Secretion is a common bottleneck in the production of industrial proteins. Although overexpression of the unfolded protein response regulator Hac1p has been widely used to enhance protein secretion, its effects on the physiology of host cells were often overlooked, which would attenuate and even neutralize its beneficial effects on overall protein production. In order to achieve high-level glucose oxidase (GOX) production in Pichia pastoris, we used a set of Hac1p homologues from Saccharomyces cerevisiae (ScHac1p), P. pastoris (PpHac1p), Trichoderma reesei (TrHac1p) and Homo sapiens (HsXbp1), to evaluate the effects of Hac1p overexpression on the secretion capacity, cell physiology and overall enzyme production in P. pastoris strains containing different copies of the GOX gene. Results showed that overexpression of Hac1ps was able to remarkably alleviate the secretion bottleneck in the 3-copy strain, to improve its GOX secretion capacity by 21.2-140.2 % and its overall enzyme production by 23.7-69.2 %. However, overexpression of ScHac1p, PpHac1p and TrHac1p led to reduced cell growth in GS-3GOX, possibly due to increased oxidative stress. Overexpression of ScHac1p and PpHac1p in the 6-copy strain (resulting in GS-6GOX-Sc and GS-6GOX-Pp, respectively) further increased the overall GOX production levels by 88.9-103.3 %, and GS-6GOX-Pp exhibited the highest overall GOX production and GOX secretion capacity among all constructed strains. Nevertheless, in addition of significantly reduced growth, loss of GOX gene was also observed for GS-6GOX-Pp and GS-6GOX-Sc during the fermentation process. With higher induction cell density and co-feeding of yeast extract, the GOX titer of GS-6GOX-Pp reached 2125.3 U/mL on 1-liter fermentor. This study not only achieves a record high GOX production level but also provides new insights into secretion pathway engineering using Hac1p overexpression strategy.

• PMID: 33051008
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Journal Article

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Yu S, Miao L, Huang H, Li Y, Zhu T

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