Pichia pastoris Protein Disulfide Isomerase (PDI1) promoter for heterologous protein production and its sequence characterization

Prattipati, Mahesh; Kamatchi, Ramakrishnan; Meenakshisundaram, S.

doi:10.1016/j.enzmictec.2020.109633

Cited by 11 publications

(3 citation statements)

References 33 publications

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“…Alternatively, one can vary the promoter strength driving the expression of these genes. Recent works have identified several constitutive promoters of various strengths that can drive gene expression in P. pastoris [ 36 , 37 , 38 ]. Finally, one can also use metabolic flux analysis to identify bottlenecks in the pathway and engineer the strain further to relieve these bottlenecks [ 39 , 40 ].…”

Section: Resultsmentioning

confidence: 99%

Engineered Production of Isobutanol from Sugarcane Trash Hydrolysates in Pichia pastoris

Bumrungtham

Promdonkoy

Prabmark

et al. 2022

JoF

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Concerns over climate change have led to increased interest in renewable fuels in recent years. Microbial production of advanced fuels from renewable and readily available carbon sources has emerged as an attractive alternative to the traditional production of transportation fuels. Here, we engineered the yeast Pichia pastoris, an industrial powerhouse in heterologous enzyme production, to produce the advanced biofuel isobutanol from sugarcane trash hydrolysates. Our strategy involved overexpressing a heterologous xylose isomerase and the endogenous xylulokinase to enable the yeast to consume both C5 and C6 sugars in biomass. To enable the yeast to produce isobutanol, we then overexpressed the endogenous amino acid biosynthetic pathway and the 2-keto acid degradation pathway. The engineered strains produced isobutanol at a titer of up to 48.2 ± 1.7 mg/L directly from a minimal medium containing sugarcane trash hydrolysates as the sole carbon source. To our knowledge, this is the first demonstration of advanced biofuel production using agricultural waste-derived hydrolysates in the yeast P. pastoris. We envision that our work will pave the way for a scalable route to this advanced biofuel and further establish P. pastoris as a versatile production platform for fuels and high-value chemicals.

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Section: Resultsmentioning

confidence: 99%

Engineered Production of Isobutanol from Sugarcane Trash Hydrolysates in Pichia pastoris

Bumrungtham

Promdonkoy

Prabmark

et al. 2022

JoF

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“…The yield of bovine lactoferrin was improved by 109.5% by HAC1 i expressed from a novel methanol-inducible promoter P 0547 , while it decreased when using the GAP promoter (Sun et al 2019 ). Recently, the UPR-inducible PDI1 promoter, whose strength was found to be equivalent to 20–25% of the GAP promoter and 4.5–5% of the AOX1 promoter, was used for moderate expression of the Candida antarctica lipase B gene (Prattipati et al 2020 ).…”

Section: Enhancing Protein Secretion By Overexpression Of Upr Genesmentioning

confidence: 99%

Engineering of the unfolded protein response pathway in Pichia pastoris: enhancing production of secreted recombinant proteins

Raschmanová

Weninger

Knejzlı́k

et al. 2021

Appl Microbiol Biotechnol

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Folding and processing of proteins in the endoplasmic reticulum (ER) are major impediments in the production and secretion of proteins from Pichia pastoris (Komagataella sp.). Overexpression of recombinant genes can overwhelm the innate secretory machinery of the P. pastoris cell, and incorrectly folded proteins may accumulate inside the ER. To restore proper protein folding, the cell naturally triggers an unfolded protein response (UPR) pathway, which upregulates the expression of genes coding for chaperones and other folding-assisting proteins (e.g., Kar2p, Pdi1, Ero1p) via the transcription activator Hac1p. Unfolded/misfolded proteins that cannot be repaired are degraded via the ER-associated degradation (ERAD) pathway, which decreases productivity. Co-expression of selected UPR genes, along with the recombinant gene of interest, is a common approach to enhance the production of properly folded, secreted proteins. Such an approach, however, is not always successful and sometimes, protein productivity decreases because of an unbalanced UPR. This review summarizes successful chaperone co-expression strategies in P. pastoris that are specifically related to overproduction of foreign proteins and the UPR. In addition, it illustrates possible negative effects on the cell’s physiology and productivity resulting from genetic engineering of the UPR pathway. We have focused on Pichia’s potential for commercial production of valuable proteins and we aim to optimize molecular designs so that production strains can be tailored to suit a specific heterologous product. Key points • Chaperones co-expressed with recombinant genes affect productivity in P. pastoris. • Enhanced UPR may impair strain physiology and promote protein degradation. • Gene copy number of the target gene and the chaperone determine the secretion rate.

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“…Although diverse regulators have been developed in recent years for synthetic biology applications in prokaryotic and eukaryotic cells ( Hartner et al, 2008 ; Bruckner et al, 2015 ; Naseri et al, 2017 ; Ji et al, 2019 ), a limited number of artificial regulators are available for P. pastoris . To express heterologous protein and multi-enzymes of a heterologous pathway in P. pastoris , its native promoters are commonly used ( Massahi and Çalık, 2018 ; Xu et al, 2018 ; Prattipati et al, 2020 ). For example, promoter libraries created by the deletion and duplication of putative transcription factor binding sites within the methanol inducible alcohol oxidase 1 ( AOX1 ) promoter ( Hartner et al, 2008 ) or the single point mutation of the constitutive glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) promoter ( Waterham et al, 1997 ) are being used in P. pastoris .…”

Section: Introductionmentioning

confidence: 99%

Artificial Transcription Factors for Tuneable Gene Expression in Pichia pastoris

Naseri

Prause

Hamdo

2021

Front. Bioeng. Biotechnol.

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The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has become a powerful eukaryotic expression platform for biopharmaceutical and biotechnological applications on both laboratory and industrial scales. Despite the fundamental role that artificial transcription factors (ATFs) play in the orthogonal control of gene expression in synthetic biology, a limited number of ATFs are available for P. pastoris. To establish orthogonal regulators for use in P. pastoris, we characterized ATFs derived from Arabidopsis TFs. The plant-derived ATFs contain the binding domain of TFs from the plant Arabidopsis thaliana, in combination with the activation domains of yeast GAL4 and plant EDLL and a synthetic promoter harboring the cognate cis-regulatory motifs. Chromosomally integrated ATFs and their binding sites (ATF/BSs) resulted in a wide spectrum of inducible transcriptional outputs in P. pastoris, ranging from as low as 1- to as high as ∼63-fold induction with only small growth defects. We demonstrated the application of ATF/BSs by generating P. pastoris cells that produce β-carotene. Notably, the productivity of β-carotene in P. pastoris was ∼4.8-fold higher than that in S. cerevisiae, reaching ∼59% of the β-carotene productivity obtained in a S. cerevisiae strain optimized for the production of the β–carotene precursor, farnesyl diphosphate, by rewiring the endogenous metabolic pathways using plant-derived ATF/BSs. Our data suggest that plant-derived regulators have a high degree of transferability from S. cerevisiae to P. pastoris. The plant-derived ATFs, together with their cognate binding sites, powerfully increase the repertoire of transcriptional regulatory modules for the tuning of protein expression levels required in metabolic engineering or synthetic biology in P. pastoris.

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Pichia pastoris Protein Disulfide Isomerase (PDI1) promoter for heterologous protein production and its sequence characterization

Cited by 11 publications

References 33 publications

Engineered Production of Isobutanol from Sugarcane Trash Hydrolysates in Pichia pastoris

Engineered Production of Isobutanol from Sugarcane Trash Hydrolysates in Pichia pastoris

Engineering of the unfolded protein response pathway in Pichia pastoris: enhancing production of secreted recombinant proteins

Artificial Transcription Factors for Tuneable Gene Expression in Pichia pastoris

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