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All Journal HAYATI Journal of Biosciences
Satya, Andreas Adhi
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Agriculture, Biological Sciences & Forestry Earth & Planetary Sciences

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Effect of Multiple Gene Copy Number of Bacterial Lipase to Increase Lipase Production in Pichia pastoris Puspitasari, Esti; Rustam, Yepy Hardi; Satya, Andreas Adhi; Suwanto, Antonius; Wahyudi, Aris Tri; Astuti, Rika Indri
HAYATI Journal of Biosciences Vol. 32 No. 2 (2025): March 2025
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.32.2.310-319

Abstract

Bacterial lipase poses potential challenges when expressed in eukaryotic protein expression systems such as Pichia pastoris. This research aims to increase extracellular T1.2RQ lipase secretion (free lipase) with multiple gene copy number strategies in Pichia pastoris and it was first performed on lipase from Geobacillus stearothermophilus T1.2. In this study, the T1.2RQ lipase gene from Geobacillus stearothermophilus T1.2 was expressed in Pichia pastoris GS115 through a strategy involving multiple copies of lipase, resulting in increased lipase activity. Three copies of the lipase gene in pPIC9K_T1.2RQ(3x) recombinant plasmid were integrated into the genome of Pichia pastoris GS115, and quantitative analysis using qPCR technique confirmed that the GS115 transformant strain contained six copies of T1.2RQ gene, indicating two integration events. Lipase activity measurement showed that the GS115/T1.2RQ(6x) strain exhibited a 111% increase compared to that containing a copy of the T1.2RQ gene. SDS-PAGE and Zymogram results showed a protein band with a size of 43kDa. Qualitative analysis in LA+TBN media of all strains containing the T1.2RQ gene showed clear zones. Lipase production in flask fermentation took at least 120 hours to produce the best lipase activity. Thus, strategies with multiple copy numbers of gene lipase have significantly increased the expression of the bacterial lipase gene in Pichia pastoris GS115.
Enhanced Lipase Production in Pichia pastoris via Multiple Copies of Bacterial Lipase Genes and Co-expression of the HAC1 Gene Puspitasari, Esti; Rustam, Yepy Hardi; Satya, Andreas Adhi; Suwanto, Antonius; Wahyudi, Aris Tri; Astuti, Rika Indri
HAYATI Journal of Biosciences Vol. 33 No. 2 (2026): March 2026
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.33.2.288-296

Abstract

The hac1 gene, a key regulator of the untranslated protein response (UPR), was co-expressed in Pichia pastoris GS115 to enhance the production of a lipase from Geobacillus stearothermophilus. Multicopy lipase constructs (1X and 4X) were transformed with pPICZAwbe_hac1, generating GS115/T1.2RQ(1X)_hac1 and GS115/T1.2RQ(4X)_hac1 strains. The GS115/T1.2RQ(1X)_hac1 strain showed an 186% lipase activity after 120 hours versus the control (100%), while the GS115/T1.2RQ(4X)_hac1 strain showed a faster initial increase (38% at 48 hours) and 28% at 120 hours, which was beneficial for efficient enzyme production. Overexpression of the hac1 gene enhances lipase production because it activates UPR genes when the endoplasmic reticulum is stressed due to a large number of recombinant proteins and forms proteins that are not appropriately folded. SDS-PAGE and tributyrin plate assays confirmed extracellular lipase expression (~43 kDa). These results demonstrate that hac1 co-expression significantly (p = 0.01)  enhances lipase production in Pichia pastoris, especially in lower-copy constructs. This is the first report of co-expressing hac1 with Geobacillus stearothermophilus lipase genes in yeast. The findings are expected to contribute to developing more efficient microbial cell factories for producing industrial enzymes.
Co-Authors Antonius Suwanto Aris Tri Wahyudi Puspitasari, Esti Rika Indri Astuti Rustam, Yepy Hardi