RHAMNOLIPID SYNTHESIS

Abstract

There is provided a method of producing at least one rhamnolipid comprising: (a) contacting a recombinant cell with a medium containing a carbon source; wherein the recombinant cell has been genetically modified such that, compared to the wild-type of the cell, the cell has an increased activity of at least one of the enzymes E.sub.1, E.sub.2 and E.sub.3, wherein the enzyme E.sub.1 is an / hydrolase (RHIA), the enzyme E.sub.2 is a rhamnosyltransferase I (RHIB) and the enzyme E.sub.3 is a rhamnosyltransferase II (RHIC), and wherein the carbon source is an alkane and/or alkanoic acid comprising 6 to 10 carbon atoms.

Claims

1-11. (canceled)

12. A method of producing at least one rhamnolipid comprising contacting a recombinant cell with a medium containing a carbon source, wherein: a) the recombinant cell has been genetically modified such that, compared to the wild-type cell, the recombinant cell has an increased activity of enzymes E.sub.1, E.sub.2 and E.sub.3, wherein: i) enzyme E.sub.1 i is an / hydrolase (RHIA); ii) enzyme E.sub.2 is a rhamnosyltransferase I (RHIB); iii) enzyme E.sub.3 is a rhamnosyltransferase II (RHIC); b) the carbon source is an alkane and/or alkanoic acid comprising 6 to 10 carbon atoms; and c) the rhamnolipid comprises the general formula (I), ##STR00004## wherein m=2, 1 or 0 n=1 R.sup.1 and R.sup.2=independently of one another, identical or different organic radicals comprising 2 to 24 carbon atoms.

13. The method of claim 12, wherein said organic radicals are alkyl radicals that are optionally branched, optionally substituted, and optionally unsaturated.

14. The method of claim 13, wherein at least one alkyl radical is hydroxy-substituted.

15. The method of claim 13, wherein at least one alkyl radical is mono-, di- or tri-unsaturated.

16. The method of claim 12, wherein said identical or different organic radicals comprise 5 to 13 carbon atoms.

17. The method of claim 12, wherein said carbon source is an alkane selected from the group consisting of: hexane; heptane; octane; nonane; and decane; and/or an alkanoic acid selected from the group consisting of: hexanoic acid; haptanoic acid; octanoic acid; nonanoic acid; and decanoic acid.

18. The method of claim 12, wherein the recombinant cell has been genetically modified such that, compared to the wild-type cell, the recombinant cell has an increased activity of enzyme E.sub.4, wherein E.sub.4 is an oxidoreductase.

19. The method according to claim 18, wherein the oxidoreductase is selected from the group consisting of: alkB-type oxidoreductase; monooxygenase; and NAD(P)H dependent alcohol dehydrogenase (ADH).

20. The method of claim 19, wherein the carbon source is hexane and/or decane.

21. The method of claim 12, wherein at least 40% by weight of the total carbon content in the medium is hexane, decane, hexanoic acid and/or decanoic acid.

22. The method of claim 12, wherein: a) enzyme E.sub.1 is able to catalyse the conversion of 3-hydroxyalkanoyl-ACP via 3-hydroxyalkanoyl-3-hydroxyalkanoic acid-ACP to hydroxyalkanoyl-3-hydroxyalkanoic acid; b) enzyme E.sub.2 is able to catalyse the conversion of dTDP-rhamnose and 3-hydroxyalkanoyl-3-hydroxyalkanoate to -L-rhamnopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate; and c) enzyme E.sub.3 is able to catalyse the conversion of dTDP-rhamnose and -L-rhamnopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate to -L-rhamnopyranosyl-(1-2)--L-rhamnopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate.

23. The method of claim 12, wherein: a) enzyme E.sub.1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; and fragments thereof; b) enzyme E.sub.2 is selected from the group consisting of: SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; and fragments thereof; and c) enzyme E.sub.3 is selected from the group consisting of: SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:15; and fragments thereof; wherein said fragments comprise a polypeptide sequences in which up to 25% of the amino acid radicals are modified by deletion, insertion, substitution or a combination thereof compared to the sequence of the respective enzyme and the fragment comprises at least 10% of the enzymatic activity of the respective enzyme.

24. The method of claim 12, wherein the recombinant cell is selected from a genus of the group consisting of: Aspergillus; Corynebacterium; Brevibacterium; Bacillus, Acinetobacter; Alcaligenes; Lactobacillus; Paracoccus; Lactococcus; Candida; Pichia; Hansenula; Kluyveromyces; Saccharomyces; Escherichia; Zymomonas; Yarrowia; Methylobacterium; Ralstonia; Pseudomonas; Rhodospirillum; Rhodobacter; Burkholderia; Clostridium; and Cupriavidus.

25. The method of claim 12, wherein the recombinant cell is selected from the group consisting of: P. putida GPp121; P. putida GPp122; P. putida GPp123; P. putida GPp124; P. putida GPp104; P. putida KT42C1; P. putida KTOY01; and P. putida KTOY02.

26. The method of claim 12, wherein the rhamnolipid is a dirhamnosyl lipid selected from the group consisting of: 2RL-C10-C10; 2RL-C8-C10; 2RL-C10-C10; and 2RL-C10-C12:1.

27. The method of claim 12, wherein the recombinant cell has been genetically modified such that, compared to the wild-type cell, the recombinant cell has an increased activity of enzyme E.sub.4, wherein E.sub.4 is an oxidoreductase.

28. The method according to claim 27, wherein the oxidoreductase is selected from the group consisting of: alkB-type oxidoreductase; monooxygenase; and NAD(P)H dependent alcohol dehydrogenase (ADH).

29. The method of claim 28, wherein the recombinant cell is selected from the group consisting of: P. putida GPp121; P. putida GPp122; P. putida GPp123; P. putida GPp124; P. putida GPp104; P. putida KT42C1; P. putida KTOY01; and P. putida KTOY02.

30. The method of claim 29, wherein the rhamnolipid may be a dirhamnosyl lipid selected from the group consisting of 2RL-C10-C10, 2RL-C8-C10, 2RL-C10-C10 and 2RL-C10-C12:1.

31. The method of claim 30, wherein at least 40% by weight of the total carbon content in the medium is hexane, decane, hexanoic acid and/or decanoic acid.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0115] No figures.

EXAMPLES

[0116] 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

[0117] Pseudomonas putida Forming Rhamnolipids from Acetate

[0118] For the biotransformation of acetate to rhamnolipids a plasmid harboring Pseudomonas putida KT2440 strain was used. The plasmid pBBR1MCS-2::ABC is described in example 2 of DE 10 2010 032 484 A1 and the transformation of Pseudomonas putida KT2440 with the vector is described in Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5): 851-854. The recombinant Pseudomonas putida KT2440 pBBR1MCS-2::ABC was cultivated on LB agar plates with 50 mg/l kanamycin.

[0119] For the preculture 10 ml of LB medium with 50 mg/I kanamycin in a 100 ml shaking flask were inoculated with a single colony from a fresh incubated agar plate and cultivated at 30 C. and 120 rpm for 15 h to an OD.sub.600nm>3.5. Then the cell suspension was centrifuged, washed with fresh M9_BS_Ac medium and centrifuged again.

[0120] For the main culture 100 ml of fresh M9_BS_Ac medium (pH 7.4; 6.81 g/L Na.sub.2HPO.sub.4, 2.4 g/L KH.sub.2PO.sub.4, 0.4 g/L NaCl, 1.4 g/L NH.sub.4Cl, 2 ml/L 1 M MgSO.sub.47 H.sub.2O, 1.63 g/L .sup.13C.sub.2-Na-acetate, 0.13 ml/L 25% HCl, 1.91 mg/L MnCl.sub.27 H.sub.2O, 1.87 mg/L ZnSO.sub.47 H.sub.2O, 0.84 mg/L Na-EDTA2 H.sub.2O, 0.3 mg/L H.sub.3BO.sub.3, 0.25 mg/L Na.sub.2MoO.sub.42 H.sub.2O, 4.7 mg/L CaCl.sub.22 H.sub.2O, 17.8 mg/L FeSO.sub.47 H.sub.2O, 0.15 mg/L CuCl.sub.22 H.sub.2O) in a 500 ml shaking flask were inoculated with centrifuged and washed cells from the preculture to an OD600nm of 0.12. This culture was incubated at 32 C. and 140 rpm for 142 h. After 6 h of cultivation, 2 g/L rhamnose was added to the culture for induction. After 7.5 h, 22.5 h, 30.5 h, 47.25 and 53 h of cultivation, 1 g/I .sup.13C.sub.2-Na-acetate were added respectively. At the start and during the culturing period, samples were taken. These were tested for optical density, pH and the different analytes (tested by NMR).

[0121] The results showed that in the main culture the amount of acetate decreased continuously from 1.63 g/I in the beginning to 1.3 g/I after 71.75 h (including the acetate feeding of 5 g/L.sup.13C.sub.2-Na-acetate). Also, the concentration of rhamnolipids (2RL-C10-C10) (a dirhamnosyl lipid) was increased from 0.0 mg/I to 332 mg/I after 71.75 h of cultivation. The formed rhamnolipids were .sup.13C-labeled (>90% in the fatty acid part). The carbon yield for .sup.13C-labeled 2RL-C10-C10 a dirhamnosyl lipid was about 12.96% related to the consumed acetate and for non-labeled 2RL-C10-C10 it was 1.44%.

Example 2

[0122] Pseudomonas putida Forming Rhamnolipids from Acetate and Decanoic Acid

[0123] For the biotransformation of acetate and decanoic acid to rhamnolipids a plasmid harboring Pseudomonas putida KT2440 strain was used. The plasmid pBBR1 MCS-2::ABC is described in example 2 of DE 10 2010 032 484 A1 and the transformation of Pseudomonas putida KT2440 with the vector is described in Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5): 851-854. The recombinant Pseudomonas putida KT2440 pBBR1MCS-2::ABC was cultivated on LB agar plates with 50 mg/I kanamycin.

[0124] For the preculture 10 ml of LB medium with 50 mg/I kanamycin in a 100 ml shaking flask were inoculated with a single colony from a fresh incubated agar plate and cultivated at 30 C. and 120 rpm for 15 h to an OD600nm>3.5. Then the cell suspension was centrifuged, washed with fresh M9_BS_Ac medium and centrifuged again.

[0125] For the main culture 100 ml of fresh M9_BS_Ac medium (pH 7.4; 6.81 g/L Na.sub.2HPO.sub.4, 2.4 g/L KH.sub.2PO.sub.4, 0.4 g/L NaCl, 1.4 g/L NH.sub.4Cl, 2 ml/L 1 M MgSO.sub.47 H.sub.2O, 1.63 g/L .sup.13C.sub.2-Na-acetate, 0.13 ml/L 25% HCl, 1.91 mg/L MnCl.sub.27 H.sub.2O, 1.87 mg/L ZnSO.sub.47 H.sub.2O, 0.84 mg/L Na-EDTA2 H.sub.2O, 0.3 mg/L H.sub.3BO.sub.3, 0.25 mg/L Na.sub.2MoO.sub.42 H.sub.2O, 4.7 mg/L CaCl.sub.22 H.sub.2O, 17.8 mg/L FeSO.sub.47 H.sub.2O, 0.15 mg/L CuCl.sub.22 H.sub.2O) in a 500 ml shaking flask were inoculated with centrifuged and washed cells from the preculture to an OD.sub.600nm of 0.12. This culture was incubated at 32 C. and 140 rpm for 142 h. After 6 h of cultivation, 2 g/L rhamnose were added to the culture for induction. After 22.5 h of cultivation, 1 g/L decanoic acid was added to the culture. After 7.5 h, 22.5 h, 30.5 h, 47.25 h and 53 h of cultivation, 1 g/I .sup.13C.sub.2-Na-acetate were added respectively. At the start and during the culturing period, samples were taken. These were tested for optical density, pH and the different analytes (tested by NMR).

[0126] The results showed that in the main culture the amount of acetate decreased continuously from 1.63 g/I in the beginning to 0 g/I after 71.75 h (including the acetate feeding of 5 g/L.sup.13C.sub.2-Na-acetate). The concentration of decanoic acid decreased from 1 g/I at 22.5 h to 0 g/L after 71.75 h. Also, the concentration of rhamnolipids (2RL-C10-C10), a dirhamnosyl lipid was increased from 0.0 mg/I to 779 mg/I after 71.75 h of cultivation. The newly formed rhamnolipids were .sup.13C-labeled (34% in the fatty acid part). The carbon yield for .sup.13C-labeled 2RL-C10-C10, the dirhamnosyl lipid was about 6.05% based on the consumed acetate and decanoic acid and for non-labeled 2RL-C10-C10 it was 11.75%. This showed that a larger percentage of the resulting rhamnolipids were formed from the unlabeled decanoic acid than the acetate.

Example 3

[0127] Pseudomonas putida Forming Rhamnolipids from Acetate and Hexanoic Acid

[0128] For the biotransformation of acetate and hexanoic acid to rhamnolipids a plasmid harboring Pseudomonas putida KT2440 strain was used. The plasmid pBBR1MCS-2::ABC is described in example 2 of DE 10 2010 032 484 A1 and the transformation of Pseudomonas putida KT2440 with the vector is described in Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5): 851-854. The recombinant Pseudomonas putida KT2440 pBBR1MCS-2::ABC was cultivated on LB agar plates with 50 mg/I kanamycin.

[0129] For the preculture 10 ml of LB medium with 50 mg/I kanamycin in a 100 ml shaking flask are inoculated with a single colony from a fresh incubated agar plate and cultivated at 30 C. and 120 rpm for 15 h to an OD.sub.600nm>3.5. Then the cell suspension is centrifuged, washed with fresh M9_BS_Ac medium and centrifuged again.

[0130] For the main culture 100 ml of fresh M9_BS_Ac medium (pH 7.4; 6.81 g/L Na.sub.2HPO.sub.4, 2.4 g/L KH.sub.2PO.sub.4, 0.4 g/L NaCl, 1.4 g/L NH.sub.4Cl, 2 ml/L 1 M MgSO.sub.47 H.sub.2O, 1.63 g/L .sup.13C.sub.2-Na-acetate, 0.13 ml/L 25% HCl, 1.91 mg/L MnCl.sub.27 H.sub.2O, 1.87 mg/L ZnSO.sub.47 H.sub.2O, 0.84 mg/L Na-EDTA2 H.sub.2O, 0.3 mg/L H.sub.3BO.sub.3, 0.25 mg/L Na.sub.2MoO.sub.42 H.sub.2O, 4.7 mg/L CaCl.sub.22 H.sub.2O, 17.8 mg/L FeSO.sub.47 H.sub.2O, 0.15 mg/L CuCl.sub.22 H.sub.2O) in a 500 ml shaking flask are inoculated with centrifuged and washed cells from the preculture to an OD.sub.600nm of 0.12. This culture is incubated at 32 C. and 140 rpm for 142 h. After 6 h of cultivation, 2 g/L rhamnose are added to the culture for induction. After 22.5 h of cultivation, 1 g/L hexanoic acid is added to the culture. After 7.5 h, 22.5 h, 30.5 h, 47.25 h and 53 h of cultivation, 1 g/l .sup.13C.sub.2-Na-acetate are added respectively. At the start and during the culturing period, samples are taken. These are tested for optical density, pH and the different analytes (tested by NMR).

[0131] In the main culture the amount of acetate decreased continuously to 0 g/I after 71.75 h (including the acetate feeding of 5 g/L.sup.13C.sub.2-Na-acetate). The concentration of hexanoic acid also decreased to 0 g/L after 71.75 h. Also, the concentration of rhamnolipid (2RL-C10-C10) increased during the cultivation. The newly formed rhamnolipids were .sup.13C-labeled (<80% in the fatty acid part). The carbon yield for .sup.13C-labeled 2RL-C10-C10, a dirhamnosyl lipid related to consumed acetate and hexanoic acid was lower and for non-labeled 2RL-C10-C10 it was higher than in cultures without hexanoic acid feeding. Again this confirmed the finding that a larger percentage of the resulting rhamnolipids were formed from the unlabeled hexanoic acid than the acetate.