Method for production of isoprenoid compounds

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.

What is claimed is: 
     
         1 . A nucleic acid molecule that encodes a variant squalene synthase enzyme, 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:
 the variant enzyme exhibits reduced cellular squalene synthase activity compared to the enzyme whose sequence is set forth in SEQ ID NO: 4;   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 such that supplementation of the eukaryotic microbial host with a sterol is not required for growth; and   the eukaryotic microbial host produces a terpene product in medium lacking a sterol.   
     
     
         2 . The nucleic acid molecule of  claim 1 , wherein the eukaryotic microbial host is a fungal microbial host. 
     
     
         3 . The nucleic acid molecule of  claim 2 , wherein the fungal microbial host is a yeast microbial host. 
     
     
         4 . The nucleic acid molecule of  claim 1 , wherein the microbial host is selected from among  Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia  and  Torulopsis.    
     
     
         5 . The nucleic acid molecule of  claim 4 , wherein the yeast microbial host is  Saccharomyces cerevisiae.    
     
     
         6 . The isolated nucleic acid molecule of  claim 1 , wherein the encoded variant squalene synthase enzyme comprises a sequence that has at least 97.5% sequence identity with the sequence of amino acids set forth in SEQ ID NO: 4. 
     
     
         7 . The isolated nucleic acid molecule of  claim 1 , wherein the encoded variant squalene synthase enzyme comprises a sequence that has at least 99% sequence identity with the sequence of amino acids set forth in SEQ ID NO: 4. 
     
     
         8 . A vector, comprising the nucleic acid molecule of  claim 1 . 
     
     
         9 . A host cell, comprising the nucleic acid molecule of  claim 1 , wherein the host cell is a prokaryotic cell. 
     
     
         10 . An isolated host cell, comprising the nucleic acid molecule of  claim 1 , wherein the host cell is a eukaryotic cell. 
     
     
         11 . A host cell, comprising the nucleic acid molecule of  claim 1 , wherein the host cell is a yeast host cell. 
     
     
         12 . The host cell of  claim 10  that is selected from among  Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia  and  Torulopsis.    
     
     
         13 . The host cell of  claim 12  that is a  Saccharomyces cerevisiae  cell. 
     
     
         14 . A variant squalene synthase enzyme, comprising 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:
 the variant enzyme exhibits reduced cellular squalene synthase activity compared to the 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;   the host produces a terpene product in medium lacking a sterol; and   the eukaryotic microbial host is a yeast microbial host.   
     
     
         15 . The variant squalene synthase enzyme of  claim 14 , wherein the yeast microbial host is  Saccharomyces cerevisiae.    
     
     
         16 . A method for producing a terpene, comprising culturing a cell of  claim 9  under conditions for production of a terpene product. 
     
     
         17 . 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;   (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) isolating a transformant from step (d) that grows in sterol-free medium and produces a terpene product in the sterol-free medium.   
     
     
         18 . The method of  claim 17 , wherein the ERG9 gene is a  Saccharomyces  ERG9 gene. 
     
     
         19 . The method of  claim 18 , wherein the  Saccharomyces  ERG9 gene is a  Saccharomyces cerevisiae  ERG9 gene. 
     
     
         20 . The method of  claim 17 , wherein the eukaryotic microbial host is a fungal microbial host. 
     
     
         21 . The method of  claim 20 , wherein the fungal microbial host is a yeast microbial host. 
     
     
         22 . The method of  claim 21 , wherein the yeast microbial host is a yeast of the genus  Saccharomyces.    
     
     
         23 . The method of  claim 22 , wherein the yeast of the genus  Saccharomyces  is  Saccharomyces cerevisiae.    
     
     
         24 . The method of  claim 17 , wherein the terpene synthase gene that produces a detectable and measurable terpene product is a  Hyoscyamus muticus  premnaspirodiene synthase (HPS) gene. 
     
     
         25 . The method of  claim 23 , wherein the strain of  S. cerevisiae  is ALX7-95 (his3, trp1, erg9::HIS3, HMGcat/TRP1::rDNA, dpp1), a leucine prototroph of strain CALI-5, that contains a plasmid expressing the  Hyoscyamus muticus  premnaspirodiene synthase (HPS) gene. 
     
     
         26 . A host cell, comprising the variant squalene synthase enzyme of  claim 14 , wherein the host cell is a bacterial cell or a yeast cell. 
     
     
         27 . An isolated host cell, comprising the variant squalene synthase enzyme of  claim 14 . 
     
     
         28 . The method of  claim 16 , wherein the terpene product is isolated. 
     
     
         29 . A method for producing a host cell that encodes a variant squalene synthase, comprising introducing a nucleic acid molecule of  claim 1  into the cell.