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. 2018 Jul 27;9:1640. doi: 10.3389/fmicb.2018.01640

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Copyright © 2018 Diderich, Weening, van den Broek, Pronk and Daran.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Construction of targeted PAD1-FDC1 deletion in Saccharomyces eubayanus CBS 12357. (A-1) The deletion cassette was amplified using the primers pair 3144/3145 and the plasmid pUG-amdSYM (Solis-Escalante et al., 2013) as template. The fragment was then transformed in S. eubayanus CBS 12357. (2) Transformants were genotyped with primers pairs 9322/9328 and 9322/9 to discriminate between wild type (1027 bp) and deleted (485 bp) loci respectively. Heterozygote diploid IMK747 (SePAD1-SeFDC1/Sepad1-Sefdc1Δ::amdSYM) was sporulated. After ascus digestion the spores were dissected resulting in a two:two segregation of the PAD1-FDC1 locus. Spores were germinated. Single colony isolates of acetamide growing spores were selected and checked for ploidy. (3) A 2n isolated homozygote (Sepad1-Sefdc1Δ::amdSYM/Sepad1-Sefdc1Δ::amdSYM) was selected and renamed IMK749. (B) PCR analysis was performed to confirm correct integration of the gene disruption cassettes using amdSYM (Solis-Escalante et al., 2013) of the PAD1-FDC1 locus in S. eubayanus. PCR was carried out on reference strain CBS 12357, IMK747, IMX749 and four segregants (S1–S4) of a single tetrad of IMK747. PCRs were performed with the primers pairs 9322/9328 and 9322/9 for PAD1-FDC1 and pad1-fdc1Δ loci, respectively. The amplification of the wild-type PAD1-FDC1 and pad1-fdc1Δ parental strain, of the loci generated fragments of 1027 and 485 bp, respectively. (C) The four segregants (S1–S4) of a single tetrad of IMK747 were grown on SM-Ac and SM. The plates were incubated at 30°C and were read after 3 days.