Cloning and disruption of the <i>Pichia pastoris ARG1, ARG2, ARG3, HIS1, HIS2, HIS5, HIS6</i> genes and their use as auxotrophic markers

Nett, Juergen H.; Hodel, Nikolai; Rausch, Sebastian; Wildt, Stefan

doi:10.1002/yea.1202

Cited by 39 publications

(33 citation statements)

References 16 publications

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“…The vectors contain restriction sites for linearization within the marker genes to target the expression cassettes to the desired locus as well as for multicopy integration (Lin-Cereghino et al 2001). Moreover, a set of integration vectors for sequential disruption of ARG1 , ARG2 , ARG3 , HIS1 , HIS2 , HIS5 and HIS6 in P. pastoris was applied to provide the host strains for engineering the protein glycosylation pathway (Nett et al 2005). …”

Section: Basic Systems For Cloning and Expression In P Pastorismentioning

confidence: 99%

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

Ahmad

Hirz

Pichler

et al. 2014

Appl Microbiol Biotechnol

819

564

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Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.

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Section: Basic Systems For Cloning and Expression In P Pastorismentioning

confidence: 99%

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

Ahmad

Hirz

Pichler

et al. 2014

Appl Microbiol Biotechnol

819

564

View full text Add to dashboard Cite

show abstract

“…1000 bp), a high variance in targeting efficiency is observed, indicating the prevalence of the non-homologous end joining (NHEJ) pathway in P. pastoris 181920. As a result, a high clonal variability is found after transformation, necessitating a time and labour-intensive screening process for the clone with the desired characteristics2122.…”

mentioning

confidence: 99%

Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing

Schwarzhans

Wibberg

Winkler

et al. 2016

Sci Rep

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The non-conventional yeast Pichia pastoris is a popular host for recombinant protein production in scientific research and industry. Typically, the expression cassette is integrated into the genome via homologous recombination. Due to unknown integration events, a large clonal variability is often encountered consisting of clones with different productivities as well as aberrant morphological or growth characteristics. In this study, we analysed several clones with abnormal colony morphology and discovered unpredicted integration events via whole genome sequencing. These include (i) the relocation of the locus targeted for replacement to another chromosome (ii) co-integration of DNA from the E. coli plasmid host and (iii) the disruption of untargeted genes affecting colony morphology. Most of these events have not been reported so far in literature and present challenges for genetic engineering approaches in this yeast. Especially, the presence and independent activity of E. coli DNA elements in P. pastoris is of concern. In our study, we provide a deeper insight into these events and their potential origins. Steps preventing or reducing the risk for these phenomena are proposed and will help scientists working on genetic engineering of P. pastoris or similar non-conventional yeast to better understand and control clonal variability.

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“…This approach has been successfully used to disrupt several genes in order to create auxotrophic mutants, e.g. URA5 [22], ARG1, ARG2, ARG3, HIS1, HIS2, HIS5 and HIS6 [23]. Recently, a CRISPR-Cas9 system was developed for K. phaffii which has greatly facilitated gene knock out in this yeast [24].…”

Section: Resultsmentioning

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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Cloning and disruption of the Pichia pastoris ARG1, ARG2, ARG3, HIS1, HIS2, HIS5, HIS6 genes and their use as auxotrophic markers

Cited by 39 publications

References 16 publications

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

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

Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing

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

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