Chromosomal DNA patterns and gene stability of Pichia pastoris

We have clearly resolved four chromosomal bands from four Pichia pastoris (Komagataella pastoris) strains by using contour-clamped homogeneous electric field gel electrophoresis. The size of the P. pastoris chromosomal bands ranged from 1.7 Mb to 3.5 Mb and total genome size was estimated to be 9.5 Mb to 9.8 Mb; however, chromosome-length polymorphisms existed among four strains. Thirteen cloned genes isolated from strain GTS115 were assigned to the separated chromosomes, revealing that different hybridization patterns were observed in the AOX2 and URA3 genes among strains. P. pastoris is frequently used as an efficient host for heterologous gene expressions. We analysed chromosomal stability of strain GTS115-derived recombinant cell expressing human serum albumin during serial cultivation under the condition of vegetative and non-selective growth. No chromosomal rearrangements were observed and the expression constructs integrated into the his4 locus on chromosome I were very stable even at 83 generations, suggesting that stable expression would be carried out even in large-scale fermentation.