Stepwise Flux Optimization for Enhanced GABA Production from Acetate in Escherichia coli.

Strategic allocation of metabolic flux is essential for achieving a higher production performance in genetically engineered organisms. Flux optimization between cell growth and chemical production has led to the establishment of cost-effective chemical production methods in microbial cell factories. This effect is amplified when utilizing a low-cost carbon source. γ-Aminobutyric acid (GABA), crucial in pharmaceuticals and biodegradable polymers, can be efficiently produced from acetate, a cost-effective substrate. However, a balanced distribution of acetate-derived flux is essential for optimizing the production without hindering growth. In this study, we demonstrated GABA production from acetate using Escherichia coli by focusing on optimizing the metabolic flux at isocitrate and α-ketoglutarate nodes. Through a series of flux optimizations, the final strain produced 2.54 g/L GABA from 5.91 g/L acetate in 24 h (0.43 g/g yield). These findings suggest that delicate flux balancing with the application of a cheap substrate can contribute to cost-effective production of GABA.

Convergent Synthesis of Two Heterogeneous Fluxes from Glucose and Acetate for High-Yield Citramalate Production.

Biological production of citramalate has garnered attention due to its wide application for food additives and pharmaceuticals, although improvement of yield is known to be challenging. When glucose is used as the sole carbon source, carbon loss through decarboxylation steps for providing acetyl-CoA from pyruvate is inevitable. To avoid this, we engineered a strain to co-utilize glucose and cost-effective acetate while preventing carbon loss for enhancing citramalate production. The production pathway diverged to independently supply the precursors required for the synthesis of citramalate from glucose and acetate, respectively. Moreover, the phosphotransferase system was inactivated and the acetate assimilation pathway and the substrate ratio were optimized to enable the simultaneous and efficient utilization of both carbon sources. This yielded results (5.0 g/L, 0.87 mol/mol) surpassing the yield and titer of the control strain utilizing glucose as the sole carbon source in flask cultures, demonstrating an economically efficient strain redesign strategy for synthesizing various products.

Systematic Engineering of Genistein Biosynthetic Pathway through Genetic Regulators and Combinatorial Enzyme Screening.

Microbial production of genistein, an isoflavonoid primarily found in soybeans, is gaining prominence in the food industry due to its significant nutritional and health benefits. However, challenges arise in redesigning strains due to intricate regulatory nodes between cell growth and genistein production and in systematically exploring core enzymes involving genistein biosynthesis. To address this, this study devised a strategy that simultaneously and precisely rewires flux at both acetyl-CoA and malonyl-CoA nodes toward genistein synthesis. In particular, naringenin, the primary precursor of genistein, was accumulated 2.6 times more than the unoptimized strain through transcriptional repressor-based genetic regulators. Building upon this, a combination of isoflavone synthase and cytochrome P450 reductase with the remarkable conversion of naringenin to genistein was screened from enzyme homologue libraries. The integrated metabolic engineering strategy yields the highest reported production (98 mg/L of genistein) to date, providing a framework for the biosynthesis of diverse flavonoids, including genistein.

Biosensor-guided discovery and engineering of metabolic enzymes.

A variety of chemicals have been produced through metabolic engineering approaches, and enhancing biosynthesis performance can be achieved by using enzymes with high catalytic efficiency. Accordingly, a number of efforts have been made to discover enzymes in nature for various applications. In addition, enzyme engineering approaches have been attempted to suit specific industrial purposes. However, a significant challenge in enzyme discovery and engineering is the efficient screening of enzymes with the desired phenotype from extensive enzyme libraries. To overcome this bottleneck, genetically encoded biosensors have been developed to specifically detect target molecules produced by enzyme activity at the intracellular level. Especially, the biosensors facilitate high-throughput screening (HTS) of targeted enzymes, expanding enzyme discovery and engineering strategies with advances in systems and synthetic biology. This review examines biosensor-guided HTS systems and highlights studies that have utilized these tools to discover enzymes in diverse areas and engineer enzymes to enhance their properties, such as catalytic efficiency, specificity, and stability.

Riboswitch-guided chalcone synthase engineering and metabolic flux optimization for enhanced production of flavonoids.

Flavonoids are a group of secondary metabolites from plants that have received attention as high value-added pharmacological substances. Recently, a robust and efficient bioprocess using recombinant microbes has emerged as a promising approach to supply flavonoids. In the flavonoid biosynthetic pathway, the rate of chalcone synthesis, the first committed step, is a major bottleneck. However, chalcone synthase (CHS) engineering was difficult because of high-level conservation and the absence of effective screening tools, which are limited to overexpression or homolog-based combinatorial strategies. Furthermore, it is necessary to precisely regulate the metabolic flux for the optimum availability of malonyl-CoA, a substrate of chalcone synthesis. In this study, we engineered CHS and optimized malonyl-CoA availability to establish a platform strain for naringenin production, a key molecular scaffold for various flavonoids. First, we engineered CHS through synthetic riboswitch-based high-throughput screening of rationally designed mutant libraries. Consequently, the catalytic efficiency (kcat/Km) of the optimized CHS enzyme was 62% higher than that of the wild-type enzyme. In addition to CHS engineering, we designed genetic circuits using transcriptional repressors to fine-tune the malonyl-CoA availability. The best mutant with synergistic effects of the engineered CHS and the optimized genetic circuit produced 98.71 mg/L naringenin (12.57 mg naringenin/g glycerol), which is the highest naringenin concentration and yield from glycerol in similar culture conditions reported to date, a 2.5-fold increase compared to the parental strain. Overall, this study provides an effective strategy for efficient production of flavonoids.

Optimum flux rerouting for efficient production of naringenin from acetate in engineered Escherichia coli.

BACKGROUND: Microbial production of naringenin has received much attention owing to its pharmaceutical applicability and potential as a key molecular scaffold for various flavonoids. In the microbial fermentation, a cheap and abundant feedstock is required to achieve an economically feasible bioprocess. From this perspective, utilizing acetate for naringenin production could be an effective strategy, with the advantages of both low-cost and abundant feedstock. For the efficient production of naringenin using acetate, identification of the appropriate regulatory node of carbon flux in the biosynthesis of naringenin from acetate would be important. While acetyl-CoA is a key precursor for naringenin production, carbon flux between the TCA cycle and anaplerosis is effectively regulated at the isocitrate node through glyoxylate shunt in acetate metabolism. Accordingly, appropriate rerouting of TCA cycle intermediates from anaplerosis into naringenin biosynthesis via acetyl-CoA replenishment would be required. RESULTS: This study identified the isocitrate and oxaloacetate (OAA) nodes as key regulatory nodes for the naringenin production using acetate. Precise rerouting at the OAA node for enhanced acetyl-CoA was conducted, avoiding extensive loss of OAA by fine-tuning the expression of pckA (encoding phosphoenolpyruvate carboxykinase) with flux redistribution between naringenin biosynthesis and cell growth at the isocitrate node. Consequently, the flux-optimized strain exhibited a significant increase in naringenin production, a 27.2-fold increase (with a 38.3-fold increase of naringenin yield on acetate) over that by the unoptimized strain, producing 97.02 mg/L naringenin with 21.02 mg naringenin/g acetate, which is a competitive result against those in previous studies on conventional substrates, such as glucose. CONCLUSIONS: Collectively, we demonstrated efficient flux rerouting for maximum naringenin production from acetate in E. coli. This study was the first attempt of naringenin production from acetate and suggested the potential of biosynthesis of various flavonoids derived from naringenin using acetate.

Effect of Oxygen Vacancy on the Conduction Modulation Linearity and Classification Accuracy of Pr0.7Ca0.3MnO3 Memristor.

An amorphous Pr0.7Ca0.3MnO3 (PCMO) film was grown on a TiN/SiO2/Si (TiN-Si) substrate at 300 °C and at an oxygen pressure (OP) of 100 mTorr. This PCMO memristor showed typical bipolar switching characteristics, which were attributed to the generation and disruption of oxygen vacancy (OV) filaments. Fabrication of the PCMO memristor at a high OP resulted in nonlinear conduction modulation with the application of equivalent pulses. However, the memristor fabricated at a low OP of 100 mTorr exhibited linear conduction modulation. The linearity of this memristor improved because the growth and disruption of the OV filaments were mostly determined by the redox reaction of OV owing to the presence of numerous OVs in this PCMO film. Furthermore, simulation using a convolutional neural network revealed that this PCMO memristor has enhanced classification performance owing to its linear conduction modulation. This memristor also exhibited several biological synaptic characteristics, indicating that an amorphous PCMO thin film fabricated at a low OP would be a suitable candidate for artificial synapses.

Multi-level rebalancing of the naringenin pathway using riboswitch-guided high-throughput screening.

Recombinant microbes have emerged as promising alternatives to natural sources of naringenin-a key molecular scaffold for flavonoids. In recombinant strains, expression levels of the pathway genes should be optimized at both transcription and the translation stages to precisely allocate cellular resources and maximize metabolite production. However, the optimization of the expression levels of naringenin generally relies on evaluating a small number of variants from libraries constructed by varying transcription efficiency only. In this study, we introduce a systematic strategy for the multi-level optimization of biosynthetic pathways. We constructed a multi-level combinatorial library covering both transcription and translation stages using synthetic T7 promoter variants and computationally designed 5'-untranslated regions. Furthermore, we identified improved strains through high-throughput screening based on a synthetic naringenin riboswitch. The most-optimized strain obtained using this approach exhibited a 3-fold increase in naringenin production, compared with the parental strain in which only the transcription efficiency was modulated. Furthermore, in a fed-batch bioreactor, the optimized strain produced 260.2 mg/L naringenin, which is the highest concentration reported to date using glycerol and p-coumaric acid as substrates. Collectively, this work provides an efficient strategy for the expression optimization of the biosynthetic pathways.

Improvement of Conductance Modulation Linearity in a Cu2+-Doped KNbO3 Memristor through the Increase of the Number of Oxygen Vacancies.

The Pt/KNbO3/TiN/Si (KN) memristor exhibits various biological synaptic properties. However, it also displays nonlinear conductance modulation with the application of identical pulses, indicating that it should be improved for neuromorphic applications. The abrupt change of the conductance originates from the inhomogeneous growth/dissolution of oxygen vacancy filaments in the KN film. The change of the filaments in a KN film is controlled by two mechanisms with different growth/dissolution rates: a redox process with a fast rate and an oxygen vacancy diffusion process with a slow rate. Therefore, the conductance modulation linearity can be improved if the growth/dissolution of the filaments is controlled by only one mechanism. When the number of oxygen vacancies in the KN film was increased through doping of Cu2+ ions, the growth/dissolution of the filaments in the Cu2+-doped KN (CKN) film was mainly influenced by the redox process of oxygen vacancies. Therefore, the CKN film exhibited improved conductance modulation linearity, confirming that the linearity of conductance modulation can be improved by increasing the number of oxygen vacancies in the memristor. This method can be applied to other memristors to improve the linearity of conductance modulation. The CKN memristor also provides excellent biological synaptic characteristics for neuromorphic computing systems.

Synaptic Plasticity and Metaplasticity of Biological Synapse Realized in a KNbO3 Memristor for Application to Artificial Synapse.

Amorphous KNbO3 (KN) films were grown on a TiN/SiO2/Si substrate to synthesize a KN memristor as a potential artificial synapse. The Pt/KN/TiN memristor exhibited typical and reliable bipolar switching behavior with multiple resistance levels. It also showed the transmission properties of a biological synapse, with a good conductance modulation linearity. Moreover, the KN memristor can emulate various biological synaptic plasticity characteristics including short-term plasticity, long-term plasticity, spike-rate dependent plasticity, paired-pulse facilitation, and post-tetanic potentiation by controlling the number and rate of the potentiation spike. Spike-timing-dependent plasticity (STDP), which is an essential property of biological synapses, is also realized in the KN memristor. The synaptic plasticity of the KN memristor can be explained by oxygen vacancy movement and oxygen vacancy filaments. The metaplasticity of biological synapses was also implemented in the KN memristor, including the metaplasticity of long-term potentiation and depression, and of STDP. Therefore, the KN memristor could be used as an artificial synapse in neuromorphic computing systems.

Low-Temperature-Grown KNbO3 Thin Films and Their Application to Piezoelectric Nanogenerators and Self-Powered ReRAM Device.

Amorphous KNbO3 (KN) film containing KN nanocrystals was grown on TiN/SiO2/Si substrate at 350 °C. This KN film showed a dielectric constant (εr) and a piezoelectric strain constant (d33) of 43 and 80 pm/V at 10 V, respectively, owing to the existence of KN nanocrystals. Piezoelectric nanogenerators (PNGs) were fabricated using KN films grown on the TiN/polyimide/poly(ethylene terephthalate) substrates. The PNG fabricated with the KN film grown at 350 °C showed an open-circuit output voltage of 2.5 V and a short-circuit current of 70 nA. The KN film grown at 350 °C exhibited a bipolar resistive switching behavior with good reliability characteristics that can be explained by the formation and rupture of the oxygen vacancy filaments. The KN resistive random access memory device powered by the KN PNG also showed promising resistive switching behavior. Moreover, the KN film shows good biocompatibility. Therefore, the KN film can be used for self-powered biomedical devices.

Optimization of industrial microorganisms: recent advances in synthetic dynamic regulators.

Production of biochemicals by industrial fermentation using microorganisms requires maintaining cellular production capacity, because maximal productivity is economically important. High-productivity microbial strains can be developed using static engineering, but these may not maintain maximal productivity throughout the culture period as culture conditions and cell states change dynamically. Additionally, economic reasons limit heterologous protein expression using inducible promoters to prevent metabolic burden for commodity chemical and biofuel production. Recently, synthetic and systems biology has been used to design genetic circuits, precisely controlling gene expression or influencing genetic behavior toward a desired phenotype. Development of dynamic regulators can maintain cellular phenotype in a maximum production state in response to factors including cell concentration, oxygen, temperature, pH, and metabolites. Herein, we introduce dynamic regulators of industrial microorganism optimization and discuss metabolic flux fine control by dynamic regulators in response to metabolites or extracellular stimuli, robust production systems, and auto-induction systems using quorum sensing.

Pathway optimization by re-design of untranslated regions for L-tyrosine production in Escherichia coli.

L-tyrosine is a commercially important compound in the food, pharmaceutical, chemical, and cosmetic industries. Although several attempts have been made to improve L-tyrosine production, translation-level expression control and carbon flux rebalancing around phosphoenolpyruvate (PEP) node still remain to be achieved for optimizing the pathway. Here, we demonstrate pathway optimization by altering gene expression levels for L-tyrosine production in Escherichia coli. To optimize the L-tyrosine biosynthetic pathway, a synthetic constitutive promoter and a synthetic 5'-untranslated region (5'-UTR) were introduced for each gene of interest to allow for control at both transcription and translation levels. Carbon flux rebalancing was achieved by controlling the expression level of PEP synthetase using UTR Designer. The L-tyrosine productivity of the engineered E. coli strain was increased through pathway optimization resulting in 3.0 g/L of L-tyrosine titer, 0.0354 g L-tyrosine/h/g DCW of productivity, and 0.102 g L-tyrosine/g glucose yield. Thus, this work demonstrates that pathway optimization by 5'-UTR redesign is an effective strategy for the development of efficient L-tyrosine-producing bacteria.

Risk assessment of the schmutzdecke of biosand filters: identification of an opportunistic pathogen in schmutzdecke developed by an unsafe water source.

The biosand filter (BSF) is widely applied in developing counties as an appropriate technology-based product for supplying "safe" water. Biosand filters exhibit relatively high purifying efficiency because of the schmutzdecke (biofilm) embedded in them. However, schmutzdecke should be cleaned or discarded on a regular basis to maintain the purifying efficiency of the BSF. Due to its role in BSFs, the purifying function of schmutzdecke, rather than its potential risk when not properly discarded, has so far been the primary focus of research. This study aims to provide a risk assessment of schmutzdecke in an attempt to draw attention to a wholly new angle of schmutzdecke usage. We conducted 16S rRNA gene sequencing and phylogenetic analysis to identify opportunistic pathogens in schmutzdecke developed using water from the Hyung-San River. The results reveal that the schmutzdecke derived from this water source contains diverse and relatively high portions of opportunistic pathogen strains; 55% of all isolates collected from schmutzdecke were identified as opportunistic pathogens. Moreover, the diversity of microorganisms is increased in the schmutzdecke compared to its water source in terms of diversity of genus, phylum and opportunistic pathogen strain. As a whole, our study indicates a potential risk associated with schmutzdecke and the necessity of a solid guideline for the after-treatment of discarded schmutzdecke.