Proteomic analysis reveals resistance mechanism against biofuel hexane in Synechocystis sp. PCC 6803

Biotechnology for Biofuels

Jie Liu¹  Lei Chen¹  Jiangxin Wang¹  Jianjun Qiao¹  Weiwen Zhang¹ 

[1] Key Laboratory of Systems Bioengineering, Ministry of Education of China, Tianjin 300072, P.R. China

关键词: Synechocystis;    Proteomics;    Tolerance;    Hexane;   

Others  :  798227
DOI  :  10.1186/1754-6834-5-68

 received in 2012-07-28, accepted in 2012-08-30,  发布年份 2012

【 摘 要 】

Background

Recent studies have demonstrated that photosynthetic cyanobacteria could be an excellent cell factory to produce renewable biofuels and chemicals due to their capability to utilize solar energy and CO₂ as the sole energy and carbon sources. Biosynthesis of carbon-neutral biofuel alkanes with good chemical and physical properties has been proposed. However, to make the process economically feasible, one major hurdle to improve the low cell tolerance to alkanes needed to be overcome.

Results

Towards the goal to develop robust and high-alkane-tolerant hosts, in this study, the responses of model cyanobacterial Synechocystis PCC 6803 to hexane, a representative of alkane, were investigated using a quantitative proteomics approach with iTRAQ - LC-MS/MS technologies. In total, 1,492 unique proteins were identified, representing about 42% of all predicted protein in the Synechocystis genome. Among all proteins identified, a total of 164 and 77 proteins were found up- and down-regulated, respectively. Functional annotation and KEGG pathway enrichment analyses showed that common stress responses were induced by hexane in Synechocystis. Notably, a large number of transporters and membrane-bound proteins, proteins against oxidative stress and proteins related to sulfur relay system and photosynthesis were induced, suggesting that they are possibly the major protection mechanisms against hexane toxicity.

Conclusion

The study provided the first comprehensive view of the complicated molecular mechanism employed by cyanobacterial model species, Synechocystis to defend against hexane stress. The study also provided a list of potential targets to engineer Synechocystis against hexane stress.

【 授权许可】

   
2012 Lui et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

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【 参考文献 】

• [1]Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY: Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 1979, 111:1-61.
• [2]Ducat D, Sachdeva G, Silver P: Rewiring hydrogenase-dependent redox circuits in cyanobacteria. Proc Natl Acad Sci USA 2011, 108:3941-3946.
• [3]Robertson DE, Jacobson SA, Morgan F, Berry D, Church GM, Afeyan NB: A new dawn for industrial photosynthesis. Photosynth Res 2011, 107:269-277.
• [4]Quintana N, Van der Kooy F, Van de Rhee MD, Voshol GP, Verpoorte R: Renewable energy from Cyanobacteria: energy production optimization by metabolic pathway engineering. Appl Microbiol Biotechnol 2011, 91:471-490.
• [5]Koksharova O, Wolk C: Genetic tools for cyanobacteria. Appl Microbiol Biotechnol 2002, 58:123-137.
• [6]Hess WR: Cyanobacterial genomics for ecology and biotechnology. Curr Opin Microbiol 2011, 14:608-614.
• [7]Rittmann BE: Opportunities for renewable bioenergy using microorganisms. Biotechnol Bioeng 2008, 100:203-212.
• [8]Angermayr SA, Hellingwerf KJ, Lindblad P, de Mattos MJ: Energy biotechnology with cyanobacteria. Curr Opin Biotechnol 2009, 20:257-263.
• [9]Sheng J, Vannela R, Rittmann BE: Evaluation of cell-disruption effects of pulsed-electric-field treatment of Synechocystis PCC 6803. Environ Sci Technol 2011, 45:3795-3802.
• [10]Sheng J, Vannela R, Rittmann BE: Evaluation of methods to extract and quantify lipids from Synechocystis PCC 6803. Bioresour Technol 2011, 102:1697-1703.
• [11]Liu X, Curtiss R 3rd: Nickel-inducible lysis system in Synechocystis sp. PCC 6803. Proc Natl Acad Sci USA 2009, 106:21550-21554.
• [12]Deng MD, Coleman JR: Ethanol synthesis by genetic engineering in cyanobacteria. Appl Environ Microb 1999, 65:523-528.
• [13]Dexter J, Fu P: Metabolic engineering of cyanobacteria for ethanol production. Energy Environmental Sci 2009, 2:857-864.
• [14]Takahama K, Matsuoka M, Nagahama K, Ogawa T: Construction and analysis of a recombinant cyanobacterium expressing a chromosomally inserted gene for an ethylene-forming enzyme at the psbAI locus. J Biosci Bioeng 2003, 95:302-305.
• [15]Lindberg P, Park S, Melis A: Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism. Metab Eng 2010, 12:70-79.
• [16]Liu X, Fallon S, Sheng J, Curtiss R: CO2-Limitation-inducible green recovery of fatty acids from cyanobacterial biomass. Proc Natl Acad Sci USA 2011, 108:6905-6908.
• [17]Tan X, Yao L, Gao Q, Wang W, Qi F, Lu X: Photosynthesis driven conversion of carbon dioxide to fatty alcohols and hydrocarbons in cyanobacteria. Metab Eng 2011, 13:169-176.
• [18]Lan EI, Liao JC: ATP drives direct photosynthetic production of 1-butanol in cyanobacteria. Proc Natl Acad Sci USA 2012, 109:6018-6023.
• [19]Lan EI, Liao JC: Metabolic engineering of cyanobacteria for 1-butanol production from carbon dioxide. Metab Eng 2011, 13:353-363.
• [20]Atsumi S, Higashide W, Liao JC: Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. Nat Biotechnol 2009, 27:1177-1180.
• [21]McNeely K, Xu Y, Bennette N, Bryant D, Dismukes G: Redirecting reductant flux into hydrogen production via metabolic engineering of fermentative carbon metabolism in a cyanobacterium. Appl Environ Microb 2010, 76:5032-5038.
• [22]Lee HS, Vermaas WF, Rittmann BE: Biological hydrogen production: prospects and challenges. Trends Biotechnol 2010, 28:262-271.
• [23]Rottig A, Wenning L, Broker D, Steinbuchel A: Fatty acid alky esters: perspectives for production of alternative biofuels. Appl Microbiol Biotechnol 2010, 85:1713-1733.
• [24]Jang YS, Park JM, Choi S, Choi YJ, Seung DY, Cho JH, Lee SY: Engineering of microorganisms for the production of biofuels and perspectives based on systems metabolic engineering approaches. Biotechnol Adv 2011. Epub ahead of print
• [25]Zhang F, Rodriguez S, Keasling JD: Metabolic engineering of microbial pathways for advanced biofuels production. Curr Opin Biotechnol 2011, 22:775-783.
• [26]Peralta-Yahya PP, Keasling JD: Advanced biofuel production in microbes. J Biotechnol 2010, 5:147-162.
• [27]Winters K, Parker PL, Van Baalen C: Hydrocarbons of blue-green algae: geochemical significance. Science 1969, 163:467-468.
• [28]Dembitsky VM, Srebnik M: Variability of hydrocarbon and fatty acid components in cultures of the filamentous cyanobacterium Scytonema sp. isolated from microbial community “black cover” of limestone walls in Jerusalem. Biochemistry (Mosc) 2002, 67:1276-1282.
• [29]Schirmer A, Rude MA, Li X, Popova E, del Cardayre SB: Microbial biosynthesis of alkanes. Science 2010, 329:559-562.
• [30]Ramos JL, Duque E, Gallegos MT, Godoy P, Ramos-Gonzalez MI, Rojas A, Teran W, Segura A: Mechanisms of solvent tolerance in gram-negative bacteria. Annu Rev Microbiol 2002, 56:743-768.
• [31]Convin H, Anderson M: The effect of intramolecular hydrophobic bonding on partition coefficients. J Org Chem 1967, 32:2583-2586.
• [32]Harnisch M, Mockel H, Schulze G: Relationship between log PO, shake-flask values and capacity factors derived from reversed-phase high-performance liquid chromatography for n-alkylbenzenes and some OECD reference substance. J Chromatogr 1983, 282:315-332.
• [33]Aono R, Negishi T, Nakajima H: Cloning of organic solvent tolerance gene ostA that determines n-hexane tolerance level in Escherichia coli. Appl Environ Microbiol 1994, 60:4624-4626.
• [34]White DG, Goldman JD, Demple B, Levy SB: Role of the acrAB locus in organic solvent tolerance mediated by expression of marA, soxS, or robA in Escherichia coli. J Bacteriol 1997, 179:6122-6126.
• [35]Hayashi S, Aono R, Hanai T, Mori H, Kobayashi T, Honda H: Analysis of organic solvent tolerance in Escherichia coli using gene expression profiles from DNA microarrays. J Biosci Bioeng 2003, 95:379-383.
• [36]Jude F, Arpin C, Brachet-Castang C, Capdepuy M, Caumette P, Quentin C: TbtABM, a multidrug efflux pump associated with tributyltin resistance in Pseudomonas stutzeri. FEMS Microbiol Lett 2004, 232:7-14.
• [37]Doukyu N, Ishikawa K, Watanabe R, Ogino H: Improvement in organic solvent tolerance by double disruptions of proV and marR genes in Escherichia coli. J Appl Microbiol 2012, 112:464-474.
• [38]Stancu MM: Effect of organic solvents on solvent-tolerant Aeromonas hydrophila IBBPo8 and pseudomonas aeruginosa IBBPo10. Indian J Biotechnol 2011, 10:352-361.
• [39]Vemuri GN, Aristidou AA: Metabolic engineering in the -omics era: elucidating and modulating regulatory networks. Microbiol Mol Biol Rev 2005, 69:197-216.
• [40]Murata M, Fujimoto H, Nishimura K, Charoensuk K, Nagamitsu H, Raina S, Kosaka T, Oshima T, Ogasawara N, Yamada M: Molecular strategy for survival at a critical high temperature in Eschierichia coli. PLoS One 2011, 6:e20063.
• [41]Shigi N, Sakaguchi Y, Asai S, Suzuki T, Watanabe K: Common thiolation mechanism in the biosynthesis of tRNA thiouridine and sulphur-containing cofactors. EMBO J 2008, 7:3267-3278.
• [42]Sun J, Daniel R, Wagner-Döbler I, Zeng AP: Is autoinducer-2 a universal signal for interspecies communication: a comparative genomic and phylogenetic analysis of the synthesis and signal transduction pathways. BMC Evol Biol 2004, 4:36-46. BioMed Central Full Text
• [43]Allakhverdiev SI, Murata N: Salt stress inhibits photosystems II and I in cyanobacteria. Photosynth Res 2008, 98:529-539.
• [44]Zhang Z, Pendse ND, Phillips KN, Cotner JB, Khodursky A: Gene expression patterns of sulfur starvation in Synechocystis sp. PCC 6803. BMC Genomics 2008, 9:344-357. BioMed Central Full Text
• [45]Qiao JJ, Wang JX, Chen L, Tian XX, Huang SQ, Ren XY, Zhang WW: Quantitative iTRAQ LC-MS/MS proteomics reveals metabolic response to biofuel ethanol in cyanobacterial Synechocystis sp. PCC 6803. J Proteome Res 2012. in revision
• [46]Seib KL, Wu HJ, Kidd SP, Apicella MA, Jennings MP, McEwan AG: Defenses against oxidative stress in Neisseria gonorrhoeae: a system tailored for a challenging environment. Microbiol Mol Biol Rev 2006, 70:344-361.
• [47]Tafforeau L, Le Blastier S, Bamps S, Dewez M, Vandenhaute J, Hermand D: Repression of ergosterol level during oxidative stress by fission yeast F-box protein Pof14 independently of SCF. EMBO J 2006, 25:4547-4556.
• [48]Takatsuka Y, Chen C, Nikaido H: Mechanism of recognition of compounds of diverse structures by the multidrug efflux pump AcrB of Escherichia coli. Proc Natl Acad Sci USA 2010, 107:6559-6565.
• [49]Kieboom J, Dennis JJ, de Bont JAM, Zylstra GJ: Identification and molecular characterization of an efflux pump involved in Pseudomonas putida S12 solvent tolerance. J Biol Chem 1998, 273:85-91.
• [50]Thiel T: Phosphate transport and arsenate resistance in the cyanobacterium Anabaena variabilis. J Bacteriol 1988, 70:1143-1147.
• [51]Verma SK, Singh HN: Evidence for energy-dependent copper efflux as a mechanism of Cu2+ resistance in the cyanobacterium Nostoc calcicola. FEMS Microbiol Lett 1991, 68:291-294.
• [52]Nomura M, Ishitani M, Takabe T, Rai AK, Takabe T: Synechococcus sp. PCC7942 transformed with Escherichia coli bet genes produces glycine betaine from choline and acquires resistance to salt stress. Plant Physiol 1995, 107:703-708.
• [53]Mikkat S, Hagemann M, Schoor A: Active transport of glucosylglycerol is involved in salt adaptation of the cyanobacterium Synechocystis sp. strain PCC 6803. Microbiol 1996, 142:1725-1732.
• [54]Omata T, Gohta S, Takahashi Y, Harano Y, Maeda S: Involvement of a CbbR homolog in low CO2-induced activation of the bicarbonate transporter operon in cyanobacteria. J Bacteriol 2001, 83:1891-1898.
• [55]Shibata M, Katoh H, Sonoda M, Ohkawa H, Shimoyama M, Fukuzawa H, Kaplan A, Ogawa T: Genes essential to sodium-dependent bicarbonate transport in cyanobacteria: function and phylogenetic analysis. J Biol Chem 2002, 277:18658-18664.
• [56]Matsuda N, Kobayashi H, Katoh H, Ogawa T, Futatsugi L, Nakamura T, Bakker EP, Uozumi N: Na+-dependent K+ uptake Ktr system from the cyanobacterium Synechocystis sp. PCC 6803 and its role in the early phases of cell adaptation to hyperosmotic shock. J Biol Chem 2004, 279:54952-54962.
• [57]Katoh H, Hagino N, Grossman AR, Ogawa T: Genes essential to iron transport in the cyanobacterium Synechocystis sp. strain PCC 6803. J Bacteriol 2001, 183:2779-2784.
• [58]Barrett CM, Freudl R, Robinson C: Twin arginine translocation (Tat)-dependent export in the apparent absence of TatABC or TatA complexes using modified Escherichia coli TatA subunits that substitute for TatB. J Biol Chem 2007, 282:36206-36213.
• [59]Oliver DB: SecA protein: autoregulated ATPase catalysing preprotein insertion and translocation across the Escherichia coli inner membrane. Mol Microbiol 1993, 7:159-165.
• [60]Stock AM, Robinson VL, Goudreau PN: Two-component signal transduction. Annu Rev Biochem 2000, 69:183-215.
• [61]Shoumskaya MA, Paithoonrangsarid K, Kanesaki Y, Los DA, Zinchenko VV, Tanticharoen M, Suzuki I, Murata N: Identical Hik-Rre systems are involved in perception and transduction of salt signals and hyperosmotic signals but regulate the expression of individual genes to different extents in Synechocystis. J Biol Chem 2005, 280:21531-21538.
• [62]Vidal R, López-Maury L, Guerrero MG, Florencio FJ: Characterization of an alcohol dehydrogenase from the cyanobacterium synechocystis sp. Strain PCC 6803 that responds to environmental stress conditions via the Hik34-Rre1 two-component system. J Bacteriol 2009, 191:4383-4391.
• [63]Kamei A, Yoshihara S, Yuasa T, Geng X, Ikeuchi M: Biochemical and functional characterization of a eukaryotic-type protein kinase, SpkB, in the cyanobacterium Synechocystis sp. PCC 6803. Curr Microbiol 2003, 46:296-301.
• [64]Kamei A, Yuasa T, Orikawa K, Geng XX, Ikeuchi M: A eukaryotic-type protein kinase, SpkA, is required for normal motility of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. J Bacteriol 2001, 183:1505-1510.
• [65]Parnell JJ, Callister SJ, Rompato G, Nicora CD, Paša-Tolić L, Williamson A, Pfrender ME: Time-course analysis of the shewanella amazonensis SB2B proteome in response to sodium chloride shock. Sci Rep 2011, 1:25-32.
• [66]Dunlop MJ: Engineering microbes for tolerance to next-generation biofuels. Biotechnol Biofuels 2011, 4:32-40. BioMed Central Full Text
• [67]Nicolaou SA, Gaida SM, Papoutsakis ET: A comparative view of metabolite and substrate stress and tolerance in microbial bioprocessing: From biofuels and chemicals, to biocatalysis and bioremediation. Metab Eng 2010, 12:307-331.
• [68]Rutherford BJ, Dahl RH, Price RE, Szmidt HL, Benke PI, Mukhopadhyay A, Keasling JD: Functional Genomic study of exogenous n-butanol stress in Escherichia coli. Appl Environ Microbiol 2010, 76:1935-1945.
• [69]Kobayashi H, Yamamoto M, Aono R: Appearance of a stress-response protein, phage-shock protein A, in Escherichia coli exposed to hydrophobic organic solvents. Microbiol 1998, 144:353-359.
• [70]McDermott C, O'Donoghue MH, Heffron JJ: n-Hexane toxicity in Jurkat T-cells is mediated by reactive oxygen species. Arch Toxicol 2008, 82:165-171.
• [71]Brynildsen MP, Liao JC: An integrated network approach identifies the isobutanol response network of Escherichia coli. Mol Syst Biol 2009, 5:277-289.
• [72]Atsumi S, Wu TY, Machado IM, Huang WC, Chen PY, Pellegrini M, Liao JC: Evolution, genomic analysis, and reconstruction of isobutanol tolerance in Escherichia coli. Mol Syst Biol 2010, 6:449-460.
• [73]Kaneko T, Nakamura Y, Sasamoto S, Watanabe A, Kohara M, Matsumoto M, Shimpo S, Yamada M, Tabata S: Structural analysis of four large plasmids harboring in a unicellular cyanobacterium, Synechocystis sp. PCC 6803. DNA Res 2003, 10:221-228.
• [74]Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G: Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 1971, 35:171-205.
• [75]Katoh A, Sonoda M, Katoh H, Ogawa T: Absence of light-induced proton extrusion in a cotA-less mutant of Synechocystis sp. strain PCC6803. J Bacteriol 1996, 178:5452-5455.
• [76]Gan CS, Chong PK, Pham TK, Wright PC: Technical, experimental, and biological variations in isobaric tags for relative and absolute quantitation (iTRAQ). J Proteome Res 2007, 6:821-827.
• [77]Unwin RD, Griffiths JR, Whetton AD: Simultaneous analysis of relative protein expression levels across multiple samples using iTRAQ isobaric tags with 2D nano LC-MS/MS. Nat Protoc 2010, 5:1574-1582.
• [78]Charbonneau ME, Girard V, Nikolakakis A, Campos M, Berthiaume F, Dumas F, Lépine F, Mourez M: O-linked glycosylation ensures the normal conformation of the autotransporter adhesin involved in diffuse adherence. J Bacteriol 2007, 189:8880-8889.