Similar articles

Abstract: The aim of this work was to construct a novel food-grade industrial arming yeast displaying β-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase (GAL1) and phosphoglycerate kinase 1 (PGK1) promoters in different orientations was constructed. The β-1,3-1,4-glucanase gene from Bacillus subtilis was fused to &alpha%29&ck%5B%5D=abstract&ck%5B%5D=keyword'>α-agglutinin and expressed under the control of the GAL1 promoter. &alpha%29&ck%5B%5D=abstract&ck%5B%5D=keyword'>α-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the &alpha%29&ck%5B%5D=abstract&ck%5B%5D=keyword'>α-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of β-1,3-1,4-glucanase. The analysis of β-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that β-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed β-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of β-1,3-1,4-glucanase displayed in the recombinant yeast cells was enhanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer.

[1] Akada, R., 2002. Genetically modified industrial yeast ready for application. J. Biosci. Bioeng., 94(6):536-544.

[2] Bamforth, C., 1994. β-glucan and β-glucanases in malting and brewing: practical aspects. Brew. Dig., 69(5):12-16.

[3] Bielecki, S., Galas, E., 1991. Microbial β-glucanase different from cellulases. Crit. Rev. Biotechnol., 10(4):275-305.

[4] Boucher, I., Parrot, M., Gaudreau, H., Champagne, C.P., Vadeboncoeur, C., Moineau, S., 2002. Novel food-grade plasmid vector based on melibiose fermentation for the genetic engineering of Lactococcus lactis. Appl. Environ. Microbiol., 68(12):6152-6161.

[5] Chen, J.L., Tsai, L.C., Wen, T.N., Tang, J.B., Yuan, H.S., Shyur, L.F., 2001. Directed mutagenesis of specific active site residues on Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase significantly affects catalysis and enzyme structural stability. J. Biol. Chem., 276(21):17895-17901.

[6] Choi, E.S., Sohn, J.H., Rhee, S.K., 1994. Optimization of the expression system using galactose-inducible promoter for the production of anticoagulant hirudin in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol., 42(4):587-594.

[7] Enevoldsen, B., 1981. Demonstration of melibiase in non-pasteurized lager beers and studies on the heat stability of the enzyme. Carlsberg Res. Comm., 46(1-2):37-42.

[8] Enevoldsen, B., 1985. Determining pasteurization units from residual melibiase activity in lager beer. J. Am. Soc. Brew. Chem. (USA), 43(4):183-189.

[9] Estruch, F., Prieto, J.A., 2003. Construction of a Trp⁻ comercial baker’s yeast strain by using food-safe-grade dominant drug resistance cassettes. FEMS Yeast Res., 4(3):329-338.

[10] Gai, S.A., Wittrup, K.D., 2007. Yeast surface display for protein engineering and characterization. Curr. Opin. Struct. Biol., 17(4):467-473.

[11] Gasent-Ramirez, J.M., Codon, A.C., Benitez, T., 1995. Characterization of genetically transformed Saccharomyces cerevisiae baker’s yeasts able to metabolize melibiose. Appl. Environ. Microbiol., 61(6):2113-2121.

[12] Gietz, R.D., Sugino, A., 1988. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene, 74(2):527-534.

[13] Guerra, O.G., Rubio, I.G.S., Filho, C.G.D.S., Bertoni, R.A., Govea, R.C.D.S., Vicente, E.J., 2006. A novel system of genetic transformation allows multiple integrations of a desired gene in Saccharomyces cerevisiae chromosomes. J. Microbiol. Methods, 67(3):437-445.

[14] Han, Z.L., Han, S.Y., Zheng, S.P., Lin, Y., 2009. Enhancing thermostability of a Rhizomucor miehei lipase by engineering a disulfide bond and displaying on the yeast cell surface. Appl. Microbiol. Biotechnol., 85(1):117-126.

[15] Harman, G.E., Kubicek, C.P., 1998. Trichoderma and Gliocladium. T.J. International Ltd., Padstow, UK, p.327-342.

[16] 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.

[17] Jeong, D.W., Lee, J.H., Kimc, K.H., Lee, H.J., 2006. A food-grade expression/secretion vector for Lactococcus lactis that uses an alpha-galactosidase gene as a selection marker. Food Microbiol., 23(5):468-475.

[18] John, R.M.H., 1995. Genetically-modified brewing yeasts for the 21st century. Progress to Date, 11(16):1613-1627.

[19] Kondo, A., Ueda, M., 2004. Yeast cell-surface display-applications of molecular display. Appl. Microbiol. Biotechnol., 64(1):28-40.

[20] Labrie, S., Bart, C., Vadeboncoeur, C., Moineau, S., 2005. Use of an α-galactosidase gene as a food-grade selection marker for Streptococcus thermophilus. J. Dairy Sci., 88:2341-2347.

[21] Li, A.M., Liu, Z.S., Li, Q.X., Yu, L., Wang, D.C., Deng, X.M., 2008. Construction and characterization of bidirectional expression vectors in Saccharomyces cerevisiae. FEMS Yeast Res., 8(1):6-9.

[22] Li, X., Huang, X., Shao, X., Li, L., 2009. Functional cell surface display of endo-beta-1,3-1,4-glucanase in Lactococcus lactis using N-acetylmuraminidase as the anchoring motif. Chin. J. Biotechnol., 25(1):89.

[23] Liljestrom-Suominen, P.L., Joutsjoki, V., Korhola, M., 1988. Construction of a stable alpha-galactosidase-producing baker’s yeast strain. Appl. Environ. Microbiol., 54(1):245-249.

[24] Mateo, C., Palomo, J.M., Fernandez-Lorente, G., Guisan, J.M., Fernandez-Lafuente, R., 2007. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol., 40(6):1451-1463.

[25] McCleary, B., 1988. α-galactosidase from luciferine and guar seed. Methods Enzymol., 160:627-632.

[26] Miller III, C.A., Martinat, M.A., Hyman, L.E., 1998. Assessment of aryl hydrocarbon receptor complex interactions using pBEVY plasmids: expression vectors with bi-directional promoters for use in Saccharomyces cerevisiae. Nucl. Acids Res., 26(15):3577-3583.

[27] Muller, J.J., Thomsen, K.K., Heinemann, U., 1998. Crystal structure of barley 1,3-1,4-beta-glucanase at 2.0-A resolution and comparison with Bacillus 1,3-1,4-beta-glucanase. J. Biol. Chem., 273(6):3438-3446.

[28] Murai, T., Ueda, M., Atomi, H., Shibasaki, Y., Kamasawa, N., Osumi, M., Kawaguchi, T., Arai, M., Tanaka, A., 1997. Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol., 48(4):499-503.

[29] Navas, L., Esteban, M., Delgado, M.A., 1991. KAR1-mediated transformation of brewing yeast. J. Inst. Brew., 97:115-118.

[30] Park, S., Xu, Y., Stowell, X.F., Gai, F., Saven, J.G., Boder, E.T., 2006. Limitations of yeast surface display in engineering proteins of high thermostability. Protein Eng. Des. Sel., 19(5):211-217.

[31] Pronk, J.T., 2002. Auxotrophic yeast strains in fundamental and applied research. Appl. Environ. Microbiol., 68(5):2095-2100.

[32] Qiao, J., Dong, B., Li, Y., Zhang, B., Cao, Y., 2009. Cloning of a β-1,3-1,4-glucanase gene from Bacillus subtilis MA139 and its functional expression in Escherichia coli. Appl. Biochem. Biotechnol., 152(2):334-342.

[33] Ruohola, H., Liljestrom, P.L., Torkkeli, T., Kopu, H., Lehtinen, P., Kalkkinen, N., Korhola, M., 1986. Expression and regulation of the yeast MEL1 gene. FEMS Microbiol. Lett., 34(2):179-185.

[34] Sakai, K., Uchiyama, T., Matahira, Y., Nanjo, F., 1991. Immobilization of chitinolytic enzymes and continuous production of N-acetylglucosamine with the immobilized enzymes. J. Ferment. Bioeng., 72(3):168-172.

[35] Shibasaki, S., Maeda, H., Ueda, M., 2009. Molecular display technology using yeast-arming technology. Anal. Sci., 25(1):41-49.

[36] Shusta, E.V., Kieke, M.C., Parke, E., Kranz, D.M., Wittrup, K.D., 1999. Yeast polypeptide fusion surface display levels predict thermal stability and soluble secretion efficiency. J. Mol. Biol., 292(5):949-956.

[37] Štagoj, M., Komel, R., 2008. The GAL induction response in yeasts with impaired galactokinase Gal1p activity. World J. Microbiol. Biotechnol., 24(10):2159-2166.

[38] Tanino, T., Fukuda, H., Kondo, A., 2006. Construction of a Pichia pastoris cell-surface display system using Flo1p anchor system. Biotechnol. Progr., 22(4):989-993.

[39] Teng, D., Wang, J.H., Fan, Y., Yang, Y.L., Tian, Z.G., Luo, J., Yang, G.P., Zhang, F., 2006. Cloning of β-1,3-1,4-glucanase gene from Bacillus licheniformis EGW039 (CGMCC 0635) and its expression in Escherichia coli BL21 (DE3). Appl. Microbiol. Biotechnol., 72(4):705-712.

[40] Teng, D., Fan, Y., Yang, Y.L., Tian, Z.G., Luo, J., Wang, J.H., 2007. Codon optimization of Bacillus licheniformis β-1,3-1,4-glucanase gene and its expression in Pichia pastoris. Appl. Microbiol. Biotechnol., 74(5):1074-1083.

[41] Thompson, J.R., Register, E., Curotto, J., Kurtz, M., Kelly, R., 1998. An improved protocol for the preparation of yeast cells for transformation by electroporation. Yeast, 14(6):565-571.

[42] Tsai, L.C., Shyur, L.F., Cheng, Y.S., Lee, S.H., 2005. Crystal structure of truncated Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase in complex with beta-1,3-1,4-cellotriose. J. Mol. Biol., 354(3):642-651.

[43] Tuan, R.S., 1997. Recombinant Gene Expression Protocols. Humana Press, Philadelphia, USA, p.131-148.

[44] van Rensburg, P., van Zyl, W.H., Pretorius, I.S., 1997. Over-expression of the Saccharomyces cerevisiae exo-beta-1,3-glucanase gene together with the Bacillus subtilis endo-beta-1,3-1,4-glucanase gene and the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase gene in yeast. J. Biotechnol., 55(1):43-53.

[45] Vis, R.B., Lorenz, K., 1997. beta-Glucans: importance in brewing and methods of analysis. LWT Food. Sci. Technol., 30(4):331-336.

[46] Wood, P.J., Erfle, J.D., Teather, R.M., 1988. Use of complex formation between Congo red and polysaccharides in detection and assay of polysaccharide hydrolases. Methods Enzymol., 160:59-74.

[47] Zhang, Q., Chen, Q.H., Fu, M.L., Wang, J.L., Zhang, H.B., He, G.Q., 2008. Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination. J. Zhejiang Univ. Sci. B, 9(7):527-535.

[48] Zhang, W., Han, S., Wei, D., Lin, Y., Wang, X., 2008. Functional display of Rhizomucor miehei lipase on surface of Saccharomyces cerevisiae with higher activity and its practical properties. J. Chem. Technol. Biotechnol., 83(3):329-335.