New generation of loxP-mutated deletion cassettes for the genetic manipulation of yeast natural isolates

Yeast
Zorana Carter, Daniela Delneri

Abstract

In this work, we developed molecular tools used in standard laboratory yeast strains, such as the cre-loxP system, so that they can be used with natural and industrial prototrophic yeast species. We constructed a new generation of dominant cassettes, with mutated loxP sites (loxLE and lox2272) and selectable drug markers, to create heterothallic strains and auxotrophic mutants without incurring in the risk of generating chromosomal rearrangements. We have shown that our newly developed loxLE-hphNT1-loxRE and lox2272-natNT2-lox2272 gene-disruption cassettes can be present in the yeast genome together with the widely used loxP-marker gene-loxP cassettes without any recombination between the lox sequences. Moreover, we also developed a new phleomycin-resistant Cre-expressing vector (to excise multiple markers simultaneously) and two new standard loxP deletion cassettes containing hygromicin B and cloNAT as selecatable markers. To validate these cassettes, we created heterothallic auxotrophic S. cerevisiae strains, without the risk of incurring gross chromosomal rearrangements, and we showed an example of a fitness study of intraspecific hybrids deriving from parents with different adaptations to carbon-limited resources.

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Citations

Oct 19, 2013·PLoS Genetics·Elzbieta M PiatkowskaDaniela Delneri
Aug 24, 2012·PloS One·Samina Naseeb, Daniela Delneri
Apr 8, 2014·Proceedings of the National Academy of Sciences of the United States of America·Alexander FarwickEckhard Boles
Aug 16, 2012·Journal of Industrial Microbiology & Biotechnology·Viviane Castelo Branco ReisFernando Araripe Gonçalves Torres
May 9, 2013·Biotechnology Journal·Chan Woo Song, Sang Yup Lee
Mar 2, 2016·Molecular Biology and Evolution·Samina NaseebDaniela Delneri
Aug 27, 2014·Biotechnology Journal·Chan Woo SongSang Yup Lee
Dec 7, 2013·Fungal Genetics and Biology : FG & B·Tiantian WangXiaomin Zhao
Mar 6, 2015·PLoS Genetics·Carrie L FloodGray F Crouse
May 29, 2015·Microbial Cell Factories·Nicholas D GoldVincent J J Martin
Jan 11, 2017·Yeast·Robert Gnügge, Fabian Rudolf
Jan 19, 2018·Yeast·Marcin G FraczekDaniela Delneri
Jan 8, 2014·Antimicrobial Agents and Chemotherapy·Sushma SharmaK Ganesan
Jan 11, 2014·Applied Microbiology and Biotechnology·Sven Krappmann
Mar 29, 2013·Applied Microbiology and Biotechnology·Lydia Warth, Josef Altenbuchner
Aug 25, 2017·Proceedings. Biological Sciences·Samina NaseebSimon C Lovell
Dec 4, 2019·Biotechnology and Bioengineering·Jidapas My An-AdirekkunMegan N McClean
Jul 3, 2016·Cold Spring Harbor Protocols·Edward J Louis
Sep 2, 2018·Applied Microbiology and Biotechnology·R SwidahM P Ashe
Oct 6, 2020·Frontiers in Microbiology·Ursula FelsPetra Van Damme
Mar 17, 2019·The Journal of Cell Biology·Mariavittoria PizzingaMark P Ashe
Jul 1, 2021·ACS Synthetic Biology·Nan HaoKeith E Shearwin
Aug 26, 2021·MSphere·Neydis Moreno MoralesMegan N McClean
Oct 3, 2021·G3 : Genes - Genomes - Genetics·Daniel P NickersonJoshua M Milnes

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