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1.
J Microbiol Biotechnol ; 33(4): 552-558, 2023 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-36775859

RESUMEN

Levulinic acid (LA) is a valuable chemical used in fuel additives, fragrances, and polymers. In this study, we proposed possible biosynthetic pathways for LA production from lignin and poly(ethylene terephthalate). We also created a genetically encoded biosensor responsive to LA, which can be used for screening and evolving the LA biosynthesis pathway genes, by employing an LvaR transcriptional regulator of Pseudomonas putida KT2440 to express a fluorescent reporter gene. The LvaR regulator senses LA as a cognate ligand. The LA biosensor was first examined in an Escherichia coli strain and was found to be non-functional. When the host of the LA biosensor was switched from E. coli to P. putida KT2440, the LA biosensor showed a linear correlation between fluorescence intensity and LA concentration in the range of 0.156-10 mM LA. In addition, we determined that 0.156 mM LA was the limit of LA detection in P. putida KT2440 harboring an LA-responsive biosensor. The maximal fluorescence increase was 12.3-fold in the presence of 10 mM LA compared to that in the absence of LA. The individual cell responses to LA concentrations reflected the population-averaged responses, which enabled high-throughput screening of enzymes and metabolic pathways involved in LA biosynthesis and sustainable production of LA in engineered microbes.


Asunto(s)
Técnicas Biosensibles , Pseudomonas putida , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Pseudomonas putida/metabolismo
2.
Oncol Rep ; 40(6): 3869-3878, 2018 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-30272370

RESUMEN

The identification of large numbers of genetic mutations in immature myeloid cells has made it difficult to identify specific targets for acute myeloid leukemia (AML) therapy. Although current pharmacological targets for controlling cancer are focused on identifying genetic mutations, it is hard to develop the specific drugs to achieve complete remission due to complex and variable genetic mutations. To overcome the failure of the genetic mutation theory, the present study targeted mitochondrial metabolism as a strategy for inducing anti­leukemic activity, based on evidence that AML cells have an abnormally high amount of mitochondria and that somatic mutations can alter metabolic flux in cancer. It was found that L­deprenyl, which is clinically available for the treatment of Parkinson's disease, exerts anti­mitochondria activity in KG­1α cells, as assessed by detection of oxygen consumption rate (OCR) and extracellular acidification (ECAR) using XF analyzer, respectively. Using a luciferase assay for detecting adenosine triphosphate (ATP) content, it was found that suppression of mitochondrial activity led to ATP depletion and was associated with potent cytotoxic activity. L­deprenyl is known to target monoamine oxidase­B (MAO­B) on the outer membrane of mitochondria, therefore, the activity of MAO­A and ­B was measured based on the fluorometric detection of H2O2 produced by the enzyme reaction. Notably, MAO­A and -B activity was low in AML cells and the present findings suggested that the anticancer effect of L­deprenyl was independent of MAO­B. Change of mitochondrial respiration­ and glycolysis­related gene expression levels were measured by reverse transcription­quantitative polymerase chain reaction. Consistent with the aforementioned results, treatment with L­deprenyl reduced the mRNA level of mitochondrial respiration­ and glycolysis­related genes. Collectively, the present results identify L­deprenyl as a novel candidate for the treatment of AML through inhibition of mitochondrial respiration.


Asunto(s)
Leucemia Mieloide Aguda/tratamiento farmacológico , Mitocondrias/metabolismo , Monoaminooxidasa/metabolismo , Selegilina/administración & dosificación , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucólisis/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Mutación , Consumo de Oxígeno/efectos de los fármacos , Selegilina/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto
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