Towards Synthetic Cells with Self-Producing Energy.

Autonomous generation of energy, specifically adenosine triphosphate (ATP), is critical for sustaining the engineered functionalities of synthetic cells constructed from the bottom-up. In this mini-review, we categorize studies on ATP-producing synthetic cells into three different approaches: photosynthetic mechanisms, mitochondrial respiration mimicry, and utilization of non-conventional approaches such as exploiting synthetic metabolic pathways. Within this framework, we evaluate the strengths and limitations of each approach and provide directions for future research endeavors. We also introduce a concept of building ATP-generating synthetic organelle that will enable us to mimic cellular respiration in a simpler way than current strategies. This review aims to highlight the importance of energy self-production in synthetic cells, providing suggestions and ideas that may help overcome some longstanding challenges in this field.

Light-based juxtacrine signaling between synthetic cells.

Cell signaling through direct physical cell-cell contacts plays vital roles in biology during development, angiogenesis, and immune response. Intercellular communication mechanisms between synthetic cells constructed from the bottom up are majorly reliant on diffusible chemical signals, thus limiting the range of responses in receiver cells. Engineering contact-dependent signaling between synthetic cells promises to unlock more complicated signaling schemes with different types of responses. Here, we design and demonstrate a light-activated contact-dependent communication tool for synthetic cells. We utilize a split bioluminescent protein to limit signal generation exclusively to contact interfaces of synthetic cells, driving the recruitment of a photoswitchable protein in receiver cells, akin to juxtacrine signaling in living cells. Our modular design not only demonstrates contact-dependent communication between synthetic cells but also provides a platform for engineering orthogonal contact-dependent signaling mechanisms.

Development of mechanosensitive synthetic cells for biomedical applications.

The ability of cells to sense and respond to their physical environment plays a fundamental role in a broad spectrum of biological processes. As one of the most essential molecular force sensors and transducers found in cell membranes, mechanosensitive (MS) ion channels can convert mechanical inputs into biochemical or electrical signals to mediate a variety of sensations. The bottom-up construction of cell-sized compartments displaying cell-like organization, behaviors, and complexity, also known as synthetic cells, has gained popularity as an experimental platform to characterize biological functions in isolation. By reconstituting MS channels in the synthetic lipid bilayers, we envision using mechanosensitive synthetic cells for several medical applications. Here, we describe three different concepts for using ultrasound, shear stress, and compressive stress as mechanical stimuli to activate drug release from mechanosensitive synthetic cells for disease treatments.

Economically viable co-production of methanol and sulfuric acid via direct methane oxidation.

The direct oxidation of methane to methanol has been spotlighted research for decades, but has never been commercialized. This study introduces cost-effective process for co-producing methanol and sulfuric acid through a direct oxidation of methane. In the initial phase, methane oxidation forms methyl bisulfate (CH3OSO3H), then transformed into methyl trifluoroacetate (CF3CO2CH3) via esterification, and hydrolyzed into methanol. This approach eliminates the need for energy-intensive separation of methyl bisulfate from sulfuric acid by replacing the former with methyl trifluoroacetate. Through the superstructure optimization, our sequential process reduces the levelized cost of methanol to nearly two-fold reduction from the current market price. Importantly, this process demonstrates adaptability to smaller gas fields, assuring its economical operation across a broad range of gas fields. The broader application of this process could substantially mitigate global warming by utilizing methane, leading to a significantly more sustainable and economically beneficial methanol industry.

Optimized Substrate Orientations for Highly Uniform Metal Halide Perovskite Film Deposition.

Uniform optoelectronic quality of metal halide perovskite (MHP) films is critical for scalable production in large-area applications, such as photovoltaics and displays. While vapor-based MHP film deposition is advantageous for this purpose, achieving film uniformity can be challenging due to uneven temperature distribution and precursor concentration over the substrate. Here, we propose optimized substrate orientations for the vapor-based fabrication of homogeneous MAPbI3 thin films, involving a PbI2 primary layer deposition and subsequent conversion using vaporized methylammonium iodide (MAI). Leveraging computational fluid dynamics (CFD) simulations, we confirm that vertical positioning during the PbI2 layer growth yields a uniform film with a narrow temperature distribution and minimal boundary layer thickness. However, during the subsequent conversion step, horizontal substrate positioning results in spatially more uniform MAPbI3 thickness and grain size compared to the vertical placement due to enhanced MAI intercalation. From this optimized substrate positioning, we observe substantial optical homogeneity across the substrate on a centimeter scale, along with uniform and enhanced optoelectronic device performance within photodetector arrays. Our results offer a potential path toward the scalable production of highly uniform perovskite films.

Hybrid Vesicles Enable Mechano-Responsive Hydrogel Degradation.

Stimuli-responsive hydrogels are intriguing biomimetic materials. Previous efforts to develop mechano-responsive hydrogels have mostly relied on chemical modifications of the hydrogel structures. Here, we present a simple, generalizable strategy that confers mechano-responsive behavior on hydrogels. Our approach involves embedding hybrid vesicles, composed of phospholipids and amphiphilic block copolymers, within the hydrogel matrix to act as signal transducers. Under mechanical stress, these vesicles undergo deformation and rupture, releasing encapsulated compounds that can control the hydrogel network. To demonstrate this concept, we embedded vesicles containing ethylene glycol tetraacetic acid (EGTA), a calcium chelator, into a calcium-crosslinked alginate hydrogel. When compressed, the released EGTA sequesters calcium ions and degrades the hydrogel. This study provides a novel method for engineering mechano-responsive hydrogels that may be useful in various biomedical applications.

Diverse Mechanisms of CRISPR-Cas9 Inhibition by Type II Anti-CRISPR Proteins.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) systems provide bacteria and archaea with an adaptive immune response against invasion by mobile genetic elements like phages, plasmids, and transposons. These systems have been repurposed as very powerful biotechnological tools for gene editing applications in both bacterial and eukaryotic systems. The discovery of natural off-switches for CRISPR-Cas systems, known as anti-CRISPR proteins, provided a mechanism for controlling CRISPR-Cas activity and opened avenues for the development of more precise editing tools. In this review, we focus on the inhibitory mechanisms of anti-CRISPRs that are active against type II CRISPR-Cas systems and briefly discuss their biotechnological applications.

Anti-CRISPR Protein AcrIIC5 Inhibits CRISPR-Cas9 by Occupying the Target DNA Binding Pocket.

Anti-CRISPR proteins inhibit CRISPR-Cas immune systems through diverse mechanisms. Previously, the anti-CRISPR protein AcrIIC5Smu was shown to potently inhibit a type II-C Cas9 from Neisseria meningitidis (Nme1Cas9). In this work, we explore the mechanism of activity of the AcrIIC5 homologue from Neisseria chenwenguii (AcrIIC5Nch) and show that it prevents Cas9 binding to target DNA. We show that AcrIIC5Nch targets the PAM-interacting domain (PID) of Nme1Cas9 for inhibition, agreeing with previous findings for AcrIIC5Smu, and newly establish that strong binding of the anti-CRISPR requires guide RNA be pre-loaded on Cas9. We determined the crystal structure of AcrIIC5Nch using X-ray crystallography and identified amino acid residues that are critical for its function. Using a protein docking algorithm we show that AcrIIC5Nch likely occupies the Cas9 DNA binding pocket, thereby inhibiting target DNA binding through a mechanism similar to that previously described for AcrIIA2 and AcrIIA4.

Engineering Functional Membrane-Membrane Interfaces by InterSpy.

Engineering synthetic interfaces between membranes has potential applications in designing non-native cellular communication pathways and creating synthetic tissues. Here, InterSpy is introduced as a synthetic biology tool consisting of a heterodimeric protein engineered to form and maintain membrane-membrane interfaces between apposing synthetic as well as cell membranes through the SpyTag/SpyCatcher interaction. The inclusion of split fluorescent protein fragments in InterSpy allows tracking of the formation of a membrane-membrane interface and reconstitution of functional fluorescent protein in the space between apposing membranes. First, InterSpy is demonstrated by testing split protein designs using a mammalian cell-free expression (CFE) system. By utilizing co-translational helix insertion, cell-free synthesized InterSpy fragments are incorporated into the membrane of liposomes and supported lipid bilayers with the desired topology. Functional reconstitution of split fluorescent protein between the membranes is strictly dependent on SpyTag/SpyCatcher. Finally, InterSpy is demonstrated in mammalian cells by detecting fluorescence reconstitution of split protein at the membrane-membrane interface between two cells each expressing a component of InterSpy. InterSpy demonstrates the power of CFE systems in the functional reconstitution of synthetic membrane interfaces via proximity-inducing proteins. This technology may also prove useful where cell-cell contacts and communication are recreated in a controlled manner using minimal components.

Analysis of S-glutathionylated proteins during adipocyte differentiation using eosin-glutathione and glutaredoxin 1.

Protein S-glutathionylation is a reversible post-translational modification on cysteine residues forming a mixed disulfide with glutathione. S-glutathionylation, not only protects proteins from oxidation but also regulates the functions of proteins involved in various cellular signaling pathways. In this study, we developed a method for the detection of S-glutathionylated proteins (ProSSG) using eosin-glutathione (E-GSH) and mouse glutaredoxin 1 (mGrx1). ProSSG was efficiently and specifically labeled with E-GSH to form ProSSG-E via thiol-disulfide exchange. ProSSG-E was readily luminescent allowing the detection of ProSSG with semi-quantitative determination. In addition, a deglutathionylation enzyme mGrx1 specifically released E-GSH from ProSSG-E, which increased fluorescence allowing a sensitive determination of ProSSG levels. Application of the method to the adipocyte differentiation of 3T3-L1 cells showed specific detection of ProSSG and its increase upon differentiation induction, which was consistent with the result obtained by conventional immunoblot analysis, but with greater specificity and sensitivity. [BMB Reports 2022; 55(3): 154-159].

Structural and Mechanistic Insight into CRISPR-Cas9 Inhibition by Anti-CRISPR Protein AcrIIC4Hpa.

Phages, plasmids, and other mobile genetic elements express inhibitors of CRISPR-Cas immune systems, known as anti-CRISPR proteins, to protect themselves from targeted destruction. These anti-CRISPR proteins have been shown to function through very diverse mechanisms. In this work we investigate the activity of an anti-CRISPR isolated from a prophage in Haemophilus parainfluenzae that blocks CRISPR-Cas9 DNA cleavage activity. We determine the three-dimensional crystal structure of AcrIIC4Hpa and show that it binds to the Cas9 Recognition Domain. This binding does not prevent the Cas9-anti-CRISPR complex from interacting with target DNA but does inhibit DNA cleavage. AcrIIC4Hpa likely acts by blocking the conformational changes that allow the HNH and RuvC endonuclease domains to contact the DNA sites to be nicked.

Synthetic Cell as a Platform for Understanding Membrane-Membrane Interactions.

In the pursuit of understanding life, model membranes made of phospholipids were envisaged decades ago as a platform for the bottom-up study of biological processes. Micron-sized lipid vesicles have gained great acceptance as their bilayer membrane resembles the natural cell membrane. Important biological events involving membranes, such as membrane protein insertion, membrane fusion, and intercellular communication, will be highlighted in this review with recent research updates. We will first review different lipid bilayer platforms used for incorporation of integral membrane proteins and challenges associated with their functional reconstitution. We next discuss different methods for reconstitution of membrane fusion and compare their fusion efficiency. Lastly, we will highlight the importance and challenges of intercellular communication between synthetic cells and synthetic cells-to-natural cells. We will summarize the review by highlighting the challenges and opportunities associated with studying membrane-membrane interactions and possible future research directions.

Development of Nano Soy Milk through Sensory Attributes and Consumer Acceptability.

Nanotechnology is currently applied in food processing and packaging in the food industry. Nano encapsulation techniques could improve sensory perception and nutrient absorption. The purpose of this study was to identify the sensory characteristics and consumer acceptability of three types of commercial and two types of laboratory-developed soy milk. A total of 20 sensory attributes of the five different soy milk samples, including appearance, smell (odor), taste, flavor, and mouthfeel (texture), were developed. The soy milk samples were evaluated by 100 consumers based on their overall acceptance, appearance, color, smell (odor), taste, flavor, mouthfeel (texture), goso flavor (nuttiness), sweetness, repeated use, and recommendation. One-way analysis of variance (ANOVA), principal component analysis (PCA), and partial least square regression (PLSR) were used to perform the statistical analyses. The SM_D sample generally showed the highest scores for overall liking, flavor, taste, mouthfeel, sweetness, repeated consumption, and recommendation among all the consumer samples tested. Consumers preferred sweet, goso (nuttiness), roasted soybean, and cooked soybean (nuttiness) attributes but not grayness, raw soybean flavor, or mouthfeel. Sweetness was closely related to goso (nuttiness) odor and roasted soybean odor and flavor based on partial least square regression (PLSR) analysis. Determination of the sensory attributes and consumer acceptance of soymilk provides insight into consumer needs and desires along with basic data to facilitate the expansion of the consumer market.

Augmented CO2 tolerance by expressing a single H+-pump enables microalgal valorization of industrial flue gas.

Microalgae can accumulate various carbon-neutral products, but their real-world applications are hindered by their CO2 susceptibility. Herein, the transcriptomic changes in a model microalga, Chlamydomonas reinhardtii, in a high-CO2 milieu (20%) are evaluated. The primary toxicity mechanism consists of aberrantly low expression of plasma membrane H+-ATPases (PMAs) accompanied by intracellular acidification. Our results demonstrate that the expression of a universally expressible PMA in wild-type strains makes them capable of not only thriving in acidity levels that they usually cannot survive but also exhibiting 3.2-fold increased photoautotrophic production against high CO2 via maintenance of a higher cytoplasmic pH. A proof-of-concept experiment involving cultivation with toxic flue gas (13 vol% CO2, 20 ppm NOX, and 32 ppm SOX) shows that the production of CO2-based bioproducts by the strain is doubled compared with that by the wild-type, implying that this strategy potentially enables the microalgal valorization of CO2 in industrial exhaust.

Comprehensive approach to improving life-cycle CO2 reduction efficiency of microalgal biorefineries: A review.

Along with the increase in global awareness of rising CO2 levels, microalgae have attracted considerable interest as a promising CO2 reduction platforms since they exhibit outstanding biomass productivity and are capable of producing numerous valuable products. At this moment, however, two major barriers, relatively low photosynthetic CO2 fixation efficiency and necessity of carbon-intensive microalgal process, obstruct them to be practically utilized. This review suggests effective approaches to improve life-cycle CO2 reduction of microalgal biorefinery. In order to enhance photosynthetic CO2 fixation, strategies to augment carbon content and to increase biomass productivity should be considered. For reducing CO2 emissions associated with the process operations, introduction of efficient process elements, designing of energy-saving process routes, reuse of waste resources and utilization of process integration can be noteworthy options. These comprehensive strategies will provide guidance for microalgal biorefineries to become a practical CO2 reduction technology in near future.

Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2.

CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The CRISPR-Cas9 system has been widely adopted as a powerful genome-editing tool, and phage-encoded inhibitors, known as anti-CRISPRs, offer a means of regulating its activity. Here, we report the crystal structures of anti-CRISPR protein AcrIIC2Nme alone and in complex with Nme1Cas9. We demonstrate that AcrIIC2Nme inhibits Cas9 through interactions with the positively charged bridge helix, thereby preventing sgRNA loading. In vivo phage plaque assays and in vitro DNA cleavage assays show that AcrIIC2Nme mediates its activity through a large electronegative surface. This work shows that anti-CRISPR activity can be mediated through the inhibition of Cas9 complex assembly.

Highly sensitive and straightforward methods for the detection of cyanide using profluorescent glutathionylcobalamin.

The extreme toxicity of cyanide and its continued use in various industries have raised concerns over environmental contamination and, therefore, considerable attention has given to develop facile and sensitive methods of cyanide detection. In this study, we developed highly sensitive and straightforward methods of cyanide detection using eosin-labeled glutathionylcobalamin (E-GSCbl) containing fluorescent eosin-labeled glutathione (E-GSH) as the upper axial ligand to the cobalt. E-GSH fluorescence was strongly quenched in E-GSCbl. The E-GSH ligand of E-GSCbl was replaced specifically by cyanide, showing recovery of the E-GSH fluorescence. This profluorescent property of E-GSCbl enabled detection of cyanide in aqueous solutions, yielding a lower detection limit of 10 nM (0.26 µg L-1). Moreover E-GSH exhibited strong luminescence under UV-light that was quenched in E-GSCbl, and this allowed naked-eye detection of cyanide at concentrations as low as 100 nM. This study demonstrates that profluorescent E-GSCbl is a highly sensitive cyanide chemosensor that can detect nanomolar concentrations of cyanide.

Meet the Anti-CRISPRs: Widespread Protein Inhibitors of CRISPR-Cas Systems.

The constant selective pressure exerted by phages, the viruses that infect bacteria, has led to the evolution of a wide range of anti-phage defenses. One of these defense mechanisms, CRISPR-Cas, provides an adaptive immune system to battle phage infection and inhibit horizontal gene transfer by plasmids, transposons, and other mobile genetic elements. Although CRISPR-Cas systems are widespread in bacteria and archaea, they appear to have minimal long-term evolutionary effects with respect to limiting horizontal gene transfer. One factor that may contribute to this may be the presence of potent inhibitors of CRISPR-Cas systems, known as anti-CRISPR proteins. Forty unique families of anti-CRISPR proteins have been described to date. These inhibitors, which are active against both Class 1 and 2 CRISPR-Cas systems, have a wide range of mechanisms of activity. Studies of these proteins have provided important insight into the evolutionary arms race between bacteria and phages, and have contributed to the development of biotechnological tools that can be harnessed for control of CRISPR-Cas genome editing.

Acidic cultivation of Haematococcus pluvialis for improved astaxanthin production in the presence of a lethal fungus.

An acidic cultivation strategy was developed to prevent contamination of a lethal fungus Paraphysoderma sedebokerensis in Haematococcus pluvialis culture for astaxanthin production. Instead of generally used neutral pH, an acidic condition (pH 4) was applied to the cultivation, resulting in a significant inhibition of the fungal contamination. This could be ascribed to the acidity-associated denaturation of a surface protein of P. sedebokerensis, which plays an important role in recognition of H. pluvialis. Stress relief strategies including stepwise light irradiation and naturally occurring nitrogen deficiency were employed in the induction stage to minimize the reduction of astaxanthin production caused by acidic pH. Accordingly, an astaxanthin titer of 84.8 mg L-1 was obtained, which is 141-fold of that from the completely contaminated culture and double of that without the stress relief methods. This strategy provides a persistent contamination control method that can be used for practical astaxanthin production by H. pluvialis.

Gender-dependent difference in serum paraoxonase 1 levels of Hanwoo, Korean native cattle, and a positive association with meat quality.

OBJECTIVE: Paraoxonase 1 (PON1), a calcium-dependent serum enzyme, has been shown to be involved in lipid metabolism. In this study, we examined the putative correlation of the serum PON1 level of Hanwoo, Korean native cattle, with gender and meat quality grade. METHODS: PON1 levels were estimated by determining the arylesterase and paraoxonase activities (AE and PO, respectively) in serum samples from Hanwoo individuals (n = 56). Serum PON1 levels were analyzed in different gender groups (female [n = 21], castrated male [n = 17], and male [n = 18]), and meat quality grades (≥1 [n = 23], 2 [n = 21], and 3 [n = 12]). RESULTS: Serum PON1 levels were similar in female (AE = 120±55 U/mL, PO = 84±43 mU/mL) and castrated male (123±44 U/mL, PO = 89±30 mU/mL), while male showed a significantly lower level (AE = 65±43 U/mL, PO = 44±34 mU/mL). Furthermore, analysis of serum PON1 levels in three different grades of meat quality showed similar levels in the grades ≥1 (AE = 118±49 U/mL, PO = 84±37 mU/mL) and 2 (AE = 116±54 U/mL, PO = 82±43 mU/mL), while the level was significantly lower in the grade 3 (AE = 58±35 U/mL, PO = 39±27 mU/mL) of lower meat quality. CONCLUSION: We discovered the gender-dependent differences in serum PON1 levels of Hanwoo and a positive association of the serum PON1 level with meat quality. Results in this study suggest that PON1 would be a useful serum marker for preliminary screening of Hanwoo individuals with high-quality meat and applicable for genetic improvement.