Isoprene synthase variants for improved microbial production of isoprene

• US 8,173,410 B2
• Filed: 04/23/2009
• Issued: 05/08/2012
• Est. Priority Date: 04/23/2008
• Status: Expired due to Fees

• Current Assignee: Goodyear Tire & Rubber Company
• Original Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Bott, Richard R., Cervin, Marguerite A., Kellis, James T. Jr., McAuliffe, Joseph C., Miasnikov, Andrei, Peres, Caroline M., Rife, Christopher Lee, Wells, Derek H., Weyler, Walter, Whited, Gregory M.
• Primary Examiner(s): RAGHU, GANAPATHIRAM
• Application Number:
  US12/429,143
• Publication Number:
  US 20100003716A1
• Time in Patent Office: 1,111 Days
• Field of Search: 435/232, 435/69.1, 435/320.1, 435/167, 435/252.3, 435/252.33, 435/254.11, 435/254.2, 435/254.21, 435/254.6, 536/23.1, 536/23.2, 536/23.6, 530/350
• US Class Current: 435/232
• 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 host cell comprising a heterologous polynucleotide sequence encoding a truncated isoprene synthase variant in operable combination with a promoter, wherein said truncated isoprene synthase variant comprises one or more amino acid substitution(s) at one or more amino acid residues corresponding to a poplar isoprene synthase having the sequence of SEQ ID NO:

120, wherein said substitution(s) are selected from the group consisting of V10M, F12S, T15A, E18G, V58I, V58F, L70Q, L70R, 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, T442A, I449V, A469S, K500R, K505Q, G507S, S509N, F511Y, and N532K; and

wherein the variant is capable of more effectively converting dimethylallyl diphosphate (DMAPP) to isoprene, as compared to an isoprene synthase variant without a substitution.

2. The host cell of claim 1 wherein at least one amino acid substitution is a L70R substitution.

3. The host cell of claim 1 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.

4. The host cell of claim 1 wherein the polynucleotide sequence is contained within a plasmid.

5. The host cell of claim 4 wherein the polynucleotide sequence is integrated into a chromosome of the host cell.

6. The host cell of claim 1 wherein the host is selected from the group consisting of gram-positive bacterial cells, gram-negative bacterial cells, filamentous fungal cells, and yeast cells.

7. The host cell of claim 1 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).

8. The host cell of claim 1 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.

9. The host cell of claim 1 wherein the host cell further comprises a heterologous or native nucleic acid encoding an isopentenyl-diphosphate delta-isomerase (IDI) polypeptide and/or a heterologous or native nucleic acid encoding a 1-Deoxyxylulose-5-phosphate synthase (DXS) polypeptide, optionally in combination with the native 1-deoxy-D-xylulose-5-phosphate (DXP) pathway.

10. The host cell of claim 1 wherein the host cell further comprises one or more nucleic acids encoding an IDI polypeptide and a DXS polypeptide.

11. The host cell of claim 1 wherein the host cell comprises one vector encoding the isoprene synthase variant, the IDI polypeptide, and the DXS polypeptide.

12. The host cell of claim 11 wherein the host cell further comprises a heterologous nucleic acid encoding a mevalonate (MVA) pathway polypeptide selected from the group consisting of an MVA pathway polypeptide from Saccharomyces cerevisia and Enterococcus faecalis.

13. The host cell of claim 1 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.

14. The host cell of claim 13 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.

15. A method of producing isoprene, comprising:

(a) culturing the host cells of claim 1 under suitable culture conditions for production of isoprene; and

(b) producing the isoprene.

16. The method of claim 15 further comprising (c) recovering the isoprene.

17. The method of claim 16 further comprising (d) polymerizing isoprene.

18. The host cell of claim 1 wherein the variant is MEA variant having the sequence of SEQ ID NO:

122.

19. The host cell of claim 18 wherein the MEA variant comprises L70R amino acid substitution.

20. The host cell of claim 1 wherein at least one amino acid substitution is a G507S substitution.

21. The host cell of claim 18 wherein the MEA variant comprises G507S amino acid substitution.

22. The host cell of claim 1 wherein the host cell further comprises one or more heterologous nucleic acid(s) encoding an MVA pathway polypeptide.