Abstract
H-OLE1, a gene encoding Δ9-fatty acid desaturase (FAD) in Hansenula polymorpha strain CBS 1976, was isolated by hybridization based upon its homology with the P-OLE1 gene cloned earlier from a related species, Pichia angusta IFO 1475. The sequence of the H-OLE1 gene revealed high structural conservation with Δ9-FADs from various organisms. A putative 451-amino acid polypeptide encoded by the gene, like all other Δ9-FADs, contained two domains: an N-terminal catalytic domain containing three conserved histidine clusters, and a C-terminal cytochrome b 5-like domain which has been suggested to be involved in electron transport in desaturation reactions. The whole H-OLE1 gene complemented a H. polymorpha fad1 mutation leading to a defect in Δ9-FAD. However, the unsaturated fatty acid requirement that the Saccharomyces cerevisiae ole1 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. polymorphaΔ9-FAD was compatible with the desaturation system of S. cerevisiae whereas the promoter of the H-OLE1 gene had no activity in heterologous cells. It was shown by Northern hybridization that transcription of the H-OLE1 gene in H. polymorpha was slightly repressed by exogenous Δ9-unsaturated fatty acid. An H. polymorpha disruption mutant (ΔH-OLE1) was created by transformation of an fad1/FAD1 diploid with disrupted H-OLE1::S-LEU2 linear DNA. It was shown by genetic and molecular analyses that input DNA was integrated in several copies into the chromosomal target to replace the mutated fad1 allele. Gas chromatography analysis showed identical fatty acid compositions in cells of both fad1 and ΔHOLE1 disruption mutants.
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Received: 22 September 2000 / Received revision: 17 January 2000 / Accepted: 4 February 2000
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Lu, SF., Tolstorukov, I., Anamnart, S. et al. Cloning, sequencing, and functional analysis of H-OLE1 gene encoding Δ9-fatty acid desaturase in Hansenula polymorpha . Appl Microbiol Biotechnol 54, 499–509 (2000). https://doi.org/10.1007/s002530000369
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DOI: https://doi.org/10.1007/s002530000369