Rewiring the metabolic flux for efficient microbial conversion requires robust, scalable gene assembly. However, conventional gene assembly approaches are labor-intensive, highly experience-dependent, and require extensive expertise to ensure reproducibility and efficiency. Even with advanced automation platforms such as biofoundries, assembling gene arrays with multiple transcriptional units (TUs) remains challenging. In this study, we present Efficient Modular Gene Assembly (EffiModular), an integrated in vitro and in vivo gene assembly platform compatible with automated workflows. EffiModular enables the assembly of up to eight TUs with 80% efficiency in a single transformation. Integrated into a biofoundry workflow, it enabled the construction of 120 distinct yeast strains with varying levels of expression of the β-carotene biosynthesis genes within 3 days. Compared with conventional approaches, it significantly reduces procedural complexity, minimizes reliance on operator expertise, and accelerates workflow timelines. These features establish EffiModular as a next-generation gene assembly platform for scalable, reproducible gene assembly in biofoundry-based genetic engineering.

Current on-site arsenic monitoring is hindered by cold-chain dependence and biosafety risks. We developed a field-deployable cell-free protein synthesis (CFPS) biosensor for the rapid, selective detection of toxic inorganic arsenic in seaweed matrices (Sargassum fusiforme and Porphyra sp.). An ArsR-regulated genetic circuit enables effective discrimination between inorganic and organic arsenic species. A tailored lyophilization strategy eliminated the need for ultra-low temperature storage, retaining ∼70% activity over four weeks at room temperature. Notably, freeze-drying serendipitously suppressed leaky expression, reducing background noise by 8-fold compared to liquid controls. Validated through spike-and-recovery tests in real seaweed extracts, this cold-chain-free, highly sensitive platform represents a significant advancement for decentralized food safety monitoring and environmental screening.

ABSTRACT Methanotrophic bacteria show significant promise for methane bioconversion. Despite their ecological and biotechnological importance, the understanding of their transcriptional regulation and genetic regulatory elements remains limited. Here, we applied high-throughput sequencing to elucidate the transcriptional regulatory landscape of Methylosinus sporium 5, a type II methanotroph. With its genome sequence completion, we identified 1,983 transcription start sites (TSSs) and 1,483 transcript 3′-ends (TEPs), which collectively defined 1,431 transcription units (TUs). This comprehensive analysis revealed diverse regulatory elements, including promoters, untranslated regions (UTRs), terminators, and regulatory RNAs in M. sporium 5. A consensus promoter motif comprizing conserved −10 (TATAHT) and −35 (TYGMSV) elements, recognized by the housekeeping sigma factor RpoD, was predominant, particularly upstream of genes involved in methane and central carbon metabolism. We also uncovered diverse cis -regulatory motifs associated with nitrogen metabolism and cell division, including binding sites for RpoN (σ 54 ) and the transcription regulator CtrA. TEP analysis identified three classes of transcript ends with distinct sequence features and termination strengths. I-shaped intrinsic terminators were most prevalent, while L-shaped terminators were enriched in highly expressed genes, ensuring efficient transcription termination. Integration of TSSs and TEPs revealed functionally related genes within polycistronic TUs and identified previously unannotated small RNAs, including EcpR1 and αr45. This study provides the first genome-scale map of transcriptional regulation in a methanotroph, offering foundational insights into regulatory architecture and enabling future strain engineering for enhanced methane bioconversion. IMPORTANCE Methanotrophic bacteria offer a sustainable solution for converting methane into valuable products. However, the molecular mechanisms governing gene expression regulation in methanotrophs remain poorly understood. In this study, we applied high-throughput sequencing approaches to elucidate gene organization and transcriptional regulation in Methylosinus sporium 5 during growth on methane. By identifying key regulatory features including promoter sequences, diverse cis -regulatory elements, and transcript boundaries, we revealed the coordinated gene expression mechanisms in this organism. This represents the first genome-wide transcriptome architecture in a methanotrophic bacterium. The regulatory elements provide a valuable resource for future genetic engineering of M. sporium 5 and related methanotrophs. Our findings significantly advance the understanding of gene regulation in methanotrophs and support their development as microbial platforms for methane-based biomanufacturing.

Cellular transport systems are key determinants of intracellular molecule concentrations and can be utilized to modulate the responsiveness of transcription factor (TF)-based biosensors. Efflux pumps facilitate the export of small molecules; therefore, their activity can directly compromise the accuracy of biosensor-based assays. We engineered a cellular export machinery to enhance the responsiveness and reduce crosstalk of a DmpR-based biosensor, which employs the phenol-responsive TF DmpR to detect intracellular phenolic ligands but is affected by ligand diffusion between cells. To overcome this limitation, efflux pump genes were knocked out to minimize ligand diffusion and improve signal fidelity. Among the efflux pumps tested, deletion of mdtA was found to promote effective intracellular accumulation of phenolic compounds. This strategy not only increased biosensor sensitivity by up to 19-fold but also reduced false positives during enzyme screening by suppressing intercellular diffusion of enzymatic products. In the mock library experiment, the proportion of false positives relative to the total positive cells was 74

Engineering enzymes to degrade solid substrates, such as crystalline cellulose from paper sludge or microplastics in sewage sludge, presents challenges for high-throughput screening (HTS), as solid substrates are not readily accessible in cell-based biosensor systems. To address this challenge, we developed a cell-free cellobiose-detectable biosensor (CB-biosensor) for rapid characterization of cellobiohydrolase (CBH) activity, enabling direct detection of hydrolysis products without cellular constraints. The CB-biosensor demonstrates higher sensitivity than conventional assays and distinguishes between CBH subtypes (CBHI and CBHII) based on their modes of action. Integration with the Echo 525 liquid handler enables precise and reproducible sample processing, with fluorescence signals from automated preparations comparable to manual experiments. Furthermore, assay volumes can be reduced to just a few microlitres-impractical with manual methods. This cell-free CB-biosensor with Echo 525 minimizes reagent consumption, accelerates testing, and facilitates reliable large-scale screening. These findings highlight its potential to overcome current HTS limitations, advancing enzyme screening and accelerating the Design-Build-Test-Learn cycle for sustainable biomanufacturing.

The interdisciplinary nature of synthetic biology merges engineering principles with biology and provides innovative solutions for issues in the biomanufacturing industry. To develop industrially applicable biocatalysts and/or microbial cell factories, a design-build-test-learn cycle-based iterative process is necessary, which is often time-consuming and labor-intensive. The integration of microfluidic technologies into synthetic biology can accelerate these processes, particularly for achieving high-throughput phenotyping and screening. In this review, we examine the potential of microfluidic technologies to revolutionize synthetic biology. Although commercial microfluidics demonstrate superior throughput for single-cell assays, their application can be limited, for example, in cases where products are retained inside the cells. Droplet microfluidics, on the other hand, is a rather flexible platform and shows high diversity in single-cell, cell-to-cell interaction-based, and cell-free reaction-based analyses. By examining previous studies, we have summarized the potential of microfluidic technologies to foster sustainable biomanufacturing and advanced biological engineering.

Although gas chromatography is the gold standard for detecting the persistent organic pollutant dichlorodiphenyltrichloroethane (DDT), a quick and easy alternative is needed to detect this highly toxic pesticide in food and household products. Therefore, we developed a monoclonal antibody (mAb)-based assay for the sensitive detection of DDT. The mAb used in our assay demonstrated favourable binding affinity in the nanomolar range against 2,2-bis(4-chlorophenyl)acetic acid (DDA), a DDT analogue, which is considered higher than commercially developed antibodies. By optimizing the assay conditions, we were able to detect DDT and its closely related compounds, including dichlorodiphenyldichloroethylene (DDE), dichlorodiphenyldichloroethane (DDD), and isomeric impurities. Our dot blotting assay demonstrated a detection limit of 0.9 ng/mL for DDT. Using computational modelling, we predicted the structure of the mAb and its interaction with DDT. Overall, this study provides insights for engineering mAb with improved sensitivity, facilitates point-of-care detection of toxic chemicals, including DDT, and offers a vital method for food safety monitoring and environmental protection.

A halotolerant consortium between microalgae and methanotrophic bacteria could effectively remediate in situ CH4 and CO2, particularly using saline wastewater sources. Herein, Methylomicrobium alcaliphilum 20Z was demonstrated to form a mutualistic association with Chlorella sp. HS2 at a salinity level above 3.0%. Co-culture significantly enhanced the growth of both microbes, independent of initial inoculum ratios. Additionally, increased methane provision in enclosed serum bottles led to saturated methane removal. Subsequent analyses suggested nearly an order of magnitude increase in the amount of carbon sequestered in biomass in methane-fed co-cultures, conditions that also maintained a suitable cultural pH suitable for methanotrophic growth. Collectively, these results suggest a robust metabolic coupling between the two microbes and the influence of the factors other than gaseous exchange on the assembled consortium. Therefore, multi-faceted investigations are needed to harness the significant methane removal potential of the identified halotolerant consortium under conditions relevant to real-world operation scenarios.

The genus Bacteroides, a predominant group in the human gut microbiome, presents significant potential for microbiome engineering and the development of live biotherapeutics aimed at treating gut diseases. Despite its promising capabilities, tools for effectively engineering Bacteroides species have been limited. In our study, we have made a breakthrough by identifying novel signal peptides in Bacteroides thetaiotaomicron and Akkermansia muciniphila. These peptides facilitate efficient protein transport across cellular membranes in Bacteroides, a critical step for therapeutic applications. Additionally, we have developed an advanced episomal plasmid system. This system demonstrates superior protein secretion capabilities compared to traditional chromosomal integration plasmids, making it a vital tool for enhancing the delivery of therapeutic proteins in Bacteroides species. Initially, the stability of this episomal plasmid posed a challenge; however, we have overcome this by incorporating an essential gene-based selection system. This novel strategy not only ensures plasmid stability but also aligns with the growing need for antibiotic-free selection methods in clinical settings. Our work, therefore, not only provides a more robust secretion system for Bacteroides but also sets a new standard for the development of live biotherapeutics.

Being able to perform modular design of artificial transcription factors is useful in bioengineering and synthetic biology, particularly in the development of biosensors and therapeutics. This study aimed to develop a two-fragment transcription factor system by splitting a lactose repressor (LacI). To fragment LacI, we screened potential split positions from transposon-based insertional libraries that we generated to identify those positions that did not disturb the intrinsic activity of LacI. The interaction of protein tags fused with fragments induces the reassembly of LacI and recovers the isopropyl-β-D-thiogalactoside-dependent regulatory function. The split LacI-based biosensor was implemented on an in vitro platform using a cell-free protein expression system to facilitate accurate analytical studies with high reproducibility. This versatile platform holds great potential to realize the rapid and simple detection of protein–protein interactions in cell-free systems; thus, it can be further extended to disease diagnosis, particularly at the point-of-care.

Isoprene has numerous industrial applications, including rubber polymer and potential biofuel. Microbial methane-based isoprene production could be a cost-effective and environmentally benign process, owing to a reduced carbon footprint and economical utilization of methane. In this study, Methylococcus capsulatus Bath was engineered to produce isoprene from methane by introducing the exogenous mevalonate (MVA) pathway. Overexpression of MVA pathway enzymes and isoprene synthase from Populus trichocarpa under the control of a phenol-inducible promoter substantially improved isoprene production. M. capsulatus Bath was further engineered using a CRISPR-base editor to disrupt the expression of soluble methane monooxygenase (sMMO), which oxidizes isoprene to cause toxicity. Additionally, optimization of the metabolic flux in the MVA pathway and culture conditions increased isoprene production to 228.1 mg/L, the highest known titer for methanotroph-based isoprene production. The developed methanotroph could facilitate the efficient conversion of methane to isoprene, resulting in the sustainable production of value-added chemicals.

Levulinic acid (LA) is a valuable chemical used in fuel additives, fragrances, and polymers. In this study, we proposed possible biosynthetic pathways for LA production from lignin and poly(ethylene terephthalate). We also created a genetically encoded biosensor responsive to LA, which can be used for screening and evolving the LA biosynthesis pathway genes, by employing an LvaR transcriptional regulator of Pseudomonas putida KT2440 to express a fluorescent reporter gene. The LvaR regulator senses LA as a cognate ligand. The LA biosensor was first examined in an Escherichia coli strain and was found to be non-functional. When the host of the LA biosensor was switched from E. coli to P. putida KT2440, the LA biosensor showed a linear correlation between fluorescence intensity and LA concentration in the range of 0.156-10 mM LA. In addition, we determined that 0.156 mM LA was the limit of LA detection in P. putida KT2440 harboring an LA-responsive biosensor. The maximal fluorescence increase was 12.3-fold in the presence of 10 mM LA compared to that in the absence of LA. The individual cell responses to LA concentrations reflected the population-averaged responses, which enabled high-throughput screening of enzymes and metabolic pathways involved in LA biosynthesis and sustainable production of LA in engineered microbes.

The engineered Methylococcus capsulatus Bath presents a promising approach for converting methane, a potent greenhouse gas, into valuable chemicals. High cell-density culture (HCDC) is necessary for high-titer growth-associated bioproducts, but it often requires time-consuming and labor-intensive optimization processes. In this study, we aimed to achieve efficient HCDC of M. capsulatus Bath by measuring the residual nutrient levels during bioreactor operations and analyzing the specific uptake of each medium component. By controlling the concentrations of nutrients, particularly calcium and phosphorus via intermittent feeding, we achieved a high cell density of 28.2 g DCW/L and a significantly elevated production of mevalonate at a concentration of 1.8 g/L from methane. Our findings demonstrate that the methanotroph HCDC approach presented herein offers a promising strategy for promoting sustainable development, with an exceptional g-scale production titer for value-added synthetic biochemicals.

Methanotrophs are promising and sustainable cell factory platforms owing to their ability to convert the most potent greenhouse gas, methane to valuable bioproducts. Genetic engineering toolkits for methanotrophs are extremely limited. Here, we present a phenol-inducible promoter for the high-level expression of exogenous genes in methanotrophs. The phenol-inducible gene expression system showed high dose-dependency and ho-mogeneity in methanotrophs. Using the phenol-inducible CRISPR-base editor (BE), we developed a highly effi-cient methanotroph genome editing system. The CRISPR-BE system efficiently introduced an early stop codon into the target gene, enabling one-step markerless genome editing in Methylococcus capsulatus Bath. We adopted this simple and efficient genome editing tool to produce mevalonate in the engineered M. capsulatus Bath. The native phosphoketolase pathway was reinforced in M. capsulatus Bath to increase the carbon flux via acetyl-CoA towards mevalonate. This engineered M. capsulatus Bath produced the maximum concentration of 2,090 mg/L mevalonate from methane, which is the highest amount of synthetic biochemicals produced from methane in methanotrophs. Here we present not only an efficient addition to the genetic engineering toolkit for methano-trophs but also a useful platform for the development of a methanotroph cell factory.

본 논문은 최근 주목을 받고 있는 공정성 인식에서의 상사 역할에 주목하여 그 효과성을 파악하고자 하였다. 연구의 목적은 상사 공정성이 조직 구성원의 조직 지향적 행동에 미치는 영향과 그 심리적 기제를 확인하는 것으로, 다중초점 공정성과 동일시 개념을 바탕으로 조직 구성원이 인식한 상사 공정성이 구성원의 조직지향 조직시민행동(OCBO)와 조직지향 반생산적 직무행동(CWBO)에 미치는 영향을 조사하였다. 상사 공정성이 OCBO 및 CWBO와 유의한 관계가 있을 것으로 보았으며, 구성원의 상사 동일시와 조직 동일시가 상사 공정성과 OCBO 및 CWBO의 관계를 매개할 것이라고 예상하였다. 마지막으로, 상사의 조직 전형성이 조직 동일시를 통한 상사 공정성의 간접적 효과를 조절할 것이라고 보았다. 연구 결과, 상사 공정성은 OCBO와 유의한 정적 관계를 보였으며, CWBO와는 유의한 관계가 나타나지 않았다. 상사공정성과 OCBO, CWBO의 관계에서 상사동일시 및 조직동일시의 매개효과의 경우 상사 공정성의 영향으로 조직 구성원이 조직에 동일했을 때 상사 공정성은 OCBO와 CWBO 모두와 유의한 관계를 갖는 것으로 나타났다. 반면, 구성원이 상사에 동일시 했을때에는 상사 공정성이 OCBO와는 간접적인 정적 관계를 보이지만 CWBO에는 유의한 간접효과가 나타나지 않는 것으로 확인됐다. 마지막으로 상사 조직 전형성의 조절된 매개효과를 확인한 결과, 상사의 조직 전형성은 조직 동일시를 매개로 한 상사 공정성과 OCBO 및 CWBO의 관계를 조절하는 것으로 나타났다. 구체적으로, OCBO를 향한 매개 효과는 정적으로 강화된 반면, CWBO를 향한 매개 효과는 부적으로 강화되었다. 본 연구 결과는 상사 공정성이 조직에 대한 행동으로 확대되는 과정에서 동일시가 형성되는 대상에 따른 차별적 효과를 보여주었으며, 이 때 상사의 조직 전형성이 이를 조절할 수 있음을 확인하였다. 본 연구 결과에 기초하여 함의 및 한계점을 논의하였다.

Enantiomerically pure d-amino acids are important intermediates as chiral building blocks for peptidomimetics and semisynthetic antibiotics. Here, a transcriptional factor-based screening strategy was used for the rapid screening of d-stereospecific amino acid amidase via an enzyme-specific amidophenol substrate. We used a d-threonine amidophenyl derivative to produce 2-aminophenol that serves as a putative enzyme indicator in the presence of d-threonine amidases. Comparative analyses of known bacterial species indicated that several Bacillus strains produce amidase and form putative indicators in culture media. The estimated amidase was cloned and subjected to rapid directed evolution through biosensor cells. Consequently, we characterized the F119A mutation that significantly improved the catalytic activity toward d-alanine, d-threonine, and d-glutamate. Its beneficial effects were confirmed by higher conversions and recurrent applications of the mutant enzyme, compared to the wild-type. This study showed that rapid directed evolution with biosensors coupled to designed substrates is useful to develop biocatalytic processes.

This study presents a novel DNA part characterization technique that increases throughput by combinatorial DNA part assembly, solid plate-based quantitative fluorescence assay for phenotyping, and barcode tagging-based long-read sequencing for genotyping. We confirmed that the fluorescence intensities of colonies on plates were comparable to fluorescence at the single-cell level from a high-end, flow-cytometry device and developed a high-throughput image analysis pipeline. The barcode tagging-based long-read sequencing technique enabled rapid identification of all DNA parts and their combinations with a single sequencing experiment. Using our techniques, forty-four DNA parts (21 promoters and 23 RBSs) were successfully characterized in 72 h without any automated equipment. We anticipate that this high-throughput and easy-to-use part characterization technique will contribute to increasing part diversity and be useful for building genetic circuits and metabolic pathways in synthetic biology.

Antibiotics have been widely used for plasmid-mediated cell engineering. However, continued use of antibiotics increases the metabolic burden, horizontal gene transfer risks, and biomanufacturing costs. There are limited approaches to maintaining multiple plasmids without antibiotics. Herein, we developed an inverter cascade using CRISPRi by building a plasmid containing a single guide RNA (sgRNA) landing pad (pSLiP); this inhibited host cell growth by repressing an essential cellular gene. Anti-sgRNAs on separate plasmids restored cell growth by blocking the expression of growth-inhibitory sgRNAs in pSLiP. We maintained three plasmids in Escherichia coli with a single antibiotic selective marker. To completely avoid antibiotic use and maintain the CRISPRi-based logic inverter cascade, we created a novel d-glutamate auxotrophic E. coli. This enabled the stable maintenance of the plasmid without antibiotics, enhanced the production of the terpenoid, (-)-α-bisabolol, and generation of an antibiotic-resistance gene-free plasmid. CRISPRi is therefore widely applicable in genetic circuits and may allow for antibiotic-free biomanufacturing.

Genetic circuits have been developed for quantitative measurement of enzyme activity, metabolic engineering of strain development, and dynamic regulation of microbial cells. A genetic circuit consists of several bio-elements, including enzymes and regulatory cassettes, that can generate the desired output signal, which is then used as a precise criterion for enzyme screening and engineering. Antagonists and inhibitors are small molecules with inhibitory effects on regulators and enzymes, respectively. In this study, an antagonist and an inhibitor were applied to a genetic circuit for a dynamic detection range. We developed a genetic circuit relying on regulators and enzymes, allowing for straightforward control of its output signal without additional genetic modification. We used para -nitrophenol and alanine as an antagonist of DmpR and inhibitor of tyrosine phenol-lyase, respectively. We show that the antagonist resets the detection range of the genetic circuit similarly to a resistor in an electrical logic circuit. These biological resistors in genetic circuits can be used as a rapid and precise controller of variable outputs with minimal circuit configuration.

Methanol dehydrogenase (Mdh), is a crucial enzyme for utilizing methane and methanol as carbon and energy sources in methylotrophy and synthetic methylotrophy. Engineering of Mdh, especially NAD-dependent Mdh, has thus been actively investigated to enhance methanol conversion. However, its poor catalytic activity and low methanol affinity limit its wider application. In this study, we applied a transcriptional factor-based biosensor for the direct evolution of Mdh from Lysinibacillus xylanilyticus (Lxmdh), which has a relatively high turnover rate and low KM value compared to other wild-type NAD-dependent Mdhs. A random mutant library of Lxmdh was constructed in Escherichia coli and was screened using formaldehyde-detectable biosensors by incubation with low methanol concentrations. Positive clones showing higher fluorescence were selected by fluorescence-activated cell sorting (FACS) system, and their catalytic activities toward methanol were evaluated. The successfully isolated mutants E396V, K318N, and K46E showed high activity, particularly at very low methanol concentrations. In kinetic analysis, mutant E396V, K318N, and K46E had superior methanol conversion efficiency, with 79-, 23-, and 3-fold improvements compared to the wild-type, respectively. These mutant enzymes could thus be useful for engineering synthetic methylotrophy and for enhancing methanol conversion to various useful products.