Proteome Reference Map and Comparative Proteomic Clostridium acetobutylicum is a low-GC-content,...

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  • Proteome Reference Map and Comparative Proteomic Analysis between a Wild Type Clostridium acetobutylicum DSM 1731 and its

    Mutant with Enhanced Butanol Tolerance and Butanol Yield

    Shaoming Mao,†,‡,§ Yuanming Luo,†,‡,| Tianrui Zhang,‡ Jinshan Li,‡ Guanhui Bao,‡,§ Yan Zhu,‡,§

    Zugen Chen,⊥ Yanping Zhang,‡ Yin Li,*,‡ and Yanhe Ma‡,|

    Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, Graduate School of Chinese Academy of Sciences, Beijing, China, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese

    Academy of Sciences, Beijing, China, and Department of Human Genetics, School of Medicine, University of California, Los Angeles, California 90095

    Received December 29, 2009

    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.

    Keywords: Clostridium acetobutylicum • proteome reference map • comparative proteome analysis • two-dimensional gel electrophoresis • acidogenesis • solventogenesis • butanol tolerance • butanol yield

    Introduction

    Clostridium acetobutylicum is a low-GC-content, Gram- positive, spore-forming, obligate anaerobe that is capable of fermenting a wide variety of sugars (e.g., glucose, galactose, cellobiose, mannose, xylose, and arabinose) to acids (acetic acid and butyric acid) and solvents (acetone, butanol, and ethanol).1

    C. acetobutylicum culture was extensively used to produce acetone and butanol from starch for industrial purposes.2

    Recently, anaerobic fermentative production of butanol using this bacterium gained remarkable interest as butanol is con- sidered as a potential superior biofuel alternative.3 Recent developments in genetics,4 genomics,5 and proteomics6,7 of C. acetobutylicum have greatly increased our understanding of the solvent production physiology, which is extremely important for improving butanol production by means of metabolic engineering or systems biotechnology.

    Butanol toxicity is the major barrier for cost-effective fer- mentative production of butanol. This can be seen from the fact that C. acetobutylicum fermentations rarely produce bu- tanol higher than 13 g/L, a level that is inhibitory for the growth of C. acetobutylicum and is generally considered as the toxic limit.8 Butanol toxicity in C. acetobutylicum is quite severe, and this has been attributed to the chaotropic effect on cell membrane9,10 and the inhibition effects on nutrient transport, glucose uptake, and membrane-bound ATPase activity.9 Mod-

    * To whom correspondence should be addressed. Yin Li, Institute of Microbiology, Chinese Academy of Sciences, No.1 West Beichen Road, Chaoyang District, Beijing 100101, China. E-mail: yli@im.ac.cn. Tel: +86- 10-64807485. Fax: +86-10-64807485.

    † These authors contributed equally to this work. ‡ Institute of Microbiology, Chinese Academy of Sciences. § Graduate School of Chinese Academy of Sciences. | State Key Laboratory of Microbial Resources, Institute of Microbiology,

    Chinese Academy of Sciences. ⊥ University of California.

    3046 Journal of Proteome Research 2010, 9, 3046–3061 10.1021/pr9012078  2010 American Chemical Society Published on Web 04/29/2010

    http://pubs.acs.org/action/showImage?doi=10.1021/pr9012078&iName=master.img-000.png&w=251&h=40

  • erate increases in butanol titers have been shown to elicit a response similar to a heat shock.11 Butanol-stressed exponen- tially growing C. acetobutylicum resulted in upregulation of numerous chaperone genes (dnaK, groES, groEL, hsp90, hsp18, clpC, and htrA), solventogenic genes, and glycerol metabolism genes glpA and glpF.12 Butanol-tolerant strains have been developed from C. acetobutylicum by using classic chemical mutagenesis, continuous culture, serial enrichment procedures, and targeted genetic/metabolic engineering.13–18 The improved butanol tolerance has different impacts on butanol production (decrease,14,18 minor improvement,13,19 or significant (>20%) improvement),15–17 suggesting the relationship between bu- tanol tolerance and butanol production is rather complex and remains largely unknown.

    The availability of the complete genome sequence of C. acetobutylicum5 enables the genome-wide investigation of the mechanism for butanol tolerance. Genome-wide transcriptome studies on C. acetobutylicum were performed.12,20,21 Charac- terization of the C. acetobutylicum Spo0A mutant SKO1 by DNA microarray analysis revealed that Spo0A inactivation triggered down-regulation of solventogenic, sporulation, and carbohy- drate metabolism genes and up-regulation of flagellar and chemotaxis genes.22 Transcriptome analysis of C. acetobutylicum mutant 824 (pGROE1) with overexpressed groESL revealed an increased expression of motility and chemotaxis genes.15

    Proteomics is a powerful tool to understand the cellular status at the protein level, which cannot be deciphered from either genome or transcriptome analysis. Proteomics approaches are increasingly employed to identify proteins that can be used as targets for metabolic engineering.23–25 Since two-dimensional electrophoresis (2-DE) was introduced as a tool to separate complex mixtures of cellular proteins,26 a large number of prokaryotic proteomes have been studied.27–29 Studies on C. acetobutylicum proteomics are underway. A proteomic analysis of the C. acetobutylicum during the transition from acidogenesis to solventogenesis discovered some changes in the protein pattern.6 A comparative proteomic analysis between C. aceto- butylicum wild type strain ATCC 824 and its mutant 824(pM- SPOA) discovered that Spo0A overexpression affected the abundance of proteins involved in glycolysis, translation, heat shock stress response, and energy production.7

    The aim of this study was to establish a comprehensive cytoplasmic proteome reference map for C. acetobutylicum so that the reference map can be used as a fundamental tool for comparative proteomics study to further increase our knowl- edge on the physiology of this species. To illustrate the significance of this reference map, we carried out a comparative proteomic analysis between two strains, C. acetobutylicum wild type strain DSM 1731 (butanol tolerance 13 g/L) and its mutant Rh8 (butanol tolerance 19 g/L), with the aim to obtain fundamental data for understanding the biological mechanism on butanol tolerance and/or butanol yield.

    Experimental Section

    Bacteria and Culture Conditions. C. acetobutylicum DSM 1731, obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), was used for generating the proteome reference map. Mutant Rh8 is a genome-shuffled strain derived from strain DSM 1731 and its butanol-tolerant mutants. Cells from a single colony were used to inoculate liquid reinforced clostridial medium (RCM),30

    which were then heat shocked at 75 °C for 10 min before growing at 37 °C. When the OD600 reached 1.0, the culture was

    stored at 30 °C for one week to produce spores.31 Spore counts were measured as following: 100 µL seed cultures were mixed with 900 µL deionized distilled water. Disposable micropipets were used to stir the mixture and drops of the suspensions were transferred to a hemacytometer slide. An estimate of the total number of spores produced in each chamber was derived arithmetically from the hemacytometer counts.32 Precultures (10 mL) of strains DSM 1731 and Rh8 were inoculated to about 106-107 spores and pasteurized for 10 min at 75 °C before incubation, then cultured in a 250 mL bottle containing 100 mL of clostridial growth medium (CGM)33 at 37 °C for 12 h. In all experiments, cell growth was monitored by measuring the absorbance of the culture broth at 600 nm (OD600) with a model DU series 800 spectrophotometer (Beckman, Fullerton, CA). For proteomic analysis, cells were harvested at exponential growth phase at OD600 of 2.0, which is corresponding to 6.5 × 108 colony forming units/mL.

    Generation of Mutant Rh8. Genome shuffling34 was used to generate C. acetobutylicum mutant Rh