One-step, PCR-mediated, gene disruption in the yeast Hansenula polymorpha

Previous evidence based on the experience of our laboratory showed that one -step gene disruption in the yeast Hansenula polymorpha is not straightforw ard. A systematic study of several factors which could affect gene disrupti on frequency was carried out. We found that the more critical factor affect ing one-step gene disruption in H. polymorpha is the length of the target g ene region flanking the marker gene. Target gene regions of about 1 kb flan king the marker gene were necessary to obtain a disruption frequency of abo ut 50%. However, the gene marker, either homologous or heterologous, the lo cus and the strain examined did not significantly affect the frequency of d isruption; the highest disruption frequency obtained for the YNR1 gene was in the strain HMI39, using the Saccharomyces cerevisiae URA3 gene as a mark er. Since long regions flanking the gene marker do not allow the easy PCR-m ediated strategies, developed for S cerevisiae, to obtain constructs to dis rupt a given gene in H. polymorpha, an alternative PCR strategy was develop ed. Copyright (C) 1999 John Wiley & Sons, Ltd.