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By exploiting versatile P450 enzymes, whole-cell biocatalysis can be performed to synthesize valuable compounds in Escherichia coli. However, the insufficient supply of heme limits the whole-cell P450 biocatalytic activity. Here a strategy for improving intracellular heme biosynthesis to enhance the catalytic efficiencies of P450s is reported. After comparing the effects of improving heme transport and biosynthesis on P450 activities, intracellular heme biosynthesis is optimized through the integrated expression of necessary synthetic genes at proper ratios and the assembly of rate-limiting enzymes using DNA-guided scaffolds. The intracellular heme level is fine-tuned by the combined use of mutated heme-sensitive biosensors and small regulatory RNA systems. The catalytic efficiencies of three different P450s, BM3, sca-2, and CYP105D7, are enhanced through fine-tuning heme biosynthesis for the synthesis of hydroquinone, pravastatin, and 7,3',4'-trihydroxyisoflavone as example products of chemical intermediate, drug, and natural product, respectively. This strategy of fine-tuned heme biosynthesis will be generally useful for developing whole-cell biocatalysts involving hemoproteins.

Microbial production of various TCA intermediates and related chemicals through the reductive TCA cycle has been of great interest. However, rumen bacteria that naturally possess strong reductive TCA cycle have been rarely studied to produce these chemicals, except for succinic acid, due to their dependence on fumarate reduction to transport electrons for ATP synthesis. In this study, malic acid (MA), a dicarboxylic acid of industrial importance, was selected as a target chemical for mass production using Mannheimia succiniciproducens, a rumen bacterium possessing a strong reductive branch of the TCA cycle. The metabolic pathway was reconstructed by eliminating fumarase to prevent MA conversion to fumarate. The respiration system of M. succiniciproducens was reconstructed by introducing the Actinobacillus succinogenes dimethylsulfoxide (DMSO) reductase to improve cell growth using DMSO as an electron acceptor. Also, the cell membrane was engineered by employing Pseudomonas aeruginosa cis-trans isomerase to enhance MA tolerance. High inoculum fed-batch fermentation of the final engineered strain produced 61 g/L of MA with an overall productivity of 2.27 g/L/h, which is the highest MA productivity reported to date. The systems metabolic engineering strategies reported in this study will be useful for developing anaerobic bioprocesses for the production of various industrially important chemicals.

Human milk oligosaccharides (HMOs) have recently attracted ever-increasing interest because of their versatile physiological functions. In HMOs, two tetrasaccharides, lacto-N-tetraose (LNT) and lac...

Development of drug-delivery systems that allow simultaneous in vivo imaging has gained much interest. We report a novel strategy to encapsulate metal nanoparticles (NPs) within alginate gel for in vivo imaging. The cell lysate of recombinant Escherichia coli strain, expressing Arabidopsis thaliana phytochelatin synthase and Pseudomonas putida metallothionein genes, was encapsulated within the alginate gel. Incubation of alginate gel with metal ion precursors followed by UV irradiation resulted in the synthesis of high concentrations of metal NPs, such as Au, Ag, CdSe, and EuSe NPs, within the gel. The alginate gel with metal NPs was used as a drug-delivery system by further co-encapsulating doxorubicin and rifampicin, the release of which was made to be pH-dependent. This system can be conveniently and safely used for in vitro and in vivo bioimaging, enabled by the metal NPs formed within the gel matrix without using toxic reducing reagents or surfactants.

Bolaamphiphile, which is a class of amphiphilic molecules, has a unique structure of two hydrophilic head groups at the ends of the hydrophobic center. Peptidic bolaamphiphiles that employ peptides or amino acids as their hydrophilic groups exhibit unique biochemical activities when they self-organize into supramolecular structures, which are not observed in a single molecule. The self-assembled peptidic bolaamphiphiles hold considerable promise for imitating proteins with biochemical activities, such as specific affinity toward heterogeneous substances, a catalytic activity similar to a metalloenzyme, physicochemical activity from harmonized amino acid segments, and the capability to encapsulate genes like a viral vector. These diverse activities give rise to large research interest in biomaterials engineering, along with the synthesis and characterization of the assembled structures. This review aims to address the recent progress in the applications of peptidic bolaamphiphile assemblies whose densely packed peptide motifs on their surface and their stacked hydrophobic centers exhibit unique protein-like activity and designer functionality, respectively.

Bio-based production of industrially important chemicals and materials from non-edible and renewable biomass has become increasingly important to resolve the urgent worldwide issues including climate change. Also, bio-based production, instead of chemical synthesis, of food ingredients and natural products has gained ever increasing interest for health benefits. Systems metabolic engineering allows more efficient development of microbial cell factories capable of sustainable, green, and human-friendly production of diverse chemicals and materials. Escherichia coli is unarguably the most widely employed host strain for the bio-based production of chemicals and materials. In the present paper, we review the tools and strategies employed for systems metabolic engineering of E. coli. Next, representative examples and strategies for the production of chemicals including biofuels, bulk and specialty chemicals, and natural products are discussed, followed by discussion on materials including polyhydroxyalkanoates (PHAs), proteins, and nanomaterials. Lastly, future perspectives and challenges remaining for systems metabolic engineering of E. coli are discussed.

The textile industry has caused severe water pollution by using many toxic chemicals for producing fabric dyes. In response to this problem, indigoidine has attracted attention as an alternative na...

Multidrug resistance (MDR) of cancer cells reduces chemotherapeutic efficacy by preventing drug accumulation in the cells through a drug efflux pump and lysosomal sequestration/exocytosis. Herein, to overcome such anticancer resistance, lysosome-targeted self-assembly of perylene diimide (PDI) derivatives is presented as a powerful strategy for effective and selective anticancer therapy. Stimulated by the lysosomal low pH, the amphiphilic PDI derivatives functionalized with amino acids (PDI-AAs) construct fibrous self-assembled structures inside the lysosomes, causing cancer cell apoptosis by lysosomal rupture. In contrast, negligible apoptosis was observed from normal cells by PDI-AA. The agglomerated fibrous assemblies were not removed by lysosomal exocytosis, thereby displaying a 10.7-fold higher anticancer efficacy on MDR cancer cells compared to a doxorubicin chemotherapeutic agent. The MDR-circumventing capability, along with high selectivity toward cancer cells, supports PDI-AAs as potential candidates for the treatment of MDR cancer cells by lysosome-targeted self-assembly.

Genome-scale metabolic modeling and simulation have been widely employed in biological studies and biotechnological applications due to their powerful capabilities of estimating metabolic fluxes at the systems level. In recent years, machine learning (ML) has been beginning to be applied to the reconstruction and analysis of genome-scale metabolic models (GEMs) to improve their quality. Also, ML has been used to diversify the utilization of information derived from genome-scale metabolic modeling and simulation. Recent studies have shown that machine learning can improve predictive performance and data coverage of GEMs. Also, genome-scale metabolic modeling and simulation provide interpretability of ML applications. Although many biological data still need to be made suitable for ML applications, it is expected that ML will be increasingly applied to GEMs to further improve the practical use and find new applications of GEMs.

Sustainable production of chemicals and materials from renewable non-food biomass using biorefineries has become increasingly important in an effort toward the vision of 'net zero carbon' that has recently been pledged by countries around the world. Systems metabolic engineering has allowed the efficient development of microbial strains overproducing an increasing number of chemicals and materials, some of which have been translated to industrial-scale production. Fermentation is one of the key processes determining the overall economics of bioprocesses, but has recently been attracting less research attention. In this Review, we revisit and discuss factors affecting the competitiveness of bacterial fermentation in connection to strain development by systems metabolic engineering. Future perspectives for developing efficient fermentation processes are also discussed.

By exploiting versatile P450 enzymes, whole-cell biocatalysis can be performed to synthesize valuable compounds in Escherichia coli. However, the insufficient supply of heme limits the whole-cell P450 biocatalytic activity. Here we report a strategy of improving intracellular heme supply for enhancing the catalytic efficiencies of P450s. After comparing the effects of enhancing heme transport and biosynthesis on P450 activities, intracellular heme supply was optimized through the integrated expression of necessary synthetic genes at proper ratio and the assembly of rate-limiting enzymes using DNA-guided scaffolds. By the combined use of mutated heme-sensitive biosensor and small regulatory RNA systems, the intracellular heme level was fine-tuned. The catalytic efficiencies of three different P450s, BM3, sca-2 and CYP105D7, were enhanced through fine-tuning heme supply for the synthesis of hydroquinone, pravastatin, and 7,3',4'-trihydroxyisoflavone as examples of chemical intermediate, drug, and natural product, respectively. This strategy will be useful to produce other hemoproteins with high activities.

Heme has recently attracted much attention due to its promising applications in the food and healthcare industries. However, the current titers and productivities of heme produced by recombinant microorganisms are not high enough for a wide range of applications. In this study, the process for the fermentation of the metabolically engineered Escherichia coli HAEM7 strain was optimized for the high-level production of heme. To improve the production of heme, different carbon sources, iron concentration in the medium, pH control strategies, induction points, and iron content in the feeding solution were examined. Moreover, strategies of increasing cell density, regular iron supplementation, and supply of excess feeding solution were developed to further improve the production of heme. In the optimized fermentation process, the HAEM7 strain produced 1.03 g/L heme with productivity of 21.5 mg/L/h. The fermentation process and strategies reported here will expedite establishing industry-level production of heme.

There is an evergrowing demand for environment-friendly processes to synthesize ammonia (NH

Exacerbation of climate change and air pollution around the world have emphasized the necessity of replacing fossil fuels with clean and sustainable energy. Metabolic engineering has provided strategies to engineer diverse organisms for the production of biofuels from renewable carbon sources. Although some of the processes are commercialized, there has been continued effort to produce advanced biofuels with higher efficiencies. In this article, metabolic engineering strategies recently exploited to enhance biofuel production and facilitate utilization of non-edible low-value carbon sources are reviewed. The strategies include engineering enzymes, exploiting new pathways, and systematically optimizing metabolism and fermentation processes, among others. In addition, metabolic and bioprocess engineering strategies to achieve competitiveness of current biofuel production systems compared with fossil fuels are discussed.

Non-ribosomal peptides (NRPs) are a family of secondary metabolites with the highest number among entire secondary metabolite types. They are biosynthesized by a multi-modular enzyme complex called non-ribosomal peptide synthetase (NRPS), which is encoded by a biosynthetic gene cluster (BGC) in plants and a special group of microorganisms. NRPs are structurally and functionally diverse with numerous industrial applications. However, native producers of these valuable NRPs have several biotechnological limitations for efficient production, including their slow growth, inefficient genetic manipulations, and silent BGCs. Heterologous expression of NRPS can address these challenges, especially using an array of model organisms with well-studied metabolic networks and readily available genetic engineering tools. Here, we review the applications of representative bacterial heterologous hosts, namely representative model Streptomyces species, Escherichia coli, Pseudomonas putida, and Bacillus subtilis, which have been engineered for the enhanced production of NRPs.

There is increasing industrial demand for five-carbon platform chemicals, particularly glutaric acid, a widely used building block chemical for the synthesis of polyesters and polyamides. Here we report the development of an efficient glutaric acid microbial producer by systems metabolic engineering of an l-lysine-overproducing Corynebacterium glutamicum BE strain. Based on our previous study, an optimal synthetic metabolic pathway comprising Pseudomonas putida l-lysine monooxygenase (davB) and 5-aminovaleramide amidohydrolase (davA) genes and C. glutamicum 4-aminobutyrate aminotransferase (gabT) and succinate-semialdehyde dehydrogenase (gabD) genes, was introduced into the C. glutamicum BE strain. Through system-wide analyses including genome-scale metabolic simulation, comparative transcriptome analysis, and flux response analysis, 11 target genes to be manipulated were identified and expressed at desired levels to increase the supply of direct precursor l-lysine and reduce precursor loss. A glutaric acid exporter encoded by ynfM was discovered and overexpressed to further enhance glutaric acid production. Fermentation conditions, including oxygen transfer rate, batch-phase glucose level, and nutrient feeding strategy, were optimized for the efficient production of glutaric acid. Fed-batch culture of the final engineered strain produced 105.3 g/L of glutaric acid in 69 h without any byproduct. The strategies of metabolic engineering and fermentation optimization described here will be useful for developing engineered microorganisms for the high-level bio-based production of other chemicals of interest to industry.

Benzoic acid (BA) is an important platform aromatic compound in chemical industry and is widely used as food preservatives in its salt forms. Yet, current manufacture of BA is dependent on petrochemical processes under harsh conditions. Here we report the de novo production of BA from glucose using metabolically engineered Escherichia coli strains harboring a plant-like β-oxidation pathway or a newly designed synthetic pathway. First, three different natural BA biosynthetic pathways originated from plants and one synthetically designed pathway were systemically assessed for BA production from glucose by in silico flux response analyses. The selected plant-like β-oxidation pathway and the synthetic pathway were separately established in E. coli by expressing the genes encoding the necessary enzymes and screened heterologous enzymes under optimal plasmid configurations. BA production was further optimized by applying several metabolic engineering strategies to the engineered E. coli strains harboring each metabolic pathway, which included enhancement of the precursor availability, removal of competitive reactions, transporter engineering, and reduction of byproduct formation. Lastly, fed-batch fermentations of the final engineered strain harboring the β-oxidation pathway and the strain harboring the synthetic pathway were conducted, which resulted in the production of 2.37 ± 0.02 g/L and 181.0 ± 5.8 mg/L of BA from glucose, respectively; the former being the highest titer reported by microbial fermentation. The metabolic engineering strategies developed here will be useful for the production of related aromatics of high industrial interest.

Fine control of the expression levels of proteins constitutes a major challenge in synthetic biology and metabolic engineering. However, the dependence of translation initiation on the downstream coding sequence (CDS) obscures accurate prediction of the protein expression levels from mRNA sequences. Here, we present a tunable gene-expression system comprising 24 expression cassettes that produce predefined relative expression levels of proteins ranging from 0.001 to 1 without being influenced by the downstream CDS. To validate the practical utility of the tunable expression system, it was applied to a synthetic circuit displaying three states of fluorescence depending on the difference in protein expression levels. To demonstrate the suitability of application to metabolic engineering, this system was used to diversify the levels of key metabolic enzymes. As a result, expression-optimized strains were capable of producing 2.25 g/L of cadaverine, 2.59 g/L of L-proline, and 95.7 mg/L of 1-propanol. Collectively, the tunable expression system could be utilized to optimize genetic circuits for desired operation and to optimize metabolic fluxes through biosynthetic pathways for enhancing production yields of bioproducts. This tunable system will be useful for studying basic and applied biological sciences in addition to applications in synthetic biology and metabolic engineering.

Inorganic nanomaterials are widely used in chemical, electronics, photonics, energy and medical industries. Preparing a nanomaterial (NM) typically requires physical and/or chemical methods that involve harsh and environmentally hazardous conditions. Recently, wild-type and genetically engineered microorganisms have been harnessed for the biosynthesis of inorganic NMs under mild and environmentally friendly conditions. Microorganisms such as microalgae, fungi and bacteria, as well as bacteriophages, can be used as biofactories to produce single-element and multi-element inorganic NMs. This Review describes the emerging area of inorganic NM biosynthesis, emphasizing the mechanisms of inorganic-ion reduction and detoxification, while also highlighting the proteins and peptides involved. We show how analysing a Pourbaix diagram can help us devise strategies for the predictive biosynthesis of NMs with high producibility and crystallinity and also describe how to control the size and morphology of the product. Here, we survey biosynthetic inorganic NMs of 55 elements and their applications in catalysis, energy harvesting and storage, electronics, antimicrobials and biomedical therapy. Furthermore, a step-by-step flow chart is presented to aid the design and biosynthesis of inorganic NMs employing microbial cells. Future research in this area will add to the diversity of available inorganic NMs but should also address scalability and purity. The biosynthesis of inorganic nanomaterials in microorganisms is an environmentally friendly alternative to chemical synthesis. This Review describes the engineering of microorganisms to rationally prepare nanomaterials for diverse applications.

Microbial production of fatty acids and derivatives from non-edible biomass has attracted much attention as an alternative to their production from plant oils and animal fats. Fatty acids and some of their derivatives are ubiquitous metabolites synthesized for membrane biosynthesis and other metabolic purposes in microorganisms. These compounds, however, are rarely produced beyond cellular demands, frequently resulting in low titers even after metabolic engineering. Recently, more advanced metabolic engineering strategies including systems metabolic engineering allowed improved production of fatty acids and their derivatives by employing non-oleaginous and oleaginous microorganisms. Here, we review metabolic engineering strategies developed for the production of fatty acids and derivative chemicals by non-oleaginous and oleaginous microorganisms in recent years.

This study sheds new light on the relationship between emotion and engagement. Specifically, we investigate how the six discrete emotions that news visuals deliver, as well as the positiveness of news text, are associated with three engagement activities: sharing, commenting, and reacting. The findings show that users are less likely to share or comment on news posts that convey positive emotions, although they tend to react to such news frequently. The most prominent kind of emotion associated with user engagement activities was “sadness.” We analyzed 12,179 news stories posted on the four major U.S. newspapers’ Facebook pages.

Bacterial cellulose (BC) has outstanding physical and chemical properties, including high crystallinity, moisture retention, and tensile strength. Currently, the major producer of BC is Komagataeibacter xylinus. However, due to limited tools of expression, this host is difficult to engineer metabolically to improve BC productivity. In this study, a regulated expression system for K. xylinus with synthetic ribosome binding site (RBS) was developed and used to engineer a BC biosynthesis pathway. A synthetic RBS library was constructed using green fluorescent protein (GFP) as a reporter, and three synthetic RBSs (R4, R15, and R6) with different strengths were successfully isolated by fluorescence-activated cell sorting (FACS). Using synthetic RBS, we optimized the expression of three homologous genes responsible for BC production, pgm, galU, and ndp, and thereby greatly increased it under both static and shaking culture conditions. The final titer of BC under static and shaking conditions was 5.28 and 3.67 g/l, respectively. Our findings demonstrate that reinforced metabolic flux towards BC through quantitative gene expression represents a practical strategy for the improvement of BC productivity.

A comprehensive method to prepare a one-dimensional (1D) metal–organic framework (MOF) has attracted research interest because the 1D MOFs are useful as precursor materials for the preparation of highly porous carbon nanorods with outstanding electrical conductivity and mechanical strength, making them particularly suitable for electrochemical applications. Herein, the synthesis of 1D zeolitic imidazolate framework-8 (ZIF-8) nanorods is reported using the metal-induced self-assembly templates of imidazole-functionalized perylenetetracarboxylic diimide (PDI-Hm). The size of PDI-Hm self-assemblies is finely tuned on the nanoscale by the method of metal-induced self-assembly whose surface-exposed metal ions were further exploited as nucleation sites for the growth of ZIF-8. Versatility of the metal-induced self-assembly template for the growth of other 1D MOFs was demonstrated using various transition-metal ions on demands. The size-controlled ZIF-8 nanorods were applied further as a precursor material to produce porous, nitrogen-doped carbon nanorods through the carbonization. The carbon nanorods show decent supercapacitor electrode material performance, with enhanced specific capacitance of 292.2 F g–1, because of their unique 1D feature with reduced charge transfer resistance and large specific surface area derived from a downscaled template size under 100 nm.

Streptomycetes are prominent sources of bioactive natural products, but metabolic engineering of the natural products of these organisms is greatly hindered by relatively inefficient genetic manipulation approaches. New advances in genome editing techniques, particularly CRISPR-based tools, have revolutionized genetic manipulation of many organisms, including actinomycetes. We have developed a comprehensive CRISPR toolkit that includes several variations of 'classic' CRISPR-Cas9 systems, along with CRISPRi and CRISPR-base editing systems (CRISPR-BEST) for streptomycetes. Here, we provide step-by-step protocols for designing and constructing the CRISPR plasmids, transferring these plasmids to the target streptomycetes, and identifying correctly edited clones. Our CRISPR toolkit can be used to generate random-sized deletion libraries, introduce small indels, generate in-frame deletions of specific target genes, reversibly suppress gene transcription, and substitute single base pairs in streptomycete genomes. Furthermore, the toolkit includes a Csy4-based multiplexing option to introduce multiple edits in a single experiment. The toolkit can be easily extended to other actinomycetes. With our protocol, it takes

Membraneless organelles formed by liquid-liquid phase separation of proteins or nucleic acids are involved in diverse biological processes in eukaryotes. However, such cellular compartments have yet to be discovered or created synthetically in prokaryotes. Here, we report the formation of liquid protein condensates inside the cells of prokaryotic Escherichia coli upon heterologous overexpression of intrinsically disordered proteins such as spider silk and resilin. In vitro reconstitution under conditions that mimic intracellular physiologically crowding environments of E. coli revealed that the condensates are formed via liquid-liquid phase separation. We also show functionalization of these condensates via targeted colocalization of cargo proteins to create functional membraneless compartments able to fluoresce and to catalyze biochemical reactions. The ability to form and functionalize membraneless compartments may serve as a versatile tool to develop artificial organelles with on-demand functions in prokaryotes for applications in synthetic biology.

Biological knowledge accumulated over the decades and advances in computational methods have facilitated the implementation of pan-genome analysis that aims at better understanding of genotype-phenotype associations of a specific group of organisms. Pan-genome analysis has been shown to be an effective approach to better understand a clade of pathogenic bacteria because it helps developing various and tailored therapeutic strategies on the basis of their biological similarities and differences. Here, we review recent progress in the pan-genome analysis of pathogenic bacteria. In particular, we focus on computational tools that allow streamlined pan-genome analysis. Also, various applications of pan-genome analysis including those relevant to devising strategies for the prevention and treatment of pathogenic bacteria are reviewed.

As climate change is an important environmental issue, the conventional petrochemical-based processes to produce valuable chemicals are being shifted toward eco-friendly biological-based processes. In this study, 3-hydroxypropionic acid (3-HP), an industrially important three carbon (C3) chemical, was overproduced by metabolically engineered Escherichia coli using glycerol as a sole carbon source. As the first step to construct a glycerol-dependent 3-HP biosynthetic pathway, the dhaB1234 and gdrAB genes from Klebsiella pneumoniae encoding glycerol dehydratase and glycerol reactivase, respectively, were introduced into E. coli to convert glycerol into 3-hydroxypropionaldehyde (3-HPA). In addition, the ydcW gene from K. pneumoniae encoding γ-aminobutyraldehyde dehydrogenase, among five aldehyde dehydrogenases examined, was selected to further convert 3-HPA to 3-HP. Increasing the expression level of the ydcW gene enhanced 3-HP production titer and reduced 1,3-propanediol production. To enhance 3-HP production, fed-batch fermentation conditions were optimized by controlling dissolved oxygen (DO) level and employing different feeding strategies including intermittent feeding, pH-stat feeding, and continuous feeding strategies. Fed-batch culture of the final engineered E. coli strain with DO control and continuous feeding strategy produced 76.2 g/L of 3-HP with the yield and productivity of 0.457 g/g glycerol and 1.89 g/L/h, respectively. To the best of our knowledge, this is the highest 3-HP productivity achieved by any microorganism reported to date.

Acrylic acid (AA) is an important industrial chemical used for several applications including superabsorbent polymers and acrylate esters. Here, we report the development of a new biosynthetic pathway for the production of AA from glucose in metabolically engineered Escherichia coli through the β-alanine (BA) route. The AA production pathway was partitioned into two modules: an AA forming downstream pathway and a BA forming upstream pathway. We first validated the operation of the downstream pathway in vitro and in vivo, and then constructed the downstream pathway by introducing efficient enzymes (Act, Acl2, and YciA) screened out of various microbial sources and optimizing the expression levels. For the direct fermentative production of AA from glucose, the downstream pathway was introduced into the BA producing E. coli strain. The resulting strain could successfully produce AA from glucose in flask cultivation. AA production was further enhanced by expressing the upstream genes (panD and aspA) under the constitutive BBa_J23100 promoter. Replacement of the native promoter of the acs gene with the BBa_J23100 promoter in the genome increased AA production to 55.7 mg/L in flask. Fed-batch fermentation of the final engineered strain allowed production of 237 mg/L of AA in 57.5 h, representing the highest AA titer reported to date.

As many regions around the world are facing water scarcity, reverse osmosis (RO) has attracted attention to supply fresh water to such areas. To design and develop energy-efficient RO processes, accurate osmotic pressure values of salt water are critical, yet conventional models of osmotic pressure have significant deviations from the actual values. In addition, absence or high charge of authentic osmotic pressure databases prevents accessing authentic data. Here, we propose combining the Chang correction factor, a polynomial in solution concentration, with traditional osmotic pressure models to approximate the osmotic pressure of dilute to concentrated solutions with high accuracy. The Chang correction factor is determined by regressing a handful of authentic osmotic pressure data divided by theo- retical values calculated using traditional models. Multiplication of resulting polynomials back to corresponding tradi- tional models enables accurate approximation of the authentic osmotic pressure of dilute to concentrated solutions with R2 approaching 1. In addition, generality of the strategy over aqueous and organic solutions is demonstrated by approx- imating osmotic pressure of NaCl and sucrose aqueous solutions and C2H4Cl2-C6H6 and C3H6Br2-C2H4Br2 organic solutions. The approximation strategy proposed and assessed here will be useful to simulate and develop processes for seawater desalination and various industries with high importance.

Nylons are widely used polyamides produced in large amounts from petrochemical-derived monomers. The production of fully biobased nylons using fermentatively produced diacids and diamines has been ...

Microbial production of chemicals and materials from renewable carbon sources is becoming increasingly important to help establish sustainable chemical industry. In this paper, we review current status of metabolic engineering for the bio-based production of linear and saturated dicarboxylic acids and diamines, important platform chemicals used in various industrial applications, especially as monomers for polymer synthesis. Strategies for the bio-based production of various dicarboxylic acids having different carbon numbers including malonic acid (C3), succinic acid (C4), glutaric acid (C5), adipic acid (C6), pimelic acid (C7), suberic acid (C8), azelaic acid (C9), sebacic acid (C10), undecanedioic acid (C11), dodecanedioic acid (C12), brassylic acid (C13), tetradecanedioic acid (C14), and pentadecanedioic acid (C15) are reviewed. Also, strategies for the bio-based production of diamines of different carbon numbers including 1,3-diaminopropane (C3), putrescine (1,4-diaminobutane; C4), cadaverine (1,5-diaminopentane; C5), 1,6-diaminohexane (C6), 1,8-diaminoctane (C8), 1,10-diaminodecane (C10), 1,12-diaminododecane (C12), and 1,14-diaminotetradecane (C14) are revisited. Finally, future challenges are discussed towards more efficient production and commercialization of bio-based dicarboxylic acids and diamines.

Supplementary information is available for this paper at https://doi.org/10.1038/s41587-020-0446-y, Reconstructing metabolic reaction networks enables the development of testable hypotheses of an organisms metabolism under different conditions1. State-of-the-art genome-scale metabolic models (GEMs) can include thousands of metabolites and reactions that are assigned to subcellular locations. Geneproteinreaction (GPR) rules and annotations using database information can add meta-information to GEMs. GEMs with metadata can be built using standard reconstruction protocols2, and guidelines have been put in place for tracking provenance and enabling interoperability, but a standardized means of quality control for GEMs is lacking3. Here we report a community effort to develop a test suite named MEMOTE (for metabolic model tests) to assess GEM quality., We acknowledge D. Dannaher and A. Lopez for their supporting work on the Angular parts of MEMOTE; resources and support from the DTU Computing Center; J. Cardoso, S. Gudmundsson, K. Jensen and D. Lappa for their feedback on conceptual details; and P. D. Karp and I. Thiele for critically reviewing the manuscript. We thank J. Daniel, T. Kristjánsdóttir, J. Saez-Saez, S. Sulheim, and P. Tubergen for being early adopters of MEMOTE and for providing written testimonials. J.O.V. received the Research Council of Norway grants 244164 (GenoSysFat), 248792 (DigiSal) and 248810 (Digital Life Norway); M.Z. received the Research Council of Norway grant 244164 (GenoSysFat); C.L. received funding from the Innovation Fund Denmark (project “Environmentally Friendly Protein Production (EFPro2)”); C.L., A.K., N. S., M.B., M.A., D.M., P.M, B.J.S., P.V., K.R.P. and M.H. received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 686070 (DD-DeCaF); B.G.O., F.T.B. and A.D. acknowledge funding from the US National Institutes of Health (NIH, grant number 2R01GM070923-13); A.D. was supported by infrastructural funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), Cluster of Excellence EXC 2124 Controlling Microbes to Fight Infections; N.E.L. received funding from NIGMS R35 GM119850, Novo Nordisk Foundation NNF10CC1016517 and the Keck Foundation; A.R. received a Lilly Innovation Fellowship Award; B.G.-J. and J. Nogales received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no 686585 for the project LIAR, and the Spanish Ministry of Economy and Competitivity through the RobDcode grant (BIO2014-59528-JIN); L.M.B. has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 633962 for project P4SB; R.F. received funding from the US Department of Energy, Offices of Advanced Scientific Computing Research and the Biological and Environmental Research as part of the Scientific Discovery Through Advanced Computing program, grant DE-SC0010429; A.M., C.Z., S.L. and J. Nielsen received funding from The Knut and Alice Wallenberg Foundation, Advanced Computing program, grant #DE-SC0010429; S.K.’s work was in part supported by the German Federal Ministry of Education and Research (de.NBI partner project “ModSim” (FKZ: 031L104B)); E.K. and J.A.H.W. were supported by the German Federal Ministry of Education and Research (project “SysToxChip”, FKZ 031A303A); M.K. is supported by the Federal Ministry of Education and Research (BMBF, Germany) within the research network Systems Medicine of the Liver (LiSyM, grant number 031L0054); J.A.P. and G.L.M. acknowledge funding from US National Institutes of Health (T32-LM012416, R01-AT010253, R01-GM108501) and the Wagner Foundation; G.L.M. acknowledges funding from a Grand Challenges Exploration Phase I grant (OPP1211869) from the Bill & Melinda Gates Foundation; H.H. and R.S.M.S. received funding from the Biotechnology and Biological Sciences Research Council MultiMod (BB/N019482/1); H.U.K. and S.Y.L. received funding from the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (grants NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) from the Ministry of Science and ICT through the National Research Foundation (NRF) of Korea; H.U.K. received funding from the Bio & Medical Technology Development Program of the NRF, the Ministry of Science and ICT (NRF-2018M3A9H3020459); P.B., B.J.S., Z.K., B.O.P., C.L., M.B., N.S., M.H. and A.F. received funding through Novo Nordisk Foundation through the Center for Biosustainability at the Technical University of Denmark (NNF10CC1016517); D.-Y.L. received funding from the Next-Generation BioGreen 21 Program (SSAC, PJ01334605), Rural Development Administration, Republic of Korea; G.F. was supported by the RobustYeast within ERA net project via SystemsX.ch; V.H. received funding from the ETH Domain and Swiss National Science Foundation; M.P. acknowledges Oxford Brookes University; J.C.X. received support via European Research Council (666053) to W.F. Martin; B.E.E. acknowledges funding through the CSIRO-UQ Synthetic Biology Alliance; C.D. is supported by a Washington Research Foundation Distinguished Investigator Award. I.N. received funding from National Institutes of Health (NIH)/National Institute of General Medical Sciences (NIGMS) (grant P20GM125503)., info:eu-repo/semantics/publishedVersion

What do citizens demand of their governing bodies to cope with the spread of emerging infectious diseases after recognizing the growing danger? What are the similarities and differences in political participation via online citizen petitions regarding COVID-19 across cities with different degrees of pandemic experience? This study aims to answer these questions by examining citizen petitions regarding the COVID-19 pandemic in urban areas of South Korea. The pattern of citizens' requests is a part of integrative socio-ecological and political systems with spatial and temporal dimensions. We compare the pattern of online citizen petitions in four Korean cities, namely Seoul, Busan, Daegu, and Incheon, some of which were epicenters of the COVID-19 outbreak. By applying relevant big data analysis techniques such as text mining, topic modeling, and network analysis, we compare the characteristics of citizen petitions on COVID-19 in the four cities, particularly whether (and how) they want financial or welfare support or COVID-19 prevention. We find that cities that experience a rapid spread are likely to have more petitions for prevention than for support. By comparison, cities without such experience are likely to have more petitions for support. This study contributes by tracing citizen and local government interactions in response to emerging infectious diseases by empirically analyzing the related big data on petitions. Policy implications suggest that urban authorities should listen to analyze and respond to the urgent needs of citizens.

Significance A gene regulatory system is an important tool for the engineering of biosynthetic pathways of organisms. Here, we report the development of an inducible-ON/OFF regulatory system using a malO operator as a key element. We identified and modulated sequence, position, numbers, and spacing distance of malO operators, generating a series of activating or repressive promoters with tunable strength. The stringency and robustness are both guaranteed in this system, a maximal induction factor of 790-fold was achieved, and nine proteins from different organisms were expressed with high yields. This system can be utilized as a gene switch, promoter enhancer, or metabolic valve in synthetic biology applications. This operator-based engineering strategy can be employed for developing similar regulatory systems in different microorganisms.