doi: 10.1111/1567-1364.12024. Epub 2012 Dec 17.
amdSYM, a new dominant recyclable marker cassette for Saccharomyces cerevisiae
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- PMID:23253382
- PMCID: PMC3563226
- DOI: 10.1111/1567-1364.12024
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amdSYM, a new dominant recyclable marker cassette for Saccharomyces cerevisiae
Daniel Solis-Escalante et al. FEMS Yeast Res.2013 Feb.
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Abstract
Despite the large collection of selectable marker genes available for Saccharomyces cerevisiae, marker availability can still present a hurdle when dozens of genetic manipulations are required. Recyclable markers, counterselectable cassettes that can be removed from the targeted genome after use, are therefore valuable assets in ambitious metabolic engineering programs. In the present work, the new recyclable dominant marker cassette amdSYM, formed by the Ashbya gossypii TEF2 promoter and terminator and a codon-optimized acetamidase gene (Aspergillus nidulans amdS), is presented. The amdSYM cassette confers S. cerevisiae the ability to use acetamide as sole nitrogen source. Direct repeats flanking the amdS gene allow for its efficient recombinative excision. As previously demonstrated in filamentous fungi, loss of the amdS marker cassette from S. cerevisiae can be rapidly selected for by growth in the presence of fluoroacetamide. The amdSYM cassette can be used in different genetic backgrounds and represents the first counterselectable dominant marker gene cassette for use in S. cerevisiae. Furthermore, using astute cassette design, amdSYM excision can be performed without leaving a scar or heterologous sequences in the targeted genome. The present work therefore demonstrates that amdSYM is a useful addition to the genetic engineering toolbox for Saccharomyces laboratory, wild, and industrial strains.
© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
Figures
pUG-amdSYM map. pUG-amdSYM was constructed by replacing the markerKanMX in pUG6 for a codon-optimized version ofAspergillus nidulans amdS. The plasmid kept all features present in the pUG family (Guldeneret al., 1996, 2002).
Growth of strains from the CEN.PK family on acetamide as sole nitrogen source. Expression ofamdS on the multicopy plasmid pUDE158 conferred toSaccharomyces cerevisiae the ability to grow on acetamide as sole nitrogen source. The strains CEN.PK113-5D (ura3-52), IME141 [ura3-52 pAG426GPD (2μ URA3 TDH3pr-CYC1ter)], and IME142 [ura3-52 pUDE158 (2μ URA3 TDH3pr-amdS-CYC1ter)] were grown on YPD, SM and SM-Ac media and incubated at 30 °C. The plates were read after 3 days.
Sequential gene deletions methodology. (A) Cassette design for targeted gene deletion and seamless marker removal. (B) Experimental procedure for the sequential deletion ofCAN1 andADE2 inSaccharomyces cerevisiae byamdSYM recycling. (C) Sequencing results of theCAN1 loci of the marker-free IMX206 (can1Δ ade2Δ).
Sequential gene deletions ofCAN1 andADE2 usingamdSYM inS. cerevisiae. (A) The strains IMX168, IMX200, and IMX201 and the parental strain CEN.PK113-7D were grown on SM-Ac, SM-Ac supplemented with adenine, and SM supplemented with adenine andl -canavanine. The plates were incubated at 30 °C and were read after 3 days. (B) PCR analysis to confirm correct integration of the gene disruption cassettes and their removal at theCAN1 andADE2 loci. PCR was carried out on reference CEN.PK113-7D, IMX168, IMX200, and IMX201. All PCRs were performed with the primer pairs CdcFW/CdcRV and AdcFW/AdcRV forCAN1 andADE2 loci, respectively. In the parental strain, amplification of theCAN1 andADE2 loci generated fragments of 2151 bp (a) and 1950 bp (d) forCAN1 andADE2, respectively. PCR on IMX168 DNA generated a fragment of 2835 bp (b) due to the incorporation ofamdSYM in theCAN1 locus. A short fragment of 333 bp (c) was obtained for IMX200 as a result ofamdSYM excision from theCAN1 locus. Similarly, the disruption ofADE2 usingamdSYM led to a large PCR product of 2691 bp in IMX201 (e) while PCR on theADE2 locus in the marker-free strain IMX206 generated a short fragment of 189 bp (f). The products obtained were then subjected to agarose gel electrophoresis.
Growth ofSaccharomyces cerevisiae laboratory strains on acetamide as sole nitrogen source. The laboratory strains CEN.PK113-7D, CBS8066, YSBN, S288c, and the modified strain IMX168 (can1Δ::amdSYM) were grown on SM-Ac and SM media and incubated at 30 °C. The plates were read after 3 days. Only the strain harboring theamdSYM module, IMX168, was able to grow when acetamide was used as nitrogen source.
amdSYM as selectable marker for laboratory, wild, and industrialSaccharomyces strains. (A) The laboratory strain CEN.PK113-7D, the industrial strains CBS1483, Scottish Ale and the wildSaccharomyces eubayanus CBS12357, and the generated strains IMK468, IMK470, IMK473, and IMK474 were grown on SM-Ac and SM media and incubated at 30 °C. The plates were read after 5 days. (B) PCR analysis to confirm correct integration of the gene disruption cassettes was carried out on CEN.PK113-7D, CBS1483, Scottish Ale, CBS12357, IMK468, IMK470, IMK473, and IMK474. Amplification ofSc.HXK1 locus in CEN.PK113-7D and CBS1483 generated fragments of 1837 bp (a, c), bigger fragments, 2836 bp (b, d) were obtained in the strains IMK468 and IMK470 due to the integration ofamdSYM. Amplification of the lociSc.ARO80 in Scottich Ale andSb.ARO80 in CBS12357 generated fragments of 4208 bp (e) and 4002 bp (g), respectively. Confirmation of the deletion ofSc.ARO80 in IMK473 andSb.ARO80 in IMK474 by PCR-generated fragments of 2992 bp (f) and 2895 bp (h), respectively. The products obtained were then subjected to agarose gel electrophoresis.
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