title: Systematic screening of glycosylation- and trafficking-associated gene knockouts in Saccharomyces cerevisiae identifies mutants with improved heterologous exocellulase activity and host secretion.
abstract: 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.
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei 115, Taiwan. whli@sinica.edu.tw.
authors.forename: Tzi-Yuan
authors.initials: TY
authors.lastname: Wang
authors.forename: Chih-Jen
authors.initials: CJ
authors.lastname: Huang
authors.forename: Hsin-Liang
authors.initials: HL
authors.lastname: Chen
authors.forename: Po-Chun
authors.initials: PC
authors.lastname: Ho
authors.forename: Huei-Mien
authors.initials: HM
authors.lastname: Ke
authors.forename: Hsing-Yi
authors.initials: HY
authors.lastname: Cho
authors.forename: Sz-Kai
authors.initials: SK
authors.lastname: Ruan
authors.forename: Kuo-Yen
authors.initials: KY
authors.lastname: Hung
authors.forename: I-Li
authors.initials: IL
authors.lastname: Wang
authors.forename: Ya-Wun
authors.initials: YW
authors.lastname: Cai
authors.forename: Huang-Mo
authors.initials: HM
authors.lastname: Sung
authors.forename: Wen-Hsiung
authors.initials: WH
authors.lastname: Li
authors.forename: Ming-Che
authors.initials: MC
authors.lastname: Shih
mesh_terms: D044602:Cellulases / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D006031:Glycosylation; D016297:Mutagenesis, Site-Directed; D020075:Phanerochaete / Q000201:enzymology; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D000431:Ethanol; D002482:Cellulose; D044602:Cellulases