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US 8,481,286 B2Granted
Method for production of isoprenoid compounds
AssigneeAllylix, Inc.
InventorsBryan Julien, Richard P. Burlingame
Life of this patent
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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.
Claims49
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.
3 more elements
A method of isolating a variant squalene synthase enzyme, comprising:
- (a) culturing host cells that express the variant squalene synthase enzyme that, 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 host cell with a sterol is not required for growth, wherein the host cell is cultured in medium lacking a sterol;
- (b) selecting a host cell that grows in medium lacking a sterol, and also produces a terpene product;
- and
- (c) isolating the variant squalene synthase enzyme from the host cell, wherein:
- the isolated variant squalene synthase enzyme comprises one or more amino acid substitution(s) compared to a wild-type squalene synthase enzyme, and exhibits reduced cellular squalene synthase activity compared to the wild-type enzyme that does not comprise the one or more amino acid substitution(s);
- and
- a host cell expressing such enzyme produces a terpene product in the absence of sterol supplementation.
4 more elements
A method of producing an isoprenoid using a eukaryotic microbial host comprising a mutated ERG9 gene, wherein:
- the mutated ERG9 gene encodes a variant squalene synthase enzyme that comprises one or more amino acid substitutions compared to a wild-type squalene synthase enzyme;
- the variant squalene synthase enzyme exhibits reduced cellular squalene synthase activity compared to the enzyme that does not comprise the one or more amino acid substitution(s), whereby:
- the variant squalene synthase enzyme, when present and expressed in vivo in the 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 method comprises the steps of:
- (a) providing a host cell that includes a mutated ERG9 gene that encodes the variant squalene synthase enzyme and at least one isoprenoid synthase gene;
- (b) allowing the host cell to produce farnesyl pyrophosphate in medium lacking a sterol and to synthesize the isoprenoid from the farnesyl pyrophosphate in the medium lacking a sterol;
- and
- (c) isolating the isoprenoid synthesized by the host cell.
5 more elements
A method of producing an isoprenoid comprising the use of a mutated ERG9 gene, wherein:
- the mutated ERG9 gene encodes a variant squalene synthase enzyme that comprises one or more amino acid substitutions compared to a wild-type squalene synthase 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), whereby:
- the variant squalene synthase 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 method comprises the steps of:
- (a) providing a host cell that includes a mutated ERG9 gene that encodes the variant squalene synthase enzyme;
- (b) allowing the host cell to produce farnesyl pyrophosphate in medium lacking a sterol;
- (c) isolating farnesyl pyrophosphate from the host cell;
- (d) reacting the farnesyl pyrophosphate in vitro with one or more isoprenoid synthases to synthesize the isoprenoid;
- and
- (e) isolating the isoprenoid.
8 more elements
A method of producing an isoprenoid using a eukaryotic microbial host comprising a mutated ERG9 gene, wherein:
- the mutated ERG9 gene encodes a variant squalene synthase enzyme that comprises one or more amino acid substitutions compared to a wild-type squalene synthase enzyme;
- the variant squalene synthase enzyme, when present and expressed in vivo in the 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 results in reduced cellular squalene synthase activity compared to wild-type enzyme;
- the method comprises the steps of:
- (a) providing a Saccharomyces cerevisiae host cell that includes a mutated ERG9 gene that encodes the variant squalene synthase enzyme;
- (b) allowing the host cell to produce farnesyl pyrophosphate;
- (c) isolating farnesyl pyrophosphate from the host cell;
- (d) reacting the farnesyl pyrophosphate in vitro with one or more isoprenoid synthases to synthesize the isoprenoid, wherein an isoprenoid synthase is the H. muticus premnaspirodiene synthase, and the isoprenoid produced is premnaspirodiene;
- and
- (e) isolating the premnaspirodiene, by:
- (i) sequestering the premnaspirodiene by binding it to a hydrophobic resin;
- and
- (ii) isolating the premnaspirodiene from the hydrophobic resin.
9 more elements
A method of producing an isoprenoid using a host cell comprising a mutated ERG9 gene, wherein:
- the mutated ERG9 gene encodes a variant squalene synthase enzyme that comprises one or more amino acid substitutions compared to a wild-type squalene synthase enzyme;
- the variant squalene synthase enzyme, when present and expressed in vivo in the 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 results in reduced cellular squalene synthase activity compared to wild-type enzyme;
- the method comprises the steps of:
- (a) providing a Saccharomyces cerevisiae host cell that includes a mutated ERG9 gene that encodes the variant squalene synthase enzyme;
- (b) allowing the host cell to produce farnesyl pyrophosphate;
- (c) isolating farnesyl pyrophosphate from the host cell;
- (d) reacting the farnesyl pyrophosphate in vitro with one or more isoprenoid synthases to synthesize the isoprenoid, wherein an isoprenoid synthase is the H. muticus premnaspirodiene synthase, and the isoprenoid produced is premnaspirodiene;
- and
- (e) isolating the premnaspirodiene, by:
- adding a non-polar solvent, substantially immiscible with an aqueous phase, to the reaction of (d);
- separating the nonpolar solvent from the aqueous phase of the reaction of (d);
- and removing the premnaspirodiene from the non-polar phase by distillation.
9 more elements
Dependencies are inferred from the claim text.