Novel Signal Peptides and Episomal Plasmid System for Enhanced Protein Secretion in Engineered Bacteroides Species.

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.

Molecular basis for the interference of the Arabidopsis WRKY54-mediated immune response by two sequence-unrelated bacterial effectors.

Arabidopsis thaliana WRKY proteins are potential targets of pathogen-secreted effectors. RESISTANT TO RALSTONIA SOLANACEARUM 1 (RRS1; AtWRKY52) is a well-studied Arabidopsis nucleotide-binding and leucine-rich repeat (NLR) immune receptor carrying a C-terminal WRKY domain that functions as an integrated decoy. RRS1-R recognizes the effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia pseudosolanacearum by direct interaction through its WRKY domain. AvrRps4 and PopP2 were previously shown to interact with several AtWRKYs. However, how these effectors selectively interact with their virulence targets remains unknown. Here, we show that several members of subgroup IIIb of the AtWRKY family are targeted by AvrRps4 and PopP2. We demonstrate that several AtWRKYs induce cell death when transiently expressed in Nicotiana benthamiana, indicating the activation of immune responses. AtWRKY54 was the only cell death-inducing AtWRKY that interacted with both AvrRps4 and PopP2. We found that AvrRps4 and PopP2 specifically suppress AtWRKY54-induced cell death. We also demonstrate that the amino acid residues required for the avirulence function of AvrRps4 and PopP2 are critical for suppressing AtWRKY54-induced cell death. AtWRKY54 residues predicted to form a binding interface with AvrRps4 were predominantly located in the DNA binding domain and necessary for inducing cell death. Notably, one AtWRKY54 residue, E164, contributes to affinity with AvrRps4 and is exclusively present among subgroup IIIb AtWRKYs, yet is located outside of the DNA-binding domain. Surprisingly, AtWRKY54 mutated at E164 evaded AvrRps4-mediated cell death suppression. Taking our observations together, we propose that AvrRp4 and PopP2 specifically target AtWRKY54 to suppress plant immune responses.

Engineered Methylococcus capsulatus Bath for efficient methane conversion to isoprene.

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.

Ptr1 and ZAR1 immune receptors confer overlapping and distinct bacterial pathogen effector specificities.

Some nucleotide-binding and leucine-rich repeat receptors (NLRs) indirectly detect pathogen effectors by monitoring their host targets. In Arabidopsis thaliana, RIN4 is targeted by multiple sequence-unrelated effectors and activates immune responses mediated by RPM1 and RPS2. These effectors trigger cell death in Nicotiana benthamiana, but the corresponding NLRs have yet not been identified. To identify N. benthamiana NLRs (NbNLRs) that recognize Arabidopsis RIN4-targeting effectors, we conducted a rapid reverse genetic screen using an NbNLR VIGS library. We identified that the N. benthamiana homolog of Ptr1 (Pseudomonas tomato race 1) recognizes the Pseudomonas effectors AvrRpt2, AvrRpm1, and AvrB. We demonstrated that recognition of the Xanthomonas effector AvrBsT and the Pseudomonas effector HopZ5 is conferred independently by the N. benthamiana homolog of Ptr1 and ZAR1. Interestingly, the recognition of HopZ5 and AvrBsT is contributed unequally by Ptr1 and ZAR1 in N. benthamiana and Capsicum annuum. In addition, we showed that the RLCK XII family protein JIM2 is required for the NbZAR1-dependent recognition of AvrBsT and HopZ5. The recognition of sequence-unrelated effectors by NbPtr1 and NbZAR1 provides an additional example of convergently evolved effector recognition. Identification of key components involved in Ptr1 and ZAR1-mediated immunity could reveal unique mechanisms of expanded effector recognition.

A plasma membrane nucleotide-binding leucine-rich repeat receptor mediates the recognition of the Ralstonia pseudosolanacearum effector RipY in Nicotiana benthamiana.

Bacterial wilt disease caused by several Ralstonia species is one of the most destructive diseases in Solanaceae crops. Only a few functional resistance genes against bacterial wilt have been cloned to date. Here, we show that the broadly conserved type III secreted effector RipY is recognized by the Nicotiana benthamiana immune system, leading to cell death induction, induction of defense-related gene expression, and restriction of bacterial pathogen growth. Using a multiplexed virus-induced gene-silencing-based N. benthamiana nucleotide-binding and leucine-rich repeat receptor (NbNLR) library, we identified a coiled-coil (CC) nucleotide-binding and leucine-rich repeat receptor (CNL) required for recognition of RipY, which we named RESISTANCE TO RALSTONIA SOLANACEARUM RIPY (RRS-Y). Genetic complementation assays in RRS-Y-silenced plants and stable rrs-y knockout mutants demonstrated that RRS-Y is sufficient to activate RipY-induced cell death and RipY-induced immunity to Ralstonia pseudosolanacearum. RRS-Y function is dependent on the phosphate-binding loop motif of the nucleotide-binding domain but independent of the characterized signaling components ENHANCED DISEASE SUSCEPTIBILITY 1, ACTIVATED DISEASE RESISTANCE 1, and N REQUIREMENT GENE 1 and the NLR helpers NB-LRR REQUIRED FOR HR-ASSOCIATED CELL DEATH-2, -3, and -4 in N. benthamiana. We further show that RRS-Y localization at the plasma membrane is mediated by two cysteine residues in the CC domain and is required for RipY recognition. RRS-Y also broadly recognizes RipY homologs across Ralstonia species. Lastly, we show that the C-terminal region of RipY is indispensable for RRS-Y activation. Together, our findings provide an additional effector/receptor pair system to deepen our understanding of CNL activation in plants.

Diversified host target families mediate convergently evolved effector recognition across plant species.

Recognition of pathogen effectors is a crucial step for triggering plant immunity. Resistance (R) genes often encode for nucleotide-binding leucine-rich repeat receptors (NLRs), and NLRs detect effectors from pathogens to trigger effector-triggered immunity (ETI). NLR recognition of effectors is observed in diverse forms where NLRs directly interact with effectors or indirectly detect effectors by monitoring host guardees/decoys (HGDs). HGDs undergo different biochemical modifications by diverse effectors and expand the effector recognition spectrum of NLRs, contributing robustness to plant immunity. Interestingly, in many cases of the indirect recognition of effectors, HGD families targeted by effectors are conserved across the plant species while NLRs are not. Notably, a family of diversified HGDs can activate multiple non-orthologous NLRs across plant species. Further investigation on HGDs would reveal the mechanistic basis of how the diversification of HGDs confers novel effector recognition by NLRs.

The Ralstonia pseudosolanacearum effector RipE1 is recognized at the plasma membrane by NbPtr1, the Nicotiana benthamiana homologue of Pseudomonas tomato race 1.

The bacterial wilt disease caused by soilborne bacteria of the Ralstonia solanacearum species complex (RSSC) threatens important crops worldwide. Only a few immune receptors conferring resistance to this devastating disease are known so far. Individual RSSC strains deliver around 70 different type III secretion system effectors into host cells to manipulate the plant physiology. RipE1 is an effector conserved across the RSSC and triggers immune responses in the model solanaceous plant Nicotiana benthamiana. Here, we used multiplexed virus-induced gene silencing of the nucleotide-binding and leucine-rich repeat receptor family to identify the genetic basis of RipE1 recognition. Specific silencing of the N. benthamiana homologue of Solanum lycopersicoides Ptr1 (confers resistance to Pseudomonas syringae pv. tomato race 1) gene (NbPtr1) completely abolished RipE1-induced hypersensitive response and immunity to Ralstonia pseudosolanacearum. The expression of the native NbPtr1 coding sequence was sufficient to restore RipE1 recognition in Nb-ptr1 knockout plants. Interestingly, RipE1 association with the host cell plasma membrane was necessary for NbPtr1-dependent recognition. Furthermore, NbPtr1-dependent recognition of RipE1 natural variants is polymorphic, providing additional evidence for the indirect mode of activation of NbPtr1. Altogether, this work supports NbPtr1 relevance for resistance to bacterial wilt disease in Solanaceae.

The broad host range pathogen Sclerotinia sclerotiorum produces multiple effector proteins that induce host cell death intracellularly.

Sclerotinia sclerotiorum is a broad host range necrotrophic fungal pathogen, which causes disease on many economically important crop species. S. sclerotiorum has been shown to secrete small effector proteins to kill host cells and acquire nutrients. We set out to discover novel necrosis-inducing effectors and characterize their activity using transient expression in Nicotiana benthamiana leaves. Five intracellular necrosis-inducing effectors were identified with differing host subcellular localization patterns, which were named intracellular necrosis-inducing effector 1-5 (SsINE1-5). We show for the first time a broad host range pathogen effector, SsINE1, that uses an RxLR-like motif to enter host cells. Furthermore, we provide preliminary evidence that SsINE5 induces necrosis via an NLR protein. All five of the identified effectors are highly conserved in globally sourced S. sclerotiorum isolates. Taken together, these results advance our understanding of the virulence mechanisms employed by S. sclerotiorum and reveal potential avenues for enhancing genetic resistance to this damaging fungal pathogen.

High Cell-Density Cultivation of Methylococcus capsulatus Bath for Efficient Methane-Derived Mevalonate Production.

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.

A Genetically Encoded Biosensor for the Detection of Levulinic Acid.

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.

CRISPRi-based programmable logic inverter cascade for antibiotic-free selection and maintenance of multiple plasmids.

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.

Novel High-Throughput DNA Part Characterization Technique for Synthetic Biology.

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.

Investigation of natural RIN4 variants reveals a motif crucial for function and provides an opportunity to broaden NLR regulation specificity.

Multiple bacterial effectors target RPM1-INTERACTING PROTEIN4 (RIN4), the biochemical modifications of which are recognized by several plant nucleotide-binding and leucine-rich repeat immune receptor (NLR) proteins. Recently, a comparative study of Arabidopsis and apple (Malus domestica) RIN4s revealed that the RIN4 specificity motif (RSM) is critical for NLR regulation. Here, we investigated the extent to which the RSM contributes to the functions of natural RIN4 variants. Functional analysis of 33 natural RIN4 variants from 28 plant species showed that the RSM is generally required yet sometimes dispensable for the RIN4-mediated suppression of NLR auto-activity or effector-triggered NLR activation. Association analysis of the sequences and fire blight resistance gene originating from Malus × robusta 5 (FB_MR5) activation functions of the natural RIN4 variants revealed H167 to be an indispensable residue for RIN4 function in the regulation of NLRs. None of the tested natural RIN4 variants could suppress RESISTANCE TO PSEUDOMONAS SYRINGAE PV. MACULICOLA1 (RPM1) auto-activity and activate FB_MR5. To engineer RIN4 to carry broader NLR compatibility, we generated chimeric RIN4 proteins, several of which could regulate RPM1, RESISTANT TO PSEUDOMONAS SYRINGAE2 (RPS2), and FB_MR5. We propose that the intrinsically disordered nature of RIN4 provides a flexible platform to broaden pathogen recognition specificity by establishing compatibility with otherwise incompatible NLRs.

Single-Cell-Based Screening and Engineering of d-Amino Acid Amidohydrolases Using Artificial Amidophenol Substrates and Microbial Biosensors.

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.

Droplet contact-based spheroid transfer technique as a multi-step assay tool for spheroid arrays.

Hanging drop plates and low-attachment well plates are suitable for a high throughput screening model of a spheroid, because each drop (or well) contains a single spheroid and the spheroid environment are separated from each other. However, uniform spheroid culture on these devices is difficult as the liquid around the spheroid is replaced by direct pipetting, which can cause spheroid damage or loss, and well-to-well variation. If spheroids need to be cultured for a long time or analyzed through chemical treatment of immunostaining, it becomes a more considerable problem as the number of pipetting action increases. To address these problems, we have developed a poly(dimethylsiloxane) (PDMS)-based drop array chip (DAC) and a pillar array chip (PAC) that can apply a droplet contact-based spheroid transfer (DCST) technique to multiple reagent change or washing steps of spheroid assays. Unlike previous DCST devices, 3D-printed mold-based DCST devices showed stable spheroid manipulation during repetitive drop contact and facile transfer of spheroid arrays to the next reagent-loaded DAC while minimizing cross-contamination of the reagents. Compared to the conventional manual or machine pipetting method, the DCST method showed lower user-to-user variation and a higher spheroid retention rate in the manipulation of the spheroid array. Live/dead staining, hypoxia staining, and immunofluorescence staining of the spheroid array were performed on a breast cancer cell line, BT-474. Furthermore, four clearing methods were applied to the spheroid array as a proof of concept, and we have identified the applicability of the DCST platform as a pretreatment platform for whole spheroid analysis.

Syntrophic co-culture of a methanotroph and heterotroph for the efficient conversion of methane to mevalonate.

As the bioconversion of methane becomes increasingly important for bio-industrial and environmental applications, methanotrophs have received much attention for their ability to convert methane under ambient conditions. This includes the extensive reporting of methanotroph engineering for the conversion of methane to biochemicals. To further increase methane usability, we demonstrated a highly flexible and efficient modular approach based on a synthetic consortium of methanotrophs and heterotrophs mimicking the natural methane ecosystem to produce mevalonate (MVA) from methane. In the methane-conversion module, we used Methylococcus capsulatus Bath as a highly efficient methane biocatalyst and optimized the culture conditions for the production of high amounts of organic acids. In the MVA-synthesis module, we used Escherichia coli SBA01, an evolved strain with high organic acid tolerance and utilization ability, to convert organic acids to MVA. Using recombinant E. coli SBA01 possessing genes for the MVA pathway, 61 mg/L (0.4 mM) of MVA was successfully produced in 48 h without any addition of nutrients except methane. Our platform exhibited high stability and reproducibility with regard to cell growth and MVA production. We believe that this versatile system can be easily extended to many other value-added processes and has a variety of potential applications.

Phylogenetic analysis of ABCG subfamily proteins in plants: functional clustering and coevolution with ABCGs of pathogens.

ABCG subfamily proteins are highly enriched in terrestrial plants. Many of these proteins secrete secondary metabolites that repel or inhibit pathogens. To establish why the ABCG subfamily proteins proliferated extensively during evolution, we constructed phylogenetic trees from a broad range of eukaryotic organisms. ABCG proteins were massively duplicated in land plants and in oomycetes, a group of agronomically important plant pathogens, which prompted us to hypothesize that plant and pathogen ABCGs coevolved. Supporting this hypothesis, full-size ABCGs in host plants (Arabidopsis thaliana and Glycine max) and their pathogens (Hyaloperonospora arabidopsidis and Phytophthora sojae, respectively) had similar divergence times and patterns. Furthermore, generalist pathogens with broad ranges of host plants have diversified more ABCGs than their specialist counterparts. The hypothesis was further tested using an example pair of ABCGs that first diverged during multiplication in a host plant and its pathogen: AtABCG31 of A. thaliana and HpaP802307 of H. arabidopsidis. AtABCG31 expression was activated following infection with H. arabidopsidis, and disrupting AtABCG31 led to increased susceptibility to H. arabidopsidis. Together, our results suggest that ABCG genes in plants and their oomycete pathogens coevolved in an arms race, to extrude secondary metabolites involved in the plant's defense response against pathogens.

Machine learning linked evolutionary biosensor array for highly sensitive and specific molecular identification.

Bacteria initiate complicated signaling cascades from the detection of intracellular metabolites or exogenous substances by hundreds of transcription factors, which have been widely investigated as genetically-encoded biosensors for molecular recognition. However, the limited number of transcription factors and their broad substrate specificity result in ambiguity in small molecule identification. This study presents a new small molecule fingerprinting technique using evolutionary biosensor arrays with a machine learning technique that can capture highly specific substrate signals. Employing multiple mutant transcription factors derived from a single transcription factor has effectively circumvented the limited availability of transcription factors induced by a small molecule of our interest. This method achieved up to 95.3% true positive rate for identifying small molecules, and the high-resolution protein engineering technique improved the limit of detection 75-fold. The signal trade-offs with background noises caused by the complex cellular biochemistry of mutant transcription factors enable the biosensor arrays to be more informative in terms of statistical variance. The machine learning technology, coupled with the single transcription factor-driven evolutionary biosensor array, will open new avenues for molecular fingerprinting technologies.

Adaptive laboratory evolution of Escherichia coli lacking cellular byproduct formation for enhanced acetate utilization through compensatory ATP consumption.

Acetate has attracted great attention as a carbon source to develop economically feasible bioprocesses for sustainable bioproducts. Acetate is a less-preferred carbon source and a well-known growth inhibitor of Escherichia coli. In this study, we carried out adaptive laboratory evolution of an E. coli strain lacking four genes (adhE, pta, ldhA, and frdA) involved in acetyl-CoA consumption, allowing the efficient utilization of acetate as its sole carbon and energy source. Four genomic mutations were found in the evolved strain through whole-genome sequencing, and two major mutations (in cspC and patZ) mainly contributed to efficient utilization of acetate and tolerance to acetate. Transcriptomic reprogramming was examined by analyzing the genome-wide transcriptome with different carbon sources. The evolved strain showed high levels of intracellular ATP by upregulation of genes involved in NADH and ATP biosynthesis, which facilitated the production of enhanced green fluorescent protein, mevalonate, and n-butanol using acetate alone. This new strain, given its high acetate tolerance and high ATP levels, has potential as a starting host for cell factories targeting the production of acetyl-CoA-derived products from acetate or of products requiring high ATP levels.

Sensitive and Rapid Phenotyping of Microbes With Soluble Methane Monooxygenase Using a Droplet-Based Assay.

Methanotrophs with soluble methane monooxygenase (sMMO) show high potential for various ecological and biotechnological applications. Here, we developed a high throughput method to identify sMMO-producing microbes by integrating droplet microfluidics and a genetic circuit-based biosensor system. sMMO-producers and sensor cells were encapsulated in monodispersed droplets with benzene as the substrate and incubated for 5 h. The sensor cells were analyzed as the reporter for phenol-sensitive transcription activation of fluorescence. Various combinations of methanotrophs and biosensor cells were investigated to optimize the performance of our droplet-integrated transcriptional factor biosensor system. As a result, the conditions to ensure sMMO activity to convert the starting material, benzene, into phenol, were determined. The biosensor signals were sensitive and quantitative under optimal conditions, showing that phenol is metabolically stable within both cell species and accumulates in picoliter-sized droplets, and the biosensor cells are healthy enough to respond quantitatively to the phenol produced. These results show that our system would be useful for rapid evaluation of phenotypes of methanotrophs showing sMMO activity, while minimizing the necessity of time-consuming cultivation and enzyme preparation, which are required for conventional analysis of sMMO activity.