Journal of Biosciences

, Volume 34, Issue 6, pp 881–890 | Cite as

Xylose reductase from the thermophilic fungus Talaromyces emersonii: cloning and heterologous expression of the native gene (Texr) and a double mutant (TexrK271R + N273D) with altered coenzyme specificity

  • Sara Fernandes
  • Maria G. Tuohy
  • Patrick G. Murray
Article

Abstract

Xylose reductase is involved in the first step of the fungal pentose catabolic pathway. The gene encoding xylose reductase (Texr) was isolated from the thermophilic fungus Talaromyces emersonii, expressed in Escherichia coli and purified to homogeneity. Texr encodes a 320 amino acid protein with a molecular weight of 36 kDa, which exhibited high sequence identity with other xylose reductase sequences and was shown to be a member of the aldoketoreductase (AKR) superfamily with a preference for reduced nicotinamide adenine dinucleotide phosphate (NADPH) as coenzyme. Given the potential application of xylose reductase enzymes that preferentially utilize the reduced form of nicotinamide adenine dinucleotide (NADH) rather than NADPH in the fermentation of five carbon sugars by genetically engineered microorganisms, the coenzyme selectivity of TeXR was altered by site-directed mutagenesis. The TeXRK271R+N273D double mutant displayed an altered coenzyme preference with a 16-fold improvement in NADH utilization relative to the wild type and therefore has the potential to reduce redox imbalance of xylose fermentation in recombinant S. cerevisiae strains. Expression of Texr was shown to be inducible by the same carbon sources responsible for the induction of genes encoding enzymes relevant to lignocellulose hydrolysis, suggesting a coordinated expression of intracellular and extracellular enzymes relevant to hydrolysis and metabolism of pentose sugars in T. emersonii in adaptation to its natural habitat. This indicates a potential advantage in survival and response to a nutrient-poor environment.

Keywords

Xylose reductase Talaromyces emersonii thermophilic co-enzyme specificity transcriptional analysis 

Abbreviations used

AKR

aldoketoreductase

BSA

bovine serum albumin

IPTG

isopropylthio-β-galactoside

LOOPP

Learning, Observing and Outputting Protein Patterns

NADH

reduced form of nicotinamide adenine dinucleotide

NADPH

reduced nicotinamide adenine dinucleotide phosphate

NCBI

National Centre for Biotechnological Information

PCR

polymerase chain reaction

RACE

rapid amplification of cDNA ends

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Altschul S F, Gish W, Miller W, Myers E W and Lipman D J 1990 Basic local alignment search tool; J. Mol. Biol. 215 403–410PubMedGoogle Scholar
  2. Amore R, Kotter P, Kuster C, Ciriacy M and Hollenberg C P 1991 Cloning and expression in Saccharomyces cerevisiae of the NAD(P)H-dependent xylose reductase-encoding gene (XYL1) from the xylose-assimilating yeast Pichia stipitis; Gene 109 89–97CrossRefPubMedGoogle Scholar
  3. Bairoch A, Apweiler R, Wu C H, Barker W C, Boeckmann B, Ferro S, Gasteiger E, Huang H, Lopez R, Magrane M, Martin M J, Natale D A, O’Donovan C, Redaschi N and Yeh L S 2005 The Universal Protein Resource (UniProt); Nucleic Acids Res. 33 154–159CrossRefGoogle Scholar
  4. Billard P, Menart S, Fleer, R and Bolotin-Fukuhara M 1995 Isolation and characterization of the gene encoding xylose reductase from Kluyveromyces lactis; Gene 162 93–97CrossRefPubMedGoogle Scholar
  5. Bradford M M 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding; Anal. Biochem. 72 248–254CrossRefPubMedGoogle Scholar
  6. Branden I C 1991 The TIM barrel — the most frequently occuring folding motif in proteins; Curr. Opin. Struct. Biol. 1 978–983CrossRefGoogle Scholar
  7. Brooks M M, Tuohy M G, Savage A V, Claeyssens M and Coughlan M P 1992 The stereochemical course of reactions catalysed by the cellobiohydrolases produced by Talaromyces emersonii; Biochem. J. 283 31–34PubMedGoogle Scholar
  8. Chomczynski P and Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction; Anal. Biochem. 162 156–159CrossRefPubMedGoogle Scholar
  9. Di Luccio E, Elling R A and Wilson D K 2006 Identification of a novel NADH-specific aldo-keto reductase using sequence and structural homologies; Biochem. J. 400 105–114CrossRefPubMedGoogle Scholar
  10. Fernandes S, Murray P G and Tuohy M G 2008 Enzyme systems from the thermophilic fungus Talaromyces emersonii for sugar beet bioconversion; Bioresources 3 898–909Google Scholar
  11. Hahn-Hägerdal B, Wahlbom C F, Gardonyi M, van Zyl W H, Cordero Otero R R and Jonsson L J 2001 Metabolic engineering of Saccharomyces cerevisiae for xylose utilization; Adv. Biochem. Eng. Biotechnol. 73 53–84PubMedGoogle Scholar
  12. Hahn-Hägerdal B, Galbe M, Gorwa-Grausland M-F, Liden G and Zacchi G 2006 Bio-ethanol — the fuel of tommorrow from the residues of today; Trends Biotechnol. 24 549–556CrossRefPubMedGoogle Scholar
  13. Hahn-Hägerdal B, Karhumaa K, Fonseca C, Spencer-Martins I and Gorwa-Grauslund M F 2007 Towards industrial pentosefermenting yeast strains; Appl. Microbiol. Biotechnol. 74 937–953CrossRefPubMedGoogle Scholar
  14. Handumrongkul C, Ma D P and Silva J L 1998 Cloning and expression of Candida guilliermondii xylose reductase gene (xyl1) in Pichia pastoris; Appl. Microbiol. Biotechnol. 49 399–404CrossRefPubMedGoogle Scholar
  15. Hasper A A, Visser J and de Graaff L H 2000 The Aspergillus niger transcriptional activator XlnR, which is involved in the degradation of the polysaccharides xylan and cellulose, also regulates D-xylose reductase gene expression; Mol. Microbiol. 36 193–200CrossRefPubMedGoogle Scholar
  16. Higgins D G 1994 CLUSTAL W: multiple alignment of DNA and protein sequences; Methods Mol. Biol. 2 307–318Google Scholar
  17. Hyndman D, Bauman, D R, Heredia V V and Penning T M 2003 The aldo-keto reductase superfamily homepage; Chem. Biol. Interact. 143 621–631CrossRefPubMedGoogle Scholar
  18. Jez J M, Bennett M J, Schlegel B P, Lewis M and Penning T M 1997 Comparative anatomy of the aldo-keto reductase superfamily; Biochem. J. 326 625–636PubMedGoogle Scholar
  19. Jez J M and Penning T M 2001 The aldo-keto reductase (AKR) superfamily: an update; Chem. Biol. Interact. 130 499–525CrossRefPubMedGoogle Scholar
  20. Jung-Kul L, Bong-seong K and Sang-Yong K 2003 Cloning and characterisation of the xyl1 gene, encoding an NADH-preferring xylose reductase from Candida parapsilosis, and its functional expression in Candida tropicalis; Appl. Environ. Microbiol. 69 6179–6188CrossRefGoogle Scholar
  21. Kavanagh K L, Klimacek M, Nidetzky B and Wilson D K 2003 Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases; Biochem. J. 373 319–326CrossRefPubMedGoogle Scholar
  22. Kostrzynska M, Sopher C R and Lee H 1998 Mutational analysis of the role of the conserved lysine-270 in the Pichia stipitis xylose reductase; FEMS Microbiol. Lett. 159 107–112CrossRefPubMedGoogle Scholar
  23. Kratzer R, Kavanagh K L, Wilson D K and Nidetzky B 2004 Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant; Biochemistry 43 4944–4954CrossRefPubMedGoogle Scholar
  24. Kratzer R, Leitgeb S, Wilson D K and Nidetzky B 2006 Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis; Biochem. J. 393 51–58CrossRefPubMedGoogle Scholar
  25. Leitgeb S, Petschacher B, Wilson D K and Nidetzky B 2005 Fine tuning of coenzyme specificity in family 2 aldo-keto reductases revealed by crystal structures of the Lys-274→Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+; FEBS Lett. 579 763–767CrossRefPubMedGoogle Scholar
  26. Liang L, Zhang J and Lin Z 2007 Altering coenzyme specificity of Pichia stipitis xylose reductase by the semi-rational approach CASTing; Microb. Cell Fact. 6 36CrossRefPubMedGoogle Scholar
  27. Liu Z, Cai R and Wang J 2006 Reviews in fluorescence 2006 — current development in the determination of intracellular NADH level (New York: Plenum Publishers)Google Scholar
  28. McCarthy T, Hanniffy O, Savage A V and Tuohy M G 2003 Catalytic properties and mode of action of three endo-betaglucanases from Talaromyces emersonii on soluble beta-1,4- and beta-1,3;1,4-linked glucans; Int. J. Biol. Macromol. 33 141–148CrossRefPubMedGoogle Scholar
  29. McCarthy T, Lalor E, Hanniffy O, Savage A V and Tuohy M G 2005 Comparison of wild-type and UV-mutant beta-glucanaseproducing strains of Talaromyces emersonii with potential in brewing applications; J. Ind. Microbiol. Biotechnol. 32 125–134CrossRefPubMedGoogle Scholar
  30. Moloney A, Considine P J and Coughlan M P 1983 Cellulose hydrolysis by Talaromyces emersonii grown on different substrates; Biotech. Bioeng. 25 1169–1173CrossRefGoogle Scholar
  31. Moloney A P, McCrae S I, Wood T M and Coughlan M P 1985 Isolation and characterization of the endoglucanases of Talaromyces emersonii; Biochem. J. 225 365–374PubMedGoogle Scholar
  32. Murray P G, Grassick A, Laffey C D, Cuffe M M, Higgins T, Savage A V, Planas A and Tuohy M G 2001 Isolation and characterization of a thermostable endo-beta-glucanase active on 1,3-1,4-beta-D-glucans from the aerobic fungus Talaromyces emersonii CBS 814.70; Enzyme Microb. Technol. 29 90–98CrossRefPubMedGoogle Scholar
  33. Murray P G, Collins C M, Grassick A and Tuohy M G 2003 Molecular cloning, transcriptional, and expression analysis of the first cellulase gene (cbh2), encoding cellobiohydrolase II, from the moderately thermophilic fungus Talaromyces emersonii and structure prediction of the gene product; Biochem. Biophys. Res. Commun. 301 280–286CrossRefPubMedGoogle Scholar
  34. Petschacher B, Leitgeb S, Kavanagh K L, Wilson D K and Nidetzky B 2005 The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography; Biochem. J. 385 75–83CrossRefPubMedGoogle Scholar
  35. Petschacher B and Nidetzky B 2005 Engineering Candida tenuis xylose reductase for improved utilization of NADH: antagonistic effects of multiple side chain replacements and performance of site-directed mutants under simulated in vivo conditions; Appl. Environ. Microbiol. 71 6390–6393CrossRefPubMedGoogle Scholar
  36. Petschacher B and Nidetzky B 2008 Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of Saccharomyces cerevisiae; Microb. Cell Fact. 7 9PubMedGoogle Scholar
  37. Raeder U and Broda P 1985 Rapid preparation of DNA from filamentous fungi; Lett. Appl. Microbiol. 1 17–20CrossRefGoogle Scholar
  38. Rost B 1996 PHD: predicting one-dimensional protein structure by profile-based neural networks; Methods Enzymol. 266 525–539CrossRefPubMedGoogle Scholar
  39. Schlegel B P, Jez J M and Penning T M 1998 Mutagenesis of 3 alpha-hydroxysteroid dehydrogenase reveals a “pushpull” mechanism for proton transfer in aldo-keto reductases; Biochemistry 37 3538–3548CrossRefPubMedGoogle Scholar
  40. Stolk A C and Sampson R A 1972 The genus Talaromyces — studies in mycology 2 (Baarn: Centraalbureau Voor Schimmelcultures Publishers)Google Scholar
  41. Stricker A R, Grosstessner-Hain K, Würleitner E and Mach R L 2006 Xyr1 (xylanase regulator 1) regulates both the hydrolytic enzyme system and D-xylose metabolism in Hypocrea jecorina; Eukaryot. Cell 5 2128–2137CrossRefPubMedGoogle Scholar
  42. Tuohy M G and Coughlan M P 1992 Production of thermostable xylan degrading enzymes by Talaromyces emersonii CBS 814.70; Bioresource Technol. 39 131–137CrossRefGoogle Scholar
  43. Tuohy M G, Puls J, Claeyssens M, Vrsanská M and Coughlan M P 1993 The xylan-degrading enzyme system of Talaromyces emersonii: novel enzymes with activity against aryl beta-Dxylosides and unsubstituted xylans; Biochem. J. 290 515–523PubMedGoogle Scholar
  44. Tuohy M G, Walsh D J, Murray P G, Claeyssens M, Cuffe M M, Savage A V and Coughlan M P 2002 Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii; Biochim. Biophys. Acta 1596 366–380PubMedGoogle Scholar
  45. van Kuyk P A, de Groot M J, Ruijter G J, de Vries R P and Visser J 2001 The Aspergillus niger D-xylulose kinase gene is coexpressed with genes encoding arabinan degrading enzymes, and is essential for growth on D-xylose and L-arabinose; Eur. J. Biochem. 268 5414–5423CrossRefGoogle Scholar
  46. van Peij N N, Gielkens M M, de Vries R P, Visser J and de Graaff L H 1998 The transcriptional activator XlnR regulates both xylanolytic and endoglucanase gene expression in Aspergillus niger; Appl. Environ. Microbiol. 64 3615–3619PubMedGoogle Scholar
  47. Watanabe S, Abu Saleh A, Pack S P, Annaluru N, Kodaki T and Makino K 2007 Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis; Microbiology 153 3044–3054CrossRefPubMedGoogle Scholar

Copyright information

© Indian Academy of Sciences 2009

Authors and Affiliations

  • Sara Fernandes
    • 1
  • Maria G. Tuohy
    • 1
  • Patrick G. Murray
    • 2
  1. 1.Molecular Glycobiotechnology Group, Biochemistry, School of Natural SciencesNational University of IrelandGalwayIreland
  2. 2.Shannon Applied Biotechnology CentreLimerick Institute TechnologyLimerickIreland

Personalised recommendations