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Tyrian purple, mainly composed of 6,6'-dibromoindigo (6BrIG), is an ancient dye extracted from sea snails and was recently demonstrated as a biocompatible semiconductor material. However, its synthesis remains limited due to uncharacterized biosynthetic pathways and the difficulty of regiospecific bromination. Here, we introduce an effective 6BrIG production strategy in Escherichia coli using tryptophan 6-halogenase SttH, tryptophanase TnaA and flavin-containing monooxygenase MaFMO. Since tryptophan halogenases are expressed in highly insoluble forms in E. coli , a flavin reductase (Fre) that regenerates FADH 2 for the halogenase reaction was used as an N-terminal soluble tag of SttH. A consecutive two-cell reaction system was designed to overproduce regiospecifically brominated precursors of 6BrIG by spatiotemporal separation of bromination and bromotryptophan degradation. These approaches led to 315.0 mg l −1 6BrIG production from tryptophan and successful synthesis of regiospecifically dihalogenated indigos. Furthermore, it was demonstrated that 6BrIG overproducing cells can be directly used as a bacterial dye. A two-cell setup containing tryptophanase, a flavin-dependent monooxygenase and a regiospecific halogenase (linked to a flavin reductase as a solubility tag) enables the production of 6,6'-dibromoindigo and other indigoid dyes in Escherichia coli .

BackgroundYarrowia lipolytica, an oleaginous yeast, is a promising platform strain for production of biofuels and oleochemicals as it can accumulate a high level of lipids in response to nitrogen limitation. Accordingly, many metabolic engineering efforts have been made to develop engineered strains of Y. lipolytica with higher lipid yields. Genome-scale model of metabolism (GEM) is a powerful tool for identifying novel genetic designs for metabolic engineering. Several GEMs for Y. lipolytica have recently been developed; however, not many applications of the GEMs have been reported for actual metabolic engineering of Y. lipolytica. The major obstacle impeding the application of Y. lipolytica GEMs is the lack of proper methods for predicting phenotypes of the cells in the nitrogen-limited condition, or more specifically in the stationary phase of a batch culture.ResultsIn this study, we showed that environmental version of minimization of metabolic adjustment (eMOMA) can be used for predicting metabolic flux distribution of Y. lipolytica under the nitrogen-limited condition and identifying metabolic engineering strategies to improve lipid production in Y. lipolytica. Several well-characterized overexpression targets, such as diglyceride acyltransferase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase, were successfully rediscovered by our eMOMA-based design method, showing the relevance of prediction results. Interestingly, the eMOMA-based design method also suggested non-intuitive knockout targets, and we experimentally validated the prediction with a mutant lacking YALI0F30745g, one of the predicted targets involved in one-carbon/methionine metabolism. The mutant accumulated 45% more lipids compared to the wild-type.ConclusionThis study demonstrated that eMOMA is a powerful computational method for understanding and engineering the metabolism of Y. lipolytica and potentially other oleaginous microorganisms.

A simple yet effective dynamic bead-based microarray is necessary for multiplexed high-throughput screening applications in the fields of biology and chemistry. This paper introduces a microfluidic-based dynamic microbead array system using pneumatically driven elastomeric valves integrated with a microchannel in a single polydimethylsiloxane (PDMS) layer that performs the following functions: single-microbead arraying with loading and trapping efficiencies of 100 %, sequential microbead release for selective retrieval of microbeads of interest, and rapid microarray resettability (<1 s). The key feature is the utilization of an elastomeric membrane as a valve for trapping and releasing single microbeads; this membrane is deformable depending on the applied pneumatic pressure, thereby simply providing a dual trap-and-release function. We propose an effective single-microbead-trapping mechanism based on a dynamic flow-change network and a mathematical model as the design criterion of a trapping site. A sequential microbead release technique via a multistep “release-retrap-and-repeat” method was developed for the selective retrieval of trapped microbeads with a simple configuration consisting of a single PDMS layer and a simple macro-to-micro connection. The proposed dynamic microbead array could be a powerful tool for high-throughput multiplex bead-based drug screening or disease diagnosis.

This paper considers the multiple path planning problem to cover a region of interest using vertical takeoff and landing (VTOL) drones. Drones are used not only to explore unknown areas but also to view areas from the air that are difficult for people to approach by cars, boats, etc. Hence, the coverage path planning for drones should work regardless of whether the drone is inside or outside the region of interest. The proposed algorithm starts by adjusting the region of interest based on the capturing area. Once the region of interest is determined, multiple path plans are created based on the pre-derived rules for generating an optimal path under the general case and the drone’s performance, such as speed, maximum flight time, etc. The aim is to minimize the time to cover the whole region of interest. Hence, the following criteria are applied to determine the appropriateness of the generated paths: (1) whether the start and end points of the path are located as close as possible to the drone’s position, (2) whether the number of generated paths is appropriate, and (3) whether the makespan differences between the paths are small. The performance of the proposed algorithm is verified by numerous simulations.

Biofilm formation protects bacteria from stresses including antibiotics and host immune responses. Carbon sources can modulate biofilm formation and host colonization in Vibrio cholerae , but the underlying mechanisms remain unclear. Here, we show that EIIA Glc , a component of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), regulates the intracellular concentration of the cyclic dinucleotide c-di-GMP, and thus biofilm formation. The availability of preferred sugars such as glucose affects EIIA Glc phosphorylation state, which in turn modulates the interaction of EIIA Glc with a c-di-GMP phosphodiesterase (hereafter referred to as PdeS). In a Drosophila model of V. cholerae infection, sugars in the host diet regulate gut colonization in a manner dependent on the PdeS-EIIA Glc interaction. Our results shed light into the mechanisms by which some nutrients regulate biofilm formation and host colonization. Carbon sources can modulate biofilm formation and host colonization in Vibrio cholerae . Here, Heo et al. show that this process is mediated by a component of the PEP:carbohydrate phosphotransferase system (PTS), which regulates c-di-GMP hydrolysis by interacting with a c-di-GMP phosphodiesterase.

Greater than a hundred genetic risk factors for Alzheimer’s disease (AD) have been identified by genome-wide association studies (GWAS), many of which are primarily expressed in microglia. However, the pathogenic role for most of them remains unclear. We sought to assess at scale how AD GWAS hits influence human microglial inflammatory responses. Thus, we conducted CRISPR inhibition (CRISPRi) screens of 119 AD GWAS hits in hiPSC-derived microglia (iMGLs), with reactive oxygen species (ROS) produced in response to the viral mimic poly(I:C) as a readout. Top hits that either decreased or increased ROS in response to poly(I:C) when knocked down were then interrogated in CROP-seq experiments, where CRISPRi was combined with single cell RNA-sequencing (scRNA-seq). These analyses identified 9 unique microglial clusters, including a poly(I:C)-driven inflammatory cluster (2). Emerging evidence supports a pathogenic role of viral infections in AD and our scRNA-seq showed significant overlap with AD-relevant microglial clusters. Knockdown (KD) of two hit genes, MS4A6A and EED , which lead to high levels of ROS in the presence of poly(I:C), increased the proportion of inflammatory cluster 2 cells and led to functionally related changes in gene expression. In addition, KD of MS4A6A reduced the proportion of iMGLs in the disease-associated microglia (DAM) cluster under all conditions, suggesting that MS4A6A may modulate the DAM response. In contrast, KD of hit genes, INPP5D or RABEP1  which led to low levels of ROS in the presence of poly(I:C), did not significantly affect the proportion of cells in inflammatory cluster 2 but rather shaped the inflammatory response. This included upregulation of a poly(I:C)-independent HLA inflammatory cluster (6) by INPP5D KD under all conditions. Despite INPP5D or RABEP1  being involved in disparate biological processes, their perturbation led to similar changes in gene expression, which included changes in genes related to metabolism such as oxidative phosphorylation, suggesting a shared conversion point by which AD GWAS hits affect cell state. Overall, our data begin to elucidate the functional roles played by various AD GWAS hits, including how they regulate and shape inflammatory responses such as those observed in AD.

Wearable electronic devices such as mobile communication devices, portable computers, and various sensors are the latest significant innovations in technology which use the Internet of Things (IoT) to track personal data. Wearable energy harvesters are required to supply electricity to such devices for the convenience of users. In this study, a textile-type triboelectric nanogenerator (T-TENG), produced using commercial electrode fibers, was fabricated to generate electrical energy using external mechanical stimulation. The commercial fiber was an electrode coated with Teflon on a copper wire with a diameter of ~ 320 μm. Using this commercial fiber, a T-TENG was easily fabricated by knitting and weaving. The performance of the T-TENG was analyzed to understand the effect of force and frequency. It was observed that the performance of the T-TENG did not degrade even under harsh conditions and treatment. The textile-type TENG possessed an energy harvesting capability with an output power density of ~ 0.36 W/m2 and could operate electronic devices by charging a capacitor.

This study presents a finger-triggered portable polydimethylsiloxane suction cup that enables equipment-free microfluidic pumping. The key feature of this method is that its operation only involves a “pressing-and-releasing” action for the cup placed at the outlet of a microfluidic device, which transports the fluid at the inlet toward the outlet through a microchannel. This method is simple, but effective and powerful. The cup is portable and can easily be fabricated from a three-dimensional printed mold, used without any pre-treatment, reversibly bonded to microfluidic devices without leakage, and applied to various material-based microfluidic devices. The effect of the suction cup geometry and fabrication conditions on the pumping performance was investigated. Furthermore, we demonstrated the practical applications of the suction cup by conducting an equipment-free pumping of thermoplastic-based microfluidic devices and water-in-oil droplet generation.

This paper introduces a simple method for trapping and releasing single particles, such as microbeads and living cells, using dual-function elastomeric valves. Our key technique is the utilization of the elastomeric valve as a dual-function removable trap instead of a fixed trap and a separate component for releasing trapped particles, thereby enabling a simple yet effective trap-and-release of particles. We designed, fabricated, and characterized a microfluidic-based device for trapping and releasing single beads by controlling elastomeric valves driven by pneumatic pressure and a fluid flow action. The fluid flow is controlled to ensure that beads flowing in a main stream enter into a branch channel. A bead is trapped by deflected elastomeric valves positioned at the entrance of a branch channel. The trapped bead is easily released by removing the applied pressure. The trapping and releasing of single beads of 21 μm in diameter were successfully performed under an optimized pressure and flow rate ratio. Moreover, we confirmed that continuous trapping and releasing of single beads by repeatedly switching elastomeric valves enables the collection of a controllable number of beads. Our simple method can be integrated into microfluidic systems that require single or multiple particle arrays for quantitative and high-throughput assays in applications within the fields of biology and chemistry.

As a means to develop oleaginous biorefinery, Yarrowia lipolytica was utilized to produce ω-hydroxy palmitic acid from glucose using evolutionary metabolic engineering and synthetic FadR promoters for cytochrome P450 (CYP) expression. First, a base strain was constructed to produce free fatty acids (FFAs) from glucose using metabolic engineering strategies. Subsequently, through ethyl methanesulfonate (EMS)-induced random mutagenesis and fluorescence-activated cell sorting (FACS) screening, improved FFA overproducers were screened. Additionally, synthetic promoters containing bacterial FadR binding sequences for CYP expression were designed to respond to the surge of the concentration of FFAs to activate the ω-hydroxylating pathway, resulting in increased transcriptional activity by 14 times from the third day of culture compared to the first day. Then, endogenous alk5 was screened and expressed using the synthetic FadR promoter in the developed strain for the production of ω-hydroxy palmitic acid. By implementing the synthetic FadR promoter, cell growth and production phases could be efficiently decoupled. Finally, in batch fermentation, we demonstrated de novo production of 160 mg/L of ω-hydroxy palmitic acid using FmeN3-TR1-alk5 in nitrogen-limited media. This study presents an excellent example of the production of ω-hydroxy fatty acids using synthetic promoters with bacterial transcriptional regulator (i.e., FadR) binding sequences in oleaginous yeasts.