Method for production of isoprenoid compounds
AssigneeAllylix, Inc.
InventorsBryan Julien, Richard Burlingame
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Abstract
The present invention is directed to variant squalene synthase enzymes, including Saccharomyces cerevisiae squalene synthase enzymes, and to nucleic acid molecules encoding these variant enzymes. These variant enzymes produce squalene at a lower rate than the wild-type enzyme, allowing more farnesyl pyrophosphate to be utilized for production of isoprenoid compounds, while still producing sufficient squalene to allow the S. cerevisiae cells to grow without the requirement for supplementation by sterols such as ergosterol. These variant enzymes, therefore, are highly suitable for the efficient production of isoprenoids.
Claims31
A method for selecting a host cell that comprises nucleic acid that encodes a defective ERG9 gene, wherein the gene encodes a protein that permits growth of the 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 in the sterol-free medium to thereby isolate a host cell that comprises the defective ERG9 gene that encodes a protein that permits growth of the host in sterol-free medium and production of terpenes by the host cell in sterol-free medium.
A method of producing an isoprenoid in a eukaryotic microbial host comprising a mutated ERG9 gene, comprising:
- (a) providing a microbial host cell that comprises:
- a defective ERG9 gene that encodes a protein that permits growth of the host in sterol-free medium and production of terpenes by the host cell in sterol-free medium;
- and
- nucleic acid encoding an isoprenoid synthase;
- and
- (b) allowing the host cell to produce farnesyl pyrophosphate in medium lacking a sterol and to synthesize an isoprenoid in the medium lacking a sterol to thereby produce the isoprenoid synthesized by the host cell.
A method of producing an isoprenoid, comprising:
- (a) providing a host cell that comprises a defective ERG9 gene that encodes a protein that permits growth of the host in sterol-free medium and production of terpenes by the host cell in sterol-free medium;
- (b) allowing the host cell to produce farnesyl pyrophosphate in medium lacking a sterol;
- (c) isolating farnesyl pyrophosphate from the host cell;
- and
- (d) reacting the farnesyl pyrophosphate in vitro with one or more isoprenoid synthases to thereby produce the isoprenoid.
A method of producing an isoprenoid, comprising:
- (a) providing a host cell that comprises a defective ERG9 gene that encodes a protein that permits growth of the host in sterol-free medium and production of terpenes by the host cell in sterol-free medium;
- (b) allowing the host cell to produce farnesyl pyrophosphate;
- (c) isolating farnesyl pyrophosphate from the host cell;
- and
- (d) reacting the farnesyl pyrophosphate in vitro with one or more isoprenoid synthases to thereby produce the isoprenoid.
A nucleic acid molecule that encodes a variant squalene synthase enzyme, wherein:
- the variant squalene synthase enzyme comprises one or more amino acid substitution(s) in a squalene synthase enzyme;
- the squalene synthase enzyme is a yeast enzyme;
- the variant enzyme exhibits reduced cellular squalene synthase activity compared to the enzyme that does not comprise the one or more amino acid substitution(s);
- the variant enzyme, when present and expressed in vivo in a eukaryotic microbial host as the only squalene synthase species, catalyzes the synthesis of squalene at a sufficiently high rate that supplementation of the eukaryotic microbial host with a sterol is not required for growth;
- and
- the eukaryotic microbial host that contains the nucleic acid molecule can produce a terpene product in medium lacking a sterol.
Dependencies are inferred from the claim text.