Isoprenoid compounds

A nucleic acid molecule that encodes a variant squalene synthase enzyme, wherein the variant enzyme comprises one or more amino acid substitutions in the squalene synthase enzyme whose sequence is set forth in SEQ ID NO: 4, and comprises a sequence of amino acid residues that is at least 95% identical to the squalene synthase enzyme whose sequence is set forth in SEQ ID NO: 4, whereby:nthe 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; andthe eukaryotic microbial host produces a terpene product in medium lacking a sterol.

An isolated nucleic acid molecule that encodes a variant squalene synthase enzyme, wherein:nthe variant enzyme comprises amino acid substitution(s) selected from among E149G/G168V/Y181N/Q290L/I323T, V186I, Y330H, Y181N/L271I/S275G/N320D, M214T/I242V/V277A/Q290L, E13V/L60P, D125V, L130P, V56L/S335P and H89Y/F110L/R224G/K251G in a wild-type Saccharomyces squalene synthase enzyme;the variant enzyme, when present and expressed in vivo in a eukaryotic microbial host as the only squalene synthase species, exhibits reduced activity compared to the wild-type squalene synthase enzyme, and 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.

An isolated nucleic acid molecule that encodes a modified squalene synthase enzyme, wherein:nthe encoded variant squalene synthase enzyme has at least 95% sequence identity with the squalene synthase whose sequence is set forth in SEQ ID NO: 4; andthe isolated nucleic acid molecule comprises the nucleic acid modifications selected from among A691G/G748T/T786A/A1114T/T1213C/T1290C, C72A/110ΔA/G801A, T989A/G1112A/G1220A/T1233C, T786A/A1025G/T1056A/A1068G/A1203G, T886C/A969G/T1075C/A1114T, T84A/A283T/T424C/A440G/T1076C, A619T/T634C/C962T, A150T/T410G/G411T/T1248C and C510T/T573C/A918G/A997G.

A variant squalene synthase enzyme, comprising one or more amino acid substitutions in the squalene synthase enzyme whose sequence is set forth in SEQ ID NO: 4, and the variant enzyme comprises a sequence of amino acid residues that is at least 95% identical to the squalene synthase enzyme whose sequence is set forth in SEQ ID NO: 4, whereby:nthe variant enzyme exhibits reduced cellular squalene synthase activity compared to wild-type enzyme, but 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; andthe host produces a terpene product in medium lacking a sterol.

A variant squalene synthase enzyme, comprising substitutions selected from among E149G/G168V/Y181N/Q290L/I323T, V186I, Y330H, Y181N/L271I/S275G/N320D, M214T/I242V/V277A/Q290L, E13V/L60P, D125V, L130P, V56L/S335P and H89Y/F110L/R224G/K251G, wherein:nthe unmodified squalene synthase comprises the sequence of amino acids set forth in SEQ ID NO:4 or a sequence of amino acids having at least 95% sequence identity therewith; andthe variant enzyme, when present and expressed in vivo in a eukaryotic microbial host as the only squalene synthase species, exhibits reduced activity compared to the enzyme without the amino acid substitutions, and 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.

A nucleic acid molecule that encodes a variant squalene synthase enzyme, wherein the variant enzyme comprises an amino acid substitution in the squalene synthase enzyme whose sequence is set forth in SEQ ID NO: 4 or in a squalene synthase enzyme having at least 95% sequence identity therewith, whereby:nthe variant enzyme exhibits reduced cellular squalene synthase activity compared to the enzyme that does not comprise the amino acid substitution;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; andthe eukaryotic microbial host produces farnesyl pyrophosphate or premnaspirodiene in medium lacking a sterol.

An isolated nucleic acid molecule encoding a Saccharomyces cerevisiae squalene synthase that comprises the nucleic acid modifications selected from among A691G/G748T/T786A/A1114T/T1213C/T1290C, C72A/110ΔA/G801A, T989A/G1112A/G1220A/T1233C, T786A/A1025G/T1056A/A1068G/A1203G, T886C/A969G/T1075C/A1114T, T84A/A283T/T424C/A440G/T1076C, A619T/T634C/C962T, A150T/T410G/G411T/T1248C and C510T/T573C/A918G/A997G, wherein the unmodified Saccharomyces cerevisiae squalene synthase comprises the sequence of amino acids set forth in SEQ ID NO:4.

A nucleic acid molecule that encodes a variant squalene synthase enzyme, wherein the variant enzyme comprises an amino acid substitution in the squalene synthase enzyme whose sequence is set forth in SEQ ID NO: 4 or in a squalene synthase enzyme having at least 95% sequence identity therewith, whereby:nthe variant enzyme exhibits reduced cellular squalene synthase activity compared to the enzyme that does not comprise the amino acid substitution;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; andthe eukaryotic microbial host produces a terpene product in medium lacking a sterol such that the level of the terpene product is at least equivalent to the level of terpene product produced by the strain of the eukaryotic microbial host that requires a sterol in the medium for growth.

A variant squalene synthase enzyme, comprising an amino acid substitution in the squalene synthase enzyme whose sequence is set forth in SEQ ID NO: 4 or in a squalene synthase enzyme having at least 95% sequence identity therewith, wherein the variant enzyme comprises a sequence of amino acid residues that is at least 95% identical to the squalene synthase enzyme whose sequence is set forth in SEQ ID NO: 4, whereby:nthe variant enzyme exhibits reduced cellular squalene synthase activity compared to the enzyme that does not comprise the amino acid substitution, but 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: andthe eukaryotic microbial host produces a terpene product in medium lacking a sterol such that the level of the terpene product is at least equivalent to the level of terpene product produced by the strain of the eukaryotic microbial host that requires a sterol in the medium for growth.