Isoprenoid compounds

• US 8,609,371 B2
• Filed: 08/12/2009
• Issued: 12/17/2013
• Est. Priority Date: 08/12/2008
• Status: Active Grant

• Current Assignee: Minnesota Specialty Yeast LLC
• Original Assignee: Allylix Inc. (Evolva Holding SA)
• Inventors: Julien, Bryan, Burlingame, Richard P.
• Primary Examiner(s): Gebreyesus, Kagnew H
• Application Number:
  US12/540,050
• Publication Number:
  US 20100151555A1
• Time in Patent Office: 1,588 Days
• Field of Search: None
• US Class Current: 435/69.1
• CPC Class Codes: C12N 15/1079 Screening libraries by altering the phenotype or phenotypic trait of the host reporter assays C12N15/1086
• CPC Class Codes: C12N 9/1085 transferring alkyl or aryl groups other than methyl groups (2.5)
• CPC Class Codes: C12P 5/002 cyclic compounds containing at least three condensed carbocyclic rings C12P15/00
• CPC Class Codes: C12P 5/007 containing one or more isoprene units, i.e. terpenes carotenes C12P23/00

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.

Claims

1. 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;

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 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 3, wherein the yeast microbial host is selected from the group consisting of 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 nucleic acid molecule of claim 1, wherein the squalene synthase enzyme encoded by the nucleic acid molecule has a reduced V_max for squalene synthesis.

7. The nucleic acid molecule of claim 1, wherein the squalene synthase enzyme encoded by the nucleic acid molecule has an increased Michaelis constant (K_m) for its farnesyl pyrophosphate (FPP) substrate.

8. The nucleic acid molecule of claim 1 that encodes a squalene synthase enzyme that, when expressed in vivo in the eukaryotic microbial host, produces squalene at a rate of less than 75% of the enzyme that does not comprise the one or more amino acid substitution(s).

9. The nucleic acid molecule of claim 8 that encodes a squalene synthase enzyme that, when expressed in vivo in the eukaryotic microbial host, produces squalene at a rate of less than 50% of the enzyme that does not comprise the one or more amino acid substitution(s).

10. The nucleic acid molecule of claim 9 that encodes a squalene synthase enzyme that, when expressed in vivo in the eukaryotic microbial host, produces squalene at a rate of less than 25% of the enzyme that does not comprise the one or more amino acid substitution(s).

11. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule, when expressed in vivo, causes a host cell to produce a greater concentration of an isoprenoid in grams of isoprenoid per liter of culture than a corresponding host cell expressing the nucleic acid molecule that does not comprise the amino acid substitutions.

20. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule further comprises at least one additional nucleotide base at the 5′

-terminus, the 3′

-terminus, or at both the 5′

-terminus and 340 -terminus of the nucleic acid molecule encoding a variant squalene synthase enzyme, to which the nucleic acid molecule encoding a squalene synthase enzyme is operatively linked.

21. The nucleic acid molecule of claim 20, wherein the at least one additional base is a linker or an adaptor, or forms a restriction endonuclease cleavage.

22. A vector, comprising the nucleic acid molecule of claim 1.

23. The vector of claim 22, wherein the vector is capable of replication in a prokaryotic organism.

24. The vector of claim 22, wherein the vector is capable of replication in a eukaryotic organism.

25. The vector of claim 22, wherein the vector is capable of integration into the genome of a mammalian cell line.

26. The vector of claim 22, wherein the vector further comprises at least one additional nucleic acid sequence at the 5′

-terminus, the 3′

-terminus, or at both the 5′

-terminus and 3′

-terminus, to which the nucleic acid molecule encoding a squalene synthase enzyme is operatively linked.

27. The vector of claim 26, wherein the at least one additional nucleic acid sequence is selected from the group consisting of linkers, adaptors, restriction endonuclease cleavage sites, regulatory sequences, and coding sequences.

28. A host cell comprising the nucleic acid molecule of claim 1.

29. The host cell of claim 28, wherein the host cell is a prokaryotic cell.

30. The host cell of claim 28, wherein the host cell is a eukaryotic cell.

31. The host cell of claim 28, wherein the host cell further comprises at least one additional nucleotide base at the 5′

-terminus, the 3′

-terminus, or at both the 5′

-terminus and 3′

-terminus of the nucleic acid molecule that encodes a variant squalene synthase enzyme, to which the nucleic acid molecule encoding a squalene synthase enzyme is operatively linked.

32. The host cell of claim 31, wherein the at least one additional base is a linker or an adaptor, or forms a restriction endonuclease cleavage site.

33. A host cell transformed or transfected with the vector of claim 22.

12. An isolated nucleic acid molecule that encodes a variant squalene synthase enzyme, wherein:

the 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.

13. The isolated nucleic acid molecule of claim 12, comprising one or more silent mutations.

14. The isolated nucleic acid molecule of claim 12 in which the isolated nucleic acid molecule is an isolated nucleic acid molecule in which the squalene synthase enzyme encoded by the isolated nucleic acid molecule differs by one or more conservative amino acid substitutions.

15. An isolated nucleic acid molecule that encodes a modified squalene synthase enzyme, wherein:

the 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; and

the isolated nucleic acid molecule comprises the nucleic acid modifications selected from among A691G/G748T/T786A/A1114T/T1213C/T1290C, C72A/110Δ

A/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.

16. The isolated nucleic acid molecule of claim 15, 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.

17. The isolated nucleic acid molecule of claim 16, 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.

18. The isolated nucleic acid molecule of claim 17, wherein the encoded variant squalene synthase enzyme comprises a sequence that has at least 99.5% sequence identity with the sequence of amino acids set forth in SEQ ID NO:

4.

19. The isolated nucleic acid molecule of claim 18, wherein the encoded variant squalene synthase enzyme comprises a sequence that has at least 99.8% sequence identity with the sequence of amino acids set forth in SEQ ID NO:

4.

34. 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;

the 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; and

the host produces a terpene product in medium lacking a sterol.

35. The valiant squalene synthase enzyme of claim 34, wherein the eukaryotic microbial host is a fungal microbial host.

36. The variant squalene synthase enzyme of claim 35, wherein the fungal microbial host is a yeast microbial host.

37. The variant squalene synthase enzyme of claim 36, wherein the yeast microbial host is selected from the group consisting of Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia and Torulopsis.

38. The variant squalene synthase enzyme of claim 37, wherein the Saccharomyces yeast microbial host is Saccharomyces cerevisiae.

39. The variant squalene synthase enzyme of claim 34, wherein the squalene synthase enzyme has a reduced V_max for squalene synthesis.

40. The variant squalene synthase enzyme of claim 34, wherein the squalene synthase enzyme has an increased K_m, for its FPP substrate.

41. The variant squalene synthase enzyme of claim 34, wherein the squalene synthase enzyme produces squalene in vivo in the eukaryotic microbial host at a rate of less than 75% of the wild-type enzyme.

42. The variant squalene synthase enzyme of claim 41, wherein the squalene synthase enzyme produces squalene in vivo in the eukaryotic microbial host at a rate of less than 50% of the wild-type enzyme.

43. The variant squalene synthase enzyme of claim 42, wherein the squalene synthase enzyme produces squalene in vivo in the eukaryotic microbial host at a rate of less than 25% of the wild-type enzyme.

44. 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:

the 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; and

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 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.

45. The variant squalene synthase enzyme of claim 44, wherein the squalene synthase enzyme comprises a conservative amino acid substitution compared to the sequence of amino acids set forth in SEQ ID NO:

4.

46. 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;

the 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; and

the eukaryotic microbial host produces farnesyl pyrophosphate or premnaspirodiene in medium lacking a sterol.

47. 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/110Δ

A/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.

48. 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;

the 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; and

the 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.

49. 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;

the 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; and

the 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.