Integrated intracellular metabolic profiling and pathway analysis approaches reveal complex metabolic regulation by Clostridium acetobutylicum

Liu et al. Microb Cell Fact Integrated intracellular metabolic profiling and pathway analysis approaches reveal complex metabolic regulation by Clostridium acetobutylicum Huanhuan Liu 0 2 3 Di Huang 1 4 5 Jianping Wen 0 2 3 0 SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University , Tianjin 300072 , People's Republic of China 1 TEDA Institute of Biological Sciences and Biotechnology, Nankai University , TEDA, Tianjin 300457 , People's Republic of China 2 SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineer- ing (Tianjin), School of Chemical Engineering and Technology , Tianjin Univer- sity, Tianjin 300072 , People's Republic of China 3 Key Laboratory of System Bioengineering (Tianjin University), Ministry of Education , Tianjin 300072 , People's Republic of China 4 Tianjin Key Laboratory of Microbial Functional Genomics , Tianjin 300457 , People's Republic of China 5 Key Laboratory of Molecular Microbiology and Technol- ogy, Ministry of Education , Tianjin 300071 , People's Republic of China Background: Clostridium acetobutylicum is one of the most important butanol producing strains. However, environmental stress in the fermentation process usually leads to a lower yield, seriously hampering its industrialization. In order to systematically investigate the key intracellular metabolites that influence the strain growth and butanol production, and find out the critical regulation nodes, an integrated analysis approach has been carried out in this study. Results: Based on the gas chromatography-mass spectrometry technology, the partial least square discriminant analysis and the pathway analysis, 40 metabolic pathways linked with 43 key metabolic nodes were identified. Indepth analysis showed that lots of amino acids metabolism promoted cell growth but exerted slight influence on butanol production, while sugar metabolism was favorable for cell growth but unfavorable for butanol synthesis. Besides, both lysine and succinic acid metabolism generated a complex effect on the whole metabolic network. Dicarboxylate metabolism exerted an indispensable role on cell growth and butanol production. Subsequently, rational feeding strategies were proposed to verify these conclusions and facilitate the butanol biosynthesis. Feeding amino acids, especially glycine and serine, could obviously improve cell growth while yeast extract, citric acid and ethylene glycol could significantly enhance both growth and butanol production. Conclusions: The feeding experiment confirmed that metabolic profiling combined with pathway analysis provided an accurate, reasonable and practical approach to explore the cellular metabolic activity and supplied a basis for improving butanol production. These strategies can also be extended for the production of other important biochemical compounds. Butanol; Metabolic profiling analysis; Pathway analysis; Clostridium acetobutylicum; GC-MS; Metabolomics Background Bio-butanol, the next generation of liquid biofuels after bio-ethanol, has gained much interest due to many distinguished advantages, such as better blending ratio with gasoline, lower vapor pressure and corrosivity, higher energy density and less fuel consumption per unit [ 1, 2 ]. More importantly, as an ideal supplement or a sustainable replacement of gasoline, butanol can directly fuel the existing engines without any modification [1]. At present the production of butanol mainly depends on chemical method by propylene oxo synthesis in the industry [ 3 ]. However, serious environmental pollution, high oil prices and the exhaustion of oil resources force us to explore the sustainable clean alternative process for butanol production [ 4 ]. Hence, bio-butanol based on the classical acetone-butanol-ethanol (ABE) fermentation by microorganisms such as Clostridium acetobutylicum, Clostridium beijerinckii and similar strains, has been brought to light again [ 5 ]. However, in the last decade the butanol production has stayed at the level of approximately 10–20 g per liter during a batch ABE fermentation owing to some disadvantages by C. acetobutylicum. As a species of strictly anaerobic bacteria, Clostridia catabolizes a variety of sugars for cellular events, accompanying with the production of toxic metabolites, such as acetic acid, butyric acid, ethanol, butanol and acetone, which seriously inhibit cell growth and butanol production due to the acidified intracellular environment and the insufficient ATP production capacity [ 6 ], and even lead to the “acid crash” [ 7 ]. Moreover, the formation of endogenous spore can result in the termination of butanol secretion with a lower yield [ 8 ]. Butanol production, on the other hand, is limited by the defect of redox system, and the cell is highly sensitive to the redox status of intracellular and extracellular conditions [ 9–1 (...truncated)