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.

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.

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.

Mobile genetic element-based gene editing and genome engineering: Recent advances and applications.

Genome engineering has revolutionized several scientific fields, ranging from biochemistry and fundamental research to therapeutic uses and crop development. Diverse engineering toolkits have been developed and used to effectively modify the genome sequences of organisms. However, there is a lack of extensive reviews on genome engineering technologies based on mobile genetic elements (MGEs), which induce genetic diversity within host cells by changing their locations in the genome. This review provides a comprehensive update on the versatility of MGEs as powerful genome engineering tools that offers efficient solutions to challenges associated with genome engineering. MGEs, including DNA transposons, retrotransposons, retrons, and CRISPR-associated transposons, offer various advantages, such as a broad host range, genome-wide mutagenesis, efficient large-size DNA integration, multiplexing capabilities, and in situ single-stranded DNA generation. We focused on the components, mechanisms, and features of each MGE-based tool to highlight their cellular applications. Finally, we discussed the current challenges of MGE-based genome engineering and provided insights into the evolving landscape of this transformative technology. In conclusion, the combination of genome engineering with MGE demonstrates remarkable potential for addressing various challenges and advancing the field of genetic manipulation, and promises to revolutionize our ability to engineer and understand the genomes of diverse organisms.

Direct and robust citramalate production from brown macroalgae using fast-growing Vibrio sp. dhg.

Brown macroalgae is a promising feedstock for biorefinery owing to its high biomass productivity and contents of carbohydrates such as alginate and mannitol. However, the limited availability of microbial platforms efficiently catabolizing the brown macroalgae sugars has restricted its utilization. In this study, the direct production of citramalate, an important industrial compound, was demonstrated from brown macroalgae by utilizing Vibrio sp. dhg, which has a remarkably efficient catabolism of alginate and mannitol. Specifically, citramalate synthase from Methanocaldococcus jannaschii was synthetically expressed, and competing pathways were removed to maximally redirect the carbon flux toward citramalate production. Notably, a resulting strain, VXHC, produced citramalate up to 9.8 g/L from a 20 g/L mixture of alginate and mannitol regardless of their ratios. Citramalate was robustly produced even when diverse brown macroalgae were provided directly. Collectively, this study showcased the high potential of brown macroalgae biorefinery using Vibrio sp. dhg.

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.

Recent Progress in Metabolic Engineering of Escherichia coli for the Production of Various C4 and C5-Dicarboxylic Acids.

As an alternative to petrochemical synthesis, well-established industrial microbes, such as Escherichia coli, are employed to produce a wide range of chemicals, including dicarboxylic acids (DCAs), which have significant potential in diverse areas including biodegradable polymers. The demand for biodegradable polymers has been steadily rising, prompting the development of efficient production pathways on four- (C4) and five-carbon (C5) DCAs derived from central carbon metabolism to meet the increased demand via the biosynthesis. In this context, E. coli is utilized to produce these DCAs through various metabolic engineering strategies, including the design or selection of metabolic pathways, pathway optimization, and enhancement of catalytic activity. This review aims to highlight the recent advancements in metabolic engineering techniques for the production of C4 and C5 DCAs in E. coli.

Molecular Complementarity of Proteomimetic Materials for Target-Specific Recognition and Recognition-Mediated Complex Functions.

As biomolecules essential for sustaining life, proteins are generated from long chains of 20 different α-amino acids that are folded into unique 3D structures. In particular, many proteins have molecular recognition functions owing to their binding pockets, which have complementary shapes, charges, and polarities for specific targets, making these biopolymers unique and highly valuable for biomedical and biocatalytic applications. Based on the understanding of protein structures and microenvironments, molecular complementarity can be exhibited by synthesizable and modifiable materials. This has prompted researchers to explore the proteomimetic potentials of a diverse range of materials, including biologically available peptides and oligonucleotides, synthetic supramolecules, inorganic molecules, and related coordination networks. To fully resemble a protein, proteomimetic materials perform the molecular recognition to mediate complex molecular functions, such as allosteric regulation, signal transduction, enzymatic reactions, and stimuli-responsive motions; this can also expand the landscape of their potential bio-applications. This review focuses on the recognitive aspects of proteomimetic designs derived for individual materials and their conformations. Recent progress provides insights to help guide the development of advanced protein mimicry with material heterogeneity, design modularity, and tailored functionality. The perspectives and challenges of current proteomimetic designs and tools are also discussed in relation to future applications.

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.

Circuit-guided population acclimation of a synthetic microbial consortium for improved biochemical production.

Microbial consortia have been considered potential platforms for bioprocessing applications. However, the complexity in process control owing to the use of multiple strains necessitates the use of an efficient population control strategy. Herein, we report circuit-guided synthetic acclimation as a strategy to improve biochemical production by a microbial consortium. We designed a consortium comprising alginate-utilizing Vibrio sp. dhg and 3-hydroxypropionic acid (3-HP)-producing Escherichia coli strains for the direct conversion of alginate to 3-HP. We introduced a genetic circuit, named "Population guider", in the E. coli strain, which degrades ampicillin only when 3-HP is produced. In the presence of ampicillin as a selection pressure, the consortium was successfully acclimated for increased 3-HP production by 4.3-fold compared to that by a simple co-culturing consortium during a 48-h fermentation. We believe this concept is a useful strategy for the development of robust consortium-based bioprocesses.

Enzyme activity engineering based on sequence co-evolution analysis.

The utility of engineering enzyme activity is expanding with the development of biotechnology. Conventional methods have limited applicability as they require high-throughput screening or three-dimensional structures to direct target residues of activity control. An alternative method uses sequence evolution of natural selection. A repertoire of mutations was selected for fine-tuning enzyme activities to adapt to varying environments during the evolution. Here, we devised a strategy called sequence co-evolutionary analysis to control the efficiency of enzyme reactions (SCANEER), which scans the evolution of protein sequences and direct mutation strategy to improve enzyme activity. We hypothesized that amino acid pairs for various enzyme activity were encoded in the evolutionary history of protein sequences, whereas loss-of-function mutations were avoided since those are depleted during the evolution. SCANEER successfully predicted the enzyme activities of beta-lactamase and aminoglycoside 3'-phosphotransferase. SCANEER was further experimentally validated to control the activities of three different enzymes of great interest in chemical production: cis-aconitate decarboxylase, α-ketoglutaric semialdehyde dehydrogenase, and inositol oxygenase. Activity-enhancing mutations that improve substrate-binding affinity or turnover rate were found at sites distal from known active sites or ligand-binding pockets. We provide SCANEER to control desired enzyme activity through a user-friendly webserver.

Kinetic compartmentalization by unnatural reaction for itaconate production.

Physical compartmentalization of metabolism using membranous organelles in eukaryotes is helpful for chemical biosynthesis to ensure the availability of substrates from competitive metabolic reactions. Bacterial hosts lack such a membranous system, which is one of the major limitations for efficient metabolic engineering. Here, we employ kinetic compartmentalization with the introduction of an unnatural enzymatic reaction by an engineered enzyme as an alternative strategy to enable substrate availability from competitive reactions through kinetic isolation of metabolic pathways. As a proof of concept, we kinetically isolate the itaconate synthetic pathway from the tricarboxylic acid cycle in Escherichia coli, which is natively separated by mitochondrial membranes in Aspergillus terreus. Specifically, 2-methylcitrate dehydratase is engineered to alternatively catalyze citrate and kinetically secure cis-aconitate for efficient production using a high-throughput screening system. Itaconate production can be significantly improved with kinetic compartmentalization and its strategy has the potential to be widely applicable.

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.

Parallel screening and cheminformatics modeling of flavonoid activated aptasensors.

A parallel screening of 27 different flavonoids and chalcones was conducted using 6 artificial naringenin-activated riboswitches (M1, M2, M3, O, L and H). A quantitative structure-property relationship approach was applied to understand the physicochemical properties of the flavonoid structures resulting in specificity differences relied on the fluorescence intensity of a green fluorescent protein reporter. Robust models of riboswitches M1, M2 and O that had good predictive power were constructed with descriptors selected for their high correlation. Increased electronegativity and hydrophilicity of the flavonoids structures were identified as two properties that increased binding affinity to RNA riboswitches. Hydroxyl groups at the C-3' and C-4' positions of the flavonoid molecule were strictly required for ligand-activation with riboswitches M1 and M2. Riboswitches O and L preferred multi-hydroxylated flavones as ligands. Substitutions on the A ring of the flavonoid molecule were not important in the molecular recognition process. O-glycosylated derivatives were not recognized by any of the riboswitches, presumably due to steric hindrances. Despite the challenges of detecting RNA conformational change after ligand binding, the resulting models elucidate important physicochemical features in the ligands for conformational structural studies of artificial aptamer complexes and for design of ligands having higher binding specificity.

Development of Synthetic Riboswitches to Guide the Evolution of Metabolite Production in Microorganisms.

The untranslated region (UTR) of prokaryotic mRNA contains riboswitches, which are gene regulating modules. Riboswitches can be used as biosensors to regulate the expression of a gene or an operon depending on the intracellular level of a target molecule and consequently modulate the cellular responses. In evolutionary engineering, riboswitch-based biosensors have been widely applied for high-throughput screening or selection of target phenotypes. Evolutionary approaches can overcome the limitations of rational approaches in metabolic engineering. Previous studies have reported synthetic riboswitches equipped with novel aptamers and marker genes based on a deep understanding of the operation mechanism of the riboswitch. Here, we introduce the development process of novel synthetic riboswitches for applications in metabolic engineering.

A Vibrio-based microbial platform for accelerated lignocellulosic sugar conversion.

BACKGROUND: Owing to increasing concerns about climate change and the depletion of fossil fuels, the development of efficient microbial processes for biochemical production from lignocellulosic biomass has been a key issue. Because process efficiency is greatly affected by the inherent metabolic activities of host microorganisms, it is essential to utilize a microorganism that can rapidly convert biomass-derived sugars. Here, we report a novel Vibrio-based microbial platform that can rapidly and simultaneously consume three major lignocellulosic sugars (i.e., glucose, xylose, and arabinose) faster than any previously reported microorganisms. RESULTS: The xylose isomerase pathway was constructed in Vibrio sp. dhg, which naturally displays high metabolic activities on glucose and arabinose but lacks xylose catabolism. Subsequent adaptive laboratory evolution significantly improved xylose catabolism of initial strain and led to unprecedently high growth and sugar uptake rate (0.67 h-1 and 2.15 g gdry cell weight-1 h-1, respectively). Furthermore, we achieved co-consumption of the three sugars by deletion of PtsG and introduction of GalP. We validated its superior performance and applicability by demonstrating efficient lactate production with high productivity (1.15 g/L/h) and titer (83 g/L). CONCLUSIONS: In this study, we developed a Vibrio-based microbial platform with rapid and simultaneous utilization of the three major sugars from lignocellulosic biomass by applying an integrated approach of rational and evolutionary engineering. We believe that the developed strain can be broadly utilized to accelerate the production of diverse biochemicals from lignocellulosic biomass.

Synthetic cellular communication-based screening for strains with improved 3-hydroxypropionic acid secretion.

Although cellular secretion is important in industrial biotechnology, its assessment is difficult due to the lack of efficient analytical methods. This study describes a synthetic cellular communication-based microfluidic platform for screening strains with the improved secretion of 3-hydroxypropionic acid (3-HP), an industry-relevant platform chemical. 3-HP-secreting cells were compartmentalized in droplets, with receiving cells equipped with a genetic circuit that converts the 3-HP secretion level into an easily detectable signal. This platform was applied to identify Escherichia coli genes that enhance the secretion of 3-HP. As a result, two genes (setA, encoding a sugar exporter, and yjcO, encoding a Sel1 repeat-containing protein) found by this platform enhance the secretion of 3-HP and its production. Given the increasing design capability for chemical-detecting cells, this platform has considerable potential in identifying efflux pumps for not only 3-HP but also many important chemicals.

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.

Plug-in repressor library for precise regulation of metabolic flux in Escherichia coli.

In metabolic engineering, enhanced production of value-added chemicals requires precise flux control between growth-essential competing and production pathways. Although advances in synthetic biology have facilitated the exploitation of a number of genetic elements for precise flux control, their use requires expensive inducers, or more importantly, needs complex and time-consuming processes to design and optimize appropriate regulator components, case-by-case. To overcome this issue, we devised the plug-in repressor libraries for target-specific flux control, in which expression levels of the repressors were diversified using degenerate 5' untranslated region (5' UTR) sequences employing the UTR Library Designer. After we validated a wide expression range of the repressor libraries, they were applied to improve the production of lycopene from glucose and 3-hydroxypropionic acid (3-HP) from acetate in Escherichia coli via precise flux rebalancing to enlarge precursor pools. Consequently, we successfully achieved optimal carbon fluxes around the precursor nodes for efficient production. The most optimized strains were observed to produce 2.59 g/L of 3-HP and 11.66 mg/L of lycopene, which were improved 16.5-fold and 2.82-fold, respectively, compared to those produced by the parental strains. These results indicate that carbon flux rebalancing using the plug-in library is a powerful strategy for efficient production of value-added chemicals in E. coli.

Synthetic biosensor accelerates evolution by rewiring carbon metabolism toward a specific metabolite.

Proper carbon flux distribution between cell growth and production of a target compound is important for biochemical production because improper flux reallocation inhibits cell growth, thus adversely affecting production yield. Here, using a synthetic biosensor to couple production of a specific metabolite with cell growth, we spontaneously evolve cells under the selective condition toward the acquisition of genotypes that optimally reallocate cellular resources. Using 3-hydroxypropionic acid (3-HP) production from glycerol in Escherichia coli as a model system, we determine that mutations in the conserved regions of proteins involved in global transcriptional regulation alter the expression of several genes associated with central carbon metabolism. These changes rewire central carbon flux toward the 3-HP production pathway, increasing 3-HP yield and reducing acetate accumulation by alleviating overflow metabolism. Our study provides a perspective on adaptive laboratory evolution (ALE) using synthetic biosensors, thereby supporting future efforts in metabolic pathway optimization.