This study characterized the transcript profile of Escherichia coli in acetate cultures using DNA... more This study characterized the transcript profile of Escherichia coli in acetate cultures using DNA microarray on glass slides. Glucose-grown cultures were used as a reference. At the 95% confidence level, 354 genes were up-regulated in acetate, while 370 genes were downregulated compared with the glucose-grown culture. Generally, more metabolic genes were up-regulated in acetate than other gene groups, while genes involved in cell replication, transcription, and translation machinery tended to be down-regulated. It appears that E. coli commits more resources to metabolism at the expense of growth when cultured in the poor carbon source. The expression profile confirms many known features in acetate metabolism such as the induction of the glyoxylate pathway, tricarboxylic acid cycle, and gluconeogenic genes. It also provided many previously unknown features, including induction of malic enzymes, ppsA, and the glycolate pathway and repression of glycolytic and glucose phosphotransferase genes in acetate. The carbon flux delivered from the malic enzymes and PpsA in acetate was further confirmed by deletion mutations. In general, the gene expression profiles qualitatively agree with the metabolic flux changes and may serve as a predictor for gene function and metabolic flux distribution.
Predicting metabolic fluxes of a genetically engineered organism is an important step toward rati... more Predicting metabolic fluxes of a genetically engineered organism is an important step toward rational pathway design. However, because of various regulatory mechanisms, which are complex, often ill-characterized, and sometimes undiscovered, predicting metabolic fluxes using kinetic simulation is difficult. We propose to incorporate regulatory constraints in flux calculation to allow prediction of the steady-state fluxes without complete kinetics. The regulatory constraint, in its linear form, is derived from the dynamic metabolic control theory and involves the flux control coefficients. It is shown that with these constraints, the responses to metabolic perturbation can be predicted. Conversely, the regulatory constraints and the control coefficients can be determined by comparing the experimental data with the prediction. Therefore, this approach may offer a practical direction toward prediction of fluxes for metabolically engineered organisms.
DNA microarray technology was applied to detect differential transcription profiles of a subset o... more DNA microarray technology was applied to detect differential transcription profiles of a subset of the Escherichia coli genome. A total of 111 E. coli genes, including those in central metabolism, key biosyntheses, and some regulatory functions, were cloned, amplified, and used as probes for detecting the level of transcripts. An E. coli strain was grown in glucose, acetate, and glycerol media, and the transcript levels of the selected genes were detected. Despite extensive studies on E. coli physiology, many new features were found in the regulation of these genes. For example, several genes were unexpectedly up-regulated, such as pps, ilvG, aroF, secA, and dsbA in acetate and asnA and asnB in glycerol, or down-regulated, such as ackA, pta, and adhE in acetate. These genes were regulated with no apparent reasons by unknown mechanisms. Meanwhile, many genes were regulated for apparent purposes but by unknown mechanisms. For example, the glucose transport genes (ptsHI, ptsG, crr) in both acetate and glycerol media were down-regulated, and the ppc, glycolytic, and biosynthetic genes in acetate were also down-regulated because of the reduced fluxes. However, their molecular mechanisms remain to be elucidated. Furthermore, a group of genes were regulated by known mechanisms, but the physiological roles of such regulation remain unclear. This group includes pckA and aspA, which are up-regulated in glycerol, and gnd and aspA, which are down-and up-regulated, respectively, in acetate. The DNA microarray technology demonstrated here is a powerful yet economical tool for characterizing gene regulation and will prove to be useful for strain improvement and bioprocess development.
The isoprenoid pathway is a versatile biosynthetic network leading to over 23,000 compounds. Simi... more The isoprenoid pathway is a versatile biosynthetic network leading to over 23,000 compounds. Similar to other biosynthetic pathways, the production of isoprenoids in microorganisms is controlled by the supply of precursors, among other factors. To engineer a host that has the capability to supply geranylgeranyl diphosphate (GGPP), a common precursor of isoprenoids, we cloned and overexpressed isopentenyl diphosphate (IPP) isomerase (encoded by idi) from Escherichia coli and GGPP synthase (encoded by gps) from the archaebacterium Archaeoglobus fulgidus. The latter was shown to be a multifunctional enzyme converting dimethylallyl diphosphate (DMAPP) to GGPP. These two genes and the gene cluster (crtBIYZW) of the marine bacterium Agrobacterium aurantiacum were introduced into E. coli to produce astaxanthin, an orange pigment and antioxidant. This metabolically engineered strain produces astaxanthin 50 times higher than values reported before. To determine the rate-controlling steps in GGPP production, the IDI-GPS pathway was compared with another construct containing idi, ispA (encoding farnesyl diphosphate (FPP) synthase in E. coli), and crtE (encoding GGPP synthase from Erwinia uredovora). Results show that the conversion from FPP to GGPP is the first bottleneck, followed sequentially by IPP isomerization and FPP synthesis. Removal of these bottlenecks results in an E. coli strain providing sufficient precursors for in vivo synthesis of isoprenoids.
DNA origami requires long scaffold DNA to be aligned with the guidance of short staple DNA strand... more DNA origami requires long scaffold DNA to be aligned with the guidance of short staple DNA strands. Scaffold DNA is produced in Escherichia coli as a form of the M13 bacteriophage by rolling circle amplification (RCA). This study shows that RCA can be reconfigured by reducing phage protein V (pV) expression, improving the production throughput of scaffold DNA by at least 5.66-fold. The change in pV expression was executed by modifying the untranslated region sequence and monitored using a reporter green fluorescence protein fused to pV. In a separate experiment, pV expression was controlled by an inducer. In both experiments, reduced pV expression was correlated with improved M13 bacteriophage production. High-cell-density cultivation was attempted for mass scaffold DNA production, and the produced scaffold DNA was successfully folded into a barrel shape without compromising structural quality. This result suggested that scaffold DNA production throughput can be significantly improv...
Polyacetylene vesicle has attracted attentions due to its unique chromatic properties, such as bl... more Polyacetylene vesicle has attracted attentions due to its unique chromatic properties, such as blue-to-red transition. The liposome-like vesicle was immobilized on solid substrates to detect small quantity analytes. In this case, fluorescence was used for detection. We successfully detected protein and bacteria using antibodies or aptamers on the vesicles as probes. Recently, we developed a different immobilization method, which provided much better immobilization efficiency. Target nucleic acid was successfully detected by hybridization to its complementary DNA probe on PDA vesicle. We introduced a stable label-free optical sensor using PDA liposome. Keywords - Polydiacetylene vesicle, Label-free biosensor, chromatic detection
The discrimination learning of multiple odors, in which multi-odor can be associated with differe... more The discrimination learning of multiple odors, in which multi-odor can be associated with different responses, is important for responding quickly and accurately to changes in the external environment. However, very few studies have been done on multi-odor discrimination by animal sniffing. Herein, we report a novel multi-odor discrimination system by detection rats based on the combination of 2-Choice and Go/No-Go (GNG) tasks into a single paradigm, in which the Go response of GNG was replaced by 2-Choice, for detection of toluene and acetone, which are odor indicators of lung cancer and diabetes, respectively. Three of six trained rats reached performance criterion, in 12 consecutive successful tests within a given set or over 12 sets with a success rate of over 90%. Through a total of 1300 tests, the trained animals (N = 3) showed multi-odor sensing performance with 88% accuracy, 87% sensitivity and 90% specificity. In addition, a dependence of behavior response time on odor conc...
Microbial Cell Bioprocessing Group, Bioprocessing Technology Institute, Agency for Science, Techn... more Microbial Cell Bioprocessing Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A∗STAR), Singapore, Singapore, Department of Chemical and Biological Engineering, Korea University, Seoul, South Korea, Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan, Department of Chemical Engineering, Feng Chia University, Taichung, Taiwan
The electron transport chain (ETC) is one of the major energy generation pathways in microorganis... more The electron transport chain (ETC) is one of the major energy generation pathways in microorganisms under aerobic condition. Higher yield of ATP can be achieved through oxidative phosphorylation with consumption of NADH than with substrate level phosphorylation. However, most value‐added metabolites are in an electrochemically reduced state, which requires reducing equivalent NADH as a cofactor. Therefore, optimal production of value‐added metabolites should be balanced with ETC in terms of energy production. In this study, we attempted to reduce the activity of ETC to secure availability of NADH. The ETC mutants exhibited poor growth rate and production of fermentative metabolites compared to parental strain. Introduction of heterologous pathways for synthesis of 2,3‐butanediol and isobutanol to ETC mutants resulted in increased titres and yields of the metabolites. ETC mutants yielded higher NADH/NAD+ ratio but similar ATP content than that by the parental strain. Furthermore, ETC mutants operated fermentative metabolism pathways independent of oxygen supply in large‐scale fermenter, resulting in increased yield and titre of 2,3‐butanediol. Thus, engineering of ETC is a useful metabolic engineering approach for production of reduced metabolites.
Protein production requires a significant amount of intracellular energy. Eliminating the flagell... more Protein production requires a significant amount of intracellular energy. Eliminating the flagella has been proposed to help Escherichia coli improve protein production by reducing energy consumption. In this study, the gene encoding a subunit of FlhC, a master regulator of flagella assembly, was deleted to reduce the expression of flagella-related genes. FlhC knockout in the ptsG-deleted strain triggered significant growth retardation with increased ATP levels and a higher NADPH/NADP+ ratio. Metabolic flux analysis using a 13C-labeled carbon substrate showed increased fluxes toward the pentose phosphate and tricarboxylic acid cycle pathways in the flhC- and ptsG-deleted strains. Introduction of a high copy number plasmid or overexpression of the recombinant protein in this strain restored growth rate without increasing glucose consumption. These results suggest that the metabolic burden caused by flhC deletion was resolved by recombinant protein production. The recombinant enhanced...
Systems and synthetic biology on Streptomyces for discovering novel secondary metabolites and enh... more Systems and synthetic biology on Streptomyces for discovering novel secondary metabolites and enhancing the production.
Resveratrol, a phytoalexin produced by plants, has several beneficial effects in humans. It can b... more Resveratrol, a phytoalexin produced by plants, has several beneficial effects in humans. It can be produced using E. coli by introducing only three heterologous genes: TAL, 4CL, and STS. However, the resveratrol synthesis pathway requires two precursors, tyrosine and acetyl-CoA, which are produced by two branched central metabolic pathways. Therefore, overexpression of these genes in E. coli results in the production of only trace amounts of resveratrol. In this study, we attempted to produce resveratrol via coculture of two engineered strains in which the two metabolic pathways are activated. The first strain was engineered to produce p-coumaric acid using tyrosine as a precursor, which can be synthesized by the pentose phosphate pathway. The second strain produced resveratrol by combining p-coumaric acid from the first strain and malonyl-CoA synthesized from acetyl-CoA, which is produced by the glycolytic pathway. In total, 55.69 mg/L of resveratrol was produced from 20 g/L of glucose via coculture of these two strains in glucose minimal medium without any supplements. The metabolic fluxes in each of the strains producing resveratrol were successfully investigated by 13C metabolic flux analysis. The results showed that the balance between the citric acid cycle and malonyl-CoA supply node was important for resveratrol production.
Background Most microorganisms have evolved to maximize growth rate, with rapid consumption of ca... more Background Most microorganisms have evolved to maximize growth rate, with rapid consumption of carbon sources from the surroundings. However, fast growing phenotypes usually feature secretion of organic compounds. For example, E. coli mainly produced acetate in fast growing condition such as glucose rich and aerobic condition, which is troublesome for metabolic engineering because acetate causes acidification of surroundings, growth inhibition and decline of production yield. The overflow metabolism can be alleviated by reducing glucose uptake rate. Results As glucose transporters or their subunits were knocked out in E. coli, the growth and glucose uptake rates decreased and biomass yield was improved. Alteration of intracellular metabolism caused by the mutations was investigated with transcriptome analysis and 13C metabolic flux analysis (13C MFA). Various transcriptional and metabolic perturbations were identified in the sugar transporter mutants. Transcription of genes related ...
Background: Formate converted from CO 2 reduction has great potential as a sustainable feedstock ... more Background: Formate converted from CO 2 reduction has great potential as a sustainable feedstock for biological production of biofuels and biochemicals. Nevertheless, utilization of formate for growth and chemical production by microbial species is limited due to its toxicity or the lack of a metabolic pathway. Here, we constructed a formate assimilation pathway in Escherichia coli and applied adaptive laboratory evolution to improve formate utilization as a carbon source in sugar-free conditions. Results: The genes related to the tetrahydrofolate and serine cycles from Methylobacterium extorquens AM1 were overexpressed for formate assimilation, which was proved by the 13 C-labeling experiments. The amino acids detected by GC/MS showed significant carbon labeling due to biomass production from formate. Then, 150 serial subcultures were performed to screen for evolved strains with improved ability to utilize formate. The genomes of evolved mutants were sequenced and the mutations were associated with formate dehydrogenation, folate metabolism, and biofilm formation. Last, 90 mg/L of ethanol production from formate was achieved using fed-batch cultivation without addition of sugars. Conclusion: This work demonstrates the effectiveness of the introduction of a formate assimilation pathway, combined with adaptive laboratory evolution, to achieve the utilization of formate as a carbon source. This study suggests that the constructed E. coli could serve as a strain to exploit formate and captured CO 2 .
Caprolactam is a monomer used for the synthesis of nylon-6, and a recombinant microbial strain fo... more Caprolactam is a monomer used for the synthesis of nylon-6, and a recombinant microbial strain for biobased production of nylon-6 was recently developed. An intracellular biosensor for caprolactam can facilitate high-throughput metabolic engineering of recombinant microbial strains. Because of the mixed production of caprolactam and valerolactam in the recombinant strain, a caprolactam biosensor should be highly specific for caprolactam. However, a highly specific caprolactam sensor has not been reported. Here, we developed an artificial riboswitch that specifically responds to caprolactam. This riboswitch was prepared using a coupled in vitro- in vivo selection strategy with a heterogeneous pool of RNA aptamers obtained from in vitro selection to construct a riboswitch library used in in vivo selection. The caprolactam riboswitch successfully discriminated caprolactam from valerolactam. Moreover, the riboswitch was activated by 3.36-fold in the presence of 50 mM caprolactam. This riboswitch enabled caprolactam-dependent control of cell growth, which will be useful for improving caprolactam production and is a valuable tool for metabolic engineering.
Journal of Photochemistry and Photobiology B: Biology
To inactivate methicillin-resistant Staphylococcus aureus (MRSA) with minimum damage to host cell... more To inactivate methicillin-resistant Staphylococcus aureus (MRSA) with minimum damage to host cells and tissue, target-oriented photofunctional nanoparticles (TOPFNs) were fabricated and characterized. MRSA is a predominant infective pathogen even in hospital and non-hospital environments due to its ability to develop high levels of resistance to several classes of antibiotics through various pathways. To solve this major problem, photodynamic inactivation (PDI) method applies to treat antibiotic-resistant bacteria. PDI involves the photosensitizer (PS) and light with a specific wavelength to be able to apply for a non-invasive therapeutic procedure to treat pathogenic bacteria by inducing apoptosis or necrosis of microorganisms. However, most current PDI researches have suffered from the instability of PDI agents in the biological environment due to the lack of selectivity and low solubility of PDI agents, which leads to the low PDI efficiency. In this study, the TOPFNs were fabricated by an esterification reaction to introduce hematoporphyrin (HP) and MRSA antibody to the surface of Fe 3 O 4 nanoparticles. The TOPFNs were designed as dispersible PDI agent in biological condition, which was effectively used for selectively capturing and killing of MRSA. The capture efficiency TOPFNs was compared with PFNs as a negative control. The results showed that the capture efficiency of TOPFNs and PFNs was 95.55% and 6.43% in MRSA and L-929 cell mixed condition, respectively. And TOPFNs have a selective killing ability for MRSA with minimum damage to L-929 cells. Furthermore, PDI effect of TOPFNs was evaluated on the mice in vivo condition in order to check the possibility of practical medical application.
This study characterized the transcript profile of Escherichia coli in acetate cultures using DNA... more This study characterized the transcript profile of Escherichia coli in acetate cultures using DNA microarray on glass slides. Glucose-grown cultures were used as a reference. At the 95% confidence level, 354 genes were up-regulated in acetate, while 370 genes were downregulated compared with the glucose-grown culture. Generally, more metabolic genes were up-regulated in acetate than other gene groups, while genes involved in cell replication, transcription, and translation machinery tended to be down-regulated. It appears that E. coli commits more resources to metabolism at the expense of growth when cultured in the poor carbon source. The expression profile confirms many known features in acetate metabolism such as the induction of the glyoxylate pathway, tricarboxylic acid cycle, and gluconeogenic genes. It also provided many previously unknown features, including induction of malic enzymes, ppsA, and the glycolate pathway and repression of glycolytic and glucose phosphotransferase genes in acetate. The carbon flux delivered from the malic enzymes and PpsA in acetate was further confirmed by deletion mutations. In general, the gene expression profiles qualitatively agree with the metabolic flux changes and may serve as a predictor for gene function and metabolic flux distribution.
Predicting metabolic fluxes of a genetically engineered organism is an important step toward rati... more Predicting metabolic fluxes of a genetically engineered organism is an important step toward rational pathway design. However, because of various regulatory mechanisms, which are complex, often ill-characterized, and sometimes undiscovered, predicting metabolic fluxes using kinetic simulation is difficult. We propose to incorporate regulatory constraints in flux calculation to allow prediction of the steady-state fluxes without complete kinetics. The regulatory constraint, in its linear form, is derived from the dynamic metabolic control theory and involves the flux control coefficients. It is shown that with these constraints, the responses to metabolic perturbation can be predicted. Conversely, the regulatory constraints and the control coefficients can be determined by comparing the experimental data with the prediction. Therefore, this approach may offer a practical direction toward prediction of fluxes for metabolically engineered organisms.
DNA microarray technology was applied to detect differential transcription profiles of a subset o... more DNA microarray technology was applied to detect differential transcription profiles of a subset of the Escherichia coli genome. A total of 111 E. coli genes, including those in central metabolism, key biosyntheses, and some regulatory functions, were cloned, amplified, and used as probes for detecting the level of transcripts. An E. coli strain was grown in glucose, acetate, and glycerol media, and the transcript levels of the selected genes were detected. Despite extensive studies on E. coli physiology, many new features were found in the regulation of these genes. For example, several genes were unexpectedly up-regulated, such as pps, ilvG, aroF, secA, and dsbA in acetate and asnA and asnB in glycerol, or down-regulated, such as ackA, pta, and adhE in acetate. These genes were regulated with no apparent reasons by unknown mechanisms. Meanwhile, many genes were regulated for apparent purposes but by unknown mechanisms. For example, the glucose transport genes (ptsHI, ptsG, crr) in both acetate and glycerol media were down-regulated, and the ppc, glycolytic, and biosynthetic genes in acetate were also down-regulated because of the reduced fluxes. However, their molecular mechanisms remain to be elucidated. Furthermore, a group of genes were regulated by known mechanisms, but the physiological roles of such regulation remain unclear. This group includes pckA and aspA, which are up-regulated in glycerol, and gnd and aspA, which are down-and up-regulated, respectively, in acetate. The DNA microarray technology demonstrated here is a powerful yet economical tool for characterizing gene regulation and will prove to be useful for strain improvement and bioprocess development.
The isoprenoid pathway is a versatile biosynthetic network leading to over 23,000 compounds. Simi... more The isoprenoid pathway is a versatile biosynthetic network leading to over 23,000 compounds. Similar to other biosynthetic pathways, the production of isoprenoids in microorganisms is controlled by the supply of precursors, among other factors. To engineer a host that has the capability to supply geranylgeranyl diphosphate (GGPP), a common precursor of isoprenoids, we cloned and overexpressed isopentenyl diphosphate (IPP) isomerase (encoded by idi) from Escherichia coli and GGPP synthase (encoded by gps) from the archaebacterium Archaeoglobus fulgidus. The latter was shown to be a multifunctional enzyme converting dimethylallyl diphosphate (DMAPP) to GGPP. These two genes and the gene cluster (crtBIYZW) of the marine bacterium Agrobacterium aurantiacum were introduced into E. coli to produce astaxanthin, an orange pigment and antioxidant. This metabolically engineered strain produces astaxanthin 50 times higher than values reported before. To determine the rate-controlling steps in GGPP production, the IDI-GPS pathway was compared with another construct containing idi, ispA (encoding farnesyl diphosphate (FPP) synthase in E. coli), and crtE (encoding GGPP synthase from Erwinia uredovora). Results show that the conversion from FPP to GGPP is the first bottleneck, followed sequentially by IPP isomerization and FPP synthesis. Removal of these bottlenecks results in an E. coli strain providing sufficient precursors for in vivo synthesis of isoprenoids.
DNA origami requires long scaffold DNA to be aligned with the guidance of short staple DNA strand... more DNA origami requires long scaffold DNA to be aligned with the guidance of short staple DNA strands. Scaffold DNA is produced in Escherichia coli as a form of the M13 bacteriophage by rolling circle amplification (RCA). This study shows that RCA can be reconfigured by reducing phage protein V (pV) expression, improving the production throughput of scaffold DNA by at least 5.66-fold. The change in pV expression was executed by modifying the untranslated region sequence and monitored using a reporter green fluorescence protein fused to pV. In a separate experiment, pV expression was controlled by an inducer. In both experiments, reduced pV expression was correlated with improved M13 bacteriophage production. High-cell-density cultivation was attempted for mass scaffold DNA production, and the produced scaffold DNA was successfully folded into a barrel shape without compromising structural quality. This result suggested that scaffold DNA production throughput can be significantly improv...
Polyacetylene vesicle has attracted attentions due to its unique chromatic properties, such as bl... more Polyacetylene vesicle has attracted attentions due to its unique chromatic properties, such as blue-to-red transition. The liposome-like vesicle was immobilized on solid substrates to detect small quantity analytes. In this case, fluorescence was used for detection. We successfully detected protein and bacteria using antibodies or aptamers on the vesicles as probes. Recently, we developed a different immobilization method, which provided much better immobilization efficiency. Target nucleic acid was successfully detected by hybridization to its complementary DNA probe on PDA vesicle. We introduced a stable label-free optical sensor using PDA liposome. Keywords - Polydiacetylene vesicle, Label-free biosensor, chromatic detection
The discrimination learning of multiple odors, in which multi-odor can be associated with differe... more The discrimination learning of multiple odors, in which multi-odor can be associated with different responses, is important for responding quickly and accurately to changes in the external environment. However, very few studies have been done on multi-odor discrimination by animal sniffing. Herein, we report a novel multi-odor discrimination system by detection rats based on the combination of 2-Choice and Go/No-Go (GNG) tasks into a single paradigm, in which the Go response of GNG was replaced by 2-Choice, for detection of toluene and acetone, which are odor indicators of lung cancer and diabetes, respectively. Three of six trained rats reached performance criterion, in 12 consecutive successful tests within a given set or over 12 sets with a success rate of over 90%. Through a total of 1300 tests, the trained animals (N = 3) showed multi-odor sensing performance with 88% accuracy, 87% sensitivity and 90% specificity. In addition, a dependence of behavior response time on odor conc...
Microbial Cell Bioprocessing Group, Bioprocessing Technology Institute, Agency for Science, Techn... more Microbial Cell Bioprocessing Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A∗STAR), Singapore, Singapore, Department of Chemical and Biological Engineering, Korea University, Seoul, South Korea, Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan, Department of Chemical Engineering, Feng Chia University, Taichung, Taiwan
The electron transport chain (ETC) is one of the major energy generation pathways in microorganis... more The electron transport chain (ETC) is one of the major energy generation pathways in microorganisms under aerobic condition. Higher yield of ATP can be achieved through oxidative phosphorylation with consumption of NADH than with substrate level phosphorylation. However, most value‐added metabolites are in an electrochemically reduced state, which requires reducing equivalent NADH as a cofactor. Therefore, optimal production of value‐added metabolites should be balanced with ETC in terms of energy production. In this study, we attempted to reduce the activity of ETC to secure availability of NADH. The ETC mutants exhibited poor growth rate and production of fermentative metabolites compared to parental strain. Introduction of heterologous pathways for synthesis of 2,3‐butanediol and isobutanol to ETC mutants resulted in increased titres and yields of the metabolites. ETC mutants yielded higher NADH/NAD+ ratio but similar ATP content than that by the parental strain. Furthermore, ETC mutants operated fermentative metabolism pathways independent of oxygen supply in large‐scale fermenter, resulting in increased yield and titre of 2,3‐butanediol. Thus, engineering of ETC is a useful metabolic engineering approach for production of reduced metabolites.
Protein production requires a significant amount of intracellular energy. Eliminating the flagell... more Protein production requires a significant amount of intracellular energy. Eliminating the flagella has been proposed to help Escherichia coli improve protein production by reducing energy consumption. In this study, the gene encoding a subunit of FlhC, a master regulator of flagella assembly, was deleted to reduce the expression of flagella-related genes. FlhC knockout in the ptsG-deleted strain triggered significant growth retardation with increased ATP levels and a higher NADPH/NADP+ ratio. Metabolic flux analysis using a 13C-labeled carbon substrate showed increased fluxes toward the pentose phosphate and tricarboxylic acid cycle pathways in the flhC- and ptsG-deleted strains. Introduction of a high copy number plasmid or overexpression of the recombinant protein in this strain restored growth rate without increasing glucose consumption. These results suggest that the metabolic burden caused by flhC deletion was resolved by recombinant protein production. The recombinant enhanced...
Systems and synthetic biology on Streptomyces for discovering novel secondary metabolites and enh... more Systems and synthetic biology on Streptomyces for discovering novel secondary metabolites and enhancing the production.
Resveratrol, a phytoalexin produced by plants, has several beneficial effects in humans. It can b... more Resveratrol, a phytoalexin produced by plants, has several beneficial effects in humans. It can be produced using E. coli by introducing only three heterologous genes: TAL, 4CL, and STS. However, the resveratrol synthesis pathway requires two precursors, tyrosine and acetyl-CoA, which are produced by two branched central metabolic pathways. Therefore, overexpression of these genes in E. coli results in the production of only trace amounts of resveratrol. In this study, we attempted to produce resveratrol via coculture of two engineered strains in which the two metabolic pathways are activated. The first strain was engineered to produce p-coumaric acid using tyrosine as a precursor, which can be synthesized by the pentose phosphate pathway. The second strain produced resveratrol by combining p-coumaric acid from the first strain and malonyl-CoA synthesized from acetyl-CoA, which is produced by the glycolytic pathway. In total, 55.69 mg/L of resveratrol was produced from 20 g/L of glucose via coculture of these two strains in glucose minimal medium without any supplements. The metabolic fluxes in each of the strains producing resveratrol were successfully investigated by 13C metabolic flux analysis. The results showed that the balance between the citric acid cycle and malonyl-CoA supply node was important for resveratrol production.
Background Most microorganisms have evolved to maximize growth rate, with rapid consumption of ca... more Background Most microorganisms have evolved to maximize growth rate, with rapid consumption of carbon sources from the surroundings. However, fast growing phenotypes usually feature secretion of organic compounds. For example, E. coli mainly produced acetate in fast growing condition such as glucose rich and aerobic condition, which is troublesome for metabolic engineering because acetate causes acidification of surroundings, growth inhibition and decline of production yield. The overflow metabolism can be alleviated by reducing glucose uptake rate. Results As glucose transporters or their subunits were knocked out in E. coli, the growth and glucose uptake rates decreased and biomass yield was improved. Alteration of intracellular metabolism caused by the mutations was investigated with transcriptome analysis and 13C metabolic flux analysis (13C MFA). Various transcriptional and metabolic perturbations were identified in the sugar transporter mutants. Transcription of genes related ...
Background: Formate converted from CO 2 reduction has great potential as a sustainable feedstock ... more Background: Formate converted from CO 2 reduction has great potential as a sustainable feedstock for biological production of biofuels and biochemicals. Nevertheless, utilization of formate for growth and chemical production by microbial species is limited due to its toxicity or the lack of a metabolic pathway. Here, we constructed a formate assimilation pathway in Escherichia coli and applied adaptive laboratory evolution to improve formate utilization as a carbon source in sugar-free conditions. Results: The genes related to the tetrahydrofolate and serine cycles from Methylobacterium extorquens AM1 were overexpressed for formate assimilation, which was proved by the 13 C-labeling experiments. The amino acids detected by GC/MS showed significant carbon labeling due to biomass production from formate. Then, 150 serial subcultures were performed to screen for evolved strains with improved ability to utilize formate. The genomes of evolved mutants were sequenced and the mutations were associated with formate dehydrogenation, folate metabolism, and biofilm formation. Last, 90 mg/L of ethanol production from formate was achieved using fed-batch cultivation without addition of sugars. Conclusion: This work demonstrates the effectiveness of the introduction of a formate assimilation pathway, combined with adaptive laboratory evolution, to achieve the utilization of formate as a carbon source. This study suggests that the constructed E. coli could serve as a strain to exploit formate and captured CO 2 .
Caprolactam is a monomer used for the synthesis of nylon-6, and a recombinant microbial strain fo... more Caprolactam is a monomer used for the synthesis of nylon-6, and a recombinant microbial strain for biobased production of nylon-6 was recently developed. An intracellular biosensor for caprolactam can facilitate high-throughput metabolic engineering of recombinant microbial strains. Because of the mixed production of caprolactam and valerolactam in the recombinant strain, a caprolactam biosensor should be highly specific for caprolactam. However, a highly specific caprolactam sensor has not been reported. Here, we developed an artificial riboswitch that specifically responds to caprolactam. This riboswitch was prepared using a coupled in vitro- in vivo selection strategy with a heterogeneous pool of RNA aptamers obtained from in vitro selection to construct a riboswitch library used in in vivo selection. The caprolactam riboswitch successfully discriminated caprolactam from valerolactam. Moreover, the riboswitch was activated by 3.36-fold in the presence of 50 mM caprolactam. This riboswitch enabled caprolactam-dependent control of cell growth, which will be useful for improving caprolactam production and is a valuable tool for metabolic engineering.
Journal of Photochemistry and Photobiology B: Biology
To inactivate methicillin-resistant Staphylococcus aureus (MRSA) with minimum damage to host cell... more To inactivate methicillin-resistant Staphylococcus aureus (MRSA) with minimum damage to host cells and tissue, target-oriented photofunctional nanoparticles (TOPFNs) were fabricated and characterized. MRSA is a predominant infective pathogen even in hospital and non-hospital environments due to its ability to develop high levels of resistance to several classes of antibiotics through various pathways. To solve this major problem, photodynamic inactivation (PDI) method applies to treat antibiotic-resistant bacteria. PDI involves the photosensitizer (PS) and light with a specific wavelength to be able to apply for a non-invasive therapeutic procedure to treat pathogenic bacteria by inducing apoptosis or necrosis of microorganisms. However, most current PDI researches have suffered from the instability of PDI agents in the biological environment due to the lack of selectivity and low solubility of PDI agents, which leads to the low PDI efficiency. In this study, the TOPFNs were fabricated by an esterification reaction to introduce hematoporphyrin (HP) and MRSA antibody to the surface of Fe 3 O 4 nanoparticles. The TOPFNs were designed as dispersible PDI agent in biological condition, which was effectively used for selectively capturing and killing of MRSA. The capture efficiency TOPFNs was compared with PFNs as a negative control. The results showed that the capture efficiency of TOPFNs and PFNs was 95.55% and 6.43% in MRSA and L-929 cell mixed condition, respectively. And TOPFNs have a selective killing ability for MRSA with minimum damage to L-929 cells. Furthermore, PDI effect of TOPFNs was evaluated on the mice in vivo condition in order to check the possibility of practical medical application.
Uploads
Papers by Min-Kyu Oh