Decrease of hirudin degradation by deleting the KEX1 gene in recombinant Pichia pastoris
Research article published in Yeast (Chichester, England) (2008)
Abstract
Our previous study on recombinant hirudin production in Pichia pastoris demonstrated that, although the total productivity of hirudin was fairly high, its degradation was still severe, even if many engineering methods were applied to improve cell viability and reduce the release of intracellular proteinases. In this work, a pop-in/pop-out method, replacing the auxotrophic marker ARG4 gene with the resistant marker sh ble gene, was used to delete the KEX1 gene to reduce hirudin degradation in P. pastoris GS115Hir. Using this strategy, hirudin degradation was greatly decreased. At the same wet cell weight and cell viability, the percentage of intact hirudin Hir65 in total hirudin in strain GS115HirDeltakex1 was always kept as high as 90% in the initial stage of the methanol fermentation phase and above 62% even in the later stage of the methanol fermentation phase, whereas the percentage for the undeleted strain GS115Hir was only about 40% in the whole methanol fermentation phase. As a result, the intact hirudin Hir65 concentration could maximally reach 2.4 g/l in GS115HirDeltakex1 while it was only 1.1 g/l in GS115Hir.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Zusammenfassung
Our previous study on recombinant hirudin production in Pichia pastoris demonstrated that, although the total productivity of hirudin was fairly high, its degradation was still severe, even if many engineering methods were applied to improve cell viability and reduce the release of intracellular proteinases.
Warum dies für die Hirudotherapie relevant ist
This study examined recombinant hirudin production in Pichia pastoris, specifically testing whether deletion of the KEX1 gene could reduce degradation of intact hirudin (Hir65) during methanol fermentation. The KEX1-deleted strain maintained intact hirudin at approximately 90% initially and above 62% in later fermentation stages, versus only ~40% in the undeleted strain, with intact hirudin concentration reaching 2.4 g/L compared to 1.1 g/L. This is highly relevant to ASH's domain because it addresses a practical biotechnological bottleneck in manufacturing a key leech-derived anticoagulant. However, this is an industrial microbiology and fermentation optimization study with no clinical or animal data; its relevance is confined to recombinant production rather than therapeutic application of hirudotherapy.
Zitation
Decrease of hirudin degradation by deleting the KEX1 gene in recombinant Pichia pastoris
Ni Z et al. · Yeast (Chichester, England), 2008
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