RESUMO
Alpha-lipoic acid (LA), a naturally occurring cofactor reported to be present in a diverse group of microorganisms, plants, and animal tissues, has been widely and successfully used as a therapy for a variety of diseases, including diabetes and heart disease. However, to date, recombinant DNA technology has not been applied for higher LA production due mainly to difficulties in the functional expression of key enzymes involved in LA production. Here, we report a study for higher LA production with the aid of chaperone plasmids, DnaKJE and trigger factor (Tf). The lipA and lplA genes encoding lipoate synthase and lipoate protein ligase in Pseudomonas fluorescens, respectively, were cloned and transformed into Escherichia coli K12. When they were overexpressed in E. coli, both LipA and LplA were expressed as inclusion bodies leading to no increase in LA production. However, when chaperone plasmids DnaKJE and Tf were coexpressed with lipA and lplA, the resulting recombinant E. coli strains showed higher LA production than the wild-type E. coli by 32-111%, respectively.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Chaperonas Moleculares/metabolismo , Peptídeo Sintases/metabolismo , Ácido Tióctico/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/genética , Chaperonas Moleculares/genética , Peptídeo Sintases/genética , Pseudomonas fluorescens/genéticaRESUMO
Using the optimal concentrations of octanoic acid (0.75 mM) and ethyl mercaptan (2 mM), as the most effective sulfur donor, Pseudomonas reptilivora produced 74 microg lipoic acid per dry cell weight at pH 7.5 and 30 degrees C in a fermenter over 9 h. The dry cell weight was 13.9 g l(-1).
Assuntos
Caprilatos/metabolismo , Pseudomonas/metabolismo , Enxofre/metabolismo , Ácido Tióctico/biossíntese , FermentaçãoRESUMO
The conversion of linoleic acid into 10-hydroxy-12(Z)-octadecenoic acid by whole cells of Stenotrophomonas nitritireducens as an isolated bacterium was optimized, and the optimal temperature, pH, and cell and substrate concentrations were 30 degrees C, 7.5, and 20 and 20 g/L, respectively. Under these conditions, whole cells in a bioreactor produced 15 g/L 10-hydroxy-12(Z)-octadecenoic acid in 2 h of reaction time without detectable byproducts. Using 2 g/L linoleic acid, the cells produced 1.92 g/L 10-hydroxy-12(Z)-octadecenoic acid. These are the highest concentration and yield of 10-hydroxy-12(Z)-octadecenoic acid ever reported.
Assuntos
Ácido Linoleico/metabolismo , Ácidos Esteáricos/metabolismo , Stenotrophomonas/metabolismo , Reatores Biológicos/microbiologia , Concentração de Íons de Hidrogênio , Microbiologia Industrial/instrumentação , Microbiologia Industrial/métodos , Ácido Linoleico/química , Ácidos Esteáricos/química , Stenotrophomonas/citologia , Stenotrophomonas/crescimento & desenvolvimento , TemperaturaRESUMO
An isolated bacterium that converted unsaturated fatty acids to hydroxy fatty acids was identified as Stenotrophomonas nitritireducens by API analysis, cellular fatty acids compositions, sequencing the full 16S ribosomal ribonucleic acid, and evaluating its nitrite reduction ability. S. nitritireducens has unique regio-specificity for C16 and C18 cis-9 unsaturated fatty acids. These fatty acids are converted to their 10-hydroxy fatty acids without detectable byproducts. Among the cis-9-unsaturated fatty acids, S. nitritireducens showed the highest specificity for linoleic acid. The cells converted 20 mM linoleic acid to 13.5 mM 10-hydroxy-12(Z)-octadecenoic acid at 30 degrees C and pH 7.5 with a yield of 67.5% (mol/mol).
