3-hydroxyisovalerate (HIV) synthase variants

Abstract

Described are 3-hydroxyisovalerate (HIV) synthase variants having improved activity in converting acetone and a compound which provides an activated acetyl group into 3-hydroxyisovalerate (HIV). Moreover, described are in particular methods for the production of 3-hydroxyisovalerate and methods for the production of isobutene from acetone utilizing the HIV synthase variants of the present invention.

Claims

1. A 3-hydroxyisovalerate (HIV) synthase variant showing an improved activity in converting acetone and a compound which provides an activated acetyl group characterized by the following formula (I): ##STR00014## into 3-hydroxyisovalerate over the corresponding HIV synthase from which it is derived, wherein X is selected from the group consisting of SCH2-CH2-NHCOCH2-CH2-NHCOCH(OH)C(CH3)2-CH2-OPO2HOPO2HC10H13N5O7P (coenzyme A), SCH2-CH2-NHCOCH2-CH2-NHCOCH(OH)C(CH3)2-CH2-OPO2H-polypeptide (acyl-carrier protein), SCH2-CH2-NHCOCH2-CH2-NHCOCH(OH)C(CH3)2-CH2-OH (pantetheine), SCH.sub.2-CH.sub.2-NHCOCH.sub.3 (N-acetyl-cysteamine), SCH.sub.3 (methane thiol), SCH2-CH(NH2)-CO2H (cysteine), SCH2-CH2-CH(NH2)-CO2H (homocysteine), SCH2-CH(NHC5H8NO3)-CONHCH2-CO2H (glutathione), SCH.sub.2-CH.sub.2-SO.sub.3H (coenzyme M) and OH (acetic acid), and wherein the HIV synthase variant is derived from the amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:1, and in which the HIV synthase variant comprises one or more amino acid residues at a position selected from the group consisting of: (1) an amino acid residue at position 33 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is substituted; and/or (2) an amino acid residue at position 74 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (3) an amino acid residue at position 171 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is substituted; and/or (4) an amino acid residue at position 221 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (5) an amino acid residue at position 222 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (6) an amino acid residue at position 338 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (7) an amino acid residue at position 345 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (8) an amino acid residue at position 394 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (9) an amino acid residue at position 396 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is substituted.

2. The HIV synthase variant of claim 1, wherein the HIV synthase variant is derived from the amino acid sequence shown in SEQ ID NO:1.

3. The HIV synthase variant of claim 1, wherein the HIV synthase variant is derived from a sequence having at least 90% sequence identity to SEQ ID NO:1.

4. The HIV synthase variant of claim 1 comprising an amino acid sequence at least 95% sequence identity to SEQ ID NO:1.

5. The HIV synthase variant of claim 1, wherein the HIV synthase variant further comprises one or more amino acid residues at a position selected from the group consisting of: (1) an amino acid residue at position 7 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (2) an amino acid residue at position 13 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (3) an amino acid residue at position 22 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (4) an amino acid residue at position 24 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (5) an amino acid residue at position 38 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (6) an amino acid residue at position 41 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (7) an amino acid residue at position 43 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (8) an amino acid residue at position 54 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (9) an amino acid residue at position 75 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (10) an amino acid residue at position 81 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (11) an amino acid residue at position 165 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (12) an amino acid residue at position 167 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (13) an amino acid residue at position 201 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (14) an amino acid residue at position 226 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (15) an amino acid residue at position 246 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (16) an amino acid residue at position 259 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (17) an amino acid residue at position 296 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (18) an amino acid residue at position 325 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (19) an amino acid residue at position 363 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (20) an amino acid residue at position 457 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (21) an amino acid residue at position 462 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (22) an amino acid residue at position 473 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (23) an amino acid residue at position 475 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (24) an amino acid residue at position 480 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (25) an amino acid residue at position 481 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (26) an amino acid residue at position 486 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (27) an amino acid residue at position 490 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (28) an amino acid residue at position 491 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (29) an amino acid residue at position 500 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (30) an amino acid residue at position 514 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (31) an amino acid residue at position 516 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (32) an amino acid residue at position 519 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (33) an amino acid residue at position 520 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted.

6. The HIV synthase variant of claim 5, wherein: (1) the amino acid residue at position 7 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with isoleucine or glycine; and/or (2) the amino acid residue at position 13 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with arginine or leucine; and/or (3) the amino acid residue at position 22 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with methionine; and/or (4) the amino acid residue at position 24 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with methionine; and/or (5) the amino acid residue at position 33 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is substituted with glutamic acid; and/or (6) the amino acid residue at position 38 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with glycine; and/or (7) the amino acid residue at position 41 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with serine; and/or (8) the amino acid residue at position 43 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with valine; and/or (9) the amino acid residue at position 54 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with glycine; and/or (10) the amino acid residue at position 74 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with glutamic acid; and/or (11) the amino acid residue at position 81 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with arginine; and/or (12) the amino acid residue at position 165 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with proline armor glutamine; and/or (13) the amino acid residue at position 167 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with alanine; and/or (14) the amino acid residue at position 171 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with alanine or glycine; and/or (15) the amino acid residue at position 201 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with threonine; and/or (16) the amino acid residue at position 221 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with leucine, valine, isoleucine or threonine; and/or (17) the amino acid residue at position 222 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with arginine, glutamine, lysine or histidine; and/or (18) the amino acid residue at position 226 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with methionine; and/or (19) the amino acid residue at position 246 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with arginine; and/or (20) the amino acid residue at position 259 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with aspartic acid; and/or (21) the amino acid residue at position 296 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with glutamine; and/or (22) the amino acid residue at position 325 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with alanine, leucine or valine; and/or (23) the amino acid residue at position 338 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with proline; and/or (24) the amino acid residue at position 345 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with phenylalanine; and/or (25) the amino acid residue at position 363 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with arginine; and/or (26) the amino acid residue at position 394 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is substituted with serine; and/or (27) the amino acid residue at position 396 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with asparagine; and/or (28) the amino acid residue at position 457 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with cysteine; and/or (29) the amino acid residue at position 462 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with tyrosine; and/or (30) the amino acid residue at position 473 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with aspartic acid or glycine; and/or (31) the amino acid residue at position 475 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with arginine; and/or (32) the amino acid residue at position 480 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with cysteine; and/or (33) the amino acid residue at position 481 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with serine; and/or (34) the amino acid residue at position 486 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with arginine; and/or (35) the amino acid residue at position 490 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with asparagine; and/or (36) the amino acid residue at position 491 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with alanine; and/or (37) the amino acid residue at position 500 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with serine; and/or (38) the amino acid residue at position 514 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with arginine, glycine or serine; and/or (39) the amino acid residue at position 516 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with asparagine; and/or (40) the amino acid residue at position 519 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with aspartic acid; and/or (41) the amino acid residue at position 520 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with serine.

7. The HIV synthase variant of claim 1, wherein said HIV synthase variant further comprises an amino acid residue at position 75 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with asparagine.

8. The HIV synthase variant of claim 1, wherein X is SCH2-CH2-NHCOCH2-CH2-NHCOCH(OH)C(CH3)2-CH2-OPO2HOPO2HC10H13N5O7P (coenzyme A).

9. The HIV synthase variant of claim 1, wherein the HIV synthase variant is capable of converting acetone and the compound into 3-hydroxyisovalerate (HIV) at a turnover rate of at least 0.9310.sup.2 s.sup.1.

10. A nucleic acid molecule encoding the HIV synthase variant of claim 1.

11. A vector comprising the nucleic acid of claim 10.

12. A host cell comprising the vector of claim 11.

13. An in vitro or in vivo method of producing 3-hydroxyisovalerate (HIV), comprising enzymatically converting by the HIV synthase variant of claim 1 acetone and a compound which provides an activated acetyl group characterized by the following formula (I): ##STR00015## into 3-hydroxyisovalerate (HIV), wherein X is selected from the group consisting of SCH2-CH2-NHCOCH2-CH2-NHCOCH(OH)C(CH3)2-CH2-OPO2HOPO2HC10H13N5O7P (coenzyme A), SCH2-CH2-NHCOCH2-CH2-NHCOCH(OH)C(CH3)2-CH2-OPO2H-polypeptide (acyl-carrier protein), SCH2-CH2-NHCOCH2-CH2-NHCOCH(OH)C(CH3)2-CH2-OH (pantetheine), SCH2-CH2-NHCOCH.sub.3 (N-acetyl-cysteamine), SCH.sub.3 (methane thiol), SCH2-CH(NH2)-CO2H (cysteine), SCH2-CH2-CH(NH2)-CO2H (homocysteine), SCH2-CH(NHC5H8NO3)-CONHCH2-CO2H (glutathione), SCH.sub.2CH.sub.2SO.sub.3H (coenzyme M) and OH (acetic acid) to produce HIV.

14. The method of claim 13, wherein the HIV synthase variant is derived from the amino acid sequence shown in SEQ ID NO:1.

15. The method of claim 13, wherein the HIV synthase variant is derived from a sequence having at least 90% sequence identity to SEQ ID NO:1.

16. The method of claim 15, wherein the HIV synthase variant comprises an amino acid sequence at least 95% sequence identity to SEQ ID NO:1.

17. The method of claim 13, the HIV synthase variant further comprises one or more amino acid residues at a position selected from the group consisting of: (1) an amino acid residue at position 7 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (2) an amino acid residue at position 13 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (3) an amino acid residue at position 22 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (4) an amino acid residue at position 24 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (5) an amino acid residue at position 38 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (6) an amino acid residue at position 41 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (7) an amino acid residue at position 43 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (8) an amino acid residue at position 54 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (9) an amino acid residue at position 75 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (10) an amino acid residue at position 81 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (11) an amino acid residue at position 165 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (12) an amino acid residue at position 167 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (13) an amino acid residue at position 201 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (14) an amino acid residue at position 226 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (15) an amino acid residue at position 246 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (16) an amino acid residue at position 259 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (17) an amino acid residue at position 296 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (18) an amino acid residue at position 325 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (19) an amino acid residue at position 363 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (20) an amino acid residue at position 457 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (21) an amino acid residue at position 462 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (22) an amino acid residue at position 473 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (23) an amino acid residue at position 475 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (24) an amino acid residue at position 480 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (25) an amino acid residue at position 481 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (26) an amino acid residue at position 486 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (27) an amino acid residue at position 490 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (28) an amino acid residue at position 491 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (29) an amino acid residue at position 500 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (30) an amino acid residue at position 514 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (31) an amino acid residue at position 516 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (32) an amino acid residue at position 519 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted; and/or (33) an amino acid residue at position 520 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted.

18. The method of claim 17, wherein: (1) the amino acid residue at position 7 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with isoleucine or glycine; and/or (2) the amino acid residue at position 13 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with arginine or leucine; and/or (3) the amino acid residue at position 22 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with methionine; and/or (4) the amino acid residue at position 24 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with methionine; and/or (5) the amino acid residue at position 33 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is substituted with glutamic acid; and/or (6) the amino acid residue at position 38 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with glycine; and/or (7) the amino acid residue at position 41 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with serine; and/or (8) the amino acid residue at position 43 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with valine; and/or (9) the amino acid residue at position 54 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with glycine; and/or (10) the amino acid residue at position 74 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with glutamic acid; and/or (11) the amino acid residue at position 81 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with arginine; and/or (12) the amino acid residue at position 165 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with proline or glutamine; and/or (13) the amino acid residue at position 167 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with alanine; and/or (14) the amino acid residue at position 171 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is substituted with alanine or glycine; and/or (15) the amino acid residue at position 201 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with threonine; and/or (16) the amino acid residue at position 221 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with leucine, valine, isoleucine or threonine; and/or (17) the amino acid residue at position 222 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with arginine, glutamine, lysine or histidine; and/or (18) the amino acid residue at position 226 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with methionine; and/or (19) the amino acid residue at position 246 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with arginine; and/or (20) the amino acid residue at position 259 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with aspartic acid; and/or (21) the amino acid residue at position 296 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with glutamine; and/or (22) the amino acid residue at position 325 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with alanine, leucine or valine; and/or (23) the amino acid residue at position 338 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with proline; and/or (24) the amino acid residue at position 345 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with phenylalanine; and/or (25) the amino acid residue at position 363 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with arginine; and/or (26) the amino acid residue at position 394 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with serine; and/or (27) the amino acid residue at position 396 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with asparagine; and/or (28) the amino acid residue at position 457 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with cysteine; and/or (29) the amino acid residue at position 462 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with tyrosine; and/or (30) the amino acid residue at position 473 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with aspartic acid or glycine; and/or (31) the amino acid residue at position 475 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with arginine; and/or (32) the amino acid residue at position 480 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with cysteine; and/or (33) the amino acid residue at position 481 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with serine; and/or (34) the amino acid residue at position 486 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with arginine; and/or (35) the amino acid residue at position 490 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with asparagine; and/or (36) the amino acid residue at position 491 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with alanine; and/or (37) the amino acid residue at position 500 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with serine; and/or (38) the amino acid residue at position 514 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with arginine, glycine or serine; and/or (39) the amino acid residue at position 516 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with asparagine; and/or (40) the amino acid residue at position 519 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with aspartic acid; and/or (41) the amino acid residue at position 520 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with serine.

19. The method of claim 13, wherein said HIV synthase variant further comprises an amino acid residue at position 75 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in the sequence from which the HIV synthase variant is derived, is deleted or substituted with asparagine.

20. The method of claim 13, wherein X is SCH2-CH2-NHCOCH2-CH2-NHCOCH(OH)C(CH3)2-CH2-OPO2HOPO2HC10H13N5O7P (coenzyme A).

21. The method of claim 13, wherein the method is carried out by culturing a host cell to express the HIV synthase variant to produce HIV.

22. The method of claim 13 wherein the method further comprises the step of converting the produced HIV into isobutene.

Description

(1) Other aspects and advantages of the invention will be described in the following examples, which are given for purposes of illustration and not by way of limitation.

(2) FIG. 1: Isobutene metabolic pathway. The HIV phosphorylase and IBN synthetase (herein, the latter is also termed PIV decarboxylase) referred to in FIG. 1 refers to enzymes which are capable of catalyzing the conversion of 3-hydroxyisovalerate (HIV) into 3-phosphonoxy-isovaleric acid (PIV) as defined above and to enzymes which are capable of catalyzing the conversion of 3-phosphonoxy-isovaleric acid (PIV) into isobutene (IBN) as defined above, respectively.

(3) FIG. 2: Schematic representation of a directed evolution approach.

(4) FIG. 3: Sample results from the primary screen of the directed evolution approach of Hmg-CoA synthase with respect to HIV production by screening in vitro.

(5) FIG. 4.: Relative activity of 4 variants identified displaying increased activity over the WT HIV synthase activity.

(6) FIG. 5: Analysis of mutants harbouring 1 to 4 recombined mutations.

(7) FIG. 6: Sample results from the primary screen of the directed evolution approach of Hmg-CoA synthase with respect to HIV production by screening in vitro.

(8) FIG. 7: Results of in vivo screening of an HIV synthase variants library. Relative activity is expressed as an IBN peak array from twelve replicates for one given mutant over the mean IBN peak area from twelve replicates for the template sequence.

(9) FIG. 8: Relative activity of mutant variants having mutations at position 221 with respect to the production of HIV.

(10) FIG. 9: Determination of kinetic constants for HIV synthases: indicated is the IBN peak area for wild type HIV synthase of SEQ ID NO:1 versus the IBN peak area of mutants [L22M; A201T; H462Y] and [L22M; A201T; S221L; H462Y] assayed for the production of HIV using acetone and acetyl-CoA as a substrate in a combined enzymatic test.

(11) FIG. 10: Results from variants exhibiting a acetate-production-reducing/HIV-production-improving mutation.

(12) FIG. 11: Characterization of HIV synthase variants in a whole cell assay demonstrating that all three variants tested produce an increased amount of isobutene compared to the wild type enzyme.

(13) Other aspects and advantages of the invention will be described in the following examples, which are given for purposes of illustration and not by way of limitation.

EXAMPLES

(14) General Outline of the Screening for HIV Synthase Variants Showing Improved Activity in Converting Acetone and a Compound which Provides an Activated Acetyl Group into HIV

(15) The screening was based on a directed evolution approach which consisted in (1) the generation of a DNA library coding for single or multiple point mutants of the HMG CoA synthase enzyme, (2) the design and validation of an assay to test the activity of these enzyme variants, (3) the use of the activity assay to screen the collection of mutants in order to identify mutants with improved activity compared to the wild type HMG CoA synthase. A schematic diagram of this approach is presented in FIG. 2. The screening method consists generally of several steps (up to 4) in order to eliminate false negatives or assay artefacts amongst the initial positive hits and thus allows to only retain true leads.

(16) The screening aimed at identifying enzyme variants with higher rates of conversion of acetone into 3-hydroxyisovalerate (HIV).

(17) A list summarizing the mutations identified which exhibit higher rates of conversion of acetone into 3-hydroxyisovalerate (HIV) is provided in the following Table 6.

(18) TABLE-US-00006 TABLE 6 WT Improving sequence Position mutations L 7 I; G W 13 L; R L 22 M I 24 M Q 33 E K 38 G G 41 S D 43 V A 54 G Q 74 E K 75 N S 81 R K 100 L T 165 P; Q N 167 A T 171 A; G A 201 T S 221 L; V; I; T I 222 Q; K; H; R L 226 M K 246 R G 259 D L 270 I; M S 296 Q E 325 A; L: V S 338 P Y 345 F Q 363 R P 394 S S 396 N R 457 C H 462 Y N 473 G; D H 475 R G 480 C M 481 S S 486 R T 490 N E 491 A V 500 S V 514 G; S; R S 516 N E 519 D H 520 S

(19) Moreover, variants obtained from the above described screening experiments bearing one or more mutations that confer increased HIV synthesis activity compared to the wild type sequence SEQ ID NO:1 are described in the following Table 7 where they have been organized according to their range of activities. Indicated in Table 7 is the mean relative activity, i.e., the ratio of Gas chromatography signal obtained for the mutant enzyme over Gas chromatography signal obtained for wild type enzyme. The fold increase was determined for one acetone concentration (125 mM or 700 mM in vitro and 500 mM in vivo). The enzyme quantity was normalized for in vitro measurements using purified enzymes but not for in vivo experiments.

(20) TABLE-US-00007 TABLE 7 Mean relative Screening Sequence Activity Assay L22M T165P A201T S221L I222Q G259D S296Q H462Y 34.90 IN VIVO M481S V500S S516N T165P I222Q S296Q M481S V500S S516N 34.90 IN VIVO L22M T165P A201T S221L I222Q G259D S296Q H462Y 34.20 IN VIVO N473G T490N L22M T165P A201T S221L I222Q G259D S296Q H462Y 33.82 IN VIVO V500S T165P I222Q S296Q V500S 33.81 IN VIVO L22M T165P A201T S221L I222Q G259D S296Q H462Y 33.04 IN VIVO N473G M481S V500S T165P I222Q S296Q N473G M481S V500S 33.04 IN VIVO L22M K75N T165Q A201T S221L I222Q G259D S296Q 31.54 IN VIVO H462Y H475R L22M I24M K75N K100L T165P A201T S221L I222Q 31.15 IN VIVO L226M K246R G259D L270I H462Y N473D G480C V500S L22M T165P A201T S221L I222Q G259D S296Q H462Y 31.10 IN VIVO N473G V500S T165P I222Q S296Q N473G V500S 31.10 IN VIVO L22M D43V T165P A201T S221L I222Q G259D H462Y 30.56 IN VIVO M481S V500S D43V T165P I222Q M481S V500S 30.55 IN VIVO L22M D43V S81R T165P A201T S221L I222Q G259D 30.48 IN VIVO S296Q H462Y V500S V514S L22M K75N T165Q A201T S221L I222Q L226M G259D 29.47 IN VIVO L270M S296Q H462Y N473D G480C V500S L22M I24M T165P A201T S221L I222K L226M G259D 29.42 IN VIVO L270M S296Q H462Y H475R L22M K75N T165P A201T S221L I222K G259D S296Q 29.37 IN VIVO H462Y N473G M481S L22M D43V T165P A201T S221L I222Q G259D S296Q 27.69 IN VIVO P394S R457C H462Y M481S V500S V514S D43V T165P I222Q S296Q P394S R457C M481S V500S 27.69 IN VIVO V514S L22M K75N T165P A201T S221L I222H L226M K246R 27.36 IN VIVO G259D H462Y N473D G480C L22M T165P A201T S221L I222Q G259D H462Y 27.00 IN VIVO L22M T165P A201T S221L I222Q G259D S296Q S396N 26.91 IN VIVO H462Y T165P I222Q S296Q S396N 26.91 IN VIVO L22M K75N A201T S221L I222H G259D S296Q H462Y 26.65 IN VIVO N473G G480C V500S L22M K75N T165P A201T S221L I222K S296Q H462Y 26.49 IN VIVO N473D H475R V500S I222Q S296Q Y345F Q363R N473G M481S V500S 26.21 IN VIVO V514S L22M A201T S221L I222Q G259D S296Q Y345F Q363R 26.21 IN VIVO H462Y N473G M481S V500S V514S L22M D43V T165P A201T S221L I222Q G259D S296Q 26.06 IN VIVO H462Y M481S V514S D43V T165P I222Q S296Q M481S V514S 26.06 IN VIVO L22M A201T S221L I222Q G259D S296Q H462Y N473G 25.75 IN VIVO M481S I222Q S296Q N473G M481S 25.75 IN VIVO L22M A201T S221L I222Q G259D S296Q H462Y S486R 25.52 IN VIVO I222Q S296Q S486R 25.51 IN VIVO L22M I24M K75N T165Q A201T S221L I222Q L226M 25.17 IN VIVO K246R G259D S296Q H462Y N473D G480C V500S L22M T165Q A201T S221L I222K L226M K246R G259D 24.95 IN VIVO S296Q H462Y N473D H475R V500S L22M A201T I24M K75N T165P S221L I222K L226M 24.68 IN VIVO H462Y L22M A201T S221L I222Q G259D H462Y N473G M481S 23.97 IN VIVO V500S I222Q N473G M481S V500S 23.96 IN VIVO L22M A201T S221L I222Q G259D S296Q H462Y N473G 23.89 IN VIVO M481S V500S V514S I222Q S296Q N473G M481S V500S V514S 23.89 IN VIVO L22M I24M K75N T165Q A201T S221L I222Q L226M 23.55 IN VIVO K246R G259D L270I S296Q H462Y N473D G480C M481S V500S L22M A201T S221L I222Q G259D S296Q H462Y N473G 23.27 IN VIVO V500S I222Q S296Q N473G V500S 23.27 IN VIVO L22M K75N T165Q A201T S221L I222K S296Q H462Y 23.21 IN VIVO G480C V500S L22M K75N T165P A201T S221L I222K K246R G259D 22.81 IN VIVO H462Y V500S L22M I24M T165Q A201T S221L I222K L226M G259D 22.20 IN VIVO H462Y H475R V500S L22M I24M K75N A201T S221L I222Q L226M G259D 21.97 IN VIVO H462Y N473D M481S L22M I24M K75N K100L T165P A201T S221L I222K 21.97 IN VIVO K246R G259D S296Q H462Y N473G M481S V500S L22M K75N A201T S221L I222Q L226M H462Y N473D 21.76 IN VIVO V500S L22M T165P A201T S221L I222Q L226M G259D L270I 21.60 IN VIVO H462Y N473G H475R G480C L7I W13L L22M K75N T165P A201T S221L I222Q 21.48 IN VIVO G259D H462Y L22M I24M K75N T165Q A201T S221L I222H G259D 21.47 IN VIVO H462Y G480C L22M I24M K75N A201T S221L I222Q K246R S296Q 20.12 IN VIVO H462Y N473G H475R L22M A201T S221L T165Q I222H G259D S296Q 20.07 IN VIVO H462Y N473G G480C L22M I24M K75N T165P A201T S221L I222K L226M 19.23 IN VIVO K246R G259D H462Y H475R V500S L22M I24M K75N T165Q A201T S221L I222H K246R 19.15 IN VIVO S296Q H462Y N473G G480C V500S L22M K75N T165Q A201T S221L I222Q S296Q H462Y 19.07 IN VIVO H475R G480C M481S L22M K75N T165P A201T S221L I222Q G259D L270I 18.77 IN VIVO S296Q H462Y H475R V500S L22M I24M K75N K100L T165P A201T S221L I222H 18.19 IN VIVO L226M K246R G259D L270M S296Q H462Y N473D H475R L22M I24M K75N A201T S221L I222H G259D L270I 18.17 IN VIVO S296Q H462Y H475R L22M I24M K75N K100L T165P A201T S221L I222K 18.13 IN VIVO L226M K246R G259D L270M H462Y N473D V500S L22M I24M K75N T165Q A201T S221L L226M K246R 17.87 IN VIVO G259D L270I S296Q H462Y H475R V500S V514S L22M K75N T165Q A201T S221L I222Q G259D S296Q 17.84 IN VIVO H462Y H475R L22M I24M K75N T165Q A201T S221L I222K L226M 17.69 IN VIVO G259D H462Y N473G L22M K75N T165P A201T S221L I222K S296Q H462Y 17.45 IN VIVO N473D H475R V500S L22M I24M K75N T165Q A201T S221L I222Q L226M 17.38 IN VIVO K246R G259D L270M S296Q H462Y N473D G480C L22M K75N A201T S221L G259D S296Q H462Y N473G 17.20 IN VIVO G480C L22M K75N T165P A201T S221L I222K G259D S296Q 17.06 IN VIVO H462Y N473G M481S L22M I24M K75N T165P A201T S221L I222K L226M 16.99 IN VIVO K246R L270M S296Q H462Y N473G G480C M481S V500S L22M K75N T165Q A201T S221L I222H K246R G259D 16.93 IN VIVO L270I S296Q H462Y N473G G480C M481S L22M I24M K75N T165Q A201T S221L I222R K246R 16.62 IN VIVO G259D H462Y N473D G480C V500S E519D L22M K75N T165P A201T S221L I222H L226M K246R 15.60 IN VIVO G259D H462Y N473D G480C L22M T165Q A201T S221L I222K L226M K246R G259D 15.55 IN VIVO S296Q H462Y N473D H475R V500S L22M A201T S221L G259D H462Y 15.51 IN VIVO L22M I24M K75N T165P A201T S221L I222K L226M 15.49 IN VIVO S296Q H462Y L22M I24M K75N K100L T165P A201T S221L I222H 15.36 IN VIVO K246R G259D L270M H462Y N473G G480C V500S L22M K75N T165Q A201T S221L H462Y 15.05 IN VIVO L22M I24M K75N T165Q A201T S221L I222Q K246R 14.73 IN VIVO G259D L270M H462Y N473G V514S L22M I24M K75N K100L T165P A201T S221L I222K 14.71 IN VIVO K246R G259D L270M S296Q H462Y N473G M481S L22M A201T S221L I222Q H462Y 14.57 IN VIVO L22M K75N T165Q A201T S221L L226M G259D L270I 14.42 IN VIVO H462Y N473D V500S L22M K75N A201T S221L I222Q L226M K246R S296Q 13.93 IN VIVO H462Y G480C V500S L22M I24M A201T S221L I222Q H462Y 13.74 IN VIVO L22M A201T S221L I222K H462Y 13.54 IN VIVO L22M I24M K75N T165Q A201T S221L I222H L226M 12.90 IN VIVO L270M H462Y H475R L22M I24M K75N T165Q A201T S221L I222Q L226M 12.54 IN VIVO K246R G259D L270M S296Q H462Y H475R V500S L22M I24M K75N T165Q A201T S221L L226M K246R 12.50 IN VIVO G259D S296Q H462Y G480C V500S L22M K75N T165P A201T S221L I222K L226M K246R 11.71 IN VIVO G259D L270M H462Y N473D H475R L22M I24M K75N A201T S221L I222H L270M S296Q 11.70 IN VIVO H462Y V500S L22M A201T S221L I222H H462Y 11.48 IN VIVO L22M A201T S221L H462Y V500S 11.14 IN VIVO L22M A201T S221L S296Q H462Y 11.06 IN VIVO L22M A201T S221L H462Y E491A 10.11 IN VIVO L22M A201T S221L H462Y H475R 9.68 IN VIVO L22M A201T S221L L226M H462Y 9.43 IN VIVO L22M A201T S221L H462Y 8.57 IN VIVO L22M A201T L270I H462Y 3.72 IN VITRO L22M A201T L270M H462Y 3.31 IN VITRO L22M K100L A201T H462Y 2.81 IN VITRO L22M A201T K246R H462Y 2.76 IN VITRO L22M A201T H462Y H520S 2.74 IN VITRO L22M A201T H462Y E519D 2.61 IN VITRO L22M A201T E325A H462Y 2.50 IN VITRO L22M G41S A201T H462Y 2.47 IN VITRO L22M H462Y 2.30 IN VITRO L22M H462Y E519D 2.23 IN VITRO L22M A201T E519D 2.07 IN VITRO L22M A201T H462Y 2.04 IN VITRO L22M A201T 1.91 IN VITRO A201T H462Y E519D 1.87 IN VITRO L22M E519D 1.66 IN VITRO H462Y E519D 1.53 IN VITRO H462Y 1.40 IN VITRO A201T 1.28 IN VITRO L22M 1.24 IN VITRO E519D 1.19 IN VITRO A201T H462Y 1.12 IN VITRO A201T E519D 1.03 IN VITRO

I. Example 1: Directed Evolution of Hmg-CoA Synthase for HIV Production by in Vitro Screening

(21) 1. Library Construction

(22) A cDNA library coding for single residue mutants of HIV synthase was constructed using standard mutagenesis techniques. The full length coding sequence of the Mus musculus HIV synthase enzyme with N-term His6 tag was subcloned into commercial peT-25b+ expression vector and used as a template for the mutagenic PCR. The quality control of the library construction consisted of two steps: (1) the amplified DNA fragments obtained were analyzed and quantified against a range of control reactions and (2) DNA sequencing was carried out on 200 randomly-selected clones. The profiles of the DNA fragments were as expected.

(23) 2. Screening Assay

(24) Plasmid DNA generated as described above and plasmid DNA containing the sequence coding for the Mus musculus wild type HIV synthase were transformed into BL21(DE3) competent cells and plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 30 C. until individual colonies reach the desired size.

(25) Colonies were then picked and individually transferred into 1 mL of liquid LB medium supplemented with the appropriate antibiotic. Cell growth is carried out with agitation for 20 hours at 30 C. LB cultures were used to inoculate 1 ml of autoinduction medium (ZYM medium, Studier F. W; Protein Expr. Purif. 41 (2005), 207-234) supplemented with the appropriate antibiotic. Cultures were grown overnight at 30 C. for 20-22 hours in shacking incubator set at 700 rpm and 85% humidity. Cells were finally pelleted and stored at 80 C. overnight.

(26) Frozen cell pellets were thawed on ice and resuspended in 200 l Bugbuster (Merck Novagen) and incubated for 10 minutes at room temperature followed by 20 minutes on ice to allow cell lysis to proceed. Raw lysates were then clarified by centrifugation.

(27) In parallel, 96 wells purification plates (Macherey Nagel) were prepared as follows. Purification matrices are wetted and equilibrated with 500 l of wash buffer (10 mM Tris, 300 mM NaCl, 10 mM Imidazole, pH7.5). Clarified lysates (200 l) were then transferred onto purification columns and allowed to flow through by gravity. Columns were washed with 1.2 ml of wash buffer and 200 l of elution buffer (50 mM Tris, 250 mM Imidazole pH7.5). Elution of purified enzyme variants was finally performed with 110 l of elution buffer.

(28) After purification enzyme preparations were desalted using Zeba Spin Desalting plates (Perbio Sciences). Plates were centrifuged to remove storage buffer (1000 g, 2 min, 19C). Enzyme preparations from the previous step were transferred onto desalting resin and collected as effluent by centrifugation (100 g, 5 minutes, 19 C.). Thus prepared enzyme variants were then ready for use in the screening assay.

(29) Screening assay consist in the in vitro coupled enzymatic conversion of acetone and acetyl-CoA into isobutene (IBN) via 3-hydroxyisovalerate (HIV) and 3-phosphonoxy-isovaleric acid (PIV) using the HIV synthase variant to be screened and purified HIV phosphorylase and PV decarboxylase enzymes prepared. As an HIV phosphorylase, i.e., an enzyme capable of catalyzing the conversion of the 3-hydroxyisovalerate (HIV) into 3-phosphonoxy-isovaleric acid (PIV), a mevalonate diphosphate decarboxylase (EC 4.1.1.33) isolated from Thermoplasma acidophilum may be used (Uniprot entry for the wildtype sequence Q9H1N1-THEAC) having an amino acid substitution at position 200 (L200E) including a N terminal His6-tag wherein the HIV phosphorylase has the amino acid sequence as shown in SEQ ID NO:2. As an PIV decarboxylase, i.e., an enzyme capable of catalyzing the conversion of PIV into isobutene (IBN), a mevalonate diphosphate decarboxylase (EC 4.1.1.33) isolated from Streptococcus mitis strain B6 may be used (Uniprot entry for the wildtype sequence D3HAT7-STRM6) having amino acid substitutions at positions 24, 118, 121, 159, 173, 177, 282, 291, and 297 (K24R C118L Y121R E159L M173C E177C K282C E291D F297L) including a N terminal His6-tag wherein the PIV decarboxylase has the amino acid sequence as shown in SEQ ID NO:3. The HIP phosphorylase and PIV decarboxylase may be produced as follows: the coding sequences of both the above described genes are sub-cloned into peT25b(+) (Merck-Novagen) and the resulting expression vectors are transformed into BL21(DE3) according to standard procedures. Single transformants are used to inoculate 1 liter of ZYM-5052 autodinduction medium (Studier F W, Prot. Exp. Pur. 41, (2005), 207-234). Cells are grown in a shaking incubator for 20-22 hours at 30 C. for the above S. mitis MDP and 8 hours at 37 C. followed by a 16 hours incubation at 28 C. for the above T. acidophilum MDP. Cells are pelleted and stored at 80 C. until further processed. For the cell lysis, cells pellets are resuspended in 40 ml of Bugbuster reagent (Merck-Novagen) supplemented with 100 l of lysonase 10 minutes at room temperature followed by a further 20-minutes incubation at 4 C. Cell lysates are clarified by centrifugation (30-40 minutes at 10,000 g) and filtered through at 0.22 m filter. Purification of the N-term His-tagged proteins of Interest from these cell lysates is carried out by IMAC (Immobilized Metal ion Affinity Chromatography) on a 5 ml HisTrap HP column using a KTA Purifier UPC 100 (GE Healthcare) according to the manufacturer's recommendations. The eluted proteins are concentrated and desalted by ultrafiltration using Millipore Amicon Ultra-15 concentrated.

(30) 70 l of enzymes preparations were mixed with 30 l of reaction buffer (final concentrations in reaction are as follows: 50 mM Tris, 10 mM MgCl2, 20 mM KCl, 4 mM Ac-CoA, 125 mM acetone, 5 mM ATP, 0.5 mM DTT, 5 g HIV phosphorylase and 85 g PIV decarboxylase as produced and purified as described above) in 2 ml crimp top glass vials. Glass vials were sealed using crimp caps and incubated for 8 hours at 37 C. to allow enzymatic conversion of substrates into isobutene (IBN) to proceed. Enzymatic reactions were finally stopped by heat shock denaturation of enzymes at 80 c. for 5 minutes.

(31) The isobutene (IBN) produced spontaneously volatilizes and can be quantified by gas chromatography (GC) analysis of reactions head space. Downstream enzymes (i.e. the HIV phosphorylase and PIV decarboxylase as produced and purified as described above) being in excess, the quantity of isobutene (IBN) produced is directly proportional with the quantity of HIV produced and therefore correlates with HIV synthase activity. It provides an indirect readout for the reaction of interest.

(32) For GC headspace analysis, 100 l of headspace gases from each enzymatic reaction were injected (Injection parameters: 250 C.; split=10) in a Brucker GC-450 system equipped with a Flame ionization detector (FID) (250 C.; 28 ml.Math.min.sup.1 H.sub.2; 30 ml.Math.min.sup.1 N.sub.2; 300 ml.Math.min.sup.1 synthetic air). Compounds present in samples were separated by chromatography using a RTX-1 column (15m x 0.32 mm; Restek, France) at 100 C. with a 1 ml.Math.min.sup.1 constant flow of carrier gaz (nitrogen 5.0, Messer, France). Upon injection, peak area of isobutene was calculated for samples and standards.

(33) Variants displaying improved activity over that of parental enzyme were identified based on increased IBN peak area as quantified by GC. An example of results as obtained from the primary screen is presented in FIG. 3.

(34) 3. Identification of Enzyme Variants with Increased Activity

(35) Of the initial HIV synthase variants library 7,392 variants were assayed as described above. Alongside the HIV synthase variants, control reactions were setup including reference controls using wild type HIV synthase enzyme. Altogether 8,064 clones were screened. Out of 7,392 HIV synthase variants, 147 positive hits were identified and represent 1.98% of the population screened. Out of the 147 variants isolated in the primary screen, 4 variants remained after two additional rounds of screening. These variants were tested in multiple replicates and in a range of conditions to ensure that the increase of activity is reproducible and not due to an artifact of the assay. Finally, each clone was subjected to DNA sequencing in order to identify the mutation responsible for the change in enzyme activity. Final results are presented in FIG. 4.

II. Example 2: Recombination of Improving Mutation by Direct Mutagenesis

(36) 1. Production of Exhaustive Recombinants Library

(37) Previously identified improving mutations (identified as described in Example 1) were recombined by single or successive standard directed mutagenesis reactions to obtain mutants containing more than one mutation.

(38) 2. Production and Screening of Purified Enzyme Variants

(39) Variants were produced and purified as follows. Plasmid DNA generated as described above and plasmid DNA containing the sequence coding for the wild type HIV synthase were transformed into BL21(DE3) competent cells and plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 30 C. until individual colonies reach the desired size.

(40) Single transformants were used to inoculate 50 ml of autoinduction medium in order to produce recombinant enzyme in bacteria. Cell pellets containing the overexpressed recombinant HIV synthase variants were stored at 80 C. overnight before being resuspended in lysis buffer (BugBuster, Merck Novagen). The suspension was incubated 10 minutes at room temperature followed by 20 minutes on ice. Cell lysates were clarified by centrifugation and His6 tagged enzymes were purified from clarified lysates by affinity chromatography (Macherey Nagel), concentrated by centrifugation on ultrafiltration membranes (Amicon ultra, Millipore) and desalted by size exclusion chromatography (Zeba spin columns, Perbio Science).

(41) Purified enzymes were characterized in vitro in a coupled, multi-step enzymatic conversion of acetone and acetyl-CoA into IBN (IBN) via 3-hydroxyisovalerate (HIV) and 3-phosphonoxy-isovaleric acid (PIV) using the HIV synthase variants and controls to be assessed and purified HIV phosphorylase and PIV decarboxylase enzymes prepared as outlined above. 40 g of pure enzyme preparations were incubated in HIV/IBN production buffer (50 mM Tris, 10 mM MgCl2, 20 mM KCl, 0.5 mM DTT, 700 mM acetone, 4 mM acetyl-CoA, 5 mM ATP, 0.5 mM DTT, 5 g HIV phosphorylase and 85 g PIV decarboxylase) in 2 ml crimp top glass vials. Glass vials were sealed using crimp caps and incubated for 8 hours at 37 C. to allow enzymatic conversion of substrates into isobutene (IBN). Enzymatic reactions were finally stopped by heat shock denaturation of enzymes at 80 c. for 5 minutes.

(42) Isobutene (IBN) production was analyzed by gas chromatography for controls and variants as described earlier and results are shown in FIG. 5.

III. Example 3: Directed Evolution of Hmg-CoA Synthase for HIV Production by in Vivo Screening

(43) 1. Library Construction

(44) A cDNA library coding for single residue mutants of HIV synthase was constructed using standard mutagenesis techniques. The full length coding sequence of a mutated HIV synthase enzyme (previously identified as described in Examples 1 and 2 and bearing mutations L22M, A201T, S221L, H462Y on a SEQ ID NO:1 backbone) with N-term His6 tag was subcloned into commercial peT-25b+ expression vector and used as a template for the mutagenic PCR. The quality control of the library construction consisted of two steps: (1) the amplified DNA fragments obtained were analyzed and quantified against a range of control reactions and (2) DNA sequencing was carried out on 200 randomly-selected clones. The profiles of the DNA fragments were as expected. In term of the DNA sequence analysis of the gene coding for the HIV synthase, approximately 75% of the clones presented single residue mutations while the rest were found wild type.

(45) 2. Screening Assay

(46) Plasmid DNA generated as described above and plasmid DNA containing the sequence coding for the wild type HIV synthase were transformed into BL21(DE3) competent cells and plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 30 C. until individual colonies reach the desired size.

(47) Colonies were then picked and individually transferred into 1 mL of liquid LB medium supplemented with the appropriate antibiotic. Cell growth is carried out with agitation for 20 hours at 30 C. LB cultures were used to inoculate 1 ml of autoinduction medium (ZYM medium, Studier F. W; Protein Expr. Purif. 41 (2005), 207-234) supplemented with the appropriate antibiotic. Cultures were grown overnight at 30 C. for 20-22 hours in shacking incubator set at 700 rpm and 85% humidity. The cells were then pelleted by centrifugation and clarified medium was discarded.

(48) Bacterial pellets were resuspended in HIV production medium (Potassium phosphate 200 mM, Citric acid 4 mM, Ammonium chloride 20 mM, NTA mix 1, glucose 45 g/L and acetone 500 mM) at OD600=10 and transferred to sealed culture vessels and incubated at 37 C. for 16 hours. Bacterial cultures were then deactivated by 5 minutes incubation at 80 C. and allowed to cool at room temperature.

(49) We have previously observed that 3-hydroxyisovalerate (HIV) can be detected in the culture medium of producing cells but that some remains intracellular. Cell lysis at high temperature therefore ensures that production is stopped and that intracellular HIV is released into the culture medium for accurate quantification. 3-hydroxyisovalerate (HIV) produced by bacterial cultures was therefore enzymatically converted to isobutene (IBN) for analysis by GC. 75 L of 3-hydroxyisovalerate (HIV) containing preparations were, therefore, supplemented with 25 L HIV revelation buffer (final concentration in reaction are as follows: KCl 20 mM, ATP 20 mM, HIV phosphorylase 5 g, PIV decarboxylase 85 g) (the production and purification of the HIV phosphorylase and PIV decarboxylase is described above) in 2 ml crimp top glass vials. Glass vials were sealed using crimp caps and incubated for 24 hours at 37 C. Enzymatic reactions were finally stopped by heat shock denaturation of enzymes at 80 C. for 5 minutes.

(50) The isobutene (IBN) produced spontaneously volatilizes and can be quantified by gas chromatography (GC) analysis of reactions head space. The quantity of IBN produced is directly proportional with the quantity of HIV in reactions and therefore with in vivo HIV synthase activity. It provides an indirect readout of the reaction of interest.

(51) For GC headspace analysis, 100 l of headspace gases from each enzymatic reaction are injected (Injection parameters: 250 C.; split=10) in a Brucker GC-450 system equipped with a Flame ionization detector (FID) (250 C.; 28 ml.Math.min.sup.1 H.sub.2; 30 ml.Math.min.sup.1 N.sub.2; 300 ml.Math.min.sup.1 synthetic air). Compounds present in samples were separated by chromatography using a RTX-1 column (15m x 0.32 mm; Restek, France) at 100 C. with a 1 ml.Math.min.sup.1 constant flow of carrier gaz (nitrogen 5.0, Messer, France). Upon injection, peak area of isobutene was calculated for samples and standards.

(52) Variants displaying improved activity over that of parental enzyme were identified based on Increased isobutene (IBN) peak area as quantified by GC. An example of screening results is presented in FIG. 6.

(53) 3. Identification of Enzyme Variants with Increased Activity

(54) Of the initial HIV synthase variants library 24,960 variants were assayed as described above. Alongside the HIV synthase variants, control reactions were setup including reference controls using wild type HIV synthase enzyme. Altogether 27,560 clones were screened. Out of 24,960 HIV synthase variants, 219 positive hits were identified and represent 0.87% of the population screened. Out of the 219 variants isolated in the primary screen, 11 variants remained after two additional rounds of screening. These variants were tested in multiple replicates and in a range of conditions to ensure that the increase of activity is reproducible and not due to an artifact of the assay. Finally each clone was subjected to DNA sequencing in order to identify the mutation responsible for the change in enzyme activity. Final results are presented in FIG. 7.

IV. Example 4: Identification of Mutations of Residue 221 of the HIV Synthase that Lead to an Increase in the Activity of HIV Production from Acetone and Acetyl-CoA

(55) Mutations S221L and S221V were identified by screening of a mutants library. An exhaustive and systematic test of all possible substitutions at position S221 was carried out in order to assess whether other substitutions could, similarly to S221L and S221V, enhance the activity of the enzyme. 48 clones were randomly selected out of a cDNA library mutagenized at position S221 and subjected to DNA sequencing in order to select as many substitutions out of the 19 amino acids possible other than WT sequence. The plasmid DNA for all expression vectors encoding these variants were transformed into BL21(DE3) and single transformants were used to inoculate 1 ml of autoinduction medium in order to produce recombinant enzyme in bacteria. Cell pellets containing the overexpressed recombinant HIV synthase variants were stored at 80 C. overnight before being resuspended in 200 l lysis buffer (BugBuster, Merck Novagen). The suspension was incubated 10 minutes at room temperature followed by 20 minutes on ice. Cell lysates were clarified by centrifugation and His6 tagged enzymes were purified from clarified by lysates by affinity chromatography (Macherey Nagel), concentrated by centrifugation over ultrafiltration membranes (Amicon ultra, Millipore) and desalted by size exclusion chromatography (Zeba spin columns, Perbio Science). Enzymatic reactions were setup in 2 ml glass vials using 70 l of enzyme preparation and 30 l of reaction mix (final concentrations are 50 mM Tris pH 7.5, 10 mM MgCl.sub.2, 20 mM KCl, 4 mM acetyl-CoA, 125 mM acetone, 5 mM ATP, 0.5 mM DTT, 5 g HIV phosphorylase and 85 g P/V decarboxylase; the production and purification of the HIV phosphorylase and PIV decarboxylase is described above). Vials were sealed and incubated at 37 C. for 8 hours followed by 5 min at 80 C. to stop the enzymatic reactions. The isobutene produced was previously shown to be directly proportional to 3-hydroxyisovalerate (HIV) production and was, therefore, quantified by gas chromatography as readout of HIV synthase activity as follows. 100 l of headspace gases from each enzymatic reaction are injected (Injection parameters: 250 C.; split=10) in a Brucker GC-450 system equipped with a Flame ionization detector (FID) (250 C.; 28 ml.Math.min.sup.1 H.sub.2; 30 ml.Math.min.sup.1 N2; 300 ml.Math.min.sup.1 synthetic air). Compounds present in samples were separated by chromatography using a RTX-1 column (15m x 0.32 mm; Restek, France) at 100 C. with a 1 ml.Math.min.sup. constant flow of carrier gas (nitrogen 5.0, Messer, France). Upon injection, peak area of isobutene was calculated for samples and standards; see FIG. 8.

V. Example 5: Determination of Kinetic Constants for HIV Synthases

(56) Michaelis-Menten apparent steady state kinetics constants for the overall reaction of HIV production from acetone and acetyl-CoAK.sub.cat.sup.app and K.sub.m.sup.appwere determined using the following protocol.

(57) Plasmid DNA containing the sequence coding for the wild type HIV synthase and variants showing increased HIV synthesis activity were transformed into BL21(DE3) competent cells and plated out onto LB agar petri dishes supplemented with the appropriate antibiotic.

(58) Cells were grown overnight at 30 C. and isolated transformants were picked and used to inoculate autoinduction medium (ZYM medium, Studier F. W; Protein Expr. Purif. 41 (2005), 207-234). The cultures were then grown overnight at 30 C. for 20-22 hours in shaking incubator. The cells were pelleted and stored at 80 C. overnight.

(59) Frozen cell pellets were thawed on ice and resuspended in adequate amounts of Bugbuster (Merck Novagen) and incubated for 10 minutes at room temperature followed by 20 minutes on ice to allow cell lysis to proceed. Raw lysates were then clarified by centrifugation and His6 tagged enzymes were purified by affinity chromatography (Macherey Nagel).

(60) 40 g of purified enzymes were then mixed with HIV production buffer (50 mM Tris, 10 mM MgCl2, 20 mM KCl, 0.5 mM DTT, 4 mM Ac-CoA) supplemented with a range of acetone concentrations (0 to 1200 mM). Enzymatic reactions were thus setup in 2 ml sealed glass vials and were incubated for 2 hours at 37 C. followed by a 5 minutes deactivation at 80 C. to spot the reaction. Reactions were clarified by centrifugation and supernatant was transferred to a fresh tube to which IBN production reagents were added (50 mM Tris pH 7.5, 5 mM ATP, 20 mM KCl, 5 g HIV phosphorylase and 85 g PIV decarboxylase; the production and purification of the HIV phosphorylase and PIV decarboxylase used is described above). 3-hydroxyisovalerate (HIV) to isobutene (IBN) conversion for reactions and standards was performed for 24 hours at 37 C. 100 l of headspace gases from each enzymatic reaction were then injected (Injection parameters: 250 C.; split=10) in a Brucker GC-450 system equipped with a Flame ionization detector (FID) (250 C.; 28 ml.Math.min.sup.1 H.sub.2; 30 ml.Math.min.sup. N.sub.2; 300 ml.Math.min.sup.1 synthetic air). Compounds present in samples were separated by chromatography using a RTX-1 column (15m x 0.32 mm; Restek, France) at 100 C. with a 1 ml.Math.min.sup. constant flow of carrier gas (nitrogen 5.0, Messer, France) and peak area of isobutene was calculated for samples and standards as shown in FIG. 9.

(61) In order to quantify absolute amounts of isobutene (IBN) and 3-hydroxyisovalerate (HIV) produced a range of concentrations of HIV (0.25 to 2 mM) were subjected to enzymatic conversion to IBN as applied to samples and a range of concentrations of pure IBN (1 to 100,000 ppm) were used to calibrate the gas chromatograph. Both calibrations curves showed the response to be linear within the selected range. The production rates of HIV (moles of HIV/mole enzyme/sec) were plotted as a function of the concentration of acetone and the curve was fitted using Michaelis Menten equation:

(62) $V=\frac{\left(\mathrm{Vmax}\left[\mathrm{substrate}\right]\right)}{\left(\mathrm{Km}+\left[\mathrm{substrate}\right]\right)}$

(63) TABLE-US-00008 TABLE 8 Kinetic data for wild type and improved mutants Kcat Kcat/Km Kcat fold Km Kcat/Km fold Enzyme (s.sup.1) increase (mM) (10.sup.2 s.sup.1/mM) increase Wild type 0.93 10.sup.2 158 0.0059 enzyme Template 2.19 10.sup.2 2.44 331 0.0066 1.12 Template + 4.97 10.sup.2 5.44 77 0.0645 10.93 S221L

VI. Example 6: Characterization of Reaction Modifying Mutation at Position S221

(64) It is of interest to note that in the presence of acetyl-CoA but in absence of the secondary substrate acetone, the enzyme effectively performs reaction (1) followed by (2) instead of (1) followed by (2).
Enzyme-S+Acetyl-CoA.fwdarw.Enzyme-S-Ac+CoA-SH(1)
Enzyme-S-Acetyl+Acetone+H.sub.2O.fwdarw.Enzyme-SH+HIV(2)
Enzyme-S-Acetyl+H.sub.2O.fwdarw.Enzyme-SH+Acetate(2)

(65) It is therefore of importance to monitor both HIV and Acetate production in WT and mutant enzymes to assess the relative efficiency of acetate production in the absence or at low acetone concentrations. Mutations have been shown to specifically suppress reaction (2) while enhancing the successive reactions (1) and (2) leading to increased HIV production when both substrates (Acetyl-CoA and Acetone) are present. Such results are shown in FIG. 10 for one mutation at position 221.

(66) Enzymatic reactions are setup as described in Example 5 but stopped before conversion of 3-hydroxyisovalerate (HIV) to isobutene (IBN). HIV and Acetate production are monitored by HPLC for each assay, control and production calibration samples in triplicate to quantify product formation (Hi Plex H 30com column set at 30 C. and 0.4 ml/min mobile phase (5.5 mM H.sub.2SO.sub.4 solution).

VII. Example 7: Characterization of HMG CoA Synthesis Activity of Variants Displaying Improved HIV Synthesis Activity

(67) Variants were produced and purified in the laboratory as follows. Plasmid DNA generated as described above and plasmid DNA containing the sequence coding for the wild type HIV synthase were transformed into BL21(DE3) competent cells and plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 30 C. until individual colonies reach the desired size.

(68) Single transformants were used to inoculate 50 ml of autoinduction medium in order to produce recombinant enzyme in bacteria. Cell pellets containing the overexpressed recombinant HIV synthase variants were stored at 80 C. overnight before being resuspended in lysis buffer (BugBuster, Merck Novagen). The suspension was incubated 10 minutes at room temperature followed by 20 minutes on ice. Cell lysates were clarified by centrifugation and His6 tagged enzymes were purified from clarified lysates by affinity chromatography (Macherey Nagel), concentrated by centrifugation on ultrafiltration membranes (Amicon ultra, Millipore) and desalted by size exclusion chromatography (Zeba spin columns, Perbio Science).

(69) HMG CoA synthase activity can be measured by methods well known in the art. One possible and preferably used assay is described, e.g. in Clinkenbeard et al. (J. Biol. Chem 250 (1975), 3108-3116). In this assay HMG-CoA synthase activity is measured by monitoring the decrease in the absorbance at 303 nm that accompanies the acetyl-CoA-dependent disappearance of the enolate form of acetoacetyl-CoA.

(70) The following three items were prepared individually on ice: Purified enzymes to be tested were diluted (1.6 mg/ml in 50 mM Tris pH 7.5 buffer) Reaction buffer (50 mM Tris pH 7.5, 20 mM MgCl.sub.2, 0.5 mM DTT, 0.2 mM AcCoA) Substrate (1 mM AcAcCoA in 50 mM Tris pH 7.5)

(71) Reagents were then mixed together on ice and immediately transferred to a spectrophotometer chamber set at 30 C. with shaking. Decrease in absorbency at 303 nm is monitored for 30 minutes for assay reactions and appropriate controls in the absence of enzymes or substrates. Enzyme activity (in mole/mg of enzyme/minute) is calculated from the slope of the curve obtained from the change in Abs(303 nm) in time. Results for WT HIV synthase (SEQ ID NO:1) and 11 variants are shown in Table 9 and expressed as the ratio of the specific HMG CoA synthase activity of each variant over the specific HMG CoA synthase activity of the WT enzyme. Corresponding HIV synthase activity for each variant also expressed relatively to that of the WT enzyme is also presented alongside.

(72) TABLE-US-00009 TABLE 9 HMG CoA synthesis activity of variants displaying improved HIV synthesis activity. Corresponding HMG CoA- and HIV synthase activities of HIV synthase variants displaying concomitantly increased HIV synthase activity and decreased HMG CoA synthase activity. Relative Relative HMG CoA HIV synthesis synthesis Sequence activity activity WT 1 1 L22M A201T H462Y 0.76 2.04 L22M A201T S221L G259D H462Y 0.54 15.51 L22M T165P A201T S221L I222Q G259D 0.46 23.91 H462Y L22M K75N T165P A201T S221L I222K 0.23 16.82 G259D S296Q H462Y N473G M481S L22M A201T S221L H462Y 0.19 8.57 L22M T165Q A201T S221L I222K L226M 0.125 15.295 K246R G259D S296Q H462Y N473D H475R V500S L22M K75N T165Q A201T S221L I222Q 0.1 18.55 G259D S296Q H462Y H475R L22M A201T S221L I222K H462Y 0 13.54 L22M K75N T165P A201T S221L I222K 0 19.75 S296Q H462Y N473D H475R V500S WT 1 1 L22M A201T H462Y 0.76 2.04

VIII. Example 8: Characterization of HMG CoA Synthesis Activity of Variants Displaying Improved HIV Synthesis Activity

(73) 1. Identification of HIV Synthase Variants with Improved HIV Synthesis Activity

(74) A combinatorial library was constructed in order to recombine a collection of amino acids mutations that had been identified in previous screens. The combinatorial library was constructed using the HIV synthase L22M A201T S221L H462Y variant as a template. This sequence was randomized at 19 positions in order to introduce 26 distinct mutations as detailed in Table 10. The construction of the combinatorial library used standard techniques of gene synthesis based on the assembly of overlapping sense and antisense oligonucleotides designed to match the targeted gene sequence (Czar et al, 2009 Trends in Biotechnology 27:63-72; Kodumal et al, 2004, Proc. Natl. Acad. Sci. USA 101:15573-15578; Smith et al 2003, Proc. Natl. Acad. Sci. USA 101:15440-15445; Xiong et al, 2008, FEMS Microbiol. Rev. 32:552-540). Briefly, a mixture of 69 34-35-mer oligonucleotides representing the HIV synthase L22M A201T S221L H462Y variant backbone was prepared at a final concentration of 50 M and spiked with oligonucleotides mutated at the targeted amino acid positions (0.05 to 0.4 M). PCR like reactions, without DNA template, were set up using 3 l of the oligonucleotide mixtures and 0.5 l of Pfx polymerase (LifeTechnologies) in order to assemble the gene from the oligonucleotides. The rate of mutations per clone was controlled by the ratio of backbone oligonucleotides to mutated oligonucleotides. A further cycle of PCR amplification of the reassembled gene was performed using primers situated at the 5 and 3 end of the gene was carried out. Finally, the amplified fragment was sub-cloned into the commercial peT-300/NT-DEST (LifeTechnologies).

(75) The combinatorial library was screened using the in vivo screening assay as follows. Plasmid DNA generated as described above and plasmid DNA containing the sequence coding for the reference HIV synthase L22M A201T S221L H462Y were transformed into BL21(DE3) competent cells and plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 30 C. until individual colonies reach the desired size. Single colonies were then picked and individually transferred into 75 L of liquid LB medium supplemented with the appropriate antibiotic. Cell growth is carried out with shaking for 20 hours at 30 C. The LB cultures were used to inoculate 300 l of Terrific broth supplemented with the appropriate antibiotic. Cultures were grown overnight at 30 C. for 20 hours in shaking incubator set at 700 rpm and 90% humidity. Cells were finally pelleted and the supernatant discarded. Bacterial pellets were resuspended in HIV production medium (Potassium phosphate 200 mM, Citric acid 4 mM, Ammonium chloride 20 mM, NTA mix 1, glucose 45 g/L and acetone 250 mM) at final OD600=10 and transferred to sealed culture vessels and incubated at 37 C. for 6 hours. Bacterial cultures were then deactivated by 5 minutes incubation at 80 C. and allowed to cool at room temperature. HIV produced by bacterial cultures was enzymatically converted to IBN for analysis by GC. The HIV containing preparations were therefore supplemented with 5 L HIV revelation buffer (final concentrations in reaction are as follows: KCl 20 mM, ATP 20 mM, HIV phosphorylase 2 g, PIV decarboxylase 50 g; HIV phosphorylase and PIV decarboxylase as produced and purified as described above). The enzymatic reactions were sealed and incubated for 24 hours at 37 C. Enzymatic reactions were finally stopped by heat shock denaturation of enzymes at 80 C. for 5 minutes. The isobutene (IBN) produced was quantified by gas chromatography (GC) according to the method described in Example 3. Table 11 presents a list of variants identified in this screen and their corresponding improvement factor compared to the control variant.

(76) TABLE-US-00010 TABLE 10 List of mutations recombined in the combinatorial library Wild type Mutations sequence Position of interest L 22 M I 24 M K 75 N K 100 L T 165 P/Q A 201 T I 222 Q/K/H/R L 226 M G 259 D L 270 I/M/H S 296 Q H 462 Y N 473 G/D H 475 R G 480 C M 481 S V 500 S V 514 S E 519 D

(77) TABLE-US-00011 TABLE 11 List of variants with increased HIV synthesis activity compared to L22M A201T S221L H462Y variant HIV production relative to template (L22M A201T S221L Variant sequence H462Y) L22M K75N T165Q A201T S221L I222Q G259D S296Q H462Y 3.7 H475R L22M I24M K75N K100L T165P A201T S221L I222Q L226M 3.6 K246R G259D L270I H462Y N473D G480C V500S L22M K75N T165Q A201T S221L I222Q L226M G259D L270M 3.4 S296Q H462Y N473D G480C V500S L22M I24M T165P A201T S221L I222K L226M G259D L270M 3.4 S296Q H462Y H475R L22M K75N T165P A201T S221L I222K G259D S296Q H462Y 3.4 N473G M481S L22M K75N T165P A201T S221L I222H L226M K246R G259D 3.2 H462Y N473D G480C L22M K75N A201T S221L I222H G259D S296Q H462Y N473G 3.1 G480C V500S L22M K75N T165P A201T S221L I222K S296Q H462Y N473D 3.1 H475R V500S L22M I24M K75N T165Q A201T S221L I222Q L226M K246R 2.9 G259D S296Q H462Y N473D G480C V500S L22M T165Q A201T S221L I222K L226M K246R G259D S296Q 2.9 H462Y N473D H475R V500S L22M A201T I24M K75N T165P S221L I222K L226M H462Y 2.9 L22M I24M K75N T165Q A201T S221L I222Q L226M K246R 2.7 G259D L270I S296Q H462Y N473D G480C M481S V500S L22M K75N T165Q A201T S221L H462Y I222K S296Q G480C 2.7 V500S L22M K75N T165P A201T S221L I222K K246R G259D H462Y 2.7 V500S L22M I24M T165Q A201T S221L I222K L226M G259D H462Y 2.6 H475R V500S L22M I24M K75N A201T S221L I222Q L226M G259D H462Y 2.6 N473D M481S L22M I24M K75N K100L T165P A201T S221L I222K K246R 2.6 G259D S296Q H462Y N473G M481S V500S L22M K75N A201T S221L I222Q L226M H462Y N473D V500S 2.5 L22M I24M K75N T165Q A201T S221L I222H G259D H462Y 2.5 G480C L22M I24M K75N A201T S221L I222Q K246R S296Q H462Y 2.3 N473G H475R L22M A201T S221L H462Y T165Q I222H G259D S296Q N473G 2.3 G480C L22M I24M K75N T165P A201T S221L I222K L226M K246R 2.2 G259D H462Y H475R V500S L22M I24M K75N T165Q A201T S221L I222H K246R S296Q 2.2 H462Y N473G G480C V500S L22M K75N T165QA201T S221L H462Y I222Q S296Q H475R 2.2 G480C M481S L22M K75N T165P A201T S221L I222Q G259D L270I S296Q 2.2 H462Y H475R V500S L22M I24M K75N K100L T165P A201T S221L I222H L226M 2.1 K246R G259D L270M S296Q H462Y N473D H475R L22M I24M K75N A201T S221L I222H G259D L270I S296Q 2.1 H462Y H475R L22M I24M K75N K100L T165P A201T S221L I222K L226M 2.1 K246R G259D L270M H462Y N473D V500S L22M I24M K75N T165Q A201T S221L L226M K246R G259D 2.1 L270I S296Q H462Y H475R V500S V514S L22M I24M K75N T165Q A201T S221L I222K L226M G259D 2.1 H462Y N473G L22M I24M K75N T165Q A201T S221L I222Q L226M K246R 2.0 G259D L270M S296Q H462Y N473D G480C L22M K75N A201T S221L G259D S296Q H462Y N473G G480C 2.0 L22M I24M K75N T165P A201T S221L I222K L226M K246R 2.0 L270M S296Q H462Y N473G G480C M481S V500S L22M K75N T165Q A201T S221L I222H K246R G259D L270I 2.0 S296Q H462Y N473G G480C M481S L22M I24M K75N T165Q A201T S221L I222R K246R G259D 1.9 H462Y N473D G480C V500S E519D L22M I24M K75N T165P A201T S221L I222K L226M S296Q 1.8 H462Y L22M I24M K75N K100L T165P A201T S221L I222H K246R 1.8 G259D L270M H462Y N473G G480C V500S L22M K75N T165Q A201T S221L H462Y 1.8 L22M I24M K75N T165Q A201T S221L I222Q K246R G259D 1.7 L270M H462Y N473G V514S L22M I24M K75N K100L T165P A201T S221L I222K K246R 1.7 G259D L270M S296Q H462Y N473G M481S L22M K75N T165Q A201T S221L L226M G259D L270I H462Y 1.7 N473D V500S L22M K75N A201T S221L I222Q L226M K246R S296Q H462Y 1.6 G480C V500S L22M I24M A201T S221L I222Q H462Y 1.6 L22M I24M K75N T165Q A201T S221L I222H L226M L270M 1.5 H462Y H475R L22M I24M K75N T165Q A201T S221L I222Q L226M K246R 1.5 G259D L270M S296Q H462Y H475R V500S L22M I24M K75N T165Q A201T S221L L226M K246R G259D 1.5 S296Q H462Y G480C V500S L22M K75N T165P A201T S221L I222K L226M K246R G259D 1.4 L270M H462Y N473D H475R L22M I24M K75N A201T S221L I222H L270M S296Q H462Y 1.4 V500S L22M A201T S221L H462Y 1.0
2. Analysis of the HMG CoA Synthesis Activity of Variants with Improved HIV Synthesis Activity

(78) A collection of 5 variants was selected out of Table 11 and their HMG CoA synthesis activity was assessed according to the assay described in Example 7. Results obtained for the 5 variants are shown in Table 12 and presented as the ratio of the specific HMG coA synthesis activity of each variant over the specific activity of the library template (L22M A201T S221L H462Y) and one of the best performing variant L22M A201T S221L H462Y G259D T165P I222Q L71 W13L K75N (variant constructed based on mutations isolated in a range of screens). Of particular interest are the variants L22M K75N T165P A201T S221L I222K S296Q H462Y N473D H475R V500S and L22M T165Q A201T S221L I222K L226M K246R G259D S296Q H462Y N473D H475R V500S that are characterized by an activity of HIV synthesis high compared to the two controls while their HMG-CoA synthesis activity is significantly decreased compared to the two controls.

(79) TABLE-US-00012 TABLE 12 HIV synthesis HMG CoA synthesis relative activity relative activity compared to L7I compared to L7I W13L L22M K75N W13L L22M K75N T165P A201T S221L T165P A201T S221L I222Q G259D H462Y I222Q G259D H462Y variant variant L7I W13L L22M K75N 1.00 1.00 T165P A201T S221L I222Q G259D H462Y L22M A201T S221L 0.40 0.23 H462Y L22M K75N T165Q 0.83 0.30 A201T S221L I222Q G259D S296Q H462Y H475R L22M K75N T165P 0.81 0.00 A201T S221L I222K S296Q H462Y N473D H475R V500S L22M K75N T165P 0.79 0.36 A201T S221L I222K G259D S296Q H462Y N473G M481S L22M K75N T165P 0.73 0.91 A201T S221L I222H L226M K246R G259D H462Y N473D G480C L22M T165Q A201T 0.72 0.00 S221L I222K L226M K246R G259D S296Q H462Y N473D H475R V500S

IX. Example 9: Mutation I222K Confers a Loss of HMG CoA Synthesis Activity

(80) The mutation I222K was of particular interest since (1) it lies in proximity with position S221 which is described as suppressing the production of acetate while enhancing the HIV production (see Example 6); (2) it is found in variants characterized with low HMGCoA synthesis ability (see Example 8). The importance of this mutation for both reactions was further tested.

(81) The coding sequences for variant L22M A201T S221L H462Y and variant L22M A201T S221L H462Y I222K were subcloned in pET25b+ bacterial expression vector (Merck-Novagen). These enzyme variants were produced and purified as described in Example 2. The HMGCoA synthesis activity and the HIV synthesis activity were measured as described in Example 7 and 2 respectively. Results are presented in Table 13 and indicate that the I222K mutation is critical to HMGCoA synthesis.

(82) TABLE-US-00013 TABLE 13 HIV synthesis HMG CoA synthesis relative activity relative activity compared to compared to L22M A201T S221L L22M A201T S221L H462Y variant H462Y variant L22M A201T S221L 1.0 1.0 H462Y L22M A201T S221L 1.6 0.0 H462Y I222K

X. Example 10: Characterization of HIV Synthase Variants in a Whole Cell Assay

(83) A selection of best performing HIV synthase variants obtained from sequential rounds of evolution were further characterized in a whole cell assay. This assay is based on the use of bacterial strain transformed with an expression vector (Merck-Novagen peT25b(+)) that contains the coding sequences and lead to the production of the 3 enzymes involved in the metabolic pathway converting acetone to isobutene; namely the HIV synthase for the production of HIV; the HIV phosphorylase for the production of PIV and the PIV decarboxylase for the conversion of PIV into isobutene (See FIG. 1); the production and purification of the HIV phosphorylase and the PIV decarboxylase is described above. The wild type HIV synthase, variant L22M A201T H462Y; variant L22M A201T S221L G259D H462Y and variant L22M T165P A201T S221L I222Q G259D H462Y were subcloned in the expression vector containing the HIV phosphorylase and PIV decarboxylase coding sequences. BL21(DE3) competent cells were transformed with these vectors and the cells were plated out and grown 24 hours at 30 C. on LB plates supplemented with the appropriate antibiotic. Single transformants were then used to inoculate 1 ml of LB culture medium and grown at 30 C. for 20 hours in a shaking incubator set at 700 rpm and 85% humidity. These starter cultures were used to inoculate 1 ml of auto-induction medium (Studier F. W; Protein Expr. Purif. 41 (2005), 207-234) and grown for a further 24 hours at 30 C. in a shaking incubator set at 700 rpm and 85% humidity. The cultures were centrifuged for 10 minutes at 4000 rpm and the supernatant discarded. The cell pellets containing the three overexpressed recombinant enzymes was then resuspended in 500 l of minimum medium supplemented with acetone and glucose (Potassium phosphate 200 mM, Citric acid 4 mM, Ammonium chloride 20 mM, NTA mix 1, glucose 45 g/L and acetone 500 mM). The cell suspensions, in sealed containers, were incubated at 37 C. for 2-4-6-16-20-24 hours in a shaking incubator. The Isobutene produced was then quantified by GC according to the method described in the previous examples. Results presented in FIG. 11 show that all 3 variants produce increased amount of isobutene compared to the wild type enzyme throughout the assay time course.

XI. Example 11: Improving HIV Synthesis Activity and Reducing HMG-CoA Synthesis Activity by Transfer of Mutation

(84) The mutation I222K has been shown in Example 9 as being beneficial for reducing the HMG-CoA synthesis activity.

(85) By transferring the mutation I222K and mutating one of the best performing variant L22M A201T S221L H462Y G259D T165P I222Q L71 W13L K75N, a new variant L22M A201T S221L I222K H462Y G259D T165P L71 W13R K75N was produced presenting both an increase in HIV synthesis and a decrease in HMG-CoA synthesis.

(86) The variants were subcloned in pET25b+ bacterial expression vector (Merck-Novagen). These enzyme variants were produced and purified as described in Example 2. The HMGCoA synthesis activities were measured as described in Example 7. The HIV synthesis activities were measured as followed. Plasmid DNA were transformed into BL21(DE3) competent cells and plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 30 C. until individual colonies reach the desired size. Single colonies were then picked and individually transferred into 1 mL of liquid LB medium supplemented with the appropriate antibiotic. Cell growth is carried out with shaking for 20 hours at 30 C. The LB cultures were used to inoculate 300 L of of auto-induction medium (Studier et al) supplemented with the appropriate antibiotic and grown for a further 24 hours at 30 C. in a shaking incubator set at 900 rpm and 85% humidity. Cells were finally pelleted and the supernatant discarded. Bacterial pellets were resuspended in 30 L HIV production medium (Potassium phosphate 200 mM, Citric acid 4 mM, Ammonium chloride 20 mM, NTA mix 1, glucose 45 g/L, magnesium sulfate 1 mM and acetone 25 mM) supplemented with the appropriate antibiotic and incubated at 30 C. for 4 hours. Bacterial cultures were then deactivated by 5 minutes incubation at 80 C. and allowed to cool at 4 C. overnight. HIV produced by bacterial cultures was enzymatically converted to IBN for analysis by GC. The HIV containing preparations were therefore supplemented with 5 L lysis buffer (Tris/HCl pH 7.5 50 mM, Lysonase 0.25% (Merck-Novagen)) and 10 L of revelation buffer prepared as followed. A plasmid vector containing the HIV phosphorylase and PIV decarboxylase was transformed into BL21(DE3) competent cells and plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 30 C. until individual colonies reach the desired size. Single colonies were then picked and individually transferred into 20 mL of liquid LB medium supplemented with the appropriate antibiotic. Cell growth is carried out with shaking for 20 hours at 30 C. The LB cultures were used to inoculate 500 mL of auto-induction medium (Studier et al) supplemented with the appropriate antibiotic and grown for a further 24 hours at 30 C. in a shaking incubator set at 900 rpm and 85% humidity. Cells were finally pelleted and the supernatant discarded. Following a 30 min freezing at 80 C., cells were resuspended in 50 mL of lysis buffer (Tris/HCl pH 7.5 50 mM, magnesium chloride 2 mM, potassium chloride 20 mM, ATP 15 mM, Lysonase 0.25% (Merck-Novagen)). The enzymatic reactions were sealed and incubated for 7 hours at 37 C. Enzymatic reactions were finally stopped by heat shock denaturation of enzymes at 80 C. for 5 minutes. The IBN produced was quantified by gas chromatography (GC) according to the method described in Example 3. Results are presented in Table 14.

(87) TABLE-US-00014 TABLE 14 HIV synthesis HMG CoA synthesis relative activity relative activity compared to compared to L22M A201T S221L L22M A201T S221L H462Y G259D T165P H462Y G259D T165P I222Q L7I W13L I222Q L7I W13L K75N variant K75N variant L22M A201T S221L 1.0 1.0 H462Y G259D T165P I222Q L7I W13L K75N L22M A201T S221L 1.29 0.25 I222K H462Y G259D T165P L7I W13R K75N

XII. Example 12: Directed Evolution of HMG-CoA Synthase for in Vivo HIV Production by in Vitro Screening

(88) Plasmid DNA containing one of the best performing variant L22M A201T S221L I222K H462Y G259D T165P L71 W13R K75N was subjected to standard mutagenesis protocols to generate a library of single or double residues mutations variants. This library was screened as described in Example 11. The best described variants are shown in Table 15.

(89) TABLE-US-00015 TABLE 15 HIV synthesis relative activity compared to L22M A201T S221L I222K H462Y G259D Mutations T165P L7I W13R K75N variant T171A-S338P 1.92 T171A-E325L 1.51 N167A-T171A 1.51 Q33E 1.51 T171A 1.44 S338P 1.25 T171G-E325V 1.16 A54G 1.14