Open AccessA tool kit for molecular genetics of Kluyveromyces lactis comprising a congenic strain series and a set of versatile vectors
Author(s) -
Heinisch Jürgen J.,
Buchwald Ulf,
Gottschlich André,
Heppeler Nele,
Rodicio Rosaura
Publication year - 2010
Publication title -
fems yeast research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.991
H-Index - 92
eISSN - 1567-1364
pISSN - 1567-1356
DOI - 10.1111/j.1567-1364.2009.00604.x
Subject(s) - biology , kluyveromyces lactis , ura3 , genetics , strain (injury) , congenic , cre recombinase , plasmid , saccharomyces cerevisiae , shuttle vector , gene , recombinase , recombination , recombinant dna , vector (molecular biology) , transgene , genetically modified mouse , anatomy
A set of different marker deletions starting with a ura3 derivative of the Kluyveromyces lactis type strain CBS2359 was constructed. After a first cross to obtain a strain with the opposite mating type that also carried a leu2 allele, continuous back‐crosses were used to obtain a congenic strain series with different marker combinations, including deletions in KlHIS3, KlADE2 and KlLAC4 . Enzymes involved in carbohydrate metabolism were shown to behave very similarly to the original type strain and other K. lactis strains investigated previously. Moreover, a vector series of Saccharomyces cerevisiae genes flanked by loxP sites was constructed to be used as heterologous deletion cassettes in K. lactis , together with two plasmids for expression of Cre‐recombinase for marker regeneration. To increase the frequency of homologous recombination, the Klku80 deletion was also introduced into the congenic strain series. A PCR‐based method for determination of mating type is provided.