A systematical investigation on the genetic stability of multi-copy Pichia pastoris strains

A battery of Pichia pastoris transformants, G1, G6, A2, A3, C3, carrying 1, 6, 12, 18 and 29 copies of porcine insulin precursor (PIP) gene, were employed to investigate the genetic stability of these multi-copy P. pastoris strains. Both G6 and C3 maintained their original copy numbers in serial culture without methanol induction for 35 generations. With methanol as an inducer and carbon source, G1 and G6 remained stable but the average copy numbers (ACNs) of PIP gene in A2, A3, C3 were decreased to 10, 10 and 15 copies, respectively, after 96 h of induction in shake-flask culture. A PIP copy number distribution analysis of fermentation samples of C3 indicated that the majority of yeast cells have partially or completely lost their PIP genes. In 5-l fermentor culture, the ACNs of PIP gene in A2, A3, C3 were also decreased to 10, 15, 21 copies, respectively, after 72 h of methanol induction.