Improvement of cellulose catabolism in Clostridium cellulolyticum by sporulation abolishment and carbon alleviation

Biotechnology for Biofuels

Yongchao Li²  Tao Xu²  Timothy J Tschaplinski³  Nancy L Engle³  Yunfeng Yang⁴  David E Graham³  Zhili He²  Jizhong Zhou¹ 

[1] Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

[2] Institute for Environmental Genomics and Department of Microbiology and Plant Biology, University of Oklahoma, 101 David L. Boren Boulevard, Norman, OK 73019, USA

[3] Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA

[4] State Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing 100084, China

关键词: Isobutanol;    Cellulose catabolism;    spo0A;    Sporulation;    Clostridium cellulolyticum;   

Others  :  793592
DOI  :  10.1186/1754-6834-7-25

 received in 2013-10-06, accepted in 2014-02-06,  发布年份 2014

【 摘 要 】

Background

Clostridium cellulolyticum can degrade lignocellulosic biomass, and ferment the soluble sugars to produce valuable chemicals such as lactate, acetate, ethanol and hydrogen. However, the cellulose utilization efficiency of C. cellulolyticum still remains very low, impeding its application in consolidated bioprocessing for biofuels production. In this study, two metabolic engineering strategies were exploited to improve cellulose utilization efficiency, including sporulation abolishment and carbon overload alleviation.

Results

The spo0A gene at locus Ccel_1894, which encodes a master sporulation regulator was inactivated. The spo0A mutant abolished the sporulation ability. In a high concentration of cellulose (50 g/l), the performance of the spo0A mutant increased dramatically in terms of maximum growth, final concentrations of three major metabolic products, and cellulose catabolism. The microarray and gas chromatography–mass spectrometry (GC-MS) analyses showed that the valine, leucine and isoleucine biosynthesis pathways were up-regulated in the spo0A mutant. Based on this information, a partial isobutanol producing pathway modified from valine biosynthesis was introduced into C. cellulolyticum strains to further increase cellulose consumption by alleviating excessive carbon load. The introduction of this synthetic pathway to the wild-type strain improved cellulose consumption from 17.6 g/l to 28.7 g/l with a production of 0.42 g/l isobutanol in the 50 g/l cellulose medium. However, the spo0A mutant strain did not appreciably benefit from introduction of this synthetic pathway and the cellulose utilization efficiency did not further increase. A technical highlight in this study was that an in vivo promoter strength evaluation protocol was developed using anaerobic fluorescent protein and flow cytometry for C. cellulolyticum.

Conclusions

In this study, we inactivated the spo0A gene and introduced a heterologous synthetic pathway to manipulate the stress response to heavy carbon load and accumulation of metabolic products. These findings provide new perspectives to enhance the ability of cellulolytic bacteria to produce biofuels and biocommodities with high efficiency and at low cost directly from lignocellulosic biomass.

【 授权许可】

   
2014 Li et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS: Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 2002, 66:506-577.
• [2]Li Y, Irwin DC, Wilson DB: Processivity, substrate binding, and mechanism of cellulose hydrolysis by Thermobifida fusca Cel9A. Appl Environ Microbiol 2007, 73:3165-3172.
• [3]Lynd LR, van Zyl WH, McBride JE, Laser M: Consolidated bioprocessing of cellulosic biomass: an update. Curr Opin Biotechnol 2005, 16:577-583.
• [4]Desvaux M: Clostridium cellulolyticum: model organism of mesophilic cellulolytic clostridia. FEMS Microbiol Rev 2005, 29:741-764.
• [5]Higashide W, Li Y, Yang Y, Liao JC: Metabolic engineering of Clostridium cellulolyticum for production of isobutanol from cellulose. Appl Environ Microbiol 2011, 77:2727-2733.
• [6]Kristensen JB, Felby C, Jorgensen H: Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose. Biotechnol Biofuels 2009, 2:11. BioMed Central Full Text
• [7]Petitdemange ECF, Giallo J, Gaudin C: Clostridium cellulolyticum sp. nov., a cellulolytic, mesophilic: species from decayed grass. Int J Syst Bacteriol 1984, 34:155-159.
• [8]Guedon E, Desvaux M, Payot S, Petitdemange H: Growth inhibition of Clostridium cellulolyticum by an inefficiently regulated carbon flow. Microbiology 1999, 145:1831-1838.
• [9]Guedon E, Desvaux M, Petitdemange H: Improvement of cellulolytic properties of Clostridium cellulolyticum by metabolic engineering. Appl Environ Microbiol 2002, 68:53-58.
• [10]Desvaux M, Petitdemange H: Sporulation of Clostridium cellulolyticum while grown in cellulose-batch and cellulose-fed continuous cultures on a mineral-salt based medium. Microb Ecol 2002, 43:271-279.
• [11]Wiegel J, Dykstra M: Clostridium thermocellum: adhesion and sporulation while adhered to cellulose and hemicellulose. Appl Microbiol Biotechnol 1984, 20:59-65.
• [12]Papoutsakis ET: Engineering solventogenic clostridia. Curr Opin Biotechnol 2008, 19:420-429.
• [13]Tracy BP, Jones SW, Papoutsakis ET: Inactivation of sigma(E) and sigma(G) in Clostridium acetobutylicum illuminates their roles in Clostridial-cell-form biogenesis, granulose synthesis, solventogenesis, and spore morphogenesis. J Bacteriol 2011, 193:1414-1426.
• [14]Jones SW, Tracy BP, Gaida SM, Papoutsakis ET: Inactivation of sigmaF in Clostridium acetobutylicum ATCC 824 blocks sporulation prior to asymmetric division and abolishes sigmaE and sigmaG protein expression but does not block solvent formation. J Bacteriol 2011, 193:2429-2440.
• [15]Bi CH, Jones SW, Hess DR, Tracy BP, Papoutsakis ET: SpoIIE is necessary for asymmetric division, sporulation, and expression of sigma(F), sigma(E), and sigma(G) but does not control solvent production in Clostridium acetobutylicum ATCC 824. J Bacteriol 2011, 193:5130-5137.
• [16]Paredes CJ, Alsaker KV, Papoutsakis ET: A comparative genomic view of clostridial sporulation and physiology. Nat Rev Microbiol 2005, 3:969-978.
• [17]Molle V, Fujita M, Jensen ST, Eichenberger P, Gonzalez-Pastor JE, Liu JS, Losick R: The Spo0A regulon of Bacillus subtilis. Mol Microbiol 2003, 50:1683-1701.
• [18]Atsumi S, Hanai T, Liao JC: Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 2008, 451:86-89.
• [19]Hoch JA: Genetic analysis of pleiotropic negative sporulation mutants in Bacillus Subtilis. J Bacteriol 1971, 105:896-901.
• [20]Huang IH, Waters M, Grau RR, Sarker MR: Disruption of the gene (spo0A) encoding sporulation transcription factor blocks endospore formation and enterotoxin production in enterotoxigenic Clostridium perfringens type A. FEMS Microbiol Lett 2004, 233:233-240.
• [21]Ravagnani A, Jennert KC, Steiner E, Grunberg R, Jefferies JR, Wilkinson SR, Young DI, Tidswell EC, Brown DP, Youngman P, Morris JG, Young M: Spo0A directly controls the switch from acid to solvent production in solvent-forming clostridia. Mol Microbiol 2000, 37:1172-1185.
• [22]Harris LM, Welker NE, Papoutsakis ET: Northern, morphological, and fermentation analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824. J Bacteriol 2002, 184:3586-3597.
• [23]Guedon E, Desvaux M, Petitdemange H: Kinetic analysis of Clostridium cellulolyticum carbohydrate metabolism: importance of glucose 1-phosphate and glucose 6-phosphate branch points for distribution of carbon fluxes inside and outside cells as revealed by steady-state continuous culture. J Bacteriol 2000, 182:2010-2017.
• [24]Graves MC, Rabinowitz JC: In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. Evidence for “extended” promoter elements in gram-positive organisms. J Biol Chem 1986, 261:11409-11415.
• [25]Gal L, Pages S, Gaudin C, Belaich A, Reverbel-Leroy C, Tardif C, Belaich JP: Characterization of the cellulolytic complex (cellulosome) produced by Clostridium cellulolyticum. Appl Environ Microbiol 1997, 63:903-909.
• [26]Abdou L, Boileau C, de Philip P, Pages S, Fierobe HP, Tardif C: Transcriptional regulation of the Clostridium cellulolyticum cip-cel operon: a complex mechanism involving a catabolite-responsive element. J Bacteriol 2008, 190:1499-1506.
• [27]Drepper T, Eggert T, Circolone F, Heck A, Krauss U, Guterl JK, Wendorff M, Losi A, Gartner W, Jaeger KE: Reporter proteins for in vivo fluorescence without oxygen. Nat Biotechnol 2007, 25:443-445.
• [28]Mader U, Homuth G, Scharf C, Buttner K, Bode R, Hecker M: Transcriptome and proteome analysis of Bacillus subtilis gene expression modulated by amino acid availability. J Bacteriol 2002, 184:4288-4295.
• [29]Knoppova M, Phensaijai M, Vesely M, Zemanova M, Nesvera J, Patek M: Plasmid vectors for testing in vivo promoter activities in Corynebacterium glutamicum and Rhodococcus erythropolis. Curr Microbiol 2007, 55:234-239.
• [30]Li Y, Tschaplinski TJ, Engle NL, Hamilton CY, Rodriguez M Jr, Liao JC, Schadt CW, Guss AM, Yang Y, Graham DE: Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations. Biotechnol Biofuels 2012, 5:2. BioMed Central Full Text
• [31]Heap JT, Kuehne SA, Ehsaan M, Cartman ST, Cooksley CM, Scott JC, Minton NP: The ClosTron: mutagenesis in Clostridium refined and streamlined. J Microbiol Methods 2009, 80:49-55.
• [32]Atsumi S, Wu TY, Eckl EM, Hawkins SD, Buelter T, Liao JC: Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes. Appl Microbiol Biotechnol 2010, 85:651-657.
• [33]Hemme CL, Fields MW, He Q, Deng Y, Lin L, Tu QC, Mouttaki H, Zhou AF, Feng XY, Zuo Z, Ramsay BD, He Z, Wu L, Van Nostrand J, Xu J, Tang YJ, Wiegel J, Phelps TJ, Zhou J: Correlation of genomic and physiological traits of thermoanaerobacter species with biofuel yields. Appl Environ Microbiol 2011, 77:7998-8008.
• [34]He Q, Huang KH, He ZL, Alm EJ, Fields MW, Hazen TC, Arkin AP, Wall JD, Zhou JZ: Energetic consequences of nitrite stress in Desulfovibrio vulgaris Hildenborough, inferred from global transcriptional analysis. Appl Environ Microbiol 2006, 72:4370-4381.
• [35]Liang YT, He ZL, Wu LY, Deng Y, Li GH, Zhou JZ: Development of a common Oligonucleotide reference standard for microarray data normalization and comparison across different microbial communities. Appl Environ Microbiol 2010, 76:1088-1094.
• [36]Yang S, Tschaplinski TJ, Engle NL, Carroll SL, Martin SL, Davison BH, Palumbo AV, Rodriguez M Jr, Brown SD: Transcriptomic and metabolomic profiling of Zymomonas mobilis during aerobic and anaerobic fermentations. BMC Genomics 2009, 10:34. BioMed Central Full Text
• [37]Stevenson DM, Weimer PJ: Expression of 17 genes in Clostridium thermocellum ATCC 27405 during fermentation of cellulose or cellobiose in continuous culture. Appl Environ Microbiol 2005, 71:4672-4678.
• [38]Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001, 29:e45.