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 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 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 histidine clusters, and a C-terminal cytochrome b5-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 delta9-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. polymorpha delta9-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 delta9-unsaturated fatty acid. An H. polymorpha disruption mutant (deltaH-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 deltaHOLE1 disruption mutants.

Isolation and characterization of mutations affecting expression of the delta9- fatty acid desaturase gene, OLE1, in Saccharomyces cerevisiae.

Expression of the delta9- fatty acid desaturase gene, OLE1, of Saccharomyces cerevisiae is negatively regulated transcriptionally and post-transcriptionally by unsaturated fatty acids. In order to isolate mutants exhibiting irregulation of OLE1 expression, we constructed an OLE1p-PHO5 fusion gene as a reporter consisting of the PHO5 gene encoding repressible acid phosphatase (rAPase) under the control of the OLE1 promoter (OLE1p). By EMS mutagenesis, we isolated three classes of mutants, pfo1, pfo2 and pfo3 positive regulatory factor for OLE1) mutants, which show decreased rAPase activity under derepression conditions (absence of oleic acid). Analysis of the transcription of OLE1 in these pfo mutants revealed that pfo1 and pfo3 mutants have a defect in the regulation of OLE1 expression at the transcriptional level while pfo2 mutants were suggested to have a mutation affecting OLE1 expression at a post-transcriptional step. In addition, four other classes of mutants, nfo1, nfo2, nfo3 and nfo4 (negative factor for OLE1) mutants that have mutations causing strong expression of the OLE1p-PHO5 fusion gene under repression conditions (presence of oleic acid), were isolated. Results of Northern analysis of OLE1 as well as OLE1p-PHO5 transcripts in nfo mutants suggested that these mutations occurred in genes encoding global repressors. We also demonstrated that TUP1 and SSN6 gene products are required for full repression of OLE1 gene expression, by showing that either tup1 or ssn6 mutations greatly increase the level of the OLE1 transcript.

The P-OLE1 gene of Pichia angusta encodes a delta 9-fatty acid desaturase and complements the ole1 mutation of Saccharomyces cerevisiae.

Three PCR-amplified DNA fragments hybridizing with the OLE1 gene encoding delta 9-fatty acid desaturase of Saccharomyces cerevisiae were obtained using, respectively, genomic DNAs of one strain each of Kluyveromyces thermotolerans, Pichia angusta and Yarrowia lipolytica as templates. A gene designated P-OLE1 was cloned from the above fragment of P. angusta and sequenced. An open reading frame of P-OLE1 encodes a 49.6-kDa protein consisting of 451 amino acid residues, which shows high identity (62%) and similarity (89%) to that deduced from the OLE1 nucleotide sequence. Expression of P-OLE1 driven by the S. cerevisiae GAP promoter or its own promoter complemented the ole1 mutation of S.cerevisiae. Transcription of P-OLE1 in the native host was suggested to be partially repressed by oleic acid in the medium, as was that of OLE1 in S. cerevisiae and a similar gene in Y. lipolytica, but that of a similar gene in K. thermotolerans was not.