Cells and methods for producing rhamnolipids using alternative glucose transporters

Abstract

The present invention relates to cells that produce rhamnolipids and that have been genetically modified to have a reduced activity of an ABC glucose transporter compared to its wild type and an increased activity of at least one non-ABC glucose transporter compared to its wild type, and to methods for producing rhamnolipids using cells according to the invention.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to cells which produce rhamnolipids and have been genetically modified to have a reduced activity of an ABC glucose transporter compared to its wild type and an increased activity of at least one non-ABC glucose transporter compared to its wild type, and to a method for producing rhamnolipids using the cells according to the invention.

[0002] Prior Art Rhamnolipids are one of the economically important substance classes, since they can potentially replace conventional petroleum-based surfactants and thus improve the environmental compatibility of the corresponding formulations. Currently, rhamnolipids are produced using wild-type isolates of various human and animal pathogenic bacteria, in particular representatives of the genera Pseudomonas and Burkholderia (see Hydrocarbon and Lipid Microbiology, 2010, pp. 3037-51).

[0003] WO 2012013554 discloses the production of rhamnolipids in non-pathogenic organisms, such as P. putida KT2440, whereby enzymes encoded by, for example, the Pseudomonas aeruginosa genes rhlA, rhlB and rhlC are expressed.

[0004] The development and establishment of the most efficient processes requires, inter alia, the achievement of the highest possible space-time yields.

[0005] The object of the present invention is to develop microbial cells and methods using said cells, which achieve high space-time yields.

[0006] Description of the invention Surprisingly, it has been found that cells which produce rhamnolipids and have been genetically modified to have a reduced activity of an ABC glucose transporter compared to their wild type and an increased activity of at least one non-ABC glucose transporter compared to their wild type provide higher space-time yields of rhamnolipid at equivalent substrate inputs than comparable strains with functional ABC transporters.

[0007] The present invention therefore provides a rhamnolipid producing cell, characterized in that it has been genetically modified to have a reduced activity of an ABC glucose transporter compared to its wild type and to have an increased activity of at least one non-ABC glucose transporter compared to its wild type.

[0008] The present invention further provides a method for producing rhamnolipids using the above cells as a biocatalyst.

[0009] One advantage of the present invention is that it allows the use of organisms that are non-pathogenic and easy to culture.

[0010] Another advantage of the present invention is that it allows for a wide range of carbon source options.

[0011] A further advantage is that it is not necessary in any case to use oil as the sole substrate or as a co-substrate.

[0012] Another advantage is that the present invention allows the production of rhamnolipids with defined and tunable properties.

[0013] A further advantage is that rhamnolipids can be produced with a higher space-time yield and a higher carbon yield than with cells whose activity is not altered.

[0014] The present invention therefore provides a rhamnolipid producing cell, advantageously an isolated rhamnolipid producing cell, characterized in that the cell has been genetically modified so as to have a reduced activity of an ABC glucose transporter compared to its wild type and to have an increased activity of at least one non-ABC glucose transporter compared to its wild type.

[0015] As used herein, the term "wild type" of a cell refers to a cell whose genome exists in the state in which it naturally arose through evolution. The term applies to both whole cells and individual genes. Thus, the term "wild type" specifically does not include cells or genes whose gene sequences have been at least partially modified by humans using recombinant techniques. The term "wild type" specifically refers to the phenotype, genotype, or gene that occurs most frequently in the natural population numbers of an organism.

[0016] In the present invention, the term "rhamnolipid" refers to a compound of the general formula (I) [ka] [In the formula, m is 2, 1 or 0, in particular 1 or 0, n is 1 or 0, in particular 1; R 1 is an optionally branched, optionally substituted, in particular hydroxy-substituted, optionally unsaturated organic radical having 2 to 24, preferably 5 to 13, carbon atoms, in particular an optionally mono-, di- or triunsaturated alkyl radical, in particular pentenyl, heptenyl, nonenyl, undecenyl and tridecenyl and (CH 2 ) o -CH 3 where o is from 1 to 23, preferably from 4 to 12; and R 2are, independently of one another, identical or different organic radicals having 2 to 24, preferably 5 to 13, carbon atoms, in particular optionally branched, optionally substituted, in particular hydroxy-substituted, optionally unsaturated, in particular optionally mono-, di- or triunsaturated alkyl radicals, preferably pentenyl, heptenyl, nonenyl, undecenyl and tridecenyl and (CH 2 ) o -CH 3 or a salt thereof, wherein o is selected from the group consisting of:

[0017] If the cell according to the invention is capable of producing rhamnolipid m=1, then R 1 and R 2 The group determined by [ka] are 3-hydroxyoctanoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydecenoic acid, 3-hydroxydecenoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydodecanoic acid, 3-hydroxydodecanoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydodeceno ...dodecanoyl-3-hydroxyoctanoic acid, 3-hydroxydodecanoyl-3-hydroxyoctanoic acid, 3-hydroxydodecanoic acid, 3-hydroxydodecanoyl-3-hydroxyoctanoic acid, 3-hydroxydodecanoic acid, 3-hydroxydodecanoyl-3-hydroxyoctanoic acid, 3-hydroxydodecanoic acid, 3-hydroxydodecanoyl-3-hydroxyoctanoic acid, 3-hydroxydodecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxydodecanoyl-3-hydroxyoctanoic acid, 3-hydroxydodecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxydodec 3-Hydroxyoctanoyl, 3-Hydroxydecanoyl, 3-Hydroxydecanoyl, 3-Hydroxydeceno ...dodecanoyl, 3-Hydroxydodecanoyl, 3-Hydroxydecanoyl, 3-Hydroxydodecanoyl, 3-Hydroxydodecenoyl, 3-Hydroxydecanoyl, 3-Hydroxytetradecenoic acid , 3-hydroxytetradecanoyl-3-hydroxydecenoic acid, 3-hydroxydodecenoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxytetradecanoic acid, 3-hydroxytetradecanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxytetradecenoic acid, 3-hydroxytetradecanoyl-3-hydroxydecanoic acid, 3-hydroxydodecanoyl-3-hydroxydodecanoic acid, 3-hydroxydodecenoyl-3-hydroxydodecanoic acid, 3-hydroxydodecenoyl-3-hydroxydodecanoic acid, 3- Advantageously, it is derived from hydroxydodecanoyl-3-hydroxydodecenoic acid, 3-hydroxydodecanoyl-3-hydroxytetradecanoic acid, 3-hydroxytetradecanoyl-3-hydroxydodecanoic acid, 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoyl-3-hydroxytetradecanoic acid, 3-hydroxytetradecanoyl-3-hydroxyhexadecanoic acid or 3-hydroxyhexadecanoyl-3-hydroxyhexadecanoic acid.

[0018] It will be clear to those skilled in the art that the cells according to the invention may also produce mixtures of different rhamnolipids of general formula (I).

[0019] In this context, the cells according to the invention are characterized in that more than 80% by weight, advantageously more than 90% by weight and particularly advantageously more than 95% by weight of the produced rhamnolipids have n=1 and R 1 and R 2 It is advantageous to be able to produce mixtures of rhamnolipids of general formula (I) characterised in that, with respect to the group determined by, less than 20% by weight, advantageously less than 15% by weight, of the rhamnolipids produced derives from 3-hydroxydecanoyl-3-hydroxyoctanoic acid or 3-hydroxyoctanoyl-3-hydroxydecanoic acid, where the indicated weight percentages are based on the sum of all rhamnolipids of general formula (I) produced.

[0020] The accession numbers listed herein correspond to entries in the NCBI protein structure database as of January 26, 2016, generally in this case where the version number of the entry is identified by a ".number", e.g. ".1".

[0021] Unless otherwise stated, all percentages given are by weight.

[0022] Therefore, the enzyme E x The expression "reduced activity of" is understood to mean an activity that is advantageously reduced by at least 0.5-fold, particularly advantageously by at least 0.1-fold, more advantageously by at least 0.01-fold, even more advantageously by at least 0.001-fold, most advantageously by at least 0.0001-fold. The expression "reduced activity" also includes undetectable activity ("activity 0").

[0023] Methods for reducing enzyme activity in microorganisms are known to those skilled in the art. Here, molecular biology techniques are particularly useful. For example, the activity of a specific enzyme can be reduced by targeted mutation or other methods known to those skilled in the art for reducing the activity of a specific enzyme. Instructions for modifying and reducing protein expression and concomitantly reducing enzyme activity, particularly for inhibiting specific genes, particularly for Pseudomonas and Burkholderia, can be found by those skilled in the art, for example, in Dubeau et al. 2009. BMC Microbiology 9:263; Singh & Roehm. Microbiology. 2008. 154:797-809 or Lee et al. FEMS Microbiol Lett. 2009. 297(1):38-48. The advantageous methods for reducing the enzyme activity of ABC glucose transporters described below can likewise be advantageously used in the present invention to reduce further enzyme activity.

[0024] An advantageous cell according to the invention is characterized in that a reduction in the enzymatic activity is achieved by genetic modification of the gene encoding the ABC glucose transporter, whereby this modification comprises, advantageously selected from the group consisting of, the insertion of foreign DNA into the gene, the deletion of at least a part of the gene, a point mutation in the gene sequence, in particular a point mutation in or in the gene sequence of a regulatory sequence, such as, for example, a promoter or a terminator, or a point mutation in the gene sequence of a ribosome binding site.

[0025] In this context, foreign DNA is understood to mean any DNA sequence that is "foreign" to the gene (but not to the organism), i.e. in this context, endogenous DNA sequences can also function as "foreign DNA". In this context, the gene is particularly advantageously disrupted by the insertion of a selection marker gene. The foreign DNA is therefore a selection marker gene, the insertion being advantageously effected by homologous recombination into the gene locus.

[0026] Alternatively, an advantageous cell according to the invention is characterized in that a reduction in the enzymatic activity is achieved by targeted transcriptional or post-transcriptional gene silencing of the gene encoding the ABC glucose transporter, in particular by means of at least one repressor binding to the promoter of the gene encoding the ABC glucose transporter, by means of nonsense codon-mediated mRNA decay (NMD) and RNA interference (RNAi), preferably by means of microRNA methodology (miRNA) or small interfering RNA methodology (siRNA), whereby the mRNA of the ABC glucose transporter is degraded.

[0027] The cells according to the invention are genetically modified to have a reduced activity of an ABC glucose transporter compared to its wild type.

[0028] In the present invention, the term "ABC glucose transporter" should be taken to mean a membrane protein that has an ATP-binding cassette (ABC) as a common structural element and actively transports a specific substrate, such as glucose, across the cell membrane. ACB transporters consist of four core domains, namely two integral membrane domains and two cytoplasmic ATP-binding domains, the so-called ATP-binding cassettes. The cassettes (functional domains / regions in a protein) are based on the energy-coupled transport of a substrate against a concentration gradient.

[0029] In the present invention, the activity of an ABC glucose transporter is defined as the ability to take up 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), which is a substitute for glucose and can be measured, into a cell.

[0030] The activity of the ABC glucose transporter can be measured using the Cayman Chemicals Glucose Uptake Cell Base Assay kit (part number 600470), specifically according to the manufacturer's instructions dated October 9, 2015. For this purpose, it is obvious to one of ordinary skill in the art to directly compare cells that differ only in terms of genetic modifications that are directly directed to reduce the activity of the ABC glucose transporter to each other and determine whether there are differences in activity.

[0031] The cells according to the invention are genetically modified to have increased activity of at least one non-ABC glucose transporter compared to the wild type.

[0032] The term "non-ABC glucose transporter" according to the present invention is defined as a glucose transporter that is not an ABC glucose transporter as defined above. Particularly advantageously, the present invention utilizes a non-ABC glucose transporter that is foreign to the cell according to the invention and therefore is not present in the wild-type genome.

[0033] Advantageous non-ABC glucose transporters are in particular selected from the group consisting of the phosphoenolpyruvate phosphotransferase system of EC 2.7.3.9, galactose permease, glucose facilitator, myo-inositol transporter, glucose permease and glucose / galactose transporter.

[0034] Transporters are classified by those skilled in the art according to the Transporter Classification Database (www.tcdb.org.) In the present invention, preferred non-ABC glucose transporters according to this classification are those selected from the TCDB family, 2.A.1 Major Facilitator superfamily, 2.A.123 The Sweet superfamily and 2.A.7 DMT superfamily, with those selected from the TCDB transporter classes 2.A.1.1.1, 2.A.1.1.4, 2.A.1.1.53, 2.A.1.7.3, 2.A.1.1.81, 2.A.123.2 and 2.A.7.5 being particularly preferred.

[0035] Advantageous non-ABC glucose transporters are especially the enzymes encoded by the galP, glf, iolT1, glcP, gluP, SemiSWEET, or glcU genes, and the PTS system, which consists of the components Enzyme I, HPr, Enzyme IIA, Enzyme IIB, and Enzyme IIC, where Enzymes IIA, IIB, and IIC can be present as fusion proteins; or Enzymes which have a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues of the enzymes encoded by the galP, glf, iolT1, glcP, gluP, SemiSWEET or glcU genes and the PTS system have been modified by deletion, insertion, substitution or a combination thereof, and which still have at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of the enzymes encoded by the galP, glf, iolT1, glcP, gluP, SemiSWEET or glcU genes and the PTS system. wherein the enzymatic activity of a non-ABC glucose transporter is understood to mean the ability to take up 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) into a cell.

[0036] With regard to said polypeptide sequences, the iolT1 gene is in particular from C. glutamicum, the glcP gene is in particular from M. smegmatis, S. frigidimarina or S. amazonensis, the gluP gene is in particular from B. abortus, the SemiSWEET gene is in particular from L. biflexa and the glcU gene is in particular from B. subtilis or S. xylosus.

[0037] The activity of non-ABC glucose transporters can be measured using the Cayman Chemicals Glucose Uptake Cell Base Assay kit (part number 600470), specifically according to the manufacturer's instructions dated October 9, 2015.

[0038] The cells according to the invention may be prokaryotic or eukaryotic. They may be mammalian cells (e.g. human cells), plant cells or microorganisms such as yeasts, fungi or bacteria, with microorganisms being particularly preferred and bacteria and yeasts being the most preferred.

[0039] Furthermore, according to the present invention, the cell according to the present invention is C 6 ~C 16It is advantageous if the cell is capable of producing polyhydroxyalkanoates having a chain length of monoalkanoates. Such cells may be, for example, Burkholderia sp., Burkholderia thailandensis, Pseudomonas sp., Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas oleovorans, Pseudomonas chlororaphis, Pseudomonas stutzeri, Pseudomonas fluorescens, Pseudomonas citronellolis, Pseudomonas resinovorans, Pseudomonas resinovorans, Comamonas testosteroni, Aeromonas hydrophila, Cupriavidus necator, Alcaligenes latus and Ralstonia eutropha. In this context, an advantageous cell of the invention is genetically modified such that the cell is capable of producing less polyhydroxyalkanoates compared to its wild type.

[0040] Among the group of bacteria, Pseudomonas putida, Escherichia coli and Burkholderia thailandensis are particularly advantageous.

[0041] The starting strain of cells according to the invention may be a natural rhamnolipid producer, which may be a cell that already produces rhamnolipid as a wild type, or it may be a cell that has merely been made capable of producing rhamnolipid by genetic engineering.

[0042] In either case, the cells advantageously have a phenotype in which the cells have a higher E 1 , E 2 and E 3 The present invention benefits from the fact that the present invention is genetically modified to have increased activity of at least one enzyme selected from the group consisting of enzyme E 1 can catalyze the conversion of 3-hydroxyalkanoyl-ACP to hydroxyalkanoyl-3-hydroxyalkanoic acid via 3-hydroxyalkanoyl-3-hydroxyalkanoic acid-ACP, and enzyme E 2 is rhamnosyltransferase I and is capable of catalyzing the conversion of dTDP-rhamnose and 3-hydroxyalkanoyl-3-hydroxyalkanoate to α-L-rhamnopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate, and is enzyme E 3 is a rhamnosyltransferase II and is capable of catalyzing the conversion of dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate.

[0043] Enzyme E 1 Advantageously, The enzyme encoded by the rhlA gene, and an enzyme having a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues have been modified by deletion, insertion, substitution or a combination thereof with respect to the enzyme encoded by the rhlA gene, and which still has at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of an enzyme having a reference sequence for the enzyme encoded by the rhlA gene. is selected from.

[0044] Enzyme E 2 Advantageously, The enzyme encoded by the rhlB gene, and an enzyme having a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues have been modified by deletion, insertion, substitution or a combination thereof with respect to the enzyme encoded by the rhlB gene, and which still has at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of an enzyme having a reference sequence for the enzyme encoded by the rhlB gene. is selected from.

[0045] Enzyme E 3 Advantageously, The enzyme encoded by the rhlC gene, and an enzyme having a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues have been modified by deletion, insertion, substitution or a combination thereof with respect to the enzyme encoded by the rhlC gene, and which still has at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of an enzyme having a reference sequence for the enzyme encoded by the rhlC gene. is selected from.

[0046] E 1 , E 2 and E 3 Particularly advantageous for: Enzyme E 1 is selected from the group consisting of: At least one enzyme E 1a And, having the polypeptide sequence ADP06387.1, or It has a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues have been modified by deletion, insertion, substitution or a combination thereof compared to the reference sequence ADP06387.1, and still has at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme having the reference sequence ADP06387.1. Enzyme E 1a where the enzyme E 1a is understood to mean the ability to convert 3-hydroxydecanoyl-ACP via 3-hydroxydecanoyl-3-hydroxydecanoic acid-ACP to hydroxydecanoyl-3-hydroxydecanoic acid; At least one enzyme E 1b And, Polypeptide sequences AIP29471.1, CBI71021.1, NP_252169.1, ABR81106.1, YP_439272.1, YP_111362.1, YP_110557.1, YP_105231.1, ZP_02461688.1, ZP_02358949.1, ZP_01769192.1, ZP_04893165.1, ZP_02265387.2, ZP_02511781.1, ZP_03456835.1, ZP_03794633.1, YP_990329.1, ZP_02408727.1, YP_0029082 43.1, ZP_04884056.1, YP_004348703.1, ZP_04905334.1, ZP_02376540.1, EGC99875.1, ZP_02907621.1, YP_001811696.1, ZP_02466678.1, ZP_02891475.1, YP_776393.1, YP_002234939.1, YP_001778804.1, YP_371314.1, ZP_04943305.1, YP_623139.1, ZP_02417235.1 or ZP_04892059.1, or having a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues for a particular said accession number have been modified by deletion, insertion, substitution or a combination thereof, and still having at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme having the particular said accession number. Enzyme E 1b where the enzyme E 1b is understood to mean the ability to convert 3-hydroxytetradecanoyl-ACP to hydroxytetradecanoyl-3-hydroxytetradecanoic acid via 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid-ACP.

[0047] Enzyme E 2 is selected from the group consisting of: At least one enzyme E 2a And, having the polypeptide sequence ADP06388.1, YP_001347032.1, CBI71029.1, YP_002439138.1, CBI71031.1, NP_252168.1, CBI71034.1, CBI71028.1, AAA62129.1 or ZP_04929750.1, or having a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues for a particular said accession number have been modified by deletion, insertion, substitution or a combination thereof, and still having at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme having the particular said accession number. Enzyme E 2a where the enzyme E 2a is understood to mean the ability to convert dTDP-rhamnose and 3-hydroxydecanoyl-3-hydroxydecanoic acid into α-L-rhamnopyranosyl-hydroxydecanoyl-3-hydroxydecanoic acid; At least one enzyme E 2b And, Polypeptide sequences AJY01590.1, ABR84881.1, NP_252168.1, FN601364.1, YP_440074.1, ZP_05590657.1, ZP_04520374.1, ZP_00438360.2, ZP_00438209.2, YP_001074761.1, ZP_04811084.1, YP_110 558.1, YP_111361.1, ZP_02492857.1, YP_337246.1, YP_001061811.1, YP_105607.1, ZP_0237 1503.1, ZP_02503962.1, ZP_03456839.1, ZP_02461690.1, ZP_03794634.1, ZP_01769736.1, Z P_01769308.1, ZP_02358948.1, ZP_02487736.1, ZP_02408758.1, YP_002234937.1, ZP_02891 477.1, YP_001778806.1, YP_623141.1, YP_838721.1, ZP_04943307.1, YP_776391.1, YP_0043 48704.1, ZP_02907619.1, YP_371316.1, ZP_02389948.1, YP_001811694.1, YP_002908244.1, ZP_02511808.1, ZP_02376542.1, EGC99877.1, ZP_02451760.1 or ZP_02414414.1, or having a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues for a particular said accession number have been modified by deletion, insertion, substitution or a combination thereof, and still having at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme having the particular said accession number. Enzyme E 2b where the enzyme E 2b is understood to mean the ability to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid into α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid.

[0048] Enzyme E 3 is selected from the group consisting of: At least one enzyme E 3a And, having the polypeptide sequence NP_249821.1, or It has a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues have been modified by deletion, insertion, substitution or a combination thereof relative to the reference sequence NP_249821.1, and still has at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme having the reference sequence NP_249821.1. Enzyme E 3a where the enzyme E 3a is understood to mean the ability to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid into α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid; One enzyme E 3b And, Polypeptide sequences AJY02981.1, FN601387.1, FN601391.1 YP_440071.1, ZP_02375899.1, ZP_02466676.1, YP_001075863.1, ZP_02408796.1, YP_335530.1, ZP_01769176.1, YP_105609.1, ZP_01770867.1, ZP_04520 873.1, YP_110560.1, YP_001024014.1, ZP_03450125.1, YP_001061813.1, YP_ 111359.1, ZP_00440994.2, ZP_03456926.1, ZP_02358946.1, ZP_00438001.2, ZP_02461478.1, ZP_02503929.1, ZP_02511832.1, YP_004348706.1, ZP_0489 8742.1, YP_002908246.1, ZP_02382844.1, EGD05167.1, YP_001778808.1, YP_ 001811692.1, YP_002234935.1, YP_371318.1, YP_623143.1, YP_776389.1, ZP_02891479.1, ZP_02907617.1, ZP_02417424.1 or ZP_04898743.1, or having a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues for a particular said accession number have been modified by deletion, insertion, substitution or a combination thereof, and still having at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme having the particular said accession number. Enzyme E 3b where the enzyme E 3b The enzymatic activity of is understood to mean the ability to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid into α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid.

[0049] Enzyme E 1a ~E 3b It will be apparent that the activities specifically listed for are merely specific illustrative choices of the wide variety of activities of the enzymes, and each listed activity is one for which a reliable measurement is available for a given enzyme. Thus, unbranched saturated C 10 Although enzymes that convert substrates bearing -alkyl groups may have reduced activity, C 6 - or C 16 It is clear that substrates bearing -alkyl groups (which may also be branched or unsaturated) will be converted similarly.

[0050] A preferred cell according to the invention is, as a wild type, unable to produce or unable to produce in detectable amounts of rhamnolipids, and more preferably, as a wild type, unable to produce the enzyme E 1 , E 2 and E 3 or has no detectable activity.

[0051] According to the invention, the following enzyme combinations are used: E 1 , E 2 , E 3 , E 1 E 2 , E 1 E 3 , E 2 E 3 and E 1 E 2 E 3 Of these, cells having increased activity of: E 2 , E 2 E 3 and E 1 E 2 E 3 , especially E 1 E 2 E 3 is particularly advantageous.

[0052] Enzyme combination E 1 E2 E 3 In a preferred embodiment of the cell according to the invention having an increased activity of n=1.

[0053] In the present invention, the term "increased activity of an enzyme" should be taken to mean advantageously increased intracellular activity.

[0054] In principle, an increase in enzyme activity can be achieved by increasing the copy number of the gene sequence encoding the enzyme, by using a strong promoter or an improved ribosome binding site, by attenuating the negative regulation of gene expression, for example by using a transcription regulator, or by promoting the positive regulation of gene expression, for example by using a transcription regulator, by changing the codon usage of the gene, by increasing the half-life of the mRNA or the enzyme in various ways, by changing the regulation of the expression of the gene, or by using genes or alleles that code for the corresponding enzymes with increased activity, and by appropriately combining these methods. The increase in activity is advantageously increased according to the present invention by increasing the copy number of the gene sequence encoding the enzyme compared to the wild type. The introduction of a copy of a gene sequence that was not previously present in the wild type corresponds trivially to an increase in the copy number from 0 to 1.

[0055] The genetically modified cells according to the invention are produced, for example by transformation, transduction, conjugation or a combination of these methods, using a vector comprising the desired gene, an allele or part of said gene, and optionally a promoter allowing expression of the gene. Heterologous expression is achieved in particular by integrating the gene or allele into the chromosome of the cell or by replicating the vector extrachromosomally.

[0056] An overview of options for increasing enzymatic activity in cells is given, by way of example, for pyruvate carboxylase in DE-A-10031999, the content of which is incorporated herein by reference and whose disclosure forms part of the disclosure of the present invention with regard to options for increasing enzymatic activity in cells.

[0057] The expression of the above enzymes or genes and all the following enzymes or genes can be detected by one-dimensional and two-dimensional protein gel separation and then optically identifying protein concentration in gel using appropriate evaluation software.If the increase in enzyme activity is based only on the increase in the expression of the corresponding gene, the increase in said enzyme activity can be easily quantified by comparing one-dimensional or two-dimensional protein separation between wild-type and genetically modified cells.The usual method of making protein gel and identifying said protein in the case of coryneform bacteria is the method described by Hermann et al. (Electrophoresis, 22:1712.23 (2001)). Protein concentration can be similarly analyzed by Western blot hybridization using antibodies specific for the protein to be detected (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989) followed by optical evaluation using appropriate software for concentration measurement (Lohaus and Meyer (1989) Biospektrum, 5:32-39; Lottspeich (1999) Angewandte Chemie 111:2630-2647). The activity of DNA-binding proteins can be measured by using a DNA band shift assay (also called gel retardation) (Wilson et al. (2001) Journal of Bacteriology, 183:2151-2155). The effect of DNA binding proteins on the expression of other genes can be detected by a variety of well-described reporter gene assays (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989).Intracellular enzyme activity can be measured by a variety of described methods (Donahue et al. (2000) Journal of Bacteriology 182(19):5624-5627; Ray et al. (2000) Journal of Bacteriology 182(8):2277-2284; Freedberg et al. (1973) Journal of Bacteriology 115(3):816-823). Unless a specific method for measuring the activity of a particular enzyme is described in the following description, increased enzyme activity and decreased enzyme activity are preferably measured using the methods described in Hermann et al., Electophoresis, 22:1712-23(2001), Lohaus et al., Biospektrum 5 32-39(1998), Lottspeich, Angewandte Chemie 111:2630-2647(1999) and Wilson et al., Journal of Bacteriology 183:2151-2155(2001).

[0058] If the increase in enzyme activity is achieved by mutation of an internal gene, such mutation may be produced indirectly by traditional methods, for example by UV irradiation or by mutagenic chemicals, or by genetic engineering methods, in particular deletion, insertion and / or nucleotide substitution. Modified cells are obtained by these mutations. Particularly advantageous mutants of enzymes are those that are no longer under feedback, product or substrate inhibition, or at least less so compared to the wild-type enzyme.

[0059] When the increase in enzyme activity is achieved by increasing the synthesis of the enzyme, for example, the copy number of the relevant gene is increased or the promoter and the control region or the ribosome binding site located upstream of the structural gene are mutated. An expression cassette integrated upstream of the structural gene shows a similar effect. Furthermore, expression can be increased at any desired time by an inducible promoter. However, in addition, so-called "enhancers" can also be provided as control sequences to the enzyme gene, which also cause increased gene expression by improving the interaction between RNA polymerase and DNA. Expression can also be improved by measures that extend the life span of the mRNA. Furthermore, enzyme activity can also be enhanced by inhibiting the degradation of the enzyme protein. Here, the gene or gene construct is present in a plasmid with various copy numbers or integrated and amplified in the chromosome. Alternatively or additionally, overexpression of the relevant gene can be achieved by modifying the medium composition and culture.Guidance in this regard can be found by those skilled in the art, inter alia, in Martin et al. (Bio / Technology 5, 137-146 (1987)), Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)), Eikmanns et al. (Gene 102, 93-98 (1991)), EP-A-0472869, US 4,601,893, Schwarzer and Puehler (Bio / Technology 9, 84-87 (1991)), Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132(1994)), LaBarre et al. (Journal of Bacteriology 175, 1001-1007(1993)), WO-A-96 / 15246, Malumbres et al. (Gene 134, 15-24(1993)), JP-A-10-229891, Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195(1998)), and known genetics and molecular biology textbooks. The above techniques, such as mutation, result in genetically modified cells.

[0060] To increase the expression of a particular gene, for example, episomal plasmids are used. In principle, as plasmids or vectors, all embodiments available to the skilled artisan for this purpose are possible. Such plasmids and vectors can be inferred, for example, from the brochures of Novagen, Promega, New England Biolabs, Clontech or Gibco BRL. Further advantageous plasmids and vectors can be found in: Glover, DM (1985) DNA cloning: a practical approach, Vol. I-III, IRL Press Ltd., Oxford; Rodriguez, RL and Denhardt, D. T (eds) (1988) Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, DV (1990) Systems for heterologous gene expression, Methods Enzymol. 185, 3-7; Sambrook, J.; Fritsch, EF and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York.

[0061] The plasmid vector containing the gene to be amplified is then transferred to the desired strain by conjugation or transformation. Methods of conjugation are described, for example, in Schaefer et al., Applied and Environmental Microbiology 60:756-759(1994). Methods of transformation are described, for example, in Thierbach et al., Applied Microbiology and Biotechnology 29:356-362(1988), Dunican and Shivnan, Bio / Technology 7:1067-1070(1989) and Tauch et al., FEMS Microbiology Letters 123:343-347(1994). After homologous recombination by "crossover" event, the resulting strain contains at least two copies of the gene of interest.

[0062] In the present invention, an increase in the activity of an enzyme is particularly advantageously achieved by increasing the copy number of the region coding for the enzyme under consideration compared to the wild-type cell, in particular by coupling it to a strong promoter, and in the case of an enzyme already present in the wild type, by using a stronger promoter compared to the one present in the wild-type gene.

[0063] As used above and in the following description, the enzyme x The expression "increased activity of a particular enzyme E compared to its wild type" refers to x It should always be understood that the term "enzyme E" means that the activity of the enzyme is preferably increased by at least 2-fold, particularly preferably by at least 10-fold, even more preferably by at least 100-fold, even more preferably by at least 1000-fold and most preferably by at least 10000-fold. x The cells according to the invention have an "increased activity of the enzyme E compared to its wild type", in particular when the wild type is x or at least has no detectable activity, and only after the enzymatic activity has been increased, for example by overexpression, this enzyme E xIn this context, the term "overexpression" or "increasing expression" as used in the following description also includes cells in which the starting cells, e.g. "wild type cells", do not express or at least do not express detectably and which express the enzyme E only by recombinant methods. x This also includes cases where detectable synthesis of is induced.

[0064] The modification of amino acid residues in a given polypeptide sequence that does not lead to significant changes in the properties and functions of the given polypeptide is well known to those skilled in the art. Thus, for example, conserved amino acids can be exchanged. Examples of such suitable amino acid substitutions are: alanine and serine; arginine and lysine; asparagine and glutamine or histidine; aspartic acid and glutamic acid; cysteine ​​and serine; glutamine and asparagine; glutamic acid and aspartic acid; glycine and proline; histidine and asparagine or glutamine; isoleucine and leucine or valine; leucine and methionine or valine; lysine and arginine or glutamine or glutamic acid; methionine and leucine or isoleucine; phenylalanine and methionine or leucine or tyrosine; serine and threonine; threonine and serine; tryptophan and tyrosine; tyrosine and tryptophan or phenylalanine; valine and isoleucine or leucine. It is also known that alterations, eg, in the form of amino acid insertions or deletions, particularly at the N- or C-terminus of a polypeptide, often do not significantly affect the function of the polypeptide.

[0065] "Amino acid identity" associated with the enzymes used in the present invention is determined using known methods, generally using special computer programs with algorithms that take into account prescribed requirements.

[0066] A preferred method for determining identity first produces a maximum alignment between the sequences to be compared. Computer programs for determining identity include, but are not limited to, the GCG program package, which includes: GAP (Deveroy, J. et al., Nucleic Acid Research 12(1984), p. 387), Genetics Computer Group University of Wisconsin, Medicine(Wi), as well as BLASTP, BLASTN and FASTA (Altschul, S. et al., Journal of Molecular Biology 215(1990), p. 403-410). BLAST programs are available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Handbuch, Altschul S. et al., NCBI NLM NIH Bethesda ND 22894; Altschul S. et al. supra).

[0067] The well-known Smith-Waterman algorithm can also be used to determine identity.

[0068] Preferred parameters for determining "amino acid identity" when using the BLASTP program are as follows (Altschul, S. et al., Journal of Molecular Biology 215 (1990), pp. 403-410): Expected Threshold: 10 Word size: 3 Matrix: BLOSUM62 Gap Cost: Existence: 11; Extension: 1 Configuration Adjustment: Conditional score matrix configuration adjustment The above parameters are the default parameters for amino acid sequence comparison. The GAP program is also suitable for use with the above parameters. In the present invention, 60% identity by the above algorithm means 60% identity. The same applies for higher identities.

[0069] The activity of the enzyme can be measured by disrupting the cells containing said activity using methods known to those skilled in the art, for example, using a bead mill, French press or ultrasonic grinder, and then removing intact cells, cell debris and disruption aids, such as glass beads, by centrifugation at 11000×g and 4° C. for 10 minutes. The resulting cell-free crude extract can then be used to perform an enzyme assay, followed by LC-ESI-MS detection of the product. The enzyme can also be enriched or purified to homogeneity by chromatographic methods (e.g., nickel-nitrilotriacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography or ion exchange chromatography) using methods known to those skilled in the art.

[0070] Although it is a minor point and mentioned only for completeness, to measure increased or decreased activity compared to the wild type of cells, a wild type reference culture exposed to the same conditions as the sample to be measured is used.

[0071] The cell-free crude extract obtained as described above was used to detect the enzyme E 1 The activity of is determined as follows: the standard assay uses 100 μM E. coli ACP, 1 mM β-mercaptoethanol, 200 μM malonyl-coenzyme A, 40 μM octanoyl-coenzyme A (E 1a ) or dodecanoyl-Coenzyme A (E 1b (for example), 100 μM NADPH, 2 μg E. coli FabD, 2 μg Mycobacterium tuberculosis FabH, 1 μg E. coli FabG, 0.1 M sodium phosphate buffer (pH 7.0), and 5 μg enzyme E 5 The ACP, β-mercaptoethanol and sodium phosphate buffer are pre-incubated at 37° C. for 30 minutes to completely reduce the ACP. 1The reaction is started by the addition of 0.1% NaCl. The reaction is stopped with 2 ml water, which has been acidified to pH 2.0 with HCl, and then extracted twice with 2 ml chloroform / methanol (2:1 (v:v)). Phase separation is carried out by centrifugation (16100 g, 5 min, RT). The lower organic phase is removed and completely evaporated in a vacuum centrifuge, and the precipitate is taken up in 50 μl methanol. The insoluble components are precipitated by centrifugation (16100 g, 5 min, RT) and the samples are analyzed by LC-ESI-MS. The corresponding mass traces and MS 2 The product is identified by spectral analysis.

[0072] The cell-free crude extract obtained above was then used to detect the enzyme E 2 The activity of 3-hydroxydecanoyl-3-hydroxydecanoic acid (E) is determined as follows: a standard assay can consist of 185 μl of 10 mM Tris-HCl (pH 7.5), 10 μl of 125 mM dTDP-rhamnose, and 50 μl of dissolved crude protein extract (approximately 1 mg total protein) or purified protein (5 μg purified protein). 2a ) or 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid (E 2b The reaction is started by the addition of 10 μl of a 10 mM ethanolic solution of 10 mM acetone (in the case of 10 mM) and incubated for 1 h at 30° C. with shaking (600 rpm). The reaction is then mixed with 1 ml of acetone. The insoluble components are precipitated by centrifugation (16100 g, 5 min, room temperature) and the samples are analyzed by LC-ESI-MS. The corresponding mass traces and MS 2 The product is identified by spectral analysis.

[0073] The cell-free crude extract obtained above was then used to detect the enzyme E 3The activity of is determined as follows: a standard assay can consist of 185 μl of 10 mM Tris-HCl (pH 7.5), 10 μl of 125 mM dTDP-rhamnose, and 50 μl of dissolved crude protein extract (approximately 1 mg total protein) or purified protein (5 μg purified protein). 3a ) or α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid (E 3b The reaction is started by the addition of 10 μl of a 10 mM ethanolic solution of 10 mM ethanol (in the case of 10 mM ethanol) and incubated for 1 h at 30° C. with shaking (600 rpm). The reaction is then mixed with 1 ml of acetone. The insoluble components are precipitated by centrifugation (16100 g, 5 min, room temperature) and the samples are analyzed by LC-ESI-MS. The corresponding mass traces and MS 2 The product is identified by spectral analysis.

[0074] An advantageous cell according to the invention has at least one enzyme E, which catalyzes the conversion of D-glucose and quinone to D-glucono-1,5-lactone and quinol, when compared to the wild type. 4 The present invention is characterized in that the antibody has been genetically modified to have reduced activity of

[0075] According to the present invention, E 4 is advantageously a glucose 1-dehydrogenase of the EC 1.1.5.2 class. A particularly advantageous enzyme E 4 teeth, The enzyme encoded by the gcd gene, and an enzyme having a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues have been modified by deletion, insertion, substitution or a combination thereof with respect to the enzyme encoded by the gcd gene, and which still has at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of an enzyme having a reference sequence for the enzyme encoded by the gcd gene. is selected from.

[0076] In particular, enzyme E 4 teeth, having the polypeptide sequence AAN67066.1, or It has a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues have been modified by deletion, insertion, substitution or a combination thereof relative to AAN67066.1, and still has at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme having the reference sequence AAN67066.1. Enzyme E 4 is selected from.

[0077] Enzyme E 1 The activity is measured using the Abcam Colorimetric Glucose Dehydrogenase Assay Kit (part number ab102532) according to the manufacturer's instructions using cell-free extracts.

[0078] According to the invention, the following enzyme combinations are used: E 4 , E 1 E 4 , E 2 E 4 , E 3 E 4 , E 1 E 2 E 4 , E 1 E 3 E 4 , E 2 E 3 E 4 and E 1 E 2 E 3 E 4 Preferred are cells having an altered activity of E 2 E 4 , E 2 E 3 E 4 and E 1 E 2 E3 E 4 , especially E 1 E 2 E 3 E 4 is particularly advantageous.

[0079] The cells according to the invention have, in comparison with their wild type counterparts, at least one enzyme E which catalyzes the excretion of the rhamnolipid of general formula (I) from the cells into the surrounding medium. 5 It is further advantageous and therefore preferred if the polypeptide has been genetically modified to have increased activity of

[0080] In the case of the preferred cells according to the invention, E 5 teeth, The following polypeptide sequences:AAG04520.1、AJY02996.1、ZP_05590661.1、YP_439278.1、YP_440069.1、ZP_04969301.1、ZP_04520234.1、YP_335528.1、YP_001075859.1 、YP_001061817.1、ZP_02487499.1、YP_337251.1、ZP_04897712.1、ZP_048 10190.1、YP_990322.1、ZP_02476924.1、ZP_04899735.1、ZP_04893873.1、Z P_02365982.1、YP_001062909.1、YP_105611.1、ZP_03794061.1、ZP_03457011.1、ZP_02385401.1、ZP_02370552.1、YP_105236.1、ZP_04905097.1、YP_776387.1、YP_001811690.1、YP_004348730.1、YP_004348708.1、YP_371320.1、YP_623145.1、YP_001778810.1、YP_002234933.1、CCE52909.1、YP_002 908248.1, ZP_04954557.1, ZP_04956038.1, ZP_02408950.1, ZP_02375897.1, ZP_02389908.1, YP_439274.1, YP_001074762.1, YP_337247.1, YP_110559.1, ZP_02495927.1, YP_111360.1, YP_105608.1, ZP_02487826.1, ZP_02358947.1, YP_001078605.1, ZP_00438000.1, ZP_00440993.1, ZP_02477260 .1、YP_371317.1、YP_001778807.1、ZP_02382843.1、YP_002234936.1、YP_623142.1、ZP_02907618.1、ZP_02891478.1、YP_776390.1、ZP_04943308.1、YP_001811693.1、ZP_02503985.1、YP_004362740.1、YP_002908245.1、YP_004348705.1、ZP_02408798.1、ZP_02417250.1、EGD05166.1、ZP_02458677.1, ZP_02465793.1, YP_001578240.1, ZP_04944344.1, YP_771932.1, ZP_02889166.1, YP_002232614.1, ZP_03 574808.1, ZP_02906105.1, YP_001806764.1, YP_619912.1, YP_001117913.1, YP_106647.1, YP_001763368.1 , ZP_02479535.1, ZP_02461743.1, YP_560998.1, YP_331651.1, ZP_04893070.1, YP_003606714.1, ZP_025039 95.1, ZP_06840428.1, YP_104288.1, ZP_02487849.1, ZP_02353848.1, YP_367475.1, ZP_02377399.1, ZP_023 72143.1, YP_001897562.1, ZP_02361066.1, YP_440582.1, ZP_03268453.1, AET90544.1, YP_003908738.1, YP _004230049.1, ZP_02885418.1, CDH72316.1, WP_001297013.1, WP_010955775.1, WP_010955671.1, WP_01095 5672.1, WP_010955673.1, WP_010952401.1, WP_010952402.1, WP_010952403.1, WP_010952855.1, WP_010954573.1, WP_010954631.1, WP_010954632.1, WP_010954404.1, WP_004575310.1 or ZP_02511831.1, or It has a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues have been modified by deletion, insertion, substitution or a combination thereof, for a particular above-mentioned accession number, and still has at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme having the above-mentioned accession number. Enzyme E 5 wherein the enzyme E is selected from the group consisting of 5is understood to mean the ability to excrete the rhamnolipid of general formula (I) from the cell into the surrounding medium.

[0081] The cell-free crude extract obtained as above was then used to produce the enzyme E 5 By measuring the amount of enzyme E 5 This means that more enzyme E can be produced per unit of biomass. 5 This quantification is based on the assumption that more rhamnolipids of general formula (I) can be exported from the cell to the surrounding medium by the enzyme E 5 This can be carried out by immunological detection using antibodies specific for (see Kurien, TB, Scofield, R. H (Eds.). Protein Blotting and Detection: Methods and Protocols. Methods in Molecular Biology, Vol. 536. 1st Ed., Humana Press. NY USA, 2009) or by mass spectrometry (see Schmidt, A., Kellermann, J. & Lottspeich, F. A novel strategy for quantitative proteornics using isotope-coded protein labels. Proteomics 5, 4-15(2005)).

[0082] Or enzyme E 5The activity of can also be determined by carrying out an uptake assay using radiolabeled rhamnolipids and inside-out vesicles produced from the cells according to the invention. A general method is described, for example, in Nies DH, The cobalt, zinc, and cadmium efflux system CzcABC from Alcaligenes eutrophus functions as a cation-proton antiporter in Escherichia coli.J Bacteriol. 1995. 177(10):2707-12, or in Lewinson O, Adler J, Poelarends GJ, Mazurkiewicz P, Driessen AJ, Bibi E. The Escherichia coli multidrug transporter MdfA catalyzes both electrogenic and electroneutral transport reactions. Proc Natl Acad Sci US A. 2003 Feb 18;100(4):1667-72.

[0083] The cells according to the invention can be advantageously used for the production of rhamnolipids.

[0084] The present invention therefore further provides the use of a cell according to the invention for producing a compound of general formula (I).

[0085] The present invention further relates to rhamnolipids, in particular those of the general formula (I) [In the formula, m is 2, 1 or 0, in particular 1 or 0, n is 1 or 0, in particular 1; R 1 and R 2are identical or different organic radicals having 2 to 24, preferably 5 to 13, carbon atoms, in particular optionally branched, optionally substituted, in particular hydroxy-substituted, optionally unsaturated, in particular optionally mono-, di- or triunsaturated alkyl groups, preferably pentenyl, heptenyl, nonenyl, undecenyl and tridecenyl and (CH 2 ) o -CH 3 (wherein o is selected from the group consisting of 1 to 23, advantageously 4 to 12), comprising the following process steps: I) contacting the cells according to the invention with a medium containing a carbon source; II) culturing the cells under conditions that allow the cells to produce rhamnolipids from the carbon source; and III) Optionally, isolating the produced rhamnolipid The present invention provides a method comprising:

[0086] The genetically modified cells according to the invention can be contacted with a culture medium and cultured continuously or discontinuously in a batch or fed-batch manner or by repeated fed-batch culture in order to produce the product.

[0087] Semi-continuous methods are also conceivable, as described in GB-A-1009370. Overviews of known culture methods are given in the texts by Chmiel ("Bioprozesstechnik 1. Einfuehrung in die Bioverfahrenstechnik" [Bioprocess technology 1. Introduction to Bioprocess Technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or by Storhas ("Bioreaktoren und periphere Einrichtungen" [Bioreactors and Peripheral Devices] (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).

[0088] The culture medium to be used must adequately meet the requirements of the particular strain. Descriptions of culture media for various yeast strains are contained, for example, in "Nonconventional yeast in biotechnology" (Ed. Klaus Wolf, Springer-Verlag Berlin, 1996).

[0089] The carbon sources used are carbohydrates, such as glucose, sucrose, arabinose, xylose, lactose, fructose, maltose, molasses, starch, cellulose and hemicellulose, vegetable and animal oils and fats, such as soybean oil, safflower oil, peanut oil, hemp seed oil, jatropha oil, coconut fat, pumpkin seed oil, linseed oil, corn oil, poppy seed oil, evening primrose oil, olive oil, palm kernel oil, palm oil, rapeseed oil, sesame oil, sunflower oil, grape seed oil, walnut oil, wheat germ oil and Coconut fat, fatty acids such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitol acid, stearic acid, arachidonic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, γ-linolenic acid and their methyl or ethyl esters and fatty acid mixtures, mono-, di- and triglycerides containing the aforementioned fatty acids, alcohols such as glycerol, ethanol and methanol, hydrocarbons such as methane, carbonaceous gases and gas mixtures such as CO, CO 2 , synthesis gas or combustion gas, amino acids such as L-glutamate or L-valine or organic acids such as acetic acid. These substances can be used individually or as a mixture. Particularly advantageous is the use of carbohydrates, in particular mono-, oligo- or polysaccharides, as well as hydrocarbons, in particular alkanes, alkenes and alkynes and the monocarboxylic acids derived therefrom and the mono-, di- and triglycerides derived therefrom and glycerol and acetate, as carbon sources as described in US Pat. No. 6,01,494 and US Pat. No. 6,136,576. Very particular preference is given to mono-, di- and triglycerides which contain esterification products of glycerol with caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, arachidonic acid, behenic acid, oleic acid, linoleic acid, linolenic acid and / or γ-linolenic acid.

[0090] The main advantage of the present invention is that the cells according to the invention are able to produce rhamnolipids from very simple carbon sources, such as glucose, sucrose or glycerol, which means that it is not necessary to provide long-chain carbon sources in the medium during the method according to the invention. It is therefore advantageous, in case of insufficient availability, for the medium in step I) of the method according to the invention to contain no or no detectable amounts of carboxylic acids with a chain length longer than 6 carbon atoms or esters or glycerides derivable therefrom.

[0091] The nitrogen sources used may be nitrogen-containing organic compounds such as peptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, ammonia, ammonium hydroxide or aqueous ammonia. The nitrogen sources may be used individually or as a mixture.

[0092] The phosphorus source used may be phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, the culture medium should contain salts of metals necessary for growth, such as magnesium sulfate or ferrous sulfate. Finally, essential growth substances such as amino acids and vitamins may be used in addition to the above substances. Furthermore, suitable precursors may be added to the culture medium. The above starting materials may be added to the culture in the form of a single batch or may be fed appropriately during the cultivation. To adjust the pH of the culture, basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acidic compounds such as phosphoric acid or sulfuric acid are suitably used. To adjust foaming, antifoaming agents such as fatty acid polyglycol esters can be used. To maintain the stability of the plasmids, suitable selection substances, such as antibiotics, can be added to the medium. To maintain aerobic conditions, oxygen or an oxygenic gas mixture, such as air, is introduced into the culture.

[0093] The temperature of the culture is usually higher than 20°C, preferably higher than 25°C and can also be higher than 40°C, preferably not exceeding a culture temperature of 95°C, particularly preferably 90°C and most preferably 80°C.

[0094] In step III) of the method according to the invention, the rhamnolipids produced by the cells can optionally be isolated from the cells and / or from the culture medium, using any method known to the skilled artisan for isolating low molecular weight substances from complex compositions for use for isolation purposes, such as filtration, extraction, adsorption (chromatography) or crystallization.

[0095] Furthermore, the product phase contains biomass residues and various impurities, such as oil, fatty acids and other culture medium components. The impurities are advantageously removed in a solvent-free manner. For example, the product phase can be diluted with water to facilitate pH adjustment. The product phase and the aqueous phase can then be homogenized by lowering or raising the pH with acid or alkali to transfer the rhamnolipids to a water-soluble form. Possibly, solubilization of the rhamnolipids in the aqueous phase can be supported by incubation at relatively high temperatures, for example 60-90°C, and constant mixing. Subsequent raising or lowering of the pH with alkali or acid can transfer the rhamnolipids again to a water-insoluble form, and thus they can be easily separated from the aqueous phase. The product phase can then be further washed one or more times with water to remove water-soluble impurities.

[0096] The oil residues can be removed, for example, by extraction with a suitable solvent, advantageously with an organic solvent. Alkanes, for example n-hexane, are preferred as solvents.

[0097] As an alternative to the above-mentioned solvent-free method, the product can be removed from the aqueous phase using a suitable solvent, for example an ester, for example ethyl acetate or butyl acetate. The described extraction steps can be carried out in any desired order. Here, a solvent, in particular an organic solvent, is advantageously used. A preferred solvent is n-pentanol. The solvent is removed, for example, by distillation. The lyophilized product can then be further purified, for example, by chromatographic methods. Examples that can be mentioned in this respect include precipitation with a suitable solvent, extraction with a suitable solvent, complexation, for example with cyclodextrins or cyclodextrin derivatives, crystallization, purification or isolation by chromatographic methods, or conversion of the rhamnolipid into an easily removable derivative.

[0098] A particularly suitable rhamnolipid isolation procedure for process step III) comprises the following sub-steps: A) transferring the rhamnolipid into an aqueous medium having a pH of less than 6; B) contacting said medium with at least one organic solvent to obtain a multiphase system and removing the aqueous phase; C) increasing the pH to a pH of 6 or greater to obtain a multi-phase organic system; D) Removing the rhamnolipid-enriched organic phase; and E) Optionally, further purifying the rhamnolipid This includes the method of:

[0099] A detailed description of how to carry out this advantageous embodiment of method step III) is given in US 20140148588.

[0100] The invention likewise provides rhamnolipids obtainable using the process according to the invention, in particular the above-mentioned rhamnolipid mixtures also obtainable using the process according to the invention.

[0101] Advantageously, the rhamnolipids and mixtures obtainable with the process according to the invention can be used in cleaning agents, in cosmetic or pharmaceutical preparations and in crop protection agents.

[0102] The invention therefore further provides the use of rhamnolipids obtained using the process according to the invention for the production of cosmetic, dermatological or pharmaceutical preparations, crop protection agents and care products and cleaning and surfactant concentrates.

[0103] The present invention will be described by way of example with reference to the examples presented below, but the present invention is not limited to the embodiments explicitly shown in the examples, and its scope of application is apparent from the entire specification and claims of the present invention.

[0104] example: Example 1 (not according to the invention): The strain P. putida KT2440 Δupp+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} was used.

[0105] Construction of strain P. putida KT2440 Δupp+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} Plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} was constructed for heterologous expression of genes rhlA, rhlB and rhlC and genes rmlB, rmlD, rmlA and rmlC (both from P. aeruginosa). This plasmid contains, firstly, a synthetic operon consisting of the genes rhlA and rhlB (encoding rhamnosyltransferase 1) and rhlC (encoding rhamnosyltransferase 2) from P. aeruginosa DSM 1128 (SEQ ID NO: 1), and, secondly, an operon consisting of the genes rmlB (encoding dTDP-D-glucose 4,6-dehydratase), rmlD (encoding dTDP-4-dehydrorhamnose reductase), rmlA (encoding glucose-1-phosphate thymidylyltransferase) and rmlC (encoding dTDP-4-dehydrorhamnose 3,5-epimerase) from P. aeruginosa DSM 19880 (SEQ ID NO: 2). The genes rhlABC encode the rhamnose-inducible P Rha The rmlBDAC gene is under the control of the arabinose-inducible P BAD The rmlBDAC gene was amplified from genomic DNA derived from P. aeruginosa DSM 19880, and a synthetic rhlABC operon was obtained by gene synthesis. Rha Promoter cassette (SEQ ID NO:3) and P BAD The promoter cassette (SEQ ID NO: 4) as well as the terminator sequence (SEQ ID NO: 5) were amplified from genomic E. coli DNA. The rhlABC genes are required for the synthesis of dirhamnolipid, whereas the rmlBDAC gene is required for the provision of activated dTDP-L-rhamnose.

[0106] The vector is based on the plasmid pACYC184 (New England Biolabs, Frankfurt am Main, Germany) and contains the p15A origin of replication for replication in E. coli and the pVS1 origin of replication for replication in P. putida. The pVS1 origin of replication was amplified from the Pseudomonas plasmid pVS1 (Itoh Y, Watson JM, Haas D, Leisinger T, Plasmid 1984, 11(3), 206-20). Vector parts and DNA fragments were cloned using commercially available in vitro DNA assembly kits (e.g. NEBuilder HiFi DNA Assembly Cloning Kit according to the manufacturer's instructions (NEB; Frankfurt am Main, Germany)). E. coli 10 beta chemically competent cells (NEB; Frankfurt am Main, Germany) were transformed using methods known to those skilled in the art. Correct insertion of the target gene was checked by restriction analysis and the authenticity of the introduced homologous region was confirmed by DNA sequencing. The size of the resulting plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} (SEQ ID NO: 6) is 17337 base pairs.

[0107] This plasmid was then introduced into P. putida KT2440 Δupp. This strain is used as the starting strain for constructing markerless gene deletions in P. putida (Graf & Altenbuchner, 2011, Applied and Environmental Microbiology, Vol 77, No. 15, 5549-5552, DOI:10.1128 / AEM.05055-11). The method is based on a negative counterselection system for P. putida, which exploits the activity of uracil phosphoribosyltransferase and the sensitivity of P. putida to the antimetabolite 5-fluorouracil. The deletion of the upp gene does not affect rhamnolipid biosynthesis. Transformation of P. putida KT2440 Δupp with vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} was performed as described by Iwasaki et al. (Iwasaki K, Uchiyama H, Yagi O, Kurabayashi, T, Ishizuka K, Takamura Y, Biosci. Biotech. Biochem. 1994. 58(5):851-854). Plasmid DNA was isolated from each of the 10 clones and analyzed. The strain harboring this plasmid was designated P. putida KT2440 Δupp pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0108] The production of biotechnological surfactants was carried out in an Eppendorf eight-fold parallel fermentation system "DASGIP".

[0109] For fermentation, a 1 L reactor was used. The pH probe was calibrated by two-point calibration with measuring solutions at pH 4.0 and pH 7.0. The reactor was filled with 300 mL of water and autoclaved at 121 °C for 20 min to ensure sterility. The next morning, the water was removed in a clean bench and replaced with sterile fermentation medium (autoclaved: 2.2 g / L of (NH 4 ) 2 SO 4 , 0.02g / L NaCl, 0.4g / L MgSO 4 7H 2 O, 0.04 g / L CaCl 2 2H 2 O, sterilized separately: 2g / L KH 2 PO 4 , 15 g / L glucose, 10 mL / L trace element solution M12 [sterile filtered: 0.2 g / L ZnSO 4 7H 2 O, 0.1 g / L MnCl 2 4H 2 O, 1.5 g / L trisodium citrate 2H 2 O, 0.1 g / L CuSO 4 5H 2 O, 0.002 g / L NiCl 2 6H 2 O, 0.003 g / L Na 2 MoO 4 2H 2 O, 0.03 g / L H 3 BO 3 , 1g / L FeSO 4 7H 2 O]). Next, pO 2 The probes were calibrated by one-point calibration (agitator: 600 rpm / aeration: 10 sL / h air) and the feed, calibrator and inducer lines were cleaned by cleaning in place: for this, the tubes were flushed with 70% ethanol, then with 1 M NaOH, then with sterile demineralized water and finally filled with the specific medium.

[0110] Using 100 μL from a frozen culture, the strain (P. putida KT2440 Δupp+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}) was first grown overnight at 30 °C and 200 rpm for approximately 18 h in 25 mL of LB1 medium (10 g / L casein hydrolysate, 5 g / L yeast extract, 1 g / L NaCl) in a 250 mL baffled flask containing 50 mg / L kanamycin. After measuring the optical density of this culture, 50 mL of sterile seed medium (autoclaved: 4.4 g / L NaCl) was added to a 500 mL baffled flask. 2 HPO 4 2H 2 O, 1.5 g / L KH 2 PO 4 , 1g / L NH 4 Cl, 10 g / L yeast extract, sterilized separately: 20 g / L glucose, 0.2 g / L MgSO 4 7H 2 O, 0.006 g / L FeCl 3 , 0.015g / L CaCl 2 , 1mL / L trace element solution SL6 [sterile filtered: 0.3g / L H 3 BO 3 , 0.2 g / L CoCl 2 6H 2 O, 0.1 g / L ZnSO 4 7H 2 O, 0.03 g / L MnCl 2 4H 2 O, 0.01 g / L CuCl 2 2H 2 O, 0.03 g / L Na 2 MoO 4 2H 2 O, 0.02 g / L NiCl 2 6H 2 O]) to a starting OD of 0.2 600 The medium was inoculated from an LB preculture using the PBS and incubated at 30° C., 200 rpm for approximately 7 hours. An optical density OD of approximately 8 was reached. 600 The main culture was incubated at a starting OD of 0.7.600 was used to inoculate.

[0111] To inoculate the reactor using an optical density of 0.7, approximately 26 mL was loaded into a 30 mL syringe and inoculated into the reactor by needle through the septum.

[0112] The following standard program was used: [Table 1]

[0113] The pH was unilaterally adjusted to pH 7.0 using ammonia (12.5%). During cultivation and biotransformation, the dissolved oxygen in the culture was kept constant at 30% by the agitator speed and aeration rate. The fermentation was performed as fed-batch, with a 2.5 g / Lh glucose feed with a 500 g / L glucose feed triggered by the DO peak from the start of the feed. Expression of the recombinantly introduced genes was induced 3 h after the start of the feed by automatic addition of 0.2% (w / v) rhamnose and 0.2% (w / v) arabinose. The required amount of induction sugars was based on the fermentation start volume. Stock solutions of 220 g / L were used for both sugars. Surfactant production started from the time of induction. All online measurement data such as pH, DO, CTR, OTR, as well as flow rates and amounts of substrates, e.g. ammonia solution for pH adjustment, glucose feed or inducer flow rates, were recorded by the DASGIP fermentation system.

[0114] For the fermentation analysis, 2 mL was withdrawn from each vessel as a forecut using a 10 mL syringe and discarded. Following this, another 6 mL was withdrawn from the reactor for the actual analysis. The rhamnolipid content was determined. The fermentation was terminated after 65 hours.

[0115] Rhamnolipid concentrations were determined using HPLC. 100 μL of fermentation sample was mixed with 900 μL of 70% (v / v) n-propanol in an Eppendorf tube and shaken in a Retsch mill at 30 Hz for 1 min. The sample was then centrifuged at 13000 rpm for 5 min and the supernatant was transferred to a new Eppendorf tube. If further dilution was required, this was done using 55% n-propanol. All tubes were immediately closed to avoid evaporation. The samples were then transferred to HPLC vials and stored at -20°C until measurement.

[0116] A 2 ml reaction tube was filled with 1 ml of acetone using a positive displacement pipette (Combitip) and the reaction tube was immediately closed to minimize evaporation. This was followed by the addition of 1 ml of culture broth. After vortexing the culture broth / acetone mixture, the mixture was centrifuged at 13000 rpm for 3 min and 800 μl of the supernatant was transferred to an HPLC vial. An evaporative light scattering detector (Sedex LT-ELSD Model 85LT) was used for detection and quantification of rhamnolipids. The actual measurements were performed using an Agilent Technologies 1200 Series (Santa Clara, California) and a Zorbax SB-C8 Rapid Resolution Column (4.6 × 150 mm, 3.5 μm, Agilent). The injection volume was 5 μl and the run time of the method was 20 min. Aqueous 0.1% TFA (trifluoroacetic acid, solution A) and methanol (solution B) were used as the mobile phase. The column temperature was 40 °C. ELSD (detector temperature 60° C.) and DAD (diode array, 210 nm) were utilized as detectors. The gradient used in the method was as follows: [Table 2]

[0117] Three experiments were carried out in parallel with each of Example 2.

[0118] Total RL concentration measured after 65 hours: 34 g / L.

[0119] Calculated space-time yield: 0.53 g / L h.

[0120] Example 2 (according to the invention) Strain P. putida KT2440 Δupp Δ[PP_1016-1018]::galP_Ec+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}{rhlABC_Pa}{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} was used.

[0121] Construction of a vector for inserting the galP gene into Pseudomonas putida KT2440 Δupp Galactose-H + A vector for inserting the galP gene from E. coli K12, which codes for the symporter GalP, is prepared by PCR amplification of the gene. The template used is the genomic DNA of E. coli K12 W3110. The aim is to exchange the galP gene with the genes PP_1016-PP_1018, which code for the ABC transporter permease, the ABC transporter binding protein and the ABC transporter ATP binding protein, in P. putida KT2440 Δupp. For this, about 680 base pairs upstream and downstream of the genes PP_1016-PP_1018 are amplified by PCR.

[0122] The following primers were used to amplify the galP gene: PCR1:galP O.BF_FM_1 * 07 5'-CCAATACATGCCTGACGCTAAAAAACA-3' (SEQ ID NO: 7) O.BF_FM_1 * 10 5'-TAGACGAGTTAATCGTGAGCGCCTATTT-3' (sequence number 8).

[0123] The following primers were used to amplify the homologous regions upstream and downstream of the PP_1016-PP_1018 genes: PCR2: Upstream region of PP_1016 O.BF_FM_1 * 03 5'-GCCGCTTTGGTCCCGGCCGCCAAGGTCATTAAC-3' (SEQ ID NO: 9) O.BF_FM_1 * 08 5'-TCAGGCATGTATTGGATCCCGAGGTAGT-3' (SEQ ID NO: 10) PCR3: downstream region of PP_1018 O.BF_FM_1 * 02 5'-GCTTGCATGCCTGCAGGCGTGATGTTGTACTTC-3' (SEQ ID NO: 11) O.BF_FM_1 * 09 5'-CGATTAACTCGTCTACACCATCAATAA-3' (sequence number 12).

[0124] The following parameters were used for PCR: Denaturation: 98℃ 30 seconds Denaturation: 98℃ 10sec 30x Annealing: 62℃ 12sec 30x Extension: 72℃ 22 seconds 30x Final extension: 72°C 5 min.

[0125] For amplification, Phusion™ High-Fidelity Master Mix from NEB (Frankfurt am Main, Germany) was used according to the manufacturer's recommendations. 50 μl of each PCR reaction was then resolved on a 1% TAE agarose gel. PCR, agarose gel electrophoresis, ethidium bromide staining of DNA and determination of PCR fragment size are performed by methods known to those skilled in the art. PCR fragments of the expected size (PCR1, 1410 base pairs, (SEQ ID NO: 13); PCR2, 675 base pairs (SEQ ID NO: 14); PCR3, 697 base pairs (SEQ ID NO: 15)) were amplified. PCR products were purified using the Qiagen “QIAquick PCR Purification kit” according to the manufacturer's specifications. The purified PCR product was cloned into the BamHI- and SbfI-cut pKOPp vector (SEQ ID NO: 16) using the NEBuilder HiFi DNA Assembly Cloning Kit according to the manufacturer's instructions (NEB; Frankfurt am Main, Germany). E. coli 10 beta chemically competent cells (NEB, Frankfurt am Main, Germany) were transformed as known to those skilled in the art. Correct insertion of the target gene was checked by restriction analysis and the authenticity of the introduced homologous regions was confirmed by DNA sequencing. The resulting knockout vector was designated pKO_PP_1016-PP_1018::galP (SEQ ID NO: 17).

[0126] Construction of strain P. putida KT2440 Δupp Δ[PP_1016-1018]::galP_Ec The construction of strain P. putida KT2440 Δupp Δ[PP_1016-1018]::galP_Ec was carried out using the plasmid pKO_PP_1016-PP_1018::galP and the method described in Graf et al., 2011 (Graf N, Altenbuchner J, Appl. Environ. Micorbiol., 2011 , 77(15):5549; DOI:10.1128 / AEM.05055-11). The DNA sequence after exchange of genes PP_1016-PP_1018 with galP is given in SEQ ID NO:18. Transformation of P. putida KT2440 Δupp Δ[ΡΡ_1016-1018]::galP_Ec with vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} was performed as described by Iwasaki et al. (Iwasaki K, Uchiyama H, Yagi O, Kurabayashi, T, Ishizuka K, Takamura Y, Biosci. Biotech. Biochem. 1994. 58(5):851-854). Cells were then plated on LB agar plates supplemented with kanamycin (50 μg / ml). The plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} (SEQ ID NO: 6) has already been described in Example 1. Plasmid DNA was isolated from each of the 10 clones and analyzed using restriction analysis. The strain carrying this plasmid was called P. putida KT2440 Δupp Δ[PP_1016-1018]::galP_Ec pACYCATh5-{PrhaSR}{rhaSR_Ec}{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}. The technical implementation was carried out as described in Example 1.

[0127] Three experiments were carried out in parallel with Example 1.

[0128] Total RL concentration measured after 64 hours: 44.5 g / L.

[0129] Calculated space-time yield: 0.70 g / L h.

[0130] Example 3 (according to the invention) Strain P. putida (P. putida) KT2440 Δupp Use Δ[PP_1016-1018]::glf_Zm(co_Pp)+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0131] Construction of a vector for inserting the glf gene into Pseudomonas putida KT2440 Δupp A vector for inserting the glf gene from Zymomonas mobilis, which codes for the glucose facilitator Glf, is prepared by PCR amplification of the gene codon optimized for P. putida KT2440. A synthetic DNA fragment is used as a template. The aim is to exchange the glf gene with genes PP_1016-PP_1018, which code for the ABC transporter permease, ABC transporter binding protein and ABC transporter ATP binding protein, in P. putida KT2440 Δupp. For this, approximately 690 base pairs upstream and downstream of genes PP_1016-PP_1018 are amplified by PCR.

[0132] The following primers are used for amplification of the glf gene: PCR4:glf MW_18_02 5'-TACCTCGGGATCCAATACATGTCCAGCGAGTCGTCCCAG-3' (SEQ ID NO: 19) MW_18_03 5'-TTACTTCTGCGAGCGCCACATC-3' (sequence number 20).

[0133] The following primers are used to amplify the homologous regions upstream and downstream of the PP_1016-PP_1018 genes: PCR5: Upstream region of PP_1016 O.BF_FM_1 * 03 5'-GCCGCTTTGGTCCCGGCCGCCAAGGTCATTAAC-3' (SEQ ID NO: 9) MW_18_01 5'-CATGTATTGGATCCCGAGGTAG-3' (SEQ ID NO: 21) PCR6: downstream region of PP_1018 MW_18_04 5'-GCTCGCAGAAGTAACAATACCTCGTCTACACCATCAATAAGAAAAAG-3' (SEQ ID NO: 22) O.BF_FM_1 * 02 5'-GCTTGCATGCCTGCAGGCGTGATGTTGTACTTC-3' (sequence number 11).

[0134] The following parameters are used for PCR: Denaturation: 98℃ 30 seconds Denaturation: 98℃ 10sec 30x Annealing: 62℃ 12sec 30x Extension: 72℃ 22 seconds 30x Final extension: 72°C 5 min.

[0135] For amplification, Phusion™ High-Fidelity Master Mix from NEB (Frankfurt am Main, Germany) is used according to the manufacturer's recommendations. 50 μl of each PCR reaction is then resolved on a 1% TAE agarose gel. PCR, agarose gel electrophoresis, ethidium bromide staining of DNA and determination of PCR fragment size are performed by methods known to those skilled in the art. PCR fragments of the expected size (PCR4 (SEQ ID NO: 23), 1440 base pairs; PCR5 (SEQ ID NO: 24), 670 base pairs; PCR6 (SEQ ID NO: 25), 710 base pairs) are amplified. PCR products are purified using the Qiagen “QIAquick PCR Purification kit” according to the manufacturer's specifications. The purified PCR product is cloned into the BamHI- and SbfI-cut pKOPp vector (SEQ ID NO: 16) using the NEBuilder HiFi DNA Assembly Cloning Kit according to the manufacturer's instructions (NEB; Frankfurt am Main, Germany). E. coli 10 beta chemically competent cells (NEB, Frankfurt am Main, Germany) are transformed as known to those skilled in the art. Correct insertion of the target gene is checked by restriction analysis and the authenticity of the introduced homologous regions is confirmed by DNA sequencing. The resulting knockout vector is called pKO_PP_1016-PP_1018::glf_Zm(co_Pp) (SEQ ID NO: 26).

[0136] Construction of strain P. putida KT2440 Δupp Δ[PP_1016-1018]::glf_Zm(co_Pp) The construction of strain P. putida KT2440 Δupp Δ[PP_1016-1018]::glf_Zm(co_Pp) is carried out using the plasmid pKO_PP_1016-PP_1018::glf_Zm(co_Pp) and the method described in Graf et al., 2011 (Graf N, Altenbuchner J, Appl. Environ. Micorbiol., 2011,77(15):5549;DOI:10.1128 / AEM.05055-11). The DNA sequence after exchanging genes PP_1016-PP_1018 with glf is given in SEQ ID NO:27. Transformation of P. putida KT2440 Δupp Δ[ΡΡ_1016-1018]::glf_Zm(co_Pp) with vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} is carried out as described in Iwasaki et al. (Iwasaki K, Uchiyama H, Yagi O, Kurabayashi, T, Ishizuka K, Takamura Y, Biosci. Biotech. Biochem. 1994. 58(5):851-854). The cells are then plated on LB agar plates supplemented with kanamycin (50 μg / ml). The plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} (SEQ ID NO: 6) has already been described in Example 1. Plasmid DNA is isolated from each of the 10 clones and analyzed by restriction analysis. The strain carrying this plasmid is called P. putida KT2440 Δupp Δ[PP_1016-1018]::glf_Zm(co_Pp)pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}. The technical implementation was carried out as described in Example 1.

[0137] Three experiments are carried out in parallel with each of Example 1.

[0138] Compared to Example 1, significantly higher total RL concentrations after 64 hours and significantly higher calculated space-time yields are observed.

[0139] Example 4 (according to the invention) Strain P. putida KT2440 Δupp Δ[PP_1016-1018]::[ptsH_Ec ptsl_Ec crr_Ec ptsG_Ec]+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0140] Construction of a vector for inserting the pts gene into Pseudomonas putida KT 2440 Δupp A vector for inserting the pts gene from E. coli K12, which codes for the phosphoenolpyruvate phosphotransferase system PEP-PTS, is prepared by PCR amplification of the genes ptsH, ptsl, crr and ptsG. ptsH codes for the phosphocarrier protein HPr, ptsl codes for the PTS enzyme I, crr codes for the enzyme IIAGlc and ptsG codes for the glucose-specific PTS enzyme IIBC. The template used is the genomic DNA of E. coli K12 W3110. The aim is to exchange the PTS system in P. putida KT2440 Δupp with the genes PP_1016-PP_1018, which code for the ABC transporter permease, the ABC transporter binding protein and the ABC transporter ATP binding protein. For this, approximately 690 base pairs upstream and downstream of the genes PP_1016-PP_1018 are amplified by PCR.

[0141] The following primers are used for amplification of the PTS gene: PCR7: operon ptsH / ptsl / crr O.BF_FM_1 * 23 5'-TCCAATACATGTTCCAGCAAGAAGTTACC-3' (SEQ ID NO: 28) O.BF_FM_1 * 22 5'-CCTGAGTTTACTTCTTGATGCGGATAACC-3' (SEQ ID NO: 29) PCR8:ptsG O.BF_FM_1 * 21 5'-AGAAGTAAACTCAGGAGCACTCTCAATT-3' (SEQ ID NO: 30) O.BF_FM_1 * 26 5'-AGACGAGTTAGTGGTTACGGATGTACTC-3' (sequence number 31).

[0142] The following primers are used to amplify the homologous regions upstream and downstream of the PP_1016-PP_1018 genes: PCR9: Upstream region of PP_1016 O.BF_FM_1 * 03 5'-GCCGCTTTGGTCCCGGCCGCCAAGGTCATTAAC-3' (SEQ ID NO: 9) O.BF_FM_1 * 24 5'-GGAACATGTATTGGATCCCGAGGTAGTG-3' (SEQ ID NO: 32) PCR10: Downstream region of PP_1018 O.BF_FM_1 * 25 5'-ACCACTAACTCGTCTACACCATCAATAAG-3' (SEQ ID NO: 33) O.BF_FM_1 * 02 5'-GCTTGCATGCCTGCAGGCGTGATGTTGTACTTC-3' (sequence number 11).

[0143] The following parameters are used for PCR: Denaturation: 98℃ 30 seconds Denaturation: 98℃ 10sec 30x Annealing: 62℃ 12sec 30x Extension: 72℃ 22 seconds 30x Final extension: 72°C 5 min.

[0144] For amplification, Phusion™ High-Fidelity Master Mix from NEB (Frankfurt am Main, Germany) is used according to the manufacturer's recommendations. 50 μl of each PCR reaction is then resolved on a 1% TAE agarose gel. PCR, agarose gel electrophoresis, ethidium bromide staining of DNA and determination of PCR fragment size are performed by methods known to those skilled in the art. PCR fragments of the expected size (PCR7 (SEQ ID NO: 34), 2595 base pairs; PCR8 (SEQ ID NO: 35), 1469 base pairs; PCR9 (SEQ ID NO: 36), 674 base pairs; PCR10 (SEQ ID NO: 37), 698 base pairs) are amplified. PCR products are purified using the Qiagen “QIAquick PCR Purification kit” according to the manufacturer's specifications. The purified PCR product is cloned into the BamHI- and SbfI-cut pKOPp vector (SEQ ID NO: 16) using the NEBuilder HiFi DNA Assembly Cloning Kit according to the manufacturer's instructions (NEB; Frankfurt am Main, Germany). Chemically competent E. coli 10beta cells (NEB, Frankfurt am Main, Germany) are transformed as known to those skilled in the art. Correct insertion of the target gene is checked by restriction analysis and the authenticity of the introduced homologous regions is confirmed by DNA sequencing. The resulting knockout vector is called pKO_PP_1016-PP_1018::ptsH_Ec ptsl_Ec crr_Ec ptsG_Ec) (SEQ ID NO: 38).

[0145] Construction of strain P. putida KT2440 Δupp Δ[PP_1016-1018]::ptsH_Ec ptsl_Ec crr_Ec ptsG_Ec The construction of strain P. putida KT2440 Δupp Δ[PP_1016-1018]::ptsH_Ec ptsl_Ec crr_Ec ptsG_Ec is carried out using the plasmid pKO_PP_1016-PP_1018::ptsH_Ec ptsl_Ec crr_Ec ptsG_Ec and the method described in Graf et al., 2011 (Graf N, Altenbuchner J, Appl. Environ. Micorbiol., 2011, 77(15):5549; DOI:10.1128 / AEM.05055-11). The DNA sequence after replacing genes PP_1016-PP_1018 with ptsH_Ec ptsl_Ec crr_Ec ptsG_Ec is set forth in SEQ ID NO:39. Transformation of P. putida KT2440 Δupp Δ[PP_1016-1018]::ptsH_Ec ptsl_Ec crr_Ec ptsG_Ec with vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} is carried out as described in Iwasaki et al. (Iwasaki K, Uchiyama H, Yagi O, Kurabayashi, T, Ishizuka K, Takamura Y, Biosci. Biotech. Biochem. 1994. 58(5):851-854). The cells are then plated on LB agar plates supplemented with kanamycin (50 μg / ml). The plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} (SEQ ID NO: 6) has already been described in Example 1. Plasmid DNA is isolated from each of the 10 clones and analyzed by restriction analysis.The strain carrying this plasmid is called P. putida KT2440 Δupp Δ[PP_1016-1018]::[ptsH_Ec ptsl_Ec crr_Ec ptsG_Ec] pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}. The technical implementation is carried out as described in Example 1.

[0146] Three experiments are carried out in parallel with each of Example 1.

[0147] Compared to Example 1, significantly higher total RL concentrations after 64 hours and significantly higher calculated space-time yields are observed.

[0148] Example 5 (according to the invention) Using strain P. putida KT2440 Δupp Δ[PP_1016-1018]::gluP_Bab+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0149] Construction of a vector for inserting the gluP gene into Pseudomonas putida KT2440 Δupp A vector for inserting the gluP gene from Brucella abortus, which codes for the glucose / galactose transporter GluP, is prepared by PCR amplification of the gene. The template used is a synthetic DNA fragment. The aim is to exchange the gulP gene with the genes PP_1016-PP_1018, which code for the ABC transporter permease, the ABC transporter binding protein and the ABC transporter ATP binding protein, in P. putida KT2440 Δupp. For this, about 680 base pairs upstream and downstream of the genes PP_1016-PP_1018 are amplified by PCR.

[0150] The following primers are used to amplify the gluP gene: PCR11:gluP MW_18_05 5'-TACCTCGGGATCCAATACATGGCAACTTCCATCCCAAC-3' (SEQ ID NO: 40) MW_18_06 5'-TCAGCTTTTGCTGCCGATGAG-3' (sequence number 41).

[0151] The following primers are used to amplify the homologous regions upstream and downstream of the PP_1016-PP_1018 genes: PCR5: Upstream region of PP_1016 O.BF_FM_1 * 03 5'-GCCGCTTTGGTCCCGGCCGCCAAGGTCATTAAC-3' (SEQ ID NO: 9) MW_18_01 5'-CATGTATTGGATCCCGAGGTAG-3' (sequence number 21). PCR12: Downstream region of PP_1018 MW_18_07 5'-TCATCGGCAGCAAAAGCTGACTCGTCTACACCATCAATAAGAAAAAG-3' (SEQ ID NO: 42) O.BF_FM_1 * 02 5'-GCTTGCATGCCTGCAGGCGTGATGTTGTACTTC-3' (sequence number 11).

[0152] The following parameters are used for PCR: Denaturation: 98℃ 30 seconds Denaturation: 98℃ 10sec 30x Annealing: 62℃ 12sec 30x Extension: 72℃ 22 seconds 30x Final extension: 72°C 5 min.

[0153] For amplification, Phusion™ High-Fidelity Master Mix from NEB (Frankfurt am Main, Germany) is used according to the manufacturer's recommendations. 50 μl of each PCR reaction is then resolved on a 1% TAE agarose gel. PCR, agarose gel electrophoresis, ethidium bromide staining of DNA and determination of PCR fragment size are performed by methods known to those skilled in the art. PCR fragments of the expected size (PCR11 (SEQ ID NO: 43), 1257 base pairs; PCR5 (SEQ ID NO: 24), 670 base pairs; PCR12 (SEQ ID NO: 44), 710 base pairs) are amplified. PCR products are purified using the Qiagen “QIAquick PCR Purification kit” according to the manufacturer's specifications. The purified PCR product is cloned into the BamHI- and SbfI-cut pKOPp vector (SEQ ID NO: 16) using the NEBuilder HiFi DNA Assembly Cloning Kit according to the manufacturer's instructions (NEB; Frankfurt am Main, Germany). E. coli 10 beta chemically competent cells (NEB, Frankfurt am Main, Germany) are transformed as known to those skilled in the art. Correct insertion of the target gene is checked by restriction analysis and the authenticity of the introduced homologous regions is confirmed by DNA sequencing. The resulting knockout vector is called pKO_PP_1016-PP_1018::gluP_Bab (SEQ ID NO: 45).

[0154] Construction of strain P. putida KT2440 Δupp Δ[PP_1016-1018]::gluP_Bab The construction of strain P. putida KT2440 Δupp Δ[PP_1016-1018]::gluP_Bab is carried out using the plasmid pKO_PP_1016-PP_1018::gluP_Bab and the method described in Graf et al., 2011 (Graf N, Altenbuchner J, Appl. Environ. Micorbiol., 2011, 77(15):5549; DOI:10.1128 / AEM.05055-11). The DNA sequence after replacing the gene PP_1016-PP_1018 with gluP is given in SEQ ID NO:46. Transformation of P. putida KT2440 Δupp Δ[PP_1016-1018]::gluP_Bab with vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} is carried out as described in Iwasaki et al. (Iwasaki K, Uchiyama H, Yagi O, Kurabayashi, T, Ishizuka K, Takamura Y, Biosci. Biotech. Biochem. 1994. 58(5):851-854). The cells are then plated on LB agar plates supplemented with kanamycin (50 μg / ml). The plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} (SEQ ID NO: 6) has already been described in Example 1. Plasmid DNA is isolated from each of the 10 clones and analyzed by restriction analysis. The strain carrying this plasmid is called P. putida KT2440 Δupp Δ[PP_1016-1018]::gluP_Bab pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}. The technical implementation is carried out as described in Example 1.

[0155] Three experiments are carried out in parallel with each of Example 1.

[0156] Compared to Example 1, significantly higher total RL concentrations after 64 hours and significantly higher calculated space-time yields are observed.

[0157] Example 6 (according to the invention) Strain P. putida (P. putida) KT2440 Δupp Use Δ[PP_1016-1018]::iolT1_Cg(co_Pp)+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0158] Construction of a vector for inserting the iolT1 gene into Pseudomonas putida KT2440 Δupp A vector for inserting the iolT1 gene from Corynebacterium glutamicum ATCC 13032, which encodes the myo-inositol facilitator IolT1, is prepared by PCR amplification of the gene codon-optimized for P. putida KT2440. A synthetic DNA fragment is used as template. The aim is to exchange the iolT1 gene with the genes PP_1016-PP_1018, which encode the ABC transporter permease, the ABC transporter binding protein and the ABC transporter ATP binding protein, in P. putida KT2440 Δupp. For this, approximately 680 base pairs upstream and downstream of the genes PP_1016-PP_1018 are amplified by PCR.

[0159] The following primers are used to amplify the iolT1 gene: PCR13:iolT1 MW_18_08 5'-TACCTCGGGATCCAATACATGGCAAGCACCTTTATCCAGGCCGACAG-3' (SEQ ID NO: 47) MW_18_09 5'-TCAATGGACCTTGCCCTTGCGAATG-3' (sequence number 48).

[0160] The following primers are used to amplify the homologous regions upstream and downstream of the PP_1016-PP_1018 genes: PCR5: Upstream region of PP_1016 O.BF_FM_1 * 03 5'-GCCGCTTTGGTCCCGGCCGCCAAGGTCATTAAC-3' (SEQ ID NO: 9) MW_18_01 5'-CATGTATTGGATCCCGAGGTAG-3' (sequence number 21). PCR14: Downstream region of PP_1018 MW_18_10 5'-GCAAGGGCAAGGTCCATTGACTCGTCTACACCATCAATAAGAAAAAG-3' (SEQ ID NO: 49) O.BF_FM_1 * 02 5'-GCTTGCATGCCTGCAGGCGTGATGTTGTACTTC-3' (sequence number 11).

[0161] The following parameters are used for PCR: Denaturation: 98℃ 30 seconds Denaturation: 98℃ 10sec 30x Annealing: 62℃ 12sec 30x Extension: 72℃ 22 seconds 30x Final extension: 72°C 5 min.

[0162] For amplification, Phusion™ High-Fidelity Master Mix from NEB (Frankfurt am Main, Germany) is used according to the manufacturer's recommendations. 50 μl of each PCR reaction is then resolved on a 1% TAE agarose gel. PCR, agarose gel electrophoresis, ethidium bromide staining of DNA and determination of PCR fragment size are performed by methods known to those skilled in the art. PCR fragments of the expected size (PCR13 (SEQ ID NO: 50), 1494 base pairs; PCR5 (SEQ ID NO: 24), 670 base pairs; PCR14 (SEQ ID NO: 51), 710 base pairs) are amplified. PCR products are purified using the Qiagen “QIAquick PCR Purification kit” according to the manufacturer's specifications. The purified PCR product is cloned into the BamHI- and SbfI-cut pKOPp vector (SEQ ID NO: 16) using the NEBuilder HiFi DNA Assembly Cloning Kit according to the manufacturer's instructions (NEB; Frankfurt am Main, Germany). Chemically competent E. coli 10beta cells (NEB, Frankfurt am Main, Germany) are transformed as known to those skilled in the art. Correct insertion of the target gene is checked by restriction analysis and the authenticity of the introduced homologous regions is confirmed by DNA sequencing. The resulting knockout vector is called pKO_PP_1016-PP_1018::iolT1_Cg(co_Pp) (SEQ ID NO: 52).

[0163] Construction of strain P. putida KT2440 Δupp Δ[ΡΡ_1016-1018]::iolT1_Cg(co_Pp) The construction of the strain P. putida KT2440 Δupp Δ[ΡΡ_1016-1018]::iolT1_Cg(co_Pp) is carried out using the plasmid pKO_PP_1016-PP_1018::iolT1_Cg(co_Pp) and the method described in Graf et al., 2011 (Graf N, Altenbuchner J, Appl. Environ. Micorbiol., 2011, 77(15):5549; DOI:10.1128 / AEM.05055-11). The DNA sequence after exchanging the gene ΡΡ_1016-PP-1018 with iolT1_Cg(co_Pp) is given in SEQ ID NO:53. Transformation of P. putida KT2440 Δupp Δ[ΡΡ_1016-1018]::iolT1_Cg(co_Pp) with vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} is carried out as described in Iwasaki et al. (Iwasaki K, Uchiyama H, Yagi O, Kurabayashi, T, Ishizuka K, Takamura Y, Biosci. Biotech. Biochem. 1994. 58(5):851-854). The cells are then plated on LB agar plates supplemented with kanamycin (50 μg / ml). The plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} (SEQ ID NO: 6) has already been described in Example 1. Plasmid DNA is isolated from each of the 10 clones and analyzed by restriction analysis. The strain carrying this plasmid is called P. putida KT2440 Δupp Δ[ΡΡ_1016-1018]::iolT1_Cg(co_Pp)pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}. The technical implementation is carried out as described in Example 1.

[0164] Three experiments are carried out in parallel with each of Example 1.

[0165] Compared to Example 1, significantly higher total RL concentrations after 64 hours and significantly higher calculated space-time yields are observed.

[0166] Example 7 (according to the invention) Using strain P. putida KT2440 Δupp Δ[PP_1016-1018]::glcP_Ms+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0167] Construction of a vector for inserting the glcP gene into Pseudomonas putida KT2440 Δupp A vector for inserting the glcP gene from Mycobacterium smegmatis, which codes for the arabinose proton symporter GlcP, is prepared by PCR amplification of the gene. The template used is a synthetic DNA fragment. The aim is to exchange the glcP gene with the genes PP_1016-PP_1018, which code for the ABC transporter permease, the ABC transporter binding protein and the ABC transporter ATP binding protein, in P. putida KT2440 Δupp. For this, approximately 680 base pairs upstream and downstream of the genes PP_1016-PP_1018 are amplified by PCR.

[0168] The following primers are used for amplification of the glcP gene: PCR15:glcP MW_18_11 5'-ACTACCTCGGGATCCAATACATGAATGTGATCGGTATCACTCTC-3' (SEQ ID NO: 54) MW_18_12 5'-TCAGTGCCCCAGCGCTTCGG-3' (sequence number 55).

[0169] The following primers are used to amplify the homologous regions upstream and downstream of the PP_1016-PP_1018 genes: PCR5: Upstream region of PP_1016 O.BF_FM_1 * 03 5'-GCCGCTTTGGTCCCGGCCGCCAAGGTCATTAAC-3' (SEQ ID NO: 9) MW_18_01 5'-CATGTATTGGATCCCGAGGTAG-3' (SEQ ID NO: 21) PCR16: Downstream region of PP_1018 MW_18_13 5'-CCGAAGCGCTGGGGCACTGACTCGTCTACACCATCAATAAGAAAAAG-3' (SEQ ID NO: 56) O.BF_FM_1 * 02 5'-GCTTGCATGCCTGCAGGCGTGATGTTGTACTTC-3' (sequence number 11).

[0170] The following parameters are used for PCR: Denaturation: 98℃ 30 seconds Denaturation: 98℃ 10sec 30x Annealing: 62℃ 12sec 30x Extension: 72℃ 22 seconds 30x Final extension: 72°C 5 min.

[0171] For amplification, Phusion™ High-Fidelity Master Mix from NEB (Frankfurt am Main, Germany) is used according to the manufacturer's recommendations. 50 μl of each PCR reaction is then resolved on a 1% TAE agarose gel. PCR, agarose gel electrophoresis, ethidium bromide staining of DNA and determination of PCR fragment size are performed by methods known to those skilled in the art. PCR fragments of the expected size (PCR15 (SEQ ID NO: 57), 1517 base pairs; PCR5 (SEQ ID NO: 24), 670 base pairs; PCR16 (SEQ ID NO: 58), 710 base pairs) are amplified. PCR products are purified using the Qiagen “QIAquick PCR Purification kit” according to the manufacturer's specifications. The purified PCR product is cloned into the BamHI- and SbfI-cut pKOPp vector (SEQ ID NO: 16) using the NEBuilder HiFi DNA Assembly Cloning Kit according to the manufacturer's instructions (NEB; Frankfurt am Main, Germany). E. coli 10 beta chemically competent cells (NEB, Frankfurt am Main, Germany) are transformed as known to those skilled in the art. Correct insertion of the target gene is checked by restriction analysis and the authenticity of the introduced homologous regions is confirmed by DNA sequencing. The resulting knockout vector is called pKO_PP_1016-PP_1018::glcP_Ms (SEQ ID NO: 59).

[0172] Construction of strain P. putida KT2440 Δupp Δ[PP_1016-1018]::glcP_Ms The construction of the strain P. putida KT2440 Δupp Δ[PP_1016-1018]::glcP_Ms is carried out using the plasmid pKO_PP_1016-PP_1018::glcP_Ms and the method described in Graf et al., 2011 (Graf N, Altenbuchner J, Appl. Environ. Micorbiol., 2011 , 77(15):5549; DOI:10.1128 / AEM.05055-11). The DNA sequence after exchanging the genes PP_1016-PP_1018 for glcP is given in SEQ ID NO:60. Transformation of P. putida KT2440 Δupp Δ[ΡΡ_1016-1018]::glcP_Ms with vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} is carried out as described by Iwasaki et al. (Iwasaki K, Uchiyama H, Yagi O, Kurabayashi, T, Ishizuka K, Takamura Y, Biosci. Biotech. Biochem. 1994. 58(5):851-854). The cells are then plated on LB agar plates supplemented with kanamycin (50 μg / ml). The plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} (SEQ ID NO: 6) has already been described in Example 1. Plasmid DNA is isolated from each of the 10 clones and analyzed by restriction analysis. The strain carrying this plasmid is called P. putida KT2440 Δupp Δ[PP_1016-1018]::glcP_Ms pACYCATh5-{PrhaSR}{rhaSR_Ec}{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}. The technical implementation is carried out as described in Example 1.

[0173] Three experiments are carried out in parallel with each of Example 1.

[0174] Compared to Example 1, significantly higher total RL concentrations after 64 hours and significantly higher calculated space-time yields are observed.

[0175] Example 8 (according to the invention) Strain P. putida (P. putida) KT2440 Δupp Use Δ[PP_1016-1018]::glcU_Bs(co_Pp)+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0176] Construction of a vector for inserting the glcU gene into Pseudomonas putida KT2440 Δupp A vector for inserting the glcU gene from Bacillus subtilis, which codes for the glucose uptake protein GlcU, is prepared by PCR amplification of a gene codon-optimized for P. putida KT2440. The template used is a synthetic DNA fragment. The aim is to exchange the glcU gene with the genes PP_1016-PP_1018, which code for the ABC transporter permease, the ABC transporter binding protein and the ABC transporter ATP binding protein, in P. putida KT2440 Δupp. For this, approximately 680 base pairs upstream and downstream of the genes PP_1016-PP_1018 are amplified by PCR.

[0177] The following primers are used to amplify the glcU gene: PCR17:glcU MW_18_14 5'-TACCTCGGGATCCAATACATGGACTTGTTGCTGGCTCTG-3' (SEQ ID NO: 61) MW_18_15 5'-CTAGCTGTTGGTCTTGGCGATG-3' (sequence number 62).

[0178] The following primers are used to amplify the upstream and downstream homologous regions of PP_1016-PP_1018: PCR5: Upstream region of PP_1016 O.BF_FM_1 * 03 5'-GCCGCTTTGGTCCCGGCCGCCAAGGTCATTAAC-3' (SEQ ID NO: 9) MW_18_01 5'-CATGTATTGGATCCCGAGGTAG-3' (SEQ ID NO: 21) PCR18: Downstream region of PP_1018 MW_18_16 5'-TCGCCAAGACCAACAGCTAGCTCGTCTACACCATCAATAAGAAAAAG-3' (SEQ ID NO: 63) O.BF_FM_1 * 02 5'-GCTTGCATGCCTGCAGGCGTGATGTTGTACTTC-3' (sequence number 11).

[0179] The following parameters are used for PCR: Denaturation: 98℃ 30 seconds Denaturation: 98℃ 10sec 30x Annealing: 62℃ 12sec 30x Extension: 72℃ 22 seconds 30x Final extension: 72°C 5 min.

[0180] For amplification, Phusion™ High-Fidelity Master Mix from NEB (Frankfurt am Main, Germany) is used according to the manufacturer's recommendations. 50 μl of each PCR reaction is then resolved on a 1% TAE agarose gel. PCR, agarose gel electrophoresis, ethidium bromide staining of DNA and determination of PCR fragment size are performed by methods known to those skilled in the art. PCR fragments of the expected size (PCR17 (SEQ ID NO: 64), 882 base pairs; PCR5 (SEQ ID NO: 24), 670 base pairs; PCR18 (SEQ ID NO: 65), 710 base pairs) are amplified. PCR products are purified using the Qiagen “QIAquick PCR Purification kit” according to the manufacturer's specifications. The purified PCR product is cloned into the BamHI- and SbfI-cut pKOPp vector (SEQ ID NO: 16) using the NEBuilder HiFi DNA Assembly Cloning Kit according to the manufacturer's instructions (NEB; Frankfurt am Main, Germany). Chemically competent E. coli 10beta cells (NEB, Frankfurt am Main, Germany) are transformed as known to those skilled in the art. Correct insertion of the target gene is checked by restriction analysis and the authenticity of the introduced homologous regions is confirmed by DNA sequencing. The resulting knockout vector is called pKO_PP_1016-PP_1018::glcU_Bs (SEQ ID NO: 66).

[0181] Construction of strain P. putida KT2440 Δupp Δ[PP_1016-1018]::glcU_Bs The construction of the strain P. putida KT2440 Δupp Δ[PP_1016-1018]::glcU_Bs is carried out using the plasmid pKO_PP_1016-PP_1018::glcU_Bs and the method described in Graf et al., 2011 (Graf N, Altenbuchner J, Appl. Environ. Micorbiol., 2011 , 77(15):5549; DOI:10.1128 / AEM.05055-11). The DNA sequence after exchanging the genes PP_1016-PP_1018 for glcU is given in SEQ ID NO:67. Transformation of P. putida KT2440 Δupp Δ[PP_1016-1018]::glcU_Bs with vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} is carried out as described in Iwasaki et al. (Iwasaki K, Uchiyama H, Yagi O, Kurabayashi, T, Ishizuka K, Takamura Y, Biosci. Biotech. Biochem. 1994. 58(5):851-854). The cells are then plated on LB agar plates supplemented with kanamycin (50 μg / ml). The plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} (SEQ ID NO: 6) has already been described in Example 1. Plasmid DNA is isolated from each of the 10 clones and analyzed by restriction analysis. The strain carrying this plasmid is called P. putida KT2440 Δupp Δ[PP_1016-1018]::glcU_Bs pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}. The technical implementation is carried out as described in Example 1.

[0182] Three experiments are carried out in parallel with each of Example 1.

[0183] Compared to Example 1, significantly higher total RL concentrations after 64 hours and significantly higher calculated space-time yields are observed.

[0184] Example 9 (according to the invention) Strain P. putida (P. putida) KT2440 Δupp Use Δ[PP_1016-1018]::SemiSWEET_Lb(co_Pp)+pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.

[0185] Construction of a vector for inserting the SemiSWEET gene into Pseudomonas putida KT2440 Δupp A vector for inserting the SemiSWEET gene from Leptospira biflexa, which codes for the sugar transporter semisweet, is prepared by PCR amplification of the gene codon-optimized for P. putida KT2440. The template used is a synthetic DNA fragment. The aim is to exchange the semiSWEET gene with the genes PP_1016-PP_1018, which code for the ABC transporter permease, the ABC transporter binding protein and the ABC transporter ATP binding protein, in P. putida KT2440 Δupp. For this, approximately 680 base pairs upstream and downstream of the genes PP_1016-PP_1018 are amplified by PCR.

[0186] The following primers are used for amplification of the semiSWEET gene: PCR19:semiSWEET MW_18_17 5'-TACCTCGGGATCCAATACATGGAAAACTTGATCGGCTATGTG-3' (SEQ ID NO: 68) MW_18_18 5'-TCAGGTTTGGTTGCCCTCGGTCAG-3' (sequence number 69).

[0187] The following primers are used to amplify the homologous regions upstream and downstream of the PP_1016-PP_1018 genes: PCR5: Upstream region of PP_1016 O.BF_FM_1 * 03 5'-GCCGCTTTGGTCCCGGCCGCCAAGGTCATTAAC-3' (SEQ ID NO: 9) MW_18_01 5'-CATGTATTGGATCCCGAGGTAG-3' (SEQ ID NO: 21) PCR20: Downstream region of PP_1018 MW_18_19 5'-CCGAGGGCAACCAAACCTGACTCGTCTACACCATCAATAAGAAAAAG-3' (SEQ ID NO: 70) O.BF_FM_1 * 02 5'-GCTTGCATGCCTGCAGGCGTGATGTTGTACTTC-3' (sequence number 11).

[0188] The following parameters are used for PCR: Denaturation: 98℃ 30 seconds Denaturation: 98℃ 10sec 30x Annealing: 62℃ 12sec 30x Extension: 72℃ 22 seconds 30x Final extension: 72°C 5 min.

[0189] For amplification, Phusion™ High-Fidelity Master Mix from NEB (Frankfurt am Main, Germany) is used according to the manufacturer's recommendations. 50 μl of each PCR reaction is then resolved on a 1% TAE agarose gel. PCR, agarose gel electrophoresis, ethidium bromide staining of DNA and determination of PCR fragment size are performed by methods known to those skilled in the art. PCR fragments of the expected size (PCR19 (SEQ ID NO: 71), 276 base pairs; PCR5 (SEQ ID NO: 24), 670 base pairs; PCR20 (SEQ ID NO: 72), 710 base pairs) are amplified. PCR products are purified using the Qiagen “QIAquick PCR Purification kit” according to the manufacturer's specifications. The purified PCR product is cloned into the BamHI- and SbfI-cut pKOPp vector (SEQ ID NO: 16) using the NEBuilder HiFi DNA Assembly Cloning Kit according to the manufacturer's instructions (NEB; Frankfurt am Main, Germany). Chemically competent E. coli 10beta cells (NEB, Frankfurt am Main, Germany) are transformed as known to those skilled in the art. Correct insertion of the target gene is checked by restriction analysis and the authenticity of the introduced homologous regions is confirmed by DNA sequencing. The resulting knockout vector is called pKO_PP_1016-PP_1018]::SemiSWEET_Lb(co_Pp) (SEQ ID NO: 73).

[0190] Construction of strain P. putida KT2440 Δupp Δ[PP_1016-1018]::semiSWEET_Lb(co_Pp) The construction of strain P. putida KT2440 Δupp Δ[PP_1016-1018]::semiSWEET_Lb(co_Pp) is carried out using the plasmid pKO_PP_1016-PP_1018::semiSWEET_Lb(co_Pp) and the method described in Graf et al., 2011 (Graf N, Altenbuchner J, Appl. Environ. Micorbiol., 2011 , 77(15):5549; DOI:10.1128 / AEM.05055-11). The DNA sequence after exchanging genes PP_1016-PP_1018 with semiSWEET is given in SEQ ID NO:74. Transformation of P. putida KT2440 Δupp Δ[PP_1016-1018]::semiSWEET_Lb(co_Pp) with vector pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} is carried out as described in Iwasaki et al. (Iwasaki K, Uchiyama H, Yagi O, Kurabayashi, T, Ishizuka K, Takamura Y, Biosci. Biotech. Biochem. 1994. 58(5):851-854). The cells are then plated on LB agar plates supplemented with kanamycin (50 μg / ml). The plasmid pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk} (SEQ ID NO: 6) has already been described in Example 1. Plasmid DNA is isolated from each of the 10 clones and analyzed using restriction analysis. The strain carrying this plasmid is designated P. putida KT2440 Δupp Δ[PP_1016-1018]::semisweet_Lb(co_Pp)pACYCATh5-{PrhaSR}[rhaSR_Ec]{PrhaBAD}[rhlABC_Pa]{Talk}[araC_Ec]{ParaBAD}[rmlBDAC_Pa]{Talk}.The technical realization is carried out as described in Example 1.

[0191] Three experiments are carried out in parallel with each of Example 1.

[0192] Compared to Example 1, significantly higher total RL concentrations after 64 hours and significantly higher calculated space-time yields are observed.

Claims

1. In a rhamnolipid producing cell, the cell is Pseudomonas putida and has been genetically modified to have a decreased activity of an ABC glucose transporter compared to its wild type and to have an increased activity of at least one non-ABC glucose transporter compared to its wild type; wherein the genetic modification comprises the insertion of foreign DNA into a gene encoding an ABC glucose transporter; wherein the non-ABC glucose transporter is An enzyme encoded by the galP, glf, iolT1, glcP, gluP, SemiSWEET or glcU gene, and a PTS system consisting of components Enzyme I, HPr, Enzyme IIA, Enzyme IIB and Enzyme IIC, wherein Enzymes IIA, IIB and IIC can exist as a fusion protein; The cells were compared to their wild type counterparts in Group E １ , E ２ and E ３ wherein E 1 is an enzyme encoded by the rhlA gene or has a polypeptide sequence modified by deletion, insertion, substitution or a combination thereof of up to 10% of the amino acid residues relative to the enzyme encoded by the rhlA gene, and still has more than 90% of the enzymatic activity of the enzyme having the reference sequence of the enzyme encoded by the rhlA gene; E 2 is an enzyme encoded by the rhlB gene or has a polypeptide sequence modified by deletion, insertion, substitution or a combination thereof of up to 10% of the amino acid residues relative to the enzyme encoded by the rhlB gene, and still has more than 90% of the enzymatic activity of the enzyme having the reference sequence of the enzyme encoded by the rhlB gene; and E 3 is an enzyme encoded by the rhlB gene or has a polypeptide sequence modified by deletion, insertion, substitution or a combination thereof of up to 10% of the amino acid residues relative to the enzyme encoded by the rhlB gene, and still has more than 90% of the enzymatic activity of the enzyme having the reference sequence of the enzyme encoded by the rhlB gene. A cell characterized in that the enzyme is encoded by the rhlC gene or has a polypeptide sequence in which up to 10% of the amino acid residues have been modified by deletion, insertion, substitution or a combination thereof relative to the enzyme encoded by the rhlC gene, and still has more than 90% of the enzymatic activity of an enzyme having a reference sequence for the enzyme encoded by the rhlC gene.

2. The cell has at least one enzyme E that catalyzes the conversion of D-glucose and a quinone to D-glucono-1,5-lactone and a quinol, as compared to its wild type. ４ The cell of claim 1, characterized in that it is genetically modified to have a reduced activity of.

3. E ４ The cell according to claim 2, characterized in that it is glucose 1-dehydrogenase of EC 1.1.5.

2.

4. The method steps are as follows: I) contacting the cell according to claim 1 with a medium containing a carbon source; II) culturing the cells under conditions that allow the cells to produce rhamnolipids from the carbon source; and III) Optionally, isolating the rhamnolipid produced. A method for producing rhamnolipids comprising the steps of:

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