Cells and methods for producing rhamnolipids
09580720 ยท 2017-02-28
Assignee
Inventors
- Steffen Schaffer (Herten, DE)
- Mirja Wessel (Bochum, DE)
- Anja Thiessenhusen (Muenster, DE)
- Nadine Stein (Recklinghausen, DE)
Cpc classification
C12N15/70
CHEMISTRY; METALLURGY
Y02P20/52
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C12P19/44
CHEMISTRY; METALLURGY
International classification
C12P19/44
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
Abstract
This invention relates to cells and nucleic acids and also use thereof for producing rhamnolipids, and also methods for producing rhamnolipids.
Claims
1. A genetically modified cell, which is able to form at least one rhamnolipid of general formula (I), ##STR00003## wherein m=2, 1 or 0, n=1 or 0, R.sup.1 and R.sup.2 are organic residues having 2 to 24 carbon atoms, said cell having been genetically modified such that, compared to its wild-type, the cell has increased activity of at least one of the enzymes E.sub.1, E.sub.2 and E.sub.3, wherein: the enzyme E.sub.1 has at least 95% amino acid identity to a sequence selected from SEQ ID NO: 18, 78, 80, 82, or 2; the enzyme E.sub.2 has at least 95% amino acid identity to a sequence selected from SEQ ID NO: 20, 84, 86, 88, or 4; the enzyme E.sub.3 has at least 95% amino acid identity to a sequence selected from SEQ ID NO: 22, 90, 92, or 6; wherein said cell, compared to its wild-type further has increased activity of an enzyme E.sub.8, which catalyses rhamnolipid export from the cell into the surrounding medium; and wherein E.sub.8 has at least 95% amino acid identity to SEQ ID NO: 8.
2. The genetically modified cell of claim 1, wherein said cell has increased activities of an enzyme combination selected from E.sub.1E.sub.2, E.sub.2E.sub.3 and E.sub.1E.sub.2E.sub.3.
3. The genetically modified cell of claim 2, wherein said cell has an increased activity of the enzyme combination E.sub.1E.sub.2E.sub.3 and n is =1.
4. The genetically modified cell of claim 1, wherein said cell is selected from a genus of the group consisting of Aspergillus, Corynebacterium, Brevibacterium, Bacillus, Acinetobacter, Alcaligenes, Lactobacillus, Paracoccus, Lactococcus, Candida, Pichia, Hansenula, Kluyveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Pseudomonas, Rhodospirillum, Rhodobacter, Burkholderia, Clostridium and Cupriavidus.
5. The genetically modified cell of claim 1, wherein said cell is a bacterial cell.
6. The genetically modified cell of claim 1, wherein the wild-type of said cell forms polyhydroxyalkanoates having chain lengths of C.sub.6 to C.sub.16.
7. The genetically modified cell of claim 6, wherein said cell, compared to its wild-type, has a decreased activity of at least one enzyme E.sub.9 or E.sub.10, wherein E.sub.9 has at least 95% identity to the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 32, and E.sub.10 has at least 95% identity to the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 36.
8. The genetically modified cell of claim 1, wherein said cell, compared to its wild-type, has increased activity of at least one enzyme selected from the group consisting of: E.sub.4, which has at least 95% amino acid identity to SEQ ID NO: 10, E.sub.5, which has at least 95% amino acid identity to SEQ ID NO: 12, E.sub.6, which has at least 95% amino acid identity to SEQ ID NO: 16, and E.sub.7, which has at least 95% amino acid identity to SEQ ID NO: 14.
9. The genetically modified cell of claim 8, wherein said cell has increased activity of each of the enzymes E.sub.4, E.sub.5, E.sub.6, and E.sub.7.
10. The genetically modified cell of claim 1, wherein said genetic modification comprises introduction into said cell of at least one vector comprising at least one nucleic acid sequence selected from: a sequence with at least 95% identity to SEQ ID NO: 17, 77, 79, 81, or 1; a sequence with at least 95% identity to SEQ ID NO: 19, 83, 85, 87, or 3; and a sequence with at least 95% identity to SEQ ID NO: 21, 89, 91, or 5.
11. A method for producing rhamnolipids of general formula (I) ##STR00004## wherein m=2, 1 or 0, n=1 or 0 R.sup.1 and R.sup.2 are organic residues having 2 to 24 carbon atoms, said method comprising: I) contacting the genetically modified cell of claim 1 with a medium containing a carbon source; and II) culturing the cell under conditions in which the cell forms rhamnolipids from the carbon source.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
EXAMPLES
1. Construction of a Vector pBBR1MCS-2::AB for the Heterologous Expression of the Pseudomonas aeruginosa 1707 Genes rhIA and rhIB in Pseudomonas putida
(4) For the heterologous expression of the Pseudomonas aeruginosa DSM1707 genes rhIA and rhIB, the plasmid pBBR1MCS-2::AB (Seq ID No. 38) was constructed. For this, the synthetic operon rhIAB (Seq ID No. 37) was synthesized by the company GeneArt AG (Regensburg) and intercloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of the Pseudomonas aeruginosa DSM1707. Starting from the vector pMA::AB, the synthetic operon was cleaved from the vector by means of BglII and XbaI and subsequently ligated into the expression vector pBBR1MCS-2 (Seq ID No. 49) cleaved with BamHI and XbaI (described in Kovach et al., 1995: Four new derivatives of the broad host range cloning vector pBBR1MCS carrying different antibiotic-resistance cassettes. Gene, 166:175-176). The resulting plasmid pBBR1MCS-2::AB (Seq ID No. 38) is 7422 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5 cells (Gibco-BRL, Karlsruhe) took place in the manner known to the person skilled in the art. The authenticity of the insert was checked by DNA sequence analysis.
(5) The transformation of Pseudomonas putida KT2440 and GPp104 using the vectors pBBR1MCS-2 (Seq ID No. 49) and pBBR1MCS-2::AB took place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of 10 clones was isolated and analyzed. The strains obtained carrying the plasmids were named P. putida KT2440 pBBR1MCS-2, P. putida GPp104 pBBR1MCS-2, P. putida KT2440 pBBR1MCS-2::AB and P. putida GPp104 pBBR1MCS-2::AB.
2. Construction of a Vector pBBR1MCS-2::ABC for the Heterologous Expression of the Pseudomonas aeruginosa DSM1707 Genes rhIA, rhIB and rhIC in Pseudomonas putida
(6) For the heterologous expression of the Pseudomonas aeruginosa DSM1707 genes rhIA, rhIB and rhIC, the plasmid pBBR1MCS-2::ABC (Seq ID No. 40) was constructed. For this, the synthetic operon rhIABC (Seq ID No. 39) was synthesized by the company GeneArt AG (Regensburg) and intercloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of the Pseudomonas aeruginosa DSM1707. Starting from the vector pMA::ABC, the synthetic operon was cleaved from the vector by means of BglII and XbaI and subsequently ligated into the expression vector pBBR1MCS-2 (Seq ID No. 49) cleaved with BamHI and XbaI (Kovach et al., 1995: Four new derivatives of the broad host range cloning vector pBBR1MCS carrying different antibiotic-resistance cassettes. Gene, 166:175-176). The resulting plasmid pBBR1MCS-2::ABC (Seq ID No. 40) is 8409 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5 cells (Gibco-BRL, Karlsruhe) took place in the manner known to the person skilled in the art. The authenticity of the insert was checked by DNA sequence analysis.
(7) The transformation of Pseudomonas putida KT2440 and GPp104 using the vector pBBR1MCS-2::ABC took place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of every 10 clones was isolated and analyzed. The strains obtained carrying the plasmids were named P. putida KT2440 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABC.
3. Construction of a Vector pBBR1MCS-2::ABM for the Heterologous Expression of the Pseudomonas aeruginosa DSM1707 Genes rhIA, rhIB and pa1131 in Pseudomonas putida
(8) For the heterologous expression of the Pseudomonas aeruginosa DSM1707 genes rhIA, rhIB and pa1131 the plasmid pBBR1MCS-2::ABM (Seq ID No. 42) was constructed. For this, the synthetic operon rhIAB-pa1131 (Seq ID No. 41) was synthesized by the company GeneArt AG (Regensburg) and intercloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of the Pseudomonas aeruginosa DSM1707. Starting from the vector pMA::ABM the synthetic operon was cleaved from the vector by means of BglII and XbaI and subsequently ligated into the expression vector pBBR1MCS-2 (Seq ID No. 49) cleaved with BamHI and XbaI (Kovach et al., 1995: Four new derivatives of the broad host range cloning vector pBBR1MCS carrying different antibiotic-resistance cassettes. Gene, 166:175-176). The resulting plasmid pBBR1MCS-2::ABM (Seq ID No. 42) is 8702 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5 cells (Gibco-BRL, Karlsruhe) took place in the manner known to the person skilled in the art. The authenticity of the insert was checked by DNA sequence analysis.
(9) The transformation of Pseudomonas putida KT2440 and GPp104 using the vector pBBR1MCS-2::ABM took place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of every 10 clones was isolated and analyzed. The strains obtained carrying the plasmids were named P. putida KT2440 pBBR1MCS-2::ABM and P. putida GPp104 pBBR1MCS-2::ABM.
4. Quantification of Rhamnolipid Production by Recombinant P. putida Strains
(10) The recombinant strains P. putida KT2440 pBBR1MCS-2; P. putida KT2440 pBBR1MCS-2::AB; P. putida KT2440 pBBR1MCS-2::ABC; P. putida KT2440 pBBR1MCS-2::ABM; P. putida GPp104 pBBR1MCS-2; P. putida GPp104 pBBR1MCS-2::AB, P. putida GPp104 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABM were cultured on LB agar kanamycin (50 g/ml) plates.
(11) For the production of the rhamnolipids, the medium designated below as CMP medium was used. This consists of 2% (w/v) glucose, 0.007% (w/v) KH.sub.2PO.sub.4, 0.11% Na.sub.2HPO.sub.42 H.sub.2O, 0.2% (w/v) NaNO.sub.3, 0.04% (w/v) MgSO.sub.4H.sub.2O, 0.01% (w/v) CaCl.sub.22 H.sub.2O and 0.2% (v/v) of a trace element solution. This consists of 0.2% (w/v) FeSO.sub.47 H.sub.2O, 0.15% (w/v) MnSO.sub.4H.sub.2O and 0.06% (w/v) (NH.sub.4)MO.sub.7O.sub.244 H.sub.2O. The pH of the medium was adjusted to 6.7 with NaOH and the medium was subsequently sterilized by means of an autoclave (121 C., 20 min). An adjustment of the pH during the culturing was not necessary.
(12) For the investigation of the rhamnolipid production in the shaker flask a preculture was first prepared. For this, an inoculation loop of a strain freshly streaked on an LB agar plate was used and 10 ml of LB medium was inoculated into a 100 ml Erlenmeyer flask. All recombinant P. putida strains were in the LB medium, to which 50 g/ml of kanamycin was added. The culturing of the strains took place overnight at 30 C. and 200 rpm.
(13) The precultures were used to inoculate 50 ml of CMP medium in the 250 ml Erlenmeyer flask (start OD.sub.600 0.1). The cultures were cultured at 200 rpm and 30 C. for at most 120 h. At intervals of 24 h, a sample of 1 ml of broth was removed from the culture flask. The sample preparation for the following chromatographic analyses took place as follows:
(14) Using a displacement pipette (Combitip), 1 ml of acetone was introduced into a 2 ml reaction vessel and the reaction vessel was immediately closed for the minimization of evaporation. The addition of 1 ml of broth followed. After vortexing of the broth/acetone mixture, this was centrifuged off for 3 min at 13,000 rpm, and 800 l of the supernatant was transferred to an HPLC vessel.
(15) For the detection and for the quantification of rhamnolipids, an evaporative light scattering detector (Sedex LT-ELSD Model 85LT) was used. The actual measurement was carried out by means of Agilent Technologies 1200 Series (Santa Clara, Calif.) and the Zorbax SB-C8 rapid resolution column (4.6150 mm, 3.5 m, Agilent). The injection volume was 5 l and the runtime of the method was 20 min. As mobile phase, aqueous 0.1% TFA (trifluoroacetic acid, solution A) and methanol (solution B) was used. 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:
(16) TABLE-US-00001 t Solution Flow [min] B vol. % [ml/min] 0.00 70% 1.00 15.00 100% 1.00 15.01 70% 1.00 20.00 70% 1.00
(17) While P. putida KT2440 pBBR1MCS-2 and GPp104 pBBR1MCS-2 produced no rhamnolipids, in the recombinant strains P. putida KT2440 pBBR1MCS-2::AB, P. putida KT2440 pBBR1MCS-2::ABC, P. putida KT2440 pBBR1MCS-2::ABM, P. putida GPp104 pBBR1MCS-2::AB, P. putida GPp104 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABM the formation of different rhamnolipid species was detectable (
(18) By the incorporation of pBBR1MCS-2::AB and pBBR1MCS-2::ABM into P. putida, it was possible to generate monorhamnosyl lipids (
(19) If rhIC (pBBR1MCS-2::ABC) was additionally incorporated into the strains, mono- and dirhamnosyl lipids were produced (
(20) The direct comparison of the rhamnolipid formation by P. putida pBBR1MCS-2::AB and P. putida pBBR1MCS-2::ABM shows that the coexpression of P. aeruginosa p3111 with P. aeruginosa rhIAB leads to an improvement in the rhamnolipid biosynthesis (
(21) If the monorhamnosyl lipid synthesis of the strains P. putida KT2440 pBBR1MCS-2::ABM and P. putida GPp104 pBBR1MCS-2::ABM was compared, it was possible in the PHA-negative mutant P. putida GPp104 pBBR1MCS-2::ABM to detect 62 peak areas/OD 600 nm (120 h culturing) and with P. putida KT2440 pBBR1MCS-2::ABM 50 area/OD 600 nm monorhamnosyl lipids (
(22) A comparative analysis of the dirhamnosyl lipid formation (mg/l/OD 600 nm) in the strains P. putida KT2440 and GPp104 likewise showed a greater formation of the dirhamnosyl lipids in the PHA-negative strain background of the P. putida GPp104. P. putida GPp104 pBBR1MCS-2::ABC formed on average 113 mg/l/OD 600 nm of dirhamnosyl lipids (96 h), whereas with P. putida KT2440 pBBR1MCS-2::ABC only 55 mg/l/OD 600 nm of dirhamnosyl lipids could be detected after 96 h (
(23) Thus it was possible to show that the use of a strain background attenuated with respect to PHA synthesis leads to an improvement in the rhamnolipid biosynthesis.
5. Construction of a Vector pBBR1MCS-2::ABMC for the Heterologous Expression of the Pseudomonas aeruginosa DSM1707 Genes rhIA, rhIB, pa1131 and rhIC in Pseudomonas putida
(24) For the heterologous expression of the Pseudomonas aeruginosa DSM1707 genes rhIA, rhIB, pa1131 and rhIC, the plasmid pBBR1MCS-2::ABMC (Seq ID No. 51) was constructed. For this, the synthetic operon rhIAB-pa1131-rhIC (Seq ID No. 50) was synthesized by the company GeneArt AG (Regensburg) and intercloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of the Pseudomonas aeruginosa DSM1707. Starting from the vector pMA::ABMC the synthetic operon was cleaved by means of BglII and XbaI from the vector and subsequently ligated into the expression vector pBBR1MCS-2 (Seq ID No. 49) cleaved with BamHI and XbaI (Kovach et al., 1995: Four new derivatives of the broad-host-range cloning vector pBBR1MCS carrying different antibiotic-resistance cassettes. Gene, 166:175-176). The resulting plasmid pBBR1MCS-2::ABMC (Seq ID No. 51) is 9663 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5 cells (Gibco-BRL, Karlsruhe) took place in a manner known to the person skilled in the art. The authenticity of the insert was checked by DNA sequence analysis.
(25) The transformation of Pseudomonas putida KT2440 and GPp104 using the vector pBBR1MCS-2::ABMC took place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of every 10 clones was isolated and analyzed. The strains obtained carrying the plasmids were named P. putida KT2440 pBBR1MCS-2::ABMC and P. putida GPp104 pBBR1MCS-2::ABMC.
6. Qualitative Comparison of the Rhamnolipid Production by Recombinant P. putida Strains and P. aeruginosa Strains
(26) The recombinant strains P. putida GPp104 pBBR1MCS-2 and P. putida GPp104 pBBR1MCS-2::ABMC and P. aeruginosa DSM 19880 were cultured on LB agar kanamycin (50 g/ml; P. putida) and LB agar plates (P. aeruginosa).
(27) For the production of the rhamnolipids the medium below designated as CMP medium was used. This consists of 2% (w/v) glucose, 0.007% (w/v) KH.sub.2PO.sub.4, 0.11% Na.sub.2HPO.sub.42 H.sub.2O, 0.2% (w/v) NaNO.sub.3, 0.04% (w/v) MgSO.sub.4H.sub.2O, 0.01% (w/v) CaCl.sub.22 H.sub.2O and 0.2% (v/v) of a trace element solution. This consists of 0.2% (w/v) FeSO.sub.47 H.sub.2O, 0.15% (w/v) MnSO.sub.4H.sub.2O and 0.06% (w/v) (NH.sub.4)MO.sub.7O.sub.244 H.sub.2O. The pH of the medium was adjusted to 6.7 using NaOH and the medium was subsequently sterilized by means of an autoclave (121 C., 20 min). An adjustment of the pH during the culturing was not necessary.
(28) For the investigation of the rhamnolipid production in the shaker flask, a preculture was first prepared. For this, an inoculation loop of a strain freshly streaked on LB agar plate was used and 10 ml of LB medium was inoculated into a 100 ml Erlenmeyer flask. The recombinant P. putida strains were cultured in the LB medium, to which 50 g/ml of kanamycin was added. P. aeruginosa was cultured in the LB medium. The culturing of the strains took place at 30 C. and 200 rpm overnight.
(29) The precultures were used to inoculate 50 ml of CMP medium in the 250 ml Erlenmeyer flask (start OD.sub.600 0.1). The cultures were cultured at 200 rpm and 30 C. for at most 120 h. At intervals of 24 h, a sample of 1 ml of broth was removed from the culture flask. The sample preparation for the following chromatographic analyses took place as follows:
(30) Using a displacement pipette (Combitip), 1 ml of acetone was introduced into a 2 ml reaction vessel and the reaction vessel was immediately closed for the minimization of evaporation. The addition of 1 ml of broth followed. After vortexing of the broth/acetone mixture, this was centrifuged off for 3 min at 13,000 rpm, and 800 l of the supernatant were transferred to an HPLC vessel.
(31) For the identification of the products formed, 5 l were injected into an Accela UPLC unit (Thermo Scientific, Dreieich). The substances to be investigated were analyzed using a semi UPLC column Pursuit XRs ULTRA (C8, 2.8 m, 2.1100 mm) (Varian, Darmstadt). The separation took place within 25 min by means of a gradient consisting of the mobile phase A1 (H.sub.2O, 0.1% (v/v) TFA) and the mobile phase B1 (methanol, 0.1% (v/v) TFA) using a flow rate of 0.3 ml/min at 40 C. The time course of the gradient was the following:
(32) TABLE-US-00002 Time Mobile phase Mobile phase [min] A1 [%] B1 [%] 0 30 70 15 0 100 25 0 100 25.01 30 70 32 30 70
(33) Detection took place by means of DAD detector in the wavelength range from 200-600 nm and mass-selectively using a high-resolution FT-ICR LTQ-FT mass spectrometer (Thermo Scientific, Dreieich) in the scanning range m/e 100-1000. Ionization took place by means of ESI (electrospray ionization). Exact masses and empirical chemical formulae were determined with the aid of the FT-ICR mass analyzer, using a resolution of R=100000 and a mass accuracy of 2 ppm. The identification of the products takes place by analysis of the corresponding mass traces and the MS.sup.2 spectra. To be able to compare the strains, the peak areas of the corresponding substances were contrasted.
(34) As shown in
7. Construction of a Vector pBBR1MCS-2::rfbBDAC and pBBR1MCS-2::ABC_rfbBDAC for Heterologous Expression in Pseudomonas putida
(35) At the company Trenzyme GmbH (Konstanz), the rhamnose biosynthesis operon rfbBDAC was amplified starting from chromosomal DNA of Pseudomonas putida KT2440. For this, the following primers were used:
(36) TABLE-US-00003 RL1: (SeqIDNo.48) 5-TATATATAGAATTCGCGTCATCTGTCTACGACAACAC-3 RL2: (SeqIDNo.43) 5-TATATATAGAATTCGGCTGCGCTACCGCAGCCCTTC-3
(37) The PCR product obtained was intercloned in Trenzyme's alligator cloning system and transformed in E. coli DH5 (New England Biolabs; Frankfurt). Vectors of different candidates were analyzed and sequenced. After successful and error-free DNA sequencing, the vector was cleaved by means of EcoRI and the target fragment rfbBDAC was isolated. For a further inter-cloning, the vector pBBR1MCS-2 (Kovach et al., 1995: Four new derivatives of the broad-host-range cloning vector pBBR1MCS carrying different antibiotic-resistance cassettes. Gene, 166:175-176) was cleaved in the same manner. The cleaved target fragment (rfbBDAC) and the cleaved vector (pBBR1MCS-2) were merged by conventional ligation. The resulting vector pBBR1MCS-2::rfbBDAC (Seq ID No. 45) was likewise transformed in E. coli DH5 (New England Biolabs; Frankfurt). Some candidates of the transformants were investigated with respect to the successful uptake of the plasmid.
(38) The vector pBBR1MCS-2::rfbBDAC served as a matrix for a PCR. The following oligonucleotides were used:
(39) TABLE-US-00004 RL_XbaI-fw: (SeqIDNo.44) 5-TATATATATCTAGAATTAATGCAGCTGGCACGAC-3 RL_Xba_rev: (SeqIDNo.46) 5-GGCCGCTCTAGAACTAGTGGA-3
(40) The PCR was carried out using the Phusion High-Fidelity Master Mix of New England Biolabs (Frankfurt) polymerase. It was carried out in the manner known to the person skilled in the art. The target sequence (lac promoter and rfbBDAC) was intercloned in the Trenzyme alligator cloning system. E. coli DH5 (New England Biolabs; Frankfurt) transformants were selected and the plasmid DNA of different candidates was isolated and sequenced. After the sequence had been checked and investigated for correctness, the vector was cleaved using XbaI. The target fragment was ligated into the pBBR1MCS-2::ABC likewise cleaved using XbaI (see above) by means of conventional ligation methods. The target vector pBBR1MCS-2::ABC_rfbBDAC obtained (Seq ID No. 47) has a size of 12249 base pairs. The insert of the vector was sequenced. The carrying-out of the PCR, the checking of the successful amplification of the PCR by means of agarose gel electrophoresis, ethidium bromide staining of the DNA, determination of the PCR fragment size, purification of the PCR products and DNA concentration determination took place in the manner known to the person skilled in the art.
(41) The transformation of Pseudomonas putida KT2440 and GPp104 using the vector pBBR1MCS-2::ABC_rfbBDAC took place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of every 10 clones was isolated and analyzed. The strains obtained carrying the plasmids are named P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC and P. putida GPp104 pBBR1MCS-2::ABC_rfbBDAC.
8. Quantification of the Rhamnolipid Production by Recombinant P. putida Strains with and without Overexpression of the rfbBDAC Operon
(42) The recombinant strains P. putida KT2440 pBBR1MCS-2; P. putida KT2440 pBBR1MCS-2::ABC, P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC, P. putida GPp104 pBBR1MCS-2, P. putida GPp104 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABC_rfbBDAC are cultured on LB agar kanamycin (50 g/ml) plates.
(43) For the production of the rhamnolipids, the medium designated below as CMP medium is used. This consists of 2% (w/v) glucose, 0.007% (w/v) KH.sub.2PO.sub.4, 0.11% Na.sub.2HPO.sub.42 H.sub.2O, 0.2% (w/v) NaNO.sub.3, 0.04% (w/v) MgSO.sub.4H.sub.2O, 0.01% (w/v) CaCl.sub.22 H.sub.2O and 0.2% (v/v) of a trace element solution. This consists of 0.2% (w/v) FeSO.sub.47 H.sub.2O, 0.15% (w/v) MnSO.sub.4H.sub.2O and 0.06% (w/v) (NH.sub.4)MO.sub.7O.sub.244 H.sub.2O. The pH of the medium is adjusted to 6.7 using NaOH and the medium is subsequently sterilized by means of an autoclave (121 C., 20 min). An adjustment of the pH during the culturing is not necessary.
(44) For the investigation of the rhamnolipid production in the shaker flask, a preculture is first prepared. For this, an inoculation loop of a strain freshly streaked on LB agar plate is used and 10 ml of LB medium are inoculated into a 100 ml Erlenmeyer flask. All recombinant P. putida strains are cultured in the LB medium, to which 50 g/ml of kanamycin is added. The culturing of the P. putida strains was carried out at 30 C. and 200 rpm overnight.
(45) The precultures are used to inoculate 50 ml of CMP medium in the 250 ml Erlenmeyer flask (start OD.sub.600 0.1). The cultures are cultured at 200 rpm and 30 C. for at most 120 h. At intervals of 24 h, a sample of 1 ml broth is removed from the culture flask. The sample preparation for the following chromatographic analyses takes place as follows:
(46) Using a displacement pipette (Combitip), 1 ml of acetone is introduced into a 2 ml reaction vessel and the reaction vessel is closed immediately for the minimization of evaporation. The addition of 1 ml of broth follows. After vortexing of the broth/acetone mixture, this is centrifuged off for 3 min at 13,000 rpm, and 800 l of the supernatant are transferred to an HPLC vessel. For the detection and for the quantification of rhamnolipids, an evaporative light scattering detector (Sedex LT-ELSD Model 85LT) is used. The actual measurement is carried out by means of Agilent Technologies 1200 Series (Santa Clara, Calif.) and the Zorbax SB-C8 rapid resolution column (4.6150 mm, 3.5 m, Agilent). The injection volume is 5 l and the runtime of the method is 20 min. As a mobile phase, aqueous 0.1% TFA (trifluoroacetic acid, solution A) and methanol (solution B) is used. The column temperature is 40 C. The ELSD (detector temperature 60 C.) and the DAD (diode array, 210 nm) serve as detectors. The gradient used in the method is:
(47) TABLE-US-00005 t Solution Flow [min] B vol. % [ml/min] 0.00 70% 1.00 15.00 100% 1.00 15.01 70% 1.00 20.00 70% 1.00
(48) While P. putida KT2440 pBBR1MCS-2 and GPp104 pBBR1MCS-2 produce no rhamnolipids, in the recombinant strains P. putida KT2440 pBBR1MCS-2::ABC, P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC; P. putida GPp104 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABC_rfbBDAC the formation of rhamnolipids is detectable.
(49) P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC shows in comparison to P. putida KT2440 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABC_rfbBDAC shows in comparison to P. putida GPp104 pBBR1MCS-2::ABC an increased formation of the di- and monorhamnosyl lipids. This clearly shows the positive influence of the amplification of the expression of rfbBDAC on the formation of mono- and dirhamnosyl lipids.
(50) If the mono- and dirhamnosyl lipid biosynthesis of the strains P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC and P. putida GPp104 pBBR1MCS-2::ABC_rfbBDAC is compared, an increased mono- and dirhamnosyl lipid synthesis is detected in the PHA-negative mutant P. putida GPp104 pBBR1MCS-2::ABC_rfbBDAC.
(51) As already described above, the rhamnolipid biosynthesis is increased with the use of a strain background inactivated in the PHA synthesis.
9. Generation of Recombinant E. coli W3110 pBBR1MCS-2::ABC and E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC
(52) The transformation of E. coli W3110 took place as previously described (Miller J H. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Plainview, N.Y.: Cold Spring Harbor Lab. Press; 1992) by means of electroporation. The plasmid DNA of every 10 clones was isolated and analyzed. The strains obtained carrying the plasmids were named E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC and E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC.
10. Quantification of the Rhamnolipid Production by Recombinant E. coli Strains with and without Overexpression of the rfbBDAC Operon
(53) The recombinant strains E. coli W3110 pBBR1MCS-2; E. coli W3110 pBBR1MCS-2::ABC and E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC are cultured on LB agar kanamycin (50 g/ml) plates.
(54) For the production of the rhamnolipids, the medium designated in the following as CMP medium is used. This consists of 2% (w/v) glucose, 0.007% (w/v) KH.sub.2PO.sub.4, 0.11% Na.sub.2HPO.sub.42 H.sub.2O, 0.2% (w/v) NaNO.sub.3, 0.04% (w/v) MgSO.sub.4H.sub.2O, 0.01% (w/v) CaCl.sub.22 H.sub.2O and 0.2% (v/v) of a trace element solution. This consists of 0.2% (w/v) FeSO.sub.47 H.sub.2O, 0.15% (w/v) MnSO.sub.4H.sub.2O and 0.06% (w/v) (NH.sub.4)MO.sub.7O.sub.244 H.sub.2O. The pH of the medium is adjusted to 6.7 using NaOH and the medium is subsequently sterilized by means of an autoclave (121 C., 20 min). An adjustment of the pH during the culturing is not necessary.
(55) For the investigation of the rhamnolipid production in the shaker flask, a preculture is first prepared. For this, an inoculation loop of a strain freshly streaked on LB agar plate is used and 10 ml of LB medium is inoculated into a 100 ml Erlenmeyer flask. All recombinant E. coli strains are cultured in the LB medium, to which 50 g/ml of kanamycin is added. The culturing of the E. coli strains took place at 37 C. and 200 rpm overnight.
(56) The precultures are used to inoculate 50 ml of CMP medium in the 250 ml Erlenmeyer flask (start OD.sub.600 0.1). The cultures are cultured at 200 rpm and 30 C. for at most 120 h. At intervals of 24 h a sample of 1 ml of broth is removed from the culture flask. The sample preparation for the following chromatographic analyses takes place as follows:
(57) Using a displacement pipette (Combitip), 1 ml of acetone is introduced into a 2 ml reaction vessel and the reaction vessel is closed immediately for the minimization of evaporation. The addition of 1 ml of broth follows. After vortexing of the broth/acetone mixture, this is centrifuged off for 3 min at 13,000 rpm, and 800 l of the supernatant are transferred to an HPLC vessel. For detection and for the quantification of rhamnolipids, an evaporative light scattering detector (Sedex LT-ELSD Model 85LT) is used. The actual measurement is carried out by means of Agilent Technologies 1200 Series (Santa Clara, Calif.) and the Zorbax SB-C8 rapid resolution column (4.6150 mm, 3.5 m, Agilent). The injection volume is 5 l and the runtime of the method is 20 min. Aqueous 0.1% TFA (trifluoroacetic acid, solution A) and methanol (solution B) is used as the mobile phase. The column temperature is 40 C. The ELSD (detector temperature 60 C.) and the DAD (diode array, 210 nm) serve as detectors. The gradient used in the method is:
(58) TABLE-US-00006 t Solution Flow [min] B vol. % [ml/min] 0.00 70% 1.00 15.00 100% 1.00 15.01 70% 1.00 20.00 70% 1.00
(59) While E. coli W3110 pBBR1MCS-2 produces no rhamnolipids, the formation of mono- and dirhamnosyl lipids is detectable in the recombinant strains E. coli W3110 pBBR1MCS-2::ABC and E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC, wherein E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC forms significantly more mono- and dirhamnosyl lipids than E. coli W3110 pBBR1MCS-2::ABC. This shows that the heterologous expression of rhIABC of Pseudomonas aeruginosa DSM1707 leads to the formation of mono- and dirhamnosyl lipids in E. coli. This furthermore shows the positive influence of the reinforcement of the expression of rfbBDAC on the formation of mono- and dirhamnosyl lipids.
11. Construction of a vector pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 for the Heterologous Expression of the Pseudomonas aeruginosa DSM1707 Genes rhIA, rhIB and rhIC and the Burkholderia thailandensis E264 Genes BTH_II1077, BT_II1080 and BT_II1081 in Pseudomonas putida
(60) For the heterologous expression of the Pseudomonas aeruginosa DSM1707 genes rhIA, rhIB and rhIC and the B. thailandensis E264 genes BTH_II1077, BT_II1080 and BT_II1081 in Pseudomonas putida, the plasmid pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 (Seq ID No. 69) is constructed. For this, the synthetic operon BTH_II1077, BT_II1080 and BT_II1081 (Seq ID No. 70) is synthesized by the company DNA 2.0 (Menlo Park, Calif., USA) and intercloned in the commercial vector pJ294 (DNA 2.0; Menlo Park, Calif., USA). The basis for the synthesis is the genomic sequence of the strain B. thailandensis E264. Starting from the vector pJ294-BTH_II1077-II1080-II1081, the synthetic operon is cleaved from this vector by means of XbaI and subsequently ligated into the vector pBBR1MCS-2::ABC (Seq ID No. 40) likewise cleaved using XbaI. The target vector pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 (Seq ID No. 69) obtained has a size of 13768 base pairs. The insert of the vector is sequenced. The carrying-out of the PCR, the checking of the successful amplification of the PCR by means of agarose gel electrophoresis, ethidium bromide staining of the DNA, determination of the PCR fragment size, purification of the PCR products and DNA concentration determination takes place in the manner known to the person skilled in the art.
(61) The transformation of Pseudomonas putida KT2440 and GPp104 using the vector pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 (Seq ID No. 69) takes place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of every 10 clones is isolated and analyzed. The strains obtained carrying the plasmids are named P. putida KT2440 pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 and P. putida GPp104 pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081.
12. Quantification of the Rhamnolipid Production by Recombinant P. putida Strains with and without Overexpression of the B. thailandensis E264 Genes BTH_II1077, BT_II1080 and BT_II1081
(62) The recombinant strains P. putida strains P. putida KT2440 pBBR1MCS-2::AB, P. putida KT2440 pBBR1MCS-2::AB-BTH_II1077-II1080-II1081, P. putida GPp104 pBBR1MCS-2::AB, P. putida GPp104 pBBR1MCS-2::AB-BTH_II1077-II1080-II1081, P. putida KT2440 pBBR1MCS-2::ABC, P. putida KT2440 pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 P. putida GPp104 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 generated in the Examples 1, 2 and 11 are cultured on LB agar kanamycin (50 g/ml) plates.
(63) For the production of the rhamnolipids, the medium designated in the following as M9 medium is used. This medium consists of 2% (w/v) glucose, 0.3% (w/v) KH.sub.2PO.sub.4, 0.679% Na.sub.2HPO.sub.4, 0.05% (w/v) NaCl, 0.2% (w/v) NH.sub.4Cl, 0.049% (w/v) MgSO.sub.47 H.sub.2O and 0.1% (v/v) of a trace element solution. This consists of 1.78% (w/v) FeSO.sub.47 H.sub.2O, 0.191% (w/v) MnCl.sub.27 H.sub.2O, 3.65% (w/v) HCl, 0.187% (w/v) ZnSO.sub.47 H.sub.2O, 0.084% (v/v) Na EDTA2H.sub.2O, 0.03% (v/v) H.sub.3BO.sub.3, 0.025% (w/v) Na.sub.2MoO.sub.42 H.sub.2O and 0.47% (w/v) CaCl.sub.22 H.sub.2O. The pH of the medium is adjusted to 7.4 using NH.sub.4OH and the medium is subsequently sterilized by means of an autoclave (121 C., 20 min). An adjustment of the pH during the culturing is not necessary. For the investigation of the rhamnolipid production in the shaker flask, a preculture is first prepared. For this, an inoculation loop of a strain freshly streaked on LB agar plate is used and 10 ml of LB medium are inoculated into a 100 ml Erlenmeyer flask. All recombinant P. putida strains are cultured in LB medium, to which 50 g/ml of kanamycin was added. The culturing of the P. putida strains takes place at 37 C. and 200 rpm overnight.
(64) The precultures are used to inoculate 50 ml of M9 medium (+50 g/ml of kanamycin) in the 250 ml Erlenmeyer flask (start OD.sub.600 0,1). The cultures are cultured at 200 rpm and 30 C. At intervals of 24 h, a sample of 1 ml of broth is removed from the culture flask. The sample preparation for the following chromatographic analyses and the chromatographic analyses themselves are carried out as described in Example 4.
(65) It is shown that the recombinant strains P. putida KT2440 pBBR1MCS-2::AB-BTH_II1077-II1080-II1081 and P. putida GPp104 pBBR1MCS-2::AB-BTH_II1077-II1080-II1081 form significantly more monorhamnosyl lipids than the strains P. putida KT2440 pBBR1MCS-2::AB and P. putida GPp104 pBBR1MCS-2::AB. This demonstrates that the amplification of BTH_II1077-II1080-II1081 from B. thailandensis E264 increases the formation of monorhamnosyl lipids in P. putida strains containing the Pseudomonas aeruginosa DSM1707 genes rhIAB.
(66) It is furthermore shown that the recombinant strains P. putida KT2440 pBBR1MCS-2::ABC BTH_II1077-II1080-II1081 and P. putida GPp104 pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 form significantly more mono- and dirhamnosyl lipids than the strains P. putida KT2440 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABC. This proves that the amplification of BTH_II1077-II1080-II1081 from B. thailandensis E264 increases the formation of mono- and dirhamnosyl lipids in P. putida strains containing the Pseudomonas aeruginosa DSM1707 genes rhIABC.
(67) It is finally shown that the reduction of the polyhydroxybutyrate formation in the strain background P. putida GPp104 compared to the strain P. putida KT2440 leads to an increased rhamnolipid formation, as the strains P. putida KT2440 pBBR1MCS-2::AB, P. putida KT2440 pBBR1MCS-2::ABC, P. putida KT2440 pBBR1MCS-2::AB-BTH_II1077-II1080-II1081 and P. putida KT2440 pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 are able to form significantly fewer mono-( ) and mono- and dirhamnosyl lipids ( ) than the corresponding control strains P. putida GPp104 pBBR1MCS-2::AB, P. putida GPp104 pBBR1MCS-2::ABC, P. putida GPp104 pBBR1MCS-2::AB-BTH_II1077-II1080-II1081 and P. putida GPp104 pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081.
13. Construction of a Vector pBBR1MCS-2::ABCM for the Heterologous Expression of the Pseudomonas aeruginosa DSM1707 Genes rhIA, rhIB, pa1131 and rhIC in Pseudomonas putida
(68) For the heterologous expression of the Pseudomonas aeruginosa DSM1707 genes rhIA, rhIB, pa1131 and rhIC, the plasmid pBBR1MCS-2::ABCM (Seq ID No. 58) was constructed. For this, the gene pa1131 (Seq ID No. 59) was amplified starting from genomic DNA of the strain Pseudomonas aeruginosa PAO1 (DSM 1707) containing the oligonucleotides
(69) TABLE-US-00007 MFS2.0_xbaI_fw: (SeqIDNo.60) 5-AGGAAATCTAGATGAGAGGCCGGCAAGGATAC-3 MFS2.0_XbaI_rev: (SeqIDNo.61) 5-CCAGGTTCTAGACGCCAGGATTGAACAGTACC-3.
(70) The amplification of the PCR product (1483 base pairs) was carried out using the Phusion High-Fidelity Master Mix from New England Biolabs (Frankfurt) polymerase. The PCR product was cleaved using XbaI and ligated in the vector pBBR1MCS-2::ABC (Seq ID No. 40) likewise cleaved using XbaI by means of Fast Link Ligation Kit (Epicentre Technologies; Madison, Wis., USA). The target vector pBBR1MCS-2::ABCM (Seq ID No. 58) obtained has a size of 9892 base pairs. The insert of the vector was sequenced. The chromosomal DNA was isolated by means of DNeasy Blood and Tissue Kit (Qiagen; Hilden) according to manufacturer's instructions. The carrying-out of the PCR, the checking of the successful amplification of the PCR by means of agarose gel electrophoresis, ethidium bromide staining of the DNA, determination of the PCR fragment size, purification of the PCR products and DNA concentration determination took place in a manner known to the person skilled in the art. The transformation of Pseudomonas putida KT2440 and GPp104 using the vector pBBR1MCS-2::ABCM took place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of every 10 clones was isolated and analyzed. The strains obtained carrying the plasmids were named P. putida KT2440 pBBR1MCS-2::ABCM and P. putida GPp104 pBBR1MCS-2::ABCM.
14. Quantification of the Rhamnolipid Production by Recombinant P. putida Strains with and without Overexpression of the Pseudomonas aeruginosa DSM1707 pa1131 Gene
(71) The recombinant strains P. putida strains P. putida KT2440 pBBR1MCS-2::ABC, P. putida KT2440 pBBR1MCS-2::ABCM, P. putida KT2440 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABCM generated in the Examples 2 and 13 were cultured on LB agar kanamycin (50 g/ml) plates. The subsequent culturing for the production of the rhamnolipids took place as described in Example 12.
(72) The sample preparation for the following chromatographic analyses and the chromatographic analyses themselves took place as described in Example 4.
(73) The results are shown in the following table.
(74) Formation of di- and monorhamnosyl lipids by P. putida strains with and without overexpression of the P. aeruginosa gene pa1131 after 48 h incubation
(75) TABLE-US-00008 Dirhamnosyl Monorhamnosyl P. putida strains lipids [mg/l] lipids [peak area] KT2440 pBBR1MCS-2::ABC 310 19 KT2440 pBBR1MCS-2::ABCM 1053 314 GPp104 pBBR1MCS-2::ABC 689 127 GPp104 pBBR1MCS-2::ABCM 960 1090
(76) The results show that the overexpression of the P. aeruginosa gene pa1131 in both strain backgrounds (KT2440: wild-type and GPp104 having inactivated polyhydroxybutyrate formation) leads to an increased formation of di- and monorhamnosyl lipids. The results furthermore show that the reduction of the polyhydroxybutyrate formation in GPp104 generally leads to an increased formation of rhamnosyl lipids.
15. Construction of a vector pEC-XT99A::AB for the heterologous expression of the genes rhIA and rhIB from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum
(77) For the heterologous expression of the genes rhIA and rhIB from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum, the plasmid pEC-XT99A::AB (Seq ID No. 52) is constructed. For this, the synthetic operon rhIAB (Seq ID No. 37) was synthesized by the company GeneArt AG (Regensburg) and intercloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of the Pseudomonas aeruginosa DSM1707. Starting from the vector pMA::AB, the synthetic operon is cleaved from the vector by means of BglII and XbaI and subsequently ligated into the expression vector pEC-XT99A (U.S. Pat. No. 7,118,904) cleaved using BamHI and XbaI. The resulting plasmid pEC-XT99A::AB (Seq ID No. 52) is 9793 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5 cells (Gibco-BRL, Karlsruhe) takes place in the manner known to the person skilled in the art. The authenticity of the insert is checked by DNA sequence analysis.
(78) The transformation of C. glutamicum ATCC13032 using the vector pEC-XT99A::AB takes place as previously described (Liebl et al., FEMS Microbiol. Lett. 53:299-303 (1989)). The selection of the transformants takes place on LBHIS agar plates (18.5 g/l of brain heart infusion broth, 0.5 M sorbitol, 5 g/l of Bacto tryptone, 2.5 g/l of Bacto yeast extract, 5 g/l of NaCl and 18 g/l of Bacto agar, supplemented with 5 mg/l of tetracycline). The plates were incubated at 33 C. for two days. The strain obtained carrying the plasmid is named C. glutamicum pEC-XT99A::AB.
16. Construction of a Vector pEC-XT99A::ABC for the Heterologous Expression of the Genes rhIA, rhIB and rhIC from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum
(79) For the heterologous expression of the genes rhIA, rhIB and rhIC from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum, the plasmid pEC-XT99A::ABC (Seq ID No. 53) is constructed. For this, the synthetic operon rhIABC (Seq ID No. 39) was synthesized by the company GeneArt AG (Regensburg) and intercloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of the Pseudomonas aeruginosa DSM1707. Starting from the vector pMA::ABC, the synthetic operon is cleaved from the vector by means of BglII and XbaI and subsequently ligated into the expression vector pEC-XT99A (U.S. Pat. No. 7,118,904) cleaved using BamHI and XbaI. The resulting plasmid pEC-XT99A::ABC (Seq ID No. 53) is 10780 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5 cells (Gibco-BRL, Karlsruhe) takes place in the manner known to the person skilled in the art. The authenticity of the insert is checked by DNA sequence analysis.
(80) The transformation of C. glutamicum ATCC13032 using the vector pEC-XT99A::ABC takes place as previously described (Liebl et al., FEMS Microbiol. Lett. 53:299-303 (1989)). The selection of the transformants takes place on LBHIS agar plates (18.5 g/l of brain heart infusion broth, 0.5 M sorbitol, 5 g/l of Bacto tryptone, 2.5 g/l of Bacto yeast extract, 5 g/l of NaCl and 18 g/l of Bacto agar, supplemented using 5 mg/l of tetracycline). The plates were incubated at 33 C. for two days. The strain obtained carrying the plasmid is named C. glutamicum pEC-XT99A::ABC.
17. Construction of a Vector pEC-XT99A::ABM for the Heterologous Expression of the Genes rhIA, rhIB and pa1131 from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum
(81) For the heterologous expression of the genes rhIA, rhIB and pa1131 from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum, the plasmid pEC-XT99A::ABM (Seq ID No. 54) is constructed. For this, the synthetic operon rhIABM (Seq ID No. 41) was synthesized by the company GeneArt AG (Regensburg) and intercloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of the Pseudomonas aeruginosa DSM1707. Starting from the vector pMA::ABM, the synthetic operon is cleaved from the vector by means of BglII and XbaI and subsequently ligated into the expression vector pEC-XT99A (U.S. Pat. No. 7,118,904) cleaved using BamHI and XbaI. The resulting plasmid pEC-XT99A::ABM (Seq ID No. 54) is 11073 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5 cells (Gibco-BRL, Karlsruhe) takes place in the manner known to the person skilled in the art. The authenticity of the insert is checked by DNA sequence analysis.
(82) The transformation of C. glutamicum ATCC13032 using the vector pEC-XT99A::ABM takes place as previously described (Liebl et al., FEMS Microbiol. Lett. 53:299-303 (1989)). The selection of the transformants takes place on LBHIS agar plates (18.5 g/l of brain heart infusion broth, 0.5 M sorbitol, 5 g/l of Bacto tryptone, 2.5 g/l of Bacto yeast extract, 5 g/l of NaCl and 18 g/l of Bacto agar, supplemented with 5 mg/l of tetracycline). The plates were incubated at 33 C. for two days. The strain obtained carrying the plasmid is named C. glutamicum pEC-XT99A::ABM.
18. Construction of a Vector pEC-XT99A::ABCM for the Heterologous Expression of the Genes rhIA, rhIB, pa1131 and rhIC from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum
(83) For the heterologous expression of the genes rhIA, rhIB, pa1131 and rhIC from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum, the plasmid pEC-XT99A::ABCM (Seq ID No. 55) is constructed. For this, the gene pa1131 (Seq ID No. 59) was amplified starting from genomic DNA of the strain Pseudomonas aeruginosa PAO1 (DSM 1707) using the oligonucleotides
(84) TABLE-US-00009 MFS2.0_xbaI_fw: (SeqIDNo.60) 5-AGGAAATCTAGATGAGAGGCCGGCAAGGATAC-3 MFS2.0_XbaI_rev: (SeqIDNo.61) 5-CCAGGTTCTAGACGCCAGGATTGAACAGTACC-3.
(85) The amplification of the PCR product (1483 base pairs) was carried out using the Phusion High-Fidelity Master Mix from New England Biolabs (Frankfurt) polymerase. The PCR product was cleaved using XbaI and ligated into the vector pBBR1MCS-2::ABC (Seq ID No. 40) likewise cleaved using XbaI by means of Fast Link Ligation Kit (Epicentre Technologies; Madison, Wis., USA). The target vector pEC-XT99A::ABCM (Seq ID No. 55) obtained has a size of 12263 base pairs. The insert of the vector was sequenced. The chromosomal DNA was isolated by means of DNeasy Blood and Tissue Kit (Qiagen; Hilden) according to manufacturer's instructions. The carrying-out of the PCR, the checking of the successful amplification of the PCR by means of agarose gel electrophoresis, ethidium bromide staining of the DNA, determination of the PCR fragment size, purification of the PCR products and DNA concentration determination took place in the manner known to the person skilled in the art.
(86) The transformation of C. glutamicum ATCC13032 using the vector pEC-XT99A::ABCM takes place as previously described (Liebl et al., FEMS Microbiol. Lett. 53:299-303 (1989)). The selection of the transformants takes place on LBHIS agar plates (18.5 g/l of brain heart infusion broth, 0.5 M sorbitol, 5 g/l of Bacto tryptone, 2.5 g/l of Bacto yeast extract, 5 g/l of NaCl and 18 g/l of Bacto agar, supplemented with 5 mg/l of tetracycline). The plates were incubated for two days at 33 C. The strain obtained carrying the plasmid is named C. glutamicum pEC-XT99A::ABCM.
19. Construction of a Vector pVWEX1::rfbBDAC for Heterologous Expression in C. glutamicum
(87) For the heterologous expression of the genes rfbBDAC from P. putida under the control of the lac promoter in C. glutamicum, the vector pVWEX1::rfbBDAC (Seq ID No. 57) is constructed. For this, the vector pBBR1MCS-2::rfbBDAC (Seq ID No. 45) is digested using XbaI and the fragment (3840 bp) containing the genes rfbBDAC from P. putida KT2440 and the lac promoter is ligated into the vector pVWEX1 (Seq ID No. 56) digested with XbaI. The resulting plasmid pVWEX1::rfbBDAC (Seq ID No. 57) is 12311 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5 cells (Gibco-BRL, Karlsruhe) takes place in the manner known to the person skilled in the art. The authenticity of the insert is checked by DNA sequence analysis.
(88) The transformation of C. glutamicum ATCC13032 pEC-XT99A, ATCC13032 pEC-XT99A::AB, ATCC13032 pEC-XT99A::ABM, ATCC13032 pEC-XT99A::ABC and ATCC13032 pEC-XT99A::ABCM using the vector pVWEX1::rfbBDAC takes place as previously described (Liebl et al., FEMS Microbiol. Lett. 53:299-303 (1989)). The selection of the transformants takes place on LBHIS agar plates (18.5 g/l of brain heart infusion broth, 0.5 M sorbitol, 5 g/l of Bacto tryptone, 2.5 g/l of Bacto yeast extract, 5 g/l of NaCl and 18 g/l of Bacto agar, supplemented with 5 mg/l of tetracycline and 25 mg/l of kanamycin). The plates were incubated at 33 C. for two days. The strains obtained carrying the plasmids are named C. glutamicum pEC-XT99A pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::AB pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::ABM pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::ABC pVWEX1::rfbBDAC and C. glutamicum pEC-XT99A::ABCM pVWEX1::rfbBDAC.
20. Quantification of the Rhamnolipid Production by Recombinant C. glutamicum Strains
(89) The recombinant strains C. glutamicum strains generated in the Examples 15 to 19 C. glutamicum pEC-XT99A, C. glutamicum pEC-XT99A::AB, C. glutamicum pEC-XT99A::ABC, C. glutamicum pEC-XT99A::ABM, C. glutamicum pEC-XT99A::ABCM, C. glutamicum pEC-XT99A pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::AB pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::ABM pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::ABC pVWEX1::rfbBDAC and C. glutamicum pEC-XT99A::ABCM pVWEX1::rfbBDAC are cultured on LBHIS agar plates using 5 mg/l of tetracycline and 5 mg/l of tetracycline and 25 mg/l of kanamycin. For the investigation of the rhamnolipid production in the shaker flask, precultures are first prepared. For this, an inoculation loop of a strain freshly streaked on an LBHIS agar plate is used and 10 ml of LBHIS medium (18.5 g/l of brain heart infusion broth, 0.5 M sorbitol, 5 g/l of Bacto tryptone, 2.5 g/l of Bacto yeast extract and 5 g/l of NaCl, supplemented with 5 mg/l of tetracycline or 5 mg/l of tetracycline and 25 mg/l of kanamycin) is inoculated into a 100 ml Erlenmeyer flask. The culturing of the strains takes place at 33 C. and 200 rpm overnight. The next morning, 50 ml of CGXII medium (containing 5 mg/l of tetracycline or 5 mg/l of tetracycline and 25 mg/l of kanamycin) are inoculated into a 500 ml Erlenmeyer flask containing baffles with 1 ml of the preculture (start OD.sub.600 0.1).
(90) CGXII Medium:
(91) 20 g/l of (NH.sub.4).sub.2SO.sub.4 (Merck) 5 g/l of urea (Merck) 1 g/l of KH.sub.2PO.sub.4 (Merck) 1 g/l of K.sub.2HPO.sub.4 (Merck) 0.25 g/l of MgSO.sub.4.7H.sub.2O (Merck) 10 mg/l of CaCl.sub.2 (Merck) 42 g/l of MOPS (Roth) 0.2 mg/l of biotin (Merck) 1 ml/l of trace salt solution adjust to pH 7 using NaOH after autoclaving add 1 ml/l of protocatechuic acid (30 g/l dissolved in dil. NaOH, sterile-filtered) and 40 g/l of glucose (Merck)
Trace Salt Solution: 10 g/l of FeSO.sub.4.7H.sub.2O (Merck) 10 g/l of MnSO.sub.4.H.sub.2O (Merck) 1 g/l of ZnSO.sub.4.7H.sub.2O (Merck) 0.2 g/l of CuSO.sub.4.5H.sub.2O (Merck) 20 mg/l of NiCl.sub.2.6H.sub.2O (Merck) to dissolve acidify to pH 1 using HCl
(92) The cultures are cultured at 200 rpm and 33 C. up to an optical density (600 nm) of 0.4-0.6. At this optical density, the cultures are induced by the addition of IPTG (isopropyl--D-thiogalactopyranoside; 1 mM final concentration). The subsequent expression likewise takes place at 33 C. and 200 rpm for 72 h. At intervals of 24 h, a sample of 1 ml of broth is removed from the culture flask. The sample preparation for the following chromatographic analyses and the chromatographic analyses themselves are carried out as described in Example 4.
(93) While C. glutamicum pEC-XT99A produces no rhamnolipids, in the recombinant strains C. glutamicum pEC-XT99A::AB, C. glutamicum pEC-XT99A::ABC, C. glutamicum pEC-XT99A::ABM and C. glutamicum pEC-XT99A::ABCM the formation of rhamnolipids is detectable. With the aid of reference materials, it is shown that C. glutamicum pEC-XT99A::AB and C. glutamicum pEC-XT99A::ABM only form monorhamnosyl lipids, while C. glutamicum pEC-XT99A::ABC, C. glutamicum pEC-XT99A::ABM and C. glutamicum pEC-XT99A::ABCM are able to form dirhamnosyl lipids and monorhamnosyl lipids. Furthermore, it is shown that C. glutamicum pEC-XT99A::ABM and C. glutamicum pEC-XT99A::ABCM are able to form more monorhamnosyl lipids or dirhamnosyl lipids and monorhamnosyl lipids than the respective reference strains C. glutamicum pEC-XT99A::AB and C. glutamicum pEC-XT99A::ABC without amplification of the pa1131 gene from Pseudomonas aeruginosa.
(94) Moreover, it is shown that the strains C. glutamicum pEC-XT99A::AB pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::ABM pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::ABC pVWEX1::rfbBDAC and C. glutamicum pEC-XT99A::ABCM pVWEX1::rfbBDAC form significantly more mono- (C. glutamicum pEC-XT99A::AB pVWEX1::rfbBDAC and C. glutamicum pEC-XT99A::ABM pVWEX1::rfbBDAC) or mono- and dirhamnosyl lipids (C. glutamicum pEC-XT99A::ABC pVWEX1::rfbBDAC and C. glutamicum pEC-XT99A::ABCM pVWEX1::rfbBDAC) than the strains, C. glutamicum pEC-XT99A::ABM, C. glutamicum pEC-XT99A::ABC and C. glutamicum pEC-XT99A::ABCM without amplification of the of the rfbBDA genes from P. putida.
21. Construction of Pseudomonas Strains that Carry the Plasmids pBBR1MCS-2, pBBR1MCS-2::AB, pBBR1MCS-2::ABC, pBBR1MCS-2::ABM and pBBR1MCS-2::ABCM
(95) The plasmids pBBR1MCS-2, pBBR1MCS-2::AB, pBBR1MCS-2::ABC, pBBR1MCS-2::ABM and pBBR1MCS-2::ABCM are incorporated in Pseudomonas fluorescens DSM 50090, Pseudomonas fluorescens DSM 9958, Pseudomonas putida DSM 6899, Pseudomonas putida DSM 50204, Pseudomonas putida 50194, P. brassicacearum DSM 13227, P. stutzeri DSM 10701, Pseudomonas stutzeri DSM 4166 and Pseudomonas fulva DSM 17717 by electroporation. The transformation of Pseudomonas strains takes place as described previously (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The selection of the transformants takes place on nutrient agar plates (5 g/l of peptone; 3 g/l of meat extract; 15 g/l of agar; pH 7; supplemented with 50 mg/l of kanamycin). The plates are incubated at 30 C. or rather 28 C. for two days. The strains obtained, carrying the plasmids, are named Pseudomonas fluorescens DSM 50090 pBBR1MCS-2, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2, Pseudomonas putida DSM 6899 pBBR1MCS-2, Pseudomonas putida DSM 50204 pBBR1MCS-2, Pseudomonas putida 50194 pBBR1MCS-2, P. brassicacearum DSM 13227 pBBR1MCS-2, P. stutzeri DSM 10701 pBBR1MCS-2, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2, Pseudomonas fulva DSM 17717 pBBR1MCS-2, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::AB, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::AB, Pseudomonas putida DSM 6899 pBBR1MCS-2::AB, Pseudomonas putida DSM 50204 pBBR1MCS-2::AB, Pseudomonas putida 50194 pBBR1MCS-2::AB, P. brassicacearum DSM 13227 pBBR1MCS-2::AB, P. stutzeri DSM 10701 pBBR1MCS-2::AB, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::AB, Pseudomonas fulva DSM 17717 pBBR1MCS-2::AB, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABC, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABC, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABC, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABC, Pseudomonas putida 50194 pBBR1MCS-2::ABC, P. brassicacearum DSM 13227 pBBR1MCS-2::ABC, P. stutzeri DSM 10701 pBBR1MCS-2::ABC, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABC, Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABC, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABCM, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABCM, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABCM, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABCM, Pseudomonas putida 50194 pBBR1MCS-2::ABCM, P. brassicacearum DSM 13227 pBBR1MCS-2::ABCM, P. stutzeri DSM 10701 pBBR1MCS-2::ABCM, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABCM, Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABCM, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABM, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABM, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABM, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABM, Pseudomonas putida 50194 pBBR1MCS-2::ABM, P. brassicacearum DSM 13227 pBBR1MCS-2::ABM, P. stutzeri DSM 10701 pBBR1MCS-2::ABM, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABM and Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABM.
22. Quantification of the Rhamnolipid Production by Recombinant Pseudomonas Strains
(96) The recombinant strains Pseudomonas strains Pseudomonas fluorescens DSM 50090, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2, Pseudomonas putida DSM 6899 pBBR1MCS-2, Pseudomonas putida DSM 50204 pBBR1MCS-2, Pseudomonas putida 50194 pBBR1MCS-2, P. brassicacearum DSM 13227 pBBR1MCS-2, P. stutzeri DSM 10701 pBBR1MCS-2, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2, Pseudomonas fulva DSM 17717 pBBR1MCS-2, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::AB, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::AB, Pseudomonas putida DSM 6899 pBBR1MCS-2::AB, Pseudomonas putida DSM 50204 pBBR1MCS-2::AB, Pseudomonas putida 50194 pBBR1MCS-2::AB, P. brassicacearum DSM 13227 pBBR1MCS-2::AB, P. stutzeri DSM 10701 pBBR1MCS-2::AB, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::AB, Pseudomonas fulva DSM 17717 pBBR1MCS-2::AB, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABC, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABC, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABC, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABC, Pseudomonas putida 50194 pBBR1MCS-2::ABC, P. brassicacearum DSM 13227 pBBR1MCS-2::ABC, P. stutzeri DSM 10701 pBBR1MCS-2::ABC, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABC, Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABC, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABCM, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABCM, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABCM, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABCM, Pseudomonas putida 50194 pBBR1MCS-2::ABCM, P. brassicacearum DSM 13227 pBBR1MCS-2::ABCM, P. stutzeri DSM 10701 pBBR1MCS-2::ABCM, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABCM, Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABCM, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABM, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABM, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABM, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABM, Pseudomonas putida 50194 pBBR1MCS-2::ABM, P. brassicacearum DSM 13227 pBBR1MCS-2::ABM, P. stutzeri DSM 10701 pBBR1MCS-2::ABM, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABM and Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABM generated in Example 21 are cultured on LB agar kanamycin (50 g/ml) plates. The subsequent culturing for the production of the rhamnolipids takes place as described in Example 12. The sample preparation for the following chromatographic analyses and the chromatographic analyses themselves are carried out as described in Example 4.
(97) While the Pseudomonas strains Pseudomonas fluorescens DSM 50090, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2, Pseudomonas putida DSM 6899 pBBR1MCS-2, Pseudomonas putida DSM 50204 pBBR1MCS-2, Pseudomonas putida 50194 pBBR1MCS-2, P. brassicacearum DSM 13227 pBBR1MCS-2, P. stutzeri DSM 10701 pBBR1MCS-2, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2, Pseudomonas fulva DSM 17717 pBBR1MCS-2 produce no rhamnolipids, in the recombinant strains Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::AB, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::AB, Pseudomonas putida DSM 6899 pBBR1MCS-2::AB, Pseudomonas putida DSM 50204 pBBR1MCS-2::AB, Pseudomonas putida 50194 pBBR1MCS-2::AB, P. brassicacearum DSM 13227 pBBR1MCS-2::AB, P. stutzeri DSM 10701 pBBR1MCS-2::AB, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::AB, Pseudomonas fulva DSM 17717 pBBR1MCS-2::AB, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABM, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABM, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABM, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABM, Pseudomonas putida 50194 pBBR1MCS-2::ABM, P. brassicacearum DSM 13227 pBBR1MCS-2::ABM, P. stutzeri DSM 10701 pBBR1MCS-2::ABM, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABM and Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABM the formation of monorhamnosyl lipids and in the strains Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABC, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABC, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABC, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABC, Pseudomonas putida 50194 pBBR1MCS-2::ABC, P. brassicacearum DSM 13227 pBBR1MCS-2::ABC, P. stutzeri DSM 10701 pBBR1MCS-2::ABC, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABC, Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABC, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABCM, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABCM, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABCM, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABCM, Pseudomonas putida 50194 pBBR1MCS-2::ABCM, P. Brassicacearum DSM 13227 pBBR1MCS-2::ABCM, P. stutzeri DSM 10701 pBBR1MCS-2::ABCM, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABCM and Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABCM the formation of mono- and dirhamnosyl lipids is detectable.
(98) Moreover, fewer monorhamnosyl lipids are formed by the recombinant Pseudomonas strains Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABM, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABM, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABM, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABM, Pseudomonas putida 50194 pBBR1MCS-2::ABM, P. brassicacearum DSM 13227 pBBR1MCS-2::ABM, P. stutzeri DSM 10701 pBBR1MCS-2::ABM, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABM Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABM and by the recombinant Pseudomonas strains Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABCM, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABCM, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABCM, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABCM, Pseudomonas putida 50194 pBBR1MCS-2::ABCM, P. brassicacearum DSM 13227 pBBR1MCS-2::ABCM, P. stutzeri DSM 10701 pBBR1MCS-2::ABCM, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABCM and Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABCM fewer mono- and dirhamnosyl lipids are formed than by the respective reference strains without the P. aeruginosa gene pa1131 Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::AB, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::AB, Pseudomonas putida DSM 6899 pBBR1MCS-2::AB, Pseudomonas putida DSM 50204 pBBR1MCS-2::AB, Pseudomonas putida 50194 pBBR1MCS-2::AB, P. brassicacearum DSM 13227 pBBR1MCS-2::AB, P. stutzeri DSM 10701 pBBR1MCS-2::AB, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::AB and Pseudomonas fulva DSM 17717 pBBR1MCS-2::AB and Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABC, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABC, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABC, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABC, Pseudomonas putida 50194 pBBR1MCS-2::ABC, P. brassicacearum DSM 13227 pBBR1MCS-2::ABC, P. stutzeri DSM 10701 pBBR1MCS-2::ABC, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABC and Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABC without amplification of the pa1131 gene from Pseudomonas aeruginosa.
23. Construction of the Vectors pBBR1MCS-2::ABPAO1-C1 and pBBR1MCS-2::ABPA7-CE264 for the Heterologous Expression of Alternative rhIA, rhIB and rhIC Genes from Pseudomonas aeruginosa PAO1, Pseudomonas aeruginosa PA7, Pseudomonas aeruginosa 1 and Burkholderia thailandensis E264 in P. putida
(99) For the heterologous expression of the genes rhIA, rhIB and rhIC from Pseudomonas aeruginosa PAO1 and Pseudomonas aeruginosa PA7, the plasmids pBBR1MCS-2::ABPAO1 (Seq ID No. 62) and pBBR1MCS-2::ABPA7 (Seq ID No. 63) are first constructed. For this, the synthetic operons rhIABPAO1 (Seq ID No. 64) and rhIABPA7 (Seq ID No. 65) are synthesized by the company DNA 2.0 (Menlo Park, Calif., U.S.A) and intercloned in the commercial vector pJ294 (DNA 2.0). The basis for the synthesis is the already known genomic sequence of the strains Pseudomonas aeruginosa PAO1 and Pseudomonas aeruginosa PA7. Starting from the vectors pJ294::ABPAO1 and pJ294::ABPA7, the synthetic operons are cleaved from the vectors by means of KpnI and XbaI and subsequently ligated into the expression vector pBBR1MCS-2 (Seq ID No. 49) (Kovach et al., 1995: Four new derivatives of the broad-host-range cloning vector pBBR1MCS carrying different antibiotic-resistance cassettes. Gene, 166:175-176) cleaved using KpnI and XbaI. The resulting plasmids pBBR1MCS-2::ABPAO1 (Seq ID No. 62) and pBBR1MCS-2::ABPA7 (Seq ID No. 63) are 7332 and 7354 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5 cells (Gibco-BRL, Karlsruhe) takes place in the manner known to the person skilled in the art. The authenticity of the insert is checked by DNA sequence analysis.
(100) In the second step, the plasmids pBBR1MCS-2::ABPAO1-C1 (Seq ID No. 66) and pBBR1MCS-2::ABPA7-CE264 (Seq ID No. 67) are produced. For this, the rhIC genes from Pseudomonas aeruginosa 1 (Seq ID No. 68) and Burkholderia thailandensis E264 (Seq ID No. 76) are synthesized by the company DNA 2.0 (Menlo Park, Calif., U.S.A) and intercloned in the commercial vector pJ294 (DNA 2.0). The basis for the synthesis is the already known genomic sequence of the strains Pseudomonas aeruginosa 1 and Burkholderia thailandensis E264. Starting from the vectors pJ294::C1 and pJ294::CE264, the rhIC genes are cleaved from the vectors by means of Xba and SacI and subsequently ligated into the vectors pBBR1MCS-2::ABPAO1 (Seq ID No. 62) and pBBR1MCS-2::ABPA7 (Seq ID No. 63) likewise cleaved using Xba and SacI. The resulting plasmids pBBR1MCS-2::ABPAO1-C1 (Seq ID No. 66) and pBBR1MCS-2::ABPA7-CE264 (Seq ID No. 67) are 8325 and 8335 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5 cells (Gibco-BRL, Karlsruhe) takes place in the manner known to the person skilled in the art. The authenticity of the insert is checked by DNA sequence analysis.
(101) The transformation of Pseudomonas putida KT2440 and GPp104 using the vectors pBBR1MCS-2, pBBR1MCS-2::ABPAO1-C1 and pBBR1MCS-2::ABPA7-CE264 takes place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of every 10 clones was isolated and analyzed. The strains obtained carrying the plasmids are named P. putida KT2440 pBBR1MCS-2, P. putida KT2440 pBBR1MCS-2::ABPAO1-C1, P. putida KT2440 pBBR1MCS-2::ABPA7-CE264, P. putida GPp104 pBBR1MCS-2, P. putida GPp104 pBBR1MCS-2::ABPAO1-C1 and P. putida GPp104 pBBR1MCS-2::ABPA7-CE264.
24. Quantification of the Rhamnolipid Production by Recombinant P. putida Strains Having Alternative rhIA, rhIB and rhIC Genes from Pseudomonas aeruginosa PAO1, Pseudomonas aeruginosa PA7, Pseudomonas aeruginosa 1 and Burkholderia thailandensis E264
(102) The recombinant strains P. putida strains generated in Example 23 are cultured on LB agar kanamycin (50 g/ml) plates. The subsequent culturing for the production of the rhamnolipids takes place as described in Example 12. The sample preparation for the following chromatographic analyses and the chromatographic analyses themselves are carried out as described in Example 4.
(103) While the strains P. putida KT2440 pBBR1MCS-2 and P. putida GPp104 pBBR1MCS-2 are not able to produce mono- and dirhamnosyl lipids, the strains P. putida KT2440 pBBR1MCS-2::ABPAO1-C1, P. putida KT2440 pBBR1MCS-2::ABPA7-CE264, P. putida GPp104 pBBR1MCS-2::ABPAO1-C1 and P. putida GPp104 pBBR1MCS-2::ABPA7-CE264 form both mono- as well as dirhamnosyl lipids. It is shown that the strains are able to produce more mono- and dirhamnosyl lipids with an attenuation of the polyhydroxybutyrate formation (P. putida GPp104 pBBR1MCS-2::ABPAO1-C1 and P. putida GPp104 pBBR1MCS-2::ABPA7-CE264) than the strains without attenuation of the polyhydroxybutyrate formation (P. putida KT2440 pBBR1MCS-2::ABPAO1-C1 and P. putida KT2440 pBBR1MCS-2::ABPA7-CE264).
25. Construction of the Vectors pBBR1MCS-2::AB_rfbBDAC, pBBR1MCS-2::ABM_rfbBDAC and pBBR1MCS-2::ABMC_rfbBDAC for the Overexpression of the P. putida rfbBDAC Operon in P. putida and E. coli
(104) For the construction of the vectors pBBR1MCS-2::AB_rfbBDAC, pBBR1MCS-2::ABM_rfbBDAC and pBBR1MCS-2::ABMC_rfbBDAC for the overexpression of the P. putida rfbBDAC operon in P. putida and E. coli, the P. putida rfbBDAC operon was first amplified by PCR. The vector pBBR1MCS-2::rfbBDAC (Seq ID No. 45) served as matrix for a PCR. The following oligonucleotides were used:
(105) TABLE-US-00010 RL_AgeI-fw: (SeqIDNo.71) 5-TATATATAACCGGTATTAATGCAGCTGGCACGAC-3 RL_AgeI_rev: (SeqIDNo.72) 5-GGCCGACCGGTACTAGTGGA-3
(106) The PCR was carried out using the Phusion High-Fidelity Master Mix of New England Biolabs (Frankfurt) polymerase. It took place in the manner known to the person skilled in the art. The target sequence (lac promoter and rfbBDAC) was intercloned in the Trenzyme alligator cloning system. E. coli DH5 (New England Biolabs; Frankfurt) transformants were selected and the plasmid DNA of different candidates was isolated and sequenced. After the sequence had been checked and examined for correctness, the vector was cleaved using AgeI. The target fragment was ligated into the vectors pBBR1MCS-2::AB (Seq ID No. 38), pBBR1MCS-2::ABM (Seq ID No. 42) and pBBR1MCS-2::ABMC (Seq ID No. 51) likewise cleaved using AgeI by means of conventional ligation methods. The resulting vectors pBBR1MCS-2::AB_rfbBDAC (Seq ID No. 73), pBBR1MCS-2::ABM_rfbBDAC (Seq ID No. 74) and pBBR1MCS-2::ABMC_rfbBDAC (Seq ID No. 75) have sizes of 11960, 13289 and 14250 base pairs. The inserts of the vectors were sequenced. The carrying-out of the PCR, the checking of the successful amplification of the PCR by means of agarose gel electrophoresis, ethidium bromide staining of the DNA, determination of the PCR fragment size, purification of the PCR products and DNA concentration determination took place in the manner known to the person skilled in the art. The transformation of Pseudomonas putida KT2440 using the vectors pBBR1MCS-2::AB_rfbBDAC, pBBR1MCS-2::ABM_rfbBDAC and pBBR1MCS-2::ABMC_rfbBDAC took place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of every 10 clones was isolated and analyzed. The strains obtained carrying the plasmids are named P. putida KT2440 pBBR1MCS-2::AB_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABM_rfbBDAC and P. putida KT2440 pBBR1MCS-2::ABMC_rfbBDAC.
26. Quantification of the Rhamnolipid Production by Recombinant P. putida KT2440 pBBR1MCS-2::AB_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABM_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABMC_rfbBDAC, P. putida KT2440 pBBR1MCS-2::AB, P. putida KT2440 pBBR1MCS-2::ABM, P. putida KT2440 pBBR1MCS-2::ABC and P. putida KT2440 pBBR1MCS-2::ABMC
(107) The recombinant strains P. putida strains generated in the Examples 2, 7 and 25 are cultured on LB agar-kanamycin (50 g/ml) plates. The subsequent culturing for the production of the rhamnolipids takes place as described in Example 12. The sample preparation for the following chromatographic analyses and the chromatographic analyses themselves take place as described in Example 4.
(108) It is shown that P. putida KT2440 pBBR1MCS-2::AB_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABM_rfbBDAC, P. putida KT2440 pBBR1MCS-2::AB and P. putida KT2440 pBBR1MCS-2::ABM are able to form monorhamnosyl lipids, while P. putida KT2440 pBBR1MCS-2::ABMC_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABC and P. putida KT2440 pBBR1MCS-2::ABMC are able to form mono- and dirhamnosyl lipids.
(109) Furthermore, it is shown that P. putida KT2440 pBBR1MCS-2::ABM_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABM, KT2440 pBBR1MCS-2::ABMC_rfbBDAC and KT2440 pBBR1MCS-2::ABMC are able to form more mono- and dirhamnosyl lipids than the corresponding control strains P. putida KT2440 pBBR1MCS-2::AB_rfbBDAC, P. putida KT2440 pBBR1MCS-2::AB, KT2440 pBBR1MCS-2::ABC_rfbBDAC and KT2440 pBBR1MCS-2::ABC without amplification of the Pseudomonas aeruginosa gene pa1131.
(110) Finally, it is shown that P. putida KT2440 pBBR1MCS-2::AB_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABM_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABMC_rfbBDAC are able to form more mono- (P. putida KT2440 pBBR1MCS-2::AB_rfbBDAC and P. putida KT2440 pBBR1MCS-2::ABM_rfbBDAC) and mono- and dirhamnosyl lipids (P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC and P. putida KT2440 pBBR1MCS-2::ABMC_rfbBDAC) than the respective control strains P. putida KT2440 pBBR1MCS-2::AB, P. putida KT2440 pBBR1MCS-2::ABM, P. putida KT2440 pBBR1MCS-2::ABC, P. putida KT2440 pBBR1MCS-2::ABMC without amplification of the P. putida genes rfbBDAC.
27. Generation of Recombinant E. coli W3110 pBBR1MCS-2::AB, E. coli W3110 pBBR1MCS-2::ABM, E. coli W3110 pBBR1MCS-2::ABC, E. coli W3110 pBBR1MCS-2::ABCM, E. coli W3110 pBBR1MCS-2::AB_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC and E. coli W3110 pBBR1MCS-2::ABCM_rfbBDAC
(111) The transformation of E. coli W3110 took place as described previously (Miller J H. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Plainview, N.Y.: Cold Spring Harbor Lab. Press; 1992) by means of electroporation. The plasmid DNA of every 10 clones was isolated and analyzed. The obtained strains carrying the plasmids were named E. coli W3110 pBBR1MCS-2::AB, E. coli W3110 pBBR1MCS-2::ABM, E. coli W3110 pBBR1MCS-2::ABC, E. coli W3110 pBBR1MCS-2::ABCM, E. coli W3110 pBBR1MCS-2::AB_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC and E. coli W3110 pBBR1MCS-2::ABCM_rfbBDAC.
28. Quantification of the Rhamnolipid Production by Recombinant E. coli W3110 pBBR1MCS-2::AB, E. coli W3110 pBBR1MCS-2::ABM, E. coli W3110 pBBR1MCS-2::ABC, E. coli W3110 pBBR1MCS-2::ABCM, E. coli W3110 pBBR1MCS-2::AB_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC and E. coli W3110 pBBR1MCS-2::ABCM_rfbBDAC
(112) The recombinant E. coli strains generated in Example 27 are cultured on LB agar kanamycin (50 g/ml) plates. The subsequent culturing for the production of the rhamnolipids takes place as described in Example 10. The sample preparation for the following chromatographic analyses and the chromatographic analyses themselves take place as described in Example 4.
(113) It is shown that E. coli W3110 pBBR1MCS-2::AB, E. coli W3110 pBBR1MCS-2::ABM, E. coli W3110 pBBR1MCS-2::AB_rfbBDAC and E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC are able to form monorhamnosyl lipids, while E. coli W3110 pBBR1MCS-2::ABC, E. coli W3110 pBBR1MCS-2::ABCM, E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC and E. coli W3110 pBBR1MCS-2::ABCM_rfbBDAC are able to form mono- and dirhamnosyl lipids. Furthermore, it is shown that E. coli W3110 pBBR1MCS-2::ABM and E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC form more monorhamnosyl lipids than E. coli W3110 pBBR1MCS-2::AB and E. coli W3110 pBBR1MCS-2::AB_rfbBDAC without amplification of the Pseudomonas aeruginosa gene pa1131.
(114) Furthermore, it is shown that E. coli W3110 pBBR1MCS-2::ABCM and E. coli W3110 pBBR1MCS-2::ABCM_rfbBDAC form more mono- and dirhamnosyl lipids than E. coli W3110 pBBR1MCS-2::ABC and E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC without amplification of the Pseudomonas aeruginosa gene pa1131. Furthermore, it is shown that E. coli W3110 pBBR1MCS-2::ABM and E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC form more monorhamnosyl lipids than E. coli W3110 pBBR1MCS-2::AB and E. coli W3110 pBBR1MCS-2::AB_rfbBDAC without amplification of the Pseudomonas aeruginosa gene pa1131.
(115) Finally, it is shown that E. coli W3110 pBBR1MCS-2::AB_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC and E. coli W3110 pBBR1MCS-2::ABCM_rfbBDAC are able to form more mono- (E. coli W3110 pBBR1MCS-2::AB_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC) and mono- and dirhamnosyl lipids (E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC and E. coli W3110 pBBR1MCS-2::ABCM_rfbBDAC) than the respective control strains E. coli W3110 pBBR1MCS-2::AB, E. coli W3110 pBBR1MCS-2::ABM, E. coli W3110 pBBR1MCS-2::ABC and E. coli W3110 pBBR1MCS-2::ABCM without amplification of the P. putida genes rfbBDAC.