Applied Microbiology and Biotechnology

, Volume 44, Issue 1–2, pp 147–156 | Cite as

Efficient expression and secretion of Aspergillus niger RH5344 polygalacturonase in Saccharomyces cerevisiae

  • C. Lang
  • A. C. Looman
Applied Genetics and Regulation

Abstract

An Aspergillus niger endopolygalacturonase (EC 3.2.1.15) cDNA was expressed in the yeast Saccharomyces cerevisiae. Secretion of the protein into the growth medium was efficiently directed by the fungal leader sequence, and processing occurred at the same site as in Aspergillus. The expression level was significantly enhanced by using a “short” version of the yeast ADHI promoter. An additional increase in the yield of heterologous protein was due to a higher plasmid stability and a rise in plasmid copy number. This was achieved by deleting most of the bacterial sequences from the expression vector. The yeast-derived enzyme showed the same enzymatic and biochemical properties as the fungal polygalacturonase, such as substrate specificity, pH and temperature optima and pI value. The yeast-derived enzyme, however, showed a higher degree of glycosylation and exhibited a more pronounced temperature stability than the fungal enzyme.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Apostol B, Greer C (1988) Copy number and stability of yeast 2μm-based plasmids carrying a transcription-conditional centromere. Gene 67:59–68Google Scholar
  2. Bennett MK, Scheller RH (1993) The molecular machinery for secretion is conserved from yeast to neurons. Proc Natl Acad Sci USA 90:2559–2563Google Scholar
  3. Bussink HJD, Kester HCM, Visser J (1990) Molecular cloning, nucleotide sequence and expression of the gene encoding pre-propolygalacturonase II of Aspergillus niger. FEBS Lett 273:127–130Google Scholar
  4. Bussink HJD, Brouwer KB, de Graaff LH, Kester HCM, Visser J (1991a) Identification and characterization of a second polygalacturonase gene of Aspergillus niger. Curr Genet 20:301–307Google Scholar
  5. Bussink HJD, Buxton FP, Visser J (1991b) Expression and sequence comparison of the Aspergillus niger and Aspergillus tubigensis genes encoding polygalacturonase II. Curr Genet 19:467–474Google Scholar
  6. Caplan S, Green R, Rocco J, Kurjan J (1991) Glycosylation and structure of the yeast MFα1 α-factor precursor is important for efficient transport through the secretory pathway. J Bacteriol 173:627–635Google Scholar
  7. Chang CN, Matteucci M, Perry J, Wulf JJ, Chen CY, Hitzeman RA (1986) Saccharomyces cerevisiae secretes and correctly processes human interferon hybrid proteins containing yeast invertase signal peptides. Mol Cell Biol 6:1812–1819Google Scholar
  8. Chen EY, Seeburg PH (1985) Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4:165–170Google Scholar
  9. Denis CL, Ferguson J, Young ET (1983) mRNA levels for the fermentative alcohol dehydrogenase of Saccharomyces cerevisiae decrease upon growth on a nonfermentable carbon source. J Biol Chem 258:1165–1171Google Scholar
  10. Devi L (1991) Consensus sequence for processing of peptide precursors at monobasic sites. FEBS Lett 280:189–194Google Scholar
  11. Ernst JF (1988) Efficient secretion and processing of heterologous proteins in Saccharomyces cerevisiae is mediated solely by the pre-segment of α-factor precursor. DNA 7:355–360Google Scholar
  12. Ferenci T, Silhavy TJ (1987) Sequence information required for protein translocation from the cytoplasm. J Bacteriol 169: 5339–5342Google Scholar
  13. Frederick KR, Tung J, Emerick RS, Masiarz FR, Chamberlain SH, Vasavada A, Rosenberg S, Chakraborty S, Schopter LM, Massey V (1990) Glucose oxidase from Aspergillus niger. J Biol Chem 265:3793–3802Google Scholar
  14. Harmsen JAM, Kusters-van Someren MA, Visser J (1990) Cloning and expression of a second Aspergillus pectin lyase gene (pelA): indications of a pectin lyase gene family in Aspergillus niger. Curr Genet 18:161–166Google Scholar
  15. Heijne G von (1983) Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem 133:17–21Google Scholar
  16. Heijne G von (1985) Signal sequences. The limits of variation. J Mol Biol 184:99–105Google Scholar
  17. Henderson ST, Petes TD (1992) Instability of simple sequence DNA in Saccharomyces cerevisiae. Mol Cell Biol 12:2749–2757Google Scholar
  18. Hiramatsu R, Horinouchi S, Uchida E, Hayakawa T, Beppu T (1991) The secretion leader of Mucor pusillus rennin which possesses an artificial Lys-Arg sequence directs the secretion of mature human growth hormone by Saccharomyces cerevisiae. Appl Environm Microbiol 57:2052–2056Google Scholar
  19. Hitzeman RA, Leung DW, Perry LJ, Kohr WJ, Levine HL, Goeddel DV (1983) Secretion of human interferons by yeast. Science 219:620–625Google Scholar
  20. Hitzeman RA, Chen CY, Dowbenko DJ, Renz ME, Liu C, Pai R, Simpson NJ, Kohr WJ, Singh A, Chisholm V, Hamilton R, Chang CN (1990) Use of heterologous and homologous signal sequences for secretion of heterologous proteins from yeast. Methods Enzymol 185:421–440Google Scholar
  21. Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272Google Scholar
  22. Innis MA, Holland MJ, McCabe PC, Cole GE, Wittman VP, Tal R, Watt KWK, Gelfand DH, Holland JP, Meade JM (1985) Expression, glycosylation and secretion of an Aspergillus glucoamylase by Saccharomyces cerevisiae. Science 228:21–26Google Scholar
  23. Kaiser CA, Preuss D, Grisafi P, Botstein D (1987) Many random sequences functionally replace the secretion signal sequence of yeast invertase. Science 235:312–317Google Scholar
  24. Kanda A, Tamaki M, Nakamura E, Teraoka H, Yoshida N (1992) Characterization of recombinant human and rat pancreatic phospholipases A2 secreted from Saccharomyces cerevisiae: difference in proteolytic processing. Biochim Biophys Acta 1171:1–10Google Scholar
  25. Kester HCM, Visser J (1990) Purification and characterization of polygalacturonases produced by the hyphal fungus Aspergillus niger. Biotechnol Appl Biochem 12:150–160Google Scholar
  26. Kitamoto N, Kimura T, Kito Y, Ohmiya K, Tsukagoshi N (1993) Structural features of a polygalacturonase gene cloned from Aspergillus oryzae KBN616. FEMS Microbiol Lett 111:37–42Google Scholar
  27. Kurjan J, Herskowitz I (1982) Structure of a yeast pheromone gene (MFα): a putative α-factor precursor contains four tandem copies of mature α-factor. Cell 30:933–943Google Scholar
  28. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132Google Scholar
  29. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  30. Lang-Hinrichs C, Berndorff D, Seefeldt C, Stahl U (1989) G418 resistance in the yeast Saccharomyces cerevisiae: comparison of the neomycin resistance genes from Tn5 and Tn903. Appl Microbiol Biotechnol 30:388–394Google Scholar
  31. Looman AC, Laude M, Stahl U (1991) Influence of the codon following the initiation codon on the expression of the lacZ gene in Saccharomyces cerevisiae. Yeast 7:157–165Google Scholar
  32. Ludwig DL, Bruschi CV (1991) The 2μm plasmid as a non-selectable, stable, high copy number yeast vector. Plasmid 25:81–95Google Scholar
  33. Ludwig DL, Ugolini S, Bruschi CV (1993) High-level heterologous gene expression in Saccharomyces cerevisiae from a stable 2 μm plasmid system. Gene 132:33–40Google Scholar
  34. Melgaard M, Svendsen I (1994) Different effects of N-glycosylation on the thermostability of highly homologous bacterial (1,3–1,4)-β-glucanases secreted from yeast. Microbiology 140:159–166Google Scholar
  35. Miller GL (1959) Use of dinitrosalicyclic acid for determination of reducing sugars. Anal Biochem 31:426–428Google Scholar
  36. Miyajima A, Bond MW, Otsu K, Arai N (1985) Secretion of mature mouse interleukin-2 by Saccharomyces cerevisiae: use of a general secretion vector containing promoter and leader sequences of the mating pheromone α-factor. Gene 37:155–161Google Scholar
  37. Nagahora H, Ishikawa K, Niwa Y, Muraki M, Jigami Y (1992) Expression and secretion of wheat germ agglutinin by Saccharomyces cerevisiae. Eur J Biochem 210:989–997Google Scholar
  38. Olsen O (1987) Analysis of the effect of dG.dC homopolymer tails on expression of a mouse α-amylase cDNA gene in yeast. Carlsberg Res Commun 52:91–97Google Scholar
  39. Parent SA, Fenimore CM, Bostian KA (1985) Vector systems for the expression, analysis and cloning of DNA sequences in S. cerevisiae. Yeast 1:83–138Google Scholar
  40. Randez-Gil F, Sanz P (1993) Expression of Aspergillus oryzae α-amylase gene in Saccharomyces cerevisiae. FEMS Microbiol Lett 112:119–124Google Scholar
  41. Romanos MA, Scorer CA, Clare JJ (1992) Foreign gene expression in yeast: a review. Yeast 8:423–488Google Scholar
  42. Rombouts FM, Pilnik W (1978) Enzymes in fruit and vegetable juice technology. Proc Biochem 8:9–13Google Scholar
  43. Ruohonen L, Hackman P, Lehtovaara P, Knowles JKC, Keränen S (1987) Efficient secretion of Bacillus amyloliquefaciens α-amylase by its own signal peptide from Saccharomyces cerevisiae host cells. Gene 59:161–170Google Scholar
  44. Ruohonen L, Pentillä M, Keränen S (1991) Optimization of Bacillus α-amylase production by Saccharomyces cerevisiae. Yeast 7:337–346Google Scholar
  45. Ruttkowski E, Labitzki R, Khanh NQ, Löffler F, Gottschalk M, Jany KD (1990) Cloning and DNA sequence analysis of a polygalacturonase cDNA from Aspergillus niger RH5344. Biochim Biophys Acta 1087:104–106Google Scholar
  46. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  47. Sidhu RS, Bollon AP (1990) Bacterial plasmid pBR322 sequences serve as upstream activating sequences in Saccharomyces cerevisiae. Yeast 6:221–229Google Scholar
  48. Singh A, Chen EY, Lugovoy JM, Chang CN, Hitzeman RA, Seeburg PH (1983) Saccharomyces cerevisiae contains two discrete genes coding for the α-factor pheromone. Nucleic Acids Res 11: 4049–4063Google Scholar
  49. Sleep D, Belfield GP, Goodey AR (1990) The secretion of human serum albumin from the yeast Saccharomyces cerevisiae using five different leader sequences. Biotechnology 8:42–46Google Scholar
  50. Stinchcomb DT, Mann C, Davis RW (1982) Centrometric DNA from Saccharomyces cerevisiae. J Mol Biol 158:157–179Google Scholar
  51. Tatsumi H, Ogawa Y, Murakami S, Ishida Y, Murakami K, Masaki A, Kawabe H, Arimura H, Nakano E, Motai H (1989) A full length cDNA clone for the alkaline protease from Aspergillus oryzae: structural analysis and expression in Saccharomyces cerevisiae. Mol Gen Genet 219:33–38Google Scholar
  52. Tatsumi H, Murakami S, Tsuji RF, Ishida Y, Murakami K, Masaki A, Kawabe H, Arimura H, Nakano E, Motai H (1991) Cloning and expression in yeast of a cDNA clone encoding Aspergillus oryzae neutral protease-II, a unique metalloprotease. Mol Gen Genet 228:97–103Google Scholar
  53. Vainio AEI, Torkkeli HT, Tuusa T, Aho SA, Fagerström BR, Korhola MP (1993) Cloning and expression of Hormoconis cresinae glucoamylase P cDNA in Saccharomyces cerevisiae. Curr Genet 24:38–44Google Scholar
  54. Vieira J, Messing J (1982) The pUC plasmid, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268Google Scholar
  55. Wittmann-Liebold B, Kimura M (1986) Microsequencing of peptides and proteins with 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate. In: Advanced methods in microsequencing analysis. Springer, Heidelberg Berlin New York, pp 78–90Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • C. Lang
    • 1
  • A. C. Looman
    • 1
  1. 1.Institut für Gärungsgewerbe und BiotechnologieAbt. Biotechnologie HülsBerlinGermany

Personalised recommendations