Increased Expression of Recombinant Chitosanase by Co-expression of
Hac1p in the Yeast Pichia pastoris

Minghai       Han; Weixian       Wang; Xun       Gong; Jianli       Zhou; Cunbin       Xu; Yinfeng       Li

doi:10.2174/0929866528666211105111155

Abstract

Background: Pichia pastoris is one of the most popular eukaryotic hosts for producing heterologous proteins, while increasing the secretion of target proteins is still a top priority for their application in industrial fields. Recently, the research effort to enhance protein production has focused on up-regulating the unfolded protein response (UPR).

Objective: We evaluated the effects of activated UPR via Hac1p co-expression with the promoter AOX1 (PAOX1) or GAP (PGAP) on the expression of recombinant chitosanase (rCBS) in P. pastoris.

Method: The DNA sequence encoding the chitosanase was chemically synthesized and cloned into pPICZαA, and the resulting pPICZαA/rCBS was transformed into P. pastoris for expressing rCBS. The P. pastorisHAC1ⁱ cDNA was chemically synthesized and cloned into pPIC3.5K to give pPIC3.5K/Hac1p. The HAC1ⁱ cDNA was cloned into PGAPZB and then inserted with the HIS4 gene from pAO815 to construct the vector PGAPZB/Hac1p/HIS4. For co-expression of Hac1p, the two plasmids pPIC3.5K/Hac1p and PGAPZB/Hac1p/HIS4 were transformed into P. pastoris harboring the CBS gene. The rCBS was assessed based on chitosanase activity and analyzed by SDSPAGE. The enhanced Kar2p was detected with western blotting to evaluate UPR.

Results: Hac1p co-expression with PAOX₁ enhanced rCBS secretion by 41% at 28°C. Although the level of UPR resulting from Hac1p co-expression with PAOX₁ was equivalent to that with PGAP in terms of the quantity of Kar2p (a hallmark of the UPR), substitution of PGAP for PAOX₁ further increased rCBS production by 21%. The methanol-utilizing phenotype of P. pastoris did not affect rCBS secretion with or without co-expression of Hac1p. Finally, Hac1p co-expression withPAOX₁ or PGAP promoted rCBS secretion from 22 to 30°C and raised the optimum induction temperature.

Conclusion: The study indicated that Hac1p co-expression with PAOX₁ or PGAP is an effective strategy to trigger UPR of P. pastoris and a feasible means for improving the production of rCBS therein.

Keywords: Unfolded protein response, Hac1p, Pichia pastoris, chitosanase, secretory expression, co-expression.

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DOI https://dx.doi.org/10.2174/0929866528666211105111155	Print ISSN 0929-8665
Publisher Name Bentham Science Publisher	Online ISSN 1875-5305

Protein & Peptide Letters

Increased Expression of Recombinant Chitosanase by Co-expression of Hac1p in the Yeast Pichia pastoris

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