RHAMNOLIPID-PRODUCING CELL HAVING REDUCED GLUCOSE DEHYDROGENASE ACTIVITY

Abstract

The invention relates to cells which make rhamnolipids and are genetically modified such that they have a decreased activity, compared to the wild type thereof, of a glucose dehydrogenase and to a method for producing rhamnolipids using the cells according to the invention.

Claims

1. A cell able to make at least one rhamnolipid, wherein it has been genetically modified such that it, compared to the wild type thereof, has a decreased activity of at least one enzyme E.sub.1, which catalyses the conversion of D-glucose and quinone to D-glucono-1,5-lactone and quinol.

2. The cell according to claim 1, wherein it is able to make, as rhamnolipid, a compound of the general formula (I), ##STR00003## where m=1, n=1 or 0, and the radical determined via R.sup.1 and R.sup.2 is ##STR00004## derived from 3-hydroxyoctanoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydecenoic acid, 3-hydroxydecenoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydodecanoic acid, 3-hydroxydodecanoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydodecenoic acid, 3-hydroxydodecenoyl-3-hydroxyoctanoic acid, 3-hydroxydecanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxydecenoic acid, 3-hydroxydecenoyl-3-hydroxydecanoic acid, 3-hydroxydecenoyl-3-hydroxydecenoic acid, 3-hydroxydecanoyl-3-hydroxydodecanoic acid, 3-hydroxydodecanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxydodecenoic acid, 3-hydroxydecanoyl-3-hydroxytetradecenoic acid, 3-hydroxytetradecanoyl-3-hydroxydecenoic acid, 3-hydroxydodecenoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxytetradecanoic acid, 3-hydroxytetradecanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxytetradecenoic acid, 3-hydroxytetradecenoyl-3-hydroxydecanoic acid, 3-hydroxydodecanoyl-3-hydroxydodecanoic acid, 3-hydroxydodecenoyl-3-hydroxydodecanoic acid, 3-hydroxydodecanoyl-3-hydroxydodecenoic acid, 3-hydroxydodecanoyl-3-hydroxytetradecanoic acid, 3-hydroxytetradecanoyl-3-hydroxydodecanoic acid, 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoyl-3-hydroxytetradecanoic acid, 3-hydroxytetradecanoyl-3-hydroxyhexadecanoic acid or 3-hydroxyhexadecanoyl-3-hydroxyhexadecanoic acid.

3. The cell according to claim 1, wherein E.sub.1 is a glucose 1-dehydrogenase of EC 1.1.5.2.

4. The cell according to claim 1, wherein the cell is selected from the group consisting of Burkholderia sp., Burkholderia thailandensis, Pseudomonas sp., Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas oleovorans, Pseudomonas stutzeri, Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas citronellolis, Pseudomonas resinovorans, Comamonas testosteroni, Aeromonas hydrophila, Cupriavidus necator, Alcaligenes latus and Ralstonia eutropha.

5. The cell according to claim 1, wherein the cell has been genetically modified such that it, compared to the wild type thereof, has an increased activity of at least one of the enzymes selected from the group E.sub.2, E.sub.3 and E.sub.4, the enzyme E.sub.2 being able to catalyse the conversion of 3-hydroxyalkanoyl-ACP via 3-hydroxyalkanoyl-3-hydroxyalkanoic acid-ACP to hydroxyalkanoyl-3-hydroxyalkanoic acid, the enzyme E.sub.3 being a rhamnosyltransferase I and being able to catalyse the conversion of dTDP-rhamnose and 3-hydroxyalkanoyl-3-hydroxyalkanoate to -L-rhamnopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate, and the enzyme E.sub.4 being a rhamnosyltransferase II and being 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.

6. The cell according to claim 5, wherein E.sub.2, E.sub.3 and E.sub.4 are encoded by an rhlA gene, an rhlB gene and an rhlC gene, respectively, or are enzymes having a polypeptide sequence in which up to 25%, of the amino acid residues are modified with respect to the enzymes encoded by an rhl gene by deletion, insertion, substitution or a combination thereof and which still has at least 10%, of the enzymatic activity of the enzyme having the reference sequence of the enzymes encoded by an rhl gene.

7. The cell according to claim 1, wherein it has increased activities of the following enzyme combinations selected from E.sub.3, E.sub.3E.sub.4 and E.sub.2E.sub.3E.sub.4.

8. The cell according to claim 1, wherein the cell, compared to the wild type thereof, has an increased activity of at least one of the enzymes selected from the group consisting of at least one enzyme E.sub.5, a dTTP:-D-glucose-1-phosphate thymidylyltransferase of EC 2.7.7.24, at least one enzyme E.sub.6, a dTDP-glucose 4,6-hydrolyase of EC 4.2.1.46, at least one enzyme E.sub.7, a dTDP-4-dehydrorhamnose 3,5-epimerase of EC 5.1.3.13, at least one enzyme E.sub.5, a dTDP-4-dehydrorhamnose reductase of EC 1.1.1.133.

9. The cell according to claim 1, wherein the cell, compared to the wild type thereof, has an increased activity of at least one enzyme E.sub.13, which catalyses the export of a rhamnolipid from the cell into the surrounding medium, selected from the group consisting of enzymes having polypeptide sequence AAG04520.1, AJY02996.1, ZP_05590661.1, YP_439278.1, YP_440069.1, ZP_04969301.1, ZP_04520234.1, YP_335528.1, YP_001075859.1, YP_001061817.1, ZP_02487499.1, YP_337251.1, ZP_04897712.1, ZP_04810190.1, YP_990322.1, ZP_02476924.1, ZP_04899735.1, ZP_04893873.1, ZP_02365982.1, YP_001062909.1, YP_105611.1, ZP_03794061.1, ZP_03457011.1, ZP_02385401.1, ZP_02370552.1, YP_105236.1, ZP_04905097.1, YP_776387.1, YP_001811690.1, YP_004348730.1, YP_004348708.1, YP_371320.1, YP_623145.1, YP_001778810.1, YP_002234933.1, CCE52909.1, YP_002908248.1, ZP_04954557.1, ZP_04956038.1, ZP_02408950.1, ZP_02375897.1, ZP02389908.1, YP_439274.1, YP_001074762.1, YP_337247.1, YP_110559.1, ZP_02495927.1, YP_111360.1, YP_105608.1, ZP_02487826.1, ZP_02358947.1, YP_001078605.1, ZP_00438000.1, ZP_00440993.1, ZP_02477260.1, YP_371317.1, YP_001778807.1, ZP_02382843.1, YP_002234936.1, YP_623142.1, ZP_02907618.1, ZP_02891478.1, YP_776390.1, ZP_04943308.1, YP_001811693.1, ZP_02503985.1, YP_004362740.1, YP_002908245.1, YP_004348705.1, ZP_02408798.1, ZP_02417250.1, EGD05166.1, ZP_02458677.1, ZP_02465793.1, YP_001578240.1, ZP_04944344.1, YP_771932.1, ZP_02889166.1, YP_002232614.1, ZP_03574808.1, ZP_02906105.1, YP_001806764.1, YP_619912.1, YP_001117913.1, YP_106647.1, YP_001763368.1, ZP_02479535.1, ZP_02461743.1, YP_560998.1, YP_331651.1, ZP_04893070.1, YP_003606714.1, ZP_02503995.1, ZP_06840428.1, YP_104288.1, ZP_02487849.1, ZP_02353848.1, YP_367475.1, ZP_02377399.1, ZP_02372143.1, YP_001897562.1, ZP_02361066.1, YP_440582.1, ZP_03268453.1, AET90544.1, YP_003908738.1, YP_004230049.1, ZP_02885418.1 or ZP_02511831.1 or having a polypeptide sequence in which up to 25% of the amino acid residues are modified with respect to the particular aforementioned accession number by deletion, insertion, substitution or a combination thereof and which still has at least 10% of the enzymatic activity of the enzyme having the particular aforementioned accession number.

10. The method for producing rhamnolipids, comprising the method steps of I) contacting the cell according to claim 1, combining these measures as appropriate, with a medium containing a carbon source II) culturing the cell under conditions allowing the cell to make rhamnolipid from the carbon source and III) optionally isolating the rhamnolipids made.

11. A formulation comprising the rhamnolipids obtained using the method according to claim 10 wherein the formulation is selected from the group consisting of cosmetic formulations, dermatological formulations, crop protection formulations, and pharmaceutical formulations.

12. A surfactant concentrate comprising the rhamnolipids obtained using the method according to claim 10.

13. A cleaning agent comprising the rhamnolipids obtained using the method according to claim 10.

14. A care product comprising the rhamnolipids obtained using the method according to claim 10.

15. The cell according to claim 2, wherein E.sub.1 is a glucose 1-dehydrogenase of EC 1.1.5.2.

16. The cell according to claim 2, wherein the cell is selected from the group consisting of Burkholderia sp., Burkholderia thailandensis, Pseudomonas sp., Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas oleovorans, Pseudomonas stutzeri, Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas citronellolis, Pseudomonas resinovorans, Comamonas testosteroni, Aeromonas hydrophila, Cupriavidus necator, Alcaligenes latus and Ralstonia eutropha.

17. The cell according to claim 2, wherein the cell has been genetically modified such that it, compared to the wild type thereof, has an increased activity of at least one of the enzymes selected from the group E.sub.2, E.sub.3 and E.sub.4, the enzyme E.sub.2 being able to catalyse the conversion of 3-hydroxyalkanoyl-ACP via 3-hydroxyalkanoyl-3-hydroxyalkanoic acid-ACP to hydroxyalkanoyl-3-hydroxyalkanoic acid, the enzyme E.sub.3 being a rhamnosyltransferase I and being able to catalyse the conversion of dTDP-rhamnose and 3-hydroxyalkanoyl-3-hydroxyalkanoate to -L-rhamnopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate, and the enzyme E.sub.4 being a rhamnosyltransferase II and being 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.

18. The cell according to claim 5, wherein E.sub.2, E.sub.3 and E.sub.4 are encoded by an rhlA gene, an rhlB gene and an rhlC gene, respectively, or are enzymes having a polypeptide sequence in which up to 15% of the amino acid residues are modified with respect to the enzymes encoded by an rhl gene by deletion, insertion, substitution or a combination thereof and which still has at least 50% of the enzymatic activity of the enzyme having the reference sequence of the enzymes encoded by an rhl gene.

19. The cell according to claim 5, wherein E.sub.2, E.sub.3 and E.sub.4 are encoded by an rhlA gene, an rhlB gene and an rhlC gene, respectively, or are enzymes having a polypeptide sequence in which up to 5% of the amino acid residues are modified with respect to the enzymes encoded by an rhl gene by deletion, insertion, substitution or a combination thereof and which still has at least 80% of the enzymatic activity of the enzyme having the reference sequence of the enzymes encoded by an rhl gene.

20. The cell according to claim 5, wherein E.sub.2, E.sub.3 and E.sub.4 are encoded by an rhlA gene, an rhlB gene and an rhlC gene, respectively, or are enzymes having a polypeptide sequence in which up to 1% of the amino acid residues are modified with respect to the enzymes encoded by an rhl gene by deletion, insertion, substitution or a combination thereof and which still has at least 90% of the enzymatic activity of the enzyme having the reference sequence of the enzymes encoded by an rhl gene.

Description

[0106] The following figures are a component of the examples:

[0107] FIG. 1: Total yields of the strains PP-155 and PP-099 [gcd] in parallel experiments (runs #1-3) and comparison of the mean values

EXAMPLES

Example 1 (not Inventive): Use was Made of Strain BS-PP-155 (P. putida KT2440 upp+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}; clone 1)

Construction of the Strain BS-PP-155

[0108] For the heterologous expression of the genes rhlA, rhlB and rhlC and of the genes rmlB, rmlD, rmlA and rmlC, both from P. aeruginosa, the plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} was constructed. The plasmid contains, firstly, a synthetic operon consisting of the genes rhlA and rhlB (encoding a rhamnosyltransferase 1) and rhlC (encoding a rhamnosyltransferase 2) from P. aeruginosa DSM1128 (SEQ ID No 1) and, secondly, an operon consisting of the genes rmlB (encoding a dTDP-D-glucose 4,6-dehydratase), rmlD (encoding a dTDP-4-dehydrorhamnose reductase), rmlA (encoding a glucose-1-phosphate thymidylyltransferase) and rmlC (encoding a dTDP-4-dehydrorhamnose 3,5-epimerase) from P. aeruginosa DSM 19880 (SEQ ID No 2). The genes rhlABC are under the control of the rhamnose-inducible P.sub.Rha promoter; the rmlBDAC genes are under the control of the arabinose-inducible P.sub.BAD promoter. Situated downstream of the two operon structures is a terminator sequence (rrnB T1T2). The rmlBDAC genes were amplified from genomic DNA from P. aeruginosa DSM19880 and the synthetic rhlABC operon was obtained by gene synthesis. The P.sub.Rha promoter cassette (SEQ ID No 3) and P.sub.BAD promoter cassette (SEQ ID No 4) and also the terminator sequence (SEQ ID No 5) were amplified from genomic E. coli DNA. Whereas the rhlABC genes are required for the synthesis of di-rhamnolipids, the rmlBDAC genes are needed for the provision of activated dTDP-L-rhamnose.

[0109] The vector is based on the plasmid pACYC184 (New England Biolabs, Frankfurt am Main, Germany) and bears a p15A origin of replication for replication in E. coli and a pVS1 origin of replication for replication in P. putida. The pVS1 origin of replication was amplified from the Pseudomonas plasmid pVS1 (Itoh Y, Watson J M, Haas D, Leisinger T, Plasmid 1984, 11(3), 206-20). The vector part and the DNA fragments were cloned using a commercially available in vitro DNA assembly kit (e.g. NEBuilder HiFi DNA Assembly Cloning Kit in accordance with the manufacturer's instructions (NEB; Frankfurt am Main, Germany)). Chemically competent E. coli 10 beta cells (NEB, Frankfurt am Main, Germany) were transformed in a manner known to a person skilled in the art. The correct insertion of the target genes was checked by restriction analysis and the authenticity of the introduced homologous regions confirmed by DNA sequencing. The size of the resulting plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} (SEQ ID No 6) is 17 337 bp. Thereafter, the plasmid was introduced into P. putida KT2440 upp. This strain is used as the starting strain for the construction of markerless gene deletions in P. putida (Graf & Altenbuchner, 2011, Applied and Environmental Microbiology, Vol 77, No. 15, 5549-5552, DOI: 10.1128/AEM.05055-11). The method is based on a negative counter-selection system for P. putida, which utilizes the activity of uracil phosphoribosyltransferase and the sensitivity of P. putida towards the antimetabolite 5-fluorouracil. The deletion of the upp gene has no effect on rhamnolipid biosynthesis.

[0110] The transformation of P. putida KT2440 upp with the vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} was carried out as described in Iwasaki et al. (Iwasaki K, Uchiyama H, Yagi O, Kurabayashi, T, Ishizuka K, Takamura Y, Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA from each of 10 clones was isolated and analysed. A strain bearing the plasmid was called P. putida KT2440 upp pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0111] The biotechnological production of surfactant was carried out in the 8-fold parallel fermentation system DASGIP from Eppendorf.

[0112] For the fermentation, 1 L reactors were used. The pH probes were calibrated by means of a two-point calibration with measurement solutions of pH 4.0 and pH 7.0. The reactors were filled with 300 mL of water and autoclaved for 20 min at 121 C. in order to ensure sterility. The water was removed the next morning in a clean bench and replaced with sterile fermentation medium (autoclaved: 2.2 g/L (NH.sub.4).sub.2SO.sub.4, 0.02 g/L NaCl, 0.4 g/L MgSO.sub.47H.sub.2O, 0.04 g/L CaCl.sub.22H.sub.2O, sterilized separately: 2 g/L KH.sub.2PO.sub.4, 15 g/L glucose, 10 mL/L trace element solution M12 [sterile-filtered: 0.2 g/L ZnSO.sub.47H.sub.2O, 0.1 g/L MnCl.sub.14H.sub.2O, 1.5 g/L Na.sub.3 citrate2H.sub.2O, 0.1 g/L CuSO.sub.45H.sub.2O, 0.002 g/L NiCl.sub.26H.sub.2O, 0.003 g/L Na.sub.2MoO.sub.42H.sub.2O, 0.03 g/L H.sub.3BO.sub.3, 1 g/L FeSO.sub.47H.sub.2O]). Subsequently, the pO.sub.2 probes were calibrated by means of a one-point calibration (stirrer: 600 rpm/aeration: 10 sL/h air), and the feed, correcting agent and induction agent lines cleaned by means of cleaning-in-place. To this end, the hoses were flushed with 70% ethanol, then with 1 M NaOH, then with sterile demineralized water and finally filled with the particular media. Using 100 L from a cryoculture, the strain (P. putida KT2440 upp+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} was first grown overnight at 30 C. and 200 rpm for approximately 18 h in 25 mL of LB 1 medium (10 g/L casein hydrolysate, 5 g/L yeast extract, 1 g/L NaCl) in a 250 mL baffled flask containing 50 mg/L kanamycin. After measurement of the optical density of the culture, 50 mL of sterile seed medium (autoclaved: 4.4 g/L Na.sub.2HPO.sub.4*2H.sub.2O, 1.5 g/L KH.sub.2PO.sub.4, 1 g/L NH.sub.4C.sub.1, 10 g/L yeast extract, sterilized separately: 20 g/L glucose, 0.2 g/L MgSO.sub.4*7H.sub.2O, 0.006 g/L FeCl.sub.3, 0.015 g/L CaCl.sub.2, 1 mL/L trace element solution SL6 [sterile-filtered: 0.3 g/L H.sub.3BO.sub.3, 0.2 g/L CoCl.sub.26H.sub.2O, 0.1 g/L ZnSO.sub.47H.sub.2O, 0.03 g/L MnCl.sub.24H.sub.2O, 0.01 g/L CuCl.sub.22H.sub.2O, 0.03 g/L Na.sub.2MoO.sub.42H.sub.2O, 0.02 g/L NiCl.sub.26H.sub.2O]) in a 500 mL baffled flask were inoculated from the LB preculture using a start OD.sub.600 of 0.2 and incubated for approximately 7 h at 30 C. and 200 rpm. At an optical density of approximately OD.sub.600 8, the main culture was inoculated using a start OD.sub.600 of 0.7.

[0113] In order to inoculate the reactors using an optical density of 0.7, approximately 26 mL were filled in a 30 mL syringe and the reactors were inoculated by means of a needle across a septum.

[0114] The following standard program was used:

TABLE-US-00002 DO regulator pH regulator 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 N (Rotation) from to XO2 (gas mixture) from to Growth and 0% 40% Growth and 0% 100% biotransformation 500 1500 biotransformation 21% 21% rpm rpm F (gas flow rate) from to Growth and biotransformation 35% 100% 9 sL/h 72 sL/h Script Trigger activated 31% DO (1/60 h) Induction, rhamnose, arabinose 3 h after feed start Feed trigger 50% DO Feed rate 1.5 [mL/h]
pH was one-sidedly adjusted to pH 7.0 using ammonia (12.5%). During cultivation and biotransformation, the dissolved oxygen in the culture was kept constant at 30% via stirrer speed and aeration rate. The fermentation was carried out as a fed batch, where, from the feed start, the feeding with 2.5 g/Lh glucose by means of a 500 g/L glucose feed was triggered via a DO peak. The expression of the recombinantly introduced genes was induced 3 h after the feed start by the automatic addition of 0.2% (w/v) rhamnose and 0.2% (w/v) arabinose. The required amounts of induction sugar are based on the fermentation starting volume. For both sugars, 220 g/L stock solutions were used. The production of surfactant started from the time of induction. All online measurement data such as pH, DO, CTR, OTR, but also the flow rates and amount of the substrates such as ammonia solution for pH adjustment, the glucose feed or the inducer flow rates, were logged by the DASGIP fermentation system.

[0115] For fermentation analysis, a 10 mL syringe was used to draw and discard 2 mL as forerun from each vessel. This was followed once more by 6 mL for the actual analysis. Rhamnolipid content, glucose concentration and dry biomass were determined. The fermentation was ended after 65 h.

[0116] Rhamnolipid concentration was determined by means of HPLC. 100 L of the fermentation sample were admixed with 900 L of 70% (v/v) n-propanol in an Eppendorf tube and shaken at 30 Hz for 1 min in a Retsch mill. Thereafter, the sample was centrifuged at 13 000 rpm for 5 min and the supernatant transferred to a fresh Eppendorf tube. In the event of a further dilution being necessary, this was done using 55% n-propanol. All tubes were closed quickly in order to avoid evaporation. The samples were then transferred to HPLC vials and stored at 20 C. until measurement.

[0117] 1 ml of acetone was charged in a 2 ml reaction tube using a positive displacement pipette (Combitip) and the reaction tube immediately closed to minimize evaporation. This was followed by the addition of 1 ml of culture broth. After vortexing of the culture broth/acetone mixture, said mixture was centrifuged for 3 min at 13 000 rpm, and 800 l of the supernatant transferred to an HPLC vial.

[0118] An evaporative light scattering detector (Sedex LT-ELSD Model 85LT) was used for detection and quantification of rhamnolipids. The actual measurement was carried out using an Agilent Technologies 1200 Series (Santa Clara, Calif.) and a Zorbax SB-C8 Rapid Resolution column (4.6150 mm, 3.5 m, Agilent). The injection volume was 5 l and the method run time was 20 min. Aqueous 0.1% TFA (trifluoroacetic acid, solution A) and methanol (solution B) was used as mobile phase. The column temperature was 40 C. The ELSD (detector temperature 60 C.) and the DAD (diode array, 210 nm) served as detectors. The gradient used in the method was:

TABLE-US-00003 t Solution B % Flow rate [min] by volume [ml/min] 0.00 70% 1.00 15.00 100% 1.00 15.01 70% 1.00 20.00 70% 1.00

[0119] Dry biomass was determined by pipetting approximately 1 ml of the sample into a pre-weighed Eppendorf tube and determining the initial weight. Thereafter, the sample was admixed with approximately 1 mL of mains water, mixed, and centrifuged at 13 000 rpm for 5 min. The supernatant was discarded and the Eppendorf tube was coarsely wiped. 1 mL of mains water was added once more and resuspension was carried out at 30 Hz for 1 min in a Retsch mill. Thereafter, centrifugation was carried out at 13 000 rpm for 10 min, the supernatant was discarded, and the Eppendorf tube was then wiped dry, for example with cotton swabs, without biomass being taken from the Eppendorf tube at the same time. The samples were dried at 105 C. for 48 h and reweighed after cooling. A duplicate determination was carried out in each case.

[0120] Dry biomass calculation was then carried out in Excel:

[00001]$\mathrm{DBM}=\frac{\mathrm{Back}.Math..Math.\mathrm{weight}-\mathrm{Tare}.Math..Math.\mathrm{weight}}{\mathrm{Initial}.Math..Math.\mathrm{weight}-\mathrm{Tare}.Math..Math.\mathrm{weight}}.Math.1000\left[\frac{g}{L}\right]$

[0121] Glucose concentration was measured with the aid of a Roche Cedex Bio HT as specified by the manufacturer after centrifugation and sterile-filtration of a fermentation sample. 3 experiments are carried out, each in parallel to Example 2.

Example 2: Use was Made of the Strain BS-PP-099 (P. putida KT2440 upp gcd+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk})

[0122] Construction of a Vector for the Deletion of the Gcd Gene in Pseudomonas putida KT2440 upp

[0123] A vector for the deletion of the gcd gene from P. putida KT2440 upp, encoding a glucose dehydrogenase, was prepared by PCR amplification of approximately 680 bp upstream and downstream of the gcd gene.

[0124] The following primers were used for the amplification of the homologous regions upstream and downstream of the gcd gene:

TABLE-US-00004 PCR1:Regionupstreamofgcd 4*54 (SEQIDNo7) 5-GCCGCTTTGGTCCCGGGTTTCAAGCTCAGCGG-3 4*57 (SEQIDNo8) 5-AAGGCGCGATCGCGGGTTAGAAACTGCTCTGG-3 PCR2:Regiondownstreamofgcd 4*56 (SEQIDNo9) 5-CCGCGATCGCGCCTTGTGTCGCGTTTC-3 4*55 (SEQIDNo10) 5-GCTTGCATGCCTGCAATGCCGTAGGCTTTGACC-3

[0125] The following parameters were used for the PCR:

TABLE-US-00005 Denaturation: 98 C. 30 s Denaturation: 98 C. 10 s 30x Annealing: 62 C. 12 s 30x Elongation: 72 C. 22 s 30x Final elongation: 72 C. 5 min

[0126] For the amplification, the Phusion High-Fidelity Master Mix from NEB (Frankfurt am Main, Germany) was used according to the manufacturer's recommendations. 50 l of each of the PCR reactions were then resolved on a 1% TAE agarose gel. The PCR, the agarose gel electrophoresis, ethidium bromide staining of the DNA and determination of the PCR fragment sizes were performed in a manner known to a person skilled in the art. PCR fragments of the expected size (PCR 1, 679 bp (SEQ ID No 11); PCR 2, 682 bp, (SEQ ID No 12)) were amplified. The PCR products were purified using the QIAquick PCR Purification Kit from Qiagen as specified by the manufacturer. Using the NEBuilder HiFi DNA Assembly Cloning Kit in accordance with the manufacturer's instructions (NEB; Frankfurt am Main, Germany), the purified PCR products were cloned into a BamHI- and SbfI-cut pKOPp vector (SEQ ID No. 13). Chemically competent E. coli 10 beta cells (NEB, Frankfurt am Main, Germany) were transformed in a manner known to a person skilled in the art. The correct insertion of the target genes was checked by restriction analysis and the authenticity of the introduced homologous regions confirmed by DNA sequencing. The resultant knock-out vector was referred to as pKOPp_gcd (SEQ ID No. 14).

[0127] Construction of the strain BS-PP-099

[0128] The construction of the strain P. putida KT2440 upp gcd was carried out with the aid of the plasmid pKOPp_gcd and a method described in Graf et al., 2011 (Graf N, Altenbuchner J, Appl. Environ. Micorbiol., 2011, 77(15):5549; DOI: 10.1128/AEM.05055-11). The DNA sequence after the deletion of gcd is described in SEQ ID No. 15. The transformation of P. putida KT2440 upp gcd with the vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} was carried out as described in Iwasaki et al. (Iwasaki K, Uchiyama H, Yagi O, Kurabayashi, T, Ishizuka K, Takamura Y, Biosci. Biotech. Biochem. 1994. 58(5):851-854). Thereafter, the cells were plated out on LB agar plates supplemented with kanamycin (50 g/ml). The plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}(SEQ ID No. 6) has already been described in Example 1. The plasmid DNA from each of 10 clones was isolated and analysed by means of restriction analysis. A strain bearing the plasmid was called P. putida KT2440 upp gcd pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0129] Technical realization was carried out as described in Example 1.

[0130] 3 experiments were carried out, each in parallel to Example 1.

[0131] What was evaluated was the total yield, determined as the sum of biomass plus rhamnolipid made divided by glucose used and consumed.

[0132] As can be seen in FIG. 1, a P. putida strain containing not only rhlA, rhlB and rhlC but also the native gene for glucose dehydrogenase achieves a total yield of 0.258 [(g biomass+g RL)/g glucose] on average. By comparison, the strain with deletion of the gcd gene achieves a total yield of 0.283 [(g biomass+g RL)/g glucose] on average.

Example 3: Construction of the Strain Pseudomonas aeruginosa PAO1 gcd

[0133] The construction of the strain P. aeruginosa PAO1 gcd is carried out with the aid of a method described in Choi & Schweizer (Choi & Schweizer, MBC Microbiology, 2005 5:30, DOI: 10.1186/1471-2180-5-30). The method allows the production of markerless gene deletions in P. aeruginosa and is based on a negative counter-selection system (sacB) using homologous recombination and an Flp-FRT recombination system for the removal of the selection marker. The DNA sequence after the deletion of gcd is described in SEQ ID No. 16. Technical realization is carried out as described in Example 1 with the exception that all cultivation steps are carried out at 37 C.

[0134] 3 experiments are carried out, each in parallel to the strain P. aeruginosa PAO1.

[0135] What is evaluated is the total yield, determined as the sum of biomass plus rhamnolipid made divided by glucose used and consumed. The P. aeruginosa strain still containing the native gene for glucose dehydrogenase achieves on average a lower total yield [(g biomass+g RL)/g glucose] compared to the strain having the deletion of the gcd gene.

Example 4 (not Inventive): Use is Made of the Strain P. putida KT2440 upp+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlAB_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}; clone 1)

Construction of the Strain.

[0136] For the heterologous expression of the genes rhlA and rhlB and of the genes rmlB, rmlD, rmlA and rmlC, both from P. aeruginosa, the plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlAB_Pa]{Talk}[araC_Ec]{ParaBAD}[rmBDAC_Pa]{Talk} is constructed. The plasmid contains, firstly, an operon consisting of the genes rhlA and rhlB (encoding a rhamnosyltransferase 1) from P. aeruginosa DSM1128 (SEQ ID No 17) and, secondly an operon consisting of the genes rmlB (encoding a dTDP-D-glucose 4,6-dehydratase), rmlD (encoding a dTDP-4-dehydrorhamnose reductase), rmlA (encoding a glucose-1-phosphate thymidylyltransferase) and rmlC (encoding a dTDP-4-dehydrorhamnose 3,5-epimerase) from P. aeruginosa DSM 19880 (SEQ ID No 2). The rhlAB genes are under control of the rhamnose-inducible P.sub.Rha promotor; the rmlBDAC genes are under the control of the arabinose-inducible P.sub.BAD promotor. Situated downstream of the two operon structures is a terminator sequence (rrnB T1T2). Whereas the rhlAB genes are required for the synthesis of monorhamnolipids, the rmlBDAC genes are needed for the provision of activated dTDP-L-rhamnose.

[0137] The vector is based on the plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} (SEQ ID No 6) (see Example 1). To remove the rhlC gene, the vector was cut with PacI and NsiI. In addition to rhlC, a section upstream and downstream of rhlC is also eliminated by the restriction. These missing regions are amplified by PCR. The template used is the plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} (SEQ ID No 6) (see Example 1). The vector part and both PCR fragments are then cloned using a commercially available in vitro DNA assembly kit (e.g. NEBuilder HiFi DNA Assembly Cloning Kit in accordance with the manufacturer's instructions (NEB; Frankfurt/Main, Germany). Chemically competent E. coli 10 beta cells (NEB, Frankfurt/Main, Germany) are transformed in a manner known to a person skilled in the art. The correct insertion of the target genes is checked by restriction analysis and the authenticity of the introduced homologous regions confirmed by DNA sequencing. The size of the resulting plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlAB_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} (SEQ ID No 18) is 16 359 bp.

[0138] Thereafter, the plasmid is introduced into P. putida KT2440 upp. This strain is used as the starting strain for the construction of markerless gene deletions in P. putida (Graf & Altenbuchner, 2011, Applied and Environmental Microbiology, Vol 77, No. 15, 5549-5552, DOI:10.1128/AEM.05055-11). The method is based on a negative counter-selection system for P. putida, which utilizes the activity of uracil phosphoribosyltransferase and the sensitivity of P. putida towards the antimetabolite 5-fluorouracil. The deletion of the upp gene has no effect on rhamnolipid biosynthesis.

[0139] The transformation of P. putida KT2440 upp with the vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlAB_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} is carried out as described in Iwasaki et al. (Iwasaki K, Uchiyama H, Yagi O, Kurabayashi, T, Ishizuka K, Takamura Y, Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA from each of 10 clones is isolated and analysed. A strain bearing the plasmid is called P. putida KT2440 upp pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlAB_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

Example 5 (Inventive): Use is Made of the Strain P. putida KT2440 upp gcd+pACYCATh5-{PrhaSR}[rhaSR_Ec]{rhaBAD}[rhlAB_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk})

[0140] Construction of the Strain P. putida KT2440 upp gcd+pACYCATh5-{PrhaSR}[rhaSR_Ec]{rhaBAD} rhlAB_Pa {Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk})

[0141] To construct the strain P. putida KT2440 upp gcd+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlAB_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}), the plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlAB_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} is introduced into the strain P. putida KT2440 upp gcd. The construction of the strain has already been described in Example 2 and the plasmid construction in Example 4.

[0142] The transformation is carried out as described in Iwasaki et al. (Iwasaki K, Uchiyama H, Yagi O, Kurabayashi, T, Ishizuka K, Takamura Y, Biosci. Biotech. Biochem. 1994. 58(5):851-854). Thereafter, the cells are plated out on LB-agar plates supplemented with kanamycin (50 g/ml). The plasmid DNA from each of 10 clones is isolated and analysed by means of restriction analysis. A strain bearing the plasmid is called P. putida KT2440 upp gcd pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}. Technical realization is carried out as described in Example 1.

3 experiments are carried out, each in parallel to Example 4.

[0143] What is evaluated is the total yield, determined as the sum of biomass plus rhamnolipid made divided by glucose used and consumed.

[0144] The P. putida strain, which besides rhlA and rhlB contains the native gene for glucose dehydrogenase, achieves on average a lower total yield [(g biomass+g RL)/g glucose] compared to the strain with the deletion of the gcd gene.