title: Molecular genetic manipulation of Pichia pastoris SEC4 governs cell growth and glucoamylase secretion.
abstract: We have previously engineered a recombinant Pichia pastoris GS115 transformant, MSPGA-7, harboring seven copies of glucoamylase (GA) fused with modified signal peptide. High yield secretion of GA was achieved as an extra copy of SEC4 was integrated to the transformant. To elucidate the physiological role of SEC4, a dominant-negative mutant of SEC4, SEC4(S28N), was overexpressed under the control of alchohol oxidase 1 (AOX1) promoter in P. pastoris strain MSPGA-7 as well as a set of host cells harboring multi-copy of wild type SEC4. We found that SEC4(S28N) mutation in the key guanine nucleotide binding domain reduced guanine nucleotide binding affinity, hence it blocked the transport of vesicles required for targeting and fusion to the plasma membrane. The inhibitory levels of cell growth and GA secretion were correlated with the dosage of SEC4(S28N) gene. In addition, overexpression of SEC4 driven by AOX1 promoter in MSPGA-7 improved the secretory production of GA, but demonstrated the delay of cell growth by increased gene dosage of SEC4. Interestingly, a limited level of Sec4p did not disturb the cell growth. It was because expression of only one copy of SEC4 resulted in delay of cell growth at an early stage while still maintaining high level Sec4p at long-term incubation. Accordingly, as glyceraldehyde-3-phosphate dehydrogenase promoter was used to substitute AOX1 promoter to drive the SEC4 expression, enhanced GA secretion but not inhibition of cell growth was achieved. Taken together, our results demonstrate that SEC4 is essential for P. pastoris in regulating cell growth and heterologous protein secretion in a dosage-dependent manner.
authors.affiliation: Institute of Molecular and Cellular Biology, Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan 30013, ROC.
authors.forename: Shi-Hwei
authors.initials: SH
authors.lastname: Liu
authors.forename: Wei-I
authors.initials: WI
authors.lastname: Chou
authors.forename: Shu-Chuan
authors.initials: SC
authors.lastname: Lin
authors.forename: Chia-Chin
authors.initials: CC
authors.lastname: Sheu
authors.forename: Margaret Dah-Tsyr
authors.initials: MD
authors.lastname: Chang
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology*; D018628:Gene Dosage; D005087:Glucan 1,4-alpha-Glucosidase / Q000378:metabolism*; D009154:Mutation; D010843:Pichia / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D011401:Promoter Regions, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D020691:rab GTP-Binding Proteins / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D005087:Glucan 1,4-alpha-Glucosidase; C064320:SEC4 protein, S cerevisiae; D020691:rab GTP-Binding Proteins