Modulating heterologous protein production in yeast: the applicability of truncated auxotrophic markers

Seresht, Ali Kazemi; Nørgaard, Per; Palmqvist, Eva; Andersen, Asser S.; Olsson, Lisbeth

doi:10.1007/s00253-012-4263-1

Cited by 17 publications

(10 citation statements)

References 36 publications

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“…In other words, the current plasmid systems may have limited applications in industrial yeast strains, where the auxotrophic selection markers are not available (Yuan and Zhao, ). Previous studies have found that plasmid copy numbers (PCNs) could be enhanced by weakening the expression of the selection marker genes (e.g., use of heterologous, regulatable, or truncated promoters) (Bao et al, ; Erhart and Hollenberg, ; Kazemi Seresht et al, ; Okkels, ) and destabilizing the marker proteins (e.g., fusion with a degradation tag) (Chen et al, ). For example, the use of partially defective promoters driving the expression of LEU2 ( LEU2 d) and URA3 ( URA3 d) significantly enhanced the copy numbers of the corresponding 2 μ‐based plasmids (Erhart and Hollenberg, ; Okkels, ).…”

Section: Introductionmentioning

confidence: 99%

Construction of plasmids with tunable copy numbers in Saccharomyces cerevisiae and their applications in pathway optimization and multiplex genome integration

Lian

Jin

Zhao

2016

Biotech & Bioengineering

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The CEN/ARS-based low-copy plasmids and 2 μ-based high-copy plasmids have been broadly used for both fundamental studies and practical applications in Saccharomyces cerevisiae. However, the relative low copy numbers and narrow dynamic range limit their applications in many cases. In this study, the expression level of the selection marker proteins was engineered to increase the plasmid copy numbers. A series of plasmids with step-wise increased copy numbers were constructed. The copy number of the plasmids with engineered dominant markers (5-100 copies per cell) showed a positive correlation with the concentration of antibiotics supplemented to the growth media. Based on this finding, we developed a simple yet highly efficient strategy, named Pathway Optimization by Tuning Antibiotic Concentrations (POTAC) to rapidly balance the flux of multi-gene pathways at the DNA level in S. cerevisiae. As proof of concept, POTAC was used to optimize the lycopene and n-butanol biosynthetic pathways, increasing the production of lycopene and n-butanol by 10- and 100-fold, respectively. Additionally, multiplex genome integration with controllable copy numbers was attempted by combining the engineered dominant markers with the CRISPR/Cas9 system. Biotechnol. Bioeng. 2016;113: 2462-2473. © 2016 Wiley Periodicals, Inc.

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Section: Introductionmentioning

confidence: 99%

Construction of plasmids with tunable copy numbers in Saccharomyces cerevisiae and their applications in pathway optimization and multiplex genome integration

Lian

Jin

Zhao

2016

Biotech & Bioengineering

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“…Defective markers are typically auxotrophic genes, which are transcriptionally impaired. In this case, prototrophy is normally obtained when several copies of the transforming vector are present to compensate for the weak transcription from the defective marker [19]. This approach has been used successfully to obtain multicopy transformants in many types of yeast, such as Saccharomyces cerevisiae [20][21][22], Yarrowia lipolytica [23,24] , and Hansenula polymorpha [25].…”

Section: Introductionmentioning

confidence: 99%

Increase of Candida antarctica lipase B production under PGK promoter in Pichia pastoris: effect of multicopies

et al. 2019

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The effect of gene dosage on the production of Candida antarctica lipase B (CalB) in the methylotrophic yeast Komagataella phaffii, at high densities in a simple medium containing crude glycerin as the sole carbon source, is described. The use of crude glycerin, the main by-product of biodiesel production from vegetable oils, will reduce the production cost of the bioprocess. Two K. phaffii strains were constructed with one or three copies of LipB, an optimized version of the gene encoding CalB under the control of the constitutive P PGK1 promoter. These two constructs were tested and compared on batches using minimal-salts medium with crude glycerin. The strain with three copies achieved a higher enzyme yield (48,760 U/L, 2.3-fold higher than the one-copy strain), with 42 g/L biomass, with no effects on growth.

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“…To compensate the low transcription levels, cells need to amplify the copy number of the defective marker in order to recover prototrophy. Consequently, copy number of the neighboring heterologous gene is also amplified [16]. An example of such defective marker is the leu2 - d allele which contains only 29 base pairs of the original promoter and is commonly used in S. cerevisiae for plasmid maintenance at high copy number under selective pressure [17].…”

Section: Introductionmentioning

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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Modulating heterologous protein production in yeast: the applicability of truncated auxotrophic markers

Cited by 17 publications

References 36 publications

Construction of plasmids with tunable copy numbers in Saccharomyces cerevisiae and their applications in pathway optimization and multiplex genome integration

Construction of plasmids with tunable copy numbers in Saccharomyces cerevisiae and their applications in pathway optimization and multiplex genome integration

Increase of Candida antarctica lipase B production under PGK promoter in Pichia pastoris: effect of multicopies

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

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