Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

Ahmad, Mudassar; Hirz, Melanie; Pichler, Harald; Schwab, Helmut

doi:10.1007/s00253-014-5732-5

Cited by 804 publications

(625 citation statements)

References 140 publications

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“…The use of P. pastoris, reclassified as Komagataella pastoris, as a producer of heterologous proteins has been reviewed by Ahmad et al 136 Among the processes developed, one of the first was the production of the plant-derived hydroxynitrile lyase at over 20 g l − 1 . 137 Fatty acids and lipids Production of polyunsaturated fatty acids by fungi and other microorganisms has been reviewed by Ratledge.…”

Section: Isoprenoidsmentioning

confidence: 99%

Production of valuable compounds by molds and yeasts

Demain

Martens

2016

J Antibiot

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where he has contributed in a major way to the reputation of this department for many years. He also served as an Adjunct Professor at the University of Geneva. Among Julian's areas of study and accomplishment are fungal toxins including α-sarcin, chemical synthesis of triterpenes, mode of action of streptomycin and other aminoglycoside antibiotics, biochemical mechanisms of antibiotic resistance in clinical isolates of bacteria harboring resistance plasmids, their origins and evolution, secondary metabolism of microorganisms, structure and function of bacterial ribosomes, antibiotic resistance mutations in yeast ribosomes, cloning of resistance genes from an antibiotic-producing microbe, gene cloning for industrial purposes, engineering of herbicide resistance in useful crops, bleomycin-resistance gene in clinical isolates of Staphylococcus aureus and many other topics. He has been an excellent teacher, lecturing in both English and French around the world, and has organized international courses. Julian has also served on the NIH study sections, as Editor for several international journals, and was one of the founders of the journal Plasmid. We expect the impact of Julian's accomplishments to continue into the future.

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Section: Isoprenoidsmentioning

confidence: 99%

Production of valuable compounds by molds and yeasts

Demain

Martens

2016

J Antibiot

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“…Nevertheless, β-glucosidase genes from T. emersonii, P. brasilianum and T. aurantiacus have been cloned and expressed in filamentous fungi such as T. reesei [187], A. oryzae [183], and P. pastoris [43], respectively. P. pastoris is known to secrete high amount of extracellular protein therefore it is preferred for heterologous expression of recombinant proteins [188,189]. Similarly, T. reesei produces cellulase component (endoglucanase and exoglucanase) in high amount with excellent properties such as thermostability and catalytic efficiency but it lacks sufficient amount of β-glucosidase.…”

Section: Cloning and Expression Of Microbial β-Glucosidasesmentioning

confidence: 99%

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

Ahmed¹,

Aslam²,

Ashraf³

et al. 2017

JAEM

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Cellulose is the most abundant biomaterial in the biosphere and the major component of plant biomass.Cellulase is an enzymatic system required for conversion of renewable cellulose biomass into free sugar for subsequent use in different applications. Cellulase system mainly consists of three individual enzymes namely: endoglucanase, exoglucanase and β-glucosidases. β-Glucosidases are ubiquitous enzymes found in all living organisms with great biological significance. β-Glucosidases have also tremendous biotechnological applications such as biofuel production, beverage industry, food industry, cassava detoxification and oligosaccharides synthesis. Microbial β-glucosidases are preferred for industrial uses because of robust activity and novel properties exhibited by them. This review aims at describing the various biochemical methods used for screening and evaluating β-glucosidases activity from microbial sources. Subsequently, it generally highlights techniques used for purification of β-glucosidases. It then elaborates various biochemical and molecular properties of this valuable enzyme such as pH and temperature optima, glucose tolerance, substrate specificity, molecular weight, and multiplicity. Furthermore, it describes molecular cloning and expression of bacterial, fungal and metagenomic β-glucosidases. Finally, it highlights the potential biotechnological applications of β-glucosidases.

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“…P. pastoris has several beneficial traits, such as suitability for high-cell-density bioreactor cultivation (5), the availability of exceptionally strong, tightly regulated promoters (6), and the capacity for secreting large amounts of heterologous proteins (1)(2)(3). Furthermore, P. pastoris is readily amenable to genetic modification, and it is relatively easy to integrate multiple copies of an expression cassette, thereby further boosting yields (7)(8)(9).…”

mentioning

confidence: 99%

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris

Vogl

Gebbie

Palfreyman

et al. 2018

Appl Environ Microbiol

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Pichia pastoris (syn. Komagataella phaffii) is one of the most common eukaryotic expression systems for heterologous protein production. Expression cassettes are typically integrated in the genome to obtain stable expression strains. In contrast to Saccharomyces cerevisiae, where short overhangs are sufficient to target highly specific integration, long overhangs are more efficient in P. pastoris and ectopic integration of foreign DNA can occur. Here, we aimed to elucidate the influence of ectopic integration by high-throughput screening of Ͼ700 transformants and whole-genome sequencing of 27 transformants. Different vector designs and linearization approaches were used to mimic the most common integration events targeted in P. pastoris. Fluorescence of an enhanced green fluorescent protein (eGFP) reporter protein was highly uniform among transformants when the expression cassettes were correctly integrated in the targeted locus. Surprisingly, most nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression. However, a few clones (Ͻ10%) harboring ectopically integrated cassettes showed a greater variation spanning a 25-fold range, surpassing specifically integrated reference strains up to 6-fold. High-expression strains showed a correlation between increased gene copy numbers and high reporter protein fluorescence levels. Our results suggest that for comparing expression levels between strains, the integration locus can be neglected as long as a sufficient numbers of transformed strains are compared. For expression optimization of highly expressible proteins, increasing copy number appears to be the dominant positive influence rather than the integration locus, genomic rearrangements, deletions, or single-nucleotide polymorphisms (SNPs).IMPORTANCE Yeasts are commonly used as biotechnological production hosts for proteins and metabolites. In the yeast Saccharomyces cerevisiae, expression cassettes carrying foreign genes integrate highly specifically at the targeted sites in the genome. In contrast, cassettes often integrate at random genomic positions in nonconventional yeasts, such as Pichia pastoris (syn. Komagataella phaffii). Hence, cells from the same transformation event often behave differently, with significant clonal variation necessitating the screening of large numbers of strains. The importance of this study is that we systematically investigated the influence of integration events in more than 700 strains. Our findings provide novel insight into clonal variation in P. pastoris and, thus, how to avoid pitfalls and obtain reliable results. The underlying mechanisms may also play a role in other yeasts and hence could be generally relevant for recombinant yeast protein production strains.KEYWORDS integration, Pichia pastoris, protein expression, genome analysis T he yeast Pichia pastoris (syn. Komagataella phaffii) is widely used for heterologous protein p...

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Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

Cited by 804 publications

References 140 publications

Production of valuable compounds by molds and yeasts

Production of valuable compounds by molds and yeasts

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris

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