Process for preparing an alpha, omega-alkanediol

Abstract

The invention relates to a process for preparing an ,-alkanediol comprising the steps of a) reacting an alkanoic acid with an alkanol to give an ester, b) oxidizing at least one terminal carbon atom of the ester by contacting with a whole-cell catalyst, which expresses an alkane hydroxylase, in aqueous solution and in the presence of molecular oxygen, to give an oxidized ester, c) hydrogenating the oxidized ester to form the alkanediol and alkanol, and d) removing the alkanol by distillation, forming a reaction mixture depleted with respect to the alkanol, and recycling the alkanol in step b).

Claims

1. A process for producing an ,-alkanediol comprising: a) reacting an alkanoic acid with an alkanol to give an ester, b) oxidizing at least one terminal carbon atom of the ester by contacting with a whole-cell catalyst, which expresses an alkane hydroxylase, in aqueous solution and in the presence of molecular oxygen, to give an oxidized ester, c) hydrogenating the oxidized ester to form an ,-alkanediol and alkanol, and d) removing the alkanol by distillation, forming a reaction mixture depleted with respect to the alkanol, and e) recycling the alkanol in a).

2. Process according to claim 1, wherein the alkanoic acid in a) is an alkanoic acid of the formula H.sub.3C(CH.sub.2).sub.nCOOH, and where n>0.

3. Process according to claim 1, wherein the alkanoic acid is butanoic acid.

4. Process according to claim 1, wherein the alkanoic acid in a) is a carboxylated cycloalkane of the formula C.sub.nH.sub.2n1COOH, where n>4.

5. Process according to claim 1, wherein the alkanol is an alkanol of the formula C.sub.nOH.sub.2n+2 and n>0.

6. Process according to claim 1, wherein the alkanoic acid and the alkanol in a) have the same carbon skeleton.

7. Process according to claim 1, wherein the whole-cell catalyst in b) expresses at least one alkane hydroxylase selected from the group consisting of AlkB from Pseudomonas putida comprising the sequence of SEQ ID NO: 1, a variant having at least 70% identity to SEQ ID NO: 1, CYP153 from Alcanivorans borkumen SK2 comprising the sequence of SEQ ID NO: 2, and a variant having at least 70% identity to SEQ ID NO: 2.

8. Process according to claim 1, wherein a) is carried out by acid-catalysed ester formation.

9. Process according to claim 1, wherein a) is carried out in aqueous solution by contacting the alkanoic acid and alkanol with an esterase, protease or lipase.

10. Process according to claim 1, wherein the alkanoic acid in a) is provided by oxidizing an alkane to the alkanoic acid.

11. Process according to claim 2, wherein n is 2 to 16.

12. Process according to claim 4, wherein n is 5 to 8.

13. Process according to claim 8, wherein a) is carried out in the presence of an organic solvent.

14. Process according to claim 9, wherein a) is carried out in aqueous solution by contacting with an esterase, protease or lipase in the form of a whole-cell catalyst expressing the esterase or lipase.

15. The process of claim 1, wherein the whole cell catalyst expresses an alkane hydroxylase that is AlkB from Pseudomonas putida comprising the sequence of SEQ ID NO: 1 or a variant having at least 95% identity to SEQ ID NO: 1.

16. The process of claim 1, wherein the whole cell catalyst expresses an alkane hydroxylase that is CYP153 from Alcanivorans borkumensis SK2 comprising the sequence of SEQ ID NO: 2 or a variant having at least 95% identity to SEQ ID NO: 2.

Description

(1) The present invention is more particularly described by the following figures and non-limiting examples from which further features, embodiments, aspects and advantages of the present invention may be discerned.

(2) FIG. 1 shows the time course of the butanoic acid and 1-butanol concentration in the biotransformation of n-butane with E. coli W3110 pBT10, resulting from the procedure of Example 1.

(3) FIG. 2 shows the mass traces of the HPLC-MS analysis of the oxidation products of butyl butyrate using E. coli W3110 pCOM10[Ab_Fd/CYP153-2/FdOR/alkL] with a comparison of the 10 min sample with the 7 h sample, resulting from the procedure of Example 2.

(4) FIG. 3 shows the mass traces of the HPLC-MS analysis of the oxidation products of butyl butyrate using E. coli W3110 pBT10 with a comparison of the 10 min sample with the 7 h sample, resulting from Example 2.

EXAMPLE 1

Oxidation of n-butane by E. coli W3110 pBT10 with the Monooxygenase (alkBGT) from P. putida GPO1

(5) a) Production of Biomass on a 10 l Scale

(6) Preseed culture: 1 liter of LB Medium (5 g/l yeast extract, 10 g/l peptone, 0.5 g/l NaCl, dissolved in 1 l of water, autoclaved for 20 minutes at 121 C.) was prepared.

(7) From this solution, 525 ml were each filled in 100 ml shaking flasks with chicanes, 25 l of a sterile-filtered kanamycin solution (50 g/l) were added to each flask and in each case inoculated with 200 l of a glycerol cryoculture of E. coli W3110 pBT10. The strain comprising the plasmid pBT10 is already described in detail in Example 4 of WO13083412. These cultures were incubated for 18 h at 37 C. and 200 rpm (amplitude 2.5 cm).

(8) Seed culture: 1 liter of high cell density medium (HCD medium) was prepared, consisting of 1.76 g/l NH.sub.4SO.sub.4, 19.08 g/l K.sub.2HPO.sub.4, 12.5 g/l KH.sub.2PO.sub.4, 6.66 g/l yeast extract, 1.96 g/l Na.sub.3-citrate, 17 ml of NH.sub.4Fe-citrate solution (1%), 5 ml of trace element solution US3 (1 liter of the trace element solution US 3 is composed of 36.5 g of HCl 37%, 1.91 g of MnCl.sub.24H.sub.2O, 1.87 g of ZnSO.sub.47H.sub.2O, 0.8 g of Na-EDTA2H.sub.2O, 0.3 g of H.sub.3BO.sub.3, 0.25 g of Na.sub.2MoO.sub.42H.sub.2O, 4.7 g of CaCl.sub.22H.sub.2O, 17.8 g of FeSO.sub.47 H.sub.2O, 0.15 g of CuCl.sub.22H.sub.2O, dissolved in 1 l of water), 30 ml of feed solution (glucose 50% w/v, MgSO.sub.47 H.sub.2O 0.5% w/v, NH.sub.4Cl 2.2% w/v), and 1 ml of kanamycin solution (50 g/l). 948 ml of the solution with NH.sub.4SO.sub.4 to Na.sub.3-citrate were autoclaved and the remainder was separately sterile filtered and then added under sterile conditions. The pH was 6.8.

(9) 575 ml of the HCD medium were added to 1000 ml shaking flasks with chicanes, each was inoculated with 25 ml of preseed culture and cultured at 37 C. and 200 rpm (amplitude 2.5 cm) for 30 h.

(10) A sterile 10 l fermenter was filled with 7 l of a sterile medium having the composition of 1.75 g/l (NH.sub.4).sub.2SO.sub.4, 19 g/l K.sub.2HPO.sub.43 H.sub.2O, 12.5 g/l KH.sub.2PO.sub.4, 6.6 g/l yeast extract, 2.24 g/l Na.sub.3-citrate2H.sub.2O, 15 g/l glucose, 0.49 g/l MgSO.sub.47 H.sub.2O, 16.6 ml/l NH.sub.4Fe-citrate solution (1% w/v), 15 ml/l trace element solution (as in the seed culture), 1 ml/l kanamycin solution (50 mg/l) and 2 ml of antifoaming agent Delamex. The feed was an autoclaved solution of glucose (50% w/v) supplemented with MgSO.sub.47H.sub.2O 10 g/l, corrected for pH with 0.5M H.sub.2SO.sub.4 and 25% NH.sub.4OH.

(11) The cultures from the shaking flasks were combined under sterile conditions and inoculated into the fermenter via a transfer bottle. The fermentation conditions were adjusted to pO.sub.2 30%, airflow 6 nlpm, stirrer 400-1200 rpm, temperature 37 C., pH 7, feed start 8 h, feed rate 150-250 g/h. After 19 h, the temperature was reduced to 30 C. and the mixture was induced with 0.4 mM DCPK. After 23 hours, the OD600 in the fermenter was ca.100, the culture broth was removed under sterile conditions and 500 ml in 1000 ml centrifuge flasks were centrifuged at 8000 rpm. The supernatant was discarded and the pellets were aliquoted into Falcon tubes each with 10 g. The pellets were frozen at 80 C. for later use.

(12) b) Oxidation of n-butane to 1-butanol and Butanoic Acid

(13) 10 g of the frozen biomass as described in a) were resuspended in 50 ml of 70 mM ammonium phosphate buffer pH 7 (composition: 8 g/l (NH.sub.4)H.sub.2PO.sub.4, 0.5 g/l NaCl, 0.49 g/l MgSO.sub.47H.sub.2O, 1 ml of trace element solution US3 and 50 g/l kanamycin). The pH was adjusted in this case with 5% NH.sub.4OH.

(14) 130 ml of 70 mM ammonium phosphate buffer pH 7 (composition: 8 g/l (NH.sub.4)H.sub.2PO.sub.4, 0.5 g/l NaCl, 0.49 g/l MgSO.sub.47H.sub.2O, 1 ml of trace element solution US3 and 50 g/l kanamycin. pH adjusted to 7.0 with 5% ammonia solution.) with ca. 3 drops of autoclaved antifoam (Delamex) were charged in a 300 ml fermenter. The fermenter was flushed with a gas mixture consisting of 25% n-butane and 75% synthetic air via a metal sinter perlator having a pore size of 0.2 m at a flow rate of 9 IN/h. The fermenter was heated to 30 C. in a water bath and stirred by means of a magnetic stirrer at 900 rpm. The pH was regulated to 7.0 with 2.5% ammonia solution. Glucose solution was fed in continuously (glucose feed rate of 0.9 g/lh). The exhaust gas was passed through an ice-cooled wash bottle containing 150 ml of water.

(15) From the biomass frozen as described in a), 10 g were resuspended in 50 ml of ammonium phosphate buffer and thawed. The fermenter was inoculated with the suspension.

(16) The biomass concentration had an optical density (600 nm) of 25.5 ml samples were removed at various time points from the fermenter. The samples were centrifuged at room temperature for 10 minutes at 10 000 g and the supernatant filtered through a 0.2 m syringe filter unit.

(17) The chromatographic analysis of butanoic acid and 1-butanol was conducted by HPLC-RID on an Agilent Technologies 1200 system. An Aminex HPX-87H column (300 mm7.8 mm) was used. The system was operated using 10 mM H.sub.2SO.sub.4 as eluent at a flow rate of 0.6 ml/min and a column temperature of 40 C. Standards for all substances to be analysed were prepared in ultra-pure water and measured under identical conditions. The evaluation was performed by comparison of retention times.

(18) After 48 h, the butanoic acid concentration was ca. 13.5 g/l and the 1-butanol concentration 0.125 g/l (see FIG. 1).

EXAMPLE 1C

Oxidation of n-butane by E. coli W3110 pBT10 with the Monooxygenase CYP153 from Alcanivorax borkumensis

(19) Three 100 ml chicane flasks containing 25 ml of LB medium with kanamycin (composition: 5 g/l yeast extract, 10 g/l peptone, 0.5 g/l NaCl, 50 mg/l kanamycin sulphate) are each inoculated with 100 l of a glycerol cryoculture of E. coli W3110 pCOM10[Ab_Fd/CYP153-2/FdOR/alkL] having the monooxygenase CYP153 from Alcanivorax borkumensis and are incubated at 37 C. and 200 rpm for 20 h. The strain used is described in detail in Example 4 in conjunction with Example 1 of PCT/EP2013/054928.

(20) Each 25 ml of the culture broth are then used as inoculum in 75 ml of modified M9 medium (composition: 15 g/l glucose, 6.8 g/l Na.sub.2PO.sub.4, 3 g/l KH.sub.2PO.sub.4, 0.5 g/l NaCl, 2 g/l NH.sub.4Cl, 15 g/l yeast extract, 0.49 g/l MgSO.sub.4*7H.sub.2O, 50 mg/l kanamycin sulphate, 15 ml/l trace element solution US3. Composition of the trace element solution: 36.5 g/l 37% hydrochloric acid, 1.91 g/l MnCl.sub.2*4H.sub.2O, 1.87 g/l ZnSO.sub.4*7H.sub.2O, 0.84 g/l Na-EDTA*2H.sub.2O, 0.3 g/l H.sub.3BO.sub.3, 0.25 g/l Na.sub.2MoO.sub.4*2H.sub.2O, 4.7 g/l CaCl.sub.2*2H.sub.2O, 17.3 g/l FeSO.sub.4*7H.sub.2O, 0.15 g/l CuCl.sub.2*2H.sub.2O) in 1000 ml chicane flasks. The flasks are incubated at 37 C. and 200 rpm for 2.5 h. The temperature is then reduced to 25 C. The culture is induced after 0.5 hours at 25 C. with 0.4 mM dicyclopropyl ketone. The cultures are incubated at 25 C. and 200 rpm for a further 16 h.

(21) The cultures are combined, transferred to 50 ml falcon tubes and centrifuged at 5500 g at 25 C. for 10 minutes. The supernatant is discarded and the pellets from 300 ml of culture are resuspended in 50 ml of conversion buffer pH 7. (Composition: 8 g/l (NH.sub.4)H.sub.2PO.sub.4, 0.5 g/l NaCl, 0.49 g/l MgSO.sub.4*7H.sub.2O, 50 mg/l kanamycin sulphate, 15 ml/l trace element solution US3; pH adjusted to 7.0 with 25% ammonia solution).

(22) The transformation, sampling and analytics are conducted with E. coli W3110 pCOM10[Ab_Fd/CYP153-2/FdOR/alkL] analogously to 1.1.

(23) The butanoic acid and the butanol from 1-b) and 1-c) are separated by distillation from the aqueous phase and used further in Example 1 d.

EXAMPLE 1D

Azeotropic Esterification of Butanoic Acid and Butanol

(24) 88 g of butanoic acid and 81.4 g of 1-butanol from Examples 1 b and 1c and in the case of butanol optionally from Example 3 and also 5 g of para-toluenesulphonic acid are mixed and heated to boiling. The reaction is carried out under reflux by means of azeotropic distillation using a water separator until the calculated quantity of water (18 mL) for quantitative conversion had been separated. The ester formed (129.6 g of butyl butyrate) is separated by distillation from excess 1-butanol and the catalyst and is transferred for the oxidation by the whole-cell catalyst in Example 2.

EXAMPLE 2

Oxidation of Butyl Butyrate

(25) Each 100 ml chicane flask containing 25 ml of LB medium with kanamycin (composition: 5 g/l yeast extract, 10 g/l peptone, 0.5 g/l NaCl, 50 mg/l kanamycin sulphate) was inoculated with 100 l of a glycerol cryoculture of E. coli W3110 pCOM10[Ab_Fd/CYP153-2/FdOR/alkL] or E. coli W3110 pBT10 and was incubated at 37 C. and 200 rpm for 20 h.

(26) Each of the complete precultures was then used as inoculum in 75 ml of modified M9 medium (composition: 15 g/l glucose, 6.8 g/l Na.sub.2PO.sub.4, 3 g/l KH.sub.2PO.sub.4, 0.5 g/l NaCl, 2 g/l NH.sub.4Cl, 15 g/l yeast extract, 0.49 g/l MgSO.sub.4*7H.sub.2O, 50 mg/l kanamycin sulphate, 15 ml/l trace element solution US3. Composition of the trace element solution: 36.5 g/l 37% hydrochloric acid, 1.91 g/l MnCl.sub.2*4H.sub.2O, 1.87 g/l ZnSO.sub.4*7H.sub.2O, 0.84 g/l Na-EDTA*2H.sub.2O, 0.3 g/l H.sub.3BO.sub.3, 0.25 g/l Na.sub.2MoO.sub.4*2H.sub.2O, 4.7 g/l CaCl.sub.2*2H.sub.2O, 17.3 g/l FeSO.sub.4*7H.sub.2O, 0.15 g/l-CuCl.sub.2*2H.sub.2O) in 1000 ml chicane flasks. The flasks were incubated at 37 C. and 200 rpm for 2.5 h. The temperature was then reduced to 25 C. The culture was induced after 0.5 hours with 0.4 mM dicyclopropyl ketone. The culture was incubated at 25 C. and 200 rpm for a further 16 h.

(27) The cultures were transferred to 50 ml falcon tubes and centrifuged at 5500 g at 25 C. for 10 minutes. The supernatant was discarded and the pellets from each strain were resuspended in 50 ml of conversion buffer pH 7 (composition: 8 g/l (NH.sub.4)H.sub.2PO.sub.4, 0.5 g/l NaCl, 0.49 g/l MgSO.sub.4*7H.sub.2O, 50 mg/l kanamycin sulphate, 15 ml/l trace element solution US3; pH adjusted to 7.0 with 25% ammonia solution) and incubated at 30 C. and 180 rpm in 500 ml chicane flasks.

(28) In order to start the reaction, 2 g/l butyl butyrate and 5 g/l glucose were added to each flask. A flask with buffer, butyl butyrate and glucose without cells was incubated as a negative control.

(29) The biomass concentration had an optical density (600 nm) of 2.5. A 2 ml sample was taken in each case after 10 min and 7 h. The samples were centrifuged at room temperature for 10 minutes at 10 000 g and the supernatant filtered through a 0.2 m syringe filter unit.

(30) Screening for oxidation products of butyl butyrate was conducted by HPLC-ESI-MS (Thermo Fisher Scientific). Due to their accurate masses and the empirical formulae derived therefrom, butyl butyrate and the oxidation products arising therefrom could be identified. In both experimental batches, the mono- and bis-terminally hydroxylated esters, inter alia, could be detected (see FIGS. 2 and 3).

EXAMPLE 3

Reductive Hydrogenation of the Oxidized Butyl Butyrate to Form 1,4-butanediol:

(31) 740 mg of lithium aluminium hydride are charged in 50 mL of dry diethyl ether and 10 g of the oxidized ester from Example 2 are slowly added dropwise at 0 C. After addition is complete, the mixture is heated to boiling and stirred under reflux until complete conversion.

(32) The resulting alcohols are separated by distillation. 7.41 g of 1,4-butanediol and 1.77 g of 1-butanol are obtained. The butanol is recycled to Example 1d and esterified therein.