Biological alkane oxidation

Abstract

The invention relates to a method for oxidizing an alkane, comprising contacting the alkane with a type alkB oxidoreductase and using a type alkB oxidoreductase to prepare a mixture of oxidation products of an alkane, wherein the ratio of carboxylic acid to alcohol in the oxidation products is preferably greater than 1:1.

Claims

1. A method for preparing a mixture of oxidation products of a C.sub.1-C.sub.5 alkane comprising: contacting a C.sub.1-C.sub.5 alkane with oxygen in the presence of an AlkB oxidoreductase, wherein the amino acid sequence of said AlkB oxidoreductase is at least 90% identical to that of the AlkB oxidoreductase of Pseudomonas putida described by SEQ ID NO: 6 for a time and under conditions sufficient to oxidize the alkane to a corresponding alcohol and carboxylic acid; and recovering a mixture of oxidation products where the ratio of carboxylic acid to alcohol is greater than 1:1.

2. The method according to claim 1, wherein the alkane is a C.sub.1-C.sub.4 alkane.

3. The method according to claim 1, wherein the C.sub.1 to C.sub.5 alkane is a branched alkane.

4. The method according to claim 1, wherein the AlkB oxidoreductase is AlkB from Pseudomonas putida GPO1.

5. The method according to claim 1, wherein the AlkB oxidoreductase is in the form of a whole-cell catalyst.

6. The method according to claim 1, wherein the AlkB oxidoreductase is in the form of a purified polypeptide.

7. The method according to claim 1, comprising recovering a mixture of oxidation products where the ratio of carboxylic acid to alcohol in the oxidation products is greater than 5:1.

8. The method according to claim 3, wherein the alkane is a C.sub.4 or C.sub.5 alkane.

9. The method according to claim 1, comprising recovering a mixture of oxidation products where the ratio of carboxylic acid to alcohol is greater than 12:1.

10. The method according to claim 1, comprising recovering a mixture of oxidation products where the ratio of carboxylic acid to alcohol is greater than 20:1.

11. The method according to claim 1, comprising recovering a mixture of oxidation products where the ratio of carboxylic acid to alcohol is greater than 40:1.

12. The method according to claim 1, wherein the C.sub.1-C.sub.5 alkane is oxidized predominantly into a carboxylic acid.

13. The method according to claim 1, wherein the C.sub.1-C.sub.5 alkane is oxidized at a terminal carbon atom.

14. A method for preparing a mixture of oxidation products of an alkane comprising: contacting a C.sub.1-C.sub.5 alkane with oxygen and with an AlkB oxidoreductase and optionally AlkG or AlkT enzyme(s), wherein the amino acid sequence of said AlkB oxidoreductase is at least 90% identical to that of the alkB oxidoreductase of Pseudomonas putida described by SEQ ID NO: 6, the amino acid sequence of AlkG is at least 90% identical to the AlkG sequence described by SEQ ID NO: 7, and the amino acid sequence of AlkT is at least 90% identical to the AlkT sequence described by SEQ ID NO: 8; and recovering a mixture of oxidation products where the ratio of carboxylic acid to alcohol is greater than 1:1.

15. The method according to claim 14, wherein the alkB oxidoreductase is expressed along with an AlkG that has a sequence that is at least 90% identical to the AlkG sequence described by SEQ ID NO: 7 and an AlkT that has a sequence that is at least 90% identical to the AlkT sequence described by SEQ ID NO: 8.

16. The method according to claim 14, wherein the alkane is contacted with oxygen and with a Pseudomonas putida enzyme consisting of an alkB oxidoreductase that is at least 90% identical to that of the AlkB oxidoreductase of Pseudomonas putida described by SEQ ID NO: 6.

17. The method according to claim 14, wherein said alkB oxidoreductase is contained within a whole cell catalyst or a crude lysate thereof.

18. The method according to claim 14, wherein said alkB oxidoreductase is in a non-cellular, purified form that when resolved on an SDS gel represents at least 99% of the visible proteins.

19. The method of claim 1, wherein the alkB oxidoreductase is a purified AlkB oxidoreductase and is in either soluble or immobilized form.

20. The method of claim 1, wherein the AlkB oxidoreductase is a form of an engineered recombinant whole cell catalyst that expresses more AlkB oxidoreductase than a corresponding non-recombinant whole cell catalyst, wherein said engineered recombinant whole cell catalyst contains an exogenous nucleic acid sequence encoding an AlkB oxidoreductase on a plasmid or integrated into its genome.

Description

(1) The present 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) FIGS. 1a), b), c) and d) show the concentration of 1-butanol (a)), 2-butanol (b)), butyraldehyde (c)) and butyric acid (d)) as a time course during conversion of butane with oxygen by means of the alkBGT monooxygenase system of P. putida GPO1 at a stirring speed of 500-800 or 900 rpm. The concentrations in the fermenter (F) and in the wash bottle (WB) are shown.

(3) FIGS. 2 a) and b) show the influence of the biomass concentration on the oxidation of butane by E. coli by the alkBGT monooxygenase system of P. putida GPO1, more precisely the concentration time course of 1-butanol (a)) and butyric acid (b)).

(4) FIGS. 3a), b), c) and d) show the influence of the concentration of trace element (TE) solution on the oxidation of butane by E. coli by the alkBGT monooxygenase system of P. putida GPO1, more precisely the concentration time course of 1-butanol (a)), 2-butanol (b)), butyraldehyde (c)) and butyric acid (d)).

(5) FIGS. 4a), b), c) and d) show a comparison of the strains E. coli BL21 and E. coli W3110 by the alkBGT monooxygenase system of P. putida GPO1 and the influence thereof on the oxidation of butane by E. coli with the monooxygenase (alkBGT) of P. putida GPO1, more precisely the concentration time course of 1-butanol (a)), 2-butanol (b)), butyraldehyde (c)) and butyric acid (d)).

(6) FIGS. 5a), b), c) show the oxidation of isobutane by E. coli by the alkBGT monooxygenase system of P. putida GPO1, more precisely the concentration time course of 2-methyl-1-propanol (a)), isobutyraldehyde (b)) and isobutyric acid (c)).

(7) FIG. 6 shows schematically the cloned vector p-LL-30 for example 7.

(8) FIG. 7 shows the oxidation of butane by E. coli with the alkBG monooxygenase system of Alcanivorax borkumensis, as carried out in example 7.

EXAMPLE 1: OXIDATION OF BUTANE BY E. COLI BY THE ALKBGT MONOOXYGENASE SYSTEM OF P. PUTIDA GPO1

(9) 100 ?l of a glycerol cryoculture of E. coli BL21 pCOM10 (empty plasmid) and E. coli BL21 pBT10 (WO 2009/077461) are plated out on an LB agar plate with 50 ?l of kanamycin and incubated at 37? C. for 24 h. The LB plates are prepared from 1 liter of a solution of yeast extract 5 g, peptone 10 g, NaCl 0.5 g, agar agar 15 g and kanamycin 50 ?g. The pH is adjusted to 7.4 with 5% NH.sub.4OH before autoclaving.

(10) From these plates (for a conversion batch), 2?25 ml of LB broth (above solution without agar agar) with 50 ?l of kanamycin in a 100 ml shaking flask with chicanes are inoculated with a full loop of an inoculating loop (capacity 10 ?l). The cultures are incubated for 24 h at 37? C. and 200 rpm (amplitude 2.5 cm).

(11) Each 25 ml of the culture broth are then used as inoculum in 75 ml of modified M9 medium (sterile filtered) with the following composition per liter: 15 g glucose, 6.79 g Na.sub.2PO.sub.4, 3 g KH.sub.2PO.sub.4, 0.5 g NaCl, 2 g NH.sub.4Cl, 15 g yeast extract, 0.49 g MgSO.sub.4*7H.sub.2O, 1 ml TE and 50 ?g kanamycin in 1000 ml shaking flasks. The trace element solution (TE) is made up per liter as follows: 36.5 g HCl 37%, 1.91 g MnCl.sub.2*4H.sub.2O, 1.87 g ZnSO.sub.4*7H.sub.2O, 0.84 g Na-EDTA*2H.sub.2O, 0.3 g H.sub.3BO.sub.3, 0.25 g Na.sub.2MoO.sub.4*2H.sub.2O, 4.7 g CaCl.sub.2*2H.sub.2O, 17.3 g FeSO.sub.4*7H.sub.2O and 0.15 g CuCl.sub.2*2H.sub.2O). The pH is adjusted to 7.4 with 5% NH.sub.4OH. In addition, 3 drops of autoclaved antifoam (Delamex) are added per flask.

(12) The flasks are incubated for 2 h at 37? C. and 180 rpm (amplitude 2.5 cm). The temperature is then reduced to 25? C. The culture is induced after 0.5 hours at 25? C. with 0.4 mM DCPK. The culture is shaken for a further 16 hours at 25? C. and 180 rpm. A microscopic examination for monosepsis is then carried out.

(13) The cultures are combined, filled into 50 ml falcon tubes and centrifuged at 10 000 g at 25? C. for 10 minutes. The supernatant is discarded. The pellets from 200 ml of culture are resuspended in 10 ml of conversion buffer. The conversion buffer consists of 70 mM Na.sup.+/K.sup.+ phosphate buffer, pH 7, adjusted with 1 M NaOH, containing 6.79 g Na.sub.2PO.sub.4, 3 g KH.sub.2PO.sub.4, 0.5 g NaCl, 0.49 g MgSO.sub.4*7H.sub.2O, 1 ml TE and 50 ?g kanamycin or consists of 70 mM (NH.sub.4)H.sub.2PO.sub.4 buffer, pH 7 containing 8 g (NH.sub.4)H.sub.2PO.sub.4, 0.5 g NaCl, 0.49 g MgSO.sub.4*7H.sub.2O, 1 ml TE and 50 ?g kanamycin per liter. The pH is adjusted in this case with 5% NH.sub.4OH.

(14) 170 ml of buffer with ca. 3 drops of autoclaved antifoam (Delamex) are placed in a 300 ml fermenter. The fermenter is flushed with a gas mixture of 25% butane and 75% synthetic air from a gas cylinder at an initial pressure of 5 bar via a sintered glass aerator having a pore size of 0.2 ?m at a flow rate of 25 l/h. The fermenter is heated to 25? C. in a water bath and stirred by means of a magnetic stirrer at 500 rpm for 2 hours, then at 800 rpm. The exhaust gas is passed through a wash bottle containing 150 ml of water.

(15) The fermenter is inoculated with 10 ml of the resuspended pellets. The OD of both cultures is approx. 10. The reaction is initiated by addition of 1% by volume glucose. The pH may optionally be regulated or unregulated during the time course of the experiment. 10 ml samples are withdrawn from the fermenter and the wash bottle after 10, 45, 135 and 240 minutes. The reaction in the samples from the fermenter is stopped with 2 ml HCl. The fermenter samples are centrifuged at room temperature for 10 minutes at 10 000 g and the supernatant filtered through a 0.2 ?m syringe filter unit. The samples are loaded into HPLC vials for analysis. The chromatographic analysis is conducted by HPLC-RID on an Agilent Technologies 1200 system. An Aminex HPX-87H column (300 mm?7.8 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 analyzed were prepared in ultra-pure water and measured under identical conditions. The evaluation was performed by comparison of retention times. In addition, a 2 ml sample is withdrawn from the fermenter at each sampling time point for the determination of pH, OD and glucose concentration. The pH is measured by an external pH-meter, the OD is determined spectrometrically at 600 nm and the glucose content with a biochemical analyzer (YSI Select 2700 from Kreienbaum).

(16) Results

(17) The results are shown in FIG. 1 a)-d). In the experiments with E. coli BL21 pCOM10 (empty plasmid), no oxidation of butane or 1-butanol occurred. In contrast, more applications of E. coli BL21pBT10 are found as oxidation products of n-butane: 1-butanol, butyric acid, 2-butanol, butyraldehyde, 1,4-butanediol (not quantifiable) and butyrolactone (traces).

(18) The concentration of all oxidation products increases with the overall experimental time period. ca. 1 g/lh of glucose is consumed, the pH decreases from 7 to ca. 5.

EXAMPLE 2: INFLUENCE OF THE STIRRING SPEED (SUBSTANCE TRANSPORT LIMITING) ON THE OXIDATION OF N-BUTANE BY E. COLI WITH THE MONOOXYGENASE (ALKBGT) OF P. PUTIDA GPO1

(19) The experiment is carried out analogously to example 1. The stirring speed is set to a constant 900 rpm from the start in a second batch. The OD is twice as high compared to example 1. The TE concentration is 15-fold. The final sampling is after 200 minutes.

(20) Results

(21) At a constant higher stirring speed, 1-butanol is formed more quickly in the fermenter (F) and reaches a maximum sooner. The concentration of 1-butanol in the wash bottles (WB) is at roughly identical low levels. The concentration of 2-butanol in the fermenter (F) increases with increasing stirrer speed over the entire experimental time course but remains low. 2-butanol is not detectable in the wash bottles until the end of the experimental time period. The concentration of butyraldehyde increases more rapidly with higher stirrer speeds, but is also driven off more rapidly since the vapour pressure is 113 hPa (20? C.). Butyraldehyde is only qualitatively, but not quantitatively, detectable.

(22) At lower stirrer speeds, n-butyric acid is not formed until the end of the experimental time period. At higher stirrer speeds, the concentration increases continuously. n-Butyric acid cannot be detected in the wash bottles.

EXAMPLE 3: INFLUENCE OF THE BIOMASS CONCENTRATION ON THE OXIDATION OF N-BUTANE BY E. COLI WITH THE MONOOXYGENASE (ALKBGT) OF P. PUTIDA GPO1

(23) The experiment is carried out analogously to example 1. The stirrer speed is constant at 900 rpm, the TE concentration is respectively 15-fold. 1? means an OD of ca. 10, 2? corresponds to 20.

(24) Results

(25) The results are shown in FIGS. 2 a) and b). The maximum concentration is reached at an earlier experimental time point at twice the OD. 1-Butanol is also more rapidly converted.

(26) Butyric acid can only be detected in the fermenter (F), not in the wash bottles. At twice the OD, the formation of butyric acid already begins at the start of the conversion. At one-fold OD, butyric acid cannot be detected under these conditions until after 240 minutes. The concentration is approximately 18% of the maximum concentration at twice the OD.

EXAMPLE 4: INFLUENCE OF THE TE CONCENTRATION ON THE OXIDATION OF N-BUTANE BY E. COLI WITH THE MONOOXYGENASE (ALKBGT) OF P. PUTIDA GPO1

(27) The experiment is carried out analogously to example 1. The stirrer speed is constant at 900 rpm. The strain used is E. coli W3110 pBT10. The concentration of TE is 1 ml/l of buffer (1?) or 15 ml/l of buffer (15?). In the experiment with the 15-fold concentration, an additional 30 mg/I MOPS are added.

(28) Results

(29) The results are shown in FIGS. 3 a)-d). In the 15-fold TE concentrations, all oxidation products are formed more rapidly and in higher concentrations.

EXAMPLE 5: COMPARISON OF THE STRAINS E. CON BL21 AND E. CON W3110 WITH THE MONOOXYGENASE (ALKBGT) OF P. PUTIDA GPO1

(30) The experiment is carried out analogously to Example 1 with fixed stirrer speed of 900 rpm. The TE concentration is 15 ml/l of conversion buffer.

(31) Results

(32) The results are shown in FIGS. 4 a)-d). The E. coli W3110 pBT10 strain forms all oxidation products more rapidly and in higher concentrations than the E. coli BL21 pBT10 strain.

EXAMPLE 6: OXIDATION OF ISOBUTANE BY E. COLI WITH THE MONOOXYGENASE (ALKBGT) OF P. PUTIDA GPO1

(33) The workflow is carried out analogously to example 1. Only the E. coli W3110 pBT10 strain is used. The conversion buffer consists of 70 mM Na.sup.+/K.sup.+ phosphate buffer, pH7, adjusted with 5% NH.sub.4OH, containing 6.79 g Na.sub.2PO.sub.4, 3 g KH.sub.2PO.sub.4, 0.5 g NaCl, 0.49 g MgSO.sub.4*7H.sub.2O, 15 ml TE and 50 ?g kanamycin per liter.

(34) The gas flushing is carried out as in example 1 but with a mixture of 25% isobutane and 75% synthetic air.

(35) Results

(36) The results are shown in FIGS. 5 a)-c). The oxidation products of isobutane found are isobutanol, isobutyric acid, tert-butanol and isobutyraldehyde.

EXAMPLE 7: OXIDATION OF BUTANE BY E. COLI WITH THE ALKBG MONOOXYGENASE SYSTEM OF ALCANIVORAX BORKUMENSIS

(37) The strain used for the oxidation comprises a plasmid with the genetic information for the alkBG monooxygenase from Alcanivorax borkumensis SK2 (Databank code CAL18155.1 and CAL18156.1). The genetic information for alkST, alkL, and the promoters for alkS and alkB originate from Pseudomonas putida GPo1.

(38) Cloning of the Target Vector

(39) For multiplication, the 2? Phusion HF Master Mix from New England Biolabs (NEB, M0531S) was used according to the manufacturer's instructions. Depending on the degree of purity, the vectors and PCR products were purified directly on a column (QiaQuick PCR Purification Kit, Qiagen, Hilden) or purified on an agarose gel and extracted (QiaQuick Gel Extraction Kit, Qiagen, Hilden). PCR, agarose gel electrophoresis, ethidium bromide staining of the DNA and determination of PCR fragment sizes were carried out in the manner known to the skilled worker. It was possible in both cases to provide PCR fragments of the expected size. For the PCR, the primers with the stated sequences SEQ ID NO 1, 2, 3, and 4 were used.

(40) The purified PCR products were cloned into the EcoRI-HF+Ac/l-cut vector pBT10_alkL after gel purification by means of recombination using the In-Fusion HD Cloning Kit according to the manufacturer's instructions (Clontech Laboratories Inc., Mountain View, Calif., USA). Chemically competent E. coli DH10?(New England Biolabs, Frankfurt) were transformed in the manner known to the skilled worker. Correct insertion of the target sequences was checked by restriction analysis and authenticity of the sequences introduced was confirmed by DNA sequencing. The resulting vector was referred to as p-LL-30 (FIG. 7). The sequence of the vector is stated in the sequence protocol under SEQ ID NO 5.

(41) Donor Organisms and Donated Genes: Pseudomonas putida GPo1 ACCESSION AJ245436 alkB gene integral membrane non-heme iron monooxygenase protein_id=CAB54050.1 alkF gene rubredoxin 1 protein_id=CAB54051.1 alkG gene rubredoxin 2 protein_id=CAB54052.1 alkH gene aldehyde dehydrogenase ACCESSION AJ245436 alkT gene rubredoxin reductase protein_id=CAB54063.1 alkL gene outer membrane protein protein_id=CAB54056.1 alkS gene Expression regulator protein_id=CAB54064.1 Alcanivorax borkumensis 1 alkB_Ab gene alkane 1-monooxygenase CAL18155.1 alkG_Ab gene rubredoxin CAL18156.1

(42) The target vector was cloned into E. coli W3110 in a manner known to the skilled worker. The resulting strain was referred to as E. coli W3110 AN-S-LL-16.

(43) Cell Culture and Biotransformation:

(44) 100 ?l of a glycerol cryoculture E. coli W3110 EN-S-LL-16 are plated out on an LB agar plate with 50 ?l of kanamycin and incubated for 24 h at 37? C. The LB plates are prepared from 1 liter of a solution of yeast extract 5 g, peptone 10 g, NaCl 0.5 g, agar agar 15 g and kanamycin 50 ?g.

(45) From these plates, 3?25 ml of LB broth (above solution without agar agar) with 50 ?l of kanamycin in a 100 ml shaking flask with chicanes are inoculated with a single colony from the plate. The cultures are incubated for 24 h at 37? C. and 200 rpm (amplitude 2.5 cm).

(46) Each 25 ml of the culture broth are then used as inoculum in 175 ml of modified M9 medium with the following composition per liter: 15 g glucose, 6.79 g Na.sub.2PO.sub.4, 3 g KH.sub.2PO.sub.4, 0.5 g NaCl, 2 g NH.sub.4Cl, 15 g yeast extract, 0.49 g MgSO.sub.4*7H.sub.2O, 1 ml TE and 50 ?g kanamycin in 1000 ml shaking flasks. The trace element solution (TE) is made up per liter as follows: 36.5 g HCl 37%, 1.91 g MnCl.sub.2*4H.sub.2O, 1.87 g ZnSO.sub.4*7H.sub.2O, 0.84 g Na-EDTA*2H.sub.2O, 0.3 g H.sub.3BO.sub.3, 0.25 g Na.sub.2MoO.sub.4*2H.sub.2O, 4.7 g CaCl.sub.2*2H.sub.2O, 17.3 g FeSO.sub.4*7H.sub.2O and 0.15 g CuCl.sub.2*2H.sub.2O). The pH is adjusted to 7.4 with 5% NH.sub.4OH. In addition, 3 drops of autoclaved antifoam (Delamex) are added per flask.

(47) The flasks are incubated for 2 h at 37? C. and 180 rpm (amplitude 2.5 cm). The temperature is then reduced to 25? C. The culture is induced after 0.5 hours at 25? C. with 0.4 mM DCPK. The culture is shaken for a further 16 hours at 25? C. and 180 rpm.

(48) The cultures are combined, filled into 50 ml falcon tubes and centrifuged at 10 000 g at 25? C. for 10 minutes. The supernatant is discarded. The pellets from 600 ml of culture are resuspended in 30 ml of conversion buffer. The conversion buffer consists of 70 mM ammonium phosphate buffer, pH 7 containing 8 g (NH.sub.4)H.sub.2PO.sub.4, 0.5 g NaCl, 0.49 g MgSO.sub.4*7H.sub.2O, 1 ml TE and 50 ?g kanamycin per liter. The pH is adjusted with 25% ammonia solution.

(49) 150 ml of buffer with ca. 3 drops of autoclaved antifoam (Delamex) are placed in a 300 ml fermenter. The fermenter is flushed with a gas mixture of 25% butane and 75% synthetic air via a sintered glass aerator having a pore size of 0.2 ?m at a flow rate of 6.5 I.sub.N/h. The fermenter is heated to 30? C. in a water bath and stirred by means of a magnetic stirrer at 900 rpm. The exhaust gas is passed through a wash bottle containing 150 ml of water.

(50) The fermenter is inoculated with the resuspended preculture pellets. The OD600 is approx. 15. The pH is regulated to 7.0 with 5% ammonia solution. The glucose feed rate is 1 g/lh. 5 ml samples are removed at various time points from the fermenter and the wash bottle. The fermenter samples are centrifuged at room temperature for 10 minutes at 10 000 g and the supernatant filtered through a 0.2 ?m syringe filter unit. The samples are loaded into HPLC vials for analysis. The chromatographic analysis is conducted by HPLC-RID on an Agilent Technologies 1200 system. An Aminex HPX-87H column (300 mm?7.8 mm) is used. The system is 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 analyzed are prepared in ultra-pure water and measured under identical conditions. The evaluation is performed by comparison of retention times. In addition, a 2 ml sample is withdrawn from the fermenter at each sampling time point for the determination of pH, OD and glucose concentration. The pH is measured by an external pH-meter, the OD is determined spectrometrically at 600 nm and the glucose content with a biochemical analyzer (YSI Select 2700 from Kreienbaum). The results are summarized in FIG. 7.

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