Patent ID: 8481286
Filing Date: 2013-07-09
Classification: C12N,C12P

Abstract:
1. A method of isolating a defective ERG9 gene that permits growth of a host in sterol-free medium and production of terpenes in sterol-free medium, comprising: (a) isolating a wild-type ERG9 gene to produce an isolated wild-type ERG9 gene; (b) subjecting the isolated wild-type ERG9 gene to mutagenesis to generate a pool of ERG9 mutants, wherein the pool comprises ERG9 mutants that encode variant squalene synthase enzymes that comprise one or more amino acid substitution(s) compared to the wild-type squalene synthase enzyme; (c) transforming mutants from the pool of erg9 mutants generated in step (b) into a strain of a eukaryotic microbial host that contains an expressed terpene synthase gene that produces a detectable and measurable terpene product, the strain of the eukaryotic microbial host being transformed in such a manner that replacement of the preexisting ERG9 allele with an erg9 mutation allows the strain to grow in a sterol-free medium; (d) growing the transformants in sterol-free medium; and (e) selecting a transformant from step (d) that grows in sterol-free medium and produces a terpene product in the sterol-free medium and isolating the defective ERG9 gene from the transformant.