Selection of yeast cells with a higher plasmid copy number in a Saccharomyces cerevisiae autoselection system

Compagno, Concetta; Porro, Danilo; Radice, Stefania; Martegani, Enzo; Ranzi, Bianca Maria

doi:10.1002/(sici)1097-0061(19960315)12:3<199::aid-yea895>3.0.co;2-u

Cited by 8 publications

(3 citation statements)

References 23 publications

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“…Production levels by the YEp‐type expression vectors were significantly higher than levels obtained with the YIp‐type vector both in batch and continuous culture. The increased gene dosage of the YEp‐type vectors thus dramatically improved heterologous protein production, as has been frequently reported (Smith et al , 1985; Bitter et al , 1987; Denis and Drouin, 1987; Kaisho et al , 1989; Janes et al , 1990; Weber et al , 1992; Mendoza‐Vega et al , 1994; Compagno et al , 1996; Lopes et al , 1996; Nacken et al , 1996; Gellissen and Hollenberg, 1997; Ljubijankic et al , 1999; Park et al , 2000; Vassileva et al , 2001; Kim et al , 2001). The previously reported saturation of the protein secretory capacity of the host strain by high‐level foreign gene expression from the multicopy YEp‐type vector (Wittrup et al , 1994; Tuite and Freedman, 1994; Parekh et al , 1995; Parekh and Wittrup, 1997) was not observed.…”

Section: Discussionmentioning

confidence: 61%

Comparison of three expression systems for heterologous xylanase production byS. cerevisiaein defined medium

Görgens

Planas

Zyl

et al. 2004

Yeast

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The influence of the auxotrophic deficiencies of the host strain and expression vector selection on the production of a heterologous protein was investigated. Heterologous xylanase production by two prototrophic S. cerevisiae transformants, containing either a plasmid-based, YEp-type expression system or an integrative, YIp-type expression system, were compared with production by an auxotrophic transformant, containing an identical YEp-type expression system, in batch and continuous cultivation, using a chemically defined medium. Heterologous xylanase production by the auxotrophic strains in defined medium was critically dependent on the availability of amino acids, as extracellular xylanase production increased dramatically when amino acids were over-consumed from the medium to the point of saturating the cell. Saturation with amino acids, indicated by an increased leakage of amino acids from the cell, was thus a prerequisite for high level of heterologous protein production by the auxotrophic strain. Maximal xylanase production levels by the auxotrophic strain corresponded to the levels obtained with a similar prototrophic strain during cultivation in defined medium without amino acids. Superfluous auxotrophic markers thus had a strong deleterious effect on heterologous protein production by recombinant yeasts, and the use of such strains should be limited to initial exploratory investigations. The increased copy number and foreign gene dosage of the YEp-based expression vector, stabilized by the ura3 fur1 autoselection system, significantly improved production levels of heterologous xylanase, compared to the YIp system, which is based on a single integration into the yeast genome. No evidence was found of the possible saturation of the host secretory capacity by multicopy overexpression. Stable production of heterologous xylanase at high levels by the prototrophic YEp-based recombinant strain, compared to the YIp system, was demonstrated.

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Section: Discussionmentioning

confidence: 61%

Comparison of three expression systems for heterologous xylanase production byS. cerevisiaein defined medium

Görgens

Planas

Zyl

et al. 2004

Yeast

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“…FCM will prove useful for environmental microbiology in general, including areas not discussed here [45–47]. Additionally, new advances in both instrument design and data processing are improving the effectiveness of FCM in environmental microbiology.…”

Section: Future Prospectsmentioning

confidence: 99%

Go with the flow - use of flow cytometry in environmental microbiology

Porter

Deere

Hardman

et al. 2006

FEMS Microbiology Ecology

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Microbiological activity in the natural world is of key importance in the integrated functioning of ecosystems, yet we remain largely ignorant of the role and relevance of the vast majority of microorganisms. This ignorance is largely due to widely acknowledged, but unresolved problems in methodologies. Application of flow cytometry to such studies has already revolutionised our understanding of marine photosynthetic planktonic microorganisms, revealed new levels of complexity in the behaviour of bacterial populations and produced a reliable screening protocol for eukaryotic water‐borne pathogens. Advances in fluorescent probe technology now offer realistic approaches for direct cell identification, viability assessment and responses to environmental changes using basic, single light‐source flow cytometers. Here we review current applications of flow cytometry in environmental microbiology and present a case for the adoption of the technique as a necessary and routine research instrument.

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“…Autoselection was first described in 1983 for an E. coli system where plasmids carrying a λ phage repressor prevented killing by λ prophage that could not make its own repressor (Rosteck and Hershberger, 1983). Since then there have been several reports of yeast autoselective systems (Loison et al, 1986(Loison et al, , 1989Unternahrer et al, 1991;Ayub et al, 1992;Napp and Da Silva, 1992;Rech et al, 2002;Compagno et al, 1993;Compagno et al, 1996;Hsieh and Da Silva, 1998;van Rensburg et al, 1998). However, only one system, relying on the rescue of lethality of fur1 ura3 mutants by URA3 plasmids (Loison et al, 1986), has any history of multiple, yet limited, usage after the initial publication (for example (Loison et al, 1989;van Rensburg et al, 1996;van Rensburg et al, 1997;van Rensburg et al, 1998;Dussossoy et al, 1999;Setati et al, 2001;La Grange et al, 2001;Gorgens et al, 2004)).…”

Section: Introductionmentioning

confidence: 99%

A Saccharomyces cerevisiae autoselection system for optimised recombinant protein expression

Geymonat¹,

Spanos²,

Sedgwick³

2007

Gene

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Selection of yeast cells with a higher plasmid copy number in a Saccharomyces cerevisiae autoselection system

Cited by 8 publications

References 23 publications

Comparison of three expression systems for heterologous xylanase production byS. cerevisiaein defined medium

Comparison of three expression systems for heterologous xylanase production byS. cerevisiaein defined medium

Go with the flow - use of flow cytometry in environmental microbiology

A Saccharomyces cerevisiae autoselection system for optimised recombinant protein expression

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