Development of Wafer-Type Plasma Monitoring Sensor with Automated Robot Arm Transfer Capability for Two-Dimensional In Situ Processing Plasma Diagnosis.

In this work, we propose our newly developed wafer-type plasma monitoring sensor based on a floating-type double probe method that can be useful for two-dimensional (2D) in situ plasma diagnosis within a semiconductor processing chamber. A key achievement of this work is the first realization of an ultra-thin plasma monitoring sensor with a system thickness of ~1.4 mm, which supports a fully automated robot arm transfer capability for in situ plasma diagnosis. To the best of our knowledge, it is the thinnest accomplishment among all wafer-type plasma monitoring sensors. Our proposed sensor is assembled with two Si wafers and SiO2-based probes; accordingly, it makes it possible to monitor the actual dynamics of processing plasmas under electrostatic chucking (ESC) conditions. Also, it allows for the prevention of chamber contamination issues after continuously exposing the radio frequency (RF) to various processing gases. Using a test-bed chamber, we successfully demonstrated the feasibility and system performance of the proposed sensor, including robot arm transfer capability, vacuum and thermal stress durability, and data integrity and reproducibility. Consequently, compared with the conventional plasma diagnostic tools, we expect that our proposed sensor will be highly beneficial for tool-to-tool matching (TTTM) and/or for studying various plasma-related items by more accurately providing the parameters of processing plasmas, further saving both time and manpower resources required for preventive maintenance (PM) routines as well.

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.

Novel evaluation of upper-limb motor performance after stroke based on normal reaching movement model.

BACKGROUND: Upper-limb rehabilitation robots provide repetitive reaching movement training to post-stroke patients. Beyond a pre-determined set of movements, a robot-aided training protocol requires optimization to account for the individuals' unique motor characteristics. Therefore, an objective evaluation method should consider the pre-stroke motor performance of the affected arm to compare one's performance relative to normalcy. However, no study has attempted to evaluate performance based on an individual's normal performance. Herein, we present a novel method for evaluating upper limb motor performance after a stroke based on a normal reaching movement model. METHODS: To represent the normal reaching performance of individuals, we opted for three candidate models: (1) Fitts' law for the speed-accuracy relationship, (2) the Almanji model for the mouse-pointing task of cerebral palsy, and (3) our proposed model. We first obtained the kinematic data of healthy (n = 12) and post-stroke (n = 7) subjects with a robot to validate the model and evaluation method and conducted a pilot study with a group of post-stroke patients (n = 12) in a clinical setting. Using the models obtained from the reaching performance of the less-affected arm, we predicted the patients' normal reaching performance to set the standard for evaluating the affected arm. RESULTS: We verified that the proposed normal reaching model identifies the reaching of all healthy (n = 12) and less-affected arm (n = 19; 16 of them showed an R2 > 0.7) but did not identify erroneous reaching of the affected arm. Furthermore, our evaluation method intuitively and visually demonstrated the unique motor characteristics of the affected arms. CONCLUSIONS: The proposed method can be used to evaluate an individual's reaching characteristics based on an individuals normal reaching model. It has the potential to provide individualized training by prioritizing a set of reaching movements.

Entropy Could Quantify Brain Activation Induced by Mechanical Impedance-Restrained Active Arm Motion: A Functional NIRS Study.

Brain activation has been used to understand brain-level events associated with cognitive tasks or physical tasks. As a quantitative measure for brain activation, we propose entropy in place of signal amplitude and beta value, which are widely used, but sometimes criticized for their limitations and shortcomings as such measures. To investigate the relevance of our proposition, we provided 22 subjects with physical stimuli through elbow extension-flexion motions by using our exoskeleton robot, measured brain activation in terms of entropy, signal amplitude, and beta value; and compared entropy with the other two. The results show that entropy is superior, in that its change appeared in limited, well established, motor areas, while signal amplitude and beta value changes appeared in a widespread fashion, contradicting the modularity theory. Entropy can predict increase in brain activation with task duration, while the other two cannot. When stimuli shifted from the rest state to the task state, entropy exhibited a similar increase as the other two did. Although entropy showed only a part of the phenomenon induced by task strength, it showed superiority by showing a decrease in brain activation that the other two did not show. Moreover, entropy was capable of identifying the physiologically important location.

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.

Process of Obtaining Social Consensus and 3-Year Functional Outcomes of the First Hand Allotransplantation in Korea.

BACKGROUND: On February 2, 2017, the surgical team of ten board-certified hand specialists of W Hospital in Korea successfully performed the nation's first hand transplantation at Yeungnam University Medical Center (YUMC). This paper reports on the legal, financial, and cultural hurdles that were overcome to open the way for hand transplantation and its functional outcomes at 36 months after the operation. METHODS: W Hospital formed a memorandum of understanding with Daegu city and YUMC to comply with government regulations regarding hand transplantation. Campaigns were initiated in the media to increase public awareness and understanding. With the city's financial and legal support and the university's medical cooperation, a surgical team performed a left distal forearm hand transplantation from a brain-dead 48-year-old man to a 35-year-old left-handed man. RESULTS: With this successful allotransplantation, the Korean Act on Organ Transplantation has now been amended to include hand transplantation. Korean national health insurance has also begun covering hand transplantation. Functional outcome at 36 months after the operation showed satisfactory progress in both motor and sensory functions. The disabilities of the arm, shoulder, and hand score were 23. The final Hand Transplantation Score was 90 points. Functional brain magnetic resonance imaging shows significant cortical reorganization of the corticospinal tract, and reinnervation of intrinsic muscle is observed. CONCLUSIONS: Hand transplantation at the distal forearm shows very satisfactory outcomes in functional, aesthetical, and psychological aspects. Legal and financial barriers against hand transplantation have long been the most burdensome issues. Despite this momentous success, there have been no other clinical applications of vascularized composite allotransplantation due to the limited acceptance by Korean doctors and people. Further public education campaigns for vascularized composite allotransplantation are needed to increase awareness and acceptance.

Multi-level engineering of Baeyer-Villiger monooxygenase-based Escherichia coli biocatalysts for the production of C9 chemicals from oleic acid.

Whole-cell biotransformation is one of the promising alternative approaches to microbial fermentation for producing high-value chemicals. Baeyer-Villiger monooxygenase (BVMO)-based Escherichia coli biocatalysts have been engineered to produce industrially relevant C9 chemicals, such as n-nonanoic acid and 9-hydroxynonanoic acid, from a renewable long-chain fatty acid. The key enzyme in the biotransformation pathway (i.e., BVMO from Pseudomonans putida KT2440) was first engineered, using structure modeling-based design, to improve oxidative and thermal stabilities. Using a stable and tunable plasmid (STAPL) system, E. coli host cells were engineered to have increased plasmid stability and homogeneity of the recombinant E. coli population, as well as to optimize the level of BVMO expression. Multi-level engineering of the key enzyme in host cells, allowed recombinant E. coli expressing a fatty acid double-bond hydratase, a long-chain secondary alcohol dehydrogenase, and the engineered BVMO from P. putida KT2440 (i.e., E6BVMO_C302L/M340L), to ultimately produce C9 chemicals (i.e., n-nonanoic acid and 9-hydroxynonanoic acid) from oleic acid, with a yield of up to 6 mmoL/g dry cells. This yield was 2.4-fold greater than the yield in the control strain before engineering. Therefore, this study will contribute to the development of improved processes for the biosynthesis of industrially relevant medium chain fatty acids via whole-cell biocatalysis.

Tools and systems for evolutionary engineering of biomolecules and microorganisms.

Evolutionary approaches have been providing solutions to various bioengineering challenges in an efficient manner. In addition to traditional adaptive laboratory evolution and directed evolution, recent advances in synthetic biology and fluidic systems have opened a new era of evolutionary engineering. Synthetic genetic circuits have been created to control mutagenesis and enable screening of various phenotypes, particularly metabolite production. Fluidic systems can be used for high-throughput screening and multiplexed continuous cultivation of microorganisms. Moreover, continuous directed evolution has been achieved by combining all the steps of evolutionary engineering. Overall, modern tools and systems for evolutionary engineering can be used to establish the artificial equivalent to natural evolution for various research applications.

Systematic optimization of L-tryptophan riboswitches for efficient monitoring of the metabolite in Escherichia coli.

Riboswitches form a class of genetically encoded sensor-regulators and are considered as promising tools for monitoring various metabolites. Functional parameters of a riboswitch, like dynamic or operational range, should be optimized before the riboswitch is implemented in a specific application for monitoring the target molecule efficiently. However, optimization of a riboswitch was not straightforward and required detailed studies owing to its complex sequence-function relationship. Here, we present three approaches for tuning and optimization of functional parameters of a riboswitch using an artificial L-tryptophan riboswitch as an example. First, the constitutive expression level was adjusted to control the dynamic range of an L-tryptophan riboswitch. The dynamic range increased as the constitutive expression level increased. Then, the function of a riboswitch-encoded protein was utilized to connect the regulatory response of the riboswitch to another outcome for amplifying the dynamic range. Riboswitch-mediated control of the host cell growth enabled the amplification of the riboswitch response. Finally, L-tryptophan aptamers with different dissociation constants were employed to alter the operational range of the riboswitch. The dose-response curve was shifted towards higher L-tryptophan concentrations when an aptamer with higher dissociation constant was employed. All strategies were effective in modifying the distinct functional parameters of the L-tryptophan riboswitch, and they could be easily applied to optimization of other riboswitches owing to their simplicity.

Naringenin-responsive riboswitch-based fluorescent biosensor module for Escherichia coli co-cultures.

The ability to design and construct combinatorial synthetic metabolic pathways has far exceeded our capacity for efficient screening and selection of the resulting microbial strains. The need for high-throughput rapid screening techniques is of upmost importance for the future of synthetic biology and metabolic engineering. Here we describe the development of an RNA riboswitch-based biosensor module with dual fluorescent reporters, and demonstrate a high-throughput flow cytometry-based screening method for identification of naringenin over producing Escherichia coli strains in co-culture. Our efforts helped identify a number of key operating parameters that affect biosensor performance, including the selection of promoter and linker elements within the sensor-actuator domain, and the effect of host strain, fermentation time, and growth medium on sensor dynamic range. The resulting biosensor demonstrates a high correlation between specific fluorescence of the biosensor strain and naringenin titer produced by the second member of the synthetic co-culture system. This technique represents a novel application for synthetic microbial co-cultures and can be expanded from naringenin to any metabolite if a suitable riboswitch is identified. The co-culture technique presented here can be applied to a variety of target metabolites in combination with the SELEX approach for aptamer design. Due to the compartmentalization of the two genetic constructs responsible for production and detection into separate cells and application as independent modules of a synthetic microbial co-culture we have subsequently reduced the need for re-optimization of the producer module when the biosensor is replaced or removed. Biotechnol. Bioeng. 2017;114: 2235-2244. © 2017 Wiley Periodicals, Inc.

Evaluation of the surface characteristics of anodic oxidized miniscrews and their impact on biomechanical stability: An experimental study in beagle dogs.

INTRODUCTION: In this study, we aimed to assess the surface characteristics and the biomechanical stability of miniscrews with an anodic oxidized surface compared with machined surface miniscrews in beagle dogs. METHODS: Self-drilled, titanium-aluminum-vanadium alloy miniscrews with an anodic oxidized surface (n = 48) or a machined surface (n = 48) were placed into the mandibles of 12 beagle dogs. The surface characteristics of both types of miniscrews were analyzed before implantation with scanning electron microscopy and atomic force microscopy. Insertion torque was measured during placement of all 96 miniscrews. Half of the implants in each group (24 specimens per subgroup) received 200 to 250 g of tensile force for 3-week or 12-week loading periods. Removal torque was measured in 12 specimens of each subgroup, and bone-implant contact and bone volume were quantified in the other 12 specimens of each subgroup. RESULTS: Atomic force microscopy measurements demonstrated that the anodic oxidized surface miniscrews had significantly higher roughness parameters than did the machined surface miniscrews (P < 0.001). The 2 types of miniscrews were not significantly different in insertion and removal torque values or in bone-implant contacts and bone volumes, regardless of the loading period. CONCLUSIONS: Anodic oxidized miniscrews have different surface roughness profiles but no clinically significant superiority in biomechanical stability compared with machined surface miniscrews.

Effectiveness of intra-articular steroid injection for atlanto-occipital joint pain.

OBJECTIVES: The aims of this study were to evaluate the role of intra-articular joint injection for atlanto-occipital (AO) joint pain and to determine pain referral sites from that joint. DESIGN: Prospective observational study. METHOD: We evaluated 29 patients with chronic refractory neck pain and/or headache, and limited range of lateral bending with rotation at the AO joint on physical examination. Of the 24 patients who consented to undergo diagnostic injections, 20 patients had at least 50% relief from pain and underwent two AO intra-articular injections of mixture of local anesthetic and steroid approximately 1 week apart. Patients completed pain drawings, visual analog scales (VASs) for pain, and neck disability index (NDI) for level of function. Patients were evaluated for 2 months after the first injection. RESULT: There was headache in 14/20 (70%), posterior neck pain (PNP) in 20, and referred pain in 17 (85%). The average VAS values for headache, PNP, and other referred pains were reduced significantly from 5.64, 5.70, and 5.41, respectively, before treatments to 0.64, 2.30, and 1.71, respectively, two months after injection (P < 0.01). The average NDI value was reduced significantly from 39.95% at pretreatment to 20.40% at 2 months after treatment (P < 0.01). CONCLUSION: AO intra-articular steroid injection appears effective for the short-term control of chronic refractory pain arising from the AO joint.

A Pilot Study on Pain and the Upregulation of Myoglobin through Low-frequency and High-amplitude Electrical Stimulation-induced Muscle Contraction.

[Purpose] It is well known that, in both in vivo and in vitro tests, muscle fatigue is produced by severe exercise, electrical stimulation, and so on. However, it is not clear whether or not low-frequency and high-amplitude modulation specifically affects serum myoglobin or urine myoglobin. The purpose of the present study was to determine the effect of low-frequency and high-amplitude modulation on serum myoglobin and urine myoglobin. [Methods] The study used whole blood samples and urine produced over 24 hours from the thirteen healthy subjects. [Results] There was a significant increase in serum myoglobin following electrical stimulation at a frequency of 10 Hz compared with the control group. Furthermore, within 24 hours, urine myoglobin also showed a significant increase for the test volunteers subjected to electrical stimulation at the 10 Hz frequency compared with the control group. However, there were no significant differences in the concentrations of hematologic results in subjects treated with electrical stimulation. [Conclusion] These results suggest that increased myoglobin related to muscle fatigue from electrical stimulation, particularly with a current of 10 Hz combined with a high-amplitude, may be partially related to increased muscle damage.

A Review of Signal Transduction of Endothelin-1 and Mitogen-activated Protein Kinase-related Pain for Nanophysiotherapy.

[Purpose] An understanding of pain is very important in the study of nanophysiotherapy. In this review, we summarize the mechanisms of endothelin-1 (ET-1)- and mitogen-activated protein kinase (MAPK)-related pain, and suggest their applications in pain physiotherapy. [Method] This review focuses on the signal transduction of pain and its mechanisms. [Results] Our reviews show that mechanisms of ET-1- and MAPK-related pain exist. [Conclusions] In this review article, we carefully discuss the signal transduction in ET-1- and MAPK-related pain with reference to pain nanophysiotherapy from the perspective of nanoparticle-associated signal transduction.

Cofilin Phosphorylation Decreased by Serum-free Starvation with Low Glucose in the L6 Myoblasts.

[Purpose] Many studies have been using cell culture models of muscle cells with exogenous cytokines or glucocorticoids to mimic atrophy in in vivo and in vitro tests. However, the changes in the phosphorylation of atrophy-related cofilin are still poorly understood in starved skeletal muscle cells. In this study, we first examined whether or not phosphorylation of cofilin is altered in L6 myoblasts after 3, 6, 12, 24, 48, and 72 hours of serum-free starvation with low glucose. [Methods] We used Western blotting to exam protein expression and phosphorylation in atrophied L6 myoblasts. [Results] L6 cell sizes and numbers were diminished as a result of serum-free starvation in a time-dependent manner. Serum-free starvation for 3, 6, 12, 24, 48, and 72 hours significantly decreased the phosphorylation of cofilin, respectively. [Conclusion] These results suggest that starvation-induced atrophy may be in part related to changes in the phosphorylation of cofilin in L6 myoblasts.

Decrease of PKB/Akt Phosphorylation is Partially Mediated by SAPK/JNK Activation in Serum-free L6 Myoblasts Starved with Low Glucose.

[Purpose] Studies have been using cell cultures of muscle cells to mimic atrophy in in vivo and in vitro tests. However, changes in the activation of atrophy-related PKB/Akt is not fully understood in serum-free starved skeletal muscle cells. The purpose of the present study was to determine the change of PKB/Akt phosphorylation in L6 myoblasts under serum-free starvation conditions. [Methods] We used western blotting to examine PKB/Akt expression and phosphorylation in atrophied L6 myoblasts. [Results] The phosphorylation of PKB/Akt was significantly lower in L6 myoblasts under serum-free starvation than that of the control group. Serum-free starvation for 6, 12, 24, 36, 48, 72, 96, and 120 hours significantly decreased the phosphorylation of PKB/Akt. Furthermore, the decrease of PKB/Akt phosphorylation under serum-free starvation was partially restored by SP600125, an inhibitor of SAPK/JNK. [Conclusion] These results suggest that decrease of PKB/Akt phosphorylation due to serum-free starvation with low glucose is partially related to the activity of SAPK/JNK in L6 myoblasts.

Somatotype analysis of elite boxing athletes compared with nonathletes for sports physiotherapy.

[Purpose] The purpose of this study was to show somatotype and physical characteristic differences between elite boxing athletes and non-athletes. [Methods] The somatotypes of 23 elite boxing athletes and 23 nonathletes were measured with the Heath-Carter method. The subjects were divided into four weight divisions as follows: lightweight, light middleweight, middleweight, and heavyweight class. [Results] The endomorphic component values of the boxing athletes were lower than those of the nonathletes. However, the mesomorphic component values of the boxing athletes were higher than those of the nonathletes. There was no significant difference in the ectomorphic component between the two groups. The higher weight divisions tended to have higher values of height, weight, and BMI than the lower weight divisions. The higher weight divisions also tended to have higher values for the endomorphic and mesomorphic components and a lower value for the ectomorphic component than the lower weight divisions. The group of nonathletes consisted of eight endomorphs, four mesomorphs, six ectomorphs, and five central types. Among the boxing athletes, there were 16 mesomorphic, four ectomorphic, and two central types and one endomorphic type. Subdividing the athletes into 13 somatotypes resulted in five balanced mesomorphs, five endomorphic mesomorphs, five mesomorph-ectomorphs, three mesomorph-endomorphs, two mesomorphic ectomorphs, two central types, and one ectomorphic mesomorph type. [Conclusion] The data from this study provides in part physical characteristics of elite boxing athletes that can be used to establish a reference for systemic study of sports physiotherapy.

Phosphorylation of Heat Shock Protein 27 is Increased by Cast Immobilization and by Serum-free Starvation in Skeletal Muscles.

[Purpose] Cast immobilization- and cell starvation-induced loss of muscle mass are closely associated with a dramatic reduction in the structural muscle proteins. Heat shock proteins are molecular chaperones that are constitutively expressed in several eukaryotic cells and have been shown to protect against various stressors. However, the changes in the phosphorylation of atrophy-related heat shock protein 27 (HSP27) are still poorly understood in skeletal muscles. In this study, we examine whether or not phosphorylation of HSP27 is changed in the skeletal muscles after cast immobilization and serum-free starvation with low glucose in a time-dependent manner. [Methods] We undertook a HSP27 expression and high-resolution differential proteomic analysis in skeletal muscles. Furthermore, we used western blotting to examine protein expression and phosphorylation of HSP27 in atrophied gastrocnemius muscle strips and L6 myoblasts. [Results] Cast immobilization and starvation significantly upregulated the phosphorylation of HSP27 in a time-dependent manner, respectively. [Conclusion] Our results suggest that cast immobilization- and serum-free starvation-induced atrophy may be in part related to changes in the phosphorylation of HSP27 in rat skeletal muscles.

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.

Rational engineering of enzyme allosteric regulation through sequence evolution analysis.

Control of enzyme allosteric regulation is required to drive metabolic flux toward desired levels. Although the three-dimensional (3D) structures of many enzyme-ligand complexes are available, it is still difficult to rationally engineer an allosterically regulatable enzyme without decreasing its catalytic activity. Here, we describe an effective strategy to deregulate the allosteric inhibition of enzymes based on the molecular evolution and physicochemical characteristics of allosteric ligand-binding sites. We found that allosteric sites are evolutionarily variable and comprised of more hydrophobic residues than catalytic sites. We applied our findings to design mutations in selected target residues that deregulate the allosteric activity of fructose-1,6-bisphosphatase (FBPase). Specifically, charged amino acids at less conserved positions were substituted with hydrophobic or neutral amino acids with similar sizes. The engineered proteins successfully diminished the allosteric inhibition of E. coli FBPase without affecting its catalytic efficiency. We expect that our method will aid the rational design of enzyme allosteric regulation strategies and facilitate the control of metabolic flux.