| Summary: | 博士 === 國立清華大學 === 生命科學系 === 94 === The methylotrophic yeast Pichia pastoris has been developed as a widely used host organism for high-level production of recombinant protein. However, we found that the secretion of Rhizopus oryzae glucoamylase (GA) encountered certain bottleneck in P. pastoris. In this study, R. oryzae GA was genetically engineered with a 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 it is to achieve a secretion level up to 100-fold in P. pastoris. Subsequently, to elucidate the physiological role of SEC4, a dominant-negative mutant of SEC4, SEC4S28N, 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 SEC4S28N 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 SEC4S28N gene. In addition, overexpression of SEC4 driven by AOX1 promoter in MSPGA-7 improved the secretory production of GA, but demonstrated a delay of cell growth by increased gene dosage of SEC4. Interestingly, a limited level of Sec4p did not disturb the cell growth. The expression of only one copy of SEC4 resulted in delay of cell growth at an early stage, while still maintained high level Sec4p after long-term incubation. Accordingly, as glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter was used to substitute AOX1 promoter to drive the SEC4 expression, enhanced GA secretion but no inhibition of cell growth were achieved. Taken together, our results demonstrate that SEC4 is substantial for regulating cell growth and heterologous protein secretion in P. pastoris.
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