ISOPRENE SYNTHASE VARIANTS FOR IMPROVED MICROBIAL PRODUCTION OF ISOPRENE

• US 20100003716A1
• Filed: 04/23/2009
• Published: 01/07/2010
• Est. Priority Date: 04/23/2008
• Status: Active Grant

• Current Assignee: Goodyear Tire & Rubber Company
• Original Assignee: Danisco US, Inc. (International Flavors & Fragrances Incorporated)
• Inventors: Bott, Richard R., Weyler, Walter, Miasnikov, Andrei, Wells, Derek H., Cervin, Marguerite A., Peres, Caroline M., Whited, Gregory M.
• Granted Patent: US 8,173,410 B2
• Time in Patent Office: Days
• US Class Current: 435/40.5
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 5/007 containing one or more isoprene units, i.e. terpenes carotenes C12P23/00
• CPC Class Codes: C12Y 402/03027 Isoprene synthase (4.2.3.27)

Abstract

The present invention provides methods and compositions comprising at least one isoprene synthase enzyme with improved catalytic activity and/or solubility. In particular, the present invention provides variant plant isoprene synthases for increased isoprene production in microbial host cells. Biosynthetically produced isoprene of the present invention finds use in the manufacture of rubber and elastomers.

Claims

1. An isolated poplar isoprene synthase variant, wherein said variant comprises a truncation in the N-terminal portion of isoprene synthase.

2. The isolated poplar isoprene synthase variant of claim 1, wherein the isoprene synthase variant has an increased specific activity compared to a full length isoprene synthase.

3. The isolated poplar isoprene synthase variant of claim 1, wherein the isoprene synthase is P. alba isoprene synthase of SEQ ID NO:

120.

4. The isolated poplar isoprene synthase variant of claim 3, wherein the variant is selected from the group consisting of:

an MEA variant (SEQ ID NO;

122), an MSV variant (SEQ ID NO;

124), an MVS variant (SEQ ID NO;

126), an MTE variant (SEQ ID NO;

128), an MNV variant (SEQ ID NO;

130).

5. The isolated poplar isoprene synthase variant of claim 4, wherein the variant is an MEA variant (SEQ ID NO:

122).

6. The isolated poplar isoprene synthase variant of claim 3, wherein the variant is selected from the group consisting of:

a TRC (−

3) variant (SEQ ID NO;

136), a TRC (−

4) variant (SEQ ID NO;

138), a TRC (−

5) variant (SEQ ID NO;

140), a TRC (−

6) variant (SEQ ID NO;

142) and a TRC (−

7) variant (SEQ ID NO;

144).

7. The isolated poplar isoprene synthase variant of claim 1, wherein the variant is a MET variant of P. tremuloides isoprene synthase (SEQ ID NO:

146).

8. The isolated poplar isoprene synthase variant of claim 1, wherein the variant is a MET variant of P. trichocharpa isoprene synthase (SEQ ID NO:

148).

9. An isolated poplar isoprene synthase variant, wherein the variant comprises a substitution of one or more amino acid residues of a wild type isoprene synthase;

and wherein the isoprene synthase variant has increased isoprene synthase activity compared to a wild type isoprene synthase.

10. The isolated poplar isoprene synthase variant of claim 9, wherein increased isoprene synthase activity is indicated by a host cell comprising the isoprene variant growing at a faster rate in the presence of dimethylallyl pyrophosphate (DMAPP) compared to a host cell comprising a parent isoprene synthase.

11. The isolated poplar isoprene synthase variant of claim 9, wherein the isoprene synthase is the P. alba isoprene synthase of SEQ ID NO:

120.

12. The isolated poplar isoprene synthase variant of claim 11, wherein the variant comprises one of more amino acid substitutions selected from the group consisting of V10M, F12S, T15A, E18G, V58I, V58F, L70Q, L70V, L70T, T71P, V79L, E89D, G94A, S119F, F120L, G127R, E175V, T212I, S257A, R262G, A266G, F280L, N297K, F305L, L319M, E323K, A328T, D342E, A359T, K366N, E368D, L374M, S396T, V418S, K438N, H440R, T442I, T442A, I449V, A469S, K500R, K505Q, G507S, S509N, F511Y, and N532K.

13. The isolated poplar isoprene synthase variant of claim 11, wherein at least one amino acid substitution is a L70R substitution.

14. The isolated poplar isoprene synthase variant of claim 11, wherein the variant comprises one of more amino acid substitutions selected from the group consisting of G127R/F511Y, L70Q/G94A/R262G/F305L, F12S/T15A/E18G/N297K, S396T/T442I, V10M/E323K, F120L/A266G, K438N/K500R, V79L/S509N, E175V/S257A/E368D/A469S, T71P/L374M, F280L/H440R, E89D/H440R, V58F/A328T/N532K, S119F/D342E/I449V, and K366N/G507S.

15. A crystalline form of a polypeptide comprising the amino acid residues of SEQ ID NO:

120 (FIG. 19).

16. A method of producing isoprene, comprising:

(a) providing a host cell comprising an expression vector comprising a polynucleotide sequence encoding an isoprene synthase variant; and

(b) culturing the host cell under conditions suitable for producing isoprene.

17. The method of claim 16 further comprising (c) recovering the isoprene.

18. The method of claim 17 further comprising (d) polymerizing the isoprene.

19. A method of detecting isoprene synthase activity, comprising:

(a) culturing a host cell comprising the expression vector under conditions suitable for producing an isoprene synthase variant;

(b) lysing the host cells with a lysis buffer comprising lysozyme to produce a cell lysate; and

(c) detecting isoprene synthase activity in the cell lysate by measuring isoprene production from dimethylallyl diphosphate (DMAPP).

20. The method of claim 19 wherein the host cell is selected from the group consisting of gram-positive bacterial cells, gram-negative bacterial cells, filamentous fungal cells, and yeast cells.

21. The method of claim 19 wherein the host cell is selected from the group consisting of Escherichia sp. (E. coli), Panteoa sp. (P. citrea), Bacillus sp. (B. subtilis), Yarrowia sp. (Y. lipolytica), and Trichoderma (T. reesei).

22. The method of claim 19 wherein the host cell is cultured in a medium that includes a carbon source selected from the group consisting of glucose, glycerol, glycerine, dihydroxyacetone, yeast extract, biomass, molasses, sucrose, and oil.

23. A method of detecting isoprene in a plurality of samples in a high-throughput screening comprising:

(a) providing;

i) a plurality of samples each comprising isoprene synthase;

ii) a glass plate comprising a plurality of wells; and

iii) a seal for the glass plate;

(b) placing the plurality of samples in the plurality of wells of the glass plate;

(c) sealing the glass plate with the seal to produce a sealed glass plate having a headspace associated with the sample in each of the plurality of wells;

(d) incubating the glass plate under conditions in which the isoprene synthase is active; and

(e) detecting isoprene in the headspace.

24. The method of claim 23 wherein the isoprene is detected by gas chromatography-mass spectrometry (GC-MS).

25. The method of claim 23 wherein the plurality of samples comprise host cells comprising an expression vector comprising a polynucleotide sequence encoding an isoprene synthase variant in operable combination with a promoter.

26. The method of claim 23 wherein the plurality of samples comprise a lysate of the host cells, lysozyme, and dimethylallyl diphosphate (DMAPP).

27. The method of claim 23 wherein the glass plate is a deep-well glass block.

28. The method of claim 23 wherein the plurality of wells comprises at least 24 wells

29. The method of claim 23 wherein the plurality of wells each comprise a volume of 2 ml or less.

30. A host cell comprising a heterologous polynucleotide sequence encoding an isoprene synthase variant in operable combination with a promoter, wherein the isoprene synthase variant comprises a substitution at a position corresponding to one or more residues (one, two, three, four, five, six, seven, eight, nine or ten) of a poplar isoprene synthase.

31. The host cell of claim 30 wherein the isoprene synthase is the P. alba isoprene synthase of SEQ ID NO:

120.

32. The host cell of claim 31 wherein the variant is selected from the group consisting of:

an MEA variant (SEQ ID NO;

122), an MSV variant (SEQ ID NO;

124), an MVS variant (SEQ ID NO;

126), an MTE variant (SEQ ID NO;

128), an MNV variant (SEQ ID NO;

130).

33. The host cell of claim 31 wherein the variant is selected from the group consisting of:

a TRC (−

3) variant (SEQ ID NO;

136), a TRC (−

4) variant (SEQ ID NO;

138), a TRC (−

5) variant (SEQ ID NO;

140), a TRC (−

6) variant (SEQ ID NO;

142) and a TRC (−

7) variant (SEQ ID NO;

144).

34. The host cell of claim 30 wherein the variant is a MET variant of P. tremuloides isoprene synthase (SEQ ID NO:

146).

35. The host cell of claim 30 wherein the variant is a MET variant of P. trichocharpa isoprene synthase (SEQ ID NO:

148).

36. The host cell of claim 31 wherein the variant comprises one of more amino acid substitutions selected from the group consisting of V10M, F12S, T15A, E18G, V58I, V58F, L70Q, L70V, L70T, T71P, V79L, E89D, G94A, S119F, F120L, G127R, E175V, T212I, S257A, R262G, A266G, F280L, N297K, F305L, L319M, E323K, A328T, D342E, A359T, K366N, E368D, L374M, S396T, V418S, K438N, H440R, T442I, T442A, I449V, A469S, K500R, K505Q, G507S, S509N, F511Y, and N532K.

37. The host cell of claim 36 wherein at least one amino acid substitution is a L70R substitution.

38. The host cell of claim 31 wherein the variant comprises one of more amino acid substitutions selected from the group consisting of G127R/F511Y, L70Q/G94A/R262G/F305L, F12S/T15A/E18G/N297K, S396T/T442I, V10M/E323K, F120L/A266G, K438N/K500R, V79L/S509N, E175V/S257A/E368D/A469S, T71P/L374M, F280L/H440R, E89D/H440R, V58F/A328T/N532K, S119F/D342E/I449V, and K366N/G507S.

39. The host cell of claim 30 wherein the polynucleotide sequence is contained within a plasmid.

40. The host cell of claim 39 wherein the polynucleotide sequence is integrated into a chromosome of the host cell.

41. The host cell of claim 30 wherein the host is selected from the group consisting of gram-positive bacterial cells, gram-negative bacterial cells, filamentous fungal cells, and yeast cells.

42. The host cell of claim 30 wherein the host is selected from the group consisting of Escherichia sp. (E. coli), Panteoa sp. (P. citrea), Bacillus sp. (B. subtilis), Yarrowia sp. (Y. lipolytica), and Trichoderma (T. reesei).

43. The host cell of claim 30 wherein the host cell is cultured in a medium comprising a carbon source selected from the group consisting of glucose, glycerol, glycerine, dihydroxyacetone, yeast extract, biomass, molasses, sucrose, and oil.

44. The host cell of claim 30 wherein the host cell further comprises a heterologous or native nucleic acid encoding an IDI polypeptide and/or a heterologous or native nucleic acid encoding a DXS polypeptide, optionally in combination with the native DXP pathway.

45. The host cell of claim 30 wherein the host cell further comprises one or more nucleic acids encoding an IDI polypeptide and a DXS polypeptide.

46. The host cell of claim 30 wherein the host cell comprises one vector encoding the isoprene synthase variant, the IDI polypeptide, and the DXS polypeptide.

47. The host cell of claim 46 wherein the host cell further comprises a heterologous nucleic acid encoding an MVA pathway polypeptide selected from the group consisting of an MVA pathway polypeptide from Saccharomyces cerevisia and Enterococcus faecalis.

48. The host cell of claim 30 wherein the host cell further comprises one or more nucleic acids encoding an MVA pathway polypeptide and a DXS polypeptide and wherein one vector encodes the isoprene synthase variant, the MVA pathway polypeptide, and the DXS polypeptide.

49. The host cell of claim 48 wherein the host cell further comprises one or more nucleic acids encoding a DXS polypeptide, an IDI polypeptide, or one or more of the rest of the DXP pathway polypeptides, and a MVA pathway polypeptide.

50. A method of producing isoprene, comprising:

(a) culturing the host cells of claim 30 under suitable culture conditions for production of isoprene; and

(b) producing the isoprene.

51. The method of claim 50 further comprising (c) recovering the isoprene.

52. The method of claim 51 further comprising (d) polymerizing isoprene.

53. A method of producing isoprene synthase, comprising:

(a) providing;

(i) a host cell; and

(ii) a nucleic acid encoding an isoprene synthase variant in operable combination with a promoter, wherein the isoprene synthase variant comprises a substitution at a position corresponding to one or more residues (one, two, three, four, five, six, seven, eight, nine or ten) of a P. alba isoprene synthase of SEQ ID NO;

120;

(b) contacting the host cell with the nucleic acid to produce a transformed host cell; and

(c) culturing the transformed host cells under suitable culture conditions for production of isoprene synthase.