Utilization of abundant and cheap carbon sources can effectively reduce the production cost and enhance the economic feasibility. Acetate is a promising carbon source to achieve cost‐effective ...microbial processes. In this study, we engineered an Escherichia coli strain to produce itaconic acid from acetate. As acetate is known to inhibit cell growth, we initially screened for a strain with a high tolerance to 10 g/L of acetate in the medium, and the W strain was selected as the host. Subsequently, the WC strain was obtained by overexpression of cad (encoding cis‐aconitate decarboxylase) using a synthetic promoter and 5′ UTR. However, the WC strain produced only 0.13 g/L itaconic acid because of low acetate uptake. To improve the production, the acetate assimilating pathway and glyoxylate shunt pathway were amplified by overexpression of pathway genes as well as its deregulation. The resulting strain, WCIAG4 produced 3.57 g/L itaconic acid (16.1% of theoretical maximum yield) after 88 hr of fermentation with rapid acetate assimilation. These efforts support that acetate can be a potential feedstock for biochemical production with engineered E. coli.
Production of itaconic acid from acetate can be promising strategy to reduce the production cost and enhance the economic feasibility. However, sole expression of synthetic pathway enzyme, cad in E. coli was inefficient in producing itaconic acid due to low acetate assimilation and insufficient glyoxylate shunt activity. Activation of acetate assimilating pathway and glyoxylate shunt could lead significantly increased acetate uptake and itaconic acid production, suggesting acetate can be a potential feedstock for biochemical production.
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•Genetically encoded biosensors are used in evolutionary metabolic engineering.•These biosensors are constructed from natural or artificial genetic parts.•Biosensor characteristics ...are optimized for efficient high-throughput screening.•Genetic parts of biosensors are modified to satisfy screening requirements.
Evolutionary engineering of microbes for the production of metabolites requires efficient screening methods to test vast mutant libraries. Genetically encoded biosensors are regarded as promising screening devices owing to their wide range of detectable ligands and great applicability to high-throughput screening and selection. Here, we reviewed the current progress in design and optimization of biosensors for high-throughput screening of chemicals. First, we summarized genetic parts of biosensors and strategies for their discovery and development. Next, we explained the properties of biosensors that are relevant to high-throughput screening. Finally, we described various methods for tuning biosensors to fulfill requirements of an efficient screening.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
A system-level framework of complex microbe-microbe and host-microbe chemical cross-talk would help elucidate the role of our gut microbiota in health and disease. Here we report a literature-curated ...interspecies network of the human gut microbiota, called NJS16. This is an extensive data resource composed of ∼570 microbial species and 3 human cell types metabolically interacting through >4,400 small-molecule transport and macromolecule degradation events. Based on the contents of our network, we develop a mathematical approach to elucidate representative microbial and metabolic features of the gut microbial community in a given population, such as a disease cohort. Applying this strategy to microbiome data from type 2 diabetes patients reveals a context-specific infrastructure of the gut microbial ecosystem, core microbial entities with large metabolic influence, and frequently produced metabolic compounds that might indicate relevant community metabolic processes. Our network presents a foundation towards integrative investigations of community-scale microbial activities within the human gut.
Ribozymes are functional RNA molecules that can catalyze biochemical reactions. Since the discovery of the first catalytic RNA, various functional ribozymes (e.g., self-cleaving ribozymes, splicing ...ribozymes, RNase P, etc.) have been uncovered, and their structures and mechanisms have been identified. Ribozymes have the advantage of possessing features of “RNA” molecules; hence, they are highly applicable for manipulating various biological systems. To fully employ ribozymes in a broad range of biological applications in synthetic biology, a variety of ribozymes have been developed and engineered. Here, we summarize the main features of ribozymes and the methods used for engineering their functions. We also describe the past and recent efforts towards exploiting ribozymes for effective and novel applications in synthetic biology. Based on studies on their significance in biological applications till date, ribozymes are expected to advance technologies in artificial biological systems.
•Unique features of catalytic RNAs over protein enzymes.•Characteristics of ribozymes and their engineering strategies.•Interesting applications of ribozymes in the field of synthetic biology.•Current challenges of ribozyme-based biotechnology and future perspectives.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Economic production of chemicals from microbes necessitates development of high-producing strains and an efficient screening technology is crucial to maximize the effect of the most popular strain ...improvement method, the combinatorial approach. However, high-throughput screening has been limited for assessment of diverse intracellular metabolites at the single-cell level. Herein, we established a screening platform that couples a microfluidic static droplet array (SDA) and an artificial riboswitch to analyse intracellular metabolite concentration from single microbial cells. Using this system, we entrapped single Escherichia coli cells in SDA to measure intracellular l-tryptophan concentrations. It was validated that intracellular l-tryptophan concentration can be evaluated by the fluorescence from the riboswitch. Moreover, high-producing strains were successfully screened from a mutagenized library, exhibiting up to 145% productivity compared to its parental strain. This platform will be widely applicable to strain improvement for diverse metabolites by developing new artificial riboswitches.
The control of viral outbreaks requires nucleic acid diagnostic tests that are sensitive, simple and fast. Here, we report a highly sensitive and specific one-pot assay for the fluorescence-based ...detection of RNA from pathogens. The assay, which can be performed within 30-50 min of incubation time and can reach a limit of detection of 0.1-attomolar RNA concentration, relies on a sustained isothermal reaction cascade producing an RNA aptamer that binds to a fluorogenic dye. The RNA aptamer is transcribed by the T7 RNA polymerase from the ligation product of a promoter DNA probe and a reporter DNA probe that hybridize with the target single-stranded RNA sequence via the SplintR ligase (a Chlorella virus DNA ligase). In 40 nasopharyngeal SARS-CoV-2 samples, the assay reached positive and negative predictive values of 95 and 100%, respectively. We also show that the assay can rapidly detect a range of viral and bacterial RNAs.
RNA interference (RNAi) is a mechanism in which small interfering RNA (siRNA) silences a target gene. Herein, we describe a DNA hydrogel capable of producing siRNA and interfering with protein ...expression. This RNAi-exhibiting gel (termed I-gel for interfering gel) consists of a plasmid carrying the gene transcribing siRNA against the target mRNA as part of the gel scaffold. The RNAi efficiency of the I-gel has been confirmed by green fluorescent protein (GFP) expression assay and RNA production quantification. The plasmid stability in the I-gel results in an 8-times higher transcription efficiency than that of the free plasmid. We further applied the I-gel to live cells and confirmed its effect in interfering with the GFP expression. The I-gel shows higher RNAi effect than plasmids in free form or complexed with Lipofectamine. This nanoscale hydrogel, which is able to produce RNA in a cell, provides a platform technology for efficient RNAi system.
For the development of clinically useful genotyping methods for SNPs, accuracy, simplicity, sensitivity, and cost‐effectiveness are the most important criteria. Among the methods currently being ...developed for SNP genotyping technology, the ligation‐dependent method is considered the simplest for clinical diagnosis. However, sensitivity is not guaranteed by the ligation reaction alone, and analysis of multiple targets is limited by the detection method. Although CE is an attractive alternative to error‐prone hybridization‐based detection, the multiplex assay process is complicated because of the size‐based DNA separation principle. In this study, we employed the ligase detection reaction coupled with high‐resolution CE‐SSCP to develop an accurate, sensitive, and simple multiplex genotyping method. Ligase detection reaction could amplify ligated products through recurrence of denaturation and ligation reaction, and SSCP could separate these products according to each different structure conformation without size variation. Thus, simple and sensitive SNP analysis can be performed using this method involving the use of similar‐sized probes, without complex probe design steps. We found that this method could not only accurately discriminate base mismatches but also quantitatively detect 37 SNPs of the tp53 gene, which are used as targets in multiplex analysis, using three‐color fluorescence‐labeled probes.
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