Systematic screening of glycosylation- and trafficking-associated gene knockouts in Saccharomyces cerevisiae identifies mutants with improved heterologous exocellulase activity and host secretion

BMC Biotechnology

Tzi-Yuan Wang¹  Chih-Jen Huang⁴  Hsin-Liang Chen¹  Po-Chun Ho¹  Huei-Mien Ke⁵  Hsing-Yi Cho²  Sz-Kai Ruan¹  Kuo-Yen Hung¹  I-Li Wang¹  Ya-Wun Cai¹  Huang-Mo Sung⁶  Wen-Hsiung Li³  Ming-Che Shih² 

[1] Biodiversity Research Center, Academia Sinica, Taipei 115, Taiwan

[2] Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan

[3] Department of Ecology and Evolution, University of Chicago, Chicago IL 60637, USA

[4] Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung 402, Taiwan

[5] Program in Microbial Genomics, National Chung Hsing University – Academia Sinica, Taipei 115, Taiwan

[6] Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan

关键词: Protein secretion;    Glycosylation;    Cellulase production;   

Others  :  1089654
DOI  :  10.1186/1472-6750-13-71

 received in 2012-12-27, accepted in 2013-08-29,  发布年份 2013

【 摘 要 】

Background

As a strong fermentator, Saccharomyces cerevisiae has the potential to be an excellent host for ethanol production by consolidated bioprocessing. For this purpose, it is necessary to transform cellulose genes into the yeast genome because it contains no cellulose genes. However, heterologous protein expression in S. cerevisiae often suffers from hyper-glycosylation and/or poor secretion. Thus, there is a need to genetically engineer the yeast to reduce its glycosylation strength and to increase its secretion ability.

Results

Saccharomyces cerevisiae gene-knockout strains were screened for improved extracellular activity of a recombinant exocellulase (PCX) from the cellulose digesting fungus Phanerochaete chrysosporium. Knockout mutants of 47 glycosylation-related genes and 10 protein-trafficking-related genes were transformed with a PCX expression construct and screened for extracellular cellulase activity. Twelve of the screened mutants were found to have a more than 2-fold increase in extracellular PCX activity in comparison with the wild type. The extracellular PCX activities in the glycosylation-related mnn10 and pmt5 null mutants were, respectively, 6 and 4 times higher than that of the wild type; and the extracellular PCX activities in 9 protein-trafficking-related mutants, especially in the chc1, clc1 and vps21 null mutants, were at least 1.5 times higher than the parental strains. Site-directed mutagenesis studies further revealed that the degree of N-glycosylation also plays an important role in heterologous cellulase activity in S. cerevisiae.

Conclusions

Systematic screening of knockout mutants of glycosylation- and protein trafficking-associated genes in S. cerevisiae revealed that: (1) blocking Golgi-to-endosome transport may force S. cerevisiae to export cellulases; and (2) both over- and under-glycosylation may alter the enzyme activity of cellulases. This systematic gene-knockout screening approach may serve as a convenient means for increasing the extracellular activities of recombinant proteins expressed in S. cerevisiae.

【 授权许可】

   
2013 Wang et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Torney F, Moeller L, Scarpa A, Wang K: Genetic engineering approaches to improve bioethanol production from maize. Current opinion in biotechnology 2007, 18(3):193-199.
• [2]Galbe M, Sassner P, Wingren A, Zacchi G: Process engineering economics of bioethanol production. Advances in biochemical engineering/biotechnology 2007, 108:303-327.
• [3]Gray KA, Zhao L, Emptage M: Bioethanol. Current opinion in chemical biology 2006, 10(2):141-146.
• [4]van Zyl WH, Lynd LR, den Haan R, McBride JE: Consolidated bioprocessing for bioethanol production using Saccharomyces cerevisiae. Advances in biochemical engineering/biotechnology 2007, 108:205-235.
• [5]Ferrarese L, Trainotti L, Gattolin S, Casadoro G: Secretion, purification and activity of two recombinant pepper endo-beta-1,4-glucanases expressed in the yeast Pichia pastoris. FEBS Lett 1998, 422(1):23-26.
• [6]Zhao XH, Wang W, Wang FQ, Wei DZ: A comparative study of beta-1, 4-endoglucanase (possessing beta-1, 4-exoglucanase activity) from Bacillus subtilis LH expressed in Pichia pastoris GS115 and Escherichia coli Rosetta (DE3). Bioresour Technol 2012, 110:539-545.
• [7]Xu Z, Escamilla-Trevino L, Zeng L, Lalgondar M, Bevan D, Winkel B, Mohamed A, Cheng CL, Shih MC, Poulton J, et al.: Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 1. Plant Mol Biol 2004, 55(3):343-367.
• [8]Wonganu B, Pootanakit K, Boonyapakron K, Champreda V, Tanapongpipat S, Eurwilaichitr L: Cloning, expression and characterization of a thermotolerant endoglucanase from Syncephalastrum racemosum (BCC18080) in Pichia pastoris. Protein Expr Purif 2008, 58(1):78-86.
• [9]Thongekkaew J, Ikeda H, Masaki K, Iefuji H: An acidic and thermostable carboxymethyl cellulase from the yeast Cryptococcus sp. S-2: purification, characterization and improvement of its recombinant enzyme production by high cell-density fermentation of Pichia pastoris. Protein Expr Purif 2008, 60(2):140-146.
• [10]Shi H, Yin X, Wu M, Tang C, Zhang H, Li J: Cloning and bioinformatics analysis of an endoglucanase gene (Aucel12A) from Aspergillus usamii and its functional expression in Pichia pastoris. J Ind Microbiol Biotechnol 2012, 39(2):347-357.
• [11]Samanta S, Basu A, Halder UC, Sen SK: Characterization of Trichoderma reesei endoglucanase II expressed heterologously in Pichia pastoris for better biofinishing and biostoning. J Microbiol 2012, 50(3):518-525.
• [12]Molhoj M, Ulvskov P, Dal Degan F: Characterization of a functional soluble form of a Brassica napus membrane-anchored endo-1,4-beta-glucanase heterologously expressed in Pichia pastoris. Plant Physiol 2001, 127(2):674-684.
• [13]Lindenmuth BE, McDonald KA: Production and characterization of Acidothermus cellulolyticus endoglucanase in Pichia pastoris. Protein Expr Purif 2011, 77(2):153-158.
• [14]Li J, Tang C, Shi H, Wu M: Cloning and optimized expression of a neutral endoglucanase gene (ncel5A) from Volvariella volvacea WX32 in Pichia pastoris. J Biosci Bioeng 2011, 111(5):537-540.
• [15]Godbole S, Decker SR, Nieves RA, Adney WS, Vinzant TB, Baker JO, Thomas SR, Himmel ME: Cloning and expression of Trichoderma reesei cellobiohydrolase I in Pichia pastoris. Biotechnol Prog 1999, 15(5):828-833.
• [16]Generoso WC, Malago-Jr W, Pereira N Jr, Henrique-Silva F: Recombinant expression and characterization of an endoglucanase III (cel12a) from Trichoderma harzianum (Hypocreaceae) in the yeast Pichia pastoris. Genet Mol Res 2012, 11(2):1544-1557.
• [17]Escamilla-Trevino LL, Chen W, Card ML, Shih MC, Cheng CL, Poulton JE: Arabidopsis thaliana beta-Glucosidases BGLU45 and BGLU46 hydrolyse monolignol glucosides. Phytochemistry 2006, 67(15):1651-1660.
• [18]Ahn YO, Zheng M, Bevan DR, Esen A, Shiu SH, Benson J, Peng HP, Miller JT, Cheng CL, Poulton JE, et al.: Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 35. Phytochemistry 2007, 68(11):1510-1520.
• [19]Baldrian P, Valaskova V: Degradation of cellulose by basidiomycetous fungi. FEMS microbiology reviews 2008, 32(3):501-521.
• [20]Watanabe H, Tokuda G: Cellulolytic systems in insects. Annu Rev Entomol 2010, 55:609-632.
• [21]Wang TY, Chen HL, Lu MY, Chen YC, Sung HM, Mao CT, Cho HY, Ke HM, Hwa TY, Ruan SK, et al.: Functional characterization of cellulases identified from the cow rumen fungus Neocallimastix patriciarum W5 by transcriptomic and secretomic analyses. Biotechnology for biofuels 2011, 4(1):24. BioMed Central Full Text
• [22]Pham TH, Quyen DT, Nghiem NM, Vu TD: Cloning, expression, purification, and properties of an endoglucanase gene (glycosyl hydrolase family 12) from Aspergillus niger VTCC-F021 in Pichia pastoris. J Microbiol Biotechnol 2011, 21(10):1012-1020.
• [23]Gary JS: Trichoderma: a review of biology and systematics of the genus. Mycol Res 1996, 100(8):923-935.
• [24]Teeri TT, Koivula A, Linder M, Wohlfahrt G, Divne C, Jones TA: Trichoderma reesei cellobiohydrolases: why so efficient on crystalline cellulose? Biochem Soc Trans 1998, 26(2):173-178.
• [25]Montenecourt BS: Trichoderma reesei cellulases. Trends Biotechnol 1983, 1(5):156-161.
• [26]Sinnott ML: The cellobiohydrolases of Trichoderma reesei: a review of indirect and direct evidence that their function is not just glycosidic bond hydrolysis. Biochem Soc Trans 1998, 26(2):160-164.
• [27]Muller S, Sandal T, Kamp-Hansen P, Dalboge H: Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis. Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica. Yeast 1998, 14(14):1267-1283.
• [28]Aho S, Arffman A, Korhola M: Saccharomyces cerevisiae mutants selected for increased production of Trichoderma reesei cellulases. Appl Microbiol Biotechnol 1996, 46(1):36-45.
• [29]Qin Y, Wei X, Liu X, Wang T, Qu Y: Purification and characterization of recombinant endoglucanase of Trichoderma reesei expressed in Saccharomyces cerevisiae with higher glycosylation and stability. Protein Expr Purif 2008, 58(1):162-167.
• [30]Wildt S, Gerngross TU: The humanization of N-glycosylation pathways in yeast. Nat Rev Microbiol 2005, 3(2):119-128.
• [31]Jigami Y: Yeast glycobiology and its application. Biosci Biotechnol Biochem 2008, 72(3):637-648.
• [32]Jeoh T, Michener W, Himmel ME, Decker SR, Adney WS: Implications of cellobiohydrolase glycosylation for use in biomass conversion. Biotechnology for biofuels 2008, 1(1):10. BioMed Central Full Text
• [33]Kruszewska JS, Butterweck AH, Kurzatkowski W, Migdalski A, Kubicek CP, Palamarczyk G: Overexpression of the Saccharomyces cerevisiae mannosylphosphodolichol synthase-encoding gene in Trichoderma reesei results in an increased level of protein secretion and abnormal cell ultrastructure. Appl Environ Microbiol 1999, 65(6):2382-2387.
• [34]Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS: Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 2002, 66(3):506-577. table of contents
• [35]Van Rensburg P, Van Zyl WH, Pretorius IS: Engineering yeast for efficient cellulose degradation. Yeast 1998, 14(1):67-76.
• [36]Liu YS, Baker JO, Zeng Y, Himmel ME, Haas T, Ding SY: Cellobiohydrolase hydrolyzes crystalline cellulose on hydrophobic faces. J Biol Chem 2011, 286(13):11195-11201.
• [37]Khan MH, Ali S, Fakhru’l-Razi A, Alam Z: Use of fungi for the bioconversion of rice straw into cellulase enzyme. Journal of environmental science and health Part 2007, 42(4):381-386.
• [38]Broda P, Birch PR, Brooks PR, Sims PF: Lignocellulose degradation by Phanerochaete chrysosporium: gene families and gene expression for a complex process. Mol Microbiol 1996, 19(5):923-932.
• [39]Vanden Wymelenberg A, Minges P, Sabat G, Martinez D, Aerts A, Salamov A, Grigoriev I, Shapiro H, Putnam N, Belinky P, et al.: Computational analysis of the Phanerochaete chrysosporium v2.0 genome database and mass spectrometry identification of peptides in ligninolytic cultures reveal complex mixtures of secreted proteins. Fungal Genet Biol 2006, 43(5):343-356.
• [40]Vanden Wymelenberg A, Gaskell J, Mozuch M, Sabat G, Ralph J, Skyba O, Mansfield SD, Blanchette RA, Martinez D, Grigoriev I, et al.: Comparative transcriptome and secretome analysis of wood decay fungi Postia placenta and Phanerochaete chrysosporium. Appl Environ Microbiol 2010, 76(11):3599-3610.
• [41]Sato S, Feltus FA, Iyer P, Tien M: The first genome-level transcriptome of the wood-degrading fungus Phanerochaete chrysosporium grown on red oak. Curr Genet 2009, 55(3):273-286.
• [42]Vallim MA, Janse BJ, Gaskell J, Pizzirani-Kleiner AA, Cullen D: Phanerochaete chrysosporium cellobiohydrolase and cellobiose dehydrogenase transcripts in wood. Appl Environ Microbiol 1998, 64(5):1924-1928.
• [43]Henriksson G, Nutt A, Henriksson H, Pettersson B, Stahlberg J, Johansson G, Pettersson G: Endoglucanase 28 (Cel12A), a new Phanerochaete chrysosporium cellulase. Eur J Biochem 1999, 259(1–2):88-95.
• [44]Huy ND, Kim SW, Park SM: Heterologous expression of endo-1,4-beta-xylanaseC from Phanerochaete chrysosporium in Pichia pastoris. J Biosci Bioeng 2011, 111(6):654-657.
• [45]Munoz IG, Ubhayasekera W, Henriksson H, Szabo I, Pettersson G, Johansson G, Mowbray SL, Stahlberg J: Family 7 cellobiohydrolases from Phanerochaete chrysosporium: crystal structure of the catalytic module of Cel7D (CBH58) at 1.32 A resolution and homology models of the isozymes. Journal of molecular biology 2001, 314(5):1097-1111.
• [46]Wymelenberg AV, Denman S, Dietrich D, Bassett J, Yu X, Atalla R, Predki P, Rudsander U, Teeri TT, Cullen D: Transcript analysis of genes encoding a family 61 endoglucanase and a putative membrane-anchored family 9 glycosyl hydrolase from Phanerochaete chrysosporium. Appl Environ Microbiol 2002, 68(11):5765-5768.
• [47]Wang TY, Chen HL, Li WH, Sung HM, Shih MC: Omics applications to biofuel research. In Biocatalysis and Biomolecular Engineering. Edited by Hou CT, Shaw JF. New York: John Wiley & Sons, Inc; 2010:265-276.
• [48]Suzuki H, Imaeda T, Kitagawa T, Kohda K: Deglycosylation of cellulosomal enzyme enhances cellulosome assembly in Saccharomyces cerevisiae. J Biotechnol 2012, 157(1):64-70.
• [49]Hou J, Tyo KEJ, Liu ZH, Petranovic D, Nielsen J: Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae. FEMS Yeast Res 2012, 12(5):491-510.
• [50]Idiris A, Tohda H, Kumagai H, Takegawa K: Engineering of protein secretion in yeast: strategies and impact on protein production. Appl Microbiol Biotechnol 2010, 86(2):403-417.
• [51]Mattanovich D, Branduardi P, Dato L, Gasser B, Sauer M, Porro D: Recombinant protein production in yeasts. Methods Mol Biol 2012, 824:329-358.
• [52]Kitagawa T, Kohda K, Tokuhiro K, Hoshida H, Akada R, Takahashi H, Lmaeda T: Identification of genes that enhance cellulase protein production in yeast. J Biotechnol 2011, 151(2):194-203.
• [53]Bartkeviciute D, Sasnauskas K: Disruption of the MNN10 gene enhances protein secretion in Kluyveromyces lactis and Saccharomyces cerevisiae. FEMS Yeast Res 2004, 4(8):833-840.
• [54]Nunes Bastos R: Functional dissection of alternative secretory pathways in the yeast S. cerevisiae. University of Helsinki: Dissertationes Biocentri Viikki Universitatis Helsingiensis; 2008.
• [55]Chen HL, Chen YC, Lu MYJ, Chang JJ, Wang HTC, Ke HM, Wang TY, Ruan SK, Wang TY, Hung KY, et al.: A highly efficient ß-glucosidase from the buffalo rumen fungus Neocallimastix patriciarum W5. Biotechnol Biofuels 2012, 5(1):24. BioMed Central Full Text
• [56]Helenius A, Aebi M: Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 2004, 73:1019-1049.
• [57]Lehle L, Strahl S, Tanner W: Protein glycosylation, conserved from yeast to man: a model organism helps elucidate congenital human diseases. Angew Chem Int Ed Engl 2006, 45(41):6802-6818.
• [58]Toikkanen J: Functional studies on components of the secretory pathway of Saccharomyces cerevisiae. Helsinki, Finland: University of Helsinki; 1999. [Ph.D. thesis]
• [59]Huffaker TC, Robbins PW: Temperature-sensitive yeast mutants deficient in asparagine-linked glycosylation. J Biol Chem 1982, 257(6):3203-3210.
• [60]Herscovics A, Orlean P: Glycoprotein biosynthesis in yeast. FASEB J 1993, 7(6):540-550.
• [61]Teixeira MC, Raposo LR, Mira NP, Lourenco AB, Sa-Correia I: Genome-wide identification of Saccharomyces cerevisiae genes required for maximal tolerance to ethanol. Appl Environ Microbiol 2009, 75(18):5761-5772.
• [62]Proszynski TJ, Simons K, Bagnat M: O-glycosylation as a sorting determinant for cell surface delivery in yeast. Mol Biol Cell 2004, 15(4):1533-1543.
• [63]Gentzsch M, Tanner W: The PMT gene family: protein O-glycosylation in Saccharomyces cerevisiae is vital. EMBO J 1996, 15(21):5752-5759.
• [64]Kruszewska JS, Perlinska-Lenart U, Gorka-Niec W, Orlowski J, Zembek P, Palamarczyk G: Alterations in protein secretion caused by metabolic engineering of glycosylation pathways in fungi. Acta Biochim Pol 2008, 55(3):447-456.
• [65]Haylock R, Broda P: Preparation and characterization of mRNA from ligninolytic fungi. Methods Enzymol 1988, 161:221-228.
• [66]Kohrer K, Domdey H: Preparation of high molecular weight RNA. Methods Enzymol 1991, 194:398-405.
• [67]Shih YP, Kung WM, Chen JC, Yeh CH, Wang AH, Wang TF: High-throughput screening of soluble recombinant proteins. Protein Sci 2002, 11(7):1714-1719.
• [68]Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P, Boeke JD: Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 1998, 14(2):115-132.
• [69]Herskowitz I, Jensen RE: Putting the HO gene to work: practical uses for mating-type switching. Methods Enzymol 1991, 194:132-146.
• [70]Gietz RD, Woods RA: Yeast transformation by the LiAc/SS Carrier DNA/PEG method. Methods Mol Biol 2006, 313:107-120.
• [71]Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A: ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 2003, 31(13):3784-3788.
• [72]Arnold K, Bordoli L, Kopp J, Schwede T: The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 2006, 22(2):195-201.
• [73]Ubhayasekera W, Munoz IG, Vasella A, Stahlberg J, Mowbray SL: Structures of Phanerochaete chrysosporium Cel7D in complex with product and inhibitors. FEBS J 2005, 272(8):1952-1964.