Factors affecting the mitotic stability of high-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae

Lopes, T. S.; Wijs, Ilse J. de; Steenhauer, S. I.; Verbakel, J. M. A.; Planta, Rudi J.

doi:10.1002/(sici)1097-0061(199604)12:5<467::aid-yea933>3.0.co;2-3

Cited by 54 publications

(31 citation statements)

References 37 publications

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“…Scattered integrations may account for the high stability of the two transformants of PO1d. The small size of the expression cassette (5.4 kb) also may be important in mitotic stability (21), and the nature of the expressed gene: LIP2 dispersed multicopy integrants are more stable than XPR2 tandem multicopy integrants, which tend to deamplify under induction conditions (20).…”

Section: Discussionmentioning

confidence: 99%

Autocloning and Amplification of LIP2 in Yarrowia lipolytica

Pignède

Wang²,

Fudalej

et al. 2000

Appl Environ Microbiol

136

115

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We synthesized a Yarrowia lipolytica strain overproducing lipase for industrial applications by using long terminal repeat () of the Y. lipolytica retrotransposon Ylt1 and an allele of URA3 with a promoter deletion to construct JMP3. JMP3 is a derivative of plasmid pHSS6 carrying a NotI-NotI cassette which contains a defective URA3 allele, a polylinker sequence, and the region for targeting to multiple sites in the genome of the recipient. We inserted the LIP2 gene (encoding extracellular lipase) under the control of the strong POX2 promoter into JMP3 to generate JMP6. The pHSS6 region was removed by NotI digestion prior to transformation. Two Y. lipolytica strains transformed with the JMP6 LIP2 cassette had a mean of 10 integrated copies devoid of the Escherichia coli region, corresponding to an autocloning event. The copy number in the transformants was stable even after 120 generations in nonselective and lipase-inducing conditions. The resulting strains could produce 0.5 g of active lipase per liter in the supernatant, 40 times more than the single-copy strain with the LIP2 promoter. This work provides a new expression system in Y. lipolytica that results in strains devoid of bacterial DNA and in strains producing a high level of lipase for industrial uses, waste treatment, and pancreatic insufficiency therapy.

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Section: Discussionmentioning

confidence: 99%

Autocloning and Amplification of LIP2 in Yarrowia lipolytica

Pignède

Wang²,

Fudalej

et al. 2000

Appl Environ Microbiol

136

115

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“…25 Briefly, Saccharomyces cerevisiae strain CEN.PK338 (gal1:URA3, leu2, ura3, pmt1) carried a multicopy integration vector, integrated at the rDNA locus, containing the protein coding sequence of Trichoderma reesei HFBII, linked to the S. cerevisiae SUC2 signal sequence and under control of the GAL7 promoter and leu2d selectable marker to maintain a high copy number. 26 The strain was grown in fed batch fermentations. 27 The cells were removed by centrifugation and filtration over a 0.2 μm filter, and the supernatant containing the HFBII was freeze-dried.…”

Section: Hydrophobin Sample Productionmentioning

confidence: 99%

Self-Assembled Bilayers from the Protein HFBII Hydrophobin: Nature of the Adhesion Energy

et al. 2011

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The hydrophobins are a class of amphiphilic proteins which spontaneously adsorb at the air/water interface and form elastic membranes of high mechanical strength as compared to other proteins. The mechanism of hydrophobin adhesion is of interest for fungal biology and for various applications in electronics, medicine, and food industry. We established that the drainage of free foam films formed from HFBII hydrophobin solutions ends with the appearance of a 6 nm thick film, which consists of two layers of protein molecules, that is, it is a self-assembled bilayer (S-bilayer), with hydrophilic domains pointing inward and hydrophobic domains pointing outward. Its formation is accompanied by a considerable energy gain, which is much greater than that typically observed with free liquid films. The experiments at different pH show that this attraction between the "hydrophilic" parts of the HFBII molecules is dominated by the short-range hydrophobic interaction rather than by the patch-charge electrostatic attraction.

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“…1b) supports this hypothesis, as the instability of the integrated construct might also be caused by an excessive length (Lopes et al, 1996). Nonetheless, insertion of foreign genes prevented the isolation of transformants (IL-1b and LDH cassettes, data not shown).…”

Section: Rrna Gene and Multiple Integrationmentioning

confidence: 59%

“…The choice of this region was based on the previously published data for S. cerevisiae as well as for other yeasts (Lopes et al, 1989(Lopes et al, , 1996Klabunde et al, 2002). For this purpose, an rRNA gene region of Z. bailii was cloned and sequenced.…”

Section: Rrna Gene and Multiple Integrationmentioning

confidence: 99%

Advances in molecular tools for the use of Zygosaccharomyces bailii as host for biotechnological productions and construction of the first auxotrophic mutant

Dato

Branduardi

Passolunghi

et al. 2010

FEMS Yeast Research

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The nonconventional yeast Zygosaccharomyces bailii has been proposed as a new host for biotechnological processes due to convenient properties such as its resistance to high sugar concentrations, relatively high temperatures and especially to acidic environments. We describe a series of new expression vectors specific for Z. bailii and the resulting improvements in production levels. By exploiting the sequences of the endogenous plasmid pSB2, 2microm-like multicopy vectors were obtained, giving a fivefold increase in production. A specific integrative vector was developed which led to 100% stability in the absence of selective pressure; a multiple-integration vector was constructed, based on an rRNA gene unit portion cloned and sequenced for this purpose, driving the insertion of up to 80 copies of the foreign construct. Moreover, we show the construction of the first stable auxotrophic mutant of Z. bailii, obtained by targeted gene deletion applied to ZbLEU2. The development of molecular tools for the Z. bailii manipulation has now reached a level that may be compatible with its industrial exploitation; the production of organic acids is a prominent field of application.

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Factors affecting the mitotic stability of high-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae

Cited by 54 publications

References 37 publications

Autocloning and Amplification of LIP2 in Yarrowia lipolytica

Autocloning and Amplification of LIP2 in Yarrowia lipolytica

Self-Assembled Bilayers from the Protein HFBII Hydrophobin: Nature of the Adhesion Energy

Advances in molecular tools for the use of Zygosaccharomyces bailii as host for biotechnological productions and construction of the first auxotrophic mutant

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