A color-based stable multi-copy integrant selection system forPichia pastorisusing the attenuatedADE1andADE2genes as auxotrophic markers

Du, Min; Battles, Michael B.; Nett, Juergen H.

doi:10.4161/bbug.3.1.17936

Cited by 19 publications

(17 citation statements)

References 17 publications

(16 reference statements)

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“…Analysis of variance followed by Tukey’s post-test showed significant difference in maximum growth rates presented in Table 1 for clones 2 and 7 when compared to X-33 (p < 0.05). Similar results were obtained when truncated ADE1 and ADE2 were used as selectable defective markers to transform K. phaffii [35]. In this case, multicopy clones were also identified as colonies with larger sizes.…”

Section: Resultssupporting

confidence: 76%

“…In a previous work, attenuated ADE1 and ADE2 genes involved in adenine biosynthesis were used to develop a color-based system for the screening of multicopy integrants in K. phaffii [35]. However, this system is based on large plasmids and, in some cases, the effects of background transcription from vector sequences were responsible for the recovery of adenine prototrophy.…”

Section: Resultsmentioning

confidence: 99%

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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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Section: Resultssupporting

confidence: 76%

Section: Resultsmentioning

confidence: 99%

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

et al. 2017

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“…The recently introduced PichiaPink™ expression kit for intracellular or secreted expression enables easy selection of multicopy integration clones by differences in colour formation based on ade2 knockout strains and truncated ADE2 promoters of varying strengths in front of the ADE2 marker gene (Du et al 2012; Nett 2010). …”

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

801

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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“…One was designed to eliminate pks4 (DDBJ/EMBL/GenBank protein accession number: EGR44538.1, JGI ID: 82208), a gene that encodes the polyketide synthase necessary for production of green conidial pigment [17]; and ade2 (DDBJ/EMBL/GenBank protein accession number: EGR49709.1, JGI ID: 105832), an ortholog of the ade2 gene in Saccharomyces cerevisiae , which encodes a phosphoribosylaminoimidazole carboxylase necessary for production of purines. Lack of this enzyme in species such as S. cerevisiae, Pichia pastoris and Aspergillus oryzae [18-20] results not only in adenine auxotrophy, but also in development of red colonies as the precursor 5-aminoimidazole ribonucleotide accumulates in the cell and polymerizes [21]. Both gene targeting substrates were based on a pyr2 selectable marker, which was flanked by 375 bp direct repeats, originating from the A. oryzae pyr4 promotor region, to allow for subsequent marker excision by direct repeat recombination, see Figure 2.…”

Section: Resultsmentioning

confidence: 99%

A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina)

et al. 2014

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BackgroundThe industrially applied filamentous fungus Trichoderma reesei has received substantial interest due to its highly efficient synthesis apparatus of cellulytic enzymes. However, the production of heterologous enzymes in T. reesei still remains low mainly due to lack of tools for genetic engineering.ResultsIn this study we present new genetic tools for T. reesei to further expand its use in industrial production. We have developed an expression platform where genes are inserted into a versatile expression vector via highly efficient uracil-excision cloning and subsequently inserted into a defined position in the T. reesei genome ensuring that enzyme production from different transformants can be directly compared. The ade2 locus was selected as integration site since ade2 mutants develop red pigment that facilitates easy and rapid detection of correctly targeted transformants. In addition, our system includes a tku70 disruption to increase gene targeting efficiency and a new bidirectional marker, pyr2, for iterative gene targeting. The dual selection system, color and prototrophism, ensures that correct transformants containing the desired gene inserted into the defined expression site can be selected with an efficiency approaching 100%.ConclusionsThe new genetic tools we have developed are suitable for high-throughput integration of genes into the genome of T. reesei and can easily be combined with techniques for generation of defined mutants. Moreover, the usability of the novel expression system with ade2 as integration site was confirmed by expression of a Thermomyces lanuginosus lipase.

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A color-based stable multi-copy integrant selection system forPichia pastorisusing the attenuatedADE1andADE2genes as auxotrophic markers

Cited by 19 publications

References 17 publications

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

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

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

A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina)

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