Applying systems biology tools to study n‐butanol degradation in Pseudomonas putida KT2440

Abstract

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 n‐butanol. 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.