Document Detail

Molecular genetic manipulation of Pichia pastoris SEC4 governs cell growth and glucoamylase secretion.
MedLine Citation:
PMID:  16176807     Owner:  NLM     Status:  MEDLINE    
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.
Shi-Hwei Liu; Wei-I Chou; Shu-Chuan Lin; Chia-Chin Sheu; Margaret Dah-Tsyr Chang
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biochemical and biophysical research communications     Volume:  336     ISSN:  0006-291X     ISO Abbreviation:  Biochem. Biophys. Res. Commun.     Publication Date:  2005 Nov 
Date Detail:
Created Date:  2005-10-03     Completed Date:  2005-12-12     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  0372516     Medline TA:  Biochem Biophys Res Commun     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1172-80     Citation Subset:  IM    
Institute of Molecular and Cellular Biology, Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan 30013, ROC.
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MeSH Terms
Alcohol Oxidoreductases / genetics,  metabolism*
Cell Proliferation
Cloning, Molecular
Fungal Proteins / genetics,  physiology*
Gene Dosage
Glucan 1,4-alpha-Glucosidase / metabolism*
Pichia / cytology,  enzymology*,  genetics
Promoter Regions, Genetic
Saccharomyces cerevisiae Proteins / genetics
rab GTP-Binding Proteins / genetics
Reg. No./Substance:
0/Fungal Proteins; 0/Saccharomyces cerevisiae Proteins; EC 1.1.-/Alcohol Oxidoreductases; EC oxidase; EC 1,4-alpha-Glucosidase; EC 3.6.1.-/rab GTP-Binding Proteins; EC 3.6.1.-./SEC4 protein, S cerevisiae

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