Biotechnological production of alcohols and derivatives thereof

Abstract

A method for oxidizing an alkyl, including a) contacting the alkyl with an aqueous solution comprising a microorganism where the microorganism has a reduced fatty acid degradation capacity compared to its wild type, wherein the fatty acid degradation capacity is reduced by deletion, inhibition or inactivation of a gene encoding an enzyme involved in the β-oxidation pathway; and the microorganism expresses a recombinant alkane oxidase, and b) contacting the aqueous solution from a) with a water-immiscible organic solvent.

Claims

1. A method for oxidizing an alkyl, comprising: a) contacting the alkyl with an aqueous solution comprising a microorganism such that the alkyl is converted to an oxidized alkyl, where the microorganism expresses a recombinant alkane oxidase and has a reduced fatty acid degradation capacity compared to its wild type, wherein the fatty acid degradation capacity is reduced by deletion, inhibition or inactivation of a gene encoding an enzyme involved in the β-oxidation pathway; b) contacting the aqueous solution from a) with a water-immiscible organic solvent and c) allowing the aqueous solution contacted with the water-immiscible organic solvent from b) to settle for 5 to 10 minutes, thereby forming an aqueous phase comprising the microorganism and an organic phase comprising the water-immiscible organic solvent, wherein the alkyl is a compound represented by the formula H—(CH.sub.2).sub.x—R, wherein x is at least 8, and R is selected from the group comprising —OH, —COH, —COOH, —COOR.sup.1, —NH.sub.2, —NO.sub.2, —CN, —OPO.sub.3H, —SO.sub.3H and —H wherein R.sup.1 is methyl or ethyl, and wherein the expressed recombinant alkane oxidase is AlkB from Pseudomonas putida GPo1 comprising the sequence of SEQ ID NO: 1 or a variant thereof having at least 90% identity to SEQ ID NO: 1, and wherein the enzyme involved in the β-oxidation pathway is selected from the group consisting of: a fatty acid importer comprising the sequence of SEQ ID NO: 2 or a variant thereof having at least 90% identity to SEQ ID NO: 2, a fatty acid-CoA ligase comprising the sequence of SEQ ID NO: 3 or a variant thereof having at least 90% identity to SEQ ID NO: 3, an acyl-CoA dehydrogenase comprising the sequence of SEQ ID NO: 4 or a variant thereof having at least 90% identity to SEQ ID NO: 4, an enoyl-CoA hydratase comprising the sequence of SEQ ID NO: 5 or a variant thereof having at least 90% identity to SEQ ID NO: 5, and a 3-ketoacyl-CoA thiolase comprising the sequence of SEQ ID NO: 6 or a variant thereof having at least 90% identity to SEQ ID NO: 6.

2. The method according to claim 1, wherein b) is carried out following completion of oxidation of the alkyl.

3. The method according to claim 1, wherein the alkyl is a linear alkane.

4. The method according to claim 1, wherein the water-immiscible organic solvent is a water-immiscible fatty acid or fatty acid ester.

5. The method according to claim 1, wherein the microorganism is a bacterial cell.

6. The method according to claim 1, wherein the microorganism is E. coli.

7. The method according to claim 1, wherein x in the formula H—(CH.sub.2).sub.x—R is 11 or more.

8. The method according to claim 1, wherein the water-immiscible organic solvent is a fatty acid represented by the formula:
CH.sub.3—(CH.sub.2).sub.y—COOR.sup.S, wherein y is an integer from 8 to 28, and R.sup.S is H, or alkyl.

9. The method according to claim 1, wherein the water-immiscible organic solvent is lauric acid methyl ester.

10. The method according to claim 1, wherein the enzyme involved in the β-oxidation pathway is a fatty acid importer comprising the sequence of SEQ ID NO: 2 or a variant thereof having at least 90% identity to SEQ ID NO: 2.

11. The method according to claim 1, wherein the enzyme involved in the β-oxidation pathway is a fatty acid-CoA ligase comprising the sequence of SEQ ID NO: 3 or a variant thereof having at least 90% identity to SEQ ID NO: 3.

12. The method according to claim 1, wherein the enzyme involved in the β-oxidation pathway is an acyl-CoA dehydrogenase comprising the sequence of SEQ ID NO: 4 or a variant thereof having at least 90% identity to SEQ ID NO: 4.

13. The method according to claim 1, wherein the enzyme involved in the β-oxidation pathway is an enoyl-CoA hydratase comprising the sequence of SEQ ID NO: 5 or a variant thereof having at least 90% identity to SEQ ID NO: 5.

14. The method according to claim 1, wherein the enzyme involved in the β-oxidation pathway is a 3-ketoacyl-CoA thiolase comprising the sequence of SEQ ID NO: 6 or a variant thereof having at least 90% identity to SEQ ID NO: 6.

15. The method according to claim 1, wherein R in the formula H—(CH.sub.2).sub.x—R is selected from the group consisting of —OH, —COH, —NH.sub.2, —NO.sub.2, —CN, —OPO.sub.3H, —SO.sub.3H and —H.

16. The method according to claim 1, wherein the water-immiscible organic solvent comprises at least one selected from the group consisting of oleic acid and hexanoic acid.

17. The method according to claim 1, wherein the alkyl comprises lauric acid methyl ester.

18. The method according to claim 1, wherein the alkyl comprises lauric acid methyl ester, and wherein the enzyme involved in the β-oxidation pathway is an acyl-CoA dehydrogenase comprising the sequence of SEQ ID NO: 4 or a variant thereof having at least 90% identity to SEQ ID NO: 4.

19. The method according to claim 1, wherein the alkyl comprises lauric acid methyl ester, wherein the water-immiscible organic solvent comprises oleic acid, and wherein the enzyme involved in the β-oxidation pathway is an acyl-CoA dehydrogenase comprising the sequence of SEQ ID NO: 4 or a variant thereof having at least 90% identity to SEQ ID NO: 4.

20. The method according to claim 19, wherein the organic phase further comprises ω-amino lauric acid methyl ester.

Description

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

(2) FIG. 1 shows different phase separation behaviour if ΔFadE mutant W3110 ΔFadE [alkB-alaD-TA] (left), also referred to as “ΔFadE”, and strain W3110 [alkB-alaD-TA] (right), also referred to as wild type (WT), the latter identical to the former strain except for the fact that is FadE is not deleted, are used to produce ALSME. The arrow points the interphase between organic and aqueous phase visible after ten minutes in case the mutant is used. No such interphase is detectable after ten minutes in case the wild type strain is used.

(3) FIG. 2 shows the results of the same experiment as described with respect to FIG. 1, except for the fact that the medium was transferred to Falcon tubes after completion of the fermentation.

(4) FIG. 3 shows the oxygen transfer rate and the carbon dioxide transfer rate of both strains used for the experiment described with respect to FIG. 1.

(5) FIG. 4 shows the concentrations of ALSME over time in the same experiment described with respect to FIG. 1.

EXAMPLE 1: ACCELERATION OF SEPARATION OF A HYDROPHOBIC PHASE FROM AN AQUEOUS MEDIUM USING A CELL WITH REDUCED ACYL COA-DEHYDROGENASE ACTIVITY FOR THE PRODUCTION OF W-AMINO LAURIC ACID METHYL ESTER (ALSME)

(6) The conversion of lauric acid methyl ester to w-amino lauric acid (ALS) methyl ester, via w-hydroxy lauric acid, was carried out in a parallel fermentation system comprising 8 vessels from DASGIP, using strains W3110 ΔFadE [alkB-alaD-TA] and W3110 [alkB-alaD-TA].

(7) N. B. that these two strains comprise a pBR322-derived plasmid comprising oxidoreductase AlkB, an alcohol dehydrogenase and a transaminase in line with international application WO 2009/077461 and are identical except for the fact that the former has a deletion in the gene encoding FadE, the E. coli acyl-CoA dehydrogenase of the β-oxidation pathway.

(8) 1 liter reaction vessels were used for the fermentation. pH electrodes were calibrated by a two-point-calibration using pH 4 and pH 7 standard solutions. Reactors containing 300 mL tap water were autoclaved for 20 minutes at 121° C. Subsequently the pO2-detectors were polarized at the DASGIP system over night (for at least 6 hours). The next morning water was removed under a clean Bench and replaced by 300 mL of high cell density medium complemented with 100 mg/L ampicillin. Subsequently, pO2 detectors were subjected to one-point-calibration (stirrer: 400 rpm, gas flow: 10 sL/h air), and the tubings associated with the feed, correction agent and induction were cleaned by clean in Place using 70% ethanol, followed by 1 M NaOH, followed by rinsing with sterile VE water.

(9) ALS and ALSME producing strains of E. coli were inoculated from the respective cryo cultures in LB medium (25 mL in a 100 mL flask with baffles) complemented with 100 mg/L ampicillin over night at 37° C. and 200 rpm for approximately 18 hours. Subsequently, 2 mL each of the cultures in high cell density medium (glucose 15 g/L (30 mL/L of a separately autoclaved 500 g/L stock solution comprising 1% MgSO.sub.4*7H.sub.2O and 2.2% NH.sub.4Cl), (NH.sub.4).sub.2SO4 1.76 g/L, K.sub.2HPO.sub.4 19.08 g/L, KH.sub.2PO.sub.4 12.5 g/L, yeast extract 6.66 g/L, trisodium dihydrate 2.24 g/L, ammonium ter iron citrate solution: 17 mL/L of a separately autoclaved 1% stock solution, trace element solution: 5 mL/L of a separately autoclaved stock solution (HCl (37%) 36.50 g/L, MnCl.sub.2*4H.sub.2O 1.91 g/L, ZnSO.sub.4*7H.sub.2O 1.87 g/L, ethylenediamintetraacetic acid dihydrate 0.84 g/L, H.sub.3BO.sub.30.30 g/L. Na.sub.2MoO.sub.4*2H.sub.2O 0.25 g/L, CaCl.sub.2*2H.sub.2O 4.70 g/L, FeSO.sub.4*7H.sub.2O 17.80 g/L, CuCl.sub.2*2H.sub.2O 0.15 g/L)) (20 mL per strain in a 100 mL flask with bethels) with 100 mg/L ampicillin were inoculated and incubated at 37° C./200 rpm for another 5.5 hours.

(10) The optical density of a culture at 600 nm was determined in the case of W3110 ΔFadE [alkB-alaD-TA] as 6.9 and 7.4 in the case of W3110 [alkB-alaD-TA]. In order to inoculate the reaction vessels to a final optical density of 0.1, 4.0 mL or 4.4 mL, respectively, were transferred into a 5 mL syringe under sterile conditions and used to inoculate the reaction using a hollow needle and a septum covered by a layer of 70% ethanol. The following standard program was used

(11) TABLE-US-00001 DO-controller pH-controller Preset 0% Preset 0 ml/h P 0.1 P 5 Ti 300 s Ti 200 s Min 0% Min 0 mlL/h Max 100%  Max 40 mL/h XO2 N (I gas F (Rotation) from to mixture) from to (gas flow) from to growth and 0% 30% growth and  0% 100% growth and 15% 80% biotrans- 400 rpm 1500 rpm biotrans- 21%  21% biotrans- 6 sL/h 72 sL/h formation formation formation script trigger sharp 31% DO (1/60 h) induction IPTG 2 h after feed start feed trigger 50% DO feed rate 3 [mL/h]

(12) The experiment carried out falls into to phases: the growth phase, wherein the aim is to attain cells at a certain optical density, and the subsequent biotransformation phase, wherein the aim is to convert the substrate lauric acid methyl ester to w-amino lauric acid methyl ester. pH values were maintained at 6.8 using ammonia (12.5%). During culture and biotransformation the dissolved oxygen in the culture was maintained via the stirrer and the gas flow rate at 30%. The fermentation was carried out as a fed batch, wherein the feed start, 5 g/Lh glucose feed (500 g/L glucose comprising 1% MgSO.sub.4*7H.sub.2O and 2.2% NH.sub.4Cl), was triggered by a DO-Peak. At the time of feed start the temperature was lowered from 37° C. to 30° C. Expression of the transaminase was induced by automatic addition of IPTG (1 mM) 2 h after feed start. alk-genes were induced by manual addition of DCPK (0.025% v/v) 10 h after feed start. The optical density of the culture broth was determined prior to starting the biotransformation.

(13) The biotransformation phase was started 14 h after feed start by adding as a batch a mixture comprising lauric acid methyl ester and oleic acid (technical grade, 90%) to the fermentation broth. In order to provide an amino group donor for the transaminase, half an hour prior to start of the biotransformation 5 mL of a 3M ammonium sulfate solution was added to the fermentation broth. 2 mL fermentation broth samples were removed from the vessel and part of it was diluted 1:20 in a mixture comprising acetone and HCl (c(HCl) 0.1 mol/L) and extracted. Samples were taken 1, 2, 3, 4, 5, 7.5, 10.5, 19.5 and 21 h following start of the biotransformation from all reaction vessels. Oxygen transfer rate (OTR) and carbon transfer rate (CTR) were determined during the fermentation via analysis of exhaust gas from the DASGIP systems. Fermentation was terminated 21 h after start of the biotransformation. The stirrer, the gas flow, the temperature control and pH control were switched of and the vessel was given the opportunity to settle for another 5-10 minutes.

(14) Results:

(15) As the biotransformation progresses, the oxygen and carbon transfer rates increase significantly in the case of W3110 [alkB-alaD-TA]. By contrast the oxygen and carbon transfer rates decrease in the case of the deletion mutant W3110 ΔFadE [alkB-alaD-TA] and approach the level observed prior to the biotransformation (FIG. 3). The amount of product formed by both strains is comparable (FIG. 4), in fact the yield is slightly better in case the mutant is used.

(16) 10 minutes after completion of the biotransformation a clear phase separation could be visually detected in the reaction vessel comprising the strain W3110 ΔFadE [alkB-alaD-TA], wherein the upper phase comprised approximately 40% and the bottom phase comprised approximately 60% of the volume. A thin inter phase could be observed between the phases. Samples were taken from the upper and lower phase, transferred into a 15 mL falcon tube and spun down at 5500×g for 10 minutes. The tube comprising the sample from the lower phase comprised approximately 95% aqueous phase and biomass. The tube comprising the sample from the upper phase comprised approximately 60% organic solution (FIG. 2). The reaction vessel comprising strain W3110 [alkB-alaD-TA] contained a homogenous emulsion after 10 minutes, and no phase separation could be observed for another 20 minutes (FIG. 1).

(17) In summary, deletion of the gene encoding FadE, the E. coli acyl-CoA dehydrogenase of the β-oxidation pathway, leads to an improved phase separation if the mutant is in an aqueous solution and contacted with a water-immiscible organic solvent as well as to a lower consumption of oxygen.