Production of auxotrophic mutants of pichia methanolica

摘要

Methods for preparing Pichia methanolica cells having auxotrophic mutations are disclosed. The methods comprise the steps of: (a) exposing P. methanolica cells to mutagenizing conditions; (b) culturing the cells from step (a) in a rich medium to allow mutations to become established and replicated in at least a portion of the cells; (c) culturing the cells from step (b) in a culture medium deficient in assimilable nitrogen to deplete cellular nitrogen stores; (d) culturing the cells from step (c) in a defined culture medium comprising an inorganic nitrogen source and an amount of nystatin sufficient to kill growing P. methanolica cells to select for cells having a deficiency in a nutritional gene; and (e) culturing the selected cells from step (d) in a rich culture medium.