Transcriptomic response to prolonged ethanol production in the cyanobacterium Synechocystis sp. PCC6803

Biotechnology for Biofuels

Dennis Dienst⁴  Jens Georg³  Thomas Abts¹  Lew Jakorew²  Ekaterina Kuchmina³  Thomas Börner⁴  Annegret Wilde³  Ulf Dühring¹  Heike Enke¹  Wolfgang R Hess³ 

[1] Algenol Biofuels Germany GmbH, Magnusstraße 1, Berlin D-12489, Germany

[2] Current address: Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C DK-1871, Denmark

[3] Faculty of Biology, Inst. Biology III, University of Freiburg, Schänzlestr 1, Freiburg D-79104, Germany

[4] Institute of Biology, Humboldt-University Berlin, Chausseestr 117, Berlin D-10115, Germany

关键词: Transcription;    Synthetic biology;    Metabolic engineering;    Synechocystis;    Ethanol production;    Cyanobacteria;    Biofuel;   

Others  :  793669
DOI  :  10.1186/1754-6834-7-21

 received in 2013-09-03, accepted in 2014-01-17,  发布年份 2014

【 摘 要 】

Background

The production of biofuels in photosynthetic microalgae and cyanobacteria is a promising alternative to the generation of fuels from fossil resources. To be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria into forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain.

Results

The transcriptome-wide response to continuous ethanol production was examined in Synechocystis sp. PCC6803 using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) ethanol d^-1 and 0.0303% (v/v) ethanol d^-1 were obtained over 18 consecutive days, measuring two sets of biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (~40%) delay in biomass accumulation, the development of a bleaching phenotype and a down-regulation of light harvesting capacity. However, microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an approximately 4 fold reduction in cpcB (sll1577) and 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional processing of the cpcBA operon mRNA leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype.

Conclusions

Changes in gene expression upon induction of long-term ethanol production in Synechocystis sp. PCC6803 are highly specific. In particular, we did not observe a comprehensive stress response as might have been expected.

【 授权许可】

   
2014 Dienst et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]McKinlay JB, Harwood CS: Photobiological production of hydrogen gas as a biofuel. Curr Opin Biotechnol 2010, 21:244-251.
• [2]Deng MD, Coleman JR: Ethanol synthesis by genetic engineering in cyanobacteria. Appl Environ Microbiol 1999, 65:523-528.
• [3]Atsumi S, Higashide W, Liao JC: Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. Nat Biotechnol 2009, 27:1177-1180.
• [4]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.
• [5]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.
• [6]Schirmer A, Rude MA, Li X, Popova E, del Cardayre SB: Microbial biosynthesis of alkanes. Sci 2010, 329:559-562.
• [7]Waltz E: Biotech’s green gold? Nat Biotechnol 2009, 27:15-18.
• [8]Qiao J, Wang J, Chen L, Tian X, Huang S, Ren X, Zhang W: Quantitative iTRAQ LC-MS/MS proteomics reveals metabolic responses to biofuel ethanol in cyanobacterial Synechocystis sp. PCC 6803. J Proteome Res 2012, 11:5286-5300.
• [9]Wang J, Chen L, Huang S, Liu J, Ren X, Tian X, Qiao J, Zhang W: RNA-seq based identification and mutant validation of gene targets related to ethanol resistance in cyanobacterial Synechocystis sp. PCC 6803. BMC Biotechnol Biofuels 2012, 5:89. BioMed Central Full Text
• [10]Gao Z, Zhao H, Li Z, Tan T, Lu X: Photosynthetic production of ethanol from carbon dioxide in genetically engineered cyanobacteria. Energy Environ Sci 2012, 5:9857-9865.
• [11]Mitschke J, Georg J, Scholz I, Sharma CM, Dienst D, Bantscheff J, Voss B, Steglich C, Wilde A, Vogel J, Hess WR: An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC6803. Proc Natl Acad Sci USA 2011, 108:2124-2129.
• [12]Georg J, Voss B, Scholz I, Mitschke J, Wilde A, Hess WR: Evidence for a major role of antisense RNAs in cyanobacterial gene regulation. Mol Syst Biol 2009, 5:305.
• [13]Voss B, Georg J, Schön V, Ude S, Hess WR: Biocomputational prediction of non-coding RNAs in model cyanobacteria. BMC Genomics 2009, 10:123. BioMed Central Full Text
• [14]Vidal R, Lopez-Maury L, Guerrero MG, Florencio FJ: Characterization of an alcohol dehydrogenase from the cyanobacterium Synechocystis sp. PCC 6803 that responds to environmental stress conditions via the Hik34-Rre1 two component system. J Bacteriol 2009, 191:4383-4391.
• [15]Hernandez-Prieto MA, Schön V, Georg J, Barreira L, Varela J, Hess WR, Futschik ME: Iron deprivation in Synechocystis: inference of pathways, non-coding RNAs, and regulatory elements from comprehensive expression profiling. G3 (Bethesda) 2012, 2:1475-1495.
• [16]Karradt A, Sobanski J, Mattow J, Lockau W, Baier K: NblA, a key protein of phycobilisome degradation, interacts with ClpC, a HSP100 chaperone partner of a cyanobacterial Clp protease. J Biol Chem 2008, 283:32394-32403.
• [17]Zengel JM, Lindahl L: Diverse mechanisms for regulating ribosomal protein synthesis in Escherichia coli. Progr Nucl Acid Res Mol Biol 1994, 47:331-370.
• [18]Møller T, Franch T, Udesen C, Gerdes K, Valentin-Hansen P: Spot 42 RNA mediates discoordinate expression of the E. coli galactose operon. Genes Dev 2002, 16:1696-1706.
• [19]Urban JH, Papenfort K, Thomsen J, Schmitz RA, Vogel J: A conserved small RNA promotes discoordinate expression of the glmUS operon mRNA to activate GlmS synthesis. J Mol Biol 2007, 373:521-528.
• [20]Na D, Yoo SM, Chung H, Park H, Park JH, Lee SY: Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nat Biotechnol 2013, 31:170-174.
• [21]Held WA, Ballou B, Mizushima S, Nomura M: Assembly mapping of 30 S ribosomal proteins from Escherichia coli. Further studies. J Biol Chem 1974, 249:3103-3111.
• [22]Cerretti DP, Mattheakis LC, Kearney KR, Vu L, Nomura M: Translational regulation of the spc operon in Escherichia coli. Identification and structural analysis of the target site for S8 repressor protein. J Mol Biol 1988, 204:309-329.
• [23]Gregory RJ, Cahill PB, Thurlow DL, Zimmermann RA: Interaction of Escherichia coli ribosomal protein S8 with its binding sites in ribosomal RNA and messenger RNA. J Mol Biol 1988, 204:295-307.
• [24]Merianos HJ, Wang J, Moore PB: The structure of a ribosomal protein S8/spc operon mRNA complex. RNA 2004, 10:954-964.
• [25]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.
• [26]Zinchenko VV, Piven IV, Melnik VA, Shestakov SV: Vectors for the complementation analysis of cyanobacterial mutants. Russ J Genet 1999, 35:228-232.
• [27]Black TA, Cai Y, Wolk CP: Spatial expression and autoregulation of hetR, a gene involved in the control of heterocyst development in Anabaena. Mol Microbiol 1993, 9:77-84.
• [28]McKinney G: Absorption of light by chlorophyll solutions. J Biol Chem 1941, 140:315-322.
• [29]Dienst D, Dühring U, Mollenkopf HJ, Vogel J, Golecki J, Hess WR, Wilde A: The cyanobacterial homologue of the RNA chaperone Hfq is essential for motility of Synechocystis sp. PCC 6803. Microbiology 2008, 154:3134-3143.
• [30]Steglich C, Futschik ME, Lindell D, Voss B, Chisholm SW, Hess WR: The challenge of regulation in a minimal photoautotroph: non-coding RNAs in Prochlorococcus. PLoS Genet 2008, 4:e1000173.
• [31]Dühring U, Irrgang KD, Lünser K, Kehr J, Wilde A: Analysis of photosynthetic complexes from a cyanobacterial ycf37 mutant. Biochim Biophys Acta 2006, 1757:3-11.
• [32]Schägger H: Tricine-SDS-PAGE. Nat Protoc 2006, 1:16-22.