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 characterized in that it comprises a step of enzymatically converting a compound responding to the general formula C.sub.nH.sub.2nO into C.sub.nH.sub.2n-2+H.sub.20, with 3<n<7, by making use of an alkenol dehydratase.

2. The method of claim 1 wherein the compound responding to the general formula C.sub.nH.sub.2nO, with 3<n<7, is a primary allyl alcohol (PRA) of the formula I: ##STR00007## wherein R.sup.1 and R.sup.2 are independently selected from H and CH.sub.3.

3. The method of claim 1 wherein the compound responding to the general formula C.sub.nH.sub.2nO, with 3<n<7, is a secondary or tertiary allyl alcohol (STA) of the formula II: ##STR00008## wherein R.sup.1 and R.sup.2 are independently selected from H and CH.sub.3.

4. The method of claim 1 wherein the compound responding to the general formula C.sub.nH.sub.2nO, with 3<n<7, is a primary homoallyl alcohol (PHA) of the formula Ill: ##STR00009## wherein R.sup.1 and R.sup.2 are independently selected from H and CH.sub.3.

5. The method of claim 1 which is carried out in vitro.

6. The method of claim 1 characterized in that the method is carried by making use of a microorganism producing an alkenol dehydratase.

7. The method of claim 6, wherein the microorganism is capable of producing a compound responding to the general formula C.sub.nH.sub.2nO, with 3<n<7.

8. Use of an alkenol dehydratase or of a microorganism producing an alkenol dehydratase for converting a compound responding to the general formula C.sub.nH.sub.2nO into C.sub.nH.sub.2n-2+H.sub.20, with 3<n<7.

9. A composition comprising an organism which produces an alkenol dehydratase and a compound responding to the general formula C.sub.nH.sub.2nO, with 3<n<7.

10. A composition comprising an alkenol dehydratase and a compound responding to the general formula C.sub.nH.sub.2nO, with 3<n<7.

11. The method of claim 1, wherein the alkenol dehydratase is a protein which comprises the amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence which is at least 30% identical to the amino acid sequence shown in SEQ ID NO: 1 and which shows the enzymatic activity of converting a compound responding to the general formula C.sub.nH.sub.2nO into C.sub.nH.sub.2n-2+H.sub.20, with 3<n<7.

12. The method of claim 1 wherein n is 4 and the produced diene compound is butadiene.

13. The method of claim 1, wherein n is 5 and the produced diene compound is isoprene.

14. The method of claim 1, wherein n is 6 and the produced diene compound is dimethyl-butadiene.

Description

[0092] As regards the preferred embodiments of the different components recited, the same applies as has been set forth above in connection with the method according to the invention.

[0093] 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

[0094] 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

[0095] 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

[0096] 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.

[0097] FIG. 5 shows an overview of the reactions catalyzed by linalool dehydratase-isomerase.

[0098] FIG. 6 shows the amino acid sequence of the linalool dehydratase-isomerase from Castellaniella defragrans (formerly Alcaligenes defragrans).

[0099] 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.

[0100] 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.

[0101] FIG. 9 shows the GC/FID chromatograms obtained for enzymatic (black) and enzyme-free (blue) assays with 80 mM isoprenol after 22 hours incubation.

[0102] 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.

[0103] 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

[0104] Cloning and Bacterial Culture

[0105] 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.

[0106] Preparation of Cell Lysate

[0107] 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)

[0108] The enzymatic assays were carried out under the following conditions:

[0109] 50 mM Tris HCl pH 7.5

[0110] 2 mM D,L-Dithiothreitol

[0111] 0-80 mM (2E)-2-buten-1-ol (trans crotyl alcohol)

[0112] The pH was adjusted to 7.5

[0113] 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

[0114] The enzymatic assays were carried out under the following conditions:

[0115] 50 mM Tris HCl pH 7.5

[0116] 2 mM D,L-Dithiothreitol

[0117] 0-80 mM 3-buten-2-ol

[0118] The pH was adjusted to 7.5

[0119] 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. 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)

[0120] The enzymatic assays were carried out under the following conditions:

[0121] 50 mM Tris HCl pH 7.5

[0122] 2 mM D,L-Dithiothreitol

[0123] 0-80 mM 3-methyl-2-buten-1-ol (prenol)

[0124] The pH was adjusted to 7.5

[0125] 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 2.0 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.

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

Example 5: 2-methyl-1,3-butadiene (isoprene) production from 3-methyl-3-buten-1-ol (isoprenol)

[0126] The enzymatic assays were carried out under the following conditions:

[0127] 50 mM Tris HCl pH 7.5

[0128] 2 mM D,L-Dithiothreitol

[0129] 0-80 mM 3-methyl-3-buten-1-ol (isoprenol)

[0130] The pH was adjusted to 7.5

[0131] 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

[0132] The enzymatic assays were carried out under the following conditions:

[0133] 50 mM Tris HCl pH 7.5

[0134] 2 mM D,L-Dithiothreitol

[0135] 0-80 mM 2-methyl-3-buten-2-ol

[0136] The pH was adjusted to 7.5

[0137] 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. 10). The turnover number for this conversion amounted to about 10.sup.3 s.sup.1 substrate molecule per enzyme active site.