Xylose isomerase from polycentric fungus Orpinomyces: gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of xylose to ethanol

  • Anjali Madhavan
  • Sriappareddy Tamalampudi
  • Kazunari Ushida
  • Daisuke Kanai
  • Satoshi Katahira
  • Aradhana Srivastava
  • Hideki Fukuda
  • Virendra S. Bisaria
  • Akihiko Kondo
Biotechnologically Relevant Enzymes and Proteins

Abstract

The cDNA sequence of the gene for xylose isomerase from the rumen fungus Orpinomyces was elucidated by rapid amplification of cDNA ends. The 1,314-nucleotide gene was cloned and expressed constitutively in Saccharomyces cerevisiae. The deduced polypeptide sequence encoded a protein of 437 amino acids which showed the highest similarity to the family II xylose isomerases. Further, characterization revealed that the recombinant enzyme was a homodimer with a subunit of molecular mass 49 kDa. Cell extract of the recombinant strain exhibited high specific xylose isomerase activity. The pH optimum of the enzyme was 7.5, while the low temperature optimum at 37°C was the property that differed significantly from the majority of the reported thermophilic xylose isomerases. In addition to the xylose isomerase gene, the overexpression of the S. cerevisiae endogenous xylulokinase gene and the Pichia stipitis SUT1 gene for sugar transporter in the recombinant yeast facilitated the efficient production of ethanol from xylose.

Keywords

Xylose isomerase Orpinomyces Xylulokinase SUT1 Recombinant Saccharomyces cerevisiae Xylose fermentation Ethanol 

References

  1. Amore R, Wilhelm M, Hollenberg CP (1989) The fermentation of xylose—an analysis of the expression of Bacillus and Actinoplanes xylose isomerase genes in yeast. Appl Microbiol Biotechnol 30:351–357CrossRefGoogle Scholar
  2. Borneman WS, Akin DE, Ljungdahl LG (1989) Fermentation products and plant cell wall-degrading enzymes produced by monocentric and polycentric anaerobic ruminal fungi. Appl Environ Microbiol 55:1066–1073Google Scholar
  3. Chandrakant P, Bisaria VS (1998) Simultaneous bioconversion of cellulose and hemicellulose to ethanol. Crit Rev Biotechnol 18:295–331CrossRefGoogle Scholar
  4. Chandrakant P, Bisaria VS (2000) Application of a compatible xylose isomerase in simultaneous bioconversion of glucose and xylose to ethanol. Biotechnol Bioprocess Eng 5:32–39CrossRefGoogle Scholar
  5. Chiang C, Knight SG (1960) Metabolism of d-xylose by moulds. Nature 188:79–81CrossRefGoogle Scholar
  6. Cregg JM, Barringer KJ, Hessler AY, Madden KR (1985) Pichia pastoris as a host system for transformations. Mol Cell Biol 5:3376–3385Google Scholar
  7. Dische Z, Borenfreund E (1951) A new spectrophotometric method for the detection and determination of keto sugars and trioses. J Biol Chem 192:583–587Google Scholar
  8. Dmytruk OV, Voronovsky AY, Abbas CA, Dmytruk KV, Ishchuk OP, Sibirny AA (2008) Overexpression of bacterial xylose isomerase and yeast host xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha. FEMS Yeast Res 8:165–173CrossRefGoogle Scholar
  9. Gárdonyi M, Hahn-Hägerdal B (2003) The Streptomyces rubiginosus xylose isomerase is misfolded when expressed in Saccharomyces cerevisiae. Enzyme Microb Technol 32:252–259CrossRefGoogle Scholar
  10. Gietz RD, Schiestl RH (2007) Large-scale high-efficiency yeast transformation using the LiAc/ss carrier DNA/PEG method. Nat Protocols 2:38–41CrossRefGoogle Scholar
  11. Harhangi HR, Akhmanova AS, Emmens R, van der Drift C, de Laat WTAM, van Dijken JP, Jetten MSM, Pronk JT, Op den Camp HJM (2003) Xylose metabolism in the anaerobic fungus Piromyces sp. strain E2 follows the bacterial pathway. Arch Microbiol 180:134–141CrossRefGoogle Scholar
  12. Hess JM, Tchernajenko V, Vieille C, Zeikus JG, Kelly RM (1998) Thermotoga neapolitana homotetrameric xylose isomerase is expressed as a catalytically active and thermostable dimer in Escherichia coli. Appl Environ Microbiol 64:2357–2360Google Scholar
  13. Jin Y, Laplaza JM, Jeffries TW (2004) Saccharomyces cerevisiae engineered for xylose metabolism exhibits a respiratory response. Appl Environ Microbiol 70:6816–6825CrossRefGoogle Scholar
  14. Katahira S, Fujita Y, Mizuike A, Fukuda H, Kondo A (2004) Construction of a xylan-fermenting yeast strain through codisplay of xylanolytic enzymes on the surface of xylose-utilizing Saccharomyces cerevisiae cells. Appl Environ Microbiol 70:5407–5414CrossRefGoogle Scholar
  15. Katahira S, Mizuike A, Fukuda H, Kondo A (2006) Ethanol fermentation from lignocellulosic hydrolysate by a recombinant xylose- and cellooligosaccharide-assimilating yeast strain. Appl Microbiol Biotechnol 72:1136–1143CrossRefGoogle Scholar
  16. Katahira S, Ito M, Takema H, Fujita Y, Tanino T, Tanaka T, Fukuda H, Kondo A (2008) Improvement of ethanol productivity during xylose and glucose co-fermentation by xylose-assimilating S. cerevisiae via expression of glucose transporter Sut1. Enzyme Microb Technol 43:115–119CrossRefGoogle Scholar
  17. Kristo P, Saarelainen R, Fagerström R, Aho S, Korhola M (1996) Protein purification, and cloning and characterization of the cDNA and gene for xylose isomerase of barley. Eur J Biochem 237:240–246CrossRefGoogle Scholar
  18. Kuyper M, Harhangi HR, Stave AK, Winkler AA, Jetten MSM, de Laat WTAM, den Ridder JJJ, Op den Camp HJM, van Dijken JP, Pronk JT (2003) High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae? FEMS Yeast Res 4:69–78CrossRefGoogle Scholar
  19. Kuyper M, Winkler AA, van Dijken JP, Pronk JT (2004) Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle. FEMS Yeast Res 4:655–664CrossRefGoogle Scholar
  20. Kuyper M, Hartog MMP, Toirkens MJ, Almering MJH, Winkler AA, van Dijken JP, Pronk JT (2005a) Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation. FEMS Yeast Res 5:399–409CrossRefGoogle Scholar
  21. Kuyper M, Toirkens MJ, Diderich JA, Winkler AA, van Dijken JP, Pronk JT (2005b) Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain. FEMS Yeast Res 5:925–934CrossRefGoogle Scholar
  22. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  23. Le MT, Vanderheyden PML, Fierens FLP, Vauquelin G (2003) Molecular characterization of the high-affinity [3H] neuropeptide Y-binding component from the venom of Conus anemone. Fund Clin Pharmacol 17:457–462CrossRefGoogle Scholar
  24. Leandro MJ, Gonçalves P, Spencer-Martins I (2006) Two glucose/xylose transporter genes from the yeast Candida intermedia: first molecular characterization of a yeast xylose-H+ symporter. Biochem J 395:543–549CrossRefGoogle Scholar
  25. Lee C, Bhatnagar L, Saha BC, Lee Y, Takagi M, Imanaka T, Bagdasarian M, Zeikus JG (1990) Cloning and expression of the Clostridium thermosulfurogenes glucose isomerase gene in Escherichia coli and Bacillus subtilis. Appl Environ Microbiol 56:2638–2643Google Scholar
  26. Lee WJ, Kim MD, Ryu YW, Bisson LF, Seo JH (2002) Kinetic studies on glucose and xylose transport in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 60:186–191CrossRefGoogle Scholar
  27. Lee TH, Kim MD, Park YC, Bae SM, Ryu YW, Seo JH (2003) Effects of xylulokinase activity on ethanol production from d-xylulose by recombinant Saccharomyces cerevisiae. J Appl Microbiol 95:847–852CrossRefGoogle Scholar
  28. Lowe SE, Theodorou MK, Trinci APJ, Hespell RB (1985) Growth of anaerobic rumen fungi on defined and semi-defined media lacking rumen fluid. J Gen Microbiol 131:2225–2229Google Scholar
  29. Meaden PG, Aduse-Opoku J, Reizer J, Reizer A, Lanceman YA, Martin MF, Mitchell WJ (1994) The xylose isomerase-encoding gene (xylA) of Clostridium thermosaccharolyticum: cloning, sequencing and phylogeny of XylA enzymes. Gene 141:97–101CrossRefGoogle Scholar
  30. Moes CJ, Pretorius IS, van Zyl WH (1996) Cloning and expression of the Clostridium thermosulfurogenes d-xylose isomerase gene (xylA) in Saccharomyces cerevisiae. Biotechnol Lett 18:269–274CrossRefGoogle Scholar
  31. Pitkänen J, Aristidou A, Salusjärvi L, Ruohonen L, Penttilä M (2003) Metabolic flux analysis of xylose metabolism in recombinant Saccharomyces cerevisiae using continuous culture. Metab Eng 5:16–31CrossRefGoogle Scholar
  32. Richard P, Toivari MH, Penttilä M (2000) The role of xylulokinase in Saccharomyces cerevisiae xylulose catabolism. FEMS Microbiol Lett 190:39–43CrossRefGoogle Scholar
  33. Saloheimo A, Rauta J, Stasyk OV, Sibirny AA, Penttilä M, Ruohonen L (2007) Xylose transport studies with xylose-utilizing Saccharomyces cerevisiae strains expressing heterologous and homologous permeases. Appl Microbiol Biotechnol 74:1041–1052CrossRefGoogle Scholar
  34. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor New York, USAGoogle Scholar
  35. Sarthy AV, McConaughy BL, Lobo Z, Sundstrom JA, Furlong CE, Hall BD (1987) Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae. Appl Environ Microbiol 53:1996–2000Google Scholar
  36. Shamanna DK, Sanderson KE (1979) Uptake and catabolism of d-xylose in Salmonella typhimurium LT2. J Bacteriol 139:64–70Google Scholar
  37. Takahashi S, Ueda M, Tanaka A (2001) Function of the prosequence for in vivo folding and secretion of active Rhizopus oryzae lipase in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 55:454–462CrossRefGoogle Scholar
  38. Toivari MH, Aristidou A, Ruohonen L, Penttilä M (2001) Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae: importance of xylulokinase (XKS1) and oxygen availability. Metab Eng 3:236–249CrossRefGoogle Scholar
  39. Träff KL, Cordero RRO, van Zyl WH, Hahn-Hägerdal B (2001) Deletion of the GRE3 aldose reductase gene and its influence on xylose metabolism in recombinant strains of Saccharomyces cerevisiae expressing the xylA and XKS1 genes. Appl Environ Microbiol 67:5668–5674CrossRefGoogle Scholar
  40. van Bastelaere P, Vangrysperre W, Kersters-Hilderson H (1991) Kinetic studies of Mg2+-, Co2+- and Mn2+-activated d-xylose isomerases. Biochem J 278:285–292Google Scholar
  41. Vieille C, Hess JM, Kelly RM, Zeikus JG (1995) xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana. Appl Environ Microbiol 61:1867–1875Google Scholar
  42. Walfridsson M, Bao X, Anderlund M, Lilius G, Bülow L, Hahn-Hägerdal B (1996) Ethanolic fermentation of xylose with Saccharomyces cerevisiae harboring the Thermus thermophilus xylA gene, which expresses an active xylose (glucose) isomerase. Appl Environ Microbiol 62:4648–4651Google Scholar
  43. Weierstall T, Hollenberg CP, Boles E (1999) Cloning and characterization of three genes (SUT1-3) encoding glucose transporters of the yeast Pichia stipitis. Mol Microbiol 31:871–883CrossRefGoogle Scholar
  44. Wong HC, Ting Y, Lin H, Reichert F, Myambo K, Watt KWK, Toy PL, Drummond RJ (1991) Genetic organization and regulation of the xylose degradation genes in Streptomyces rubiginosus. J Bacteriol 173:6849–6858Google Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Anjali Madhavan
    • 1
  • Sriappareddy Tamalampudi
    • 2
  • Kazunari Ushida
    • 3
  • Daisuke Kanai
    • 4
  • Satoshi Katahira
    • 2
  • Aradhana Srivastava
    • 1
  • Hideki Fukuda
    • 2
  • Virendra S. Bisaria
    • 1
  • Akihiko Kondo
    • 5
  1. 1.Department of Biochemical Engineering and BiotechnologyIndian Institute of Technology DelhiDelhiIndia
  2. 2.Organization of Advanced Science and TechnologyKobe UniversityKobeJapan
  3. 3.Laboratory of Animal ScienceKyoto Prefectural UniversityKyotoJapan
  4. 4.Research and Development Laboratories for Sustainable Value CreationAsahi Breweries, LtdIbarakiJapan
  5. 5.Department of Chemical Science and Engineering, Graduate School of EngineeringKobe UniversityKobeJapan

Personalised recommendations