Construction of a chassis for hydrogen production: physiological and molecular characterization of a Synechocystis sp. PCC 6803 mutant lacking a functional bidirectional hydrogenase.

Cyanobacteria are photosynthetic prokaryotes that are promising 'low-cost' microbial cell factories due to their simple nutritional requirements and metabolic plasticity, and the availability of tools for their genetic manipulation. The unicellular non-nitrogen-fixing Synechocystis sp. PCC 6803 is the best studied cyanobacterial strain and its genome was the first to be sequenced. The vast amount of physiological and molecular data available, together with a relatively small genome, makes Synechocystis suitable for computational metabolic modelling and to be used as a photoautotrophic chassis in synthetic biology applications. To prepare it for the introduction of a synthetic hydrogen producing device, a Synechocystis sp. PCC 6803 deletion mutant lacking an active bidirectional hydrogenase (ΔhoxYH) was produced and characterized at different levels: physiological, proteomic and transcriptional. The results showed that, under conditions favouring hydrogenase activity, 17 of the 210 identified proteins had significant differential fold changes in comparisons of the mutant with the wild-type. Most of these proteins are related to the redox and energy state of the cell. Transcriptional studies revealed that only six genes encoding those proteins exhibited significant differences in transcript levels. Moreover, the mutant exhibits similar growth behaviour compared with the wild-type, reflecting Synechocystis plasticity and metabolic adaptability. Overall, this study reveals that the Synechocystis ΔhoxYH mutant is robust and can be used as a photoautotrophic chassis for the integration of synthetic constructs, i.e. molecular constructs assembled from well characterized biological and/or synthetic parts (e.g. promoters, regulators, coding regions, terminators) designed for a specific purpose.

[1]  Paulette M. Vignais,et al.  Sustained Photoevolution of Molecular Hydrogen in a Mutant of Synechocystis sp. Strain PCC 6803 Deficient in the Type I NADPH-Dehydrogenase Complex , 2004, Journal of bacteriology.

[2]  Takakazu Kaneko,et al.  Structural analysis of four large plasmids harboring in a unicellular cyanobacterium, Synechocystis sp. PCC 6803. , 2003, DNA research : an international journal for rapid publication of reports on genes and genomes.

[3]  John G. K. Williams.  [85] Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803 , 1988 .

[4]  Paula Tamagnini,et al.  Diversity of Cyanobacterial Hydrogenases, a Molecular Approach , 2000, Current Microbiology.

[5]  D. Mount,et al.  The SOS regulatory system of Escherichia coli , 1982, Cell.

[6]  Trong Khoa Pham,et al.  Technical, experimental, and biological variations in isobaric tags for relative and absolute quantitation (iTRAQ). , 2007, Journal of proteome research.

[7]  Peter Lindblad,et al.  Energy biotechnology with cyanobacteria. , 2009, Current opinion in biotechnology.

[8]  P. Wright,et al.  Methods in Quantitative Proteomics: Setting iTRAQ on the Right Track , 2011 .

[9]  A. Spirin,et al.  Ribosome‐associated protein that inhibits translation at the aminoacyl‐tRNA binding stage , 2001, EMBO reports.

[10]  Sarah Dubrac,et al.  Fur Positive Regulation of Iron Superoxide Dismutase in Escherichia coli: Functional Analysis of thesodB Promoter , 2000, Journal of bacteriology.

[11]  Peter Lindblad,et al.  An AbrB-Like Protein Regulates the Expression of the Bidirectional Hydrogenase in Synechocystis sp. Strain PCC 6803 , 2007, Journal of bacteriology.

[12]  Brendan Halpin,et al.  Sequence Analysis , 2020, Definitions.

[13]  R. Schulz,et al.  HYDROGEN METABOLISM IN ORGANISMS WITH OXYGENIC PHOTOSYNTHESIS : HYDROGENASES AS IMPORTANT REGULATORY DEVICES FOR A PROPER REDOX POISING? , 1998 .

[14]  Lucas J. Stal,et al.  Fermentation in cyanobacteria , 1997 .

[15]  Y. Nakamura,et al.  Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions (supplement). , 1996, DNA research : an international journal for rapid publication of reports on genes and genomes.

[16]  Arthur R. Grossman,et al.  Isolation of Regulated Genes of the CyanobacteriumSynechocystis sp. Strain PCC 6803 by Differential Display , 2000, Journal of bacteriology.

[17]  Laurent Cournac,et al.  Limiting steps of hydrogen production in Chlamydomonas reinhardtii and Synechocystis PCC 6803 as analysed by light-induced gas exchange transients , 2002 .

[18]  P. Lindblad,et al.  Cyanobacterial H2 production — a comparative analysis , 2004, Planta.

[19]  Trong Khoa Pham,et al.  Isobaric tags for relative and absolute quantitation (iTRAQ) reproducibility: Implication of multiple injections. , 2006, Journal of proteome research.

[20]  M. Stumpp,et al.  Comprehensive survey of proteins targeted by chloroplast thioredoxin , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[21]  J. Sjöholm,et al.  Transcription and Regulation of the Bidirectional Hydrogenase in the Cyanobacterium Nostoc sp. Strain PCC 7120 , 2007, Applied and Environmental Microbiology.

[22]  B. McFadden,et al.  The complete DNA sequence and replication analysis of the plasmid pCB2.4 from the cyanobacterium Synechocystis PCC 6803. , 1994, Plasmid.

[23]  P. Manow.  ‚The Good, the Bad, and the Ugly‘ , 2002 .

[24]  M. Hagemann,et al.  Repression by Fur is not the main mechanism controlling the iron-inducible isiAB operon in the cyanobacterium Synechocystis sp. PCC 6803. , 2003, FEMS microbiology letters.

[25]  Hong Li,et al.  Differential Gene Expression in Response to Hydrogen Peroxide and the Putative PerR Regulon of Synechocystis sp. Strain PCC 6803 , 2004, Journal of bacteriology.

[26]  Ishtiaq Rehman,et al.  iTRAQ underestimation in simple and complex mixtures: "the good, the bad and the ugly". , 2009, Journal of proteome research.

[27]  Tatsuro Watanabe,et al.  A small heat-shock protein confers stress tolerance and stabilizes thylakoid membrane proteins in cyanobacteria under oxidative stress , 2009, Archives of Microbiology.

[28]  P. Lindblad,et al.  Hydrogenases in Nostoc sp. Strain PCC 73102, a Strain Lacking a Bidirectional Enzyme , 1997, Applied and environmental microbiology.

[29]  J Devillers,et al.  Prediction of biological activity profiles of cyanobacterial secondary metabolites , 2007, SAR and QSAR in environmental research.

[30]  Mark Johnson,et al.  NCBI BLAST: a better web interface , 2008, Nucleic Acids Res..

[31]  Gregory Stephanopoulos,et al.  Prediction of transcriptional profiles of Synechocystis PCC6803 by dynamic autoregressive modeling of DNA microarray data , 2003, Biotechnology and bioengineering.

[32]  Laura M Patterson-Fortin,et al.  A LexA-related protein regulates redox-sensitive expression of the cyanobacterial RNA helicase, crhR , 2006, Nucleic acids research.

[33]  P. Wright,et al.  Current trends in high throughput proteomics in cyanobacteria , 2009, FEBS letters.

[34]  Valery M. Dembitsky,et al.  Anticancer activity of natural and synthetic acetylenic lipids , 2006, Lipids.

[35]  Cloning of Fe-superoxide dismutase gene from the diazotroph Azotobacter vinelandii and the stimulation of its expression under anaerobic conditions in Escherichia coli , 2008 .

[36]  R. Abed,et al.  Applications of cyanobacteria in biotechnology , 2009, Journal of applied microbiology.

[37]  R. Schulz,et al.  Hydrogenase(s) in Synechocystis , 1998 .

[38]  P. Lindblad,et al.  Transcriptional regulation of the cyanobacterial bidirectional Hox-hydrogenase. , 2009, Dalton transactions.

[39]  F. Pinto,et al.  Transcription profiles of hydrogenases related genes in the cyanobacterium Lyngbya majuscula CCAP 1446/4 , 2009, BMC Microbiology.

[40]  J. Popot,et al.  An atypical haem in the cytochrome b6f complex , 2003, Nature.

[41]  M. Hagemann,et al.  Transcriptional analysis of the isiAB operon in salt-stressed cells of the cyanobacterium Synechocystis sp. PCC 6803. , 1998, FEMS microbiology letters.

[42]  J. Appel,et al.  Inhibition of respiration and nitrate assimilation enhances photohydrogen evolution under low oxygen concentrations in Synechocystis sp. PCC 6803. , 2007, Biochimica et biophysica acta.

[43]  Gregory Stephanopoulos,et al.  Reevaluating synthesis by biology. , 2010, Current opinion in microbiology.

[44]  Kai A Reidegeld,et al.  An easy‐to‐use Decoy Database Builder software tool, implementing different decoy strategies for false discovery rate calculation in automated MS/MS protein identifications , 2008, Proteomics.

[45]  M. Ludwig,et al.  Occurrence of Hydrogenases in Cyanobacteria and Anoxygenic Photosynthetic Bacteria: Implications for the Phylogenetic Origin of Cyanobacterial and Algal Hydrogenases , 2006, Journal of Molecular Evolution.

[46]  L. Sherman,et al.  Localization of Membrane Proteins in the Cyanobacterium Synechococcus sp. PCC7942 (Radial Asymmetry in the Photosynthetic Complexes) , 1994, Plant physiology.

[47]  R. Schulz,et al.  The [NiFe]-hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 works bidirectionally with a bias to H2 production. , 2011, Journal of the American Chemical Society.

[48]  A. Khodursky,et al.  Gene expression patterns of sulfur starvation in Synechocystis sp. PCC 6803 , 2008, BMC Genomics.

[49]  Guido Sanguinetti,et al.  Systems biology meets synthetic biology: a case study of the metabolic effects of synthetic rewiring. , 2009, Molecular bioSystems.

[50]  P. Lindblad,et al.  The bidirectional hydrogenase in the cyanobacterium Synechocystis sp. strain PCC 6803 , 2006 .

[51]  Paulo Oliveira.  Regulation of the Cyanobacterial Bidirectional Hydrogenase , 2008 .

[52]  Genes involved in the maturation of hydrogenase(s) in the nonheterocystous cyanobacterium Lyngbya majuscula CCAP 1446/4 , 2006 .

[53]  Janet L. Smith,et al.  Cytochrome b6f Complex , 2019, Encyclopedia of Biophysics.

[54]  T. Antal,et al.  Production of H2 by sulphur‐deprived cells of the unicellular cyanobacteria Gloeocapsa alpicola and Synechocystis sp. PCC 6803 during dark incubation with methane or at various extracellular pH , 2005, Journal of applied microbiology.

[55]  I. Vass,et al.  Transcriptional regulation of the bidirectional hydrogenase in the cyanobacterium Synechocystis 6803. , 2009, Journal of biotechnology.

[56]  Paula Tamagnini,et al.  Hydrogenases and Hydrogen Metabolism of Cyanobacteria , 2002, Microbiology and Molecular Biology Reviews.

[57]  L. Sherman,et al.  Gene expression under low-oxygen conditions in the cyanobacterium Synechocystis sp. PCC 6803 demonstrates Hik31-dependent and -independent responses. , 2011, Microbiology.

[58]  O. Koksharova,et al.  Genetic tools for cyanobacteria , 2001, Applied Microbiology and Biotechnology.

[59]  M. Kanehisa,et al.  Response to oxidative stress involves a novel peroxiredoxin gene in the unicellular cyanobacterium Synechocystis sp. PCC 6803. , 2004, Plant & cell physiology.

[60]  Josselin Noirel,et al.  A quantitative proteomic analysis of biofilm adaptation by the periodontal pathogen Tannerella forsythia , 2010, Proteomics.

[61]  Kenneth F Reardon,et al.  Shotgun proteomics of cyanobacteria--applications of experimental and data-mining techniques. , 2006, Briefings in functional genomics & proteomics.

[62]  F. Blattner,et al.  Recombinant protein production in an Escherichia coli reduced genome strain. , 2007, Metabolic engineering.

[63]  G. Dismukes,et al.  Redirecting Reductant Flux into Hydrogen Production via Metabolic Engineering of Fermentative Carbon Metabolism in a Cyanobacterium , 2010, Applied and Environmental Microbiology.

[64]  H. Sakurai,et al.  Disruption of the uptake hydrogenase gene, but not of the bidirectional hydrogenase gene, leads to enhanced photobiological hydrogen production by the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 , 2002, Applied Microbiology and Biotechnology.

[65]  L. Sherman,et al.  Global Transcriptional Response of the Alkali-Tolerant Cyanobacterium Synechocystis sp. Strain PCC 6803 to a pH 10 Environment , 2008, Applied and Environmental Microbiology.

[66]  R. Schulz,et al.  The bidirectional hydrogenase of Synechocystis sp. PCC 6803 works as an electron valve during photosynthesis , 2000, Archives of Microbiology.

[67]  J. Cate,et al.  Structural basis for the control of translation initiation during stress , 2004, Nature Structural &Molecular Biology.

[68]  Phillip C. Wright,et al.  Marine cyanobacteria—a prolific source of natural products , 2001 .

[69]  C. Bashor,et al.  Rewiring cells: synthetic biology as a tool to interrogate the organizational principles of living systems. , 2010, Annual review of biophysics.

[70]  Guido Sanguinetti,et al.  Identifying differentially expressed subnetworks with MMG , 2008, Bioinform..

[71]  F. Blattner,et al.  Emergent Properties of Reduced-Genome Escherichia coli , 2006, Science.

[72]  Carrie Eckert,et al.  The role of the bidirectional hydrogenase in cyanobacteria. , 2011, Bioresource technology.

[73]  H. Hayashi,et al.  No coordinated transcriptional regulation of the sod-kat antioxidative system in Synechocystis sp. PCC 6803 , 2002 .

[74]  Minli Xu,et al.  Computational analysis of LexA regulons in Cyanobacteria , 2010, BMC Genomics.

[75]  Masahiro Ito,et al.  Alkaline pH homeostasis in bacteria: new insights. , 2005, Biochimica et biophysica acta.

[76]  G. Ananyev,et al.  Optimization of Metabolic Capacity and Flux through Environmental Cues To Maximize Hydrogen Production by the Cyanobacterium “Arthrospira (Spirulina) maxima” , 2008, Applied and Environmental Microbiology.

[77]  J. Kalinowski,et al.  Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. , 1994, Gene.

[78]  P. Wright,et al.  Comparison of protein and peptide prefractionation methods for the shotgun proteomic analysis of Synechocystis sp. PCC 6803 , 2005, Proteomics.

[79]  Jae-Hyun Kim,et al.  Light-dependent expression of superoxide dismutase from cyanobacterium Synechocystis sp. strain PCC 6803 , 2005, Archives of Microbiology.

[80]  F. Florencio,et al.  Thioredoxin-linked processes in cyanobacteria are as numerous as in chloroplasts, but targets are different , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[81]  A. Serrano,et al.  Concurrent transcriptional activation of ppa and ppx genes by phosphate deprivation in the cyanobacterium Synechocystis sp. strain PCC 6803. , 2003, Biochemical and biophysical research communications.

[82]  Guido Sanguinetti,et al.  MMG: a probabilistic tool to identify submodules of metabolic pathways , 2008, Bioinform..

[83]  L. Sherman,et al.  The Mechanism of Iron Homeostasis in the Unicellular Cyanobacterium Synechocystis sp. PCC 6803 and Its Relationship to Oxidative Stress1[C][W] , 2009, Plant Physiology.

[84]  Paula Tamagnini,et al.  Cyanobacterial hydrogenases: diversity, regulation and applications. , 2007, FEMS microbiology reviews.

[85]  I. Suzuki,et al.  The heat shock response of Synechocystis sp. PCC 6803 analysed by transcriptomics and proteomics. , 2006, Journal of experimental botany.

[86]  W. Widger,et al.  Cloning and sequencing of the petBD operon from the cyanobacterium Synechococcus sp. PCC 7002 , 1992, Plant Molecular Biology.

[87]  B. McFadden,et al.  A small plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC 6803 encodes a rep protein and replicates by a rolling circle mechanism , 1993, Journal of bacteriology.

[88]  H. Osiewacz,et al.  Nucleotide sequence of the petB gene encoding cytochrome b6 from the mesophilic cyanobacterium Synechocystis PCC 6803: implications for evolution and function. , 1994, Biochimica et biophysica acta.

[89]  William E. Newton,et al.  Nitrogen Fixation and Hydrogen Metabolism in Cyanobacteria , 2010, Microbiology and Molecular Biology Reviews.

[90]  L. Nefedova,et al.  Mutants of Cyanobacterium Synechocystis sp. PCC 6803 with Insertional Inactivation of the sodB Gene Encoding Fe-Superoxide Dismutase , 2003, Russian Journal of Genetics.

[91]  J. Zehr,et al.  Unicellular cyanobacteria with a new mode of life: the lack of photosynthetic oxygen evolution allows nitrogen fixation to proceed , 2010, Archives of Microbiology.

[92]  Steven P Gygi,et al.  Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry , 2007, Nature Methods.

[93]  Angelicque E. White,et al.  Unicellular Cyanobacterial Distributions Broaden the Oceanic N2 Fixation Domain , 2010, Science.

[94]  P. Lindblad,et al.  LexA, a transcription regulator binding in the promoter region of the bidirectional hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. , 2005, FEMS microbiology letters.

[95]  F. Florencio,et al.  Photosynthetic regulation of the cyanobacterium Synechocystis sp. PCC 6803 thioredoxin system and functional analysis of TrxB (Trx x) and TrxQ (Trx y) thioredoxins. , 2009, Molecular plant.

[96]  D. Endy.  Foundations for engineering biology , 2005, Nature.

[97]  J. van der Oost,et al.  Fermentation metabolism of the unicellular cyanobacterium Cyanothece PCC 7822 , 1989, Archives of Microbiology.

[98]  R. Schulz,et al.  Sequence analysis of an operon of a NAD(P)-reducing nickel hydrogenase from the cyanobacterium Synechocystis sp. PCC 6803 gives additional evidence for direct coupling of the enzyme to NAD(P)H-dehydrogenase (complex I). , 1996, Biochimica et biophysica acta.

[99]  Yasukazu Nakamura,et al.  A Large-scale Protein–protein Interaction Analysis in Synechocystis sp. PCC6803 , 2007, DNA research : an international journal for rapid publication of reports on genes and genomes.

[100]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[101]  J. Foster,et al.  The role of fur in the acid tolerance response of Salmonella typhimurium is physiologically and genetically separable from its role in iron acquisition , 1996, Journal of bacteriology.

[102]  M. Kanehisa,et al.  Positive Regulation of Sugar Catabolic Pathways in the Cyanobacterium Synechocystis sp. PCC 6803 by the Group 2 σ Factor SigE* , 2005, Journal of Biological Chemistry.

[103]  J. Appel,et al.  LexA regulates the bidirectional hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 as a transcription activator , 2005, Molecular microbiology.

[104]  J. W. Campbell,et al.  Expression of two recombinant chloramphenicol acetyltransferase variants in highly reduced genome Escherichia coli strains , 2007, Biotechnology and bioengineering.

[105]  Takakazu Kaneko,et al.  CyanoBase: The Genome Database for Synechocystis sp. strain PCC6803 , 1996 .

[106]  T. Nunoshiba,et al.  Role of Iron and Superoxide for Generation of Hydroxyl Radical, Oxidative DNA Lesions, and Mutagenesis in Escherichia coli * , 1999, The Journal of Biological Chemistry.

[107]  Y. Hihara,et al.  A cyanobacterial gene encoding an ortholog of Pirin is induced under stress conditions , 2004, FEBS letters.

[108]  H. Bothe,et al.  The diaphorase subunit HoxU of the bidirectional hydrogenase as electron transferring protein in cyanobacterial respiration? , 1996, Naturwissenschaften.

[109]  Nooruddin Thajuddin,et al.  Cyanobacterial biodiversity and potential applications in biotechnology , 2005 .

[110]  R. Woodgate,et al.  Identification of additional genes belonging to the LexA regulon in Escherichia coli , 2000, Molecular microbiology.

[111]  Ahmad S. Khalil,et al.  Synthetic biology: applications come of age , 2010, Nature Reviews Genetics.

[112]  M. Ikeuchi,et al.  Location of PsbY in oxygen‐evolving photosystem II revealed by mutagenesis and X‐ray crystallography , 2007, FEBS letters.

[113]  Peter Lindblad,et al.  Production of H2 by the unicellular cyanobacterium Gloeocapsa alpicola CALU 743 during fermentation , 2002 .

[114]  W. Xu,et al.  Sequence analysis of plasmid pCC5.2 from cyanobacterium Synechocystis PCC 6803 that replicates by a rolling circle mechanism. , 1997, Plasmid.

[115]  Sayaka,et al.  Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. , 1996, DNA research : an international journal for rapid publication of reports on genes and genomes.

[116]  F. Florencio,et al.  Electron transport controls transcription of the thioredoxin gene (trxA) in the cyanobacterium Synechocystis sp. PCC 6803 , 2000, Plant Molecular Biology.

[117]  J. Keasling.  Synthetic biology for synthetic chemistry , 2014 .