CHROMOSOMAL DNA PATTERNS AND GENE STABILITY OF PICHIA-PASTORIS

We have clearly resolved four chromosomal bands from four Pichia pasto ris (Komagataella pastoris) strains by using contour-clamped homogeneo us electric field gel electrophoresis. The size of the P. pastoris chr omosomal 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 polymorph isms existed among four strains. Thirteen cloned genes isolated from s train GTS115 were assigned to the separated chromosomes, revealing tha t different hybridization patterns were observed in the AOX2 and URA3 genes among strains. P. pastoris is frequently used as an efficient ho st for heterologous gene expressions. We analysed chromosomal stabilit y of strain GTS115-derived recombinant cell expressing human serum alb umin during serial cultivation under the condition of vegetative and n on-selective growth. No chromosomal rearrangements were observed and t he expression constructs integrated into the his4 locus on chromosome I were very stable even at 83 generations, suggesting that stable expr ession would be carried out even in large-scale fermentation. (C) 1998 John Wiley & Sons, Ltd.