Lipid production

Abstract

The present invention relates to at least one cell for producing at least one lipid with general formula II from at least one carbon substrate, ##STR00001##
wherein R.sup.1 and R.sup.2 independently of one another comprises identical or different organic radicals each with 5 to 13 carbon atoms,
wherein the cell is a non-pathogenic cell that is genetically modified to increase the heterologous expression relative to the wild type cell of: an enzyme (E.sub.2) capable of converting 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA/ACP or 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) and NDP-glucose into β-D-glucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate.

Claims

1. A microbial cell for producing at least one lipid with general formula II from at least one carbon substrate, ##STR00009## wherein R.sup.1 and R.sup.2 independently of one another comprise identical or different organic radicals each with 5 to 13 carbon atoms; and wherein the cell is a non-pathogenic cell that is genetically modified to increase the heterologous expression relative to the wild type cell of an enzyme (E.sub.2) capable of converting 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA/ACP and/or 3-(3-hydroxy-alkanoyloxy)alkanoic acid (HAA) in combination with NDP-glucose into β-D-glucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate wherein the enzyme E.sub.2 is a glycosyltransferase (EC 2.4) comprising SEQ ID NO: 4 or variant thereof, wherein the variant comprises at least 90% sequence identity to SEQ ID NO: 4.

2. The microbial cell of claim 1, wherein the R in the lipid with general formula II is a monounsaturated alkyl radical.

3. The microbial cell of claim 2, wherein the alkyl radical is selected from the group consisting of nonenyl, undecenyl and tridecenyl.

4. The microbial cell of claim 1, wherein the cell is further genetically modified to increase the heterologous expression relative to the wild type cell of an enzyme (E.sub.1) capable of converting 3-hydroxyalkanoyl-CoA/ACP into 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA/ACP and further to 3-(3-hydroxy-alkanoyloxy)alkanoic acid (HAA).

5. The microbial cell of claim 3, wherein the cell is further genetically modified to increase the heterologous expression relative to the wild type cell of an enzyme (E.sub.1) capable of converting 3-hydroxyalkanoyl-CoA/ACP into 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA/ACP and further to 3-(3-hydroxy-alkanoyloxy)alkanoic acid (HAA).

6. The microbial cell of claim 4, wherein the enzyme E.sub.1 is a 3-(3-hydroxy alkanoyloxy)alkanoic acid (HAA) synthase.

7. The microbial cell of claim 4, wherein the enzyme E.sub.1 comprises SEQ ID NO: 2 or variant thereof, wherein the variant comprises at least 90% sequence identity to SEQ ID NO: 2.

8. The microbial cell of claim 6, wherein the enzyme E.sub.1 comprises SEQ ID NO: 2 or variant thereof, wherein the variant comprises at least 90% sequence identity to SEQ ID NO: 2.

9. The microbial cell of claim 4, wherein the enzyme E.sub.1 comprises a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and variants thereof, wherein the variants comprise at least 90% sequence identity to SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14 respectively.

10. The microbial cell of claim 6, wherein the enzyme E.sub.1 comprises a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and variants thereof, wherein the variants comprise at least 90% sequence identity to SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14 respectively.

11. The microbial cell of claim 1, wherein the cell is genetically modified to increase the expression of: enzyme E.sub.2 comprising SEQ ID NO: 4 or a variant thereof, wherein the variant comprises at least 90% sequence identity to SEQ ID NO: 4; and —enzyme E.sub.1 comprising SEQ ID NO: 2 or a variant thereof, wherein the variant comprises at least 90% sequence identity to SEQ ID NO: 2.

12. The microbial cell of claim 10, wherein the cell is genetically modified to increase the expression of: enzyme E.sub.2 comprising SEQ ID NO: 4 or a variant thereof, wherein the variant comprises at least 90% sequence identity to SEQ ID NO: 4; and —enzyme E.sub.1 comprising SEQ ID NO: 2 or a variant thereof, wherein the variant comprises at least 90% sequence identity to SEQ ID NO: 2.

13. The microbial cell claim 1, wherein the cell produces a further lipid with general formula I from the carbon substrate, ##STR00010## wherein R.sup.1 and R.sup.2 independently of one another comprise identical or different organic radicals each with 5 to 13 carbon atoms.

14. The microbial cell of claim 13, wherein the R in the lipid with general formula I is a monounsaturated alkyl radical.

15. The microbial cell of claim 1, wherein the carbon source is selected from the group consisting of glucose, dextrose, sucrose, polysaccharides, vegetal oils, animal fats, fatty acids, fatty acid esters, carbonaceous gases, alkanes, glycerol, acetate, ethanol and methanol.

16. The microbial cell of claim 14, wherein the carbon source is selected from the group consisting of glucose, dextrose, sucrose, polysaccharides, vegetal oils, animal fats, fatty acids, fatty acid esters, carbonaceous gases, alkanes, glycerol, acetate, ethanol and methanol.

17. The microbial cell of claim 1, wherein the cell is selected from the group consisting of Acinetobacter sp., Bacillus sp., Brevibacterium sp., Burkholderia sp., Chlorella sp., Clostridium sp., Corynebacterium sp., Cyanobakterien, Escherichia sp., Pseudomonas sp., Klebsiella sp., Salmonella sp., Rhizobium sp., Saccharomyces sp., Pichia sp., and Nostoc sp.

18. A method of producing at least one lipid with general formula II and/or general formula I: ##STR00011## wherein IV and R.sup.2 independently of one another comprise identical or different organic radicals each with 5 to 13 carbon atoms; and wherein the method comprises a step of contacting at least one microbial cell of claim 1 with at least one carbon source.

Description

EXAMPLES

(1) The foregoing describes preferred embodiments, which, as will be understood by those skilled in the art, may be subject to variations or modifications in design, construction or operation without departing from the scope of the claims. These variations, for instance, are intended to be covered by the scope of the claims.

Example 1

(2) Construction of an expression vector for the Serratia rubidaea genes rbwAB For the heterologous expression of the genes rbwA (SEQ ID NO. 1) as enzyme E.sub.1 and rbwB (SEQ ID NO. 3) as enzyme E.sub.2 from Serratia rubidaea the plasmid pACYC_rbwAB_Srub was constructed. The synthetic operon consisting of rbwAB_Srub (SEQ ID NO: 15) which encode an 3-(3′-hydroxyalkanoyloxy)alkanoic acids (HAAs) synthase (RbwA, SEQ ID NO: 2) and a glucosyltransferase (RbwB, SEQ ID NO: 4), respectively, was cloned under the control of the rhamnose inducible promoter P.sub.rha into the vector pACYCATh-5, which is based on pAYCY184 (New England Biolabs, Frankfurt/Main, Germany). Downstream of the synthetic operon a terminator sequence is located. The genes were amplified from genomic DNA of S. rubidaea via PCR. The P.sub.Rha promoter cassette (SEQ ID NO: 16) and the terminator sequence (SEQ ID NO: 17) were amplified from E. coli K12 genomic DNA. The plasmid pACYCATh-5 carries a p15A origin of replication for E. coli and a pVS1 origin of replication for the replication in P. putida KT2440. The pVS1 origin comes from the Pseudomonas plasmid pVS1 (Itoh Y, et al. Plasmid 1984, 11(3), 206-20). rbwA and rbwB were fused via cross-over PCR to generate an optimized operon. For amplification the Phusion™ High-Fidelity Master Mix from New England Biolabs (Frankfurt/Main, Germany) was used according to manufacturer's manual. In the next step the fusion construct was cloned into the vector pACYCATh-5 using the restriction sites ApaI/PspXI. The ligated product was transformed into chemically competent E. coli DH5c cells (New England Biolabs, Frankfurt/Main, Germany). Procedure of PCR purification, cloning and transformation were carried out according to manufacturer's manual. The correct insertion of the target genes was checked by restriction analysis and the authenticity of the introduced DNA fragments was verified by DNA sequencing. The resulting plasmid was named pACYC_rbwAB_Srub (SEQ ID NO: 18). The P. putida strain KT2440 was transformed with the plasmid pACYC_rbwAB_Srub by means of electroporation (Iwasaki K, et al., Biosci. Biotech. Biochem. 1994. 58(5):851-854)) and plated onto LB-agar plates supplemented with kanamycin (50 μg/mL). Transformants were checked for the presence of the correct plasmid by plasmid preparation and analytic restriction analysis. The resulting strain was named BS-PP-360 (P. putida KT2440 pACYC_rbwAB_Srub).

Example 2

(3) Construction of an expression vector for the Serratia rubidaea gene rbwA For the heterologous expression of the gene rbwA (SEQ ID NO: 1) from S. rubidaea the plasmid pACYC_rbwA_Srub was constructed. For this approach the plasmid pACYC_rbwAB_Srub (see Example 1) was cut with the restriction enzymes NsiI and XhoI to eliminate rbwB. To re-ligate the modified vector, the plasmid was treated with T4 DNA polymerase (New England Biolabs, Frankfurt/Main, Germany) in order to remove 3′ overhangs and to fill-in of 5′ overhangs to form blunt ends. The religated product was transformed into chemically competent E. coli DH5a cells (New England Biolabs, Frankfurt/Main, Germany). Procedure of PCR purification, cloning and transformation were carried out according to manufacturer's manual. The correct insertion of the target genes was checked by restriction analysis and the authenticity of the introduced DNA fragments was verified by DNA sequencing. The resulting plasmid was named pACYC_rbwA_Srub (SEQ ID NO: 19). The P. putida strain KT2440 was transformed with the plasmid pACYC_rbwA_Srub by means of electroporation (Iwasaki K, et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854) and plated onto LB-agar plates supplemented with kanamycin (50 μg/mL). Transformants were checked for the presence of the correct plasmid by plasmid preparation and analytic restriction analysis. The resulting strain was named BS-PP-433 (P. putida KT2440 pACYC_rbwA_Srub).

Example 3

(4) Construction of an expression vector for the P. aeruginosa gene rh/A and S. rubidaea gene rbwB For the heterologous expression of the gene rhIA (SEQ ID NO: 5) from P. aeruginosa and rbwB (SEQ ID NO: 3) from S. rubidaea the plasmid pACYC_rhIA_Pa rbwB_Srub was constructed. The synthetic operon consisting of rhIA_Pa (SEQ ID NO: 20) which encodes a 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAAs) synthase (RhIA, SEQ ID NO: 6) and a glucosyltransferase (RbwB, SEQ ID NO: 4), respectively, was cloned under the control of the rhamnose inducible promoter P.sub.rha into the vector pACYCATh-5. Downstream of the synthetic operon a terminator sequence is located. The genes were amplified from genomic DNA of P. aeruginosa and S. rubidaea respectively via PCR. The P.sub.Rha promoter cassette (SEQ ID NO: 16) and the terminator sequence (SEQ ID NO: 17) were amplified from E. coli K12 genomic DNA. The vector is based on pACYC184 (New England Biolabs, Frankfurt/Main, Germany) and carries a p15A origin of replication for E. coli and a pVS1 origin of replication for the replication in P. putida KT2440. The pVS1 origin comes from the Pseudomonas plasmid pVS1 (Itoh Y, Watson J M, Haas D, Leisinger T, Plasmid 1984, 11(3), 206-20). rh/A and rbwB were fused via cross-over PCR to generate an optimized operon. For amplification the Phusion™ High-Fidelity Master Mix from New England Biolabs (Frankfurt/Main, Germany) was used according to manufacturer's manual. In the next step the fusion construct was cloned into the vector pACYCATh-5 using the restriction sites ApaI/PspXI. The ligated product was transformed into chemically competent E. coli DH5c cells (New England Biolabs, Frankfurt/Main, Germany). Procedure of PCR purification, cloning and transformation were carried out according to manufacturer's manual. The correct insertion of the target genes was checked by restriction analysis and the authenticity of the introduced DNA fragments was verified by DNA sequencing. The resulting plasmid was named pACYC_rhIA_Pa rbwB_Srub (SEQ ID NO: 21).

(5) The P. putida strain KT2440 was transformed with the plasmid pACYC_rhIA_Pa rbwB_Srub by means of electroporation (Iwasaki K, et al., Biosci. Biotech. Biochem. 1994. 58(5):851-854)) and plated onto LB-agar plates supplemented with kanamycin (50 μg/mL). Transformants were checked for the presence of the correct plasmid by plasmid preparation and analytic restriction analysis. The resulting strain was named BS-PP-368 (P. putida KT2440 pACYC_rhIA_Pa rbwB_S rub).

Example 4

(6) Production of Lipid R1 with Strain BS-PP-433 (P. putida KT2440 pACYC_rbwA_Srub)

(7) For the production of lipid R1, DASGIP® parallel bioreactor system from Eppendorf (Hamburg, Germany) is used. The fermentation is performed using 1 L reactors. pH and pO2 are measured online for process monitoring. OTR/CTR measurements serve for estimating the metabolic activity and cell fitness, inter alia.

(8) The pH electrodes are calibrated by means of a two-point calibration using standard solutions of pH 4.0 and pH 7.0, as specified in DASGIP's technical instructions. The reactors are equipped with the necessary sensors and connections as specified in the technical instructions, and the agitator shaft is fitted. The reactors are then filled with 300 ml water and autoclaved for 20 min at 121° C. to ensure sterility. The pO2 electrodes are connected to the measuring amplifiers and polarized overnight (for at least 6 h). Thereafter, the water is removed under a clean bench and replaced by fermentation medium (2.2 g/L (NH.sub.4).sub.2SO.sub.4, 0.02 g/L NaCl, 0.4 g/L MgSO.sub.4×7H.sub.2O, 0.04 g/L CaCl.sub.2×2H.sub.2O, sterilized separately: 2 g/L KH.sub.2PO.sub.4, 8.51 g/L KH.sub.2PO.sub.4, 20 g/L glucose, 10 mL/L trace elements solution M12 (sterile-filtered: 0.2 g/L ZnSO4×7H.sub.2O, 0.1 g/L MnCl.sub.2×4H.sub.2O, 1.5 g/L Na.sub.3-Citrat×2H.sub.2O, 0.1 g/L CuSO.sub.4×5H.sub.2O, 0.002 g/L NiCl.sub.2×6H.sub.2O, 0.003 g/L Na.sub.2MoO.sub.4×2H.sub.2O, 0.03 g/L H.sub.3BO.sub.3, 1 g/L FeSO.sub.4×7H.sub.2O). Thereafter, the pO2 electrodes are calibrated to 100% with a one-point calibration (stirrer: 600 rpm/aeration 10 sl/h air), and the feed, correction agent and induction agent lines are cleaned by “cleaning in place” as specified in the technical instructions. To this end, the tubes are rinsed first with 70% ethanol, then with 1 M NaOH, then with sterile fully-demineralized water and, finally, filled with the respective media.

(9) Using the BS-PP-433 (P. putida strain KT2440 pACYC_rbwA_Srub), 25 ml LB1 medium (10 g/L tryptone, 5 g/L yeast extract, 1 g/L NaCl, pH 7.0) supplemented with kanamycin (50 μg/mL) in a baffeled shake flask are inoculated with 100 μl of a glycerol stock solution and incubated for ˜18 h over night at 30° C. and 200 rpm. The first preculture is used to inoculate 50 ml 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 elements solution SL6 (sterile-filtered: 0.3 g/L H.sub.3BO.sub.3, 0.2 g/L CoCl.sub.2×H.sub.2O, 0.1 g/L ZnSO.sub.4×7 H.sub.2O, 0.03 g/L MnCl.sub.2×4H.sub.2O, 0.01 g/L CuCl.sub.2×2H.sub.2O, 0.03 g/L Na.sub.2MoO.sub.4×2H.sub.2O, 0.02 g/L NiCl.sub.2×6H.sub.2O) in a 500 ml baffeled shake flask (starting OD.sub.600 0.2). The culture is incubated for ˜7 h at 200 rpm and 30° C. In order to inoculate the reactors with an optical density of 0.7, the 00600 of the second preculture stage is measured and the amount of culture required for the inoculation is calculated.

(10) The required amount of culture is added with the help of a 30 ml syringe through a septum into the heat-treated and aerated reactor.

(11) The standard program shown in Table 1 is used:

(12) TABLE-US-00001 TABLE 1 Standard program used for heated and aerated reactor a) DO controller pH controller Preset 0% Preset 0 mL/h P 0.1 P 5 Ti 300 s Ti 200 s Min 0% Min 0 mL/h Max 100%  Max 40 mL/h b) N XO2 F (Rotation) From To (gas mixture) from to (gas flow) from to Growth and 0% 40% Growth and  0% 100% Growth and 35% 100% biotrans- biotrans- biotrans- formation 500 rpm 1500 rpm formation 21%  21% formation 9 sl/h 72 sL/h c) Script Trigger fires 31% DO (1/60 h) Temperature 37° C. Induction 3 h after the rhamnose feed start Feed trigger 50% DO Feed rate 1.5 [mL/h]

(13) The pH is adjusted unilaterally to pH 7.0 with 12.5% strength ammonia solution. During the growth phase and the biotransformation, the dissolved oxygen (pO2 or DO) in the culture is adjusted to at least 30% via the stirrer speed and the aeration rate. After the inoculation, the DO dropped from 100% to these 30%, where it is maintained permanently for the rest of the fermentation.

(14) The fermentation is carried out as a fed batch. The feed starts with a 2.5 g/L*h glucose feed, composed of 500 g/L glucose, and was triggered via the DO peak which indicates the end of the batch phase. 3 h after the feed start, the expression of lipid R1 production was induced with 0.2% (w/v) rhamnose. The inducer concentration refers to the volume at the beginning of fermentation.

(15) The production of lipid R1 starts with the induction. At specified time points samples are taken from the fermenter to determine the concentration of lipid R1 produced.

(16) The strain BS-PP-433 produces more 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) than the reference strain with an empty plasmid.

Example 5

(17) Production of Rubiwettin RG1 with Strain BS-PP-360 (P. putida KT2440 pACYC_rbwAB_Srub)

(18) For the production of rubiwettin RG1 the DASGIP® parallel bioreactor system from Eppendorf (Hamburg, Germany) was used. The fermentation was performed using 1 L reactors. pH and pO2 were measured online for process monitoring. OTR/CTR measurements served for estimating the metabolic activity and cell fitness, inter alia.

(19) The pH electrodes were calibrated by means of a two-point calibration using standard solutions of pH 4.0 and pH7.0, as specified in DASGIP's technical instructions. The reactors were equipped with the necessary sensors and connections as specified in the technical instructions, and the agitator shaft was fitted. The reactors were then filled with 300 ml water and autoclaved for 20 min at 121° C. to ensure sterility. The pO2 electrodes were connected to the measuring amplifiers and polarized overnight (for at least 6 h). Thereafter, the water was removed under a clean bench and replaced by fermentation medium (2.2 g/L (NH.sub.4).sub.2SO.sub.4, 0.02 g/L NaCl, 0.4 g/L MgSO.sub.4×7H.sub.2O, 0.04 g/L CaCl.sub.2×2H.sub.2O, sterilized separately: 2 g/L KH.sub.2PO.sub.4, 8.51 g/L KH.sub.2PO.sub.4, 20 g/L glucose, 10 mL/L trace elements solution M12 (sterile-filtered: 0.2 g/L ZnSO.sub.4×7H.sub.2O, 0.1 g/L MnCl.sub.2×H.sub.2O, 1.5 g/L Na.sub.3-Citrat×2H.sub.2O, 0.1 g/L CuSO.sub.4×5H.sub.2O, 0.002 g/L NiCl.sub.2×6H.sub.2O, 0.003 g/L Na.sub.2MoO.sub.4×2H.sub.2O, 0.03 g/L H.sub.3BO.sub.3, 1 g/L FeSO.sub.4×7H.sub.2O). thereafter, the pO2 electrodes were calibrated to 100% with a one-point calibration (stirrer: 600 rpm/aeration 10 sl/h air), and the feed, correction agent and induction agent lines were cleaned by “cleaning in place” as specified in the technical instructions. To this end, the tubes were rinsed first with 70% ethanol, then with 1 M NaOH, then with sterile fully-demineralized water and, finally, filled with the respective media.

(20) Using the P. putida strain BS-PP-360, 25 ml LB1 medium (10 g/L tryptone, 5 g/L yeast extract, 1 g/L NaCl, pH 7.0) supplemented with kanamycin (50 μg/mL) in a baffeled shake flask were inoculated with 100 μl of a glycerol stock solution and incubated for ˜18 h over night at 30° C. and 200 rpm. The first preculture was used to inoculate 50 ml 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 elements solution SL6 (sterile-filtered: 0.3 g/L H.sub.3BO.sub.3, 0.2 g/L CoCl.sub.2×6H.sub.2O, 0.1 g/L ZnSO.sub.4×7H.sub.2O, 0.03 g/L MnCl.sub.2×4H.sub.2O, 0.01 g/L CuCl.sub.2×2H.sub.2O, 0.03 g/L Na.sub.2MoO.sub.4×2H.sub.2O, 0.02 g/L NiCl.sub.2×6H.sub.2O) in a 500 ml baffeled shake flask (starting OD.sub.600 0.2). The culture were incubated for ˜7 h at 200 rpm and 30° C. In order to inoculate the reactors with an optical density of 0.7, the 00600 of the second preculture stage was measured and the amount of culture required for the inoculation was calculated.

(21) The required amount of culture was added with the help of a 30 ml syringe through a septum into the heat-treated and aerated reactor. The standard program shown in example 4 of table 1 was used.

(22) The pH was adjusted unilaterally to pH 7.0 with 12.5% strength ammonia solution. During the growth phase and the biotransformation, the dissolved oxygen (pO2 or DO) in the culture was adjusted to at least 30% via the stirrer speed and the aeration rate. After the inoculation, the DO dropped from 100% to these 30%, where it was maintained stably for the remainder of the fermentation.

(23) The fermentation was carried out as a fed batch. The feed starts with a 2.5 g/L*h glucose feed, composed of 500 g/L glucose, and was triggered via the DO peak which indicates the end of the batch phase. 3 h after the feed start, the expression of rubiwettin production was induced with 0.2% (w/v) rhamnose. The inducer concentration referred to the volume at the beginning of fermentation. For both sugars stock solution of 220 g/L was used. The production of rubiwettin RG1 started with the induction. At specified time points samples were taken from the fermenter to determine the concentration of rubiwettins produced. After 65 h fermentation 0.53 g/L rubiwettin RG1 was produced.

Example 6

(24) Production of Rubiwettin RG1 with Strain BS-PP-368 (P. putida KT2440 pACYC_rhIA_Pa rbwB_Srub)

(25) For the production of rubiwettin RG1 the DASGIP® parallel bioreactor system from Eppendorf (Hamburg, Germany) was used. The fermentation was performed using 1 L reactors. pH and pO2 were measured online for process monitoring. OTR/CTR measurements served for estimating the metabolic activity and cell fitness, inter alia.

(26) The pH electrodes were calibrated by means of a two-point calibration using standard solutions of pH 4.0 and pH 7.0, as specified in DASGIP's technical instructions. The reactors were equipped with the necessary sensors and connections as specified in the technical instructions, and the agitator shaft was fitted. The reactors were then filled with 300 ml water and autoclaved for 20 min at 121° C. to ensure sterility. The pO2 electrodes were connected to the measuring amplifiers and polarized overnight (for at least 6 h). Thereafter, the water was removed under a clean bench and replaced by fermentation medium (2.2 g/L (NH.sub.4).sub.2SO.sub.4, 0.02 g/L NaCl, 0.4 g/L MgSO.sub.4×7H.sub.2O, 0.04 g/L CaCl.sub.2×2H.sub.2O, sterilized separately: 2 g/L KH.sub.2PO.sub.4, 8.51 g/L KH.sub.2PO.sub.4, 20 g/L glucose, 10 mL/L trace elements solution M12 (sterile-filtered: 0.2 g/L ZnSO.sub.4×7H.sub.2O, 0.1 g/L MnCl.sub.2×4H.sub.2O, 1.5 g/L Na.sub.3-Citrat×2H.sub.2O, 0.1 g/L CuSO.sub.4×5H.sub.2O, 0.002 g/L NiCl.sub.2×6H.sub.2O, 0.003 g/L Na.sub.2MoO.sub.4×2H.sub.2O, 0.03 g/L H.sub.3BO.sub.3, 1 g/L FeSO.sub.4×7H.sub.2O). Thereafter, the pO2 electrodes were calibrated to 100% with a one-point calibration (stirrer: 600 rpm/aeration 10 sl/h air), and the feed, correction agent and induction agent lines were cleaned by “cleaning in place” as specified in the technical instructions.

(27) To this end, the tubes were rinsed first with 70% ethanol, then with 1 M NaOH, then with sterile fully-demineralized water and, finally, filled with the respective media.

(28) Using the P. putida strain BS-PP-368, 25 ml LB1 medium (10 g/L tryptone, 5 g/L yeast extract, 1 g/L NaCl, pH 7.0) supplemented with kanamycin (50 μg/mL) in a baffeled shake flask were inoculated with 100 μl of a glycerol stock solution and incubated for ˜18 h over night at 30° C. and 200 rpm. The first preculture was used to inoculate 50 ml 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 elements solution SL6 (sterile-filtered: 0.3 g/L H.sub.3BO.sub.3, 0.2 g/L CoCl.sub.2×6H.sub.2O, 0.1 g/L ZnSO.sub.4×7H.sub.2O, 0.03 g/L MnCl.sub.2×4H.sub.2O, 0.01 g/L CuCl.sub.2×2H.sub.2O, 0.03 g/L Na.sub.2MoO.sub.4×2H.sub.2O, 0.02 g/L NiCl.sub.2×6H.sub.2O) in a 500 ml baffeled shake flask (starting OD600 0.2). The culture were incubated for ˜7 h at 200 rpm and 30° C. In order to inoculate the reactors with an optical density of 0.7, the 00600 of the second preculture stage was measured and the amount of culture required for the inoculation was calculated.

(29) The required amount of culture was added with the help of a 30 ml syringe through a septum into the heat-treated and aerated reactor. The standard program shown in table 1 of example 4 was used for the heated and aerated reactor.

(30) The pH was adjusted unilaterally to pH 7.0 with 12.5% strength ammonia solution. During the growth phase and the biotransformation, the dissolved oxygen (pO2 or DO) in the culture was adjusted to at least 30% via the stirrer speed and the aeration rate. After the inoculation, the DO dropped from 100% to these 30%, where it was maintained stably for the remainder of the fermentation.

(31) The fermentation was carried out as a fed batch. The feed starts with a 2.5 g/L*h glucose feed, composed of 500 g/L glucose, and was triggered via the DO peak which indicates the end of the batch phase. 3 h after the feed start, the expression of rubiwettin production was induced with 0.2% (w/v) rhamnose. The inducer concentration referred to the volume at the beginning of fermentation. For both sugars stock solution of 220 g/L was used. The production of rubiwettin RG1 started with the induction. At specified time points samples were taken from the fermenter to determine the concentration of rubiwettins produced. After 65 h fermentation 11.1 g/L rubiwettin RG1 was produced.

Example 7

(32) HPLC-Based Quantification of Rubiwettins

(33) Quantification of lipids R1 and RG1 was carried out by means of HPLC. Using a displacement pipette (Combitip), 900 μl of 70% (v/v) n-propanol was introduced into a 2 ml reaction vessel and the reaction vessel was immediately closed for minimization of evaporation. The addition of 100 μl fermentation broth followed. After shaking for 1 min in a Retsch mill at a frequency of 30 Hz, the resulting crude extract mixture was centrifuged for 5 min at 13,000 rpm, and 800 μl of the clear supernatant was transferred into an HPLC vial. Further dilutions of cell broth were carried out in 55% (v/v) propanol. Samples were stored at −20° C. before measurement.

(34) For the detection and quantification of lipids an evaporation light scattering detector (Sedex LT-ELSD Model 85LT) was used. The measurement was carried out by means of Agilent Technologies 1200 Series (Santa Clara, Calif.) and a Zorbax SB-C8 Rapid Resolution column (4,6×150 mm, 3.5 μm, Agilent). The injection volume was 5.0 μl and the run time was 20 min. Mobile phase A: aqueous 0.1% TFA (trifluoracetic acid, solution); mobile phase B: methanol. The column temperature was 40° C. The ELSD (detector temperature 60° C.) and the DAD (diode array, 210 nm) were used as detectors.

(35) Gradient:

(36) TABLE-US-00002 TABLE 2 Gradient of mobile phases of A and B over time t [min] Flow [1 ml/min] 0.00 70% 1.00 15.00 100%  1.00 15.01 70% 1.00 20.00 70% 1.00

(37) The gradient used starts with 70% B in A to 100% B within 15 minutes at a flow rate of 1 mL/min followed by 5 minutes of re-equilibration with 70% B in A (see Table 2). Reference materials were used whose identity and purity were checked by HPLC-MS/MS and NMR.

Example 8

(38) Construction of Agrobacterium tumefaciens Strains for Production of Rubiwettin R1 and Rubiwettin RG1

(39) In order to show production of rubiwettins with yet another microbial species, Agrobacterium tumefaciens, we prepare electrocompetent cells of Agrobacterium tumefaciens LBA 4404 and transformed it with plasmids pACYC_rbwA_Srub (SEQ ID NO: 19), pACYC_rbwAB_Srub (SEQ ID NO: 18) and pACYC_rhIA_Pa rbwB_Srub (SEQ ID NO: 21).

(40) To that end, freshly growing cells (1-2 days old) of A. tumefaciens LBA 4404 are spread on LB agar plates (diameter 90 mm, 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, and 15 g/L agar, supplemented with 50 μg/mL rifampicin) and incubated overnight (˜16 h) at 27° C. to produce a bacterial lawn that covers the surface of the plate completely.

(41) Bacterial cells are carefully washed off the plate with 4 mL ice-cold 10% (v/v) sterile glycerol. Cells growing on the surface of the plate are scraped off with an inoculation loop avoiding damages of the agar medium and suspended in the glycerol solution. The bacterial suspension is then transferred into two sterile 2 mL centrifuge tubes.

(42) Suspensions in the two tubes are centrifuged at 14,000 rpm (18,000 g) for 1 min at 4° C.; the supernatant is discarded.

(43) 1 mL ice-cold 10% (v/v) sterile glycerol is added to each tube containing the bacterial pellet. The tubes are thoroughly vortexed afterwards to resuspend the cells and this washing step is repeated one more time.

(44) After the two centrifugation steps, the supernatant is removed and discarded again and the bacterial pellets in the two tubes are resuspended in 200 μl ice-cold 10% (v/v) sterile glycerol each and combined in one tube (yielding 400 μl in total).

(45) The tube with the Agrobacterium cell suspension is kept on ice until electroporation.

(46) For electroporation 70-80 μl of the ice-cold suspension of electrocompetent bacterial cells is mixed with 1-3 μl plasmid DNA (1-100 ng) in a sterile centrifuge tube. This mixture is loaded into a chilled electroporation cuvette (gap=2 mm) and placed into the cuvette holder. The electroporator (Gene Pulser Xcell™ Microbial Electroporation Systems; Bio-Rad) is used with the following parameters: 2.5 kV, 25 μF capacitance, and 400 Ohm resistance. One mL SOC medium (20 mM glucose, 20 g/L tryptone, 5 g/L yeast extract, 10 mM NaCl, 2.5 mM MgCl.sub.2, and 10 mM MgSO.sub.4) is added immediately to the electroporation cuvette and the resulting bacterial suspension transferred into a 15 mL centrifuge tube, and the tube is incubated at 27° C. for 1 h with rotating.

(47) After incubation 100 μl from each suspension of electroporated cells is spread onto LB plates supplemented with kanamycin (50 μg/mL). The plates are incubated for 2 days at 27° C. and successfully transformed colonies verified by plasmid isolation and analytical restriction digests. The following strains are generated: A. tumefaciens LBA 4404 pACYC_rbwA_Srub for production of rubiwettin R1 A. tumefaciens LBA 4404 pACYC_rbwAB_Srub for production of rubiwettin RG1 A. tumefaciens LBA 4404 pACYC_rhIA_Pa rbwB_Srub for production of rubiwettin RG1

Example 9

(48) Production of rubiwettin R1 with Agrobacterium tumefaciens LBA 4404 pACYC_rbwA_Srub and rubiwettin RG1 with Agrobacterium tumefaciens LBA 4404 pACYC_rbwAB_Srub and Agrobacterium tumefaciens LBA 4404 pACYC_rhIA_Pa rbwB_Srub

(49) For the production of rubiwettins R1 and RG1 the DASGIP® parallel bioreactor system from Eppendorf (Hamburg, Germany) is used. The fermentation is performed using 1 L reactors. pH and pO2 are measured online for process monitoring. OTR/CTR measurements served for estimating the metabolic activity and cell fitness, inter alia.

(50) The pH probes are calibrated by means of a two-point calibration with measurement solutions of pH 4.0 and pH 7.0 according to technical reference of DASGIP. The reactors are provided according to technical reference with the required sensors and connections and the stirrer shaft is installed. The reactors are then filled with 300 ml of water and autoclaved for 20 min at 121° C. in order to ensure sterility. The pO2 probes are polarized overnight (at least 6 h) following connection to the measurement amplifier. The water is then removed under the clean bench and replaced by high-cell-density medium consisting of (NH.sub.4)2504 1.76 g/l, K.sub.2HPO.sub.4 19.08 g/l, KH2PO.sub.4 12.5 g/l, yeast extracts 6.66 g/l, trisodium citrate dihydrate 11.2 g/l, 17 ml/l of a filter-sterilized 1% strength ammonium iron citrate solution, and 5 ml/l of a filter-sterilized trace element stock solution (consisting of HCl (37%) 36.50 g/l, MnCl.sub.2*4H2O 1.91 g/l, ZnSO.sub.4*7H2O 1.87 g/l, ethylenediaminetetraacetic acid dihydrate 0.84 g/l, H3BO3 0.30 g/l, Na2MoO4*2H2O0.25 g/l, CaCl2*2H2O 4.70 g/l, FeSO4*7H2O 17.80 g/l, CuCl2*2H2O 0.15 g/l) with 15 g/l glucose as carbon source (added by metered addition of 30 ml/l of a sterile feed solution consisting of 500 g/l glucose, 1% (w/v) MgSO4*7H2O and 2.2% (w/v) NH4Cl) with 50 mg/l kanamycin.

(51) Subsequently, the pO2 probes are calibrated using a single-point calibration (stirrer: 600 rpm/gassing: 10 sL/h air) to 100% and the feed, correction agent and induction agent stretches are cleaned by means of cleaning-in-place according to technical reference. For this, the tubes are firstly flushed with 70% ethanol, then with 1 M NaOH, then with sterile demineralized water and finally filled with the respective media.

(52) For production of rubiwettin R1 with Agrobacterium tumefaciens LBA 4404 pACYC_rbwA_Srub as well as production of rubiwettin RG1 with Agrobacterium tumefaciens LBA 4404 pACYC_rbwAB_Srub and Agrobacterium tumefaciens LBA 4404 pACYC_rhIA_Pa rbwB_Srub, the three strains are cultured firstly from a cryoculture in LB medium (25 ml in a 100 ml baffled shake flask) with 50 mg/l kanamycin overnight at 28° C. and 200 rpm for about 18 h. Then, 2 ml of this culture is transferred for a second preculture stage into 25 ml of high-cell-density medium consisting of (NH4)2504 1.76 g/L, K2HPO4 19.08 g/l, KH2PO4 12.5 g/l, yeast extract 6.66 g/l, trisodium citrate dihydrate 11.2 g/l, 17 ml/l of a filter-sterilized 1% strength ammonium iron citrate solution, and 5 ml/l of a filter-sterilized trace element stock solution (consisting of HCl (37%) 36.50 g/l, MnCl2*4H2O1.91 g/l, ZnSO4*7H2O 1.87 g/l, ethylenediaminetetraacetic acid dihydrate 0.84 g/l, H3BO3 0.30 g/l. Na2MoO4*2H2O 0.25 g/l, CaCl2*2H2O 4.70 g/l, FeSO4*7H2O 17.80 g/l, CuCl2*2H2O 0.15 g/l) with 15 g/l glucose as carbon source (added by metered addition of 30 ml/l of a sterile feed solution consisting of 500 g/l glucose, 1% (w/v) MgSO4*7H2O and 2.2% (w/v) NH4Cl) with the already described antibiotics in a 100 ml shake flask and incubated at 28° C./200 rpm fora further 6 h.

(53) In order to inoculate the reactors with an optical density of 0.1, the 00600 of the second preculture stage is measured and the amount of culture required for the inoculation is calculated. The required amount of culture is added with the help of a 5 ml syringe through a septum into the heat-treated and aerated reactor.

(54) The standard program used is shown in Table 3:

(55) TABLE-US-00003 TABLE 3 The standard program for production of rubiwettins with Agrobacterium strains 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 ml/h max 100%  max 40 ml/h XO2 F N (Rotation) from to (gas mixture) from to (gas flow rate) from to growth and 0% 30% growth and  0% 100% growth and 15% 80% biotransformation 400 rpm 1500 rpm biotransformation 21%  21% biotransformation 6 sL/h 72 sL/h Script Trigger sharp 31% DO (1/60 h) Induction 3 h after feed start Rhamnose Feed trigger 50% DO Feed rate 1 [ml/h]

(56) The pH is regulated to pH 6.8 on one side with 12.5% strength ammonia solution. During cultivation and biotransformation, the dissolved oxygen (pO2 or DO) in the culture is regulated to at least 30% by means of stirrer feed and gassing rate. Following inoculation, the DO drops from 100% to this 30%, where it is kept stable for the remainder of the fermentation. The temperature is kept stable at 28° C.

(57) The fermentation is carried out as fed-batch, where the feed start is triggered as delivery to the feed phase with 1.5 g/l*h glucose feed, consisting of 500 g/l glucose, 1% (w/v) MgSO.sub.4*7H.sub.2O and 2.2% (w/v) NH.sub.4Cl, via the DO peak inducing the end of the batch phase. 3 h after the feed start, rubiwettin production is induced with 0.2% (w/v) rhamnose. The inducer concentration refers to the volume at the beginning of fermentation. A rhamnose stock solution of 220 g/L is used. Quantification of formation of rubiwettins R1 and RG1 is performed as described in Example 7.

(58) It is shown that Agrobacterium tumefaciens LBA 4404 pACYC_rbwA_Srub produces rubiwettin R1.

(59) It is also shown that Agrobacterium tumefaciens LBA 4404 pACYC_rbwAB_Srub and Agrobacterium tumefaciens LBA 4404 pACYC_rhIA_Pa rbwB_Srub both produce rubiwettins RG1.

Example 10

(60) Construction of E. coli strains for production of rubiwettin R1 and rubiwettin RG1 The plasmids pACYC_rbwA_Srub (SEQ ID NO: 19), pACYC_rbwAB_Srub (SEQ ID NO: 18) and pACYC_rhIA_Pa rbwB_Srub (SEQ ID NO: 21) are transformed via electroporation into E. coli W3110 and plated onto LB agar plates with kanamycin (50 μg/ml). Transformants are screened for presence and authenticity of the plasmids by plasmid preparation and restriction digest analysis. The following strains are generated: E. coli W3110 pACYC_rbwA_Srub for production of rubiwettin R1 E. coli W3110 pACYC_rbwAB_Srub for production of rubiwettin RG1 E. coli W3110 pACYC_rhIA_Pa rbwB_Srub for production of rubiwettin RG1

Example 11

(61) Production of Rubiwettin R1 with E. coli W3110 pACYC_rbwA_Srub and Rubiwettin RG1 with E. coli W3110 pACYC_rbwAB_Srub and E. coli W3110 pACYC_rhIA_Pa rbwB_Srub

(62) For the production of rubiwettins R1 and RG1 the DASGIP® parallel bioreactor system from Eppendorf (Hamburg, Germany) is used. The fermentation is performed using 1 L reactors. pH and pO2 are measured online for process monitoring. OTR/CTR measurements served for estimating the metabolic activity and cell fitness, inter alia.

(63) The pH probes are calibrated by means of a two-point calibration with measurement solutions of pH 4.0 and pH 7.0 according to technical reference of DASGIP. The reactors are provided according to technical reference with the required sensors and connections and the stirrer shaft is installed. The reactors are then filled with 300 ml of water and autoclaved for 20 min at 121° C. in order to ensure sterility. The pO2 probes are polarized overnight (at least 6 h) following connection to the measurement amplifier. The water is then removed under the clean bench and replaced by high-cell-density medium consisting of (NH.sub.4).sub.2SO.sub.4 1.76 g/l, K.sub.2HPO.sub.4 19.08 g/l, KH.sub.2PO.sub.4 12.5 g/l, yeast extracts 6.66 g/l, trisodium citrate dihydrate 11.2 g/l, 17 ml/l of a filter-sterilized 1% strength ammonium iron citrate solution, and 5 ml/l of a filter-sterilized trace element stock solution (consisting of HCl (37%) 36.50 g/l, MnCl.sub.2*4H.sub.2O 1.91 g/l, ZnSO.sub.4*7H.sub.2O 1.87 g/l, ethylenediaminetetraacetic acid dihydrate 0.84 g/l, H.sub.3BO.sub.3 0.30 g/l, Na.sub.2MoO.sub.4*2H.sub.2O 0.25 g/l, CaCl.sub.2*2H.sub.2O 4.70 g/l, FeSO.sub.4*7H.sub.2O 17.80 g/l, CuCl.sub.2*2H.sub.2O 0.15 g/l) with 15 g/l glucose as carbon source (added by metered addition of 30 ml/l of a sterile feed solution consisting of 500 g/l glucose, 1% (w/v) MgSO.sub.4*7H.sub.2O and 2.2% (w/v) NH.sub.4Cl) with 50 mg/l kanamycin.

(64) Subsequently, the pO2 probes are calibrated using a single-point calibration (stirrer: 600 rpm/gassing: 10 sL/h air) to 100% and the feed, correction agent and induction agent stretches are cleaned by means of cleaning-in-place according to technical reference. For this, the tubes are firstly flushed with 70% ethanol, then with 1 M NaOH, then with sterile demineralized water and finally filled with the respective media.

(65) For production of rubiwettin R1 with E. coli W3110 pACYC_rbwA_Srub as well as production of rubiwettin RG1 with E. coli W3110 pACYC_rbwAB_Srub and E. coli W3110 pACYC_rhIA_Pa rbwB_Srub, the three strains are cultured firstly from a cryoculture in LB medium (25 ml in a 100 ml baffled shake flask) with 50 mg/l kanamycin overnight at 37° C. and 200 rpm for about 18 h. Then, 2 ml of this culture is transferred for a second preculture stage into 25 ml of high-cell-density medium consisting of (NH.sub.4)2SO.sub.4 1.76 g/L, K.sub.2HPO.sub.4 19.08 g/l, KH.sub.2PO.sub.4 12.5 g/l, yeast extract 6.66 g/l, trisodium citrate dihydrate 11.2 g/l, 17 ml/l of a filter-sterilized 1% strength ammonium iron citrate solution, and 5 ml/l of a filter-sterilized trace element stock solution (consisting of HCl (37%) 36.50 g/l, MnCl.sub.2*4H.sub.2O 1.91 g/l, ZnSO.sub.4*7H.sub.2O 1.87 g/l, ethylenediaminetetraacetic acid dihydrate 0.84 g/l, H.sub.3BO.sub.3 0.30 g/l. Na.sub.2MoO.sub.4*2H.sub.2O 0.25 g/l, CaCl.sub.2*2H.sub.2O 4.70 g/l, FeSO.sub.4*7H.sub.2O 17.80 g/l, CuCl.sub.2*2H.sub.2O 0.15 g/l) with 15 g/l glucose as carbon source (added by metered addition of 30 ml/l of a sterile feed solution consisting of 500 g/l glucose, 1% (w/v) MgSO.sub.4*7H.sub.2O and 2.2% (w/v) NH.sub.4Cl) with the already described antibiotics in a 100 ml shake flask and incubated at 37° C./200 rpm for a further 6 h.

(66) In order to inoculate the reactors with an optical density of 0.1, the 00600 of the second preculture stage is measured and the amount of culture required for the inoculation is calculated. The required amount of culture is added with the help of a 5 ml syringe through a septum into the heat-treated and aerated reactor.

(67) The standard program used is shown in Table 4:

(68) TABLE-US-00004 TABLE 4 The standard program for production of rubiwettins with E. coli strains 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 ml/h max 100%  max 40 ml/h XO2 F N (Rotation) from to (gas mixture) from to (gas flow rate) from to growth and 0% 30% growth and  0% 100% growth and 15% 80% biotransformation 400 rpm 1500 rpm biotransformation 21%  21% biotransformation 6 sL/h 72 sL/h Script Trigger sharp 31% DO (1/60 h) Induction 3 h after feed start Rhamnose Feed trigger 50% DO Feed rate 3 [ml/h]

(69) The pH is regulated to pH 6.8 on one side with 12.5% strength ammonia solution. During cultivation and biotransformation, the dissolved oxygen (pO2 or DO) in the culture is regulated to at least 30% by means of stirrer feed and gassing rate. Following inoculation, the DO drops from 100% to this 30%, where it is kept stable for the remainder of the fermentation. The temperature is kept stable at 37° C. The fermentation is carried out as fed-batch, where the feed start is triggered as delivery to the feed phase with 5 g/l*h glucose feed, consisting of 500 g/l glucose, 1% (w/v) MgSO.sub.4*7H.sub.2O and 2.2% (w/v) NH.sub.4Cl, via the DO peak inducing the end of the batch phase. 3 h after the feed start, rubiwettin production is induced with 0.2% (w/v) rhamnose. The inducer concentration refers to the volume at the beginning of fermentation. A rhamnose stock solution of 220 g/L is used. Quantification of formation of rubiwettins R1 and RG1 is performed as described in Example 7.

(70) It is shown that E. coli W3110 pACYC_rbwA_Srub produces rubiwettin R1.

(71) It is also shown that E. coli W3110 pACYC_rbwAB_Srub and E. coli W3110 pACYC_rhIA_Pa rbwB_Srub both produce rubiwettins RG1.