Cloning, sequencing, and functional analysis of H-OLE1 gene encoding Delta9-fatty acid desaturase in Hansenula polymorpha

H-OLE1 a gene encoding Delta9-fatty acid desaturase (FAD) in Hansenula poly morpha strain CBS 1976, was isolated by hybridization based upon its homolo gy with the P-OLE1 gene cloned earlier from a related species, Pichia angus ta IFO 1475. The sequence of the H-OLE1 gene revealed high structural conse rvation With Delta9-FADs from various organisms. A putative 451-amino acid polypeptide encoded by the gene, like all other Delta9-FADs, contained two domains: an N-terminal catalytic domain containing three conserved histidin e clusters, and a C-terminal cytochrome b(5)-like domain which has been sug gested to be involved in electron transport in desaturation reactions. The whole H-OLE1 gene complemented a H. polymorpha fad1 mutation leading to a d efect in Delta9-FAD. However, the unsaturated fatty acid requirement that t he Saccharomyces cerevisiae ole 1 mutant displays was complemented by only the open reading frame of H-OLE1 driven by S. cerevisiae glyceroaldehyde-3- phosphate dehydrogenase promoter, but not by the intact H-OLE1, suggesting that the H. polymorpha Delta9-FAD was compatible with the desaturation syst em of S. cerevisiae whereas the promoter of the H-OLE1 gene had no activity in heterologous cells. It was shown by Northern hybridization that transcr iption of the H-OLE1 gene in H. polymorpha was slightly repressed by exogen ous Delta9-unsaturated fatty acid. An H. polymorpha disruption mutant (Delt aH-OLE1) was created by transformation of an fad1/FAD1 diploid with disrupt ed H-OLEI1::S-LEU2 linear DNA. It was shown by genetic and molecular analys es that input DNA was integrated in several copies into the chromosomal tar get to replace the mutated fad1 allele. Gas chromatography analysis showed identical fatty acid compositions in cells of both fad1 and Delta HOLE1 dis ruption mutants.