Methods of producing rhamnolipids

Abstract

The present invention relates to a method of preparing at least one rhamnolipid comprising: contacting a recombinant cell with a medium containing a carbon source; and culturing the cell under suitable conditions for preparation of the rhamnolipid from the carbon source by the cell, 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, the enzyme E.sub.2 is a rhamnosyltransferase I and the enzyme E.sub.3 is a rhamnosyl-transferase II, and wherein the carbon source is a C.sub.4 molecule.

Claims

1. A method of preparing 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, said recombinant cell has increased activity of all three of enzymes E.sub.1, E.sub.2 and E.sub.3, and wherein: i) enzyme E.sub.1 comprises the sequence of SEQ ID NO:2 or an enzyme comprising a sequence in which up to 10% of the amino acids of SEQ ID NO:2 have been modified and for which more than 50% of the enzymatic activity of SEQ ID NO:2 in converting 3-hydroxy-decanoyl-ACP via 3-hydroxydecanoyl-3-hydroxydecanoic acid-ACP to hydroxydecanoyl-3-hydroxydecanoic acid is maintained; ii) enzyme E.sub.2 comprises the sequence of SEQ ID NO:7 or an enzyme comprising a sequence in which up to 10% of the amino acids of SEQ ID NO:7 have been modified and for which more than 50% of the enzymatic activity of SEQ ID NO:7 in converting dTDP-rhamnose and 3-hydroxydecanoyl-3-hydroxydecanoic acid to a-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid is maintained; ii) enzyme E.sub.3 comprises the sequence of SEQ ID NO:12 or an enzyme comprising a sequence in which up to 10% of the amino acids of SEQ ID NO:12 have been modified and for which more than 50% of the enzymatic activity of SEQ ID NO:12 in converting dTDP rhamnose and a-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid to a-L-rhamnopyranosyl-(1-2)-a-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid is maintained; b) culturing the recombinant cell under suitable conditions for preparation of the rhamnolipid from the carbon source by the cell; c) optionally isolating rhamnolipids from the cells and/or the medium of step b); wherein at least 70% of the total carbon content of the medium in which the recombinant cells are cultured is in the form of C.sub.4 molecules having exactly four carbon atoms.

2. The method of claim 1, wherein the C.sub.4 molecules have no atoms other than carbon, oxygen and hydrogen.

3. The method of claim 1, wherein the C.sub.4 molecule is selected from the group consisting of: butane; 1-butanol; 2-butanol; 1-butanal; butanone; butyric acid; and combinations thereof.

4. The method of claim 3, wherein at least 90% of the total carbon content of the medium in which the recombinant cells are cultured is in the form of butane; 1-butanol; 2-butanol; 1-butanal; butanone; butyric acid; or combinations thereof.

5. The method of claim 3, wherein at least 90% of the total carbon content of the medium in which the recombinant cells are cultured is in the form of butyric acid or butane.

6. The method of claim 3, wherein 100% of the total carbon content of the medium in which the recombinant cells are cultured is in the form of butane; 1-butanol; 2-butanol; 1-butanal; butanone; butyric acid; or combinations thereof.

7. The method of claim 1, wherein the recombinant cell has been genetically modified such that, compared to the wild-type cell, there is increased activity of an oxidoreductase.

8. The method of claim 7, wherein the oxidoreductase is selected from the group consisting of: alkB-type oxidoreductase; monooxygenase; and NAD(P)H dependent alcohol dehydrogenase (ADH).

9. The method according of claim 1, wherein said 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.

10. The method of claim 1, wherein said 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.

11. The method of claim 1, wherein the rhamnolipid comprises the general formula (I), ##STR00003## wherein: m=2, 1 or 0; n=1 or 0; and R.sup.1 and R.sup.2=independently of one another, identical or different organic radicals having 2 to 24 carbons.

12. The method of claim 11, wherein, in formula I, one or both of the organic radicals are branched and/or substituted.

13. The method of claim 11, wherein, in formula I, one or both of the organic radicals are unsaturated.

14. The method of claim 11, wherein, in formula I, m=1 or 0 and n=1.

15. The method of claim 1, wherein enzyme E.sub.1 consists of the amino acid sequence of SEQ ID NO:2.

16. The method of claim 15, wherein enzyme E.sub.2 consists of the amino acid sequence of SEQ ID NO:7.

17. The method of claim 16, wherein enzyme E.sub.3 consists of the amino acid sequence of SEQ ID NO:12.

18. The method of claim 17, wherein said 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.

19. The method of claim 18, wherein at least 90% of the total carbon content of the medium in which the recombinant cells are cultured is in the form of butane; 1-butanol; 2-butanol; 1-butanal; butanone; butyric acid; or combinations thereof.

20. The method of claim 18, wherein 100% of the total carbon content of the medium in which the recombinant cells are cultured is in the form of butane; 1-butanol; 2-butanol; 1-butanal; butanone; butyric acid; or combinations thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a graph illustrating the composition by percentage of the rhamnolipid products formed depending on the different carbon sources.

EXAMPLES

(2) 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.

(3) In the production of rhamnolipids by Pseudomonas putida, before the product concentration in the different examples is determined, the reaction samples were diluted immediately after fermentation with acetone at a volume ratio of 1:1 and centrifuged at 21,000 g and 4 C. for 2 min. The sample supernatant was then measured by HPLC.

Example 1 (A Comparative Example, not of the Invention)

Production of Rhamnolipids with BS-PP-001 from Glucose

(4) On an LB agar plate containing 50 mg/l kanamycin an inoculation loop of glycerol cryoculture of the strain Pseudomonas putida KT2440 pBBR1MCS-2::ABC (BS-PP-001) was streaked. The method of producing the vector pBBR1 MCS-2::ABC is provided in Example 2 of DE102010032484A1. The Pseudomonas putida is then transformed with the vector and stored. The agar plate was incubated for 24 h at 30 C. A 100 ml flask with baffles containing 25 ml of LB medium with kanamycin was inoculated with a single culture of overgrown agar plate and incubated in a shaking incubator for 24 h at 30 C. and 200 rpm to produce a preculture. The preculture was centrifuged at 5500 g at room temperature for 10 minutes. The supernatant was then discarded. The pellet was resuspended in 25 ml of M9 medium (composition: 6.8 g/l Na.sub.2PO.sub.4.2H.sub.2O, 2.4 g/l KH.sub.2PO.sub.4, 0.4 g/l NaCl, 1.6 g/l NH.sub.4Cl, 0.5 g/l MgSO.sub.4.7H.sub.2O, 1 ml of trace element solution US3, consisting of 36.5 g/l of 37% strength 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). This washing step was then repeated.

(5) In a 300 ml fermenter, 180 ml of M9 medium as described above was added with 20 g/l glucose and with 50 mg/l kanamycin. The fermenter was inoculated with a large volume of preculture suspension to reach a start OD.sub.600 of 0.4. The following parameters were set during fermentation: gassing with air 2 NL/h, dissolved oxygen concentration adjusted to 30% by adjusting the stirrer speed. This measurement is carried out using a standard oxygen sensor, temperature of 30 C., initial pH value of 7.4 (not regulated throughout the experiment). After 40 h of fermentation, glucose solution (concentration in the fermenter of: 15 g/l) was fed via a syringe. At specified times, samples were taken from the fermenter to determine the concentration of rhamnolipids and fatty acid dimers produced.

(6) The results are shown in Table 2 below and FIG. 1.

Example 2

Production of Rhamnolipids with BS-PP-001 from Butyric Acid

(7) The preculture was made analogously to Example 1 with glucose.

(8) In a 300 ml fermenter, 180 ml of M9 medium as described in Example 1 was added with 6.5 g/l sodium butyrate and 50 mg/l kanamycin. The fermenter was inoculated with a large volume of preculture suspension to reach a start OD.sub.600 of 0.4. The following parameters were set during fermentation: gassing with air 2 NL/h, stirrer speed set at 300 rpm, temperature 30 C., initial pH value of 7.4 (not regulated throughout the experiment). After 40 h of fermentation, sodium butyrate solution (concentration in the fermenter: 5 g/l butyric acid) was fed via a syringe. The stirrer speed was increased to 900 rpm.

(9) At specified times, samples were taken from the fermenter to determine the concentration of rhamnolipids and fatty acid dimers produced.

(10) The results are shown in Table 2 below and FIG. 1.

Example 3

Production of Rhamnolipids Using BS-PP-001+alkB from n-Butane

(11) On an LB agar plate containing 50 mg/l kanamycin, an inoculation loop full of glycerol cryoculture of the strain P. putida pBBR1 MCS-2::ABC pBT10 was (BS-PP001+alkB) streaked. This strain was produced by adding to the strain of Example 1 the gene construct pBT10 as described on pages 36 and 37 (SEQ-ID 31) of WO2009/077461A1. The agar plate was incubated for 24 h at 30 C.

(12) Three 100 ml flasks with baffles was filed with 25 ml of LB medium containing kanamycin and each inoculated with a single culture of the overgrown agar plate and incubated in a shaking incubator for 24 h at 30 C. and 200 rpm.

(13) Three 1-liter flasks with baffles were each used to mix 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.47H.sub.2O, 50 mg/l kanamycin sulfate, 15 ml trace element solution US3 consisting of 36.5 g/l of 37% strength hydrochloric acid, 11.91 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) and the preculture from the 100 ml flasks. The cultures were incubated at 30 C. and 200 rpm. After 3 hours of incubation alkBGT genes was activated by adding 0.4 mM of dicyclopropylketone. The cultures were incubated for a further 16 h at 25 C. and 200 rpm.

(14) The cultures in the three flasks were combined and centrifuged at 5500 g at room temperature for 10 minutes. The supernatant was discarded. The pellet was resuspended in 25 ml of M9 medium (composition of which is provided above). This washing step was repeated for the removal of glucose and other possible carbon sources.

(15) In a 300 ml fermenter, 180 ml of M9 medium (composition of which is provided above without a carbon source), 50 mg/l 50 mg/l kanamycin were added. The fermenter was inoculated with a large volume of preculture suspension from the earlier step to reach a start OD.sub.600 of 10. The following parameters were set during fermentation: gassing with butane/air mixture (25%/75%) 2 NL/h, stirrer speed set at 900 rpm, temperature 30 C., initial pH value of 7.4 (not regulated throughout the experiment). At specified times, samples were taken from the fermenter to determine the concentration of rhamnolipids and fatty acid dimers produced.

(16) The results are shown in Table 2 below and FIG. 1.

(17) TABLE-US-00002 TABLE 2 Final concentrations of rhamnolipids and fatty acid dimers produced based on the substrate used. Rhamno- Rhamno- Fatty lipid-2 lipid-1 Acid dimers 2RL 1RL (FA-dimer) Total Strain, Substrate [mg/l] [mg/l] [mg/l] [mg/l] BS-PP-001, Glucose 110 81 793 983 BS-PP-001, Butyrate 29 343 538 910 BS-PP-001 + AlkB, 438 58 0 496 Butane BS-PP-001 + AlKB, 1.146 142 0 1.288 1-Butanol

(18) As can be seen, Table 2 shows that the strain equipped with the genes rhlA, rhlB and rhlC from P. aeruginosa of the species P. putida KT2440 (BS-PP-001) was able to produce about 1 g/l of products, of which about 110 mg/l were dirhamnolipid and 81 mg/l were monorhamnolipid, as well as almost 800 mg of unwanted fatty acid dimers when glucose was used as a substrate.

(19) When butyrate was used as the sole carbon source, the amount of dirhamnolipid significantly increased, while only about one-third of unwanted fatty acid dimers were formed. In another example, a strain was genetically modified to introduce oxidoreductase AlkB from Pseudomonas putida GPO1 and fed with butane as the sole carbon source. The results provided in Table 2 showed that up to over 1000 mg/l of dirhamnolipid was formed and no measurable amounts of undesirable fatty acid dimers were produced.

(20) The results in FIG. 1 also illustrate composition by percentage of the product formed depending on the different carbon sources. It can be seen that the use of butyrate reduces the amount of unwanted fatty acid dimers from 81% to 64%, and with the use of butane and butanol, the amount of fatty acid dimers formed is not measurable.

Example 4

Production of Rhamnolipids Using BS-PP-001+alkB from 1-Butanol

(21) Three 100 ml flasks with baffles were filled with 25 ml of LB medium containing kanamycin and tetracyclin and each inoculated with 100 l of a glycerol cryoculture of the strain P. putida pBBR1MCS-2::ABC pBT10 (BS-PP001+alkB). This strain was produced by adding to the strain of Example 1 the gene construct pBT10 as described on pages 36 and 37 (SEQ-ID 31) of WO2009/077461A1. The flasks were incubated in a shaking incubator for 24 h at 30 C. and 200 rpm.

(22) Three 1-liter flasks with baffles were each used to mix 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.47H.sub.2O, 50 mg/l kanamycin sulfate, 10 mg/l tetracycline, 15 mill trace element solution US3 consisting of 36.5 g/l of 37% strength hydrochloric acid, 11.91 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) and the preculture from the 100 ml flasks. The cultures were incubated at 30 C. and 200 rpm. After 3 hours of incubation alkBGT genes was activated by adding 0.4 mM of dicyclopropylketone. The cultures were incubated for a further 4 h at 30 C. and 200 rpm.

(23) The cultures in the three flasks were combined and centrifuged at 5500 g at room temperature for 10 minutes. The supernatant was discarded. The pellet was resuspended in 25 ml of M9 medium (composition of which is provided above). This washing step was repeated for the removal of glucose and other possible carbon sources.

(24) In a 300 ml fermenter, 180 ml of M9 medium (composition of which is provided above without a carbon source), 50 mg/l kanamycin were added. The fermenter was inoculated with 10 ml of preculture suspension from the earlier step to reach a start OD.sub.600 of 5. The following parameters were set during fermentation: gassing with air 3 NI/h, stirrer speed set at 700 rpm, temperature 30 C., pH value of 7.0 (regulated throughout the experiment with 5% ammonia solution). Butanol solution was fed via a syringe (feed rate 0.2 g/l (1 h)). At specified times, samples were taken from the fermenter to determine the concentration of rhamnolipids and fatty acid dimers produced.

(25) The results are provided in Table 2 above.

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