CELLS AND METHOD FOR PRODUCING RHAMNOLIPIDS USING ALTERNATIVE GLUCOSE TRANSPORTERS

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 an ABC glucose transporter and, compared to the wild type thereof, an increased activity of at least one non-ABC glucose transporter and to a method for producing rhamnolipids using the cells according to the invention.

Claims

1. A rhamnolipid-making cell, wherein the rhamnolipid-making cell is genetically modified such that the rhamnolipid-making cell has a decreased activity, compared to the wild type thereof, of an ABC glucose transporter and an increased activity, compared to the wild type thereof, of at least one non-ABC glucose transporter.

2. The rhamnolipid-making cell according to claim 1, wherein the non-ABC glucose transporter is selected from the group consisting of phosphoenolpyruvate phosphotransferase systems of EC 2.7.3.9, galactose permeases, glucose facilitators, myo-inositol transporters, glucose permeases, and glucose/galactose transporters.

3. The rhamnolipid-making cell according to claim 1, wherein the non-ABC glucose transporter is selected from the group consisting of enzymes encoded by a galP, glf, iolT1, glcP, gluP, SemiSWEET or glcU gene and PTS systems consisting of the components enzyme I, HPr, enzyme IIA, enzyme IIB and enzyme IIC, it being possible for enzymes IIA, IIB and IIC to be present as fusion proteins, or enzymes having a polypeptide sequence in which up to 25% of the amino acid residues of the galP, glf, iolT1, glcP, gluP, SemiSWEET or glcU gene-encoded enzymes and PTS systems are modified by deletion, insertion, substitution or a combination thereof and which still has at least 10% of the enzymatic activity of the galP, glf, iolT1, glcP, gluP, SemiSWEET or glcU gene-encoded enzyme and PTS system.

4. The rhamnolipid-making cell according to claim 1, wherein the rhamnolipid-making 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 testosterone, Aeromonas hydrophila, Cupriavidus necator, Alcaligenes latus and Ralstonia eutropha.

5. The rhamnolipid-making cell according to claim 1, wherein the rhamnolipid-making cell has been genetically modified such that the rhamnolipid-making cell, compared to the wild type thereof, has an increased activity of at least one of the enzymes selected from the group E.sub.1, E.sub.2 and E.sub.3, the enzyme E.sub.1 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.2 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.3 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 rhamnolipid-making cell according to claim 5, wherein E.sub.1, E.sub.2 and E.sub.3 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 rhamnolipid-making cell according to at claim 1, wherein the rhamnolipid-making cell has been genetically modified such that the rhamnolipid-making cell, compared to the wild type thereof, has a decreased activity of at least one enzyme E.sub.4, which catalyses the conversion of D-glucose and quinone to D-glucono-1,5-lactone and quinol.

8. The rhamnolipid-making cell according to claim 7, wherein E.sub.4 is a glucose 1-dehydrogenase of EC 1.1.5.2.

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

10. A method for making a product selected from the group consisting of cosmetic formulation, dermatological formulation, pharmaceutical formulation, crop-protection formulation, care product, cleaning agent, and surfactant concentrate, the method comprising the method according to claim 9.

11. The rhamnolipid-making cell according to claim 2, wherein the non-ABC glucose transporter is selected from the group consisting of enzymes encoded by a galP, glf, iolT1, glcP, gluP, SemiSWEET or glcU gene and PTS systems consisting of the components enzyme I, HPr, enzyme IIA, enzyme JIB and enzyme IIC, it being possible for enzymes IIA, IIB and IIC to be present as fusion proteins, or enzymes having a polypeptide sequence in which up to 25% of the amino acid residues of the galP, glf, iolT1, glcP, gluP, SemiSWEET or glcU gene-encoded enzymes and PTS systems are modified by deletion, insertion, substitution or a combination thereof and which still has at least 10% of the enzymatic activity of the galP, glf, iolT1, glcP, gluP, SemiSWEET or glcU gene-encoded enzyme and PTS system.

12. The rhamnolipid-making cell according to claim 2, wherein the rhamnolipid-making 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.

13. The rhamnolipid-making cell according to claim 3, wherein the rhamnolipid-making 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.

14. The rhamnolipid-making cell according to claim 2, wherein the rhamnolipid-making cell has been genetically modified such that the rhamnolipid-making cell, compared to the wild type thereof, has an increased activity of at least one of the enzymes selected from the group E.sub.1, E.sub.2 and E.sub.3, the enzyme E.sub.1 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.2 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.3 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.

15. The rhamnolipid-making cell according to claim 3, wherein the rhamnolipid-making cell has been genetically modified such that the rhamnolipid-making cell, compared to the wild type thereof, has an increased activity of at least one of the enzymes selected from the group E.sub.1, E.sub.2 and E.sub.3, the enzyme E.sub.1 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.2 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.3 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.

16. The rhamnolipid-making cell according to claim 14, wherein E.sub.1, E.sub.2 and E.sub.3 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.

17. The rhamnolipid-making cell according to claim 15, wherein E.sub.1, E.sub.2 and E.sub.3 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.

18. The rhamnolipid-making cell according to claim 2, wherein the rhamnolipid-making cell has been genetically modified such that it, compared to the wild type thereof, has a decreased activity of at least one enzyme E.sub.4, which catalyses the conversion of D-glucose and quinone to D-glucono-1,5-lactone and quinol.

19. The rhamnolipid-making cell according to claim 3, wherein the rhamnolipid-making cell has been genetically modified such that it, compared to the wild type thereof, has a decreased activity of at least one enzyme E.sub.4, which catalyses the conversion of D-glucose and quinone to D-glucono-1,5-lactone and quinol.

20. The rhamnolipid-making cell according to claim 18, wherein E.sub.4 is a glucose 1-dehydrogenase of EC 1.1.5.2.

Description

EXAMPLES

Example 1 (Not Inventive)

[0134] Use was made of strain P. putida KT2440 upp+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Tal k}

[0135] Construction of the strain P. putida KT2440 upp+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Tal k}

[0136] 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 PBAD 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.

[0137] 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.

[0138] 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.

[0139] 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}.

[0140] The biotechnological production of surfactant was carried out in the 8-fold parallel fermentation system DASGIP from Eppendorf.

[0141] 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.24H.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.2probes 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.

[0142] 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]{Tal k} was first grown overnight at 30 C. and 200 rpm for approximately 18 h in 25 mL of LB1 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.4Cl, 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.

[0143] 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.

[0144] 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 XO2 (gas N (Rotation) from to 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 31% DO (1/60 h) activated Induction, 3 h after feed rhamnose, start arabinose Feed trigger 50% DO Feed rate 1.5 [mL/h]

[0145] 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.

[0146] 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 being removed from the reactor for the actual analysis. Rhamnolipid content was determined. The fermentation was ended after 65 h.

[0147] 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.

[0148] 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.

[0149] 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 Solution B % Flow rate t [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

[0150] 3 experiments were carried out, each in parallel to Example 2.

[0151] Determined total RL concentration after 65 h: 34 g/L.

[0152] Calculated space-time yield: 0.53 g/L*h

Example 2 (Inventive)

[0153] Use was made of the strain P. putida KT2440 upp [PP_1016-1018]::galP_Ec +pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Tal k})

[0154] Construction of a vector for the integration of the galP gene in Pseudomonas putida KT2440 upp

[0155] A vector for the integration of the galP gene from E. coli K12, encoding a galactose-H.sup.+ symporter GalP, is prepared by PCR amplification of the gene. The template used is genomic DNA of E. coli K12 W3110. It is intended that the galP gene replace, in P. putida KT2440 upp, the genes PP_1016-PP_1018, encoding an ABC transporter permease, an ABC transporter binding protein and an ABC transporter ATP-binding protein. To this end, approximately 680 bp upstream and downstream of the genes PP_1016-PP_1018 are amplified by means of PCR.

[0156] The following primers were used for the amplification of the galP gene:

TABLE-US-00004 PCR1:galP O.BF_FM_1*07 (SEQIDNo7) 5-CCAATACATGCCTGACGCTAAAAAACA-3 O.BF_FM_1*10 (SEQIDNo8) 5-TAGACGAGTTAATCGTGAGCGCCTATTT-3

[0157] The following primers were used for the amplification of the homologous regions upstream and downstream of the PP_1016-PP_1018 genes:

TABLE-US-00005 PCR2:RegionupstreamofPP_1016 O.BF_FM_1*03 (SEQIDNo9) 5-GCCGCTTTGGTCCCGGCCGCCAAGGTCATTAAC-3 O.BF_FM_1*08 (SEQIDNo10) 5-TCAGGCATGTATTGGATCCCGAGGTAGT-3 PCR3:RegiondownstreamofPP_1018 O.BF_FM_1*02 (SEQIDNo11) 5-GCTTGCATGCCTGCAGGCGTGATGTTGTACTTC-3 O.BF_FM_1*09 (SEQIDNo12) 5-CGATTAACTCGTCTACACCATCAATAA-3

[0158] The following parameters were used for the PCR:

TABLE-US-00006 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

[0159] 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 (1410 bp, SEQ ID No 13); PCR 2, 675 bp (SEQ ID No 14); PCR 3, 697 bp (SEQ ID No 15)) 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. 16). 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 pKO_PP_1016-PP_1018::galP (SEQ ID No. 17).

[0160] Construction of the strain P. putida KT2440 upp [PP_1016-1018]::galP_Ec

[0161] The construction of the strain P. putida KT2440 upp [PP_1016-1018]::galP_Ec was carried out with the aid of the plasmid pKO_PP_1016-PP_1018::galP 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 replacement of the genes PP_1016-PP_1018 with galP is described in SEQ ID No. 18. The transformation of P. putida KT2440 upp [PP_1016-1018]::galP_Ec with the vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Tal k} 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 [PP_1016-1018]::galP_Ec pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0162] Technical realization was carried out as described in Example 1.

[0163] 3 experiments were carried out, each in parallel to Example 1.

[0164] Determined total RL concentration after 64 h: 44.5 g/L.

[0165] Calculated space-time yield: 0.70 g/L*h

Example 3 (Inventive)

[0166] Use is made of strain P. putida KT2440 upp [PP_1016-1018]::glf Zm (co Pp)+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk})

[0167] Construction of a vector for integrating the glf gene in Pseudomonas putida KT2440 upp

[0168] A vector for the integration of the glf gene from Zymomonas mobilis, encoding a glucose facilitator Glf, is prepared by PCR amplification of the gene codon-optimized for P. putida KT2440. A synthetic DNA fragment is used as template. It is intended that the glf gene replace, in P. putida KT2440 upp, the genes PP_1016-PP_1018, encoding an ABC transporter permease, an ABC transporter binding protein and an ABC transporter ATP-binding protein. To this end, approximately 690 bp upstream and downstream of the genes PP_1016PP_1018 are amplified by means of PCR.

[0169] The following primers are used for the amplification of the glf gene:

TABLE-US-00007 PCR4:glf MW_18_02 (SEQIDNo19) 5-TACCTCGGGATCCAATACATGTCCAGCGAGTCGTCCCAG-3 MW_18_03 (SEQIDNo20) 5-TTACTTCTGCGAGCGCCACATC-3

[0170] The following primers are used for the amplification of the homologous regions upstream and downstream of the PP_1016-PP_1018 genes:

TABLE-US-00008 PCR5:RegionupstreamofPP_1016 O.BF_FM_1*03 (SEQIDNo9) 5-GCCGCTTTGGTCCCGGCCGCCAAGGTCATTAAC-3 MW_18_01 (SEQIDNo21) 5-CATGTATTGGATCCCGAGGTAG-3 PCR6:RegiondownstreamofPP_1018 MW_18_04 (SEQIDNo22) 5-GCTCGCAGAAGTAACAATACCTCGTCTACACCATCAATAAGAAAAA G-3 O.BF_FM_1*02 (SEQIDNo11) 5-GCTTGCATGCCTGCAGGCGTGATGTTGTACTTC-3

[0171] The following parameters are used for the PCR:

TABLE-US-00009 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

[0172] For the amplification, the Phusion High-Fidelity Master Mix from NEB (Frankfurt am Main, Germany) is used according to the manufacturer's recommendations. 50 l of each of the PCR reactions are 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 are performed in a manner known to a person skilled in the art. PCR fragments of the expected size (PCR 4 (SEQ ID No 23), 1440 bp; PCR 5 (SEQ ID No 24), 670 bp; PCR 6 (SEQ ID No 25), 710 bp) are amplified. The PCR products are 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 are cloned into a BamHI- and SbfI-cut pKOPp vector (SEQ ID No. 16). Chemically competent E. coli 10 beta cells (NEB, Frankfurt am 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 resultant knock-out vector is referred to as pKO_PP_1016-PP_1018::glf_Zm (co_Pp) (SEQ ID No. 26).

[0173] Construction of the strain P. putida KT2440 upp [PP_1016-1018]::glf_Zm (co_Pp)

[0174] The construction of the strain P. putida KT2440 upp [PP_1016-1018]::glf_Zm (co_Pp) is carried out with the aid of the plasmid pKO_PP_1016-PP_1018::glf_Zm (co_Pp) 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 replacement of the genes PP_1016-PP_1018 with glf is described in SEQ ID No. 27. The transformation of P. putida KT2440 upp [PP_1016-1018]::glf_Zm (co_Pp) with the vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Tal k} 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 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 is isolated and analysed by means of restriction analysis. A strain bearing the plasmid is called P. putida KT2440 upp [PP_1016-1018]::glf_Zm (co_Pp) pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0175] Technical realization was carried out as described in Example 1.

[0176] 3 experiments are carried out, each in parallel to Example 1.

[0177] A significantly higher total RL concentration after 64 h and a significantly higher calculated space-time yield is observed compared to Example 1.

Example 4 (Inventive)

[0178] Use is made of the strain P. putida KT2440 upp [PP_1016-1018]::[ptsH_Ec ptsI_Ec crr_Ec ptsG_Ec]+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk})

[0179] Construction of a vector for the integration of the pts genes in Pseudomonas putida KT2440 upp

[0180] A vector for the integration of the pts genes from E. coli K12, encoding a phosphoenolpyruvate phosphotransferase system PEP-PTS, is prepared by PCR amplification of the genes ptsH, ptsI, crr and ptsG. ptsH encodes the phosphocarrier protein HPr, ptsI encodes the PTS enzyme I, crr encodes the enzyme IIAGlc and ptsG encodes the glucose-specific PTS enzyme IIBC. The template used is genomic DNA of E. coli K12 W3110. It is intended that the PTS system replace, in P. putida KT2440 upp, the genes PP _1016-PP_1018, encoding an ABC transporter permease, an ABC transporter binding protein and an ABC transporter ATP-binding protein. To this end, approximately 690 bp upstream and downstream of the genes PP_1016-PP_1018 are amplified by means of PCR.

[0181] The following primers are used for the amplification of the PTS genes:

TABLE-US-00010 PCR7:OperonptsH/ptsI/crr O.BF_FM_1*23 (SEQIDNo28) 5-TCCAATACATGTTCCAGCAAGAAGTTACC-3 O.BF_FM_1*22 (SEQIDNo29) 5-CCTGAGTTTACTTCTTGATGCGGATAACC-3 PCR8:ptsG O.BF_FM_1*21 (SEQIDNo30) 5-AGAAGTAAACTCAGGAGCACTCTCAATT-3 O.BF_FM_1*26 (SEQIDNo31) 5-AGACGAGTTAGTGGTTACGGATGTACTC-3

[0182] The following primers are used for the amplification of the homologous regions upstream and downstream of the PP_1016-PP_1018 genes:

TABLE-US-00011 PCR9:RegionupstreamofPP_1016 O.BF_FM_1*03 (SEQIDNo9) 5-GCCGCTTTGGTCCCGGCCGCCAAGGTCATTAAC-3 O.BF_FM_1*24 (SEQIDNo32) 5-GGAACATGTATTGGATCCCGAGGTAGTG-3 PCR10:RegiondownstreamofPP_1018 O.BF_FM_1*25 (SEQIDNo33) 5-ACCACTAACTCGTCTACACCATCAATAAG-3 O.BF_FM_1*02 (SEQIDNo11) 5-GCTTGCATGCCTGCAGGCGTGATGTTGTACTTC-3

[0183] The following parameters are used for the PCR:

TABLE-US-00012 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

[0184] For the amplification, the Phusion High-Fidelity Master Mix from NEB (Frankfurt am Main, Germany) is used according to the manufacturer's recommendations. 50 l of each of the PCR reactions are 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 are performed in a manner known to a person skilled in the art. PCR fragments of the expected size (PCR 7 (SEQ ID No 34), 2595 bp; PCR 8 (SEQ ID No 35), 1469 bp; PCR 9 (SEQ ID No 36), 674 bp; PCR 10 (SEQ ID No 37), 698 bp) are amplified. The PCR products are 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 are cloned into a BamHI- and SbfI-cut pKOPp vector (SEQ ID No. 16). Chemically competent E. coli 10 beta cells (NEB, Frankfurt am 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 resultant knock-out vector is referred to as pKO_PP_1016-PP_1018::ptsH_Ec ptsI_Ec crr_Ec ptsG_Ec) (SEQ ID No 38).

[0185] Construction of the strain P. putida KT2440 upp [PP_1016-1018]::ptsH_Ec ptsI_Ec crr_Ec ptsG_Ec

[0186] The construction of the strain P. putida KT2440 upp [PP_1016-1018]::ptsH_Ec ptsI_Ec crr_Ec ptsG_Ec is carried out with the aid of the plasmid pKO_PP_1016-PP_1018::ptsH_Ec ptsI_Ec crr_Ec ptsG_Ec and a method described in Graf et al., 2011 (Graf N, Altenbuchner J, Appl. Environ. Microbiol., 2011, 77(15):5549; DOI: 10.1128/AEM.05055-11). The DNA sequence after replacement of the genes PP_1016-PP_1018 with ptsH_Ec ptsI_Ec crr_Ec ptsG_Ec is described in SEQ ID No 39. The transformation of P. putida KT2440 upp A[PP_1016-1018]::ptsH_Ec ptsI_Ec crr_Ec ptsG_Ec with the vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_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). Thereafter, the cells are 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 is isolated and analysed by means of restriction analysis. A strain bearing the plasmid is called P. putida KT2440 upp [PP_1016-1018]::[ptsH_Ec ptsI_Ec crr_Ec ptsG_Ec]pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0187] Technical realization is carried out as described in Example 1.

[0188] 3 experiments are carried out, each in parallel to Example 1.

[0189] A significantly higher total RL concentration after 64 h and a significantly higher calculated space-time yield is observed compared to Example 1.

Example 5 (Inventive)

[0190] Use is made of the strain P. putida KT2440 upp [PP_1016-1018]::gluP_Bab+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk})

[0191] Construction of a vector for the integration of the gluP gene in Pseudomonas putida KT2440 upp

[0192] A vector for the integration of the gluP gene from Brucella abortus, encoding a glucose/galactose transporter GluP, is prepared by PCR amplification of the gene. The template used is a synthetic DNA fragment. It is intended that the gluP gene replace, in P. putida KT2440 upp, the genes PP_1016-PP_1018, encoding an ABC transporter permease, an ABC transporter binding protein and an ABC transporter ATP-binding protein. To this end, approximately 680 bp upstream and downstream of the genes PP_1016-PP_1018 are amplified by means of PCR.

[0193] The following primers are used for the amplification of the gluP gene:

TABLE-US-00013 PCR11:gluP MW_18_05 (SEQIDNo40) 5-TACCTCGGGATCCAATACATGGCAACTTCCATCCCAAC-3 MW_18_06 (SEQIDNo41) 5-TCAGCTTTTGCTGCCGATGAG-3

[0194] The following primers are used for the amplification of the homologous regions upstream and downstream of the PP_1016-PP_1018 genes:

TABLE-US-00014 PCR5:RegionupstreamofPP_1016 O.BF_FM_1*03 (SEQIDNo9) 5-GCCGCTTTGGTCCCGGCCGCCAAGGTCATTAAC-3 MW_18_01 (SEQIDNo21) 5-CATGTATTGGATCCCGAGGTAG-3 PCR12:RegiondownstreamofPP_1018 MW_18_07 (SEQIDNo42) 5-TCATCGGCAGCAAAAGCTGACTCGTCTACACCATCAATAAGAAAAA G-3 O.BF_FM_1*02 (SEQIDNo11) 5-GCTTGCATGCCTGCAGGCGTGATGTTGTACTTC-3

[0195] The following parameters are used for the PCR:

TABLE-US-00015 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

[0196] For the amplification, the Phusion High-Fidelity Master Mix from NEB (Frankfurt am Main, Germany) is used according to the manufacturer's recommendations. 50 l of each of the PCR reactions are 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 are performed in a manner known to a person skilled in the art. PCR fragments of the expected size (PCR 11 (SEQ ID No 43), 1257 bp; PCR 5 (SEQ ID No 24), 670 bp; PCR 12 (SEQ ID No 44), 710 bp) are amplified. The PCR products are 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 are cloned into a BamHI- and SbfI-cut pKOPp vector (SEQ ID No. 16). Chemically competent E. coli 10 beta cells (NEB, Frankfurt am 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 resultant knock-out vector is referred to as pKO_PP_1016-PP_1018::gluP_Bab (SEQ ID No 45).

[0197] Construction of the strain P. putida KT2440 upp [PP_1016-1018]::gluP_Bab

[0198] The construction of the strain P. putida KT2440 upp [PP_1016-1018]::gluP_Bab is carried out with the aid of the plasmid pKO_PP_1016-PP_1018::gluP_Bab and a method described in Graf et al., 2011 (Graf N, Altenbuchner J, Appl. Environ. Microbiol., 2011, 77(15):5549; DOI: 10.1128/AEM.05055-11). The DNA sequence after replacement of the genes PP_1016-PP_1018 with gluP is described in SEQ ID No 46. The transformation of P. putida KT2440 upp [PP_1016-1018]::gluP_Bab with the vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_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). Thereafter, the cells are 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 is isolated and analysed by means of restriction analysis. A strain bearing the plasmid is called P. putida KT2440 upp [PP_1016-1018]::gluP_Bab pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0199] Technical realization is carried out as described in Example 1.

[0200] 3 experiments are carried out, each in parallel to Example 1.

[0201] A significantly higher total RL concentration after 64 h and a significantly higher calculated space-time yield is observed compared to Example 1.

Example 6 (Inventive)

[0202] Use is made of the strain P. putida KT2440 upp [PP_1016-1018]::iolT1_Cg (co Pp)+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk})

[0203] Construction of a vector for the integration of the iolT1 gene in Pseudomonas putida KT2440 upp

[0204] A vector for the integration of the iolT 1 gene from Corynebacterium glutamicum ATCC 13032, encoding a myoinositol facilitator IolT1, is prepared by PCR amplification of the gene codon-optimized for P. putida KT2440. A synthetic DNA fragment is used as template. It is intended that the iolT1 gene replace, in P. putida KT2440 upp, the genes PP_1016-PP_1018, encoding an ABC transporter permease, an ABC transporter binding protein and an ABC transporter ATP-binding protein. To this end, approximately 680 bp upstream and downstream of the genes PP_1016-PP_1018 are amplified by means of PCR.

[0205] The following primers are used for the amplification of the iolT1 gene:

TABLE-US-00016 PCR13:iolT1 MW_18_08 (SEQIDNo47) 5-TACCTCGGGATCCAATACATGGCAAGCACCTTTATCCAGGCCGACA G-3 MW_18_09 (SEQIDNo48) 5-TCAATGGACCTTGCCCTTGCGAATG-3

[0206] The following primers are used for the amplification of the homologous regions upstream and downstream of the PP_1016-PP_1018 genes:

TABLE-US-00017 PCR5:RegionupstreamofPP_1016 O.BF_FM_1*03 (SEQIDNo9) 5-GCCGCTTTGGTCCCGGCCGCCAAGGTCATTAAC-3 MW_18_01 (SEQIDNo21) 5-CATGTATTGGATCCCGAGGTAG-3 PCR14:RegiondownstreamofPP_1018 MW_18_10 (SEQIDNo49) 5-GCAAGGGCAAGGTCCATTGACTCGTCTACACCATCAATAAGAAAAA G-3 O.BF_FM_1*02 (SEQIDNo11) 5-GCTTGCATGCCTGCAGGCGTGATGTTGTACTTC-3

[0207] The following parameters are used for the PCR:

TABLE-US-00018 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

[0208] For the amplification, the Phusion High-Fidelity Master Mix from NEB (Frankfurt am Main, Germany) is used according to the manufacturer's recommendations. 50 l of each of the PCR reactions are 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 are performed in a manner known to a person skilled in the art. PCR fragments of the expected size (PCR 13 (SEQ ID No 50), 1494 bp; PCR 5 (SEQ ID No 24), 670 bp; PCR 14 (SEQ ID No 51), 710 bp) are amplified. The PCR products are 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 are cloned into a BamHI- and SbfI-cut pKOPp vector (SEQ ID No. 16). Chemically competent E. coli 10 beta cells (NEB, Frankfurt am 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 resultant knock-out vector is referred to as pKO_PP_1016-PP_1018::iolT1_Cg (co_Pp) (SEQ ID No 52).

[0209] Construction of the strain P. putida KT2440 upp [PP_1016-1018]::iolT1_Cg (co_Pp)

[0210] The construction of the strain P. putida KT2440 upp [PP_1016-1018]::iolT1_Cg (co_Pp) is carried out with the aid of the plasmid pKO_PP_1016-PP_1018::iolT1_Cg (co_Pp) and a method described in Graf et al., 2011 (Graf N, Altenbuchner J, Appl. Environ. Microbiol., 2011, 77(15):5549; DOI: 10.1128/AEM.05055-11). The DNA sequence after replacement of the genes PP_1016-PP_1018 with iolT1_Cg (co_Pp) is described in SEQ ID No 53. The transformation of P. putida KT2440 upp [PP_1016-1018]::iolT1_Cg (co_Pp) with the vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_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). Thereafter, the cells are 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 is isolated and analysed by means of restriction analysis. A strain bearing the plasmid is called P. putida KT2440 upp [PP_1016-1018]::iolT1_Cg (co_Pp) pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0211] Technical realization is carried out as described in Example 1.

[0212] 3 experiments are carried out, each in parallel to Example 1.

[0213] A significantly higher total RL concentration after 64 h and a significantly higher calculated space-time yield is observed compared to Example 1.

Example 7 (Inventive)

[0214] Use is made of the strain P. putida KT2440 upp [PP_1016-1018]::glcP_Ms+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk})

[0215] Construction of a vector for the integration of the glcP gene in Pseudomonas putida KT2440 upp

[0216] A vector for the integration of the glcP gene from Mycobacterium smegmatis, encoding an arabinose-proton symporter GlcP, is prepared by PCR amplification of the gene. The template used is a synthetic DNA fragment. It is intended that the glcP gene replace, in P. putida KT2440 upp, the genes PP_1016-PP_1018, encoding an ABC transporter permease, an ABC transporter binding protein and an ABC transporter ATP-binding protein. To this end, approximately 680 bp upstream and downstream of the genes PP_1016-PP_1018 are amplified by means of PCR.

[0217] The following primers are used for the amplification of the glcP gene:

TABLE-US-00019 PCR15:glcP MW_18_11 (SEQIDNo54) 5-ACTACCTCGGGATCCAATACATGAATGTGATCGGTATCACTCTC-3 MW_18_12 (SEQIDNo55) 5-TCAGTGCCCCAGCGCTTCGG-3

[0218] The following primers are used for the amplification of the homologous regions upstream and downstream of the PP_1016-PP_1018 genes:

TABLE-US-00020 PCR5:RegionupstreamofPP_1016 O.BF_FM_1*03 (SEQIDNo9) 5-GCCGCTTTGGTCCCGGCCGCCAAGGTCATTAAC-3 MW_18_01 (SEQIDNo21) 5-CATGTATTGGATCCCGAGGTAG-3 PCR16:RegiondownstreamofPP_1018 MW_18_13 (SEQIDNo56) 5-CCGAAGCGCTGGGGCACTGACTCGTCTACACCATCAATAAGAAAAA G-3 O.BF_FM_1*02 (SEQIDNo11) 5-GCTTGCATGCCTGCAGGCGTGATGTTGTACTTC-3

[0219] The following parameters are used for the PCR:

TABLE-US-00021 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

[0220] For the amplification, the Phusion High-Fidelity Master Mix from NEB (Frankfurt am Main, Germany) is used according to the manufacturer's recommendations. 50 l of each of the PCR reactions are 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 are performed in a manner known to a person skilled in the art. PCR fragments of the expected size (PCR 15 (SEQ ID No 57), 1517 bp; PCR 5 (SEQ ID No 24), 670 bp; PCR 16 (SEQ ID No 58), 710 bp) are amplified. The PCR products are 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 are cloned into a BamHI- and SbfI-cut pKOPp vector (SEQ ID No. 16). Chemically competent E. coli 10 beta cells (NEB, Frankfurt am 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 resultant knock-out vector is referred to as pKO_PP_1016-PP_1018::glcP_Ms (SEQ ID No 59).

[0221] Construction of the strain P. putida KT2440 upp [PP_1016-1018]::glcP_Ms

[0222] The construction of the strain P. putida KT2440 upp [PP_1016-1018]::glcP_Ms is carried out with the aid of the plasmid pKO_PP_1016-PP_1018::glcP_Ms and a method described in Graf et al., 2011 (Graf N, Altenbuchner J, Appl. Environ. Microbiol., 2011, 77(15):5549; DOI: 10.1128/AEM.05055-11). The DNA sequence after replacement of the genes PP_1016-PP_1018 with glcP is described in SEQ ID No 60. The transformation of P. putida KT2440 upp [PP_1016-1018]::glcP_Ms with the vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_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). Thereafter, the cells are 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 is isolated and analysed by means of restriction analysis. A strain bearing the plasmid is called P. putida KT2440 upp [PP_1016-1018]::glcP_Ms pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0223] Technical realization is carried out as described in Example 1.

[0224] 3 experiments are carried out, each in parallel to Example 1.

[0225] A significantly higher total RL concentration after 64 h and a significantly higher calculated space-time yield is observed compared to Example 1.

Example 8 (Inventive)

[0226] Use is made of the strain P. putida KT2440 upp [PP_1016-1018]::glcU_Bs (co_Pp)+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk})

[0227] Construction of a vector for the integration of the glcU gene in Pseudomonas putida KT2440 upp

[0228] A vector for the integration of the glcU gene from Bacillus subtilis, encoding a glucose uptake protein GlcU, is prepared by PCR amplification of the gene codon-optimized for P. putida KT2440. The template used is a synthetic DNA fragment. It is intended that the glcU gene replace, in P. putida KT2440 upp, the genes PP_1016-PP_1018, encoding an ABC transporter permease, an ABC transporter binding protein and an ABC transporter ATP-binding protein. To this end, approximately 680 bp upstream and downstream of the genes PP_1016-PP_1018 are amplified by means of PCR.

[0229] The following primers are used for amplification of the glcU gene:

TABLE-US-00022 PCR17:glcU MW_18_14 (SEQIDNo61) 5-TACCTCGGGATCCAATACATGGACTTGTTGCTGGCTCTG-3 MW_18_15 (SEQIDNo62) 5-CTAGCTGTTGGTCTTGGCGATG-3

[0230] The following primers are used for the amplification of the homologous regions upstream and downstream of the PP_1016-PP_1018 genes:

TABLE-US-00023 PCR5:RegionupstreamofPP_1016 O.BF_FM_1*03 (SEQIDNo9) 5-GCCGCTTTGGTCCCGGCCGCCAAGGTCATTAAC-3 MW_18_01 (SEQIDNo21) 5-CATGTATTGGATCCCGAGGTAG-3 PCR18:RegiondownstreamofPP_1018 MW_18_16 (SEQIDNo63) 5-TCGCCAAGACCAACAGCTAGCTCGTCTACACCATCAATAAGAAAAA G-3 O.BF_FM_1*02 (SEQIDNo11) 5-GCTTGCATGCCTGCAGGCGTGATGTTGTACTTC-3

[0231] The following parameters are used for the PCR:

TABLE-US-00024 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

[0232] For the amplification, the Phusion High-Fidelity Master Mix from NEB (Frankfurt am Main, Germany) is used according to the manufacturer's recommendations. 50 l of each of the PCR reactions are 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 are performed in a manner known to a person skilled in the art. PCR fragments of the expected size (PCR 17 (SEQ ID No 64), 882 bp; PCR 5 (SEQ ID No 24), 670 bp; PCR 18 (SEQ ID No 65), 710 bp) are amplified. The PCR products are 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 are cloned into a BamHI- and SbfI-cut pKOPp vector (SEQ ID No. 16). Chemically competent E. coli 10 beta cells (NEB, Frankfurt am 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 resultant knock-out vector is referred to as pKO_PP_1016-PP_1018::glcU_Bs (SEQ ID No 66).

[0233] Construction of the starin P. putida KT2440 upp [PP_1016-1018]::glcU_Bs

[0234] The construction of the strain P. putida KT2440 upp [PP_1016-1018]::glcU_Bs is carried out with the aid of the plasmid pKO_PP_1016-PP_1018::glcU_Bs and a method described in Graf et al., 2011 (Graf N, Altenbuchner J, Appl. Environ. Microbiol., 2011, 77(15):5549; DOI: 10.1128/AEM.05055-11). The DNA sequence after replacement of the genes PP_1016-PP_1018 with glcU is described in SEQ ID No 67. The transformation of P. putida KT2440 upp [PP_1016-1018]::glcU_Bs with the vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_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). Thereafter, the cells are 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 is isolated and analysed by means of restriction analysis. A strain bearing the plasmid is called P. putida KT2440 upp [PP_1016-1018]::glcU_Bs pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0235] Technical realization is carried out as described in Example 1.

[0236] 3 experiments are carried out, each in parallel to Example 1.

[0237] A significantly higher total RL concentration after 64 h and a significantly higher calculated space-time yield is observed compared to Example 1.

Example 9 (Inventive)

[0238] Use is made of the strain P. putida KT2440 upp [PP_1016-1018]:SemiSWEET_Lb (co Pp)+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk})

[0239] Construction of a vector for the integration of the SemiSWEET gene in Pseudomonas putida KT2440 upp

[0240] A vector for the integration of the semiSWEET gene from Leptospira biflexa, encoding a sugar transporter semisweet, is prepared by PCR amplification of the gene codon-optimized for P. putida KT2440. The template used is a synthetic DNA fragment. It is intended that the semiSWEET gene replace, in P. putida KT2440 upp, the genes PP_1016-PP_1018, encoding an ABC transporter permease, an ABC transporter binding protein and an ABC transporter ATP-binding protein. To this end, approximately 680 bp upstream and downstream of the genes PP_1016-PP_1018 are amplified by means of PCR.

[0241] The following primers are used for the amplification of the semiSWEET gene:

TABLE-US-00025 PCR19:semiSWEET MW_18_17 (SEQIDNo68) 5-TACCTCGGGATCCAATACATGGAAAACTTGATCGGCTATGTG-3 MW_18_18 (SEQIDNo69) 5-TCAGGTTTGGTTGCCCTCGGTCAG-3

[0242] The following primers are used for the amplification of the homologous regions upstream and downstream of the PP_1016-PP_1018 genes:

TABLE-US-00026 PCR5:RegionupstreamofPP_1016 O.BF_FM_1*03 (SEQIDNo9) 5-GCCGCTTTGGTCCCGGCCGCCAAGGTCATTAAC-3 MW_18_01 (SEQIDNo21) 5-CATGTATTGGATCCCGAGGTAG-3 PCR20:RegiondownstreamofPP_1018 MW_18_19 (SEQIDNo70) 5-CCGAGGGCAACCAAACCTGACTCGTCTACACCATCAATAAGAAAAA G-3 O.BF_FM_1*02 (SEQIDNo11) 5-GCTTGCATGCCTGCAGGCGTGATGTTGTACTTC-3

[0243] The following parameters are used for the PCR:

TABLE-US-00027 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
For the amplification, the Phusion High-Fidelity Master Mix from NEB (Frankfurt am Main, Germany) is used according to the manufacturer's recommendations. 50 l of each of the PCR reactions are 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 are performed in a manner known to a person skilled in the art. PCR fragments of the expected size (PCR 19 (SEQ ID No 71), 276 bp; PCR 5 (SEQ ID No 24), 670 bp; PCR 20 (SEQ ID No 72), 710 bp) are amplified. The PCR products are 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 are cloned into a BamHI- and SbfI-cut pKOPp vector (SEQ ID No. 16). Chemically competent E. coli 10 beta cells (NEB, Frankfurt am 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 resultant knock-out vector is referred to as pKO_PP_1016-PP_1018::SemiSWEET_Lb (co_Pp) (SEQ ID No 73).

[0244] Construction of the strain P. putida KT2440 upp [PP_1016-1018]::semiSWEET_Lb (co_Pp)

[0245] The construction of the strain P. putida KT2440 upp [PP_1016-1018]::semiSWEET_Lb (co_Pp) is carried out with the aid of the plasmid pKO_PP_1016-PP_1018::semiSWEET_Lb (co_Pp) and a method described in Graf et al., 2011 (Graf N, Altenbuchner J, Appl. Environ. Microbiol., 2011, 77(15):5549; DOI: 10.1128/AEM.05055-11). The DNA sequence after replacement of the genes PP_1016-PP_1018 with semiSWEET is described in SEQ ID No 74. The transformation of P. putida KT2440 upp APP_1016-10181: semiSWEET_Lb (co_Pp) with the vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_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). Thereafter, the cells are 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 is isolated and analysed by means of restriction analysis. A strain bearing the plasmid is called P. putida KT2440 upp [PP_1016-1018]::semiSWEET_Lb (co_Pp) pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0246] Technical realization is carried out as described in Example 1.

[0247] 3 experiments are carried out, each in parallel to Example 1.

[0248] A significantly higher total RL concentration after 64 h and a significantly higher calculated space-time yield is observed compared to Example 1.