Cloning and characterization of theYarrowia lipolytica squalene synthase (SQS1) gene and functional complementation of theSaccharomyces cerevisiae erg9 mutation
✍ Scribed by Merkulov, Sergei; van Assema, Friso; Springer, Jan; Fernandez del Carmen, Asun; Mooibroek, H.
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 300 KB
- Volume
- 16
- Category
- Article
- ISSN
- 0749-503X
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✦ Synopsis
The squalene synthase (SQS) gene encodes a key regulatory enzyme, farnesyl-diphosphate farnesyltransferase (EC 2.5.1.21), in sterol biosynthesis. The SQS1 gene was isolated from a subgenomic library of the industrially important yeast Yarrowia lipolytica, using PCR-generated probes. Probes were based on conserved regions of homologues from different organisms. The complete nucleotide sequence of the coding region and the corresponding amino acid sequence were determined. The sequences showed extensive homologies with squalene synthase genes and enzymes from a number of other organisms and extreme amino acid conservation within the binding and catalytic domains. Direct cloning of a 4.3 kb genomic Y. lipolytica fragment, also comprising its own promoter and terminator sequences, into autonomously replicating plasmid YEp352 and subsequent transformation of a Saccharomyces cerevisiae mutant strain with relevant erg9: ura3±1 markers, resulted in functional complementation of these de®ciencies, although Northern blot analyses did not reveal a unique full-length messenger. The availability of the Y. lipolytica SQS1 gene (GenBank Accession No. AF092497) offers prospects for metabolic engineering of the isoprenoid and sterol biosynthetic pathways.