A constitutive expression system for Pichia pastoris based on the PGK1 promoter

Arruda, Andrelisse; Reis, Viviane Castelo Branco; Batista, Vinícius Daniel Ferreira; Daher, Bruno S.; Piva, Luiza Cesca; Marco, Janice Lisboa De; Moraes, Lídia Maria Pepe de; Torres, Fernando Araripe Gonçalves

doi:10.1007/s10529-015-2002-2

Cited by 12 publications

(6 citation statements)

References 18 publications

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“…Promoter is a key factor for heterologous lipase production in P. pastoris (Vogl and Glieder 2013;Arruda et al 2016). Until now, P AOX1 is the most commonly used promoter for the efficient expression of heterologous lipase.…”

Section: Discussionmentioning

confidence: 99%

High-level expression of Thermomyces dupontii thermo-alkaline lipase in Pichia pastoris under the control of different promoters

Wang

Zhang

Li³

et al. 2019

3 Biotech

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In this study, 15 methanol-inducible and 9 constitutive promoters were used to drive the expression of Thermomyces dupontii lipase (TDL) in Pichia pastoris. Of the 15 methanol-inducible promoters, formaldehyde dehydrogenase promoter (P FLD1 ) showed the highest efficiency in driving lipase production, followed by alcohol oxidase 1 (P AOX1 ) and dihydroxyacetone synthase (P DAS1 ) promoters. The maximum lipase activity of transformants with P FLD1 , P AOX1 and P DAS1 promoters in 5-l bioreactor was 27,076, 24,159 and 22,342 U/ml, respectively. For the nine constitutive promoters, glycosyl phosphatidyl inositol-anchored protein promoter (P GCW14 ) produced the highest amount of lipases in a medium containing glucose or glycerol as the only carbon source, followed by mitochondrial alcohol dehydrogenase isozyme (P 0472 ) and glyceraldehyde-3-phosphate dehydrogenase (P GAP ) promoters. The maximum lipase yields in 5-l bioreactors under the control of P GCW14 , P 0472 and P GAP promoters were 17,353, 15,046 and 14,276 U/ml, respectively. The result of this study not only identifies a few highly efficient promoters for the heterologous expression of TDL in P. pastoris, but also casts some insight into the optimization of protein production in heterologous systems.

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Section: Discussionmentioning

confidence: 99%

High-level expression of Thermomyces dupontii thermo-alkaline lipase in Pichia pastoris under the control of different promoters

Wang

Zhang

Li³

et al. 2019

3 Biotech

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“…The amplified 1.4 kb fragment included the LEU2 coding region with its transcription termination region and only 29 bp of its promoter [17]. The leu2 - d amplicon was cloned into pBlueScript SK II (Agilent Technologies) and then liberated after BglII digestion for subcloning into BamHI-linearized pPICK2 [50] resulting in pK-ld vector. This vector was digested with SacI and NotI to remove the α-factor secretory sequence.…”

Section: Methodsmentioning

confidence: 99%

Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker

et al. 2017

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BackgroundA commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug-resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele.ResultsA K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth.ConclusionsIn this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0715-8) contains supplementary material, which is available to authorized users.

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“…This novel integrative vector (pPICK2) has the potential to facilitate protein production and provides an alternative platform for recombinant protein expression and metabolic engineering. 80 A study introduced double plasmid co-expression strategy, by inserting two expression vectors at different loci to improve xylanase production. From A. niger BE-2, a GH10 xylanase gene (XynC) was optimized and constitutively expressed in P. pastoris.…”

Section: Gene Dosagementioning

confidence: 99%

Recent developments in heterologous production of cellulases and xylanases using the Pichia pastoris expression system

Ullah,

Madadi,

Asadollahi

et al. 2023

J of Chemical Tech & Biotech

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The yeast expression system, Pichia pastoris, is one of the most robust and versatile expression systems in biotechnology and molecular biology, generally considered as a safe host for heterologous protein expression, especially for producing cellulases and xylanases at the industrial level. Despite the high recombinant protein expression rate, the potential of the P. pastoris expression system has still not been fully explored. Cultivation of P. pastoris under optimized conditions greatly relies on the strain and is associated with certain problems such as promoter strength, sensitivity to methanol, and oxygen demand. To address these issues, different genetic engineering strategies have been employed. Advancements in promoter engineering, optimization of gene dosage and codon usage, recombinant plasmid engineering using CRISPR/Cas9 system, and directed evolution strategies have proven beneficial to the yield of cellulase expression levels. This study will systemically review recent progress in various genetic engineering strategies to enhance cellulase and xylanase expression in the P. pastoris expression system. The utilization of alcohol oxidase 1 promoter (pAOX1), methanol‐free system, and recombinant plasmid engineering for improved production of these enzymes are highlighted. Additionally, we discuss the recent advancements in the P. pastoris expression system toolbox for improved cellulase and xylanase production, thus providing a deep insight into how P. pastoris is becoming the indispensable platform for heterologous protein production. © 2023 Society of Chemical Industry (SCI).

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A constitutive expression system for Pichia pastoris based on the PGK1 promoter

Abstract: A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.

Cited by 12 publications

References 18 publications

High-level expression of Thermomyces dupontii thermo-alkaline lipase in Pichia pastoris under the control of different promoters

High-level expression of Thermomyces dupontii thermo-alkaline lipase in Pichia pastoris under the control of different promoters

Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker

Recent developments in heterologous production of cellulases and xylanases using the Pichia pastoris expression system

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