Applied Microbiology and Biotechnology

, Volume 79, Issue 1, pp 77–86

Protein engineering of hydrogenase 3 to enhance hydrogen production

  • Toshinari Maeda
  • Viviana Sanchez-Torres
  • Thomas K. Wood
Biotechnologically Relevant Enzymes and Proteins

Abstract

The large subunit (HycE, 569 amino acids) of Escherichia coli hydrogenase 3 produces hydrogen from formate via its Ni–Fe-binding site. In this paper, we engineered HycE for enhanced hydrogen production by an error-prone polymerase chain reaction (epPCR) using a host that lacked hydrogenase activity via the hyaB hybC hycE mutations. Seven enhanced HycE variants were obtained with a novel chemochromic membrane screen that directly detected hydrogen from individual colonies. The best epPCR variant contained eight mutations (S2T, Y50F, I171T, A291V, T366S, V433L, M444I, and L523Q) and had 17-fold higher hydrogen-producing activity than wild-type HycE. In addition, this variant had eightfold higher hydrogen yield from formate compared to wild-type HycE. Deoxyribonucleic acid shuffling using the three most-active HycE variants created a variant that has 23-fold higher hydrogen production and ninefold higher yield on formate due to a 74-amino acid carboxy-terminal truncation. Saturation mutagenesis at T366 of HycE also led to increased hydrogen production via a truncation at this position; hence, 204 amino acids at the carboxy terminus may be deleted to increase hydrogen production by 30-fold. This is the first random protein engineering of a hydrogenase.

Keywords

Protein engineering Hydrogenase 3 Error-prone PCR DNA shuffling Saturation mutagenesis 

References

  1. Andrews SC, Berks BC, McClay J, Ambler A, Quail MA, Golby P, Guest JR (1997) A 12-cistron Escherichia coli operon (hyf) encoding a putative proton-translocating formate hydrogenlyase system. Microbiology 143:3633–3647CrossRefGoogle Scholar
  2. Axley MJ, Grahame DA, Stadtman TC (1990) Escherichia coli formate-hydrogen lyase. Purification and properties of the selenium-dependent formate dehydrogenase component. J Biol Chem 265:18213–18218Google Scholar
  3. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:2006.0008CrossRefGoogle Scholar
  4. Bagramyan K, Trchounian A (2003) Structural and functional features of formate hydrogen lyase, an enzyme of mixed-acid fermentation from Escherichia coli. Biochemistry (Mosc) 68:1159–1170CrossRefGoogle Scholar
  5. Ballantine SP, Boxer DH (1986) Isolation and characterisation of a soluble active fragment of hydrogenase isoenzyme 2 from the membranes of anaerobically grown Escherichia coli. Eur J Biochem 156:277–284CrossRefGoogle Scholar
  6. Bisaillon A, Turcot J, Hallenbeck PC (2006) The effect of nutrient limitations on hydrogen production by batch cultures of Escherichia coli. Int J Hydrogen Energy 31:1504–1508CrossRefGoogle Scholar
  7. Blattner FR, Plunkett G 3rd, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y (1997) The complete genome sequence of Escherichia coli K-12. Science 277:1453–1474CrossRefGoogle Scholar
  8. Blokesch M, Paschos A, Theodoratou E, Bauer A, Hube M, Huth S, Böck A (2002) Metal insertion into NiFe-hydrogenases. Biochem Soc Trans 30:674–680CrossRefGoogle Scholar
  9. Blokesch M, Albracht SP, Matzanke BF, Drapal NM, Jacobi A, Böck A (2004a) The complex between hydrogenase-maturation proteins HypC and HypD is an intermediate in the supply of cyanide to the active site iron of [NiFe]-hydrogenases. J Mol Biol 344:155–167CrossRefGoogle Scholar
  10. Blokesch M, Paschos A, Bauer A, Reissmann S, Drapal N, Böck A (2004b) Analysis of the transcarbamoylation–dehydration reaction catalyzed by the hydrogenase maturation proteins HypF and HypE. Eur J Biochem 271:3428–3436CrossRefGoogle Scholar
  11. Blokesch M, Böck A (2006) Properties of the [NiFe]-hydrogenase maturation protein HypD. FEBS Lett 580:4065–4068CrossRefGoogle Scholar
  12. Boyington JC, Gladyshev VN, Khangulov SV, Stadtman TC, Sun PD (1997) Crystal structure of formate dehydrogenase H: catalysis involving Mo, molybdopterin, selenocysteine, and an Fe4S4 cluster. Science 275:1305–1308CrossRefGoogle Scholar
  13. Burgdorf T, De Lacey AL, Friedrich B (2002) Functional analysis by site-directed mutagenesis of the NAD+-reducing hydrogenase from Ralstonia eutropha. J Bacteriol 184:6280–6288CrossRefGoogle Scholar
  14. Cadwell RC, Joyce GF (1992) Randomization of genes by PCR mutagenesis. PCR Methods Appl 2:28–33Google Scholar
  15. Canada KA, Iwashita S, Shim H, Wood TK (2002) Directed evolution of toluene ortho-monooxygenase for enhanced 1-naphthol synthesis and chlorinated ethene degradation. J Bacteriol 184:344–349CrossRefGoogle Scholar
  16. Carr R, Alexeeva M, Enright A, Eve TS, Dawson MJ, Turner NJ (2003) Directed evolution of an amine oxidase possessing both broad substrate specificity and high enantioselectivity. Angew Chem Int Ed Engl 42:4807–4810CrossRefGoogle Scholar
  17. Castle LA, Siehl DL, Gorton R, Patten PA, Chen YH, Bertain S, Cho HJ, Duck N, Wong J, Liu D, Lassner MW (2004) Discovery and directed evolution of a glyphosate tolerance gene. Science 304:1151–1154CrossRefGoogle Scholar
  18. Das D, Veziroglu TN (2001) Hydrogen production by biological processes: a survey of literature. Int J Hydrogen Energy 26:13–28CrossRefGoogle Scholar
  19. Drapal N, Böck A (1998) Interaction of the hydrogenase accessory protein HypC with HycE, the large subunit of Escherichia coli hydrogenase 3 during enzyme maturation. Biochemistry 37:2941–2948CrossRefGoogle Scholar
  20. Fishman A, Tao Y, Rui L, Wood TK (2005) Controlling the regiospecific oxidation of aromatics via active site engineering of toluene para-monooxygenase of Ralstonia pickettii PKO1. J Biol Chem 280:506–514Google Scholar
  21. Forzi L, Hellwig P, Thauer RK, Sawers RG (2007) The CO and CN- ligands to the active site Fe in [NiFe]-hydrogenase of Escherichia coli have different metabolic origins. FEBS Lett 581:3317–3321CrossRefGoogle Scholar
  22. Glieder A, Farinas ET, Arnold FH (2002) Laboratory evolution of a soluble, self-sufficient, highly active alkane hydroxylase. Nat Biotechnol 20:1135–1139CrossRefGoogle Scholar
  23. Hüttenhofer A, Heider J, Böck A (1996) Interaction of the Escherichia coli fdhF mRNA hairpin promoting selenocysteine incorporation with the ribosome. Nucleic Acids Res 24:3903–3910CrossRefGoogle Scholar
  24. King PW, Przybyla AE (1999) Response of hya expression to external pH in Escherichia coli. J Bacteriol 181:5250–5256Google Scholar
  25. Klibanov AM, Alberti BN, Zale SE (1982) Enzymatic synthesis of formic acid from H2 and CO2 and production of hydrogen from formic acid. Biotechnol Bioeng 24:25–36CrossRefGoogle Scholar
  26. Lenz O, Zebger I, Hamann J, Hildebrandt P, Friedrich B (2007) Carbamoylphosphate serves as the source of CN, but not of the intrinsic CO in the active site of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha. FEBS Lett 581:3322–3326CrossRefGoogle Scholar
  27. Leungsakul T, Keenan BG, Yin H, Smets BF, Wood TK (2005) Saturation mutagenesis of 2,4-DNT dioxygenase of Burkholderia sp. strain DNT for enhanced dinitrotoluene degradation. Biotechnol Bioeng 92:416–426CrossRefGoogle Scholar
  28. Leungsakul T, Johnson GR, Wood TK (2006) Protein engineering of the 4-methyl-5-nitrocatechol monooxygenase from Burkholderia sp. strain DNT for enhanced degradation of nitroaromatics. Appl Environ Microbiol 72:3933–3939CrossRefGoogle Scholar
  29. Maeda T, Sanchez-Torres V, Wood TK (2007a) Escherichia coli hydrogenase 3 is a reversible enzyme possessing hydrogen uptake and synthesis activities. Appl Microbiol Biotechnol 76:1035–1042CrossRefGoogle Scholar
  30. Maeda T, Sanchez-Torres V, Wood TK (2007b) Enhanced hydrogen production from glucose by metabolically engineered Escherichia coli. Appl Microbiol Biotechnol 77:879–890CrossRefGoogle Scholar
  31. Maeda T, Vardar G, Self WT, Wood TK (2007c) Inhibition of hydrogen uptake in Escherichia coli by expressing the hydrogenase from the cyanobacterium Synechocystis sp. PCC 6803. BMC Biotechnol 7:25CrossRefGoogle Scholar
  32. Maeda T, Sanchez-Torres V, Wood TK (2008) Metabolic engineering to enhance bacterial hydrogen production. Microb Biotechnol 1:30–39Google Scholar
  33. Maeda T, Wood TK (2008) Formate detection by potassium permanganate for enhanced hydrogen production in Escherichia coli. Int J Hydrogen Energy (in press)Google Scholar
  34. Magalon A, Böck A (2000) Analysis of the HypC–hycE complex, a key intermediate in the assembly of the metal center of the Escherichia coli hydrogenase 3. J Biol Chem 275:21114–21120CrossRefGoogle Scholar
  35. Maier T, Jacobi A, Sauter M, Böck A (1993) The product of the hypB gene, which is required for nickel incorporation into hydrogenases, is a novel guanine nucleotide-binding protein. J Bacteriol 175:630–635Google Scholar
  36. Maier T, Lottspeich F, Böck A (1995) GTP hydrolysis by HypB is essential for nickel insertion into hydrogenases of Escherichia coli. Eur J Biochem 230:133–138CrossRefGoogle Scholar
  37. Nagy LE, Meuser JE, Plummer S, Seibert M, Ghirardi ML, King PW, Ahmann D, Posewitz MC (2007) Application of gene-shuffling for the rapid generation of novel [FeFe]-hydrogenase libraries. Biotechnol Lett 29:421–430CrossRefGoogle Scholar
  38. Paschos A, Glass RS, Böck A (2001) Carbamoylphosphate requirement for synthesis of the active center of [NiFe]-hydrogenases. FEBS Lett 488:9–12CrossRefGoogle Scholar
  39. Patnaik R, Louie S, Gavrilovic V, Perry K, Stemmer WP, Ryan CM, del Cardayré S (2002) Genome shuffling of Lactobacillus for improved acid tolerance. Nat Biotechnol 20:707–712CrossRefGoogle Scholar
  40. Penfold DW, Sargent F, Macaskie LE (2006) Inactivation of the Escherichia coli K-12 twin-arginine translocation system promotes increased hydrogen production. FEMS Microbiol Lett 262:135–137CrossRefGoogle Scholar
  41. Richard DJ, Sawers G, Sargent F, McWalter L, Boxer DH (1999) Transcriptional regulation in response to oxygen and nitrate of the operons encoding the [NiFe] hydrogenases 1 and 2 of Escherichia coli. Microbiology 145(Pt 10):2903–2912Google Scholar
  42. Rossmann R, Sauter M, Lottspeich F, Böck A (1994) Maturation of the large subunit (HYCE) of Escherichia coli hydrogenase 3 requires nickel incorporation followed by C-terminal processing at Arg537. Eur J Biochem 220:377–384CrossRefGoogle Scholar
  43. Rui L, Kwon Y-M, Fishman A, Reardon KF, Wood TK (2004) Saturation mutagenesis of toluene ortho-monooxygenase of Burkholderia cepacia G4 for enhanced 1-naphthol synthesis and chloroform degradation. Appl Environ Microbiol 70:3246–3252CrossRefGoogle Scholar
  44. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  45. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467CrossRefGoogle Scholar
  46. Sauter M, Böhm R, Böck A (1992) Mutational analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli. Mol Microbiol 6:1523–1532CrossRefGoogle Scholar
  47. Sawers RG, Ballantine SP, Boxer DH (1985) Differential expression of hydrogenase isoenzymes in Escherichia coli K-12: evidence for a third isoenzyme. J Bacteriol 164:1324–1331Google Scholar
  48. Seibert M, Flynn T, Benson D, Tracy E, Ghirardi M (1998) Biohydrogen. Plenum, New York, pp 227–234Google Scholar
  49. Self WT, Hasona A, Shanmugam KT (2004) Expression and regulation of a silent operon, hyf, coding for hydrogenase 4 isoenzyme in Escherichia coli. J Bacteriol 186:580–587CrossRefGoogle Scholar
  50. van Loo B, Spelberg JH, Kingma J, Sonke T, Wubbolts MG, Janssen DB (2004) Directed evolution of epoxide hydrolase from A. radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling. Chem Biol 11:981–990CrossRefGoogle Scholar
  51. Vardar-Schara G, Maeda T, Wood TK (2008) Metabolically-engineered bacteria for producing hydrogen via fermentation. Microb Biotechnol 1:107–125Google Scholar
  52. Vardar G, Wood TK (2005) Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for enhanced chlorinated ethene degradation and o-xylene oxidation. Appl Microbiol Biotechnol 68:510–517CrossRefGoogle Scholar
  53. Volbeda A, Charon MH, Piras C, Hatchikian EC, Frey M, Fontecilla-Camps JC (1995) Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas. Nature 373:580–587CrossRefGoogle Scholar
  54. Woods DD (1936) Hydrogenlyases: the synthesis of formic acid by bacteria. Biochem J 30:515–527Google Scholar
  55. Yoshida A, Nishimura T, Kawaguchi H, Inui M, Yukawa H (2005) Enhanced hydrogen production from formic acid by formate hydrogen lyase-overexpressing Escherichia coli strains. Appl Environ Microbiol 71:6762–6768CrossRefGoogle Scholar
  56. Yoshida A, Nishimura T, Kawaguchi H, Inui M, Yukawa H (2006) Enhanced hydrogen production from glucose using ldh- and frd-inactivated Escherichia coli strains. Appl Microbiol Biotechnol 73:67–72CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Toshinari Maeda
    • 1
  • Viviana Sanchez-Torres
    • 1
  • Thomas K. Wood
    • 1
    • 2
    • 3
  1. 1.Artie McFerrin Department of Chemical EngineeringTexas A & M UniversityCollege StationUSA
  2. 2.Department of BiologyTexas A & M UniversityCollege StationUSA
  3. 3.Zachry Department of Civil and Environmental EngineeringTexas A & M UniversityCollege StationUSA

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