Combination of Genetic Manipulation Improved Saccharomycopsis fibuligera α-Amylase Secretion by Pichia pastoris

https://doi.org/10.22146/ijc.33140

Shabarni Gaffar(1*), Dessy Natalia(2), Toto Subroto(3), Oo Suprijana(4), Soetijoso Soemitro(5)

(1) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Jl. Raya Bandung-Sumedang Km 21 Jatinangor, Sumedang, 45363 Jawa Barat, Indonesia
(2) Department of Chemistry, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung, 40132, Jawa Barat, Indonesia
(3) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Jl. Raya Bandung-Sumedang Km 21 Jatinangor, Sumedang, 45363 Jawa Barat, Indonesia
(4) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Jl. Raya Bandung-Sumedang Km 21 Jatinangor, Sumedang, 45363 Jawa Barat, Indonesia
(5) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Jl. Raya Bandung-Sumedang Km 21 Jatinangor, Sumedang, 45363 Jawa Barat, Indonesia
(*) Corresponding Author

Abstract


This study assessed the combinations of genetic manipulation; signal peptide modification, gene dosage increment and co-expression of folding component, to increase Saccharomycopsis fibuligera R64 α-amylase (Sfamy) secretion in Pichia pastoris. Sfamy native signal peptide was replaced with modified signal peptide which contained 15 amino acid of mouse salivary α-amylase signal peptide fused to the pro-region of the signal peptide of Saccharomyces cerevisiae α-mating factor (α-MF). Increase in gene dosage was identified by screening for P. pastoris harboring multicopies of the Sfamy gene. Whereas, co-expression of folding component was done by addition of Protein Disulfide Isomerase (PDI). Expression plasmids harboring Sfamy containing modified signal sequence (pPICZA-MS-Sfamy) was used to transform P. pastoris GS115, and gene dosage increment was screened using zeocin. Effect of PDI co-expression on secretion levels of Sfamy was assessed by constructing the pPIC3.5K-Pdi1 plasmid and introducing into P. pastoris harboring multicopies of MS-Sfamy for expression of Sfamy. Signal peptide modification consequently increased Sfamy secretion by P. pastoris by 3.3-fold compared to native signal peptide. Gene dosage increment had improved Sfamy secretion by 11-fold in P. pastoris [MS-Sfamy] resistant to 2000 μg/mL zeocin, compared to P. pastoris harboring one copy of WT-Sfamy. Hence, PDI co-expression increased the secretion of Sfamy by 2-fold as compared without PDI co-expression. In summary, the combination of genetic manipulation successfully increased Sfamy secretion by 20-fold compared to P. pastoris harboring one copy of WT-Sfamy.

Keywords


Sfamy; signal peptide; gene dosage; folding; PDI; Pichia pastoris

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DOI: https://doi.org/10.22146/ijc.33140

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