Design of an effective small expression tag to enhance GPCR production in E. coli-based cell-free and whole cell expression systems.

G protein-coupled receptors (GPCRs) play crucial roles in sensory, immune, and tumor metastasis processes, making them valuable targets for pharmacological and sensing applications in various industries. However, most GPCRs have low production yields in Escherichia coli (E. coli) expression systems. To overcome this limitation, we introduced AT10 tag, an effective fusion tag that could significantly enhance expression levels of various GPCRs in E. coli and its derived cell-free protein synthesis (CFPS) system. This AT10 tag consisted of an A/T-rich gene sequence designed via optimization of translation initiation rate. It is translated into a short peptide sequence of 10 amino acids at the N-terminus of GPCRs. Additionally, effector proteins could be utilized to suppress cytotoxicity caused by membrane protein expression, further boosting GPCR production in E. coli. Enhanced expression of various GPCRs using this AT10 tag is a promising approach for large-scale production of functional GPCRs in E. coli-based CFPS and whole cell systems, enabling their potential utilization across a wide range of industrial applications.

Hydrogel Micropillar Array for Temperature Sensing in Fluid.

Localized temperature sensing and control on a micron-scale have diverse applications in biological systems. We present a micron-sized hydrogel pillar array as potential temperature probes and actuators by exploiting sensitive temperature dependence of their volume change. Soft lithography-based molding processes were presented to fabricate poly N-isopropyl acrylamide (p-NIPAAm)-based hydrogel pillar array on a glass substrate. Au nanorods as light-induced heating elements were embedded within the hydrogel pillars, and near-infrared (NIR) light was used to modulate temperature in a local area. First, static responses of the micro-pillar array were characterized as a function of its temperature. It was shown that the hydrogel had a sensitive volume transition near its low critical solution temperature (LCST). Furthermore, we showed that LCST could be readily adjusted by utilizing copolymerizing with acrylamide (AAM). To demonstrate the feasibility of spatiotemporal temperature mapping and modulation using the presented pillar array, pulsed NIR light was illuminated on a local area of the hydrogel pillar array, and its responses were recorded. Dynamic temperature change in water was mapped based on the abrupt volume change characteristics of the hydrogel pillar, and its potential actuation using NIR light was successfully demonstrated. Considering that the structure can be arrayed in a two-dimensional pixel format with high spatial resolution and high sensitive temperature characteristics, the presented method and the device structure can have diverse applications to change and sense local temperatures in liquid. This is particularly useful in biological systems, where their physiological temperature can be modulated and mapped with high spatial resolution.

Lipid extract derived from newly isolated Rhodotorula toruloides LAB-07 for cosmetic applications.

Rhodotorula toruloides is a non-conventional yeast with a natural carotenoid pathway. In particular, R. toruloides is an oleaginous yeast that can accumulate lipids in high content, thereby gaining interest as a promising industrial host. In this study, we isolated and taxonomically identified a new R. toruloides LAB-07 strain. De novo genome assembly using PacBio and Illumina hybrid platforms yielded 27 contigs with a 20.78 Mb genome size. Subsequent genome annotation analysis based on RNA-seq predicted 5296 protein-coding genes, including the fatty acid production pathway. We compared lipid production under different media; it was highest in the yeast extract salt medium with glycerol as a carbon source. Polyunsaturated α-linolenic acid was detected among the fatty acids, and docking phosphatidylcholine as a substrate to modeled Fad2, which annotated as Δ12-fatty acid desaturase showed bifunctional Δ12, 15-desaturation is structurally possible in that the distances between the diiron center and the carbon-carbon bond in which desaturation occurs were similar to those of structurally identified mouse stearoyl-CoA desaturase. Finally, the applicability of the extracted total lipid fraction of R. toruloides was investigated, demonstrating an increase in filaggrin expression and suppression of heat-induced MMP-1 expression when applied to keratinocytes, along with the additional antioxidant activity. This work presents a new R. toruloides LAB-07 strain with genomic and lipidomic data, which would help understand the physiology of R. toruloides. Also, the various skin-related effect of R. toruloides lipid extract indicates its potential usage as a promising cosmetic ingredient.

CRISPRi-mediated tunable control of gene expression level with engineered single-guide RNA in Escherichia coli.

Precise control of gene expression is essential for flux redistribution in metabolic pathways. Although the CRISPR interference (CRISPRi) system can effectively repress gene expression at the transcriptional level, it has still been difficult to precisely control the level without loss of specificity or an increase in cell toxicity. In this study, we developed a tunable CRISPRi system that performs transcriptional regulation at various levels. We constructed a single-guide RNA (sgRNA) library targeting repeat, tetraloop, and anti-repeat regions to modulate the binding affinity against dCas9. Each screened sgRNA could regulate the gene expression at a certain level between fully-repressing and non-repressing states (>45-fold). These sgRNAs also enabled modular regulation with various target DNA sequences. We applied this system to redistribute the metabolic flux to produce violacein derivatives in a predictable ratio and optimize lycopene production. This system would help accelerate the flux optimization processes in metabolic engineering and synthetic biology.

Synthetic biology tools for engineering Corynebacterium glutamicum.

Corynebacterium glutamicum is a promising organism for the industrial production of amino acids, fuels, and various value-added chemicals. From the whole genome sequence release, C. glutamicum has been valuable in the field of industrial microbiology and biotechnology. Continuous discovery of genetic manipulations and regulation mechanisms has developed C. glutamicum as a synthetic biology platform chassis. This review summarized diverse genomic manipulation technologies and gene expression tools for static, dynamic, and multiplex control at transcription and translation levels. Moreover, we discussed the current challenges and applicable tools to C. glutamicum for future advancements.

dCas9-Mediated PCR-Free Detection of Oncogenic Mutation by Nonequilibrium Nanoelectrokinetic Selective Preconcentration.

Cutting-edge nanoelectrokinetic technology in this work provides a breakthrough for the present clinical demands of molecular diagnosis to detect a trace amount of oncogenic mutation of DNA in a short time without an erroneous PCR procedure. In this work, we combined the sequence-specific labeling scheme of CRISPR/dCas9 and ion concentration polarization (ICP) mechanism to separately preconcentrate target DNA molecules for rapid detection. Using the mobility shift caused by dCas9's specific binding to the mutant, the mutated DNA and normal DNA were distinguished in the microchip. Based on this technique, we successfully demonstrated the dCas9-mediated 1-min detection of single base substitution (SBS) in EGFR DNA, a carcinogenesis indicator. Moreover, the presence/absence of target DNA was identified at a glance like a commercial pregnancy test kit (two lines for positive and one line for negative) by the distinct preconcentration mechanisms of ICP, even at the 0.1% concentration of the target mutant.

Model-driven experimental design workflow expands understanding of regulatory role of Nac in Escherichia coli.

The establishment of experimental conditions for transcriptional regulator network (TRN) reconstruction in bacteria continues to be impeded by the limited knowledge of activating conditions for transcription factors (TFs). Here, we present a novel genome-scale model-driven workflow for designing experimental conditions, which optimally activate specific TFs. Our model-driven workflow was applied to elucidate transcriptional regulation under nitrogen limitation by Nac and NtrC, in Escherichia coli. We comprehensively predict alternative nitrogen sources, including cytosine and cytidine, which trigger differential activation of Nac using a model-driven workflow. In accordance with the prediction, genome-wide measurements with ChIP-exo and RNA-seq were performed. Integrative data analysis reveals that the Nac and NtrC regulons consist of 97 and 43 genes under alternative nitrogen conditions, respectively. Functional analysis of Nac at the transcriptional level showed that Nac directly down-regulates amino acid biosynthesis and restores expression of tricarboxylic acid (TCA) cycle genes to alleviate nitrogen-limiting stress. We also demonstrate that both TFs coherently modulate α-ketoglutarate accumulation stress due to nitrogen limitation by co-activating amino acid and diamine degradation pathways. A systems-biology approach provided a detailed and quantitative understanding of both TF's roles and how nitrogen and carbon metabolic networks respond complementarily to nitrogen-limiting stress.

Programmable synthetic biology tools for developing microbial cell factories.

Microbial conversion to generate value-added chemicals from diverse biomass is one of the keystones of energy biotechnology. Programmable synthetic biology tools offer versatile, standardized options for developing microbial cell factories. These tools thus can be reprogrammed in a user-defined manner for flexible wiring of stimuli and response, highly efficient genome engineering, and extensive perturbation of metabolic flux and genetic circuits. They also can be modularly assembled to construct elaborate and unprecedented biological systems with unique features. This review highlights recent advances in programmable synthetic biology tools based on biosensors, CRISPR-Cas, and RNA devices for developing microbial cell factories that have the potential to be utilized for energy biotechnology.

Revealing Causes for False-Positive and False-Negative Calling of Gene Essentiality in Escherichia coli Using Transposon Insertion Sequencing.

The massive sequencing of transposon insertion mutant libraries (Tn-Seq) represents a commonly used method to determine essential genes in bacteria. Using a hypersaturated transposon mutant library consisting of 400,096 unique Tn insertions, 523 genes were classified as essential in Escherichia coli K-12 MG1655. This provided a useful genome-wide gene essentiality landscape for rapidly identifying 233 of 301 essential genes previously validated by a knockout study. However, there was a discrepancy in essential gene sets determined by conventional gene deletion methods and Tn-Seq, although different Tn-Seq studies reported different extents of discrepancy. We have elucidated two causes of this discrepancy. First, 68 essential genes not detected by Tn-Seq contain nonessential subgenic domains that are tolerant to transposon insertion, which leads to the false assignment of an essential gene as a nonessential or dispensable gene. These genes exhibited a high level of transposon insertion in their subgenic nonessential domains. In contrast, 290 genes were additionally categorized as essential by Tn-Seq, although their knockout mutants were available. The comparative analysis of Tn-Seq and high-resolution footprinting of nucleoid-associated proteins (NAPs) revealed that a protein-DNA interaction hinders transposon insertion. We identified 213 false-positive genes caused by NAP-genome interactions. These two limitations have to be considered when addressing essential bacterial genes using Tn-Seq. Furthermore, a comparative analysis of high-resolution Tn-Seq with other data sets is required for a more accurate determination of essential genes in bacteria. IMPORTANCE Transposon mutagenesis is an efficient way to explore gene essentiality of a bacterial genome. However, there was a discrepancy between the essential gene set determined by transposon mutagenesis and that determined using single-gene knockout strains. In this study, we generated a hypersaturated Escherichia coli transposon mutant library comprising approximately 400,000 different mutants. Determination of transposon insertion sites using next-generation sequencing provided a high-resolution essentiality landscape of the E. coli genome. We identified false negatives of essential gene discovery due to the permissive insertion of transposons in the C-terminal region. Comparisons between the transposon insertion landscape with binding profiles of DNA-binding proteins revealed interference of nucleoid-associated proteins to transposon insertion, generating false positives of essential gene discovery. Consideration of these findings is required to avoid the misinterpretation of transposon mutagenesis results.

A Vibrio-based microbial platform for accelerated lignocellulosic sugar conversion.

BACKGROUND: Owing to increasing concerns about climate change and the depletion of fossil fuels, the development of efficient microbial processes for biochemical production from lignocellulosic biomass has been a key issue. Because process efficiency is greatly affected by the inherent metabolic activities of host microorganisms, it is essential to utilize a microorganism that can rapidly convert biomass-derived sugars. Here, we report a novel Vibrio-based microbial platform that can rapidly and simultaneously consume three major lignocellulosic sugars (i.e., glucose, xylose, and arabinose) faster than any previously reported microorganisms. RESULTS: The xylose isomerase pathway was constructed in Vibrio sp. dhg, which naturally displays high metabolic activities on glucose and arabinose but lacks xylose catabolism. Subsequent adaptive laboratory evolution significantly improved xylose catabolism of initial strain and led to unprecedently high growth and sugar uptake rate (0.67 h-1 and 2.15 g gdry cell weight-1 h-1, respectively). Furthermore, we achieved co-consumption of the three sugars by deletion of PtsG and introduction of GalP. We validated its superior performance and applicability by demonstrating efficient lactate production with high productivity (1.15 g/L/h) and titer (83 g/L). CONCLUSIONS: In this study, we developed a Vibrio-based microbial platform with rapid and simultaneous utilization of the three major sugars from lignocellulosic biomass by applying an integrated approach of rational and evolutionary engineering. We believe that the developed strain can be broadly utilized to accelerate the production of diverse biochemicals from lignocellulosic biomass.

Efficient Production of Naringin Acetate with Different Acyl Donors via Enzymatic Transesterification by Lipases.

Naringin, one of the citrus flavonoids and known as a natural antioxidant, has limited bioavailability owing to its low stability and solubility. However, naringin esters formed via acylation have recently been reported to possess improved physical and chemical properties. The development of these compounds has a great potential in the food, cosmetic and pharmaceutical industries, but low conversion and productivity are barriers to industrial applications. This study aimed to improve the conversion of naringin acetate, which is formed via the enzymatic reaction between naringin and an acyl donor. An optimal reaction condition was determined by evaluating the effect of various variables (enzyme type, enzyme concentration, acyl donor, molar ratio of reactants, reaction temperature, and solvent) on the synthesis of naringin acetate. The optimal condition was as follows: 3 g/L of Lipozyme TL IM, molar ratio of 1:5 (naringin:acyl donor), reaction temperature of 40 °C, and acetonitrile as the reaction solvent. Under this condition, the maximum conversion to naringin acetate from acetic anhydride and vinyl acetate was achieved at approximately 98.5% (8 h) and 97.5% (24 h), respectively. Compared to the previously reported values, a high conversion was achieved within a short time, confirming the commercial potential of the process.

Effects of Caffeine Intake on Cardiopulmonary Variables and QT Interval after a Moderate-Intensity Aerobic Exercise in Healthy Adults: A Randomized Controlled Trial.

Caffeine is considered a widely consumed natural and legal psychoactive stimulant with several effects on the body. The present study attempted to investigate the effects of caffeine consumed before and after a physical exercise on cardiovascular and cardiorespiratory functions in healthy adults. 36 healthy adult males were recruited and randomly allocated to one of the three (3) groups: group I (exercise without caffeine consumption), group II (caffeine beverage intake before exercise), and group III (caffeine beverage intake immediately after exercise). The heart rate (HR), QTc interval, blood pressure (BP), respiratory rate (RR), oxygen consumption (VO2), and carbon dioxide emission (VCO2) were measured at 0, 5, 10, and 15 min after the exercise. We observed a significant difference in all measured outcomes during the different recovery times in all the groups (p < 0.05). HR, RR, SBP, VO2, and VCO2 gradually decreased with time, DBP contrarily increased with time, and the QTc showed an irregular pattern. We can affirm that ingestion of caffeine before and after moderate aerobic exercise slows down the parasympathetic stimulation, heart rate recovery, and the recovery of HR and QTc with no major effects on BP, RR, VO2, and VCO2 in healthy adult men.

Synthetic cellular communication-based screening for strains with improved 3-hydroxypropionic acid secretion.

Although cellular secretion is important in industrial biotechnology, its assessment is difficult due to the lack of efficient analytical methods. This study describes a synthetic cellular communication-based microfluidic platform for screening strains with the improved secretion of 3-hydroxypropionic acid (3-HP), an industry-relevant platform chemical. 3-HP-secreting cells were compartmentalized in droplets, with receiving cells equipped with a genetic circuit that converts the 3-HP secretion level into an easily detectable signal. This platform was applied to identify Escherichia coli genes that enhance the secretion of 3-HP. As a result, two genes (setA, encoding a sugar exporter, and yjcO, encoding a Sel1 repeat-containing protein) found by this platform enhance the secretion of 3-HP and its production. Given the increasing design capability for chemical-detecting cells, this platform has considerable potential in identifying efflux pumps for not only 3-HP but also many important chemicals.

Synthetic biosensor accelerates evolution by rewiring carbon metabolism toward a specific metabolite.

Proper carbon flux distribution between cell growth and production of a target compound is important for biochemical production because improper flux reallocation inhibits cell growth, thus adversely affecting production yield. Here, using a synthetic biosensor to couple production of a specific metabolite with cell growth, we spontaneously evolve cells under the selective condition toward the acquisition of genotypes that optimally reallocate cellular resources. Using 3-hydroxypropionic acid (3-HP) production from glycerol in Escherichia coli as a model system, we determine that mutations in the conserved regions of proteins involved in global transcriptional regulation alter the expression of several genes associated with central carbon metabolism. These changes rewire central carbon flux toward the 3-HP production pathway, increasing 3-HP yield and reducing acetate accumulation by alleviating overflow metabolism. Our study provides a perspective on adaptive laboratory evolution (ALE) using synthetic biosensors, thereby supporting future efforts in metabolic pathway optimization.

Engineering Vibrio sp. SP1 for the production of carotenoids directly from brown macroalgae.

Macroalgae is regarded as a promising third-generation marine biomass that can be utilized as a sustainable feedstock for bio-industry due to the high sugar level and absence of lignin. Alginate, composed of 1,4-linked D-mannuronate (M) and L-guluronate (G), is one of the major carbohydrates in brown macroalgae. It is difficult to be assimilated by most industrial microorganisms. Therefore, developing engineered microorganisms that can utilize alginate as a feedstock in order to produce natural products from marine biomass is critical. In this study, we isolated, characterized, and sequenced Vibrio sp. SP1 which rapidly grows using alginate as a sole carbon source. We further engineered this strain by introducing genes encoding enzymes under the control of synthetic expression cassettes to produce lycopene and ß-carotene which are attractive phytochemicals owing to the antioxidant property. We confirmed that the engineered Vibrio sp. SP1 could successfully produce 2.13 ± 0.37 mg L-1 of lycopene, 2.98 ± 0.43 mg L-1 of ß -carotene, respectively, from 10 g L-1 of alginate as a sole carbon source. Furthermore, our engineered strain could directly convert 60 g L-1 of brown macroalgae Sargassum fusiforme into 1.23 ± 0.21 mg L-1 of lycopene without any pretreatment which had been vitally required for the previous productions. As the first demonstrated strain to produce high-value product from Sargassum, Vibrio sp. SP1 is evaluated to be a desirable platform for the brown macroalgae-based biorefinery.

Synthetic protein quality control to enhance full-length translation in bacteria.

Coupled transcription and translation processes in bacteria cause indiscriminate translation of intact and truncated messenger RNAs, inevitably generating nonfunctional polypeptides. Here, we devised a synthetic protein quality control (ProQC) system that enables translation only when both ends of mRNAs are present and followed by circularization based on sequence-specific RNA-RNA hybridization. We demonstrate that the ProQC system dramatically improved the fraction of full-length proteins among all synthesized polypeptides by selectively translating intact mRNA and reducing abortive translation. As a result, full-length protein synthesis increased up to 2.5-fold without changing the transcription or translation efficiency. Furthermore, we applied the ProQC system for 3-hydroxypropionic acid, violacein and lycopene production by ensuring full-length expression of enzymes in biosynthetic pathways, resulting in 1.6- to 2.3-fold greater biochemical production. We believe that our ProQC system can be universally applied to improve not only the quality of recombinant protein production but also efficiencies of metabolic pathways.

Rapid photothermal actuation of light-addressable, arrayed hydrogel columns in a macroporous silicon membrane.

This paper presents rapid photothermal actuation of light-addressable, arrayed hydrogel columns in a macroporous silicon membrane. Au nanorods are incorporated into thermo-responsive p-NIPAAm hydrogel to utilize surface plasmon-induced local heating by near-infrared light. By measuring optical transmission through the fabricated membrane structure with Au nanorod embedded hydrogel, we have demonstrated that photothermal actuation of hydrogel can be done in two-dimensional, pixel-like configuration with high spatial and temporal resolutions. Benefiting from the hydrogel volume confinement within micron-sized pores, we have achieved sub-second response time of hydrogel photothermal actuation and its repeatable photothermal actuation on highly localized illuminated area. Considering that each hydrogel column is confined within each pore and different wavelength of light can be used to induce photothermal actuation of hydrogel's deswelling characteristics by modifying physical dimensions of Au nanorods, it has a potential for optically-addressable, multiplexed drug release systems with rapid response time.

Transcriptional Profiling of the Probiotic Escherichia coli Nissle 1917 Strain under Simulated Microgravity.

Long-term space missions affect the gut microbiome of astronauts, especially the viability of some pathogens. Probiotics may be an effective solution for the management of gut microbiomes, but there is a lack of studies regarding the physiology of probiotics in microgravity. Here, we investigated the effects of microgravity on the probiotic Escherichia coli Nissle 1917 (EcN) by comparing transcriptomic data during exponential and stationary growth phases under simulated microgravity and normal gravity. Microgravity conditions affected several physiological features of EcN, including its growth profile, biofilm formation, stress responses, metal ion transport/utilization, and response to carbon starvation. We found that some changes, such as decreased adhesion ability and acid resistance, may be disadvantageous to EcN relative to gut pathogens under microgravity, indicating the need to develop probiotics optimized for space flight.

Engineering Tools for the Development of Recombinant Lactic Acid Bacteria.

Lactic acid bacteria (LAB) is mainly used in food fermentation. In addition, LAB fermentation technology has been studied in the development of industrial food additives, nutrients, or enzymes used in food processing. In the field of red biotechnology, LAB is approved and is generally recognized as a safe organism and is considered safe for biotherapeutic treatments. Recent clinical trials have demonstrated the medicinal value of therapeutic recombinant LAB and the suitability of innate mechanisms of secretion and anchoring for therapeutic applications such as antibody or vaccine production. However, the gram-positive phenotypic trait of LAB creates challenges for genetic modifications when compared to other conventional workhorse bacteria, resulting in exclusive developments of genetic tools for engineering LAB. In this review, several distinct approaches in gene expression for engineering LAB are discussed.

Fabrication of Troponin I Biosensor Composed of Multi-Functional DNA Structure/Au Nanocrystal Using Electrochemical and Localized Surface Plasmon Resonance Dual-Detection Method.

In the present study, we fabricated a dual-mode cardiac troponin I (cTnI) biosensor comprised of multi-functional DNA (MF-DNA) on Au nanocrystal (AuNC) using an electrochemical method (EC) and a localized surface plasmon resonance (LSPR) method. To construct a cTnI bioprobe, a DNA 3 way-junction (3WJ) was prepared to introduce multi-functionality. Each DNA 3WJ arm was modified to possess a recognition region (Troponin I detection aptamer), an EC-LSPR signal generation region (methylene blue: MB), and an anchoring region (Thiol group), respectively. After an annealing step, the multi-functional DNA 3WJ was assembled, and its configuration was confirmed by Native-TBM PAGE for subsequent use in biosensor construction. cTnI was also expressed and purified for use in biosensor experiments. To construct an EC-LSPR dual-mode biosensor, AuNCs were prepared on an indium-tin-oxide (ITO) substrate using an electrodeposition method. The prepared multi-functional (MF)-DNA was then immobilized onto AuNCs by covalent bonding. Field emission scanning electron microscope (FE-SEM) and atomic force microscopy (AFM) were used to analyze the surface morphology. LSPR and electrochemical impedance spectroscopy (EIS) experiments were performed to confirm the binding between the target and the bioprobe. The results indicated that cTnI could be effectively detected in the buffer solution and in diluted-human serum. Based on the results of these experiments, the loss on drying (LOD) was determined to be 1.0 pM in HEPES solution and 1.0 pM in 10% diluted human serum. Additionally, the selectivity assay was successfully tested using a number of different proteins. Taken together, the results of our study indicate that the proposed dual-mode biosensor is applicable for use in field-ready cTnI diagnosis systems for emergency situations.