Production of volatile dienes by enzymatic dehydration of light alkenols

Abstract

Described is a method for generating conjugated dienes through a biological process. More specifically, the application describes a method for producing conjugated dienes (for example butadiene, isoprene or dimethylbutadiene) from light alkenols via enzymatic dehydration, in particular by making use of an alkenol dehydratase.

Claims

1. A method for producing a conjugated diene comprising enzymatically converting a C.sub.nH.sub.2nO into C.sub.nH.sub.2n-2+H.sub.2O, wherein 3<n<7, by an enzyme at least 70% identical to the amino acid sequence of SEQ ID NO:1 wherein said enzyme has alkenol dehydratase activity.

2. The method of claim 1, wherein said method is carried out in vitro.

3. The method of claim 1, wherein said method is carried out in a microorganism.

4. The method of claim 1, wherein C.sub.nH.sub.2nO is C.sub.4H.sub.8O.

5. The method of claim 4, wherein the C.sub.4H.sub.8O is but-2-en-1-ol, but-3-en-2-ol, or but-3-en-1-ol.

6. The method of claim 5, wherein 1,3-butadiene is recovered.

7. The method of claim 1, wherein C.sub.nH.sub.2nO is C.sub.5H.sub.10O.

8. The method of claim 7, wherein the C.sub.5H.sub.10O is 3-methylbut-3-en-1-ol, 2-methylbut-2-en-1-ol, 3-methylbut-3-en-2-ol, or 2-methylbut-3-en-1-ol.

9. The method of claim 8, wherein isoprene is recovered.

10. The method of claim 1, wherein C.sub.nH.sub.2nO is C.sub.6H.sub.12O.

11. The method of claim 10, wherein the C.sub.6H.sub.12O is are 2,3-dimethylbut-2-en-1-ol, 2,3-dimethylbut-3-en-2-ol and 2,3-dimethylbut-3-en-1-ol.

12. The method of claim 11, wherein dimethyl-butadiene is recovered.

13. The method of claim 10, wherein the enzyme is at least 75% identical to the amino acid sequence of SEQ ID NO:1.

14. The method of claim 10, wherein the enzyme is at least 80% identical to the amino acid sequence of SEQ ID NO:1.

15. The method of claim 10, wherein the enzyme is at least 90% identical to the amino acid sequence of SEQ ID NO:1.

16. The method of claim 10, wherein the enzyme is the amino acid sequence of SEQ ID NO:1.

17. A composition comprising a microorganism comprising an enzyme at least 70% identical to the amino acid sequence of SEQ ID NO:1, wherein said enzyme has alkenol dehydratase activity, and a C.sub.nH.sub.2nO alcohol, wherein 3<n<7.

18. The composition of claim 17, wherein C.sub.nH.sub.2nO is C.sub.4H.sub.8O.

19. The composition of claim 18, wherein the C.sub.4H.sub.8O is but-2-en-1-ol, but-3-en-2-ol, or but-3-en-1-ol.

20. The composition of claim 17, wherein C.sub.nH.sub.2nO is C.sub.5H.sub.10O.

21. The composition of claim 20, wherein the C.sub.5H.sub.10O is 3-methylbut-3-en-1-ol, 2-methylbut-2-en-1-ol, 3-methylbut-3-en-2-ol, or 2-methylbut-3-en-1-ol.

22. The composition of claim 17, wherein C.sub.nH.sub.2nO is C.sub.6H.sub.12O.

23. The composition of claim 10, wherein the C.sub.6H.sub.12O is are 2,3-dimethylbut-2-en-1-ol, 2,3-dimethylbut-3-en-2-ol and 2,3-dimethylbut-3-en-1-ol.

24. The composition of claim 22, wherein the enzyme is at least 75% identical to the amino acid sequence of SEQ ID NO:1.

25. The composition of claim 22, wherein the enzyme is at least 80% identical to the amino acid sequence of SEQ ID NO:1.

26. The composition of claim 22, wherein the enzyme is at least 90% identical to the amino acid sequence of SEQ ID NO:1.

27. The composition of claim 22, wherein the enzyme is the amino acid sequence of SEQ ID NO:1.

28. A composition comprising an enzyme at least 70% identical to the amino acid sequence of SEQ ID NO:1, wherein said enzyme has alkenol dehydratase activity, and a C.sub.nH.sub.2nO alcohol, wherein 3<n<7.

29. The composition of claim 28, wherein C.sub.nH.sub.2nO is C.sub.4H.sub.8O.

30. The composition of claim 29, wherein the C.sub.4H.sub.8O is but-2-en-1-ol, but-3-en-2-ol, or but-3-en-1-ol.

31. The composition of claim 28, wherein C.sub.nH.sub.2nO is C.sub.5H.sub.10O.

32. The composition of claim 31, wherein the C.sub.5H.sub.10O is 3-methylbut-3-en-1-ol, 2-methylbut-2-en-1-ol, 3-methylbut-3-en-2-ol, or 2-methylbut-3-en-1-ol.

33. The composition of claim 28, wherein C.sub.nH.sub.2nO is C.sub.6H.sub.12O.

34. The composition of claim 33, wherein the C.sub.6H.sub.12O is are 2,3-dimethylbut-2-en-1-ol, 2,3-dimethylbut-3-en-2-ol and 2,3-dimethylbut-3-en-1-ol.

35. The composition of claim 28, wherein the enzyme is at least 75% identical to the amino acid sequence of SEQ ID NO:1.

36. The composition of claim 28, wherein the enzyme is at least 80% identical to the amino acid sequence of SEQ ID NO:1.

37. The composition of claim 28, wherein the enzyme is at least 90% identical to the amino acid sequence of SEQ ID NO:1.

38. The composition of claim 28, wherein the enzyme is the amino acid sequence of SEQ ID NO:1.

Description

(1) FIG. 1 shows schematically the primary allyl alcohols (PRA) responding to the general formula C.sub.nH.sub.2nO, with 3<n<7. In particular shown are: Substrate/Systematic name/Formula/Category/R.sup.1/R.sup.2/Product

(2) FIG. 2 shows schematically the secondary and tertiary allyl alcohols (STA) responding to the general formula C.sub.nH.sub.2nO, with 3<n<7.Substrate/Systematic name/Formula/Category/R.sup.1/R.sup.2/Product

(3) FIG. 3 shows schematically the primary homoallyl alcohols (PHA) responding to the general formula C.sub.nH.sub.2nO, with 3<n<7.Substrate/Systematic name/Formula/Category/R.sup.1/R.sup.2/Product

(4) FIG. 4 shows a schematic overview over the conversion of the above mentioned PRA, PHA and STA compounds into a conjugated diene according to the method of the present invention.

(5) FIG. 5 shows an overview of the reactions catalyzed by linalool dehydratase-isomerase.

(6) FIG. 6 shows the amino acid sequence of the linalool dehydratase-isomerase from Castellaniella defragrans (formerly Alcaligenes defragrans).

(7) FIG. 7 shows the GC/FID chromatograms obtained for enzymatic (black) and enzyme-free (red) assays with 80 mM trans crotyl alcohol after 22 hours incubation.

(8) FIG. 8 shows the GC/FID chromatograms obtained for enzymatic (black) and enzyme-free (red) assays with 80 mM 3-buten-2-ol after 22 hours incubation.

(9) FIG. 9 shows the GC/FID chromatograms obtained for enzymatic (black) and enzyme-free (blue) assays with 80 mM isoprenol after 22 hours incubation.

(10) FIG. 10 shows the GC/FID chromatograms obtained for enzymatic (red) and enzyme-free (black) assays with 80 mM 2-methyl-3-buten-2-ol after 22 hours incubation.

(11) 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

Example 1

Cloning and Expression in E. coli of the Gene for Linalool Dehydratase-Isomerase

(12) Cloning and Bacterial Culture

(13) The sequence of linalool dehydratase-isomerase inferred from the genome of Castellaniella defragrans (formerly Alcaligenes defragrans) was generated by oligonucleotide concatenation to fit the codon usage of E. coli. A stretch of 6 histidine codons was inserted after the methionine initiation codon to provide an affinity tag for purification. The gene thus synthesized was cloned in a pET25b(+) expression vector (the vector was constructed by GeneArt AG). Competent E. coli BL21(DE3) cells (Novagen) were transformed with this vector according to the heat shock procedure. As negative control, E. coli BL21(DE3) strain was transformed with empty vector. The transformed cells were grown with shaking (160 rpm) on ZYM-5052 auto-induction medium (Studier F W, Prot. Exp. Pur. 41 (2005), 207-234) for 6 hours at 37 C. and protein expression was continued at 18 C. overnight (approximately 12 hours). The cells were collected by centrifugation at 4 C., 10,000 rpm for 20 min and the pellets were frozen at 80 C.

(14) Preparation of Cell Lysate

(15) The pellets from 100 ml of culture cells were thawed on ice and resuspended in 4 ml of 50 mM Tris-HCl pH 7.5. 10 l of lysonase (Novagen) were then added. Cells were incubated for 10 minutes at room temperature and then returned to ice for 20 minutes. Protein concentration was determined using the Bradford method (Biorad).

Example 2

1,3-butadiene Production from (2E)-2-buten-1-ol (trans crotyl alcohol)

(16) The enzymatic assays were carried out under the following conditions: 50 mM Tris HCl pH 7.5 2 mM D,L-Dithiothreitol 0-80 mM (2E)-2-buten-1-ol (trans crotyl alcohol) The pH was adjusted to 7.5

(17) 0.25 ml of cell lysate containing recombinant linalool dehydratase-isomerase was added to 0.5 ml of reaction mixture. An enzyme-free control reaction containing lysate of E. coli cells transformed with empty vector was carried out in parallel. Assays were incubated at 37 C. for 1-22 hours in a 2 ml sealed glass vial (Interchim) with shaking. One ml of the headspace phase was then collected and injected into a gas chromatograph Varian 450-GC equipped with a flame ionization detector (FID). Nitrogen was used as carrier gas with a flow rate of 1.5 ml/min. Volatile compounds were chromatographically separated on Rt-Alumina Bond/Na.sub.2SO.sub.4 column (Restek) using an isothermal mode at 130 C. The enzymatic reaction product was identified by comparison with 1,3-butadiene standard (Sigma). Under these GC conditions, the retention time for butadiene was 7.6 min. A significant production of 1,3-butadiene was observed in enzymatic assay with linalool dehydratase-isomerase. No butadiene signal was observed in enzyme-free control assay (FIG. 7). The turnover number for this conversion amounted to about 310.sup.5 s.sup.1 substrate molecule per enzyme active site.

Example 3

1,3-butadiene Production from 3-buten-2-ol

(18) The enzymatic assays were carried out under the following conditions: 50 mM Tris HCl pH 7.5 2 mM D,L-Dithiothreitol 0-80 mM 3-buten-2-ol The pH was adjusted to 7.5

(19) 0.25 ml of cell lysate containing recombinant linalool dehydratase-isomerase was added to 0.5 nil of reaction mixture. An enzyme-free control reaction containing lysate of E. coli cells transformed with empty vector was carried out in parallel. Assays were incubated at 37 C. for 1-22 hours in a 2 ml sealed glass vial (Interchim) with shaking. 1,3-butadiene production was analyzed by GC/FID procedure as described in example 2. A significant production of 1,3-butadiene was observed in enzymatic assay with linalool dehydratase-isomerase. No butadiene signal was observed in enzyme-free control assay (FIG. 8). The turnover number for this conversion amounted to about 10.sup.4 s.sup.1 substrate molecule per enzyme active site.

Example 4

2-methyl-1,3-butadiene (isoprene) Production from 3-methyl-2-buten-1-ol (prenol)

(20) The enzymatic assays were carried out under the following conditions: 50 mM Tris HCl pH 7.5 2 mM D,L-Dithiothreitol 0-80 mM 3-methyl-2-buten-1-ol (prenol) The pH was adjusted to 7.5

(21) 0.25 ml of cell lysate containing recombinant linalool dehydratase-isomerase was added to 0.5 nil of reaction mixture. An enzyme-free control reaction containing lysate of E. coli cells transformed with empty vector was carried out in parallel. Assays were incubated at 37 C. for 1-22 hours in a 2.0 ml sealed glass vial (Interchim) with shaking. 100 l of the headspace phase was then collected and injected into a gas chromatograph Varian 450-GC equipped with a flame ionization detector (FID). Volatiles compounds from headspace phase were separated on Rtx-1 column (Restek) using nitrogen as carrier gas with a flow rate of 1.5 ml/min. The oven cycle for each sample was 100 C. for 4 minutes, increasing temperature at 20 C./minute to a temperature of 130 C., and hold at 130 C. for 1.5 minutes. The total run time was 7 min. The enzymatic reaction product was identified by comparison with isoprene standard (Sigma). Under these GC conditions, the retention time for isoprene was 3.08 min. A significant production of isoprene was observed in enzymatic assay with linalool dehydratase-isomerase. An insignificant signal of isoprene corresponding to spontaneous decomposition of prenol was observed in enzyme-free control assay (Table 1). The turnover number for this conversion amounted to about 310.sup.4 s.sup.1 substrate molecule per enzyme active site.

(22) TABLE-US-00001 TABLE 1 Isoprene production after 22 hours incubation in assays with 80 mM prenol . . . Isoprene Assay peak area, arbitrary units Enzymatic assay with linalool dehydratase- 29705.4 isomerase Enzyme-free control assay 7.5

Example 5

2-methyl-1,3-butadiene (isoprene) Production from 3-methyl-3-buten-1-ol (isoprenol)

(23) The enzymatic assays were carried out under the following conditions: 50 mM Tris HCl pH 7.5 2 mM D,L-Dithiothreitol 0-80 mM 3-methyl-3-buten-1-ol (isoprenol) The pH was adjusted to 7.5

(24) 0.25 ml of cell lysate containing recombinant linalool dehydratase-isomerase was added to 0.5 ml of reaction mixture. An enzyme-free control reaction containing lysate of E. coli cells transformed with empty vector was carried out in parallel. Assays were incubated at 37 C. for 1-22 hours in a 2 ml sealed glass vial (Interchim) with shaking. Isoprene production was analyzed by GC/FID procedure as described in example 4. A significant production of isoprene was observed in enzymatic assay with linalool dehydratase-isomerase. No isoprene signal was observed in enzyme-free control assay (FIG. 9). The turnover number for this conversion amounted to about 310.sup.5 s.sup.1 substrate molecule per enzyme active site.

Example 6

2-methyl-1,3-butadiene (isoprene) production from 2-methyl-3-buten-2-ol

(25) The enzymatic assays were carried out under the following conditions: 50 mM Tris HCl pH 7.5 2 mM D,L-Dithiothreitol 0-80 mM 2-methyl-3-buten-2-ol The pH was adjusted to 7.5

(26) 0.25 ml of cell lysate containing recombinant linalool dehydratase-isomerase was added to 0.5 ml of reaction mixture. An enzyme-free control reaction containing lysate of E. coli cells transformed with empty vector was carried out in parallel.

(27) Assays were incubated at 37 C. for 1-22 hours in a 2 ml sealed glass vial (Interchim) with shaking. Isoprene production was analyzed by GC/FID procedure as described in example 4. A significant production of isoprene was observed in enzymatic assay with linalool dehydratase-isomerase. No isoprene signal was observed in enzyme-free control assay (FIG. 10). The turnover number for this conversion amounted to about 10.sup.3 s.sup.1 substrate molecule per enzyme active site.