Metabolic perturbations in mutants of glucose transporters and their applications in metabolite production in Escherichia coli.

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 to glycolysis, chemotaxis, and flagella synthesis was downregulated, and that of gluconeogenesis, Krebs cycle, alternative transporters, quorum sensing, and stress induced proteins was upregulated in the sugar transporter mutants. The specific production yields of value-added compounds (enhanced green fluorescent protein, γ-aminobutyrate, lycopene) were improved significantly in the sugar transporter mutants. CONCLUSIONS: The elimination of sugar transporter resulted in alteration of global gene expression and redirection of carbon flux distribution, which was purposed to increase energy yield and recycle carbon sources. When the pathways for several valuable compounds were introduced to mutant strains, specific yield of them were highly improved. These results showed that controlling the sugar uptake rate is a good strategy for ameliorating metabolite production.

Evaluation of the performance of two real-time PCR assays for detecting Mycobacterium species.

BACKGROUNDS: Rapid discrimination between Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) is critical for patient treatment and to avoid unnecessary expenditure on infection control. Because real-time PCR assays distinguish MTB from NTM, we evaluated the performance of two real-time PCR assays (AdvanSure and PowerChek). METHODS: This study used 143 DNA samples from respiratory specimens which were collected based on routine PCR results using Anyplex kit. A total of 87 positive samples (65 MTB and 22 NTM) and 56 negative samples were collected consecutively during 6 months and 1 month, respectively. The diagnostic performance of PCR assays (AdvanSure and PowerChek) was retrospectively analyzed based on the results of conventional mycobacterial tests and routine PCR assay. RESULTS: Based on culture results, the sensitivities/specificities of AdvanSure and PowerChek were 90.7%/87.6% and 92.6%/85.4%, respectively, for MTB detection. For PCR-positive specimens, the quantification cycle (Cq) values of smear-negative specimens were higher than those of the smear-positive specimens (P < 0.001). As expected, the two PCR assays had the same sensitivities for NTM detection, viz. 90.0%, and their specificities were 99.2% and 98.4%, respectively. The overall agreement rate between the three PCR assays was 96.5% for MTB and 97.9% for NTM. CONCLUSION: The sensitivities of PCR assays in our study might be overestimated, because this study enrolled relatively lower number of PCR-negative samples which potentially missed PCR-negative but culture-positive specimens. However, the two real-time PCR assays for detecting MTB and NTM perform equally well in relative performance evaluation and their Cq values can be considered suitable for predicting smear-positive specimens.

Optimization of hexanoic acid production in recombinant Escherichia coli by precise flux rebalancing.

The aim of this study is to demonstrate that rebalancing of metabolic fluxes at acetyl-CoA branch node can substantially improve the titer and productivity of hexanoic acid in recombinant Escherichia coli strains. First, a hexanoic acid-producing E. coli strain was constructed by expressing genes encoding ß-ketothiolase (BktB) from Cupriavidus necator and acetyl-CoA transferase (ACT) from Megasphaera sp. MH in a butyric acid producer strain. Next, metabolic flux was optimized at the acetyl-CoA branch node by fine-tuning the expression level of the gene for acetyl-CoA acetyltransferase (AtoB). Four synthetic 5'-untranslated regions were designed for atoB using UTR Designer to modulate the expression level of the gene. Notably, the productivity of the optimized strain (14.7 mg/L/h) was the highest among recombinant E. coli strains in literature when using a similar inoculum size for fermentation. These results show that fine-tuning the expression level of atoB is critical for production of hexanoic acid.

A simple method to control glycolytic flux for the design of an optimal cell factory.

BACKGROUND: A microbial cell factory with high yield and productivity are prerequisites for an economically feasible bio-based chemical industry. However, cell factories that show a kinetic imbalance between glycolysis and product formation pathways are not optimal. Glycolysis activity is highly robust for survival in nature, but is not optimized for chemical production. RESULTS: Here, we propose a novel approach to balance glycolytic activity with the product formation capacity by precisely controlling expression level of ptsG (encoded glucose transporter) through UTR engineering. For various heterologous pathways with different maximum production rates, e.g., n-butanol, butyrate, and 2,3-butanediol, glycolytic fluxes could be successfully modulated to maximize yield and productivity, while minimizing by-product formation in Escherichia coli. CONCLUSIONS: These results support the application of this simple method to explore the maximum yield and productivity when designing optimal cell factories for value-added products in the fields of metabolic engineering and synthetic biology.

Implant Ball Attachment Fabricated with CAD/CAM to Overcome an Unfavorable Clinical Situation: A Case Report.

The most common complication of implant ball attachments is loss of retention via structural wear. Hence, parallel placement of implants was identified as a prerequisite for long-term success. In this case, although severe angulation was formed between implants due to severe alveolar bone resorption, parallel ball attachments on the implants could be fabricated using computer-aided design/computer-assisted manufacture. This procedure can be a solution when implants are placed with angulation and offers additional advantages such as long-term stability.

Precise precursor rebalancing for isoprenoids production by fine control of gapA expression in Escherichia coli.

Biosynthesis of isoprenoids via the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway requires equimolar glyceraldehyde 3-phosphate and pyruvate to divert carbon flux toward the products of interest. Here, we demonstrate that precursor balancing is one of the critical steps for the production of isoprenoids in Escherichia coli. First, the implementation of the synthetic lycopene production pathway as a model system and the amplification of the native DXP pathway were accomplished using synthetic constitutive promoters and redesigned 5'-untranslated regions (5'-UTRs). Next, fine-controlled precursor balancing was investigated by tuning phosphoenolpyruvate synthase (PpsA) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The results showed that tuning-down of gapA improved the specific lycopene content by 45% compared to the overexpression of ppsA. The specific lycopene content in the strains with down-regulated gapA increased by 97% compared to that in the parental strain. Our results indicate that gapA is the best target for precursor balancing to increase biosynthesis of isoprenoids.

Bony window approach for a traumatic bone cyst on the mandibular condyle: a case report with long-term follow-up.

Traumatic bone cyst (TBC) occurs preferentially on the mandibular symphysis and body, but rarely on the mandibular condyle. When TBC occurs in the condylar area, it can usually be related with or misdiagnosed as a temporomandibular joint disorder. A 15-year-old female patient visited the Temporomandibular Joint Clinic with a 5-year history of pain and noise localized in the left temporomandibular joint. On imaging, a well demarked oval-shaped radiolucent lesion was observed on the left condyle head. The patient underwent cyst enucleation and repositioning of the bony window on the lateral cortex of the affected condyle head under the impression of subchondral cyst or TBC; however, no cystic membrane was found. The bone defect resolved and showed no recurrence on the serial radiographic postoperative follow-up for 43 months after surgery.

Modular design of metabolic network for robust production of n-butanol from galactose-glucose mixtures.

BACKGROUND: Refactoring microorganisms for efficient production of advanced biofuel such as n-butanol from a mixture of sugars in the cheap feedstock is a prerequisite to achieve economic feasibility in biorefinery. However, production of biofuel from inedible and cheap feedstock is highly challenging due to the slower utilization of biomass-driven sugars, arising from complex assimilation pathway, difficulties in amplification of biosynthetic pathways for heterologous metabolite, and redox imbalance caused by consuming intracellular reducing power to produce quite reduced biofuel. Even with these problems, the microorganisms should show robust production of biofuel to obtain industrial feasibility. Thus, refactoring microorganisms for efficient conversion is highly desirable in biofuel production. RESULTS: In this study, we engineered robust Escherichia coli to accomplish high production of n-butanol from galactose-glucose mixtures via the design of modular pathway, an efficient and systematic way, to reconstruct the entire metabolic pathway with many target genes. Three modular pathways designed using the predictable genetic elements were assembled for efficient galactose utilization, n-butanol production, and redox re-balancing to robustly produce n-butanol from a sugar mixture of galactose and glucose. Specifically, the engineered strain showed dramatically increased n-butanol production (3.3-fold increased to 6.2 g/L after 48-h fermentation) compared to the parental strain (1.9 g/L) in galactose-supplemented medium. Moreover, fermentation with mixtures of galactose and glucose at various ratios from 2:1 to 1:2 confirmed that our engineered strain was able to robustly produce n-butanol regardless of sugar composition with simultaneous utilization of galactose and glucose. CONCLUSIONS: Collectively, modular pathway engineering of metabolic network can be an effective approach in strain development for optimal biofuel production with cost-effective fermentable sugars. To the best of our knowledge, this study demonstrated the first and highest n-butanol production from galactose in E. coli. Moreover, robust production of n-butanol with sugar mixtures with variable composition would facilitate the economic feasibility of the microbial process using a mixture of sugars from cheap biomass in the near future.

Autotransplantation of an impacted premolar using collagen sponge after cyst enucleation.

INTRODUCTION: We report a case of successful autotransplantation of a premolar impacted with a dentigerous cyst and transplanted with collagen plugs for initial support. METHODS: An 18-year-old man had an impacted premolar accompanied with a large dentigerous cyst. The tooth was extracted surgically and transplanted to an edentulous alveolar ridge, and a collagen sponge was inserted to ensure proper healing and initial support. Root canal treatment was performed 3 weeks after the surgery. RESULTS: The previous lesion was healed, and the transplanted tooth was functional without any pathologic signs. CONCLUSIONS: Our protocol provides a viable option for saving an impacted tooth in the case of cyst enucleation.

A biosynthetic pathway for hexanoic acid production in Kluyveromyces marxianus.

Hexanoic acid can be used for diverse industrial applications and is a precursor for fine chemistry. Although some natural microorganisms have been screened and evolved to produce hexanoic acid, the construction of an engineered biosynthetic pathway for producing hexanoic acid in yeast has not been reported. Here we constructed hexanoic acid pathways in Kluyveromyces marxianus by integrating 5 combinations of seven genes (AtoB, BktB, Crt, Hbd, MCT1, Ter, and TES1), by which random chromosomal sites of the strain are overwritten by the new genes from bacteria and yeast. One recombinant strain, H4A, which contained AtoB, BktB, Crt, Hbd, and Ter, produced 154mg/L of hexanoic acid from galactose as the sole substrate. However, the hexanoic acid produced by the H4A strain was re-assimilated during the fermentation due to the reverse activity of AtoB, which condenses two acetyl-CoAs into a single acetoacetyl-CoA. This product instability could be overcome by the replacement of AtoB with a malonyl CoA-acyl carrier protein transacylase (MCT1) from Saccharomyces cerevisiae. Our results suggest that Mct1 provides a slow but stable acetyl-CoA chain elongation pathway, whereas the AtoB-mediated route is fast but unstable. In conclusion, hexanoic acid was produced for the first time in yeast by the construction of chain elongation pathways comprising 5-7 genes in K. marxianus.

Diagnosis and treatment of teeth with primary endodontic lesions mimicking periodontal disease: three cases with long-term follow ups.

A tooth with primary endodontic disease that demonstrates a periodontal defect might be extracted because of misdiagnosis as severe periodontal disease or a vertical root fracture. The aim of this case report was to demonstrate the long-term survival of endodontically treated teeth, which had been initially considered unsavable. With meticulous evaluation including the patient's dental history, clinical and radiographic examinations, teeth with primary endodontic lesions could be differentiated and saved after proper root canal treatment. Pain history, vitality test, and radiographic examinations, as well as a general periodontal condition check with periodontal probing on an affected tooth, might be the key methods to differentiate endodontic pathosis from that of periodontal disease.

Model-driven rebalancing of the intracellular redox state for optimization of a heterologous n-butanol pathway in Escherichia coli.

The intracellular redox state plays an important role in the cellular physiology that determines the efficiency of chemical and biofuel production by microbial cell factories. However, it is difficult to achieve optimal redox rebalancing of synthetic pathways owing to the sensitive responses of cellular physiology according as the intracellular redox state changes. Here, we demonstrate optimal rebalancing of the intracellular redox state by model-driven control of expression using n-butanol production in Escherichia coli as a model system. The synthetic n-butanol production pathway was constructed by implementing synthetic constitutive promoters and designing synthetic 5'-untranslated regions (5'-UTR) for each gene. Redox rebalancing was achieved by anaerobically activating the pyruvate dehydrogenase (PDH) complex and additionally tuning the expression level of NAD(+)-dependent formate dehydrogenase (fdh1 from Saccharomyces cerevisiae) through rational UTR engineering. Interestingly, efficient production of n-butanol required different amounts of reducing equivalents depending on whether the substrate was glucose or galactose. One intriguing implication of this work is that additional strain improvement can be achieved, even within given genetic components, through rebalancing intracellular redox state according to target products and substrates.

Butyrate production in engineered Escherichia coli with synthetic scaffolds.

Butyrate pathway was constructed in recombinant Escherichia coli using the genes from Clostridium acetobutylicum and Treponema denticola. However, the pathway constructed from exogenous enzymes did not efficiently convert carbon flux to butyrate. Three steps of the productivity enhancement were attempted in this study. First, pathway engineering to delete metabolic pathways to by-products successfully improved the butyrate production. Second, synthetic scaffold protein that spatially co-localizes enzymes was introduced to improve the efficiency of the heterologous pathway enzymes, resulting in threefold improvement in butyrate production. Finally, further optimizations of inducer concentrations and pH adjustment were tried. The final titer of butyrate was 4.3 and 7.2 g/L under batch and fed-batch cultivation, respectively. This study demonstrated the importance of synthetic scaffold protein as a useful tool for optimization of heterologous butyrate pathway in E. coli.

Synthetic biology: tools to design microbes for the production of chemicals and fuels.

The engineering of biological systems to achieve specific purposes requires design tools that function in a predictable and quantitative manner. Recent advances in the field of synthetic biology, particularly in the programmable control of gene expression at multiple levels of regulation, have increased our ability to efficiently design and optimize biological systems to perform designed tasks. Furthermore, implementation of these designs in biological systems highlights the potential of using these tools to build microbial cell factories for the production of chemicals and fuels. In this paper, we review current developments in the design of tools for controlling gene expression at transcriptional, post-transcriptional and post-translational levels, and consider potential applications of these tools.

Refactoring redox cofactor regeneration for high-yield biocatalysis of glucose to butyric acid in Escherichia coli.

In this study, the native redox cofactor regeneration system in Escherichia coli was engineered for the production of butyric acid. The synthetic butyrate pathway, which regenerates NAD(+) from NADH using butyrate as the only final electron acceptor, enabled high-yield production of butyric acid from glucose (83.4% of the molar theoretical yield). The high selectivity for butyrate, with a butyrate/acetate ratio of 41, suggests dramatically improved industrial potential for the production of butyric acid from nonnative hosts compared to the native producers (Clostridium species). Furthermore, this strategy could be broadly utilized for the production of various other useful chemicals in the fields of metabolic engineering and synthetic biology.

Sturge-Weber syndrome: ear, nose, and throat issues and neurologic status.

The pathophysiology of Sturge-Weber syndrome is poorly understood, and ear, nose, and throat involvement is possible. These issues can result in frequent illnesses or airway obstruction, affecting patients' neurologic status. Patients with definite brain involvement who reported potential ear, nose, and throat issues on intake questionnaires underwent retrospective reviews of their medical records. We examined the relationships between these issues, secondary surgical interventions, and patients' neurologic status. The most common complaints involved the sinuses and frequent ear infections. Six patients underwent placement of ear tubes, leading to improvements in migraines and stroke-like episodes in one patient, and improved seizure control in four others. Obstructive sleep apnea was confirmed in three patients who underwent sleep studies. Tonsil or adenoid removal occurred in another three patients. Surgery resulted in marked improvements regarding excessive drooling, daytime sleepiness, and breathing problems. These findings suggest that ear, nose, and throat problems occur frequently in patients with Sturge-Weber Syndrome, and when repeated ear infections are associated with uncontrolled seizures, early placement of ear tubes may be beneficial. Furthermore, patients with facial tissue hypertrophy may be at risk for obstructive sleep apnea, and should be appropriately evaluated.