Inhibition of hydrogen uptake in Escherichia coli by expressing the hydrogenase from the cyanobacterium Synechocystis sp. PCC 6803

BackgroundMolecular hydrogen is an environmentally-clean fuel and the reversible (bi-directional) hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 as well as the native Escherichia coli hydrogenase 3 hold great promise for hydrogen generation. These enzymes perform the simple reaction 2H+ + 2e- ↔ H2 (g).ResultsHydrogen yields were enhanced up to 41-fold by cloning the bidirectional hydrogenase (encoded by hoxEFUYH) from the cyanobacterium into E. coli. Using an optimized medium, E. coli cells expressing hoxEFUYH also produced twice as much hydrogen as the well-studied Enterobacter aerogenes HU-101, and hydrogen gas bubbles are clearly visible from the cultures. Overexpression of HoxU alone (small diaphorase subunit) accounts for 43% of the additional hydrogen produced by HoxEFUYH. In addition, hydrogen production in E. coli mutants with defects in the native formate hydrogenlyase system show that the cyanobacterial hydrogenase depends on both the native E. coli hydrogenase 3 as well as on its maturation proteins. Hydrogen absorption by cells expressing hoxEFUYH was up to 10 times lower than cells which lack the cloned cyanobacterial hydrogenase; hence, the enhanced hydrogen production in the presence of hoxEFUYH is due to inhibition of hydrogen uptake activity in E. coli. Hydrogen uptake by cells expressing hoxEFUYH was suppressed in three wild-type strains and in two hycE mutants but not in a double mutant defective in hydrogenase 1 and hydrogenase 2; hence, the active cyanobacterial locus suppresses hydrogen uptake by hydrogenase 1 and hydrogenase 2 but not by hydrogenase 3. Differential gene expression indicated that overexpression of HoxEFUYH does not alter expression of the native E. coli hydrogenase system; instead, biofilm-related genes are differentially regulated by expression of the cyanobacterial enzymes which resulted in 2-fold elevated biofilm formation. This appears to be the first enhanced hydrogen production by cloning a cyanobacterial enzyme into a heterologous host.ConclusionEnhanced hydrogen production in E. coli cells expressing the cyanobacterial HoxEFUYH is by inhibiting hydrogen uptake of both hydrogenase 1 and hydrogenase 2.

[1]  R. Sawers,et al.  Differential expression of hydrogenase isoenzymes in Escherichia coli K-12: evidence for a third isoenzyme , 1985, Journal of bacteriology.

[2]  B. Friedrich,et al.  Functional Analysis by Site-Directed Mutagenesis of the NAD+-Reducing Hydrogenase from Ralstonia eutropha , 2002, Journal of bacteriology.

[3]  Thomas K. Wood,et al.  YdgG (TqsA) Controls Biofilm Formation in Escherichia coli K-12 through Autoinducer 2 Transport , 2006, Journal of bacteriology.

[4]  T. Wood,et al.  Differential Gene Expression for Investigation of Escherichia coli Biofilm Inhibition by Plant Extract Ursolic Acid , 2005, Applied and Environmental Microbiology.

[5]  You-Kwan Oh,et al.  Fermentative biohydrogen production by a new chemoheterotrophic bacterium Citrobacter sp. Y19 , 2003 .

[6]  Ayelet Fishman,et al.  Controlling the Regiospecific Oxidation of Aromatics via Active Site Engineering of Toluene para-Monooxygenase of Ralstonia pickettii PKO1* , 2005, Journal of Biological Chemistry.

[7]  A. Emili,et al.  A Role for SlyD in the Escherichia coli Hydrogenase Biosynthetic Pathway* , 2005, Journal of Biological Chemistry.

[8]  Huijuan Xu,et al.  Isolation and characterization of a high H2-producing strain Klebsiella oxytoca HP1 from a hot spring. , 2005, Research in microbiology.

[9]  K. Shanmugam,et al.  Gene-product relationships of fhlA and fdv genes of Escherichia coli , 1988, Journal of bacteriology.

[10]  A. Böck,et al.  Mutational analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli , 1992, Molecular microbiology.

[11]  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.

[12]  Thomas K. Wood,et al.  Directed Evolution of Toluene ortho-Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation , 2002, Journal of bacteriology.

[13]  R. Sawers,et al.  Purification and properties of membrane-bound hydrogenase isoenzyme 1 from anaerobically grown Escherichia coli K12. , 1986, European journal of biochemistry.

[14]  T. Stadtman,et al.  Direct detection of potential selenium delivery proteins by using an Escherichia coli strain unable to incorporate selenium from selenite into proteins , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[15]  K. Bagramyan,et al.  Hydrogenase 3 but not hydrogenase 4 is major in hydrogen gas production by Escherichia coli formate hydrogenlyase at acidic pH and in the presence of external formate , 2007, Cell Biochemistry and Biophysics.

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

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

[18]  M. Schembri,et al.  Global gene expression in Escherichia coli biofilms , 2003, Molecular microbiology.

[19]  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.

[20]  H. Bothe,et al.  Unusual Gene Arrangement of the Bidirectional Hydrogenase and Functional Analysis of Its Diaphorase Subunit HoxU in Respiration of the Unicellular Cyanobacterium Anacystis nidulans , 1998, Current Microbiology.

[21]  M. Casadaban,et al.  Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. , 1976, Journal of molecular biology.

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

[23]  P. King,et al.  Response of hya Expression to External pH in Escherichia coli , 1999, Journal of bacteriology.

[24]  C. Sasikala,et al.  Augmentation of H2 photoproduction in Rhodopseudomonas palustris by N-heterocyclic aromatic compounds , 2004, Biotechnology Letters.

[25]  Thomas K. Wood,et al.  YliH (BssR) and YceP (BssS) Regulate Escherichia coli K-12 Biofilm Formation by Influencing Cell Signaling , 2006, Applied and Environmental Microbiology.

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

[27]  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.

[28]  L. Enquist,et al.  Experiments With Gene Fusions , 1984 .

[29]  S. Cosnier,et al.  Tolerance to oxygen of hydrogen enzyme electrodes , 2006 .

[30]  William T. Self,et al.  Expression and Regulation of a Silent Operon, hyf, Coding for Hydrogenase 4 Isoenzyme in Escherichia coli , 2004, Journal of bacteriology.

[31]  D. Boxer,et al.  Isolation and characterisation of a soluble active fragment of hydrogenase isoenzyme 2 from the membranes of anaerobically grown Escherichia coli. , 1986, European journal of biochemistry.

[32]  T. Happe,et al.  HoxE--a subunit specific for the pentameric bidirectional hydrogenase complex (HoxEFUYH) of cyanobacteria. , 2002, Biochimica et biophysica acta.

[33]  W. Wackernagel,et al.  Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. , 1995, Gene.

[34]  T. Wood,et al.  Gene expression in Escherichia coli biofilms , 2004, Applied Microbiology and Biotechnology.

[35]  Debabrata Das,et al.  Hydrogen production by biological processes: a survey of literature , 2001 .

[36]  H. Mori,et al.  Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection , 2006, Molecular systems biology.

[37]  Yutaka Nakashimada,et al.  Hydrogen production of Enterobacter aerogenes altered by extracellular and intracellular redox states , 2002 .

[38]  K. Fiebig,et al.  Purification of the F420-reducing hydrogenase from Methanosarcina barkeri (strain Fusaro). , 1989, European journal of biochemistry.

[39]  D. Hall,et al.  Photoproduction of hydrogen by cyanobacteria under partial vacuum in batch culture or in a photobioreactor , 1997 .

[40]  Dennis B. Troup,et al.  NCBI GEO: mining millions of expression profiles—database and tools , 2004, Nucleic Acids Res..

[41]  K. Shanmugam,et al.  Isolation and characterization of mutated FhlA proteins which activate transcription of the hyc operon (formate hydrogenlyase) of Escherichia coli in the absence of molybdate , 2000 .

[42]  T. Wood,et al.  Temporal gene-expression in Escherichia coli K-12 biofilms. , 2007, Environmental microbiology.

[43]  M. Inui,et al.  Enhanced Hydrogen Production from Formic Acid by Formate Hydrogen Lyase-Overexpressing Escherichia coli Strains , 2005, Applied and Environmental Microbiology.

[44]  Andrew Dicks,et al.  Hydrogen generation from natural gas for the fuel cell systems of tomorrow , 1996 .

[45]  C. F. Forster,et al.  Increased hydrogen production by Escherichia coli strain HD701 in comparison with the wild-type parent strain MC4100 , 2003 .

[46]  P. Lindblad,et al.  Towards optimization of cyanobacteria as biotechnologically relevant producers of molecular hydrogen, a clean and renewable energy source , 1998, Applied Microbiology and Biotechnology.

[47]  Yutaka Nakashimada,et al.  Enhanced hydrogen production in altered mixed acid fermentation of glucose by Enterobacter aerogenes , 1997 .

[48]  F. Tabita,et al.  Positive and negative selection of mutant forms of prokaryotic (cyanobacterial) ribulose-1,5-bisphosphate carboxylase/oxygenase. , 2003, Journal of molecular biology.

[49]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[50]  J. R. Kim,et al.  Isolation and characterization of Rhodopseudomonas palustris P4 which utilizes CO with the production of H2 , 1999, Biotechnology Letters.

[51]  N. Drapal,et al.  Interaction of the hydrogenase accessory protein HypC with HycE, the large subunit of Escherichia coli hydrogenase 3 during enzyme maturation. , 1998, Biochemistry.

[52]  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.

[53]  W. G. Martin,et al.  Identification and partial characterization of an Escherichia coli mutant with altered hydrogenase activity. , 1980, Canadian journal of biochemistry.