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To smoothen the process of n-butanol formation in Pseudomonas putida KT2440, detailed knowledge of the impact of this organic solvent on cell physiology and regulation is of outmost importance.Here,we conducted a detailed systems biology study to elucidate cellular responses at the metabolic, proteomic, and transcriptional level. Pseudomonas putida KT2440 was cultivated in multiple chemostat fermentations using n-butanol either as sole carbon source or together with glucose. Pseudomonas putida KT2440 revealed maximum growth rates (μ) of 0.3 h-1 with n-butanol as sole carbon source and of 0.4 h-1 using equal C-molar amounts of glucose and nbutanol. While C-mole specific substrate consumption and biomass/substrate yields appeared equal at these growth conditions, the cellular physiology was found to be substantially different: adenylate energy charge levels of 0.85 were found when n-butanol served as sole carbon source (similar to glucose as sole carbon source), but were reduced to 0.4 when n-butanol was coconsumed at stable growth conditions. Furthermore, characteristic maintenance parameters changed with increasing n-butanol consumption. 13C flux analysis revealed that central metabolism was split into a glucose-fueled Entner-Doudoroff/pentose-phosphate pathway and an n-butanol-fueled tricarboxylic acid cycle when both substrates were coconsumed. With the help of transcriptome and proteome analysis, the degradation pathway of n-butanol could be unraveled, thus representing an important basis for rendering P. putida KT2440 from an n-butanol consumer to a producer in future metabolic engineering studies. [ABSTRACT FROM AUTHOR]

A two-phase biotransformation process for selective hydroxylation of n-octane to 1-octanol via Pseudomonas putida KT2440 harboring heterologously expressed P450 monooxygenase from Mycobacterium marinum is presented. Maximum cell-specific conversion rates of 12.7 mgoctanol gCDWh-1 were observed not only in shaking flasks but also in 3.7-L-bioreactor studies. The bioreactor experiments were performed avoiding explosive gas mixtures by lowering volumetric power input, aeration rates and substrate concentrations. Based on a stoichiometric network of P. putida KT2440 topological studies were carried out. As a conclusion, potential limitations of NAD(P)H and/or ATP supply at production conditions can be excluded. Hence, the great potential of the host for further increasing conversion is outlined. [ABSTRACT FROM AUTHOR]