Proteome Reference Map and Comparative Proteomic Analysis between a Wild TypeClostridium acetobutylicumDSM 1731 and its Mutant with Enhanced Butanol Tolerance and Butanol Yield

Abstract

The solventogenic bacterium Clostridium acetobutylicum is an important species of the Clostridium community. To develop a fundamental tool that is useful for biological studies of C. acetobutylicum, we established a high resolution proteome reference map for this species. We identified 1206 spots representing 564 different proteins by mass spectrometry, covering approximately 50% of major metabolic pathways. To better understand the relationship between butanol tolerance and butanol yield, we performed a comparative proteomic analysis between the wild type strain DSM 1731 and the mutant Rh8, which has higher butanol tolerance and higher butanol yield. Comparative proteomic analysis of two strains at acidogenic and solventogenic phases revealed 102 differentially expressed proteins that are mainly involved in protein folding, solvent formation, amino acid metabolism, protein synthesis, nucleotide metabolism, transport, and others. Hierarchical clustering analysis revealed that over 70% of the 102 differentially expressed proteins in mutant Rh8 were either upregulated (e.g., chaperones and solvent formation related) or downregulated (e.g., amino acid metabolism and protein synthesis related) in both acidogenic and solventogenic phase, which, respectively, are only upregulated or downregulated in solventogenic phase in the wild type strain. This suggests that Rh8 cells have evolved a mechanism to prepare themselves for butanol challenge before butanol is produced, leading to an increased butanol yield. This is the first report on the comparative proteome analysis of a mutant strain and a base strain of C. acetobutylicum. The fundamental proteomic data and analyses will be useful for further elucidating the biological mechanism of butanol tolerance and/or enhanced butanol production.

Coenzyme A-transferase-independent butyrate re-assimilation in Clostridium acetobutylicum—evidence from a mathematical model

“…In addition, it facilitates the first step in the formation of acetone (Andersch et al 1983;. This view is supported by several experimental studies which had observed an increased transcription of the encoding genes, ctfA and ctfB, which are part of the sol operon (Grimmler et al 2011;Jones et al 2008), and an increased intracellular concentration of the protein during the pH-induced shift and during solventogenesis (Janssen et al 2010;Mao et al 2010). However, recent experimental evidence indicates that an alternative re-assimilation mechanism for butyrate could exist which is independent of the CoA-transferase (Desai et al 1999;Lehmann et al 2012a) and may rely on a pH-dependent reverse activity of the butyrate forming enzymes (Desai et al 1999;Lehmann et al 2012b).…”

“…During exponential growth acetate and butyrate are produced (acidogenic phase). This type of anaerobic metabolism enables the organism to gain the maximal amount of energy (ATP) per mole glucose using substrate-level phosphorylation (Madigan et al 2009). During the transition to the stationary phase the metabolism switches to the formation primarily of acetone and butanol (solventogenic phase).…”

The Function of Snodprot in the Cerato-Platanin Family fromDactylellina cionopagain Nematophagous Fungi

“…The gel was stained with Coomassie Blue after electrophoresis. The putative recombinant protein in the SDS-PAGE gel was cut out, and then matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis was performed as described by Mao et al 31) For protein purification, the selected transformant and the transformant with an empty vector were fermented under the same culture conditions. The proteins in the supernatant fluid, the volume of which was normalized according to the OD600 value, were precipitated with 50% ammonium sulphate overnight.…”

Genome Replication Engineering Assisted Continuous Evolution (GREACE) to Improve Microbial Tolerance for Biofuels Production

“…We applied a similar procedure to engineering n-butanol tolerance -E. coli cells carrying pQ-lib were serially transferred in media containing gradually increased n-butanol concentrations. Considering that improvement of complex traits such as n-butanol tolerance might require accumulation of much more adaptive mutations [31,32], the pQ-lib carrying cells were transferred in each n-butanol concentrations for three times, upon full growth, before transferring to the next higher butanol concentration. Figure 4A showed that pQ-lib carrying cells exhibited a better adaptability to the increased n-butanol concentrations.…”