GENETIC SELECTION MARKERS BASED ON ENZYMATIC ACTIVITIES OF THE PYRIMIDINE SALVAGE PATHWAY

摘要

The present invention relates to a method of site-directed integration into a genetic locus encoding at least one activity of the pyrimidine salvage pathway in a host cell, wherein said activity of the pyrimidine salvage pathway is purine/cytosine permease (FcyB), cytosine deaminase (FcyA), uracil-phosphoribosyl-transferase (Uprt), concentrative nucleoside transporter (CntA) or uridine kinase (UK), comprising: a) providing a host cell comprising a functional copy of the genetic locus encoding at least one activity of the pyrimidine salvage pathway; (b) introducing a gene or sequence of interest into said host cell via transformation of an integrative nucleic acid construct which comprises 3' and/or 5' of the gene or sequence of interest flanks being homologous to said genetic locus or which carries a sequence being homologous to said genetic locus of the pyrimidine salvage pathway and thus allowing for a homologous recombination at said genetic locus, wherein said homologous recombination is capable of causing an inactivation or reduction of the activity encoded by said genetic locus; (c) growing a transformed host cell under selective medium conditions, wherein said medium comprises an efficient amount of 5-flucytosine (5-FC), 5-fluorouracil (5-FU) or 5-fluorouridine (5-FUR); and (d) selecting a host cell which is capable of growing under the medium conditions of step (c). Also envisaged is a host cell, comprising at least one gene or sequence of interest in one or more genetic loci encoding an activity of the pyrimidine salvage pathway wherein said gene or sequence of interest replaces or partially replaces the sequence encoding said at least one activity of the pyrimidine salvage pathway at said locus, the use of such a host cell for the production of several activities, as well as the use of a genetic locus encoding at least one activity of the pyrimidine salvage pathway in a host cell in a process of transforming said host cell or a process of genetically modifying said host cell.