20 research outputs found

    Coenzyme B-12 can be produced by engineered Escherichia coli under both anaerobic and aerobic conditions

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    Coenzyme B-12 (Vitamin B-12) is one of the most complex biomolecules and an essential cofactor required for the catalytic activity of many enzymes. Pseudomonas denitrificans synthesizes coenzyme B-12 in an oxygen-dependent manner using a pathway encoded by more than 25 genes that are located in six different operons. Escherichia coli, a robust and suitable host for metabolic engineering was used to produce coenzyme B-12. These genes were cloned into three compatible plasmids and expressed heterologously in E. coli BL21 (DE3). Real-time PCR, SDS-PAGE analysis and bioassay showed that the recombinant E. coli expressed the coenzyme B-12 synthetic genes and successfully produced coenzyme B-12. However, according to the quantitative determination by inductively coupled plasma-mass spectrometry, the amount of coenzyme B-12 produced by the recombinant E. coli (0.21 +/- 0.02 mu g/g cdw) was approximately 13-fold lower than that by P. denitrificans (2.75 +/- 0.22 mu g/g cdw). Optimization of the culture conditions to improve the production of coenzyme B-12 by the recombinant E. coli was successful, and the highest titer (0.65 +/- 0.03 mu g/g cdw) of coenzyme B-12 was obtained. Interestingly, although the synthesis of coenzyme B-12 in P. denitrificans is strictly oxygen-dependent, the recombinant E. coli could produce coenzyme B-12 under anaerobic conditions

    Association between Continuity of Care and the Onset of Thyroid Disorder among Diabetes Patients in Korea

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    Objectives: As the relationship between diabetes mellitus and thyroid dysfunction is well known, it is important to investigate the factors influencing this association. Continuity of care is associated with better quality of care and outcomes, such as reduced complications, among diabetes patients. Therefore, the purpose of this study was to investigate the association between continuity of care and the onset of thyroid dysfunction among diabetes patients. Methods: We used Korean National Health Insurance Service National Sample Cohort data from 2002 to 2013. Our final study population included 16,806 newly diagnosed diabetes patients who were older than 45 years of age. Continuity of care was measured using the Continuity of Care index. The dependent variable was the onset of thyroid disorder. Cox proportional hazard regression models were used for statistical analyses. Results: Diabetes patients with low continuity of care were at increased risk of the onset of thyroid disorder compared with those with high continuity of care (hazard ratio (HR): 1.28, 95% confidence interval (CI): 1.07–1.54). Subgroup analyses showed that this association was significant within patients with type 2 diabetes (HR: 1.24, 95% CI: 1.01–1.52) or whose main attending site was a local clinic (HR: 1.32, 95% CI: 1.07–1.64). Conclusions: Our results show that diabetes patients with low continuity of care are more likely to experience the onset of thyroid disorder. Therefore, improving continuity of care could be a reasonable method of preventing complications or comorbidities, including thyroid disorder, among diabetes patients

    Transcriptomics analysis of Escherichia coli W mutant tolerant to high concentration of 3-hydroxypropionic acid

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    3-Hydroxypropionic acid (3-HP) is an important platform chemical which can be produced from glycerol by recombinant Escherichia coli. One of the challenges for commercialization of 3-HP production is its toxic effect at high concentration. Recently, a novel transcriptional regulator (designated as 3HPT) was found to be related with 3-HP tolerance in E. coli. However, the mechanism of its regulation is still unknown. To address this issue, transcriptome analysis of several E. coli W mutants which is highly tolerant to 3-HP was conducted. Interestingly, there was more than hundreds of genes are regulated by 3HPT. Among them, many genes are related with acid tolerance mechanisms, some genes have not been well studied. Functional analysis and further experiments to elucidate the mechanism are in progress

    Development of 3-hydroxypropionic-acid-tolerant strain of Escherichia coli W and role of minor global regulator yieP

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    3-Hydroxypropionic acid (3-HP) is an important platform chemical, but its toxic effect at high concentrations (> 200 mM) is a serious challenge for commercial production. In this study, a highly 3-HP-tolerant strain of Escherichia coli W (tolerance concentration: 400 mM in M9 minimal medium and 800 mM when yeast extract was added) was developed by adaptive laboratory evolution (ALE) with glycerol as the carbon source. Genome analysis of the adapted strain (designated as E. coli WA) indicated the presence of mutations in 13 genes, including glpK (glycerol kinase) and yieP (a less-studied global regulator). The mutant GlpK (K67T) exhibited a higher activity than the wild-type enzyme, but it was not beneficial for 3-HP production due to its causing carbon overflow metabolism. Interestingly, among the other 12 genes, the mutation in yieP alone was almost fully responsible for the improved tolerance to 3-HP. When the mutant yieP was substituted with the wild-type counterpart, the adapted E. coli WA strain completely lost its tolerance to 3-HP, showing a tolerance similar to the wild-type W strain. Deletion of yieP conferred 3-HP tolerance to several other E. coli strains including K-12 W3110, K-12 MG1655, and B except BL21 (DE3). The E. coli WA with wild-type glpK showed, as compared with its parental strain, better 3-HP production, indicating that improved tolerance is beneficial for 3-HP production

    Deletion of a minor global regulator yieP greatly improves the tolerance of Escherichia coli to 3-hydroxypropionic acid

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    3-Hydroxypropionic acid (3-HP) is an important platform chemical which can be converted to many useful chemicals. The toxic effects of 3-HP on cell growth and viability is a serious challenge for its industrial biological production. In this study, a highly 3-HP tolerant strain was developed from Escherichia coli W by adaptive laboratory evolution (ALE). Genome analysis indicated that 13 genes including glpK and yieP were mutated during ALE. For the mutated genes, contribution to 3-HP tolerance was studied by multiplex automated genome engineering (MAGE). Interestingly, a nonsense mutation in yieP alone was fully responsible for the improved tolerance to 3-HP. Mutation or complete deletion of yieP conferred a similar 3-HP tolerance to other E. coli strains. The study on 3-HP tolerance mechanism conferred by ??yieP is in progres
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