Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination

  • Qiang Zhang
  • Qi-he Chen
  • Ming-liang Fu
  • Jin-ling Wang
  • Hong-bo Zhang
  • Guo-qing He


The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone α-factor (MFα1 s ), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-βG) was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/(h·ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.

Key words

Endo-1,3-1,4-β-glucanase (bglSGene replacement Homologous recombination Bacillus subtilis PEP4 gene Saccharomyces cerevisiae 

CLC number



  1. Akada, R., 2002. Genetically modified industrial yeast ready for application. J. Biosci. Bioeng., 94(6):536–544. [doi:10.1016/S1389-1723(02)80192-X]PubMedGoogle Scholar
  2. Ammerer, G., Hunter, C.P., Rothman, J.H., Saari, G.C., Valls, L.A., Stevens, T.H., 1986. PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors. Mol. Cell Biol., 6(7):2490–2499.PubMedGoogle Scholar
  3. Antoni, P., 2000. Bacterial 1,3-1,4-β-glucanases: structure, function and protein engineering. Biochimi Biophysica Acta, 1543(2):361–382.Google Scholar
  4. 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(1–2):248–254. [doi:10.1016/0003-2697(76)90527-3]PubMedCrossRefGoogle Scholar
  5. Cantwell, B.A., McConnell, D.J., 1983. Molecular cloning and expression of a Bacillus subtilis β-glucanase gene in Escherichia coli. Gene, 23(2):211–219. [doi:10.1016/0378-1119(83)90053-7]PubMedCrossRefGoogle Scholar
  6. Cantwell, B.A., Brazil, G., Murphy, N., McConnell, D.J., 1986. Comparison of expression of the endo-β-1,3-1,4-glucanase gene from Bacillus subtilis in Saccharomyces cerevisiae from the CYCI and ADHI promoters. Curr. Genet., 11(1):65–70. [doi:10.1007/BF00389427]PubMedCrossRefGoogle Scholar
  7. Cooper, D.J., Stewart, G.G., Bryce, J.H., 2000. Yeast proteolytic activity during high and low gravity wort fermentations and its effect on head retention. J. Inst. Brew., 1066(4):197–201.Google Scholar
  8. Court, D.L., Sawitzke, J.A., Thomason, L.C., 2002. Genetic engineering using homologous recombination. Ann. Rev. Genet., 36(1):361–388. [doi:10.1146/annurev.genet.36.061102.093104]PubMedCrossRefGoogle Scholar
  9. Gaiser, O.J., Piotukh, K., Ponnuswamy, M.N., Planas, A., Borriss, R., Heinemann, U., 2006. Structural basis for the substrate specificity of a Bacillus 1,3-1,4-β-glucanase. J. Mol. Biol., 357(4):1211–1225. [doi:10.1016/j.jmb.2006.01.014]PubMedCrossRefGoogle Scholar
  10. Grujic, O., 1998. Application of a commercial enzyme preparation in the barley malting process. J. Inst. Brew., 104(5):249–253.Google Scholar
  11. He, G.Q., Wang, Z.Y., Liu, Z.S., Chen, Q.H., Ruan, H., Schwarz, P.B., 2006. Relationship of proteinase activity, foam proteins and head retention in unpasteurized beer. J. Am. Soc. Brew. Chem., 64(1):33–38.Google Scholar
  12. Hinchliffe, E., Box, W.G., 1984. Expression of the cloned endo-1,3-1,4-β-glucanase gene of Bacillus subtilis in Saccharomyces cerevisiae. Curr. Genet., 8(6):471–475. [doi:10.1007/BF00433914]CrossRefGoogle Scholar
  13. Jin, Y.L., Speers, R.A., Paulson, A.T., Stewart, R.J., 2004. Barley β-glucans and their degradation during malting and brewing. Technical Quarterly-Master Brewers Association of the Americas, 41(3):231–240.Google Scholar
  14. Jones, E.W., 1991. Tackling the protease problem in Saccharomyces cerevisiae. Methods Enzymol., 194:429–453.Google Scholar
  15. Jones, E.W., Zubenko, G.S., Parker, R.R., 1982. PEP4 gene function is required for expression of several vacuolar hydrolases in Sacchromyces cerevisiae. Genetics, 102(4):665–677.PubMedGoogle Scholar
  16. Kettunen, A., Hamalainen, J.J., Stenholm, K., Pietilä, K., 1996. A model for the prediction of β-glucan concentration during mashing. J. Food Eng., 29(2):185–200. [doi:10.1016/0260-8774(95)00070-4]CrossRefGoogle Scholar
  17. Marques, M., Mojzita, D., Amorim, M.A., Almeida, T., Hehmann, S., Moradas, F.P., Costa, V., 2006. The Pep4p vacuolar proteinase contributes to the turnover of oxidized proteins but PEP4 overexpression is not sufficient to increase chronological lifespan in Saccharomyces cerevisiae. Microbiology, 152(12):3595–3605. [doi:10.1099/mic.0.29040-0]PubMedCrossRefGoogle Scholar
  18. Muldbjerg, M., Meldal, M., Breddam, K., Sigsgaard, P., 1993. Protease activity in beer and correlation of foam. Proc. Congr. Eur. Brew. Conv., 25:357–364.Google Scholar
  19. Müller, J.J., Thomsen, K.K., Heinemann, U., 1998. Crystal structure of barley 1,3-1,4-β-glucanase at 2.0-Å resolution and comparison with Bacillus 1,3-1,4-β-glucanase. J. Biol. Chem., 273(6):3438–3446. [doi:10.1074/jbc.273.6.3438]PubMedCrossRefGoogle Scholar
  20. Olsen, O., Thomsen, K.K., 1989. Processing and secretion of barley (1-3,1-4)-β-glucanase in yeast. Carlsberg. Res. Commun., 54(2):29–39. [doi:10.1007/BF02907583]PubMedCrossRefGoogle Scholar
  21. Palmer, G.H., 1989. Cereals in Malting and Brewing. In: Palmer, G.H. (Ed.), Cereal Science and Technology. Aberdeen University Press, Aberdeen, UK, p.61–242.Google Scholar
  22. Panttilä, M.E., André, L., Saloheimo, M., Lehtovaara, P., Knowles, J.K., 1987a. Expression of two Trichoderma reesei endoglucanases in the yeast Saccharomyces cerevisiae. Yeast, 3(3):175–185. [doi:10.1002/yea.320030305]CrossRefGoogle Scholar
  23. Panttilä, M.E., Suihko, U., Lehtinen, M., Nikkola, M., Knowles, J.K.C., 1987b. Construction of brewer’s yeasts secreting fungal endo-β-glucanase. Curr. Genet., 12(6):413–420. [doi:10.1007/BF00434818]CrossRefGoogle Scholar
  24. Rothman, J.H., Hunter, C.P., Valls, L.A., Stevens, T.H., 1986. Overproduction-induced mislocalization of a yeast vacuolar protein allows isolation of its structural gene. Proc. Natl. Acad. Sci. USA, 83(10):3248–3252. [doi:10.1073/pnas.83.10.3248]PubMedCrossRefGoogle Scholar
  25. Rupp, S., Wolf, D.H., 1995. The use of active-site mutants of proteinase yscA to determine the necessity of the enzyme for vacuolar protemase maturation and proteinase yscB stability. Eur. J. Biochem., 231(1):115–125. [doi:10.1111/j.1432-1033.1995.tb20677.x]PubMedCrossRefGoogle Scholar
  26. Stevens, T.H., Esmon, B., Schekman, R., 1982. Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell, 30(2):439–448. [doi:10.1016/0092-8674(82)90241-0]PubMedCrossRefGoogle Scholar
  27. Sudarmana, D.L., Goldsmith, M.R., Hinh, A.H., Pecar, M.A., Hawthorne, D.B., Kavanagh, T.E., 1996. Microfiltration studies with a modified membrane filterability procedure. Technical Quarterly-Master Brewers Association of the Americas, 33(1):63–72.Google Scholar
  28. Todo, V., Carbonell, J.V., Sendra, J.M., 1989. Kinetics of β-glucan degradation in wort by exogenous β-glucanases treatment. J. Inst. Brew., 95(6):419–422.Google Scholar
  29. van Rensburg, P., van Zyl, W.H., Pretorius, I.S., 1997. Over-expression of the Saccharomyces cerevisiae exo-β-1,3-glucanase gene together with the Bacillus subtilis endo-β-1,3-1,4-glucanase gene and the Butyrivibrio fibrisolvens endo-β-1,4-glucanase gene in yeast. J. Biotechnol., 55(1):43–53. [doi:10.1016/S0168-1656(97)00059-X]PubMedCrossRefGoogle Scholar
  30. Wang, Z.Y., He, G.Q., Liu, Z.S., Ruan, H., Chen, Q.H., Xiong, H.P., 2005. Purification of yeast proteinase A from fresh beer and its specificity on foam proteins. Int. J. Food Sci. Tech., 40(8):835–840. [doi:10.1111/j.1365-2621.2005.01000.x]CrossRefGoogle Scholar
  31. Wang, Z.Y., He, G.Q., Ruan, H., Liu, Z.S., Yang, L.F., Zhang, B.R., 2007a. Construction of proteinase A deficient transformant of industrial brewing yeast. Eur. Food Res. Technol., 225(5–6):831–835. [doi:10.1007/s00217-006-0488-5]CrossRefGoogle Scholar
  32. Wang, Z.Y., He, X.P., Zhang, B.R., 2007b. Over-expression of GSHI gene and disruption of PEP4 gene in self-cloning industrial brewer’s yeast. Int. J. Food Microbiol., 119(3):192–199. [doi:10.1016/j.ijfoodmicro.2007.07.015]PubMedCrossRefGoogle Scholar
  33. Wolf, M., Attila, G., Ortwin, S., Rainer, B., 1995. Genes encoding xylan and β-glucan hydrolyzing enzymes in Bacillus subtilis: characterization, mapping and construction of strain deficient in lichenase, cellulose and xylanase. Microbiology, 141(2):281–290.PubMedCrossRefGoogle Scholar
  34. Wood, P.J., Erfle, J.D., Teather, R.M., 1988. Use of complex formation between Congo Red and polysaccharides hydrolases in detection and assay of polysaccharide hydrolases. Methods Enzymol., 160:59–74.CrossRefGoogle Scholar
  35. Woolford, C.A., Daniels, L.B., Park, F.J., Jones, E.W., Arsdell, J.V., Innis, M.A., 1986. The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisae vacuolar hydrolases. Mol. Cell Biol., 6(7):2500–2510.PubMedGoogle Scholar
  36. Yokoi, S., Shigyo, T., Tamaki, T., 1996. A fluorometric assay for proteinase A in beer and its application for investigation of enzymatic effects on foam stability. J. Inst. Brew., 1022(1):33–37.Google Scholar
  37. Zubenko, G.S., Park, F.J., Jones, E.W., 1983. Mutations in PEP4 locus of Saccharomyces cerevisiae block final step in maturation of two vacuolar hydrolases. Proc. Natl. Acad. Sci. USA, 80(2):510–514.PubMedCrossRefGoogle Scholar

Copyright information

© Zhejiang University and Springer-Verlag GmbH 2008

Authors and Affiliations

  • Qiang Zhang
    • 1
  • Qi-he Chen
    • 1
  • Ming-liang Fu
    • 1
  • Jin-ling Wang
    • 1
  • Hong-bo Zhang
    • 1
  • Guo-qing He
    • 1
  1. 1.Department of Food Science and NutritionZhejiang UniversityHangzhouChina

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