Alkene production

Abstract

The present invention provides a microbial cell capable of producing at least one terminal alkene from at least one short chain fatty acid, wherein the cell is genetically modified to comprise at least a first genetic mutation that increases the expression relative to the wild type cell of an enzyme (E.sub.1) selected from the CYP152 peroxygenase family, and at least a second genetic mutation that increases the expression relative to the wild type cell of at least one NAD(P)+ oxidoreductase (E.sub.2) and the corresponding mediator protein, wherein the short chain fatty acid is a C4-C10 fatty acid.

Claims

1. A microbial cell capable of producing at least one terminal alkene from at least one C.sub.4-C.sub.10 fatty acid, wherein the microbial cell is genetically modified to comprise: at least one first genetic modification that increases the expression relative to the wild type cell of an enzyme (E.sub.1) selected from the group consisting of CYP.sub.sp (E.sub.1a), CYP.sub.BSB (E.sub.1b) and OleT (E.sub.1c); and at least one second genetic modification that increases the expression relative to the wild type cell of at least one NAD(P)+ oxidoreductase (E.sub.2) and the corresponding mediator protein, wherein the NAD(P)+ oxidoreductase (E.sub.2) and the corresponding mediator protein are: (a) ferredoxin reductase (E.sub.2a) and ferredoxin; or (b) putidaredoxin reductase (E.sub.2b) and putidaredoxin wherein CYP.sub.sp (E.sub.1a), CYP.sub.BSB (E.sub.1b) and OleT (E.sub.1c) are members of the CYP152 peroxygenase family.

2. The microbial cell according to claim 1, wherein the enzyme (E.sub.1) is CYP.sub.SP (E.sub.1a).

3. The microbial cell according to claim 1, wherein the enzyme (E.sub.1) is OleT (E.sub.1c) and has at least 60% sequence identity to SEQ ID NO:1.

4. The microbial cell according to claim 1, wherein the NAD(P)+ oxidoreductase (E.sub.2) and the corresponding mediator protein are: (a) ferredoxin reductase (E.sub.2a) and ferredoxin.

5. The microbial cell according to claim 1, wherein the NAD(P)+ oxidoreductase (E.sub.2) has 60% sequence identity to SEQ ID NO:2 and the corresponding mediator protein has 60% sequence identity to SEQ ID NO:3.

6. The microbial cell according to claim 1, wherein the cell further comprises at least one third genetic modification that increases the expression relative to the wild type cell of at least one enzyme (E.sub.3) capable of NAD(P)H regeneration.

7. The microbial cell according to claim 6, wherein the enzyme (E.sub.3) is selected from the group consisting of glucose dehydrogenase, phosphite dehydrogenase and formate dehydrogenase.

8. The microbial cell according to claim 1, wherein the cell further comprises a reduced fatty acid degradation capacity relative to the wild type cell.

9. The microbial cell according to claim 8, wherein the fatty acid degradation capacity is reduced by deletion of a gene encoding an enzyme selected from the group consisting of fatty acid importer, fatty acid-CoA ligase, acyl-CoA dehydrogenase, 2,4-dienoyl-CoA reductase, enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase.

10. The microbial cell according to claim 1, wherein the microbial cell is a prokaryotic or a lower eukaryotic cell.

11. A method of producing at least one terminal alkene, comprising: contacting the microbial cell of claim 1 with the at least one C.sub.4-C.sub.10 fatty acid and collecting the alkene produced.

12. The cell according to claim 1, wherein the enzyme (E.sub.1) is CYP.sub.BSB (E.sub.1b).

13. The microbial cell according to claim 1, wherein the NAD(P)+ oxidoreductase (E.sub.2) and the corresponding mediator protein are: (b) putidaredoxin reductase (E.sub.2b) and putidaredoxin.

14. The microbial cell according to claim 1, wherein the enzyme (E.sub.1) is OleT (E.sub.1c) and has at least 60% sequence identity to SEQ ID NO:1, and the NAD(P)+ oxidoreductase (E.sub.2) has 60% sequence identity to SEQ ID NO:2 and the corresponding mediator protein has 60% sequence identity to SEQ ID NO:3.

15. The microbial cell according to claim 1, wherein the enzyme (E.sub.1) is OleT (E.sub.1c) and has at least 95% sequence identity to SEQ ID NO:1, and the NAD(P)+ oxidoreductase (E.sub.2) has 95% sequence identity to SEQ ID NO:2 and the corresponding mediator protein has 95% sequence identity to SEQ ID NO:3.

16. The microbial cell according to claim 1, wherein the enzyme (E.sub.1), the NAD(P)+ oxidoreductase (E.sub.2) and the corresponding mediator protein are heterologous to the microbial cell.

17. The microbial cell according to claim 14, wherein the enzyme (E.sub.1), the NAD(P)+ oxidoreductase (E.sub.2) and the corresponding mediator protein are heterologous to the microbial cell.

18. The microbial cell according to claim 15, wherein the enzyme (E.sub.1), the NAD(P)+ oxidoreductase (E.sub.2) and the corresponding mediator protein are heterologous to the microbial cell.

19. The microbial cell according to claim 18, wherein the cell further comprises at least one third genetic modification that increases the expression relative to the wild type cell of at least one enzyme (E.sub.3) capable of NAD(P)H regeneration.

20. The method according to claim 11, wherein the at least one C.sub.4-C.sub.10 fatty acid is at least one selected from the group consisting of isobutyric acid, butyric acid, isovaleric acid, and valeric acid.

Description

BRIEF DESCRIPTION OF FIGURES

(1) The inventions are further illustrated by the following figures and non-limiting examples from which further embodiments, aspects and advantages of the present invention may be taken.

(2) FIG. 1 is a chromatogram obtained from a conversion of heptanoic acid with the OleT-CamABFDH (cascade, a=CO2; b=EtOH; c=1-hexene; d=MIBK (derived from EtOH). Compounds were injected manually from the reaction vessel headspace into a GC-MS chromatograph.

(3) FIG. 2 is a chromatogram after manual headspace GC-MS injection of an analytical standard of 1-hexene.

(4) FIG. 3 is a graph of the GC-MS spectrum for peak c in FIG. 1 corresponding to 1-hexene (84.1 g/mol).

(5) FIG. 4 is a graph of the GC-MS spectrum for peak c in FIG. 2 corresponding to the analytical standard of 1-hexene (84.1 g/mol).

(6) FIG. 5 is a chromatogram obtained after ion extraction of ion 84 from conversion of heptanoic acid with the OleT-CamAB-FDH cascade.

(7) FIG. 6 is a calibration curve for manual headspace GC-MS injection of 1-hexene using peak area of extracted ion 84.

(8) FIG. 7 is a picture of a novel reaction set up for trapping volatile -olefins, such as 1-propene and 1-butene. A small impinger containing 10 ml of a 20 mM Br2/DCM solution was placed on top of the flask serving as sole gas outlet and trap for -olefin products. The functionality of this system was successfully confirmed using an analytical standard of 1-heptene. The concept for derivatization and trapping of volatile -olefins is shown in Scheme 2.

(9) FIG. 8A-8D are GC-FID chromatograms from the production of 1-butene from valeric acid by the OleTCamAB-FDH cascade. 8A=impinger solution (DCM/Br2); 8B=content of the impinger solution after 24 h conversion of valeric acid by OleT-CamAB-FDH cascade; 8C=analytical standard of 1,2-dibromobutane; 8D=content of the impinger solution after 24 h conversion of valeric acid without OleT. a=solvent peak (DCM); b=1,2-dibromobutane; c=impurity from DCM solvent.

(10) FIG. 9A-9D are GC-FID chromatograms from the production of 1-propene from valeric acid by the OleT-CamAB-FDH cascade. 9A=analytical standard of 1,2-dibromopropane; 9B=content of the impinger solution after 24 h conversion of valeric acid by OleT-CamAB-FDH cascade. 9C=content of the impinger solution after 24 h conversion of butyric acid by OleT-CamAB-FDH cascade; 9D=impinger solution (DCM/Br2); a=solvent peak (DCM); b=1,2-dibromopropane; c=1,2-dibromobutane; d=impurity from DCM solvent.

(11) Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

EXAMPLES

(12) The foregoing describes preferred embodiments, which, as will be understood by those skilled in the art, may be subject to variations or modifications in design, construction or operation without departing from the scope of the claims. These variations, for instance, are intended to be covered by the scope of the claims.

Example 1

Cloning, Expression and Quantification of OleT

(13) All chemicals were obtained from Sigma Aldrich. Spinach ferredoxin, spinach ferredoxin reductase, catalase from bovine liver, lysozyme, cytochrome c from bovine heart and glucose dehydrogenase (NADPH-dependent, clarify commercial source were obtained from Sigma Aldrich. Formate dehydrogenase (NADH-dependent) was obtained from Evocatal. Phosphite dehydrogenase (PDH) (Dudek, H. M., 2013) was prepared according) a standard protocol, the plasmid was obtained from M. Fraaije (Groningen).

(14) OleT was ordered from Life Technologies as codon-optimized synthetic gene for optimal expression in E. coli. The synthetic construct was cloned via restriction/ligation (NdeI and XhoI) into the pET28a expression vector downstream (pET28a-OleT) of an N-terminal His.sub.6-tag prior to chemical transformation into E. coli Snuffle T7 strain (New England Biolabs). Successful cloning was verified by colony PCR, restriction analysis and sequencing. Cells containing pET28a-OleT were grown overnight (140 rpm, 37 C.) as pre-culture in lysogeny broth (LB) media supplemented with 50 kanamycin. Expression of OleT was achieved by transferring 2 mL of pre-culture into 200 mL terrific broth (TB) media according to a published protocol (Nazor, J, 2009). Efficient expression of OleT was achieved after 20 h incubation at 25 C. and 140 rpm shaking. Cells were separated from expression media by centrifugation and pellets were stored for 24 h at 20 C., Frozen cell pellets were resuspended in purification buffer (KPi; 0.1 M; pH 7.0; 20% glycerol, 0.3 M KCl; 50 mM imidazol). Lysozyme was added (1 mg mL.sup.1) followed by incubation at 37 C. for 1 h. Cells were finally disrupted by sonication (1 min, 30% amplitude) using an ultra sonicator. Cell debris was pelleted by centrifugation and cell free lysates were pressed through a 0.45 m filter to remove residual particles. Filtered cell free lysate was loaded onto a His-Trap column connected to a Biorad FPLC pumping system with UV-detector. OleT was purified according to the protocol of Matsunaga et al., 2001 but using 400 mM (instead of 200 mM) imidazol for the final elution step. During dialysis of purified OleT with the reported dialysis buffer (KPi, 0.1 M, pH 7.0, 20% glycerol), large amounts of the protein precipitated (active protein concentrations of only 1 to 2 M OleT retained). In order to remove residual imidazole and to prevent precipitation, dialysis was performed against phosphate buffer (KPi, 0.1 M, pH 7.0, 20% glycerol) containing 300 mM KCl. The protein lysates were filled in in a dialysis tubing cellulose membrane (14 kDa cut off, Sigma Aldrich, Steinheim, Germany a dialysis bag and dialyzed three times against 300 ml of dialysis buffer (312 h) at 4 C. and continuous stirring. No visible precipitation occurred during the dialysis procedure and active P450 concentrations of >10 M were commonly obtained. Concentrations of active P450 protein in cell free lysates or purified OleT were determined by recording CO difference spectra.

(15) Cloning, Expression and Quantification of CamAB System

(16) Expression of CamA and CamB (CamAB) was done according to a published protocol employing the reported plasmid construct and E. coli host (Schallmey, A., 2011).

(17) Product Extraction, Derivatization of Fatty and Hydroxy Acids

(18) Enzymatic reactions (1 mL scale) were quenched by addition of 100 L 5 N Extraction of substrate and products was performed with 500 L EtOAc containing 0.1% 1-decanol as internal standard. The organic phase was dried over anhydrous Na.sub.2SO.sub.4. Derivatization of carboxylic acids (fatty acids and hydroxy acids) to the corresponding methyl esters was achieved by mixing the organic phase with MeOH (2.5:1.5 v/v) followed by supplementation of 5 to 15 L of 2 M TMSCHN.sub.2 (trimethylsilyldiazomethane in diethyl ether). The mixture was incubated for 30 min, 25 C. and 750 rpm before injection into GC-FID or GC-MS, Extraction and derivatization of products and substrates from conversion of short chain FAs (C12-C9) was performed using the same protocol but all steps were performed on ice to prevent undesired loss of volatile products.

(19) Preparation of Samples for Headspace GC-MS Analysis

(20) Products from FA substrates with chain lengths ranging from C22 to C10 were separated on a HP5 column (Programm) and detected by GC-FID or GC-MS. Extracted products from conversion of nonanoic acid (I-octene as product) were separated on a DB1702 column (Programm) and detected by GC-FID. Authentic standards of substrates and terminal olefins were treated as described above and were used as references for identification and preparation of calibration curves. Detection of volatile short chain terminal olefins (1-butene/1-propene [ongoing] to 1-heptene) was accomplished by manual injection from vial headspaces into GC-MS, in order to prevent loss of products, the vials containing the reaction mixtures were sealed with a PTFE septum. After conversion the closed vial was heated for 10 min at 80 C., together with a 10 ml syringe (TYPE). From the headspace 2 to 9 l volumes were injected splitless into a GC-MS chromatograph and analytes were separated on a HP5 column (Programm). Calibration curves for 1-pentene, 1-hexene and 1-heptene were prepared in the same way at 4 C. and using 5% DMF as cosolvent.

Example 2

Conversion of FAs with the CamAB-OleT Redox Cascade and O2 as Oxidant

(21) An optimized purification protocol allowed the improved production of OleT in active form (10-20 M from 400 mL culture broth), avoiding protein precipitation during dialysis (ESI). Bacterial CamAB (putidaredoxin; class 1) was used as an electron transfer system (Koga H., 1989 and Roome P. W., 1983) (Scheme 1). Initial experiments confirmed that OleT can accept electrons from CamAB allowing the decarboxylation of stearic acid with O.sub.2 as sole oxidant, whereas control reactions did not lead to any product formation (Table 1). Further, lower CamAB concentration was found to be optimum for higher conversions. No formation of H.sub.2O.sub.2 (formed via reduction of O.sub.2) could be detected by using the highly sensitive HRP/ABTS assay (Sigma Aldrich) according to manufacturer's protocol after full oxidation of 1 mM NADH, further indicating a direct electron transfer from CamAB to OleT and ruling out potential inactivation of OleT by H.sub.2O.sub.2.

(22) TABLE-US-00001 TABLE 1 Catalytic characterization of OleT using either O.sub.2/CamAB or H.sub.2O.sub.2 as oxidant for decarboxylation of stearic and palmitic acid. Oxidant/ Reaction - Electron Time Olefin TOF Coupling TON Entry Source [min] [M] [h.sup.1] [%] [] Stearic Acid 1 200 M 60 53 5 53 25 53 NADPH + O.sub.2 2 1 mM 150 118 23 47 11 118 NADPH + O.sub.2 3 200 M 6 15 1 150 7 15 NADH + O.sub.2 4 1 mM 60 20 2 20 2 20 NADH + O.sub.2 5 200 M 60 170 7 170 85 170 H.sub.2O.sub.2 6 1 mM 150 53 15 21 5 53 H.sub.2O.sub.2* Palmitic Acid 7 200 M 60 21 21 10 21 NADPH + O.sub.2 8 1 mM 150 33 13 3 33 NADPH + O.sub.2 9 200 M 6 6 60 3 60 NADH + O.sub.2 10 1 mM 60 9 9 1 9 NADH + O.sub.2 11 200 M 60 65 65 33 65 H.sub.2O.sub.2 12 1 mM 150 73 29 7 29 H.sub.2O.sub.2* 13 Control 150 0 0 0 0 reactions Reaction conditions: 1 M OleT, 0.05 U ml.sup.1 CamAB, 1200 U ml.sup.1 catalase, 1 mM substrate, 2.5% EtOH, KPi buffer (pH 7.5, 0.1M). Conversions were performed in plastic cuvettes at room temperature and without agitation. Reactions with H.sub.2O.sub.2 as oxidant contained only OleT, substrate, buffer and hydrogen peroxide. *Strong precipitation observed. .sup.1One of the following components was left out of the reaction: OleT, H.sub.2O.sub.2, CamAB, NAD(P)H or substrate.

Example 3

Conversion of FAs with the CamAB-OleT Redox Cascade and NAD(P)H Regeneration System

(23) The use of a glucose dehydrogenase (GDH)-based system provided a substantial increase in conversion (36% conversion with 1 mM stearic acid) and TIN (up to 389 with 10 mM stearic acid), with comparable conversions of both stearic and palmitic acid. After 24 h reaction time, product concentrations of 1.16 mM (0.27 g L-1) 1-heptadecene and 1 mM (0.21 g L-1) 1-pentadecene were obtained but could not be further enhanced. The inhibitory effect of gluconic acid, formed as side-produce from NAD(P)H regeneration by GDH and glucose, was therefore investigated and already a concentration of 10 mM was found to reduce OleT productivity significantly (28% reduction). Alternative systems based on phosphite dehydrogenase (PDH; NADPH-dependent) (Dudek, 2013 and Vrtis J. M., 2002) and formate dehydrogenase (EDIT; NADH-dependent)-based (Busto E., 2014) proved to be more efficient: 2.6 mM (0.62 g L-1) and 3.1 mM (0.75 g L-1) 1-heptadecene, respectively, were obtained from 5 mM stearic acid (52% and 62% conversion, respectively), yielding the highest TIN values so far (1739 and 2096, respectively) by using the above mentioned regeneration systems. Compared to a previously published whole cell reaction system, this FDH-driven system allows 6 times higher product titer and a 21 times higher volumetric productivity with 42.5 mg L-1 h-1 of -olefin after 8 h reaction time vs: 98 mg L-1 in 48 h) (Table 2).

(24) TABLE-US-00002 TABLE 2 Decarboxylation of stearic acid by OleT employing various redox partner systems using O.sub.2 as oxidant. Electron -Olefin Product TTN Source Redoxpartners OleT [M] [g L.sup.1] [] Glucose GDH-CamAB Purified [1.5 M] 1020 63 0.24 680 Phosphite PDH-CamAB Purified [1.5 M] 2609 256 0.62 1739 Formate FDH-CamAB Purified [1.5 M] 3144 121 0.75 2096 Formate FDH-CamAB Purified [3 M] 1419 0.34 473 [8 h] Formate FDH-CamAB-Fdr/Fdx).sup.a Cell free lysate [3 M] 1565 20 0.37 522 Formate FDH-(Fdr/Fdx).sup.a Cell free lysate [3 M] 452 52 0.1 151 Reaction conditions: 0.05 U ml.sup.1 CamAB, 1200 U ml.sup.1 catalase, 12 U ml.sup.1 GDH or 2 U ml.sup.1 GDH or 0.2 U ml.sup.1 PDH, 100 mM D-glucose or 100 mM ammonium formate or 100 mM sodium phosphite, 5 mM stearic acid, 2.5% EtOH, KPi buffer (pH 7.5, 0.1M) and 200 M NAD(P)H. Reactions were performed at 1 ml scale at room temperature and 170 rpm shaking for 24 h in closed glass vials. .sup.aFdr/Fdx is naturally present in E. coli and allows transfer of electrons from NAD(P)H to OleT. .sup.bReactions contained 53 mg of freeze-dried lysates of cells expressing OleT.

Example 4

Decarboxylation Fatty Acids (FAs) Employing the PDH-CamAB-OleT Redox-Cascade

(25) The substrate scope of OleT was further investigated by varying the FA chain length from C3 to C22. In addition to the standard setup at RT, the reaction was also investigated at 4 C. to prevent loss of highly volatile short chain -olefins, which resulted in surprisingly high activity levels (Table 3). For the first time, short EAs could be decarboxylated to the corresponding olefins, covering the whole spectrum from C11 down to C4. Interestingly, the conversion of short chain FAs (<C10) was recognized by a strong gasoline odor released from reaction tubes. Due to the high volatility of short chain olefins, product work up was done at 4 C. Yields were strongly dependent on the reaction temperature and the substrate chain length, with a major drop in reactivity at RT below C18 (maximum product concentration 2.45 mM from 5 mM stearic acid), while C12 appears the best substrate at 4 C. (2.53 mM product from 5 mM lauric acid). Head-space analysis by GC-MS from reactions with short chain FAs (C8-C5) confirmed the formation of the respective -olefins, allowing the first biotechnological access to 1-heptene, 1-hexene and 1-pentene and 1-butene with OleT.

(26) TABLE-US-00003 TABLE 3 Decarboxylation of FAs employing the FDH-CamAB-OleT redox-cascade. - Conver- Selectivity [% GC-area] Temp. Olefin sion - - - n- FA [ C.] [M] [%] Olefin OH OH Alkanone C22 25 n.q. 16* >99 n.d. Traces n.d. 4 93 n.d. 7 n.d. C20 25 n.q. 3* >99 n.d. Traces n.d. 4 >99 n.d. n.d. n.d. C18 25 3886 39 86 2 8 6 4 2451 25 93 <1 5 5 C16 25 1111 11 72 3 23 8 4 2059 21 80 <1 14 6 C14 25 552 6 62 4 31 n.d. 4 1247 12 87 n.d. 13 n.d. C12 25 273 3 n.d. n.d. n.d. n.d. 4 3260 33 73 n.d. 30 n.d. C11 25 n.d. 0 n.d. n.d. n.d. n.d. 4 191 2 >99 n.d. n.d. n.d. C10 25 44 0.4 >99 n.d. Traces n.d. 4 71 0.7 >99 n.d. n.d. n.d. C9 25 1829 18 53 17 30 n.d. 4 1347 13 on- on- on- going going going C8 25 364 3.6 n.i. n.i. n.i. n.i. 4 141 1.4 C7 25 1298 13 n.i. n.i. n.i. n.i. 4 946 9.5 C6 25 1170 12 n.i. n.i. n.i. n.i. 4 772 7.7 C5 25 834 8.3 n.i. n.i. n.i. n.i. C4 25 504 5 n.i. n.i. n.i. n.i. Reaction conditions: Conversion of substrates C22-C14 was performed using 3 M OleT, 6 M OleT for substrates C12-C3. All reactions mixtures contained 0.05 U ml1 CamAB, 1200 U ml1 catalase, 2 U ml1 FDH, 100 mM ammonium formate, 5% EtOH, 10 mM substrate, KPi buffer (pH 7.5, 0.1M) and 200 M NADH. Conversion of substrates C8, C7 and C6 were done in presence of 5% DMSO as cosolvent. Detection and quantification of short-chain terminal olefins was achieved using manual headspace GC-MS injection. All conversions were performed at 1 ml scale and 170 rpm shaking in closed glass vials (RT samples/24 h) or stirring (4 C. samples/72 h). *relative to conversions with C18/only GC-areas. n.i. = not investigated.

Example 5

Identification and Quantification of 1-Hexene Produced from Heptanoic Acid by OleT

(27) Conversion of heptanoic acid was carried out as described in Example 4 using the OleTCamAB-FDH reaction cascade. Decarboxylation of heptanoic acid proceeded reliably as shown in FIG. 1.

(28) An analytical standard of 1-hexene was used to confirm formation of the target product. The analytical standard eluted at the same retention time (2.617 min; FIGS. 1 and 2, peak c) and displayed the identical m/z pattern as the reaction product formed by OleT (FIGS. 3 and 4).

(29) Calibration curves for 1-hexene were prepared accordingly ranging from 0.05 to 2 mM. Due to a strong overlap of 1-hexane with the peak areas of CO.sub.2 and EtOH (FIG. 1, peaks a, b and c) the ion extraction mode of the GC-MS to generate reliable calibration curves was used. For 1-hexene the characteristic ion 84 for quantification as shown in FIG. 5 was used. Neither CO.sub.2 nor EtOH interfered with the product peak area. This can be seen comparing FIGS. 1 and 5 which allow reliable and linear quantification of 1-hexene (FIG. 6) using manual headspace GC-MS injection.

(30) Manual headspace injection into GC-MS was successfully performed for all substrates with chain lengths of C11 to C6. Obtained product concentrations are summarized in Table 3. Conversion of 10 mM heptanoic acid allowed reliable formation of up to 1.3 mM 1-hexene without further optimization of the reaction conditions.

Example 6

Trapping, Derivatization and Quantification of 1-Butene and 1-Propene

(31) Production of 1-propene and 1-butene was initially confirmed using manual headspace GC-MS injection. Since the quantification of 1-propene and 1-butene using analytical standards was not feasible, a novel trapping/derivatization system for highly volatile short-chain -olefins derived from biocatalytic reactions was designed. Thus, the established reaction set-up (see reaction conditions in Table 3) was upscaled to 10 ml (using 25 ml two-neck flasks) and the decarboxylation of butyric (C4) and valeric acid (C5) was performed in the set-up as shown in FIG. 7.

(32) ##STR00002##

(33) The obtained products were analyzed by GC-FID and GC-MS using analytical standards of 1,2-dibromobutane and 1,2-dibromopropane. Due to the (visible) gas exhaust (CO.sub.2 production by OleT and FDH) and strong evaporation of DCM (70% volume lost after 24 h), the residual volume in the impinger was determined to allow reliable product quantification. Calibration curves for 1,2-dibromobutane and 1,2-dibromopropane were prepared to allow quantification of produced 1-propene and 1-butene (not shown).

(34) Further, the solution containing 1,2-dibromobutane was concentrated to 700 l by dry air flow and analysed by 1H-NMR (FIG. 11) which serves as a further proof of 1-butene formation by OleT, Similarly, the solution containing 1,2-dibromopropane was concentrated to 700 l by dry air flow and analysed by .sup.1H-NMR (FIG. 16) which serves as a further proof of propene formation by OleT.

(35) The analytical standard of 1,2-di-bromobutane is .sup.1H NMR (300 MHz, CDCl.sub.3) 4.23-4.09 (m, 1H), 3.87 (dd, J=10.2, 4.4 Hz, 1H), 3.66 (t, J=10.1 Hz, 1H), 2.22 (dqd, J=14.6, 7.3, 3.3 Hz, 1H), 1.97-1.74 (m, 1H), 1.09 (t, 7.2 Hz, 3H).

(36) The analytical standard of 1,2-dibromopropane used was .sup.1H NMR (300 MHz, CDCl.sub.3) 4.34-4.20 (m, 1H), 3.87 (dt, J=10.1, 5.0 Hz, 1H), 3.58 (t, J=10.1 Hz, 1H), 1.85 (d, J=6.6 Hz, 3H).

(37) Control reactions (CamAB-FDH cascade without OleT) were performed to confirm production of 1-propene and 1-butene only when the full cascade is re-constituted in the reaction vessel (FIGS. 8 and 9), The formation of 1,2-dibromobutane and 1,2-dibromopropane was also confirmed by GC-MS (not shown). With the novel trapping strategy, it was possible to identify and quantify both 1-propene and 1-butene. The conversion of 10 mM valeric acid led to the formation of 834 M 1-butene, whereas 504 M 1-propene were determined for conversion of 10 mM butyric acid. All reactions were performed at least in triplicate, and the corresponding -olefins were the only reaction products detected in the impinger solution.

(38) European patent application EPI 5156699 filed Feb. 26, 2015, is incorporated herein by reference.

(39) Numerous modifications and variations on the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

REFERENCES

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