atomic force microscopy (AFM) confirmed that the stiffness and Young's elastic modulus of GPVs increase as the blending molar ratio of ZR-ELP-X to ZR-ELP increases to make vesicles. The results obtained in this study suggest a rational design to make GPVs with tunable mechanical properties for target applications by simply varying the blending ratio of ZR-ELP and ZR-ELP-X in the vesicle self-assembly.

Understanding the Coacervate-to-Vesicle Transition of Globular Fusion Proteins to Engineer Protein Vesicle Size and Membrane Heterogeneity.

Protein-rich coacervates are liquid phases separate from the aqueous bulk phase that are used by nature for compartmentalization and more recently have been exploited by engineers for delivery and formulation applications. They also serve as an intermediate phase in an assembly path to more complex structures, such as vesicles. Recombinant fusion protein complexes made from a globular protein fused with a glutamic acid-rich leucine zipper (globule-ZE) and an arginine-rich leucine zipper fused with an elastin-like polypeptide (ZR-ELP) show different phases from soluble, through an intermediate coacervate phase, and finally to vesicles with increasing temperature of the aqueous solution. We investigated the phase transition kinetics of the fusion protein complexes at different temperatures using dynamic light scattering and microscopy, along with mathematical modeling. We controlled coacervate growth by aging the solution at an intermediate temperature that supports coacervation and confirmed that the size of the coacervate droplets dictates the size of vesicles formed upon further heating. With this understanding of the phase transition, we developed strategies to induce heterogeneity in the organization of globular proteins in the vesicle membrane through simple mixing of coacervates containing two different globular fusion proteins prior to the vesicle transition. This study gives fundamental insights and practical strategies for development of globular protein-rich coacervates and vesicles for drug delivery, microreactors, and protocell applications.

Xylodon flaviporus-Derived Drimane Sesquiterpenoids That Inhibit Osteoclast Differentiation.

The presence of excessive osteoclasts is a major factor in skeletal diseases. The present study aimed to discover osteoclast differentiation inhibitors from the basidiomycete Xylodon flaviporus. Seven new drimane sesquiterpenoids (1-7) and 7-ketoisodrimenin-5-ene (8) were obtained and characterized by various spectroscopic methods. The isolated compounds were evaluated for their inhibitory effects against receptor activator of nuclear factor-kappa-B ligand-induced osteoclastogenesis in mouse bone marrow macrophages. Compounds 1, 3, and 6 showed potent activities with IC50 values of 1.6, 0.9, and 2.1 µM, respectively, while 4, 5, and 7 exhibited relatively weak activities with IC50 values of 10.7, 10.1, and 8.5 µM, respectively.

Comprehensive genomic and transcriptomic analysis of polycyclic aromatic hydrocarbon degradation by a mycoremediation fungus, Dentipellis sp. KUC8613.

The environmental accumulation of polycyclic aromatic hydrocarbons (PAHs) is of great concern due to potential carcinogenic and mutagenic risks, as well as their resistance to remediation. While many fungi have been reported to break down PAHs in environments, the details of gene-based metabolic pathways are not yet comprehensively understood. Specifically, the genome-scale transcriptional responses of fungal PAH degradation have rarely been reported. In this study, we report the genomic and transcriptomic basis of PAH bioremediation by a potent fungal degrader, Dentipellis sp. KUC8613. The genome size of this fungus was 36.71 Mbp long encoding 14,320 putative protein-coding genes. The strain efficiently removed more than 90% of 100 mg/l concentration of PAHs within 10 days. The genomic and transcriptomic analysis of this white rot fungus highlights that the strain primarily utilized non-ligninolytic enzymes to remove various PAHs, rather than typical ligninolytic enzymes known for playing important roles in PAH degradation. PAH removal by non-ligninolytic enzymes was initiated by both different PAH-specific and common upregulation of P450s, followed by downstream PAH-transforming enzymes such as epoxide hydrolases, dehydrogenases, FAD-dependent monooxygenases, dioxygenases, and glycosyl- or glutathione transferases. Among the various PAHs, phenanthrene induced a more dynamic transcriptomic response possibly due to its greater cytotoxicity, leading to highly upregulated genes involved in the translocation of PAHs, a defense system against reactive oxygen species, and ATP synthesis. Our genomic and transcriptomic data provide a foundation of understanding regarding the mycoremediation of PAHs and the application of this strain for polluted environments.

Nature-Inspired and "Water-Skating" Paper and Polyester Substrates Enabled by the Molecular Structure of Poly(γ-stearyl-α,l-glutamate) Homopolypeptide.

We demonstrate that the molecular structure of a synthetic homopolypeptide that resembles the leg architecture of water strider insects is effective to impart flexible polymeric surfaces with superhydrophobic behavior. Filter paper (FP) and polyester (PET) were modified with a coating consisting of low-molecular weight α-helical poly(γ-stearyl-α,l-glutamate) (PSLG, Mw = 4500 Da) homopolypeptide. PSLG-coated substrates displayed near to and superhydrophobic behavior (≥150°) as reflected by the contact angle values. Despite being physically adsorbed, the PSLG coating uniformly covered and was strongly adhered to the substrate surfaces. The thin coating layer displayed remarkable mechanical abrasion resistance and was insensitive to long-time exposure to ambient conditions. PLSG-coated textile fibers exhibited useful and interesting properties. Under an iron-containing load, PSLG-coated PET was able to float and "walk" on water when exposed to a magnet. The PSLG coating was able to reduce the adhesion of Escherichia coli, model Gram-negative bacteria. The results indicated that the molecular geometry of PSLG homopolypeptide, which possesses a α-helical backbone sprouting out of highly hydrophobic stearyl side chains, was the key feature responsible for the observed behaviors. This study is relevant for a broad range of potential applications: from crop and drinking water management in arid geographic areas to biomedical devices and implants.

Cooperative Catechol-Functionalized Polypept(o)ide Brushes and Ag Nanoparticles for Combination of Protein Resistance and Antimicrobial Activity on Metal Oxide Surfaces.

Prevention of biofouling and microbial contamination of implanted biomedical devices is essential to maintain their functionality and biocompatibility. For this purpose, polypept(o)ide block copolymers have been developed, in which a protein-resistant polysarcosine (pSar) block is combined with a dopamine-modified poly(glutamic acid) block for surface coating and silver nanoparticles (Ag NPs) formation. In the development of a novel, versatile, and biocompatible antibacterial surface coating, block lengths pSar were varied to derive structure-property relationships. Notably, the catechol moiety performs two important tasks in parallel; primarily it acts as an efficient anchoring group to metal oxide surfaces, while it furthermore induces the formation of Ag NPs. Attributing to the dual function of catechol moieties, antifouling pSar brush and antimicrobial Ag NPs can not only adhere stably on metal oxide surfaces, but also display passive antifouling and active antimicrobial activity, showing good biocompatibility simultaneously. The developed strategy seems to provide a promising platform for functional modification of biomaterials surface to preserve their performance while reducing the risk of bacterial infections.

Engineering Globular Protein Vesicles through Tunable Self-Assembly of Recombinant Fusion Proteins.

Vesicles assembled from folded, globular proteins have potential for functions different from traditional lipid or polymeric vesicles. However, they also present challenges in understanding the assembly process and controlling vesicle properties. From detailed investigation of the assembly behavior of recombinant fusion proteins, this work reports a simple strategy to engineer protein vesicles containing functional, globular domains. This is achieved through tunable self-assembly of recombinant globular fusion proteins containing leucine zippers and elastin-like polypeptides. The fusion proteins form complexes in solution via high affinity binding of the zippers, and transition through dynamic coacervates to stable hollow vesicles upon warming. The thermal driving force, which can be tuned by protein concentration or temperature, controls both vesicle size and whether vesicles are single or bi-layered. These results provide critical information to engineer globular protein vesicles via self-assembly with desired size and membrane structure.

Self-Assembled Materials Made from Functional Recombinant Proteins.

Proteins are potent molecules that can be used as therapeutics, sensors, and biocatalysts with many advantages over small-molecule counterparts due to the specificity of their activity based on their amino acid sequence and folded three-dimensional structure. However, they also have significant limitations in their stability, localization, and recovery when used in soluble form. These opportunities and challenges have motivated the creation of materials from such functional proteins in order to protect and present them in a way that enhances their function. We have designed functional recombinant fusion proteins capable of self-assembling into materials with unique structures that maintain or improve the functionality of the protein. Fusion of either a functional protein or an assembly domain to a leucine zipper domain makes the materials design strategy modular, based on the high affinity between leucine zippers. The self-assembly domains, including elastin-like polypeptides (ELPs) and defined-sequence random coil polypeptides, can be fused with a leucine zipper motif in order to promote assembly of the fusion proteins into larger structures upon specific stimuli such as temperature and ionic strength. Fusion of other functional domains with the counterpart leucine zipper motif endows the self-assembled materials with protein-specific functions such as fluorescence or catalytic activity. In this Account, we describe several examples of materials assembled from functional fusion proteins as well as the structural characterization, functionality, and understanding of the assembly mechanism. The first example is zipper fusion proteins containing ELPs that assemble into particles when introduced to a model extracellular matrix and subsequently disassemble over time to release the functional protein for drug delivery applications. Under different conditions, the same fusion proteins can self-assemble into hollow vesicles. The vesicles display a functional protein on the surface and can also carry protein, small-molecule, or nanoparticle cargo in the vesicle lumen. To create a material with a more complex hierarchical structure, we combined calcium phosphate with zipper fusion proteins containing random coil polypeptides to produce hybrid protein-inorganic supraparticles with high surface area and porous structure. The use of a functional enzyme created supraparticles with the ability to degrade inflammatory cytokines. Our characterization of these protein materials revealed that the molecular interactions are complex because of the large size of the protein building blocks, their folded structures, and the number of potential interactions including hydrophobic interactions, electrostatic interactions, van der Waals forces, and specific affinity-based interactions. It is difficult or even impossible to predict the structures a priori. However, once the basic assembly principles are understood, there is opportunity to tune the material properties, such as size, through control of the self-assembly conditions. Our future efforts on the fundamental side will focus on identifying the phase space of self-assembly of these fusion proteins and additional experimental levers with which to control and tune the resulting materials. On the application side, we are investigating an array of different functional proteins to expand the use of these structures in both therapeutic protein delivery and biocatalysis.

Comparison of the Diversity of Basidiomycetes from Dead Wood of the Manchurian fir (Abies holophylla) as Evaluated by Fruiting Body Collection, Mycelial Isolation, and 454 Sequencing.

In this study, three different methods (fruiting body collection, mycelial isolation, and 454 sequencing) were implemented to determine the diversity of wood-inhabiting basidiomycetes from dead Manchurian fir (Abies holophylla). The three methods recovered similar species richness (26 species from fruiting bodies, 32 species from mycelia, and 32 species from 454 sequencing), but Fisher's alpha, Shannon-Wiener, Simpson's diversity indices of fungal communities indicated fruiting body collection and mycelial isolation displayed higher diversity compared with 454 sequencing. In total, 75 wood-inhabiting basidiomycetes were detected. The most frequently observed species were Heterobasidion orientale (fruiting body collection), Bjerkandera adusta (mycelial isolation), and Trichaptum fusco-violaceum (454 sequencing). Only two species, Hymenochaete yasudae and Hypochnicium karstenii, were detected by all three methods. This result indicated that Manchurian fir harbors a diverse basidiomycetous fungal community and for complete estimation of fungal diversity, multiple methods should be used. Further studies are required to understand their ecology in the context of forest ecosystems.

Enhanced removal of PAHs by Peniophora incarnata and ascertainment of its novel ligninolytic enzyme genes.

The hazardous effects of the PAHs should be managed by removal using white rot fungal ligninolytic enzymes. The white rot fungus Peniophora incarnataKUC8836 was stimulated to produce ligninolytic enzymes in a liquid medium by the addition of four substances: 0.5 g L(-1) Tween 80, 70 mg L(-1) CuSO4·5H2O, 10 mg L(-1) MnSO4·H2O, and 0.3 g L(-1) veratryl alcohol. The experiments were carried out in two different media: basal salt and 2% malt extract (ME) liquid medium. Under the experimental conditions, both laccase and manganese-dependent peroxidase (MnP) demonstrated with the highest activities in 2% ME liquid medium following the addition of Tween 80. The biodegradation of anthracene and pyrene was significantly enhanced by the induced ligninolytic enzymes when Tween 80 was added. Tween 80 is a viable co-substrate for P. incarnata, as it enhances the ability of P. incarnata to manage effective biodegradation of PAHs. Most of all, the novel laccase and MnP genes ascertained in this study, showed that the genes were involved in the production of ligninolytic enzymes from P. incarnataKUC8836.

Novel Sarcoscypha Species from National Parks in Korea: Sarcoscypha humida sp. nov.

Sarcoscypha (Sarcoscyphaceae, Pezizales) is a saprobic fungus characterized by the cup or disc-shaped blight red apothecium and oblong to ellipsoid ascospores. The 18 species of Sarcoscypha were known to occur in Europe, North America, and tropical Asia. However, up to date, only two Sarcoscypha species have been reported in Korea. In this study, novel Sarcoscypha specimens were collected from Juwangsan, Odaesan, and Taebaeksan National Parks from September to October in Korea. This species is well distinguished from other Sarcoscypha species according to the molecular and phylogenetic analysis based on internal transcribed spacer (ITS) region. Here, we provided detailed descriptions with illustrations and a phylogenetic tree to report our specimens as novel Sarcoscypha species.

Cloning and Expression Analysis of Bioluminescence Genes in Omphalotus guepiniiformis Reveal Stress-Dependent Regulation of Bioluminescence.

Bioluminescence is a type of chemiluminescence that arises from a luciferase-catalyzed oxidation reaction of luciferin. Molecular biology and comparative genomics have recently elucidated the genes involved in fungal bioluminescence and the evolutionary history of their clusters. However, most studies on fungal bioluminescence have been limited to observing the changes in light intensity under various conditions. To understand the molecular basis of bioluminescent responses in Omphalotus guepiniiformis under different environmental conditions, we cloned and sequenced the genes of hispidin synthase, hispidin-3-hydroxylase, and luciferase enzymes, which are pivotal in the fungal bioluminescence pathway. Each gene showed high sequence similarity to that of other luminous fungal species. Furthermore, we investigated their transcriptional changes in response to abiotic stresses. Wound stress enhanced the bioluminescence intensity by increasing the expression of bioluminescence pathway genes, while temperature stress suppressed the bioluminescence intensity via the non-transcriptional pathway. Our data suggested that O. guepiniiformis regulates bioluminescence to respond differentially to specific environmental stresses. To our knowledge, this is the first study on fungal bioluminescence at the gene expression level. Further studies are required to address the biological and ecological meaning of different bioluminescence responses in changing environments, and O. quepiniiformis could be a potential model species.

White-rot fungus Merulius tremellosus KUC9161 identified as an effective degrader of polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) have a highly recalcitrant structure; however, they can be degraded by white-rot fungi which have the potential to biodegrade recalcitrant organic compounds. Four fungal isolates were selected from 23 newly isolated basidiomycetes, based on their dye decolorization rate, and they were evaluated for their ability to degrade 50 ppm of pyrene. The isolate phylogenetically affiliated to Merulius tremellosus KUC9161 demonstrated the highest degradation rate of pyrene, regardless of the production of ligninolytic enzyme activities. The selected isolates were tested for their ability to degrade pyrene and other PAHs in creosote-contaminated soil. The results of the degradation tests indicated that M. tremellosus KUC9161 degraded a larger variety of PAH compounds than Phanerochaete chrysosporium, a known PAH degrader. On the basis of our results, the isolate M. tremellosus KUC9161 has a high potential to be used in the large-scale biodegradation of PAHs, and the species may also be used to degrade recalcitrant materials in creosote-contaminated soil.

Rational design and engineering of polypeptide/protein vesicles for advanced biological applications.

Synthetic vesicles have gained considerable popularity in recent years for numerous biological and medical applications. Among the various types of synthetic vesicles, the utilization of polypeptides and/or proteins as fundamental constituents has garnered significant interest for vesicle construction owing to the unique bio-functionalities inherent in rationally designed amino acid sequences. Especially the incorporation of functional proteins onto the vesicle surface facilitates a wide range of advanced biological applications that are not easily attainable with traditional building blocks, such as lipids and polymers. The main goal of this review is to provide a comprehensive overview of the latest advancements in polypeptide/protein vesicles. Moreover, this review encompasses the rational design and engineering strategies employed in the creation of polypeptide/protein vesicles, including the synthesis of building blocks, the modulation of their self-assembly, as well as their diverse applications. Furthermore, this work includes an in-depth discussion of the key challenges and opportunities associated with polypeptide/protein vesicles, providing valuable insights for future research. By offering an up-to-date review of this burgeoning field of polypeptide/protein vesicle research, this review will shed light on the potential applications of these biomaterials.

Advances in cell coculture membranes recapitulating in vivo microenvironments.

Porous membranes play a critical role in in vitro heterogeneous cell coculture systems because they recapitulate the in vivo microenvironment to mediate physical and biochemical crosstalk between cells. While the conventionally available Transwell® system has been widely used for heterogeneous cell coculture, there are drawbacks to precise control over cell-cell interactions and separation for implantation. The size and numbers of the pores and the thickness of the porous membranes are crucial in determining the efficiency of paracrine signaling and direct junctions between cocultured cells, and significantly impact on the performance of heterogeneous cell cultures. These opportunities and challenges have motivated the design of advanced coculture platforms through improvement of the structural and functional properties of porous membranes.

Determination and Analysis of Hyper-Variable A Mating Types in Wild Strains of Lentinula edodes in Korea.

The diversity of A mating type in wild strains of Lentinula edodes was extensively analyzed to characterize and utilize them for developing new cultivars. One hundred twenty-three A mating type alleles, including 67 newly discovered alleles, were identified from 106 wild strains collected for the past four decades in Korea. Based on previous studies and current findings, a total of 130 A mating type alleles have been found, 124 of which were discovered from wild strains, indicating the hyper-variability of A mating type alleles of L. edodes. About half of the A mating type alleles in wild strains were found in more than two strains, whereas the other half of the alleles were found in only one strain. About 90% of A mating type combinations in dikaryotic wild strains showed a single occurrence. Geographically, diverse A mating type alleles were intensively located in the central region of the Korean peninsula, whereas only allele A17 was observed throughout Korea. We also found the conservation of the TCCCAC motif in addition to the previously reported motifs, including ATTGT, ACAAT, and GCGGAG, in the intergenic regions of A mating loci. Sequence comparison among some alleles indicated that accumulated mutation and recombination would contribute to the diversification of A mating type alleles in L. edodes. Our data support the rapid evolution of A mating locus in L. edodes, and would help to understand the characteristics of A mating loci of wild strains in Korea and help to utilize them for developing new cultivars.

A novel combination therapy for multidrug resistant pathogens using chitosan nanoparticles loaded with ß-lactam antibiotics and ß-lactamase inhibitors.

Antimicrobial resistance is one of the greatest global threats. Particularly, multidrug resistant extended-spectrum ß-lactamase (ESBL)-producing pathogens confer resistance to many commonly used medically important antibiotics, especially beta-lactam antibiotics. Here, we developed an innovative combination approach to therapy for multidrug resistant pathogens by encapsulating cephalosporin antibiotics and ß-lactamase inhibitors with chitosan nanoparticles (CNAIs). The four combinations of CNAIs including two cephalosporin antibiotics (cefotaxime and ceftiofur) with two ß-lactamase inhibitors (tazobactam and clavulanate) were engineered as water-oil-water emulsions. Four combinations of CNAIs showed efficient antimicrobial activity against multidrug resistant ESBL-producing Enterobacteriaceae. The CNAIs showed enhanced antimicrobial activity compared to naïve chitosan nanoparticles and to the combination of cephalosporin antibiotics and ß-lactamase inhibitors. Furthermore, CNAIs attached on the bacterial surface changed the permeability to the outer membrane, resulting in cell damage that leads to cell death. Taken together, CNAIs have provided promising potential for treatment of diseases caused by critically important ESBL-producing multidrug resistant pathogens.