title: Improved secretory production of glucoamylase in Pichia pastoris by combination of genetic manipulations.
abstract: Rhizopus oryzae glucoamylase (GA) has been genetically engineered with modified signal peptide (MSP), increased copy number of the gene, and coexpression of SEC4, a gene encoding a Rab protein associated with secretory vesicles, and its secretion level has been successfully raised up to 100-fold in Pichia pastoris. The MSP was designed to contain the signal peptide of mouse salivary alpha-amylase (S8L) fused to the pro-region of the signal peptide of Saccharomyces cerevisiae alpha-mating factor to replace the wild type signal peptide (WTSP) of GA. The P. pastoris transformant MSPGA-1 containing a single copy of MSPGA gene showed a 3.6-fold increase in GA secretion as compared to that of WTSPGA-1. Moreover, the P. pastoris transformant MSPGA-7 harboring seven copies of the MSPGA inserts was identified and showed 56-fold higher secreted GA than WTSPGA-1. In addition, we found that overexpression of SEC4 further doubled the secretion level of GA in each MSPGA/P. pastoris transformant. Taken together, the MSPGA-7-SEC4 clone showed as much as 100-fold secretion level of GA when compared to WTSPGA-1. In summary, we have demonstrated that combination of the aforementioned genetic manipulations resulted in high level secretion of R. oryzae GA in P. pastoris.
authors.affiliation: Department of Life Science, Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC.
authors.forename: Shi-Hwei
authors.initials: SH
authors.lastname: Liu
authors.forename: Wei-I
authors.initials: WI
authors.lastname: Chou
authors.forename: Chia-Chin
authors.initials: CC
authors.lastname: Sheu
authors.forename: Margaret Dah-Tsyr
authors.initials: MD
authors.lastname: Chang
mesh_terms: D003001:Cloning, Molecular / Q000379:methods; D018628:Gene Dosage; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis* / Q000235:genetics*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D021382:Protein Sorting Signals / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D005087:Glucan 1,4-alpha-Glucosidase; C064320:SEC4 protein, S cerevisiae; D020691:rab GTP-Binding Proteins