RESUMEN
Here, Corynebacterium glutamicum ATCC13032 expressing Baeyer-Villiger monooxygenase from Pseudomonas putida KT2440 was designed to produce 9-(nonanoyloxy) nonanoic acid from 10-ketostearic acid. Diverse parameters including cultivation and reaction temperatures, type of detergent, and pH were found to improve biotransformation efficiency. The optimal temperature of cultivation for the production of 9-(nonanoyloxy) nonanoic acid from 10-ketostearic acid using whole cells of recombinant C. glutamicum was 15 °C, but the reaction temperature was optimal at 30 °C. Enhanced conversion efficiency was obtained by supplying 0.05 g/L of Tween 80 at pH 7.5. Under these optimal conditions, recombinant C. glutamicum produced 0.28 mM of 9-(nonanoyloxy) nonanoic acid with a 75.6% (mol/mol) conversion yield in 2 h. This is the first report on the biotransformation of 10-ketostearic acid to 9-(nonanoyloxy) nonanoic acid with a recombinant whole-cell C. glutamicum-based biocatalyst and the results demonstrate the feasibility of using C. glutamicum as a whole-cell biocatalyst.
Asunto(s)
Biocatálisis , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Ácidos Grasos/biosíntesis , Biotransformación , Ácidos Grasos/metabolismo , Concentración de Iones de Hidrógeno , Oxigenasas de Función Mixta/metabolismo , Pseudomonas putida/enzimología , Pseudomonas putida/genética , Ácidos Esteáricos/metabolismo , TemperaturaRESUMEN
OBJECTIVE: To produce 10-ketostearic acid from oleic acid. RESULTS: Oleic acid was converted to 10-ketostearic acid by a recombinant Corynebacterium glutamicum ATCC 13032 expressing oleate hydratase from Stenotrophomonas maltophilia and a secondary alcohol dehydrogenase from Micrococcus luteus under the control of a synthetic constitutive promoter. Optimal conditions for 10-ketostearic acid production were pH 7.5 and 30 °C with 5 g cells l(-1) and 2.5 g oleic acid l(-1). Under these conditions, the cells produced 1.96 g 10-ketostearic acid l(-1) from oleic acid in 6 h, with a conversion yield of 78 % (w) and a maximum volumetric productivity of 1.67 g l(-1) h(-1). CONCLUSION: This is the first report of 10-ketostearic acid production using a recombinant C. glutamicum.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Hidroliasas/metabolismo , Ácido Oléico/metabolismo , Ácidos Esteáricos/metabolismo , Oxidorreductasas de Alcohol/genética , Biotransformación , Corynebacterium glutamicum/enzimología , Hidroliasas/genética , Concentración de Iones de Hidrógeno , Micrococcus luteus/enzimología , Micrococcus luteus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Stenotrophomonas maltophilia/enzimología , Stenotrophomonas maltophilia/genética , TemperaturaRESUMEN
The biocatalytic efficiency of recombinant Corynebacterium glutamicum ATCC 13032 expressing the secondary alcohol dehydrogenase of Micrococcus luteus NCTC2665 was studied. Recombinant C. glutamicum converts ricinoleic acid to a product, identified by gas chromatography/mass spectrometry as 12-ketooleic acid (12-oxo-cis-9-octadecenoic acid). The effects of pH, reaction temperature, and non-ionic detergent on recombinant C. glutamiucm whole cell bioconversion were examined. The determined optimal conditions for production of 12-ketooleic acid are pH 8.0, 35°C, and 0.05 g/l Tween80. Under these conditions, recombinant C. glutamicum produces 3.3 mM 12-ketooleic acid, with a 72% (mol/mol) maximum conversion yield, and 1.1 g/l/h volumetric productivity in 2 h; and 3.9 mM 12- ketooleic acid, with a 74% (mol/mol) maximum conversion yield, and 0.69 g/l/h maximum volumetric productivity in 4 h of fermentation. This study constitutes the first report of significant production of 12-ketooleic acid using a recombinant Corynebacterium glutamicum-based biocatalyst.