# Patent

## Title
Cells and methods for producing rhamnolipids

## Abstract

This invention relates to cells and nucleic acids and also use thereof for producing rhamnolipids, and also methods for producing rhamnolipids.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of U.S. patent application Ser. No. 13/812,625, filed Jan. 28, 2013, which is the National Phase of PCT/EP2011/062441, filed Jul. 20, 2011, which claims the benefit of German Application No. DE 102010032484 filed on Jul. 28, 2010, the contents of each of which is herein incorporated by reference in its entirety.


INCORPORATION BY REFERENCE OF SEQUENCE LISTING
The Sequence Listing in the ASCII text file, named as SequenceListing.txt of 547 KB, created on Dec. 10, 2012, and submitted to the United States Patent and Trademark Office via EFS-Web on Jan. 28, 2013, is incorporated herein by reference.
FIELD OF THE INVENTION
The invention relates to cells and nucleic acids and also use thereof for producing rhamnolipids, and also methods for producing rhamnolipids.
PRIOR ART
Surfactants are nowadays produced essentially based on the basis of petrochemical raw materials. The use of surfactants based on renewable raw materials is a suitable alternative on account of the foreseeable shortage of petrochemical raw materials and increasing demand for products that are based on renewable raw materials or are biodegradable.
Rhamnolipids consist of one (monorhamnosyl lipids) or two rhamnose radicals (dirhamnosyl lipids) and one or two 3-hydroxy fatty acid residues (see Handbook of Hydrocarbon and Lipid Microbiology, 2010, pages 3037-51). They have surface-active properties, which are needed in all sorts of applications for use as a surfactant (see Leitermann et al., 2009).
These lipids are nowadays produced using wild-type isolates of different human- and animal-pathogenic bacteria, in particular representatives of the genera Pseudomonas and Burkholderia (see Handbook of Hydrocarbon and Lipid Microbiology, 2010, pages 3037-51). The fact that these production organisms are able to cause diseases reduces the customer acceptance for the conventionally produced rhamnolipids very considerably. Moreover, higher safety requirements also have an effect on the production costs owing to increased capital expenditure and possibly additional working-up steps.
Although to some extent high product titers, and also space-time and/or carbon yields can be achieved with the aid of these production organisms, this requires the use of vegetable oils as the sole or co-substrate (see Handbook of Hydrocarbon and Lipid Microbiology, 2010, pages 3037-51). Vegetable oils, however, are comparatively expensive raw materials in comparison to other carbon sources, such as, for example, glucose, sucrose or polysaccharides such as, for example, starch, cellulose and hemicellulose, glycerol, CO, CO2 or CH4. Moreover, rhamnolipids distinguish themselves on account of their surfactant character in that they are susceptible to heavy foaming in fermentation processes. This is in particular the case if lipophilic substrates are employed. This problem is markedly reduced on use of water-soluble substrates such as, for example, glucose, sucrose, polysaccharides (starch, cellulose, hemicellulose) or glycerol. Finally, the properties of the rhamnolipids produced by the wild-type isolates can only be influenced to a restricted extent. Up to now, this takes place exclusively via the optimization of the process management (pH, oxygen supply, media composition, feeding strategies, nitrogen supply, temperature, choice of substrate, etc.). However, a very specific influence of certain product properties, such as, for example, the ratio of the various rhamnolipid species (number of rhamnose and 3-hydroxy fatty acid radicals) or chain length and degree of saturation of the 3-hydroxy fatty acid radicals would be desirable to be able to modulate the product properties relevant for the application.
Rhamnolipids, if they are to be employed in a large extent as surfactants in household, cleaning, cosmetic, food processing, pharmaceutical, plant protection and other applications, must appear to be in competition with the surfactants employed nowadays. These are high volume chemicals, which can be produced at very low costs, without obvious health risks for the customer and with clearly defined and modulatable product specifications. Therefore rhamnolipids must also be able to be produced at costs as low as possible, without health risks for the customer and with defined properties as far as possible.
Although rhamnolipids have already been produced in GRAS organisms (generally regarded as save) based on convenient carbon sources, such as, for example, glucose or glycerol, these are in this case exclusively monorhamnosyl lipids (Ochsner et al. Appl. Environ. Microbiol. 1995. 61(9):3503-3506).
Cha et al. in Bioresour Technol. 2008. 99(7):2192-9, on the other hand, describe the production of monorhamnosyl lipids from soybean oil in P. putida by introduction of the genes rhIA and rhIB from Pseudomonas aeruginosa. 
There is therefore an increasing need for the inexpensive and, from the health point of view, safe production of mono- and dirhamnosyl lipids having defined and modulatable properties. This modulation can be carried out, for example, by means of a balanced supply of the individual enzyme activities, which reduces the enrichment of monorhamnosyl lipids. This modulation, however, can also be carried out, for example, by the use of enzymes having certain properties, e.g. with respect to substrate specificity and thus, for example, the chain length of the hydroxy fatty acids incorporated in rhamnolipids.
The present invention therefore has the object of providing a possibility of producing rhamnolipids from readily accessible carbon sources using safe production hosts.
DESCRIPTION OF THE INVENTION
Surprisingly, it has been found that the cells and methods described below, in which these cells are employed, make a contribution to solving the stated object of the invention.
The present invention therefore relates to cells, which are able to form rhamnolipids and compared to their wild-type have at least one increased activity of a gene product of homologs of the gene products rhIA, rhIB and rhIC.
The invention further relates to a method for producing rhamnolipids using the aforementioned cells as a biocatalyst and simple carbon sources.
It is an advantage of the present invention that organisms can be employed that are non-pathogenic and simple to culture.
It is a further advantage that use of oils as the sole or co-substrate is not necessary. Another advantage is that with the aid of the invention rhamnolipids having defined and modulatable properties can be produced.
It is another advantage of the present invention that dirhamnosyl lipids can be produced.
A further advantage is that rhamnolipids can be produced with higher space-time and carbon yields than with cells without enhancement of these activities.
A contribution to achieving the object mentioned at the outset is made by a cell, preferably an isolated cell, which is able to form at least one rhamnolipid of the general formula (I) or its salt,




wherein

m=2, 1 or 0, in particular 1 or 0,

n=1 or 0, in particular 1,

R1 and R2=independently of one another identical or different organic radical 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 tri-unsaturated, alkyl radical, preferably that selected from the group consisting of pentenyl, heptenyl, nonenyl, undecenyl and tridecenyl and (CH2)o—CH3 with o=1 to 23, preferably 4 to 12, characterized in that it has been genetically modified such that, compared to its wild-type, it has an increased activity of at least one of the enzymes E1, E2 and E3, wherein the enzyme E1 is able to catalyze the conversion of 3-hydroxyalkanoyl-ACP via 3-hydroxyalkanoyl-3-hydroxyalkanoic acid-ACP to hydroxyalkanoyl-3-hydroxyalkanoic acid, the enzyme E2 is a rhamnosyltransferase I and is able to catalyze the conversion of dTDP-rhamnose and 3-hydroxyalkanoyl-3-hydroxyalkanoate to α-L-rhamnopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate and the enzyme E3 is a rhamnosyltransferase II and is able to catalyze 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, wherein these enzymes E1, E2 and E3 preferably are selected from the group consisting of

at least one enzyme E1 selected from

an enzyme E1a having polypeptide sequence Seq ID No. 2 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 radicals are modified compared to the reference sequence Seq ID No. 2 by deletion, insertion, substitution or a combination thereof and that 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 Seq ID No. 2, wherein enzymatic activity for an enzyme E1a is understood as meaning the ability preferably to convert 3-hydroxydecanoyl-ACP via 3-hydroxydecanoyl-3-hydroxydecanoic acid-ACP to hydroxydecanoyl-3-hydroxydecanoic acid,

an enzyme E1b having polypeptide sequence Seq ID No. 18 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 radicals are modified compared to the reference sequence Seq ID No. 18 by deletion, insertion, substitution or a combination thereof and that 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 Seq ID No. 18, wherein enzymatic activity for an enzyme E1b is understood as meaning the ability preferably to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid-ACP to hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

an enzyme E1c having polypeptide sequence Seq ID No. 78 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 radicals are modified compared to the reference sequence Seq ID No. 78 by deletion, insertion, substitution or a combination thereof and that 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 Seq ID No. 78, wherein enzymatic activity for an enzyme E1c is understood as meaning the ability preferably to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid-ACP to hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

an enzyme E1d having polypeptide sequence Seq ID No. 80 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 radicals are modified compared to the reference sequence Seq ID No. 80 by deletion, insertion, substitution or a combination thereof and that 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 Seq ID No. 80, wherein enzymatic activity for an enzyme E1d is understood as meaning the ability preferably to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid-ACP to hydroxytetradecanoyl-3-hydroxytetradecanoic acid, and

an enzyme E1e having polypeptide sequence Seq ID No. 82 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 radicals are modified compared to the reference sequence Seq ID No. 82 by deletion, insertion, substitution or a combination thereof and that 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 Seq ID No. 82, wherein enzymatic activity for an enzyme E1e is understood as meaning the ability preferably to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid-ACP to hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

at least one enzyme E2 having polypeptide sequence selected from

an enzyme E2a having polypeptide sequence Seq ID No. 4 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 radicals are modified compared to the reference sequence Seq ID No. 4 by deletion, insertion, substitution or a combination thereof and that 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 Seq ID No. 4, wherein enzymatic activity for an enzyme E2a is understood as meaning the ability preferably to convert dTDP-rhamnose and 3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,

an enzyme E2b having polypeptide sequence Seq ID No. 20 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 radicals are modified compared to the reference sequence Seq ID No. 20 by deletion, insertion, substitution or a combination thereof and that 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 Seq ID No. 20, wherein enzymatic activity for an enzyme E2b is understood as meaning the ability preferably to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

an enzyme E2c having polypeptide sequence Seq ID No. 84 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 radicals are modified compared to the reference sequence Seq ID No. 84 by deletion, insertion, substitution or a combination thereof and that 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 Seq ID No. 84, wherein enzymatic activity for an enzyme E2c is understood as meaning the ability preferably to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

an enzyme E2d having polypeptide sequence Seq ID No. 86 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 radicals are modified compared to the reference sequence Seq ID No. 86 by deletion, insertion, substitution or a combination thereof and that 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 Seq ID No. 86, wherein enzymatic activity for an enzyme E2d is understood as meaning the ability preferably to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, and

an enzyme E2e having polypeptide sequence Seq ID No. 88 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 radicals are modified compared to the reference sequence Seq ID No. 88 by deletion, insertion, substitution or a combination thereof and that 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 Seq ID No. 88, wherein enzymatic activity for an enzyme E2e is understood as meaning the ability preferably to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, and

at least one enzyme E3 selected from

an enzyme E3a having polypeptide sequence Seq ID No. 6 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 radicals are modified compared to the reference sequence Seq ID No. 6 by deletion, insertion, substitution or a combination thereof and that 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 Seq ID No. 6, wherein enzymatic activity for an enzyme E3a is understood as meaning the ability preferably to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid, an enzyme E3b having polypeptide sequence Seq ID No. 22 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 radicals are modified compared to the reference sequence Seq ID No. 22 by deletion, insertion, substitution or a combination thereof and that 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 Seq ID No. 22, wherein enzymatic activity for an enzyme E3b is understood as meaning the ability preferably to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

an enzyme E3c having polypeptide sequence Seq ID No. 90 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 radicals are modified compared to the reference sequence Seq ID No. 90 by deletion, insertion, substitution or a combination thereof and that 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 Seq ID No. 90, wherein enzymatic activity for an enzyme E3c is understood as meaning the ability preferably to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, and

an enzyme E3d having polypeptide sequence Seq ID No. 92 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 radicals are modified compared to the reference sequence Seq ID No. 92 by deletion, insertion, substitution or a combination thereof and that still has at least 10%, preferably 50%, particularly preferably 80%, in particular more than 92% of the enzymatic activity of the enzyme having the reference sequence Seq ID No. 92, wherein enzymatic activity for an enzyme E3d is understood as meaning the ability preferably to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid.

For general survey compare FIG. 1.
“Wild-type” of a cell herein designates a cell, the genome of which is present in a state as is formed naturally by evolution. The term is used both for the entire cell as well as for individual genes. The term “wild-type” therefore in particular does not include those cells or those genes, the gene sequences of which have been modified at least partially by man by means of recombinant methods.
The term “rhamnolipid” is understood in connection with the present invention as meaning a compound of the general formula (I) or its salt.
It is obvious that the activities actually indicated above for the enzymes E1a to E3b is only a special exemplary choice of a broader activity spectrum of the aforementioned enzymes; the respective activity mentioned is that for which a reliable measuring method is available in the case of a given enzyme. Thus it is obvious that an enzyme which a substrate having an unbranched, saturated C10-alkyl radical likewise—even though optionally with decreased activity—will convert those substrates that contain a C6- or C16-alkyl radical, which can optionally also be branched or unsaturated.
The term “increased activity of an enzyme” is preferably to be understood as meaning increased intracellular activity.
The embodiments now following for increasing the enzyme activity in cells apply both for the increase in the activity of the enzyme E1 to E3 as well as for all subsequently mentioned enzymes, the activity of which can optionally be increased.
In principle, an increase in the enzymatic activity can be achieved by increasing the copy number of the gene sequence or the gene sequences which code for the enzyme, using a strong promoter or an improved ribosome binding site, attenuating a negative regulation of gene expression, for example by transcription regulators, or amplifying a positive regulation of gene expression, modifying the codon usage of the gene, in various ways increasing the half-life of the mRNA or of the enzyme, modifying the regulation of the expression of the gene or utilizing a gene or allele that codes for an appropriate enzyme having an increased activity and optionally combining these measures. According to the invention, genetically modified cells are produced, for example, by transformation, transduction, conjugation or a combination of these methods using a vector that contains the desired gene, an allele of this gene or parts thereof and optionally contains a promoter making possible the expression of the gene. Heterologous expression is in particular achieved by integration of the gene or the alleles in the chromosome of the cell or an extrachromosomally replicating vector.
DE-A-100 31 999 gives a general survey of the possibilities for increasing the enzyme activity in cells as exemplified by pyruvate carboxylase, which is inserted hereby as a reference and whose disclosure content with respect to the possibilities for increasing the enzyme activity in cells forms a part of the disclosure of the present invention.
The expression of the above and all subsequently mentioned enzymes or genes is detectable with the aid of 1- and 2-dimensional protein gel separation and subsequent optical identification of the protein concentration in the gel using appropriate analytical software. If the increase in an enzyme activity is based exclusively on an increase in the expression of the corresponding gene, the quantification of the increase in the enzyme activity can be determined in a simple manner by a comparison of the 1- or 2-dimensional protein separations between wild-type and genetically modified cell. A customary method for the preparation of the protein gels in the case of coryneforme bacteria and for the identification of the proteins is the procedure described by Hermann et al. (Electrophoresis, 22: 1712.23 (2001)). The protein concentration can likewise be analyzed by Western Blot hybridization using an antibody 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, N.Y. USA, 1989) and subsequent optical analysis using appropriate software for the concentration determination (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 means of DNA band shift assays (also called gel retardation) (Wilson et al. (2001) Journal of Bacteriology, 183: 2151-2155). The action of DNA-binding proteins on the expression of other genes can be detected by various well-described methods of the reporter gene assay (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. USA, 1989). The intracellular enzymatic activities can be determined according to various 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). If in the following embodiments no practical methods are indicated for the determination of the activity of a certain enzyme, the determination of the increase in the enzyme activity and also the determination of the decrease of an enzyme activity preferably take place by means of 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).
If the increase in the enzyme activity is accomplished by mutation of the endogenous gene, such mutations can be randomly produced either by conventional methods, such as, for example, by UV irradiation or by mutagenic chemicals, or selectively by means of genetic engineering methods such as deletion(s), insertion(s) and/or nucleotide exchange(s). Modified cells are obtained by these mutations. Particularly preferred mutants of enzymes are in particular also those enzymes that are no longer feedback-, product- or substrate-inhibitable or are so to a reduced degree at least in comparison to the wild-type enzyme.
If the increase in the enzyme activity is accomplished by increase in the synthesis of an enzyme, the copy number of the corresponding genes is increased or the promoter and regulation region or the ribosome binding site, which is situated upstream of the structural gene, is mutated. Expression cassettes, which are incorporated upstream of the structural gene, act in the same manner. It is additionally possible, by means of inducible promoters, to increase the expression at any desired point in time. In addition, however, also “enhancers” can be assigned to the enzyme gene as regulatory sequences, which likewise bring about increased gene expression by means of an improved interaction between RNA polymerase and DNA. As a result of measures for the prolongation of the lifetime of the mRNA, the expression is likewise improved. Furthermore, by prevention of the degradation of the enzyme protein the enzyme activity is likewise increased. The genes or gene constructs are present here either in plasmids having a different copy number or are integrated and amplified in the chromosome.
Alternatively, an overexpression of the genes concerned can furthermore be achieved by modification of the media composition and culture management. The person skilled in the art finds directions for this, inter alia, in Martin et al. (Bio/Technology 5, 137-146 (1987)), in Guerrero et al. (Genes 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), in Eikmanns et al. (Genes 102, 93-98 (1991)), in EP-A-0 472 869, in U.S. Pat. No. 4,601,893, in Schwarzer and Pühler (Bio/Technology 9, 84-87 (1991)), in Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in WO-A-96/15246, in Malumbres et al. (Genes 134, 15-24 (1993)), in JP-A-10-229891, in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)) and in known textbooks of genetics and molecular biology. The measures described above likewise lead, like the mutations, to genetically modified cells.
Episomal plasmids, for example, are employed for increasing the expression of the respective genes. Suitable plasmids or vectors are in principle all embodiments available for this purpose to the person skilled in the art. Such plasmids and vectors can be taken, for example, from the brochures of the companies Novagen, Promega, New England Biolabs, Clontech or Gibco BRL. Further preferred plasmids and vectors can be found in: Glover, D. M. (1985) DNA cloning: a practical approach, Vol. IRL Press Ltd., Oxford; Rodriguez, R. L. and Denhardt, D. T (eds) (1988) Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, D. V. (1990) Systems for heterologous gene expression, Methods Enzymol. 185, 3-7; Sambrook, J.; Fritsch, E. F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York.
The plasmid vector, which contains the gene to be amplified, is then converted to the desired strain by conjugation or transformation. The method of conjugation is described, for example, in Schäfer et al., Applied and Environmental Microbiology 60: 756-759 (1994). Methods for 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 means of a “cross-over” event, the resulting strain contains at least two copies of the gene concerned.
Under the formulation used above and in the following embodiments “an activity of an enzyme Ex increased in comparison to its wild-type” is preferably always to be understood as meaning an activity of the respective enzyme Ex increased by a factor of at least 2, particularly preferably of at least 10, moreover preferably of at least 100, moreover still more preferably of at least 1,000 and most preferably of at least 10,000. Furthermore the cell according to the invention, which has “an increased activity of an enzyme Ex compared to its wild-type”, in particular also comprises a cell, whose wild-type contains no or at least no detectable activity of this enzyme Ex and which shows a detectable activity of this enzyme Ex only after increasing the enzyme activity, for example by overexpression. In this connection, the term “overexpression” or the formulation used in the following embodiments “increasing the expression” also comprises the case where a starting cell, for example a wild-type cell, has no or at least no detectable expression and a detectable synthesis of the enzyme Ex is induced only by recombinant methods.
Changes of amino acid radicals of a given polypeptide sequence, which lead to no significant changes in the properties and function of the given polypeptide, are known to the person skilled in the art. Thus, for example, “conserved amino acids” can be mutually exchanged; examples of such suitable amino acid substitutions are: Ala for Ser; Arg for Lys; Asn for Gln or His; Asp for Glu; Cys for Ser; Gln for Asn; Glu for Asp; Gly for Pro; His for Asn or Gln; Ile for Leu or Val; Leu for Met or Val; Lys for Arg or Gln or Glu; Met for Leu or Ile; Phe for Met or Leu or Tyr; Ser for Thr; Thr for Ser; Trp for Tyr; Tyr for Trp or Phe; Val for Ile or Leu. It is likewise known that changes, particularly at the N- or C-terminus of a polypeptide, in the form of, for example, amino acid insertions or deletions often exert no significant influence on the function of the polypeptide.
The activity of an enzyme can be determined by disrupting cells which contain this activity in a manner known to the person skilled in the art, for example with the aid of a ball mill, a French press or of an ultrasonic disintegrator and subsequently separating off cells, cell debris and disruption aids, such as, for example, glass beads, by centrifugation for 10 minutes at 13,000 rpm and 4° C. Using the resulting cell-free crude extract, enzyme assays with subsequent LC-ESI-MS detection of the products can then be carried out. Alternatively, the enzyme can be enriched in the manner known to the person skilled in the art by chromatographic methods (such as nickel-nitrilotriacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography or ion-exchange chromatography) or else purified to homogeneity.
The activity of the enzyme E1 is then determined using the samples obtained as described above in the following manner: A standard assay contains 100 μM E. coli ACP, 1 mM β-mercaptoethanol, 200 μM malonyl-coenzyme A, 40 μM octanoyl-coenzyme A (for E1a) or dodecanoyl-coenzyme A (for E1b), 100 μM NADPH, 2 μg of E. coli FabD, 2 μg of Mycobacterium tuberculosis FabH, 1 μg of E. coli FabG, 0.1 M sodium phosphate buffer, pH 7.0, and 5 μg of enzyme E1 in a final volume of 120 μL. ACP, β-mercaptoethanol and sodium phosphate buffer are preincubated for 30 min at 37° C. to reduce the ACP completely. The reaction is started by addition of enzyme E1. The reactions are stopped using 2 ml of water, which has been acidified with HCl to pH 2.0, and subsequently extracted twice with 2 ml of chloroform/methanol (2:1 (v:v)). Phase separation takes place by centrifugation (16,100 g, 5 min, RD. The lower organic phase is removed, evaporated completely in the vacuum centrifuge and the sediment is taken up in 50 μl of methanol. Undissolved constituents are sedimented by centrifugation (16,100 g, 5 min, RT) and the sample is analyzed by means of LC-ESI-MS. The identification of the products takes place by analysis of the corresponding mass traces and the MS2 spectra.
The activity of the enzyme E2 is then determined as follows using the samples obtained as described above: 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 protein crude extract (about 1 mg of total protein) or purified protein in solution (5 μg of purified protein). The reaction is started by the addition of 10 μl of 10 mM ethanolic solution of 3-hydroxydecanoyl-3-hydroxydecanoic acid (for E2a) or 3-hydroxy-tetradecanoyl-3-hydroxytetradecanoic acid (for E2b) and incubated for 1 h at 30° C. with shaking (600 rpm). Subsequently, the reaction is treated with 1 ml of acetone. Undissolved constituents are sedimented by centrifugation (16,100 g, 5 min, RD and the sample is analyzed by means of LC-ESI-MS. The identification of the products takes place by analysis of the corresponding mass traces and the MS2 spectra.
The activity of the enzyme E3 is then determined as follows using the samples obtained as described above: a standard assay can consist of 185 μl of 10 mM tris-HCl (pH 7.5), 10 μl of 125 mM of dTDP-rhamnose and 50 μl of protein crude extract (about 1 mg of total protein) or purified protein in solution (5 μg of purified protein). The reaction is started by the addition of 10 μl of 10 mM ethanolic solution of α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid (for E3a) or α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid (for E3b) and incubated for 1 h at 30° C. with shaking (600 rpm). Subsequently, the reaction is treated with 1 ml of acetone. Undissolved constituents are sedimented by centrifugation (16,100 g, 5 min, RT) and the sample is analyzed by means of LC-ESI-MS. The identification of the products takes place by analysis of the corresponding mass traces and the MS2 spectra.
Cells according to the invention are preferred that have increased activities of the following enzyme combinations:
E1, E2, E3, E1E2, E1E3, E2E3 and E1E2E3,
of which the combination
E2, E2E3 and E1E2E3, in particular E1E2E3 
is particularly preferred.
In a preferred embodiment of the cell according to the invention that has an increased activity of the enzyme combination E1E2E3, n is preferably =1.
The cells according to the invention can be prokaryotes or eukaryotes. These can be mammalian cells (such as, for example, cells from man), plant cells or microorganisms such as yeasts, fungi or bacteria, wherein microorganisms are particularly preferred and bacteria and yeasts are most preferred.
Suitable bacteria, yeasts or fungi are in particular those bacteria, yeasts or fungi that are deposited in the Deutsche Sammlung von Mikroorganismen and Zellkulturen (German Collection of Microorganisms and Cell Cultures) GmbH (DSMZ), Brunswick, Germany, as listed on the DSMZ website.
Preferred cells according to the invention are those of the genera Aspergillus, Corynebacterium, Brevibacterium, Bacillus, Acinetobacter, Alcaligenes, Lactobacillus, Paracoccus, Lactococcus, Candida, Pichia, Hansenula, Kluyveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Pseudomonas, Rhodospirillum, Rhodobacter, Burkholderia, Clostridium and Cupriavidus, wherein Aspergillus nidulans, Aspergillus niger, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Burkholderia andropogonis, B. brasilensis, B. caledonica, B. caribensis, B. caryophylli, B. fungorum, B. gladioli, B. glathei, B. glumae, B. graminis, B. hospita, B. kururiensis, B. phenazinium, B. phymatum, B. phytofirmans, B. plantarii, B. sacchari, B. singaporensis, B. sordidicola, B. terricola, B. tropica, B. tuberum, B. ubonensis, B. unamae, B. xenovorans, B. anthina, B. pyrrocinia, B. thailandensis, Candida blankii, Candida rugosa, Corynebacterium glutamicum, Corynebacterium efficiens, Escherichia coli, Hansenula polymorphs, Kluveromyces lactis, Methylobacterium extorquens, Paracoccus versutus, Pseudomonas argentinensis, P. borbori, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea, P. aurantiaca, P. aureofaciens, P. chlororaphis, P. fragi, P. lundensis, P. taetrolens, P. antarctica, P. azotoformans, ‘P. blatchfordae’, P. brassicacearum, P. brenneri, P. cedrina, P. corrugata, P. fluorescens, P. gessardii, P. libanensis, P. mandelii, P. marginalis, P. mediterranea, P. meridiana, P. migulae, P. mucidolens, P. orientalis, P. panacis, P. proteolytica, P. rhodesiae, P. synxantha, P. thivervalensis, P. tolaasii, P. veronii, P. denitrificans, P. pertucinogena, P. cremoricolorata, P. fulva, P. monteilii, P. mosselii, P. parafulva, P. putida, P. balearica, P. stutzeri, P. amygdali, P. avellanae, P. caricapapayae, P. cichorii, P. coronafaciens, P. ficuserectae, ‘P. helianthi’, P. meliae, P. savastanoi, P. syringae, P. tomato, P. viridiflava, P. abietaniphila, P. acidophila, P. agarici, P. alcaliphila, P. alkanolytica, P. amyloderamosa, P. asplenii, P. azotifigens, P. cannabina, P. coenobios, P. congelans, P. costantinii, P. cruciviae, P. delhiensis, P. excibis, P. extremorientalis, P. frederiksbergensis, P. fuscovaginae, P. gelidicola, P. grimontii, P. indica, P. jessenfi, P. jinjuensis, P. kilonensis, P. knackmussii, P. koreensis, P. lini, P. lutea, P. moraviensis, P. otitidis, P. pachastrellae, P. palleroniana, P. papaveris, P. peli, P. perolens, P. poae, P. pohangensis, P. psychrophila, P. psychrotolerans, P. rathonis, P. reptilivora, P. resiniphila, P. rhizosphaerae, P. rubescens, P. salomonii, P. segitis, P. septica, P. simiae, P. suis, P. thermotolerans, P. aeruginosa, P. tremae, P. trivialis, P. turbinellae, P. tuticorinensis, P. umsongensis, P. vancouverensis, P. vranovensis, P. xanthomarina, Ralstonia eutropha, Rhodospirillum rubrum, Rhodobacter sphaeroides, Saccharomyces cerevisiae, Yarrowia lipolytica and Zymomonas mobilis, 
in particular Pseudomonas putida, Escherichia coli and Burkholderia thailandensis are particularly preferred.
Preferred cells according to the invention are able as the wild-type to form no or no detectable amounts of rhamnolipids and as the wild-type moreover have preferably no or no detectable activity of the enzymes E1, E2 and E3.
It is advantageous according to the invention if the cell according to the invention is a cell which is able as the wild-type to form polyhydroxyalkanoates having chain lengths of the mono-alkanoate of C6 to C16. Such cells are, for example, Burkholderia sp., Burkholderia thailandensis, Pseudomonas sp., Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas oleovorans, Pseudomonas stutzeri, Pseudomonas fluorescens, Pseudomonas citronellolis, Pseudomonas resinovorans, Comamonas testosteroni, Aeromonas hydrophila, Cupriavidus necator, Alcaligenes latus and Ralstonia eutropha. In this connection, preferred cells according to the invention are genetically modified such that, compared to their wild-type, they are able to form fewer polyhydroxyalkanoates.
Such cells are described, for example, in De Eugenio et al., Environ Microbiol. 2010. 12(1):207-21 and Rehm et al., Appl Environ Microbiol. 2001. 67(7):3102-9.
Such a cell, able to form fewer polyhydroxyalkanoates compared to its wild-type, is in particular characterized in that, compared to its wild-type, it has a decreased activity of at least one enzyme E9 or E10,
wherein E9 represents a polyhydroxyalkanoate synthase, EC:2.3.1.-, in particular having polypeptide sequence Seq ID No. 30 or Seq ID No. 32 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 radicals compared to the respective reference sequence Seq ID No. 30 or Seq ID No. 32 are modified by deletion, insertion, substitution or a combination thereof and that still has at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme having the respective reference sequence Seq ID No. 30 or Seq ID No. 32, wherein enzymatic activity for an enzyme E9 is understood as meaning the ability to convert 3-hydroxyalkanoyl-coenzyme A to poly-3-hydroxyalkanoic acid, in particular 3-hydroxytetradecanoyl-coenzyme A to poly-3-hydroxytetradecanoic acid, and

E10 represents a 3-hydroxyalkanoyl-ACP:coenzyme A transferase, in particular having polypeptide sequence Seq ID No. 34 or Seq ID No. 36 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 radicals are modified compared to the respective reference sequence Seq ID No. 34 or Seq ID No. 36 by deletion, insertion, substitution or a combination thereof and that still has at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme having the respective reference sequence Seq ID No. 34 or Seq ID No. 36, wherein enzymatic activity for an enzyme E10 is understood as meaning the ability to convert 3-hydroxyalkanoyl-ACP to 3-hydroxy-alkananoyl-coenzyme A, in particular 3-hydroxyalkananoyl-ACP to 3-hydroxytetradecanoyl-coenzyme A.

For a general survey compare FIG. 1.
The activity of the enzyme E9 is then determined using the samples obtained as described above for the enzymes E1 to E3, by first mixing 560 μl of 100 mM tris/HCl, pH 7.5, 20 μl of 35 mM DTNB in DMSO and 20 μl of 41 mM 3-hydroxydecanoyl-coenzyme A. Subsequently, 5 μg of purified enzyme E9 in 100 μl of tris/HCl, pH 7.5 are added, and subsequently the increase in the extinction at 412 nm (caused by addition of 5,5′-dithiobis(2-nitrobenzoate) (DTNB) to free SH groups) over time (ΔE/min) is recorded continuously for 1 min in a spectrophotometer.
The activity of the enzyme E10 is then determined using the samples obtained as described above for the enzymes E1 to E3. The standard assay contains 3 mM MgCl2, 40 μM hydroxydecanoyl-coenzyme A and 20 μM E. coli ACP in 50 mM tris-HCl, pH 7.5, in a total volume of 200 μl. The reaction is started by addition of 5 μg of purified enzyme E10 in 50 μl of tris/HCl, pH 7.5 and incubated for 1 h at 30° C. The reaction is stopped by addition of 50% (w/v) trichloroacetic acid and 10 mg/ml of BSA (30 μl). Released coenzyme A is determined spectrophotometrically by recording the increase in the extinction at 412 nm, caused by addition of 5,5′-dithiobis(2-nitrobenzoate) (DTNB) to free SH groups, over time.
The formulation “decreased activity of an enzyme Ex” used is accordingly preferably understood as meaning an activity decreased by a factor of at least 0.5, particularly preferably of at least 0.1, moreover preferably of at least 0.01, moreover even more preferably of at least 0.001 and most preferably of at least 0.0001. The formulation “decreased activity” also comprises no detectable activity (“activity of zero”). The decrease in the activity of a certain enzyme can be effected, for example, by selective mutation or by other measures known to the person skilled in the art for decreasing the activity of a certain enzyme.
Methods for decreasing enzymatic activities in microorganisms are known to the person skilled in the art.
In particular, molecular biological techniques offer themselves here. The person skilled in the art finds instructions for the modification and decrease of protein expression and concomitant lowering of enzyme activity especially for Pseudomonas and Burkholderia, in particular for interrupting specific genes, for example, in Dubeau et al. 2009. BMC Microbiology 9:263; Singh & Röhm. Microbiology. 2008. 154:797-809 or Lee et al. FEMS Microbiol Lett. 2009. 297(1):38-48.
Cells preferred according to the invention are characterized in that the decrease in the enzymatic activity is achieved by modification of a gene comprising one of the said nucleic acid sequences, wherein the modification is selected from the group comprising, preferably consisting of, insertion of foreign DNA in the gene, deletion of at least parts of the gene, point mutations in the gene sequence, RNA interference (siRNA), antisense RNA or modification (insertion, deletion or point mutations) of regulatory sequences, such as, for example, promoters and terminators or of ribosome binding sites, which flank the gene.
Foreign DNA is to be understood in this connection as meaning any DNA sequence which is “foreign” to the gene (and not to the organism), i.e. endogenous DNA sequences can also function in this connection as “foreign DNA”.
In this connection it is particularly preferred that the gene is interrupted by insertion of a selection marker gene, thus the foreign DNA is a selection marker gene, wherein preferably the insertion was effected by homologous recombination in the gene locus.
In a preferred embodiment of the cell according to the invention, the cells concerned are Pseudomonas putida cells, which have a decreased polyhydroxyalkanoate synthesis compared to their wild-type. Such cells are described, for example, in Ren et al., Journal Applied Microbiology and Biotechnology 1998 June, 49(6):743-50 as GPp121, GPp122, GPp123 and GPp124, in Huisman et al., J Biol Chem. 1991 Feb. 5; 266(4):2191-8 as GPp104 as well as in De Eugenio et al., Environ Microbial. 2010. 12(1):207-21 as KT42C1 and in Ouyang et al. Macromol Biosci. 2007. 7(2):227-33 as KTOY01 and KTOY02 and are preferred cells according to the invention.
For the case where the cell according to the invention is able to form a rhamnolipid having m=1, it is preferred that the radical




defined by means of R1 and R2 is derived from 3-hydroxyoctanoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydecenoic acid, 3-hydroxydecenoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydodecanoic acid, 3-hydroxydodecanoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydodecenoic acid, 3-hydroxydodecenoyl-3-hydroxyoctanoic acid, 3-hydroxydecanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxydecenoic acid, 3-hydroxydecenoyl-3-hydroxydecanoic acid, 3-hydroxydecenoyl-3-hydroxydecenoic acid, 3-hydroxydecanoyl-3-hydroxydodecanoic acid, 3-hydroxydodecanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxydodecenoic acid, 3-hydroxydecanoyl-3-hydroxytetradecenoic acid, 3-hydroxytetradecanoyl-3-hydroxydecenoic acid, 3-hydroxydodecenoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxytetradecanoic acid, 3-hydroxytetradecanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxytetradecenoic acid, 3-hydroxytetradecenoyl-3-hydroxydecanoic acid, 3-hydroxydodecanoyl-3-hydroxydodecanoic acid, 3-hydroxydodecenoyl-3-hydroxydodecanoic acid, 3-hydroxydodecanoyl-3-hydroxydodecenoic acid, 3-hydroxydodecanoyl-3-hydroxytetradecanoic acid, 3-hydroxytetradecanoyl-3-hydroxydodecanoic acid, 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoyl-3-hydroxytetradecanoic acid, 3-hydroxytetradecanoyl-3-hydroxyhexadecanoic acid or 3-hydroxyhexadecanoyl-3-hydroxyhexadecanoic acid.

It is obvious to the person skilled in the art that a cell according to the invention is also able to form mixtures of different rhamnolipids of the general formula (I).
In this connection, it is preferred that the cells according to the invention are able to form mixtures of rhamnolipids of the general formula (I), which are characterized in that in more than 80% by weight, preferably more than 90% by weight, particularly preferably more than 95% by weight of the rhamnolipids formed n is =1 and the radical defined by means of R1 and R2 is derived in less than 10% by weight, preferably less than 5% by weight, particularly preferably less than 2% by weight of the rhamnolipids formed, from 3-hydroxydecanoyl-3-hydroxyoctanoic acid or 3-hydroxyoctanoyl-3-hydroxydecanoic acid,
wherein the % by weight indicated refers to the sum of all rhamnolipids of the general formula (I) formed.
It is advantageous if the cell according to the invention has additionally been genetically modified with respect to E1 to E3 such that, compared to its wild-type, it has an increased activity as in each case specified below of at least one of the enzymes selected from the group consisting of
at least one enzyme E4, a dTTP:α-D-glucose-1-phosphate thymidylyl transferase, EC 2.7.7.24, in particular one having polypeptide sequence Seq ID No. 10 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 radicals compared to the reference sequence Seq ID No. 10 are modified by deletion, insertion, substitution or a combination thereof and that 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 Seq ID No. 10, wherein enzymatic activity for an enzyme E4 is understood as meaning the ability to convert α-D-glucose-1-phosphate and dTTP to dTDP-glucose,

at least one enzyme E5, a dTTP-glucose-4,6-hydrolyase, EC 4.2.1.46, in particular one having polypeptide sequence Seq ID No. 12 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 radicals are modified compared to the reference sequence Seq ID No. 12 by deletion, insertion, substitution or a combination thereof and that 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 Seq ID No. 12, wherein enzymatic activity for an enzyme E5 is understood as meaning the ability to convert dTDP-glucose to dTDP-4-dehydro-6-deoxy-D-glucose,

at least one enzyme E6, a dTDP-4-dehydrorhamnose-3,5-epimerase, EC 5.1.3.13, in particular one having polypeptide sequence Seq ID No. 14 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 radicals compared to the reference sequence Seq ID No. 14 are modified by deletion, insertion, substitution or a combination thereof and that 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 Seq ID No. 14, wherein enzymatic activity for an enzyme E6 is understood as meaning the ability to convert dTDP-4-dehydro-6-deoxy-D-glucose to dTDP-4-dehydro-6-deoxy-L-mannose and

at least one enzyme E7, a dTDP-4-dehydrorhamnose reductase, EC 1.1.1.133, in particular one having polypeptide sequence Seq ID No. 16 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 radicals compared to the reference sequence Seq ID No. 16 are modified by deletion, insertion, substitution or a combination thereof and that 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 Seq ID No. 16, wherein enzymatic activity for an enzyme E7 is understood as meaning the ability to convert dTDP-4-dehydro-6-deoxy-L-mannose to dTDP-6-deoxy-L-mannose.

The activity of the enzyme E4 is determined using the samples obtained as above for the enzymes E1 to E3, by incubating α-D-glucose-1-phosphate (1.3 mM) with dTTP (5 mM) and 5 μg of purified enzyme E4 in 50 μl of sodium phosphate buffer, pH 8.5 and stopping the reaction after 5, 10 and 20 min incubation at 30° C. by addition of 20 μl of chloroform. The mixture is then vortexed and centrifuged for 5 min at 16,000 g and room temperature. The aqueous phase is transferred to a new reaction vessel and the organic phase is extracted again with 80 μl of water. Both aqueous phases are combined and analyzed by means of HPLC. A Phenosphere ODS2 column (250×4.6 mm; Phenomenex, Torrance, USA) or a Spheresorb ODS2 column (250×4.6 mm; Waters, Milford, USA) is used here. The elution of the analytes takes place at a flow rate of 1 ml min−1 using 0.5 M KH2PO4 (eluent A) for 15 min, followed by a linear gradient up to 80% eluent A and 20% methanol over a period of 14 min at a flow rate of 0.7 ml min−1. Analytes which elute from the ODS2 columns are then injected into a Phenosphere SAX ion exchanger column (250×4.6 mm; Phenomenex, Torrance, USA) and the analytes are eluted using a flow rate of 1 ml min−1 and a linear ammonium formate gradient (2 to 600 mM over 25 min). The quantification of dTDP-glucose then takes place by means of its UV absorption using a photodiode array detector (DAD). The absorption maximum of thymidine is at 267 nm. The calibration takes place by means of authentic nucleotide sugar (Sigma-Aldrich, Munich, USA).
The activity of the enzyme E5 is then determined using the samples obtained as described above for the enzymes E1 to E3 by incubating dTDP-α-D-glucose (1.3 mM) with 5 μg of purified enzyme E5 in 50 μl of sodium phosphate buffer, pH 8.5, and stopping the reaction after 5, 10 and 20 min incubation at 30° C. by addition of 20 μl of chloroform. The mixture is then vortexed and centrifuged for 5 min at 16,000 g and room temperature. The aqueous phase is transferred to a new reaction vessel and the organic phase is again extracted with 80 μl of water. Both aqueous phases are combined and analyzed by means of HPLC. A Phenosphere ODS2 column (250×4.6 mm; Phenomenex, Torrance, USA) or a Spheresorb ODS2 column (250×4.6 mm; Waters, Milford, USA) is used here. The elution of the analytes takes place at a flow rate of 1 ml min−1 using 0.5 M KH2PO4 (eluent A) for 15 min, followed by a linear gradient of up to 80% eluent A and 20% methanol over a period of 14 min at a flow rate of 0.7 ml min−1. Analytes which elute from the ODS2 columns are then injected into a Phenosphere SAX ion exchanger column (250×4.6 mm; Phenomenex, Torrance, USA) and the analytes are eluted using a flow rate of 1 ml min−1 and a linear ammonium formate gradient (2 to 600 mM over 25 min). The quantification of dTDP-glucose and dTDP-4-dehydro-6-deoxy-D-glucose then takes place by means of their UV absorption using a photodiode array detector (DAD). The absorption maximum of thymidine is at 267 nm. The calibration takes place by means of authentic nucleotide sugar (Sigma-Aldrich, Munich, USA).
The activity of the enzyme E6 is then determined using the samples obtained as described above for the enzymes E1 to E3, by first incubating dTDP-α-D-glucose (1.3 mM) with 5 μg of purified enzyme E5 in 50 μl of sodium phosphate buffer, pH 8.5, for 10 min at 30° C. Subsequently, 0.5 μg of purified enzyme E6 are added, and after 5, 10 and 20 min incubation at 30° C. the reaction is stopped by addition of 20 μl of chloroform. The mixture is then vortexed and centrifuged for 5 min at 16,000 g and room temperature. The aqueous phase is transferred to a new reaction vessel and the organic phase is again extracted with 80 μl of water. Both aqueous phases are combined and analyzed by means of HPLC. A Phenosphere ODS2 column (250×4.6 mm; Phenomenex, Torrance, USA) or a Spheresorb ODS2 column (250×4.6 mm; Waters, Milford, USA) is used here. The elution of the analytes takes place at a flow rate of 1 ml min−1 using 0.5 M KH2PO4 (eluent A) for 15 min, followed by a linear gradient of up to 80% eluent A and 20% methanol over a period of 14 min at a flow rate of 0.7 ml min−1. Analytes which elute from the ODS2 columns are then injected into a Phenosphere SAX ion exchanger column (250×4.6 mm; Phenomenex, Torrance, USA) and the analytes are eluted using a flow rate of 1 ml min−1 and a linear ammonium formate gradient (2 to 600 mM over 25 min). The quantification of dTDP-glucose, dTDP-4-dehydro-6-deoxy-D-glucose and dTDP-6-deoxy-L-mannose then takes place by means of their UV absorption using a photodiode array detector (DAD). The absorption maximum of thymidine is at 267 nm. The calibration takes place by means of authentic nucleotide sugar (Sigma-Aldrich, Munich, USA).
The activity of the enzyme E7 is then determined using the samples obtained as described above for the enzymes E1 to E3, by first incubating dTDP-α-D-glucose (1.3 mM) with 5 μg of purified enzyme E5 in 50 μl of sodium phosphate buffer, pH 8.5, for 10 min at 30° C. Subsequently, 5 μg of purified enzyme E6 and 0.5 μg of purified enzyme E7 as well as NADPH (10 mM) are added, and after incubation at 30° C. for 5, 10 and 20 min the reaction is stopped by addition of 20 μl chloroform. The mixture is then vortexed and centrifuged for 5 min at 16,000 g and room temperature. The aqueous phase is transferred to a new reaction vessel and the organic phase is again extracted with 80 μl of water. Both aqueous phases are combined and analyzed by means of HPLC. A Phenosphere ODS2 column (250×4.6 mm; Phenomenex, Torrance, USA) or a Spheresorb ODS2 column (250×4.6 mm; Waters, Milford, USA) is used here. The elution of the analytes takes place at a flow rate of 1 ml min−1 using 0.5 M KH2PO4 (eluent A) for 15 min, followed by a linear gradient of up to 80% eluent A and 20% methanol over a period of 14 min at a flow rate of 0.7 ml min−1. Analytes which elute from the ODS2 columns are then injected into a Phenosphere SAX ion exchanger column (250×4.6 mm; Phenomenex, Torrance, USA) and the analytes are eluted using a flow rate of 1 ml min−1 and a linear ammonium formate gradient (2 to 600 mM over 25 min). The quantification of dTDP-glucose, dTDP-4-dehydro-6-deoxy-D-glucose, dTDP-6-deoxy-L-mannose and dTDP-4-dehydro-6-deoxy-L-mannose then takes place by means of their UV absorption using a photodiode array detector (DAD). The absorption maximum of thymidine is 267 nm. The calibration takes place by means of authentic nucleotide sugar (Sigma-Aldrich, Munich, USA).
Cells according to the invention are preferred, which have increased activities of the following enzyme combinations:
E4E5, E4E6, E4E7, E5E6, E5E7, E6E7, E4E5E6, E4E5E7, E5E6E7, E4E6E7, E4E5E6E7,
of which the combination
E4E5E6E7 
is particularly preferred.
It can be advantageous according to the invention if the cell according to the invention has been genetically modified in the fatty acid biosynthesis such that the enzymatic reactions, which lead to the conversion of acyl-ACP and malonyl-coenzyme A to 3-ketoacyl-ACP and/or to the conversion of 3-ketoacyl-ACP to (R)-3-hydroxyalkanoyl-ACP, are increased. Additionally or alternatively it can be advantageous according to the invention if the cell according to the invention has been genetically modified in the fatty acid biosynthesis such that the enzymatic reactions, which lead to the conversion of (R)-3-hydroxyalkanoyl-ACP to trans-2-enoyl-ACP and/or to the conversion of trans-2-enoyl-ACP to acyl-ACP, are attenuated.
It can be just as advantageous if the cell according to the invention has been genetically modified in the β-oxidation of fatty acids such that the enzymatic reactions, which lead to the conversion of acyl-coenzyme A to trans-2-enoyl-coenzyme A and/or to the conversion of trans-2-enoyl-coenzyme A to (S)-3-hydroxyalkanoyl-coenzyme A, are increased. Additionally or alternatively, it can be advantageous according to the invention if the cell according to the invention in the β-oxidation of fatty acids has been genetically modified such that the enzymatic reactions, which lead to the conversion of (S)-3-hydroxyalkanoyl-coenzyme A to 3-ketoacyl-coenzyme A and/or to the conversion of 3-ketoacyl-coenzyme A to acyl-coenzyme A and acetyl-coenzyme A, are diminished.
For a general survey compare FIG. 1.
Since the cells according to the invention can be used advantageously for the production of rhamnolipids and since these lipids are subsequently optionally purified, it is advantageous if the cells according to the invention have an increased activity compared to their wild-type of at least an enzyme E8, which catalyzes the export of a rhamnolipid of the general formula (I) from the cell into the surrounding medium.
Preferably, in this connection proteins E8 are selected from the group consisting of an enzyme E8 having polypeptide sequence Seq ID No. 8, Seq ID No. 24, Seq ID No. 26 or Seq ID No. 28 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 radicals are modified by deletion, insertion, substitution or a combination thereof compared to the respective reference sequence Seq ID No. 8, Seq ID No. 24, Seq ID No. 26 or Seq ID No. 28 and that still has at least 50%, preferably 65%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme having the respective reference sequence Seq ID No. 8, Seq ID No. 24, Seq ID No. 26 or Seq ID No. 28, wherein enzymatic activity for an enzyme E8 is understood as meaning the ability to export a rhamnolipid of the general formula (I) from the cell into the surrounding medium.
A further, preferred embodiment of cells according to the invention is characterized in that it contains at least one of the nucleic acids or vectors according to the invention mentioned below.
Cells according to the invention can advantageously be used for the production of rhamnolipids. Thus a further subject of the invention is the use of cells according to the invention for the production of compounds of the general formula (I).
A further subject of the present invention is a method for producing rhamnolipids of the general formula (I),
wherein
m=2, 1 or 0, in particular 1 or 0,
n=1 or 0, in particular 1,
R1 and R2=independently of one another identical or different organic radical 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 tri-unsaturated, alkyl radical, preferably that selected from the group consisting of pentenyl, heptenyl, nonenyl, undecenyl and tridecenyl and (CH2)o—CH3 having o=1 to 23, preferably 4 to 12,

comprising the process steps

I) bringing into contact the cell according to the invention with a medium containing a carbon source

II) culturing the cell under conditions that make it possible for the cell to form rhamnolipid from the carbon source and

III) optionally isolating the rhamnolipids formed.

The genetically modified cells according to the invention can be brought into contact with the nutrient medium continuously or discontinuously in the batch process (batch culture) or in the fed-batch process (feed process) or repeated fed-batch process (repetitive feed process) for the purpose of the production of the abovementioned products and thus cultured. A semi-continuous process is also conceivable, as is described in GB-A-1009370. A summary of known culturing methods are described in the textbook of Chmiel (“Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik” [Bioprocess Technology 1. Introduction to the Bioprocess Technique] (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook of Storhas (“Bioreaktoren and periphere Einrichtungen” [Bioreactors and Peripheral Devices], Vieweg Verlag, Brunswick/Wiesbaden, 1994).
The culture medium to be used must satisfy in a suitable manner the demands of the respective strains. Descriptions of culture media of different yeast strains are contained, for example, in “Nonconventional yeast in biotechnology” (Ed. Klaus Wolf, Springer-Verlag Berlin, 1996). The carbon source used can be carbohydrates such as, for example, glucose, sucrose, arabinose, xylose, lactose, fructose, maltose, molasses, starch, cellulose and hemicellulose, vegetable and animal oils and fats such as, for example, soybean oil, safflower oil, peanut oil, hempseed oil, jatropha oil, coconut fat, calabash oil, linseed oil, corn oil, poppyseed oil, evening primrose oil, olive oil, palm kernel oil, palm oil, rapeseed oil, sesame oil, sunflower oil, grapeseed oil, walnut oil, wheat germ oil and coconut oil, fatty acids, such as, for example, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, arachidonic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, gamma-linolenic acid and its methyl or ethyl ester as well as fatty acid mixtures, mono-, di- and triglycerides containing the fatty acids just mentioned, alcohols such as, for example, glycerol, ethanol and methanol, hydrocarbons such as methane, carbon-containing gases and gas mixtures, such as CO, CO2, synthesis or flue gas, amino acids such as L-glutamate or L-valine or organic acids such as, for example, acetic acid. These substances can be used individually or as a mixture. The use of carbohydrates, in particular of monosaccharides, oligosaccharides or polysaccharides, as the carbon source as is described in U.S. Pat. No. 601,494 and U.S. Pat. No. 6,136,576 as well as of hydrocarbons, in particular of alkanes, alkenes and alkynes as well as the monocarboxylic acids derived therefrom and the mono-, di and triglycerides derived from these monocarboxylic acids, as well as of glycerol and acetate, is particularly preferred. Mono-, di- and triglycerides containing the 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 gamma-linolenic acid are very particularly preferred.
It is a great advantage of the present invention that the cells according to the invention are able to form rhamnolipids from the simplest carbon sources such as, for example, glucose, sucrose or glycerol, such that a provision of longer-chain C sources in the medium during the method according to the invention is not necessary. Thus it is advantageous in the case of lack of availability that the medium in step I) of the method according to the invention contains no or no detectable amounts of carboxylic acids having a chain length of greater than six carbon atoms or esters or glycerides derivable from these.
The nitrogen source used can be organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, cornsteep water, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, ammonia, ammonium hydroxide or ammonia water. The nitrogen sources can be used individually or as a mixture.
The phosphorus source used can be phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts. The culture medium must furthermore contain salts of metals such as, for example, magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth promoters such as amino acids and vitamins can be employed additionally to the abovementioned substances. Suitable precursors can moreover be added to the culture medium. The said feedstocks can be added to the culture in the form of a single batch or fed in a suitable manner during culturing.
Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid are suitably employed for pH control of the culture. Antifoam agents such as, for example, fatty acid polyglycol esters can be employed for the control of the foam development. Suitable selectively acting substances such as, for example, antibiotics can be added to the medium for maintaining the stability of plasmids. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures such as, for example, air are incorporated into the culture.
The temperature of the culture is normally more than 20° C., preferably more than 25° C., it can also be more than 40° C., wherein advantageously a culturing temperature of 95° C., particularly preferably 90° C. and most preferably 80° C. is not exceeded.
In step III) of the method according to the invention, the rhamnolipids formed by the cells can optionally be isolated from the cells and/or the nutrient medium, wherein for the isolation all methods known to the person skilled in the art for the isolation of low molecular weight substances from complex compositions are possible, such as, for example, filtration, extraction, adsorption (chromatography) or crystallization.
Moreover, the product phase contains residues of biomass and various impurities, such as oils, fatty acids and other nutrient media constituents. The separation of the impurities preferably takes place in a solvent-free process. Thus, for example, the product phase can be diluted with water to facilitate the adjustment of the pH. The product and aqueous phases can then be homogenized by converting the rhamnolipids into a water-soluble form by lowering or raising the pH by acids or alkalis. Potentially, the solubilization of the rhamnolipids in the aqueous phase can be assisted by incubation at higher temperatures, e.g. at 60 to 90° C., and constant mixing. By subsequent raising or lowering of the pH by alkalis or acids the rhamnolipids can then again be converted into a water-insoluble form, such that they can easily be separated from the aqueous phase. The product phase can then be washed once or several times with water to remove the water-soluble impurities.
Oil residues can be separated off, for example by extraction by means of suitable solvents advantageously by means of organic solvents. An alkane such as, for example, n-hexane is preferred as a solvent.
The separation of the product from the aqueous phase can be effected alternatively to the solvent-free process described above using a suitable solvent, e.g. an ester such as, for example, ethyl acetate or butyl acetate. The said extraction steps can be carried out in any desired sequence.
In this connection, solvents are preferably employed, in particular organic solvents. n-Pentanol is preferred as a solvent. A distillation, for example, takes place for the removal of the solvent. Subsequently, the lyophilized product can be further purified, for example by means of chromatographic methods. By way of example, at this point precipitation by means of suitable solvents, extraction by means of suitable solvents, complexation, for example by means of cyclodextrins or cyclodextrin derivatives, crystallization, purification or isolation by means of chromatographic methods or conversion of the rhamnolipids into easily separable derivatives may be mentioned.
The rhamnolipids that can be produced using the method according to the invention are likewise a subject of the present invention, in particular also the rhamnolipid mixtures described above, that can be produced using the method according to the invention.
The rhamnolipids and mixtures that can be produced using the method according to the invention can advantageously be employed in cleaning agents, in cosmetic or pharmaceutical formulations as well as in plant protection formulations.
Thus a further subject of the present invention is the use of the rhamnolipids obtained using the method according to the invention for the production of cosmetic, dermatological or pharmaceutical formulations, of plant protection formulations and of care and cleaning agents and surfactant concentrates.
The term “care agents” is understood here as meaning a formulation that fulfills the purpose of maintaining an article in its original form, reducing or avoiding the effects of external influences (e.g. time, light, temperature, pressure, pollution, chemical reaction with other reactive compounds coming into contact with the article) such as, for example, aging, pollution, material fatigue, or even improving desired positive properties of the article. For the last point, for example, an improved hair gloss or a greater elasticity of the article considered may be mentioned.
“Plant protection formulations” are to be understood as meaning those formulations that by the nature of their preparation are obviously used for plant protection; this is in particular the case if at least one compound from the classes consisting of the herbicides, fungicides, insecticides, acaricides, nematicides, protective substances against bird damage, plant nutrients and soil structure-improving agents is contained in the formulation.
According to the invention, rhamnolipids produced using the method according to the invention are preferably used in care and cleaning agents for housekeeping, industry, in particular for hard surfaces, leather or textiles.
A contribution to achieving the object is provided by an isolated nucleic acid, which contains at least in each case a sequence selected from the three groups [A1 to G1], [A2 to G2] and [A3 to G3],
wherein
the group [A1 to G1] consists of the following sequences:
A1a) a sequence according to Seq ID No. 1, wherein this sequence codes for a protein, which is able
to convert 3-hydroxydecanoyl-ACP via 3-hydroxydecanoyl-3-hydroxydecanoyl-ACP to 3-hydroxydecanoyl-3-hydroxydecanoic acid,
B1a) an intron-free sequence that is derived from a sequence according to A1a) and that encodes the same protein or peptide as the sequence according to Seq ID No. 1,
C1a) a sequence that encodes a protein or peptide that comprises the amino acid sequence according to Seq ID No. 2, and that is preferably able
to convert 3-hydroxydecanoyl-ACP via 3-hydroxydecanoyl-3-hydroxydecanoyl-ACP to 3-hydroxydecanoyl-3-hydroxydecanoic acid,
D1a) a sequence that is identical with a sequence according to one of the groups A1a) to
C1a), particularly preferably according to group A1a), to at least 70%, particularly preferably to at least 90%, moreover preferably to at least 95% and most preferably to at least 99%, wherein this sequence preferably codes for a protein or peptide, which is able to convert 3-hydroxydecanoyl-ACP via 3-hydroxydecanoyl-3-hydroxydecanoyl-ACP to 3-hydroxydecanoyl-3-hydroxydecanoic acid,

E1a) a sequence that hybridizes or, taking into consideration the degeneracy of the genetic code, would hybridize with the complementary strand of a sequence according to one of the groups A1a) to D1a), particularly preferably according to group A1a), wherein this sequence preferably codes for a protein or peptide, which is able

to convert 3-hydroxydecanoyl-ACP via 3-hydroxydecanoyl-3-hydroxydecanoyl-ACP to 3-hydroxydecanoyl-3-hydroxydecanoic acid,

F1a) a derivative of a sequence according to one of the groups A1a) to E1a), particularly preferably according to group A1a), obtained by substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, moreover preferably of at least 5 bases and most preferably of at least 10 bases, but preferably of not more than 100 bases, particularly preferably of not more than 50 bases and most preferably of not more than 25 bases, wherein this derivative preferably codes for a protein or peptide, which is able

to convert 3-hydroxydecanoyl-ACP via 3-hydroxydecanoyl-3-hydroxydecanoyl-ACP to 3-hydroxydecanoyl-3-hydroxydecanoic acid,

G1a) a complementary sequence to a sequence according to one of the groups A1a) to F1a), particularly preferably according to group A1a),

A1b) a sequence according to Seq ID No. 17, wherein this sequence codes for a protein, which is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

B1b) an intron-free sequence that is derived from a sequence according to A1b) and that encodes the same protein or peptide as the sequence according to Seq ID No. 17,

C1b) a sequence that encodes a protein or peptide that comprises the amino acid sequence according to Seq ID No. 18, and that preferably is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

D1b) a sequence that is identical with a sequence according to one of the groups A1b) to C1b), particularly preferably according to group A1b), to at least 70%, particularly preferably to at least 90%, moreover preferably to at least 95% and most preferably to at least 99%, wherein this sequence preferably codes for a protein or peptide, which is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

E1b) a sequence that hybridizes or, taking into consideration the degeneracy of the genetic code, would hybridize with the complementary strand of a sequence according to one of the groups A1b) to D1b), particularly preferably according to group A1b), wherein this sequence preferably codes for a protein or peptide, which is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

F1b) a derivative obtained by substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, moreover preferably of at least 5 bases and most preferably at least 10 bases, but preferably of not more than 100 bases, particularly preferably of not more than 50 bases and most preferably of not more than 25 bases of a sequence according to one of the groups A1b) to E1b), particularly preferably according to group A1b), wherein this derivative preferably codes for a protein or peptide, which is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, and

G1b) a complementary sequence to a sequence according to one of the groups A1b) to F1b), particularly preferably according to group A1b), and

A1c) a sequence according to Seq ID No. 77, wherein this sequence codes for a protein, which is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

B1c) an intron-free sequence that is derived from a sequence according to A1c) and that encodes the same protein or peptide as the sequence according to Seq ID No. 77,

C1c) a sequence that encodes a protein or peptide that comprises the amino acid sequence according to Seq ID No. 78, and that preferably is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

D1c) a sequence that is identical with a sequence according to one of the groups A1c) to C1c), particularly preferably according to group A1c), to at least 70%, particularly preferably to at least 90%, moreover preferably to at least 95% and most preferably to at least 99%, wherein this sequence preferably codes for a protein or peptide, which is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

E1c) a sequence that hybridizes or, taking into consideration the degeneracy of the genetic code, would hybridize with the complementary strand of a sequence according to one of the groups A1c) to D1c), particularly preferably according to group A1c), wherein this sequence preferably codes for a protein or peptide, which is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

F1c) a derivative obtained by substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, moreover preferably of at least 5 bases and most preferably of at least 10 bases, but preferably of not more than 100 bases, particularly preferably of not more than 50 bases and most preferably of not more than 25 bases of a sequence according to one of the groups A1c) to E1c), particularly preferably according to group A1c), wherein this derivative preferably codes for a protein or peptide, which is able to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, and

G1c) a complementary sequence to a sequence according to one of the groups A1c) to F1c), particularly preferably according to group A1c), and

A1d) a sequence according to Seq ID No. 79, wherein this sequence codes for a protein, which is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

B1d) an intron-free sequence that is derived from a sequence according to A1d) and that encodes the same protein or peptide as the sequence according to Seq ID No. 79,

C1d) a sequence that encodes a protein or peptide that comprises the amino acid sequence according to Seq ID No. 80, and that preferably is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

D1d) a sequence that is identical with a sequence according to one of the groups A1d) to C1d), particularly preferably according to group A1d), to at least 70%, particularly preferably to at least 90%, moreover preferably to at least 95% and most preferably to at least 99%, wherein this sequence preferably codes for a protein or peptide, which is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

E1d) a sequence that hybridizes or, taking into consideration the degeneracy of the genetic code, would hybridize with the complementary strand of a sequence according to one of the groups A1d) to Did), particularly preferably according to group A1d), wherein this sequence preferably codes for a protein or peptide, which is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

F1d) a derivative obtained by substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, moreover preferably of at least 5 bases and most preferably at least 10 bases, but preferably of not more than 100 bases, particularly preferably of not more than 50 bases and most preferably of not more than 25 bases of a sequence according to one of the groups A1d) to E1d), particularly preferably according to group A1d), wherein this derivative preferably codes for a protein or peptide, which is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, and

G1d) a complementary sequence to a sequence according to one of the groups A1d) to F1d), particularly preferably according to group A1d), and

A1e) a sequence according to Seq ID No. 81, wherein this sequence codes for a protein, which is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

B1e) an intron-free sequence that is derived from a sequence according to A1e) and that encodes the same protein or peptide as the sequence according to Seq ID No. 81,

C1e) a sequence that encodes a protein or peptide that comprises the amino acid sequence according to Seq ID No. 82, and that preferably is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

D1e) a sequence that is identical with a sequence according to one of the groups A1e) to C1e), particularly preferably according to group A1e), to at least 70%, particularly preferably to at least 90%, moreover preferably to at least 95% and most preferably to at least 99%, wherein this sequence preferably codes for a protein or peptide, which is able to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

E1e) a sequence that hybridizes or, taking into consideration the degeneracy of the genetic code, would hybridize with the complementary strand of a sequence according to one of the groups A1e) to D1e), particularly preferably according to group A1e), wherein this sequence preferably codes for a protein or peptide, which is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

F1e) a derivative obtained by substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, moreover preferably of at least 5 bases and most preferably at least 10 bases, but preferably of not more than 100 bases, particularly preferably of not more than 50 bases and most preferably of not more than 25 bases of a sequence according to one of the groups A1e) to E1e), particularly preferably according to group A1e), wherein this derivative preferably codes for a protein or peptide, which is able

to convert 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, and

G1e) a complementary sequence to a sequence according to one of the groups A1e) to F1e), particularly preferably according to group A1e), and

the group [A2 to G2] consists of the following sequences:

A2a) a sequence according to Seq ID No. 3, wherein this sequence codes for a protein, which is able

to convert dTDP-rhamnose and 3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,

B2a) an intron-free sequence that is derived from a sequence according to A2a) and that encodes the same protein or peptide as the sequence according to Seq ID No. 3,

C2a) a sequence that encodes a protein or peptide that comprises the amino acid sequence according to Seq ID No. 4, and which preferably is able

to convert dTDP-rhamnose and 3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,

D2a) a sequence that is identical with a sequence according to one of the groups A2a) to C2a), particularly preferably according to group A2a), to at least 80%, particularly preferably to at least 90%, moreover preferably to at least 95% and most preferably to at least 99%, wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and 3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,

E2a) a sequence that hybridizes or, taking into consideration the degeneracy of the genetic code, would hybridize with the complementary strand of a sequence according to one of the groups A2a) to D2a), particularly preferably according to group A2a), wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and 3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,

F2a) a derivative obtained by substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, moreover preferably of at least 5 bases and most preferably at least 10 bases, but preferably of not more than 100 bases, particularly preferably of not more than 50 bases and most preferably of not more than 25 bases of a sequence according to one of the groups A2a) to E2a), particularly preferably according to group A2a), wherein this derivative preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and 3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,

G2a) a complementary sequence to a sequence according to one of the groups A2a) to F2a), particularly preferably according to group A2a),

A2b) a sequence according to Seq ID No. 19, wherein this sequence codes for a protein, which is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

B2b) an intron-free sequence that is derived from a sequence according to A2b) and that encodes the same protein or peptide as the sequence according to Seq ID No. 19,

C2b) a sequence that encodes a protein or peptide that comprises the amino acid sequence according to Seq ID No. 20, and which preferably is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

D2b) a sequence that is identical with a sequence according to one of the groups A2b) to

C2b), particularly preferably according to group A2b), to at least 70%, particularly preferably to at least 90%, moreover preferably to at least 95% and most preferably to at least 99%, wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

E2b) a sequence that hybridizes or, taking into consideration the degeneracy of the genetic code, would hybridize with the complementary strand of a sequence according to one of the groups A2b) to D2b), particularly preferably according to group A2b), wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

F2b) a derivative obtained by substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, moreover preferably of at least 5 bases and most preferably at least 10 bases, but preferably of not more than 100 bases, particularly preferably of not more than 50 bases and most preferably of not more than 25 bases of a sequence according to one of the groups A2b) to E2b), particularly preferably according to group A2b), wherein this derivative preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, and

G2b) a complementary sequence to a sequence according to one of the groups A2b) to F2b), particularly preferably according to group A2b),

A2c) a sequence according to Seq ID No. 83, wherein this sequence codes for a protein, which is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

B2c) an intron-free sequence that is derived from a sequence according to A2c) and that encodes the same protein or peptide as the sequence according to Seq ID No. 83,

C2c) a sequence that encodes a protein or peptide that comprises the amino acid sequence according to Seq ID No. 84, and which preferably is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

D2c) a sequence that is identical with a sequence according to one of the groups A2c) to

C2c), particularly preferably according to group A2c), to at least 70%, particularly preferably to at least 90%, moreover preferably to at least 95% and most preferably to at least 99%, wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

E2c) a sequence that hybridizes or, taking into consideration the degeneracy of the genetic code, would hybridize with the complementary strand of a sequence according to one of the groups A2c) to D2c), particularly preferably according to group A2c), wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

F2c) a derivative obtained by substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, moreover preferably of at least 5 bases and most preferably at least 10 bases, but preferably of not more than 100 bases, particularly preferably of not more than 50 bases and most preferably of not more than 25 bases of a sequence according to one of the groups A2c) to E2c), particularly preferably according to group A2c), wherein this derivative preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, and

G2c) a complementary sequence to a sequence according to one of the groups A2c) to F2c), particularly preferably according to group A2c),

A2d) a sequence according to Seq ID No. 85, wherein this sequence codes for a protein, which is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

B2d) an intron-free sequence that is derived from a sequence according to A2d) and that encodes the same protein or peptide as the sequence according to Seq ID No. 85,

C2d) a sequence that encodes a protein or peptide that comprises the amino acid sequence according to Seq ID No. 86, and which preferably is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

D2d) a sequence that is identical with a sequence according to one of the groups A2d) to C2d), particularly preferably according to group A2d), to at least 70%, particularly preferably to at least 90%, moreover preferably to at least 95% and most preferably to at least 99%, wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

E2d) a sequence that hybridizes or, taking into consideration the degeneracy of the genetic code, would hybridize with the complementary strand of a sequence according to one of the groups A2d) to D2d), particularly preferably according to group A2d), wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

F2d) a derivative obtained by substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, moreover preferably of at least 5 bases and most preferably at least 10 bases, but preferably of not more than 100 bases, particularly preferably of not more than 50 bases and most preferably of not more than 25 bases of a sequence according to one of the groups A2d) to E2d), particularly preferably according to group A2d), wherein this derivative preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, and

G2d) a complementary sequence to a sequence according to one of the groups A2d) to F2d), particularly preferably according to group A2d), and

A2e) a sequence according to Seq ID No. 87, wherein this sequence codes for a protein, which is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

B2e) an intron-free sequence that is derived from a sequence according to A2e) and that encodes the same protein or peptide as the sequence according to Seq ID No. 87,

C2e) a sequence that encodes a protein or peptide that comprises the amino acid sequence according to Seq ID No. 88, and which preferably is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

D2e) a sequence that is identical with a sequence according to one of the groups A2e) to C2e), particularly preferably according to group A2e), to at least 70%, particularly preferably to at least 90%, moreover preferably to at least 95% and most preferably to at least 99%, wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

E2e) a sequence that hybridizes or, taking into consideration the degeneracy of the genetic code, would hybridize with the complementary strand of a sequence according to one of the groups A2e) to D2e), particularly preferably according to group A2e), wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

F2e) a derivative obtained by substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, moreover preferably of at least 5 bases and most preferably at least 10 bases, but preferably of not more than 100 bases, particularly preferably of not more than 50 bases and most preferably of not more than 25 bases of a sequence according to one of the groups A2e) to E2e), particularly preferably according to group A2e), wherein this derivative preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, and

G2e) a complementary sequence to a sequence according to one of the groups A2e) to F2e), particularly preferably according to group A2e),

and

the group [A3 to G3] consists of the following sequences:

A3a) a sequence according to Seq ID No. 5, wherein this sequence codes for a protein, which is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,

B3a) an intron-free sequence that is derived from a sequence according to A3a) and that encodes the same protein or peptide as the sequence according to Seq ID No. 5,

C3a) a sequence that encodes a protein or peptide that comprises the amino acid sequence according to Seq ID No. 6, and which preferably is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,

D3a) a sequence that is identical with a sequence according to one of the groups A3a) to C3a), particularly preferably according to group A3a), to at least 80%, particularly preferably to at least 90%, moreover preferably to at least 95% and most preferably to at least 99%, wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,

E3a) a sequence that hybridizes or, taking into consideration the degeneracy of the genetic code, would hybridize with the complementary strand of a sequence according to one of the groups A3a) to D3a), particularly preferably according to group A3a), wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,

F3a) a derivative obtained by substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, moreover preferably of at least 5 bases and most preferably at least 10 bases, but preferably of not more than 100 bases, particularly preferably of not more than 50 bases and most preferably of not more than 25 bases of a sequence according to one of the groups A3a) to E3a), particularly preferably according to group A3a), wherein this derivative preferably codes for a protein or peptide,

which is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,

G3a) a complementary sequence to a sequence according to one of the groups A3a) to F3a), particularly preferably according to group A3a),

A3b) a sequence according to Seq ID No. 21, wherein this sequence codes for a protein, which is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

B3b) an intron-free sequence that is derived from a sequence according to A3b) and that encodes the same protein or peptide as the sequence according to Seq ID No. 21,

C3b) a sequence that encodes a protein or peptide that comprises the amino acid sequence according to Seq ID No. 22, and which preferably is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

D3b) a sequence that is identical with a sequence according to one of the groups A3b) to C3b), particularly preferably according to group A3b), to at least 60%, particularly preferably to at least 90%, moreover preferably to at least 95% and most preferably to at least 99%, wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

E3b) a sequence that hybridizes or, taking into consideration the degeneracy of the genetic code, would hybridize with the complementary strand of a sequence according to one of the groups A3b) to D3b), particularly preferably according to group A3b), wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

F3b) a derivative obtained by substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, moreover preferably of at least 5 bases and most preferably at least 10 bases, but preferably of not more than 100 bases, particularly preferably of not more than 50 bases and most preferably of not more than 25 bases of a sequence according to one of the groups A3b) to E3b), particularly preferably according to group A3b), wherein this derivative preferably codes for a protein or peptide,

which is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, and

G3b) a complementary sequence to a sequence according to one of the groups A3b) to F3b), particularly preferably according to group A3b),

A3c) a sequence according to Seq ID No. 89, wherein this sequence codes for a protein, which is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

B3c) an intron-free sequence that is derived from a sequence according to A3c) and that encodes the same protein or peptide as the sequence according to Seq ID No. 89,

C3c) a sequence that encodes a protein or peptide that comprises the amino acid sequence according to Seq ID No. 90, and which preferably is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

D3c) a sequence that is identical with a sequence according to one of the groups A3c) to C3c), particularly preferably according to group A3c), to at least 60%, particularly preferably to at least 90%, moreover preferably to at least 95% and most preferably to at least 99%, wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

E3c) a sequence that hybridizes or, taking into consideration the degeneracy of the genetic code, would hybridize with the complementary strand of a sequence according to one of the groups A3c) to D3c), particularly preferably according to group A3c), wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

F3c) a derivative obtained by substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, moreover preferably of at least 5 bases and most preferably at least 10 bases, but preferably of not more than 100 bases, particularly preferably of not more than 50 bases and most preferably of not more than 25 bases of a sequence according to one of the groups A3c) to E3c), particularly preferably according to group A3c), wherein this derivative preferably codes for a protein or peptide,

which is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, and

G3c) a complementary sequence to a sequence according to one of the groups A3c) to F3c), particularly preferably according to group A3c) and

A3d) a sequence according to Seq ID No. 91, wherein this sequence codes for a protein, which is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

B3d) an intron-free sequence that is derived from a sequence according to A3d) and that encodes the same protein or peptide as the sequence according to Seq ID No. 91,

C3d) a sequence that encodes a protein or peptide that comprises the amino acid sequence according to Seq ID No. 92, and which preferably is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

D3d) a sequence that is identical with a sequence according to one of the groups A3d) to C3d), particularly preferably according to group A3d), to at least 60%, particularly preferably to at least 90%, moreover preferably to at least 95% and most preferably to at least 99%, wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

E3d) a sequence that hybridizes or, taking into consideration the degeneracy of the genetic code, would hybridize with the complementary strand of a sequence according to one of the groups A3d) to D3d), particularly preferably according to group A3d), wherein this sequence preferably codes for a protein or peptide, which is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,

F3d) a derivative obtained by substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, moreover preferably of at least 5 bases and most preferably at least 10 bases, but preferably of not more than 100 bases, particularly preferably of not more than 50 bases and most preferably of not more than 25 bases of a sequence according to one of the groups A3d) to E3d), particularly preferably according to group A3d), wherein this derivative preferably codes for a protein or peptide,

which is able

to convert dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, and

G3d) a complementary sequence to a sequence according to one of the groups A3d) to F3d), particularly preferably according to group A3d).

The “nucleotide identity” or “amino acid identity” is determined here with the aid of known methods. Generally, specific computer programs having algorithms taking into consideration special requirements are used.
Preferred methods for the determination of the identity for the present produce the greatest agreement between the sequences to be compared. Computer programs for the determination of the identity comprise, but are not restricted to, the GCG program package, including GAP (Deveroy, J. et al., Nucleic Acid Research 12 (1984), page 387, Genetics Computer Group University of Wisconsin, Medicine (Wi)), and BLASTP, BLASTN and FASTA (Altschul, S. et al., Journal of Molecular Biology 215 (1990), pages 403-410). The BLAST program can be obtained from the National Center for Biotechnology Information (NCBI) and from further sources (BLAST handbook, Altschul S. et al., NCBI NLM NIH Bethesda ND 22894; Altschul S. et al., above).
The known Smith-Waterman algorithm can likewise be used for the determination of the nucleotide identity.
Preferred parameters for the determination of the “nucleotide identity” are, when using the BLASTN program (Altschul, S. et a, Journal of Molecular Biology 215 (1990), pages 403-410:
Expect Threshold: 10
Word size: 28
Match Score: 1
Mismatch Score: −2
Gap costs: Linear
The above parameters are the default parameters in the nucleotide sequence comparison.
The GAP program is likewise suitable for use with the above parameters.
Preferred parameters for the determination of the “amino acid identity” are, when using the BLASTP program (Altschul, S. et al., Journal of Molecular Biology 215 (1990), pages 403-410:
Expect Threshold: 10
Word size: 3
Matrix: BLOSUM62
Gap costs: Existence: 11; Extension: 1
Compositional adjustments: Conditional compositional score matrix adjustment
The above parameters are the default parameters in the amino acid sequence comparison. The GAP program is likewise suitable for use with the above parameters.
An identity of 60% according to the above algorithm means 60% identity in connection with the present invention. The same applies for higher identities.
The feature “sequence that hybridizes or, taking into consideration the degeneracy of the genetic code,” would hybridize with the complementary strand of a sequence indicates a sequence that under preferably stringent conditions hybridizes, or would hybridize taking into consideration the degeneracy of the genetic code, with the complementary strand of a reference sequence. For example, the hybridizations can be carried out at 68° C. in 2×SSC or according to the protocol of the digoxigenin labeling kits of the company Boehringer (Mannheim). Preferred hybridization conditions are, for example, incubation at 65° C. overnight in 7% SDS, 1% BSA, 1 mM EDTA, 250 mM sodium phosphate buffer (pH 7.2) and subsequent washing at 65° C. with 2×SSC; 0.1% SDS.
The derivatives of the DNA isolated according to the invention, which according to alternatives F1), F2) or F3) can be obtained by substitution, addition, inversion and/or deletion of one or more bases of a sequence according to one of the groups A1) to E1), A2) to E2) and A3) to E3), include in particular those sequences which lead to conservative amino acid exchanges in the protein which they encode, such as, for example, to the exchange of glycine for alanine or of aspartic acid for glutamic acid. Such functionally neutral mutations are described as sense mutations and lead to no fundamental modification of the activity of the polypeptide. Furthermore, it is known that changes at the N- and/or C-terminus of a polypeptide do not significantly impair its function or can even stabilize this, so that also DNA sequences in which bases are attached at the 3′-end or at the 5′-end of the sequence containing the nucleic acids according to the invention are accordingly comprised by the present invention. The person skilled in the art finds information on this, inter alia, in Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), in O'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) and in known textbooks of genetics and molecular biology.
The nucleic acid according to the invention is preferably a vector, in particular an expression vector or a gene overexpression cassette. Suitable vectors are all vectors known to the person skilled in the art that are customarily employed for the inclusion of DNA into a host cell. These vectors can both replicate autonomously, as they have replication origins, such as, for example, those of the 2μ plasmid or ARS (autonomously replicating sequences) or integrate into the chromosomes (non-replicative plasmids). Vectors are also understood as meaning linear DNA fragments that have no replication origins at all, such as, for example, gene insertion or gene overexpression cassettes. Gene overexpression cassettes customarily consist of a marker, the genes to be overexpressed as well as regulatory regions relevant for the expression of the genes, such as, for example, promoters and terminators. Preferred vectors are selected from the group comprising plasmids and cassettes, such as, for example, E. coli yeast shuttle plasmids; expression vectors, gene insertion or gene overexpression cassettes are particularly preferred, in particular the vectors Seq ID No. 38, Seq ID No. 40, Seq ID No. 42, Seq ID No. 45 and Seq ID No. 47 described below.
According to a preferred embodiment of the vector according to the invention, the sequences of the groups [A1 to G1], [A2 to G2] and [A3 to G3] are under the control of at least one constitutive or regulatable promoter, which is suitable for the expression of the polypeptide encoded by these DNA sequences in the cell of a microorganism, preferably a bacteria, yeast or fungal cell, wherein Aspergillus nidulans, Aspergillus niger, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Burkholderia andropogonis, B. brasilensis, B. caledonica, B. caribensis, B. caryophylli, B. fungorum, B. gladioli, B. glathei, B. glumae, B. graminis, B. hospita, B. kururiensis, B. phenazinium, B. phymatum, B. phytofirmans, B. plantarii, B. sacchari, B. singaporensis, B. sordidicola, B. terricola, B. tropica, B. tuberum, B. ubonensis, B. unamae, B. xenovorans, B. anthina, B. pyrrocinia, B. thailandensis, Candida blankii, Candida rugosa, Corynebacterium glutamicum, Corynebacterium efficiens, Escherichia coli, Hansenula polymorpha, Kluveromyces lactis, Methylobacterium extorquens, Paracoccus versutus, Pseudomonas argentinensis, P. borbori, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea, P. aurantiaca, P. aureofaciens, P. chlororaphis, P. fragi, P. lundensis, P. taetrolens, P. antarctica, P. azotoformans, ‘P. blatchfordae’, P. brassicacearum, P. brenneri, P. cedrina, P. corrugata, P. fluorescens, P. gessardii, P. libanensis, P. mandelii, P. marginalis, P. mediterranea, P. meridiana, P. migulae, P. mucidolens, P. orientalis, P. panacis, P. proteolytica, P. rhodesiae, P. synxantha, P. thivervalensis, P. tolaasii, P. veronii, P. denitrificans, P. pertucinogena, P. cremoricolorata, P. fulva, P. monteilii, P. mosselii, P. parafulva, P. putida, P. balearica, P. stutzeri, P. amygdali, P. avellanae, P. caricapapayae, P. cichorii, P. coronafaciens, P. Ficuserectae, ‘P. helianthi’, P. meliae, P. savastanoi, P. syringae, P. tomato, P. viridiflava, P. abietaniphila, P. acidophila, P. agarici, P. alcaliphila, P. alkanolytica, P. amyloderamosa, P. asplenii, P. azotifigens, P. cannabina, P. coenobios, P. congelans, P. costantinii, P. cruciviae, P. delhiensis, P. excibis, P. extremorientalis, P. frederiksbergensis, P. fuscovaginae, P. gelidicola, P. grimontii, P. indica, P. jessenii, P. jinjuensis, P. kilonensis, P. knackmussii, P. koreensis, P. lini, P. lutea, P. moraviensis, P. otitidis, P. pachastrellae, P. palleroniana, P. papaveris, P. peli, P. perolens, P. poae, P. pohangensis, P. psychrophila, P. psychrotolerans, P. rathonis, P. reptilivora, P. resiniphila, P. rhizosphaerae, P. rubescens, P. salomonii, P. segitis, P. septica, P. simiae, P. suis, P. thermotolerans, P. aeruginosa, P. tremae, P. trivialis, P. turbinellae, P. tuticorinensis, P. umsongensis, P. vancouverensis, P. vranovensis, P. xanthomarina, Ralstonia eutropha, Rhodospirillum rubrum, Rhodobacter sphaeroides, Saccharomyces cerevisiae, Yarrowia lipolytica, Zymomonas mobilis, 
in particular Pseudomonas putida, Escherichia coli and Burkholderia thailandensis, are particularly preferred. Examples of constitutive promoters are lac, lacUV5, tac, trc (in each case in the absence of the LacI repressor in the cells according to the invention), Ltet-O1 (in the absence of the TetR repressor in the cells according to the invention), T5 and gap. Examples of inducible promoters are lac, lacUV5, tac, trc (in each case in the presence of the LacI repressor in the cells according to the invention), Ltet-O1 (in the presence of the TetR repressor in the cells according to the invention), T5 (in combination with a lac operator and the presence of the LacI repressor in the cells according to the invention), SP6 and T7 (in the presence of the gene encoding the cognate RNA polymerase, whose expression, for its part, is regulated). The vector according to the invention should in addition to a promoter preferably comprise a ribosome binding site as well as a terminator. It is particularly preferred here that the nucleic acid according to the invention is incorporated in an expression cassette of the vector comprising the promoter, the ribosome binding site and the terminator. In addition to the abovementioned structural elements, the vector can additionally comprise selection genes known to the person skilled in the art.
All percentages (%) indicated are percentages by mass if not indicated otherwise. In the examples presented below, the present invention is described by way of example, without the invention, whose range of application results from the entire description and the claims, being intended to be restricted to the embodiments mentioned in the examples.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: Fatty acid biosynthesis, β-oxidation of fatty acids and linkage of these metabolic pathways with the biosynthesis of rhamnolipids (enzymes E1, E2 and E3) and polyhydroxyalkanoates (enzymes E9 and E10). The carbon flows in fatty acid biosynthesis, β-oxidation of fatty acids, rhamnolipid biosynthesis and polyhydroxyalkanoate biosynthesis are shown. Consumption and formation of coenzymes, redox equivalents as well as nucleotides are not shown.
FIG. 2: Dirhamnosyl lipid formation (mg/l/OD 600 nm) of the recombinant strains P. putida KT2440 pBBR1MCS-2 and pBBR1MCS-2::ABC as well as GPp104 pBBR1MCS-2 and pBBR1MCS-2::ABC after 48 h, 72 h and 96 h culturing in CMP medium. The analysis of the rhamnolipid concentration took place by means of HPLC.
FIG. 3: Monorhamnosyl lipid formation (peakl area/OD 600 nm) of the recombinant strains P. putida KT2440 pBBR1MCS-2, pBBR1MCS-2::AB and pBBR1MCS-2::ABM as well as GPp104 pBBR1MCS-2, pBBR1MCS-2::AB and pBBR1MCS-2::ABM after 48 h, 72 h and 96 h culturing in CMP medium. The analysis of the rhamnolipid concentration took place by means of HPLC.



EXAMPLES
1. Construction of a Vector pBBR1MCS-2::AB for the Heterologous Expression of the Pseudomonas aeruginosa 1707 Genes rhIA and rhIB in Pseudomonas putida 
For the heterologous expression of the Pseudomonas aeruginosa DSM1707 genes rhIA and rhIB, the plasmid pBBR1MCS-2::AB (Seq ID No. 38) was constructed. For this, the synthetic operon rhIAB (Seq ID No. 37) was synthesized by the company GeneArt AG (Regensburg) and intercloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of the Pseudomonas aeruginosa DSM1707. Starting from the vector pMA::AB, the synthetic operon was cleaved from the vector by means of BglII and XbaI and subsequently ligated into the expression vector pBBR1MCS-2 (Seq ID No. 49) cleaved with BamHI and XbaI (described in Kovach et al., 1995: Four new derivatives of the broad host range cloning vector pBBR1MCS carrying different antibiotic-resistance cassettes. Gene, 166:175-176). The resulting plasmid pBBR1MCS-2::AB (Seq ID No. 38) is 7422 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) took place in the manner known to the person skilled in the art. The authenticity of the insert was checked by DNA sequence analysis.
The transformation of Pseudomonas putida KT2440 and GPp104 using the vectors pBBR1MCS-2 (Seq ID No. 49) and pBBR1MCS-2::AB took place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of 10 clones was isolated and analyzed. The strains obtained carrying the plasmids were named P. putida KT2440 pBBR1MCS-2, P. putida GPp104 pBBR1MCS-2, P. putida KT2440 pBBR1MCS-2::AB and P. putida GPp104 pBBR1MCS-2::AB.
2. Construction of a Vector pBBR1MCS-2::ABC for the Heterologous Expression of the Pseudomonas aeruginosa DSM1707 Genes rhIA, rhIB and rhIC in Pseudomonas putida 
For the heterologous expression of the Pseudomonas aeruginosa DSM1707 genes rhIA, rhIB and rhIC, the plasmid pBBR1MCS-2::ABC (Seq ID No. 40) was constructed. For this, the synthetic operon rhIABC (Seq ID No. 39) was synthesized by the company GeneArt AG (Regensburg) and intercloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of the Pseudomonas aeruginosa DSM1707. Starting from the vector pMA::ABC, the synthetic operon was cleaved from the vector by means of BglII and XbaI and subsequently ligated into the expression vector pBBR1MCS-2 (Seq ID No. 49) cleaved with BamHI and XbaI (Kovach et al., 1995: Four new derivatives of the broad host range cloning vector pBBR1MCS carrying different antibiotic-resistance cassettes. Gene, 166:175-176). The resulting plasmid pBBR1MCS-2::ABC (Seq ID No. 40) is 8409 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) took place in the manner known to the person skilled in the art. The authenticity of the insert was checked by DNA sequence analysis.
The transformation of Pseudomonas putida KT2440 and GPp104 using the vector pBBR1MCS-2::ABC took place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of every 10 clones was isolated and analyzed. The strains obtained carrying the plasmids were named P. putida KT2440 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABC.
3. Construction of a Vector pBBR1MCS-2::ABM for the Heterologous Expression of the Pseudomonas aeruginosa DSM1707 Genes rhIA, rhIB and pa1131 in Pseudomonas putida 
For the heterologous expression of the Pseudomonas aeruginosa DSM1707 genes rhIA, rhIB and pa1131 the plasmid pBBR1MCS-2::ABM (Seq ID No. 42) was constructed. For this, the synthetic operon rhIAB-pa1131 (Seq ID No. 41) was synthesized by the company GeneArt AG (Regensburg) and intercloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of the Pseudomonas aeruginosa DSM1707. Starting from the vector pMA::ABM the synthetic operon was cleaved from the vector by means of BglII and XbaI and subsequently ligated into the expression vector pBBR1MCS-2 (Seq ID No. 49) cleaved with BamHI and XbaI (Kovach et al., 1995: Four new derivatives of the broad host range cloning vector pBBR1MCS carrying different antibiotic-resistance cassettes. Gene, 166:175-176). The resulting plasmid pBBR1MCS-2::ABM (Seq ID No. 42) is 8702 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) took place in the manner known to the person skilled in the art. The authenticity of the insert was checked by DNA sequence analysis.
The transformation of Pseudomonas putida KT2440 and GPp104 using the vector pBBR1MCS-2::ABM took place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of every 10 clones was isolated and analyzed. The strains obtained carrying the plasmids were named P. putida KT2440 pBBR1MCS-2::ABM and P. putida GPp104 pBBR1MCS-2::ABM.
4. Quantification of Rhamnolipid Production by Recombinant P. putida Strains
The recombinant strains P. putida KT2440 pBBR1MCS-2; P. putida KT2440 pBBR1MCS-2::AB; P. putida KT2440 pBBR1MCS-2::ABC; P. putida KT2440 pBBR1MCS-2::ABM; P. putida GPp104 pBBR1MCS-2; P. putida GPp104 pBBR1MCS-2::AB, P. putida GPp104 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABM were cultured on LB agar kanamycin (50 μg/ml) plates.
For the production of the rhamnolipids, the medium designated below as CMP medium was used. This consists of 2% (w/v) glucose, 0.007% (w/v) KH2PO4, 0.11% Na2HPO4×2 H2O, 0.2% (w/v) NaNO3, 0.04% (w/v) MgSO4×H2O, 0.01% (w/v) CaCl2×2 H2O and 0.2% (v/v) of a trace element solution. This consists of 0.2% (w/v) FeSO4×7 H2O, 0.15% (w/v) MnSO4×H2O and 0.06% (w/v) (NH4)MO7O24×4 H2O. The pH of the medium was adjusted to 6.7 with NaOH and the medium was subsequently sterilized by means of an autoclave (121° C., 20 min). An adjustment of the pH during the culturing was not necessary.
For the investigation of the rhamnolipid production in the shaker flask a preculture was first prepared. For this, an inoculation loop of a strain freshly streaked on an LB agar plate was used and 10 ml of LB medium was inoculated into a 100 ml Erlenmeyer flask. All recombinant P. putida strains were in the LB medium, to which 50 μg/ml of kanamycin was added. The culturing of the strains took place overnight at 30° C. and 200 rpm.
The precultures were used to inoculate 50 ml of CMP medium in the 250 ml Erlenmeyer flask (start OD600 0.1). The cultures were cultured at 200 rpm and 30° C. for at most 120 h. At intervals of 24 h, a sample of 1 ml of broth was removed from the culture flask. The sample preparation for the following chromatographic analyses took place as follows:
Using a displacement pipette (Combitip), 1 ml of acetone was introduced into a 2 ml reaction vessel and the reaction vessel was immediately closed for the minimization of evaporation. The addition of 1 ml of broth followed. After vortexing of the broth/acetone mixture, this was centrifuged off for 3 min at 13,000 rpm, and 800 μl of the supernatant was transferred to an HPLC vessel.
For the detection and for the quantification of rhamnolipids, an evaporative light scattering detector (Sedex LT-ELSD Model 85LT) was used. The actual measurement was carried out by means of Agilent Technologies 1200 Series (Santa Clara, Calif.) and the Zorbax SB-C8 rapid resolution column (4.6×150 mm, 3.5 μm, Agilent). The injection volume was 5 μl and the runtime of the method was 20 min. As mobile phase, aqueous 0.1% TFA (trifluoroacetic acid, solution A) and methanol (solution B) was used. The column temperature was 40° C. The ELSD (detector temperature 60° C.) and the DAD (diode array, 210 nm) served as detectors. The gradient used in the method was:












t
Solution
Flow


[min]
B vol. %
[ml/min]

















0.00
70%
1.00


15.00
100% 
1.00


15.01
70%
1.00


20.00
70%
1.00









While P. putida KT2440 pBBR1MCS-2 and GPp104 pBBR1MCS-2 produced no rhamnolipids, in the recombinant strains P. putida KT2440 pBBR1MCS-2::AB, P. putida KT2440 pBBR1MCS-2::ABC, P. putida KT2440 pBBR1MCS-2::ABM, P. putida GPp104 pBBR1MCS-2::AB, P. putida GPp104 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABM the formation of different rhamnolipid species was detectable (FIGS. 2 and 3).
By the incorporation of pBBR1MCS-2::AB and pBBR1MCS-2::ABM into P. putida, it was possible to generate monorhamnosyl lipids (FIG. 3). Since no reference material for monorhamnosyl lipids was present, the identification of the products took place by analysis of the corresponding mass traces and the MS2 spectra in LC-MS.
If rhIC (pBBR1MCS-2::ABC) was additionally incorporated into the strains, mono- and dirhamnosyl lipids were produced (FIG. 2).
The direct comparison of the rhamnolipid formation by P. putida pBBR1MCS-2::AB and P. putida pBBR1MCS-2::ABM shows that the coexpression of P. aeruginosa p3111 with P. aeruginosa rhIAB leads to an improvement in the rhamnolipid biosynthesis (FIG. 3). While the strains P. putida KT2440 pBBR1MCS-2::AB and P. putida GPp104 pBBR1MCS-2::AB had produced about 39 (P. putida KT2440 pBBR1MCS-2::AB) and 23 peak areas rhamnolipids/OD 600 nm (P. putida GPp104 pBBR1MCS-2::AB) after 120 h, the strains P. putida KT2440 pBBR1MCS-2::ABM and P. putida GPp104 pBBR1MCS-2::ABM formed about 50 (P. putida KT2440 pBBR1MCS-2::ABM) and 62 peak areas rhamnolipids/OD 600 nm (P. putida GPp104 pBBR1MCS-2::ABM) after 120 h.
If the monorhamnosyl lipid synthesis of the strains P. putida KT2440 pBBR1MCS-2::ABM and P. putida GPp104 pBBR1MCS-2::ABM was compared, it was possible in the PHA-negative mutant P. putida GPp104 pBBR1MCS-2::ABM to detect 62 peak areas/OD 600 nm (120 h culturing) and with P. putida KT2440 pBBR1MCS-2::ABM 50 area/OD 600 nm monorhamnosyl lipids (FIG. 3).
A comparative analysis of the dirhamnosyl lipid formation (mg/l/OD 600 nm) in the strains P. putida KT2440 and GPp104 likewise showed a greater formation of the dirhamnosyl lipids in the PHA-negative strain background of the P. putida GPp104. P. putida GPp104 pBBR1MCS-2::ABC formed on average 113 mg/l/OD 600 nm of dirhamnosyl lipids (96 h), whereas with P. putida KT2440 pBBR1MCS-2::ABC only 55 mg/l/OD 600 nm of dirhamnosyl lipids could be detected after 96 h (FIG. 2).
Thus it was possible to show that the use of a strain background attenuated with respect to PHA synthesis leads to an improvement in the rhamnolipid biosynthesis.
5. Construction of a Vector pBBR1MCS-2::ABMC for the Heterologous Expression of the Pseudomonas aeruginosa DSM1707 Genes rhIA, rhIB, pa1131 and rhIC in Pseudomonas putida 
For the heterologous expression of the Pseudomonas aeruginosa DSM1707 genes rhIA, rhIB, pa1131 and rhIC, the plasmid pBBR1MCS-2::ABMC (Seq ID No. 51) was constructed. For this, the synthetic operon rhIAB-pa1131-rhIC (Seq ID No. 50) was synthesized by the company GeneArt AG (Regensburg) and intercloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of the Pseudomonas aeruginosa DSM1707. Starting from the vector pMA::ABMC the synthetic operon was cleaved by means of BglII and XbaI from the vector and subsequently ligated into the expression vector pBBR1MCS-2 (Seq ID No. 49) cleaved with BamHI and XbaI (Kovach et al., 1995: Four new derivatives of the broad-host-range cloning vector pBBR1MCS carrying different antibiotic-resistance cassettes. Gene, 166:175-176). The resulting plasmid pBBR1MCS-2::ABMC (Seq ID No. 51) is 9663 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) took place in a manner known to the person skilled in the art. The authenticity of the insert was checked by DNA sequence analysis.
The transformation of Pseudomonas putida KT2440 and GPp104 using the vector pBBR1MCS-2::ABMC took place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of every 10 clones was isolated and analyzed. The strains obtained carrying the plasmids were named P. putida KT2440 pBBR1MCS-2::ABMC and P. putida GPp104 pBBR1MCS-2::ABMC.
6. Qualitative Comparison of the Rhamnolipid Production by Recombinant P. putida Strains and P. aeruginosa Strains
The recombinant strains P. putida GPp104 pBBR1MCS-2 and P. putida GPp104 pBBR1MCS-2::ABMC and P. aeruginosa DSM 19880 were cultured on LB agar kanamycin (50 μg/ml; P. putida) and LB agar plates (P. aeruginosa).
For the production of the rhamnolipids the medium below designated as CMP medium was used. This consists of 2% (w/v) glucose, 0.007% (w/v) KH2PO4, 0.11% Na2HPO4×2 H2O, 0.2% (w/v) NaNO3, 0.04% (w/v) MgSO4×H2O, 0.01% (w/v) CaCl2×2 H2O and 0.2% (v/v) of a trace element solution. This consists of 0.2% (w/v) FeSO4×7 H2O, 0.15% (w/v) MnSO4×H2O and 0.06% (w/v) (NH4)MO7O24×4 H2O. The pH of the medium was adjusted to 6.7 using NaOH and the medium was subsequently sterilized by means of an autoclave (121° C., 20 min). An adjustment of the pH during the culturing was not necessary.
For the investigation of the rhamnolipid production in the shaker flask, a preculture was first prepared. For this, an inoculation loop of a strain freshly streaked on LB agar plate was used and 10 ml of LB medium was inoculated into a 100 ml Erlenmeyer flask. The recombinant P. putida strains were cultured in the LB medium, to which 50 μg/ml of kanamycin was added. P. aeruginosa was cultured in the LB medium. The culturing of the strains took place at 30° C. and 200 rpm overnight.
The precultures were used to inoculate 50 ml of CMP medium in the 250 ml Erlenmeyer flask (start OD600 0.1). The cultures were cultured at 200 rpm and 30° C. for at most 120 h. At intervals of 24 h, a sample of 1 ml of broth was removed from the culture flask. The sample preparation for the following chromatographic analyses took place as follows:
Using a displacement pipette (Combitip), 1 ml of acetone was introduced into a 2 ml reaction vessel and the reaction vessel was immediately closed for the minimization of evaporation. The addition of 1 ml of broth followed. After vortexing of the broth/acetone mixture, this was centrifuged off for 3 min at 13,000 rpm, and 800 μl of the supernatant were transferred to an HPLC vessel.
For the identification of the products formed, 5 μl were injected into an Accela UPLC unit (Thermo Scientific, Dreieich). The substances to be investigated were analyzed using a semi UPLC column “Pursuit XRs ULTRA (C8, 2.8 μm, 2.1×100 mm) (Varian, Darmstadt). The separation took place within 25 min by means of a gradient consisting of the mobile phase A1 (H2O, 0.1% (v/v) TFA) and the mobile phase B1 (methanol, 0.1% (v/v) TFA) using a flow rate of 0.3 ml/min at 40° C. The time course of the gradient was the following:












Time
Mobile phase
Mobile phase


[min]
A1 [%]
B1 [%]

















0
30
70


15
0
100


25
0
100


25.01
30
70


32
30
70









Detection took place by means of DAD detector in the wavelength range from 200-600 nm and mass-selectively using a high-resolution FT-ICR LTQ-FT mass spectrometer (Thermo Scientific, Dreieich) in the scanning range m/e 100-1000. Ionization took place by means of ESI (electrospray ionization). Exact masses and empirical chemical formulae were determined with the aid of the FT-ICR mass analyzer, using a resolution of R=100000 and a mass accuracy of ≦2 ppm. The identification of the products takes place by analysis of the corresponding mass traces and the MS2 spectra. To be able to compare the strains, the peak areas of the corresponding substances were contrasted.
As shown in FIG. 4, the strain P. putida GPp104 pBBR1MCS-2 formed no rhamnolipids at all. P. putida GPp104 pBBR1MCS-2::ABMC and P. aeruginosa DSM 19880 formed rhamnolipids, wherein the ratio between di- and monorhamnosyl lipids formed with P. putida GPp104 pBBR1MCS-2::ABMC was, for example, 4:1, with P. aeruginosa DSM 19880, for example, 2:1. Moreover, the strain P. putida GPp104 pBBR1MCS-2::ABMC in contrast to P. aeruginosa DSM 19880 formed no or only very few rhamnolipids having a radical determined by means of R1 and R2 derived from 3-hydroxyoctanoyl-3-hydroxydecanoic acid or 3-hydroxydecanoyl-3-hydroxyoctanoic acid.
7. Construction of a Vector pBBR1MCS-2::rfbBDAC and pBBR1MCS-2::ABC_rfbBDAC for Heterologous Expression in Pseudomonas putida 
At the company Trenzyme GmbH (Konstanz), the rhamnose biosynthesis operon rfbBDAC was amplified starting from chromosomal DNA of Pseudomonas putida KT2440. For this, the following primers were used:









RL1:



(Seq ID No. 48)



5′-TATATATAGAATTCGCGTCATCTGTCTACGACAACAC-3′



 



RL2:



(Seq ID No. 43)



5′-TATATATAGAATTCGGCTGCGCTACCGCAGCCCTTC-3′






The PCR product obtained was intercloned in Trenzyme's alligator cloning system and transformed in E. coli DH5α (New England Biolabs; Frankfurt). Vectors of different candidates were analyzed and sequenced. After successful and error-free DNA sequencing, the vector was cleaved by means of EcoRI and the target fragment rfbBDAC was isolated. For a further inter-cloning, the vector pBBR1MCS-2 (Kovach et al., 1995: Four new derivatives of the broad-host-range cloning vector pBBR1MCS carrying different antibiotic-resistance cassettes. Gene, 166:175-176) was cleaved in the same manner. The cleaved target fragment (rfbBDAC) and the cleaved vector (pBBR1MCS-2) were merged by conventional ligation. The resulting vector pBBR1MCS-2::rfbBDAC (Seq ID No. 45) was likewise transformed in E. coli DH5α (New England Biolabs; Frankfurt). Some candidates of the transformants were investigated with respect to the successful uptake of the plasmid.
The vector pBBR1MCS-2::rfbBDAC served as a matrix for a PCR. The following oligonucleotides were used:









RL_XbaI-fw:



(Seq ID No. 44)



5′-TATATATATCTAGAATTAATGCAGCTGGCACGAC-3′



 



RL_Xba_rev: 



(Seq ID No. 46)



5′-GGCCGCTCTAGAACTAGTGGA-3′






The PCR was carried out using the PhusionTM High-Fidelity Master Mix of New England Biolabs (Frankfurt) polymerase. It was carried out in the manner known to the person skilled in the art. The target sequence (lac promoter and rfbBDAC) was intercloned in the Trenzyme alligator cloning system. E. coli DH5α (New England Biolabs; Frankfurt) transformants were selected and the plasmid DNA of different candidates was isolated and sequenced. After the sequence had been checked and investigated for correctness, the vector was cleaved using XbaI. The target fragment was ligated into the pBBR1MCS-2::ABC likewise cleaved using XbaI (see above) by means of conventional ligation methods. The target vector pBBR1MCS-2::ABC_rfbBDAC obtained (Seq ID No. 47) has a size of 12249 base pairs. The insert of the vector was sequenced. The carrying-out of the PCR, the checking of the successful amplification of the PCR by means of agarose gel electrophoresis, ethidium bromide staining of the DNA, determination of the PCR fragment size, purification of the PCR products and DNA concentration determination took place in the manner known to the person skilled in the art.
The transformation of Pseudomonas putida KT2440 and GPp104 using the vector pBBR1MCS-2::ABC_rfbBDAC took place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of every 10 clones was isolated and analyzed. The strains obtained carrying the plasmids are named P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC and P. putida GPp104 pBBR1MCS-2::ABC_rfbBDAC.
8. Quantification of the Rhamnolipid Production by Recombinant P. putida Strains with and without Overexpression of the rfbBDAC Operon
The recombinant strains P. putida KT2440 pBBR1MCS-2; P. putida KT2440 pBBR1MCS-2::ABC, P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC, P. putida GPp104 pBBR1MCS-2, P. putida GPp104 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABC_rfbBDAC are cultured on LB agar kanamycin (50 μg/ml) plates.
For the production of the rhamnolipids, the medium designated below as CMP medium is used. This consists of 2% (w/v) glucose, 0.007% (w/v) KH2PO4, 0.11% Na2HPO4×2 H2O, 0.2% (w/v) NaNO3, 0.04% (w/v) MgSO4×H2O, 0.01% (w/v) CaCl2×2 H2O and 0.2% (v/v) of a trace element solution. This consists of 0.2% (w/v) FeSO4×7 H2O, 0.15% (w/v) MnSO4×H2O and 0.06% (w/v) (NH4)MO7O24×4 H2O. The pH of the medium is adjusted to 6.7 using NaOH and the medium is subsequently sterilized by means of an autoclave (121° C., 20 min). An adjustment of the pH during the culturing is not necessary.
For the investigation of the rhamnolipid production in the shaker flask, a preculture is first prepared. For this, an inoculation loop of a strain freshly streaked on LB agar plate is used and 10 ml of LB medium are inoculated into a 100 ml Erlenmeyer flask. All recombinant P. putida strains are cultured in the LB medium, to which 50 μg/ml of kanamycin is added. The culturing of the P. putida strains was carried out at 30° C. and 200 rpm overnight.
The precultures are used to inoculate 50 ml of CMP medium in the 250 ml Erlenmeyer flask (start OD600 0.1). The cultures are cultured at 200 rpm and 30° C. for at most 120 h. At intervals of 24 h, a sample of 1 ml broth is removed from the culture flask. The sample preparation for the following chromatographic analyses takes place as follows:
Using a displacement pipette (Combitip), 1 ml of acetone is introduced into a 2 ml reaction vessel and the reaction vessel is closed immediately for the minimization of evaporation. The addition of 1 ml of broth follows. After vortexing of the broth/acetone mixture, this is centrifuged off for 3 min at 13,000 rpm, and 800 μl of the supernatant are transferred to an HPLC vessel. For the detection and for the quantification of rhamnolipids, an evaporative light scattering detector (Sedex LT-ELSD Model 85LT) is used. The actual measurement is carried out by means of Agilent Technologies 1200 Series (Santa Clara, Calif.) and the Zorbax SB-C8 rapid resolution column (4.6×150 mm, 3.5 μm, Agilent). The injection volume is 5 μl and the runtime of the method is 20 min. As a mobile phase, aqueous 0.1% TFA (trifluoroacetic acid, solution A) and methanol (solution B) is used. The column temperature is 40° C. The ELSD (detector temperature 60° C.) and the DAD (diode array, 210 nm) serve as detectors. The gradient used in the method is:












t
Solution
Flow


[min]
B vol. %
[ml/min]

















0.00
70%
1.00


15.00
100% 
1.00


15.01
70%
1.00


20.00
70%
1.00









While P. putida KT2440 pBBR1MCS-2 and GPp104 pBBR1MCS-2 produce no rhamnolipids, in the recombinant strains P. putida KT2440 pBBR1MCS-2::ABC, P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC; P. putida GPp104 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABC_rfbBDAC the formation of rhamnolipids is detectable.
P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC shows in comparison to P. putida KT2440 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABC_rfbBDAC shows in comparison to P. putida GPp104 pBBR1MCS-2::ABC an increased formation of the di- and monorhamnosyl lipids. This clearly shows the positive influence of the amplification of the expression of rfbBDAC on the formation of mono- and dirhamnosyl lipids.
If the mono- and dirhamnosyl lipid biosynthesis of the strains P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC and P. putida GPp104 pBBR1MCS-2::ABC_rfbBDAC is compared, an increased mono- and dirhamnosyl lipid synthesis is detected in the PHA-negative mutant P. putida GPp104 pBBR1MCS-2::ABC_rfbBDAC.
As already described above, the rhamnolipid biosynthesis is increased with the use of a strain background inactivated in the PHA synthesis.
9. Generation of Recombinant E. coli W3110 pBBR1MCS-2::ABC and E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC
The transformation of E. coli W3110 took place as previously described (Miller J H. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Plainview, N.Y.: Cold Spring Harbor Lab. Press; 1992) by means of electroporation. The plasmid DNA of every 10 clones was isolated and analyzed. The strains obtained carrying the plasmids were named E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC and E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC.
10. Quantification of the Rhamnolipid Production by Recombinant E. coli Strains with and without Overexpression of the rfbBDAC Operon
The recombinant strains E. coli W3110 pBBR1MCS-2; E. coli W3110 pBBR1MCS-2::ABC and E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC are cultured on LB agar kanamycin (50 μg/ml) plates.
For the production of the rhamnolipids, the medium designated in the following as CMP medium is used. This consists of 2% (w/v) glucose, 0.007% (w/v) KH2PO4, 0.11% Na2HPO4×2 H2O, 0.2% (w/v) NaNO3, 0.04% (w/v) MgSO4×H2O, 0.01% (w/v) CaCl2×2 H2O and 0.2% (v/v) of a trace element solution. This consists of 0.2% (w/v) FeSO4×7 H2O, 0.15% (w/v) MnSO4×H2O and 0.06% (w/v) (NH4)MO7O24×4 H2O. The pH of the medium is adjusted to 6.7 using NaOH and the medium is subsequently sterilized by means of an autoclave (121° C., 20 min). An adjustment of the pH during the culturing is not necessary.
For the investigation of the rhamnolipid production in the shaker flask, a preculture is first prepared. For this, an inoculation loop of a strain freshly streaked on LB agar plate is used and 10 ml of LB medium is inoculated into a 100 ml Erlenmeyer flask. All recombinant E. coli strains are cultured in the LB medium, to which 50 μg/ml of kanamycin is added. The culturing of the E. coli strains took place at 37° C. and 200 rpm overnight.
The precultures are used to inoculate 50 ml of CMP medium in the 250 ml Erlenmeyer flask (start OD600 0.1). The cultures are cultured at 200 rpm and 30° C. for at most 120 h. At intervals of 24 h a sample of 1 ml of broth is removed from the culture flask. The sample preparation for the following chromatographic analyses takes place as follows:
Using a displacement pipette (Combitip), 1 ml of acetone is introduced into a 2 ml reaction vessel and the reaction vessel is closed immediately for the minimization of evaporation. The addition of 1 ml of broth follows. After vortexing of the broth/acetone mixture, this is centrifuged off for 3 min at 13,000 rpm, and 800 μl of the supernatant are transferred to an HPLC vessel. For detection and for the quantification of rhamnolipids, an evaporative light scattering detector (Sedex LT-ELSD Model 85LT) is used. The actual measurement is carried out by means of Agilent Technologies 1200 Series (Santa Clara, Calif.) and the Zorbax SB-C8 rapid resolution column (4.6×150 mm, 3.5 μm, Agilent). The injection volume is 5 μl and the runtime of the method is 20 min. Aqueous 0.1% TFA (trifluoroacetic acid, solution A) and methanol (solution B) is used as the mobile phase. The column temperature is 40° C. The ELSD (detector temperature 60° C.) and the DAD (diode array, 210 nm) serve as detectors. The gradient used in the method is:












t
Solution
Flow


[min]
B vol. %
[ml/min]

















0.00
70%
1.00


15.00
100% 
1.00


15.01
70%
1.00


20.00
70%
1.00









While E. coli W3110 pBBR1MCS-2 produces no rhamnolipids, the formation of mono- and dirhamnosyl lipids is detectable in the recombinant strains E. coli W3110 pBBR1MCS-2::ABC and E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC, wherein E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC forms significantly more mono- and dirhamnosyl lipids than E. coli W3110 pBBR1MCS-2::ABC. This shows that the heterologous expression of rhIABC of Pseudomonas aeruginosa DSM1707 leads to the formation of mono- and dirhamnosyl lipids in E. coli. This furthermore shows the positive influence of the reinforcement of the expression of rfbBDAC on the formation of mono- and dirhamnosyl lipids.
11. Construction of a vector pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 for the Heterologous Expression of the Pseudomonas aeruginosa DSM1707 Genes rhIA, rhIB and rhIC and the Burkholderia thailandensis E264 Genes BTH_II1077, BT_II1080 and BT_II1081 in Pseudomonas putida 
For the heterologous expression of the Pseudomonas aeruginosa DSM1707 genes rhIA, rhIB and rhIC and the B. thailandensis E264 genes BTH_II1077, BT_II1080 and BT_II1081 in Pseudomonas putida, the plasmid pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 (Seq ID No. 69) is constructed. For this, the synthetic operon BTH_II1077, BT_II1080 and BT_II1081 (Seq ID No. 70) is synthesized by the company DNA 2.0 (Menlo Park, Calif., USA) and intercloned in the commercial vector pJ294 (DNA 2.0; Menlo Park, Calif., USA). The basis for the synthesis is the genomic sequence of the strain B. thailandensis E264. Starting from the vector pJ294-BTH_II1077-II1080-II1081, the synthetic operon is cleaved from this vector by means of XbaI and subsequently ligated into the vector pBBR1MCS-2::ABC (Seq ID No. 40) likewise cleaved using XbaI. The target vector pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 (Seq ID No. 69) obtained has a size of 13768 base pairs. The insert of the vector is sequenced. The carrying-out of the PCR, the checking of the successful amplification of the PCR by means of agarose gel electrophoresis, ethidium bromide staining of the DNA, determination of the PCR fragment size, purification of the PCR products and DNA concentration determination takes place in the manner known to the person skilled in the art.
The transformation of Pseudomonas putida KT2440 and GPp104 using the vector pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 (Seq ID No. 69) takes place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of every 10 clones is isolated and analyzed. The strains obtained carrying the plasmids are named P. putida KT2440 pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 and P. putida GPp104 pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081.
12. Quantification of the Rhamnolipid Production by Recombinant P. putida Strains with and without Overexpression of the B. thailandensis E264 Genes BTH_II1077, BT_II1080 and BT_II1081
The recombinant strains P. putida strains P. putida KT2440 pBBR1MCS-2::AB, P. putida KT2440 pBBR1MCS-2::AB-BTH_II1077-II1080-II1081, P. putida GPp104 pBBR1MCS-2::AB, P. putida GPp104 pBBR1MCS-2::AB-BTH_II1077-II1080-II1081, P. putida KT2440 pBBR1MCS-2::ABC, P. putida KT2440 pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 P. putida GPp104 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 generated in the Examples 1, 2 and 11 are cultured on LB agar kanamycin (50 μg/ml) plates.
For the production of the rhamnolipids, the medium designated in the following as M9 medium is used. This medium consists of 2% (w/v) glucose, 0.3% (w/v) KH2PO4, 0.679% Na2HPO4, 0.05% (w/v) NaCl, 0.2% (w/v) NH4Cl, 0.049% (w/v) MgSO4×7 H2O and 0.1% (v/v) of a trace element solution. This consists of 1.78% (w/v) FeSO4×7 H2O, 0.191% (w/v) MnCl2×7 H2O, 3.65% (w/v) HCl, 0.187% (w/v) ZnSO4×7 H2O, 0.084% (v/v) Na EDTA×2H2O, 0.03% (v/v) H3BO3, 0.025% (w/v) Na2MoO4×2 H2O and 0.47% (w/v) CaCl2×2 H2O. The pH of the medium is adjusted to 7.4 using NH4OH and the medium is subsequently sterilized by means of an autoclave (121° C., 20 min). An adjustment of the pH during the culturing is not necessary. For the investigation of the rhamnolipid production in the shaker flask, a preculture is first prepared. For this, an inoculation loop of a strain freshly streaked on LB agar plate is used and 10 ml of LB medium are inoculated into a 100 ml Erlenmeyer flask. All recombinant P. putida strains are cultured in LB medium, to which 50 μg/ml of kanamycin was added. The culturing of the P. putida strains takes place at 37° C. and 200 rpm overnight.
The precultures are used to inoculate 50 ml of M9 medium (+50 μg/ml of kanamycin) in the 250 ml Erlenmeyer flask (start OD600 0,1). The cultures are cultured at 200 rpm and 30° C. At intervals of 24 h, a sample of 1 ml of broth is removed from the culture flask. The sample preparation for the following chromatographic analyses and the chromatographic analyses themselves are carried out as described in Example 4.
It is shown that the recombinant strains P. putida KT2440 pBBR1MCS-2::AB-BTH_II1077-II1080-II1081 and P. putida GPp104 pBBR1MCS-2::AB-BTH_II1077-II1080-II1081 form significantly more monorhamnosyl lipids than the strains P. putida KT2440 pBBR1MCS-2::AB and P. putida GPp104 pBBR1MCS-2::AB. This demonstrates that the amplification of BTH_II1077-II1080-II1081 from B. thailandensis E264 increases the formation of monorhamnosyl lipids in P. putida strains containing the Pseudomonas aeruginosa DSM1707 genes rhIAB.
It is furthermore shown that the recombinant strains P. putida KT2440 pBBR1MCS-2::ABC BTH_II1077-II1080-II1081 and P. putida GPp104 pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 form significantly more mono- and dirhamnosyl lipids than the strains P. putida KT2440 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABC. This proves that the amplification of BTH_II1077-II1080-II1081 from B. thailandensis E264 increases the formation of mono- and dirhamnosyl lipids in P. putida strains containing the Pseudomonas aeruginosa DSM1707 genes rhIABC.
It is finally shown that the reduction of the polyhydroxybutyrate formation in the strain background P. putida GPp104 compared to the strain P. putida KT2440 leads to an increased rhamnolipid formation, as the strains P. putida KT2440 pBBR1MCS-2::AB, P. putida KT2440 pBBR1MCS-2::ABC, P. putida KT2440 pBBR1MCS-2::AB-BTH_II1077-II1080-II1081 and P. putida KT2440 pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 are able to form significantly fewer mono-( ) and mono- and dirhamnosyl lipids ( ) than the corresponding control strains P. putida GPp104 pBBR1MCS-2::AB, P. putida GPp104 pBBR1MCS-2::ABC, P. putida GPp104 pBBR1MCS-2::AB-BTH_II1077-II1080-II1081 and P. putida GPp104 pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081.
13. Construction of a Vector pBBR1MCS-2::ABCM for the Heterologous Expression of the Pseudomonas aeruginosa DSM1707 Genes rhIA, rhIB, pa1131 and rhIC in Pseudomonas putida 
For the heterologous expression of the Pseudomonas aeruginosa DSM1707 genes rhIA, rhIB, pa1131 and rhIC, the plasmid pBBR1MCS-2::ABCM (Seq ID No. 58) was constructed. For this, the gene pa1131 (Seq ID No. 59) was amplified starting from genomic DNA of the strain Pseudomonas aeruginosa PAO1 (DSM 1707) containing the oligonucleotides









MFS2.0_xbaI_fw:



(Seq ID No. 60)



5′-AGGAAATCTAGATGAGAGGCCGGCAAGGATAC-3′



 



MFS2.0_XbaI_rev:



(Seq ID No. 61)



5′-CCAGGTTCTAGACGCCAGGATTGAACAGTACC-3′.






The amplification of the PCR product (1483 base pairs) was carried out using the PhusionTM High-Fidelity Master Mix from New England Biolabs (Frankfurt) polymerase. The PCR product was cleaved using XbaI and ligated in the vector pBBR1MCS-2::ABC (Seq ID No. 40) likewise cleaved using XbaI by means of Fast Link Ligation Kit (Epicentre Technologies; Madison, Wis., USA). The target vector pBBR1MCS-2::ABCM (Seq ID No. 58) obtained has a size of 9892 base pairs. The insert of the vector was sequenced. The chromosomal DNA was isolated by means of DNeasy Blood and Tissue Kit (Qiagen; Hilden) according to manufacturer's instructions. The carrying-out of the PCR, the checking of the successful amplification of the PCR by means of agarose gel electrophoresis, ethidium bromide staining of the DNA, determination of the PCR fragment size, purification of the PCR products and DNA concentration determination took place in a manner known to the person skilled in the art. The transformation of Pseudomonas putida KT2440 and GPp104 using the vector pBBR1MCS-2::ABCM took place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of every 10 clones was isolated and analyzed. The strains obtained carrying the plasmids were named P. putida KT2440 pBBR1MCS-2::ABCM and P. putida GPp104 pBBR1MCS-2::ABCM.
14. Quantification of the Rhamnolipid Production by Recombinant P. putida Strains with and without Overexpression of the Pseudomonas aeruginosa DSM1707 pa1131 Gene
The recombinant strains P. putida strains P. putida KT2440 pBBR1MCS-2::ABC, P. putida KT2440 pBBR1MCS-2::ABCM, P. putida KT2440 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABCM generated in the Examples 2 and 13 were cultured on LB agar kanamycin (50 μg/ml) plates. The subsequent culturing for the production of the rhamnolipids took place as described in Example 12.
The sample preparation for the following chromatographic analyses and the chromatographic analyses themselves took place as described in Example 4.
The results are shown in the following table.
Formation of di- and monorhamnosyl lipids by P. putida strains with and without overexpression of the P. aeruginosa gene pa1131 after 48 h incubation













Dirhamnosyl
Monorhamnosyl


P. putida strains
lipids [mg/l]
lipids [peak area]

















KT2440 pBBR1MCS-2::ABC
310
19


KT2440 pBBR1MCS-2::ABCM
1053
314


GPp104 pBBR1MCS-2::ABC
689
127


GPp104 pBBR1MCS-2::ABCM
960
1090









The results show that the overexpression of the P. aeruginosa gene pa1131 in both strain backgrounds (KT2440: wild-type and GPp104 having inactivated polyhydroxybutyrate formation) leads to an increased formation of di- and monorhamnosyl lipids. The results furthermore show that the reduction of the polyhydroxybutyrate formation in GPp104 generally leads to an increased formation of rhamnosyl lipids.
15. Construction of a vector pEC-XT99A::AB for the heterologous expression of the genes rhIA and rhIB from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum 
For the heterologous expression of the genes rhIA and rhIB from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum, the plasmid pEC-XT99A::AB (Seq ID No. 52) is constructed. For this, the synthetic operon rhIAB (Seq ID No. 37) was synthesized by the company GeneArt AG (Regensburg) and intercloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of the Pseudomonas aeruginosa DSM1707. Starting from the vector pMA::AB, the synthetic operon is cleaved from the vector by means of BglII and XbaI and subsequently ligated into the expression vector pEC-XT99A (U.S. Pat. No. 7,118,904) cleaved using BamHI and XbaI. The resulting plasmid pEC-XT99A::AB (Seq ID No. 52) is 9793 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) takes place in the manner known to the person skilled in the art. The authenticity of the insert is checked by DNA sequence analysis.
The transformation of C. glutamicum ATCC13032 using the vector pEC-XT99A::AB takes place as previously described (Liebl et al., FEMS Microbiol. Lett. 53:299-303 (1989)). The selection of the transformants takes place on LBHIS agar plates (18.5 g/l of brain heart infusion broth, 0.5 M sorbitol, 5 g/l of Bacto tryptone, 2.5 g/l of Bacto yeast extract, 5 g/l of NaCl and 18 g/l of Bacto agar, supplemented with 5 mg/l of tetracycline). The plates were incubated at 33° C. for two days. The strain obtained carrying the plasmid is named C. glutamicum pEC-XT99A::AB.
16. Construction of a Vector pEC-XT99A::ABC for the Heterologous Expression of the Genes rhIA, rhIB and rhIC from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum 
For the heterologous expression of the genes rhIA, rhIB and rhIC from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum, the plasmid pEC-XT99A::ABC (Seq ID No. 53) is constructed. For this, the synthetic operon rhIABC (Seq ID No. 39) was synthesized by the company GeneArt AG (Regensburg) and intercloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of the Pseudomonas aeruginosa DSM1707. Starting from the vector pMA::ABC, the synthetic operon is cleaved from the vector by means of BglII and XbaI and subsequently ligated into the expression vector pEC-XT99A (U.S. Pat. No. 7,118,904) cleaved using BamHI and XbaI. The resulting plasmid pEC-XT99A::ABC (Seq ID No. 53) is 10780 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) takes place in the manner known to the person skilled in the art. The authenticity of the insert is checked by DNA sequence analysis.
The transformation of C. glutamicum ATCC13032 using the vector pEC-XT99A::ABC takes place as previously described (Liebl et al., FEMS Microbiol. Lett. 53:299-303 (1989)). The selection of the transformants takes place on LBHIS agar plates (18.5 g/l of brain heart infusion broth, 0.5 M sorbitol, 5 g/l of Bacto tryptone, 2.5 g/l of Bacto yeast extract, 5 g/l of NaCl and 18 g/l of Bacto agar, supplemented using 5 mg/l of tetracycline). The plates were incubated at 33° C. for two days. The strain obtained carrying the plasmid is named C. glutamicum pEC-XT99A::ABC.
17. Construction of a Vector pEC-XT99A::ABM for the Heterologous Expression of the Genes rhIA, rhIB and pa1131 from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum 
For the heterologous expression of the genes rhIA, rhIB and pa1131 from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum, the plasmid pEC-XT99A::ABM (Seq ID No. 54) is constructed. For this, the synthetic operon rhIABM (Seq ID No. 41) was synthesized by the company GeneArt AG (Regensburg) and intercloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of the Pseudomonas aeruginosa DSM1707. Starting from the vector pMA::ABM, the synthetic operon is cleaved from the vector by means of BglII and XbaI and subsequently ligated into the expression vector pEC-XT99A (U.S. Pat. No. 7,118,904) cleaved using BamHI and XbaI. The resulting plasmid pEC-XT99A::ABM (Seq ID No. 54) is 11073 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) takes place in the manner known to the person skilled in the art. The authenticity of the insert is checked by DNA sequence analysis.
The transformation of C. glutamicum ATCC13032 using the vector pEC-XT99A::ABM takes place as previously described (Liebl et al., FEMS Microbiol. Lett. 53:299-303 (1989)). The selection of the transformants takes place on LBHIS agar plates (18.5 g/l of brain heart infusion broth, 0.5 M sorbitol, 5 g/l of Bacto tryptone, 2.5 g/l of Bacto yeast extract, 5 g/l of NaCl and 18 g/l of Bacto agar, supplemented with 5 mg/l of tetracycline). The plates were incubated at 33° C. for two days. The strain obtained carrying the plasmid is named C. glutamicum pEC-XT99A::ABM.
18. Construction of a Vector pEC-XT99A::ABCM for the Heterologous Expression of the Genes rhIA, rhIB, pa1131 and rhIC from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum 
For the heterologous expression of the genes rhIA, rhIB, pa1131 and rhIC from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum, the plasmid pEC-XT99A::ABCM (Seq ID No. 55) is constructed. For this, the gene pa1131 (Seq ID No. 59) was amplified starting from genomic DNA of the strain Pseudomonas aeruginosa PAO1 (DSM 1707) using the oligonucleotides









MFS2.0_xbaI_fw:



(Seq ID No. 60)



5′-AGGAAATCTAGATGAGAGGCCGGCAAGGATAC-3′



 



MFS2.0_XbaI_rev:



(Seq ID No. 61)



5′-CCAGGTTCTAGACGCCAGGATTGAACAGTACC-3′.






The amplification of the PCR product (1483 base pairs) was carried out using the PhusionTM High-Fidelity Master Mix from New England Biolabs (Frankfurt) polymerase. The PCR product was cleaved using XbaI and ligated into the vector pBBR1MCS-2::ABC (Seq ID No. 40) likewise cleaved using XbaI by means of Fast Link Ligation Kit (Epicentre Technologies; Madison, Wis., USA). The target vector pEC-XT99A::ABCM (Seq ID No. 55) obtained has a size of 12263 base pairs. The insert of the vector was sequenced. The chromosomal DNA was isolated by means of DNeasy Blood and Tissue Kit (Qiagen; Hilden) according to manufacturer's instructions. The carrying-out of the PCR, the checking of the successful amplification of the PCR by means of agarose gel electrophoresis, ethidium bromide staining of the DNA, determination of the PCR fragment size, purification of the PCR products and DNA concentration determination took place in the manner known to the person skilled in the art.
The transformation of C. glutamicum ATCC13032 using the vector pEC-XT99A::ABCM takes place as previously described (Liebl et al., FEMS Microbiol. Lett. 53:299-303 (1989)). The selection of the transformants takes place on LBHIS agar plates (18.5 g/l of brain heart infusion broth, 0.5 M sorbitol, 5 g/l of Bacto tryptone, 2.5 g/l of Bacto yeast extract, 5 g/l of NaCl and 18 g/l of Bacto agar, supplemented with 5 mg/l of tetracycline). The plates were incubated for two days at 33° C. The strain obtained carrying the plasmid is named C. glutamicum pEC-XT99A::ABCM.
19. Construction of a Vector pVWEX1::rfbBDAC for Heterologous Expression in C. glutamicum 
For the heterologous expression of the genes rfbBDAC from P. putida under the control of the lac promoter in C. glutamicum, the vector pVWEX1::rfbBDAC (Seq ID No. 57) is constructed. For this, the vector pBBR1MCS-2::rfbBDAC (Seq ID No. 45) is digested using XbaI and the fragment (3840 bp) containing the genes rfbBDAC from P. putida KT2440 and the lac promoter is ligated into the vector pVWEX1 (Seq ID No. 56) digested with XbaI. The resulting plasmid pVWEX1::rfbBDAC (Seq ID No. 57) is 12311 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) takes place in the manner known to the person skilled in the art. The authenticity of the insert is checked by DNA sequence analysis.
The transformation of C. glutamicum ATCC13032 pEC-XT99A, ATCC13032 pEC-XT99A::AB, ATCC13032 pEC-XT99A::ABM, ATCC13032 pEC-XT99A::ABC and ATCC13032 pEC-XT99A::ABCM using the vector pVWEX1::rfbBDAC takes place as previously described (Liebl et al., FEMS Microbiol. Lett. 53:299-303 (1989)). The selection of the transformants takes place on LBHIS agar plates (18.5 g/l of brain heart infusion broth, 0.5 M sorbitol, 5 g/l of Bacto tryptone, 2.5 g/l of Bacto yeast extract, 5 g/l of NaCl and 18 g/l of Bacto agar, supplemented with 5 mg/l of tetracycline and 25 mg/l of kanamycin). The plates were incubated at 33° C. for two days. The strains obtained carrying the plasmids are named C. glutamicum pEC-XT99A pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::AB pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::ABM pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::ABC pVWEX1::rfbBDAC and C. glutamicum pEC-XT99A::ABCM pVWEX1::rfbBDAC.
20. Quantification of the Rhamnolipid Production by Recombinant C. glutamicum Strains
The recombinant strains C. glutamicum strains generated in the Examples 15 to 19 C. glutamicum pEC-XT99A, C. glutamicum pEC-XT99A::AB, C. glutamicum pEC-XT99A::ABC, C. glutamicum pEC-XT99A::ABM, C. glutamicum pEC-XT99A::ABCM, C. glutamicum pEC-XT99A pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::AB pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::ABM pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::ABC pVWEX1::rfbBDAC and C. glutamicum pEC-XT99A::ABCM pVWEX1::rfbBDAC are cultured on LBHIS agar plates using 5 mg/l of tetracycline and 5 mg/l of tetracycline and 25 mg/l of kanamycin. For the investigation of the rhamnolipid production in the shaker flask, precultures are first prepared. For this, an inoculation loop of a strain freshly streaked on an LBHIS agar plate is used and 10 ml of LBHIS medium (18.5 g/l of brain heart infusion broth, 0.5 M sorbitol, 5 g/l of Bacto tryptone, 2.5 g/l of Bacto yeast extract and 5 g/l of NaCl, supplemented with 5 mg/l of tetracycline or 5 mg/l of tetracycline and 25 mg/l of kanamycin) is inoculated into a 100 ml Erlenmeyer flask. The culturing of the strains takes place at 33° C. and 200 rpm overnight. The next morning, 50 ml of CGXII medium (containing 5 mg/l of tetracycline or 5 mg/l of tetracycline and 25 mg/l of kanamycin) are inoculated into a 500 ml Erlenmeyer flask containing baffles with 1 ml of the preculture (start OD600 0.1).
CGXII Medium:


    
    
        20 g/l of (NH4)2SO4 (Merck)
        5 g/l of urea (Merck)
        1 g/l of KH2PO4 (Merck)
        1 g/l of K2HPO4 (Merck)
        0.25 g/l of MgSO4.7H2O (Merck)
        10 mg/l of CaCl2 (Merck)
        42 g/l of MOPS (Roth)
        0.2 mg/l of biotin (Merck)
        1 ml/l of trace salt solution
        adjust to pH 7 using NaOH
        after autoclaving add 1 ml/l of protocatechuic acid (30 g/l dissolved in dil. NaOH, sterile-filtered) and 40 g/l of glucose (Merck)

Trace Salt Solution:

        10 g/l of FeSO4.7H2O (Merck)
        10 g/l of MnSO4.H2O (Merck)
        1 g/l of ZnSO4.7H2O (Merck)
        0.2 g/l of CuSO4.5H2O (Merck)
        20 mg/l of NiCl2.6H2O (Merck)
        to dissolve acidify to pH 1 using HCl
    
    


The cultures are cultured at 200 rpm and 33° C. up to an optical density (600 nm) of 0.4-0.6. At this optical density, the cultures are induced by the addition of IPTG (isopropyl-β-D-thiogalactopyranoside; 1 mM final concentration). The subsequent expression likewise takes place at 33° C. and 200 rpm for 72 h. At intervals of 24 h, a sample of 1 ml of broth is removed from the culture flask. The sample preparation for the following chromatographic analyses and the chromatographic analyses themselves are carried out as described in Example 4.
While C. glutamicum pEC-XT99A produces no rhamnolipids, in the recombinant strains C. glutamicum pEC-XT99A::AB, C. glutamicum pEC-XT99A::ABC, C. glutamicum pEC-XT99A::ABM and C. glutamicum pEC-XT99A::ABCM the formation of rhamnolipids is detectable. With the aid of reference materials, it is shown that C. glutamicum pEC-XT99A::AB and C. glutamicum pEC-XT99A::ABM only form monorhamnosyl lipids, while C. glutamicum pEC-XT99A::ABC, C. glutamicum pEC-XT99A::ABM and C. glutamicum pEC-XT99A::ABCM are able to form dirhamnosyl lipids and monorhamnosyl lipids. Furthermore, it is shown that C. glutamicum pEC-XT99A::ABM and C. glutamicum pEC-XT99A::ABCM are able to form more monorhamnosyl lipids or dirhamnosyl lipids and monorhamnosyl lipids than the respective reference strains C. glutamicum pEC-XT99A::AB and C. glutamicum pEC-XT99A::ABC without amplification of the pa1131 gene from Pseudomonas aeruginosa. 
Moreover, it is shown that the strains C. glutamicum pEC-XT99A::AB pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::ABM pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::ABC pVWEX1::rfbBDAC and C. glutamicum pEC-XT99A::ABCM pVWEX1::rfbBDAC form significantly more mono- (C. glutamicum pEC-XT99A::AB pVWEX1::rfbBDAC and C. glutamicum pEC-XT99A::ABM pVWEX1::rfbBDAC) or mono- and dirhamnosyl lipids (C. glutamicum pEC-XT99A::ABC pVWEX1::rfbBDAC and C. glutamicum pEC-XT99A::ABCM pVWEX1::rfbBDAC) than the strains, C. glutamicum pEC-XT99A::ABM, C. glutamicum pEC-XT99A::ABC and C. glutamicum pEC-XT99A::ABCM without amplification of the of the rfbBDA genes from P. putida. 
21. Construction of Pseudomonas Strains that Carry the Plasmids pBBR1MCS-2, pBBR1MCS-2::AB, pBBR1MCS-2::ABC, pBBR1MCS-2::ABM and pBBR1MCS-2::ABCM
The plasmids pBBR1MCS-2, pBBR1MCS-2::AB, pBBR1MCS-2::ABC, pBBR1MCS-2::ABM and pBBR1MCS-2::ABCM are incorporated in Pseudomonas fluorescens DSM 50090, Pseudomonas fluorescens DSM 9958, Pseudomonas putida DSM 6899, Pseudomonas putida DSM 50204, Pseudomonas putida 50194, P. brassicacearum DSM 13227, P. stutzeri DSM 10701, Pseudomonas stutzeri DSM 4166 and Pseudomonas fulva DSM 17717 by electroporation. The transformation of Pseudomonas strains takes place as described previously (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The selection of the transformants takes place on nutrient agar plates (5 g/l of peptone; 3 g/l of meat extract; 15 g/l of agar; pH 7; supplemented with 50 mg/l of kanamycin). The plates are incubated at 30° C. or rather 28° C. for two days. The strains obtained, carrying the plasmids, are named Pseudomonas fluorescens DSM 50090 pBBR1MCS-2, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2, Pseudomonas putida DSM 6899 pBBR1MCS-2, Pseudomonas putida DSM 50204 pBBR1MCS-2, Pseudomonas putida 50194 pBBR1MCS-2, P. brassicacearum DSM 13227 pBBR1MCS-2, P. stutzeri DSM 10701 pBBR1MCS-2, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2, Pseudomonas fulva DSM 17717 pBBR1MCS-2, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::AB, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::AB, Pseudomonas putida DSM 6899 pBBR1MCS-2::AB, Pseudomonas putida DSM 50204 pBBR1MCS-2::AB, Pseudomonas putida 50194 pBBR1MCS-2::AB, P. brassicacearum DSM 13227 pBBR1MCS-2::AB, P. stutzeri DSM 10701 pBBR1MCS-2::AB, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::AB, Pseudomonas fulva DSM 17717 pBBR1MCS-2::AB, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABC, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABC, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABC, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABC, Pseudomonas putida 50194 pBBR1MCS-2::ABC, P. brassicacearum DSM 13227 pBBR1MCS-2::ABC, P. stutzeri DSM 10701 pBBR1MCS-2::ABC, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABC, Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABC, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABCM, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABCM, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABCM, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABCM, Pseudomonas putida 50194 pBBR1MCS-2::ABCM, P. brassicacearum DSM 13227 pBBR1MCS-2::ABCM, P. stutzeri DSM 10701 pBBR1MCS-2::ABCM, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABCM, Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABCM, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABM, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABM, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABM, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABM, Pseudomonas putida 50194 pBBR1MCS-2::ABM, P. brassicacearum DSM 13227 pBBR1MCS-2::ABM, P. stutzeri DSM 10701 pBBR1MCS-2::ABM, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABM and Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABM.
22. Quantification of the Rhamnolipid Production by Recombinant Pseudomonas Strains
The recombinant strains Pseudomonas strains Pseudomonas fluorescens DSM 50090, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2, Pseudomonas putida DSM 6899 pBBR1MCS-2, Pseudomonas putida DSM 50204 pBBR1MCS-2, Pseudomonas putida 50194 pBBR1MCS-2, P. brassicacearum DSM 13227 pBBR1MCS-2, P. stutzeri DSM 10701 pBBR1MCS-2, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2, Pseudomonas fulva DSM 17717 pBBR1MCS-2, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::AB, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::AB, Pseudomonas putida DSM 6899 pBBR1MCS-2::AB, Pseudomonas putida DSM 50204 pBBR1MCS-2::AB, Pseudomonas putida 50194 pBBR1MCS-2::AB, P. brassicacearum DSM 13227 pBBR1MCS-2::AB, P. stutzeri DSM 10701 pBBR1MCS-2::AB, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::AB, Pseudomonas fulva DSM 17717 pBBR1MCS-2::AB, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABC, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABC, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABC, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABC, Pseudomonas putida 50194 pBBR1MCS-2::ABC, P. brassicacearum DSM 13227 pBBR1MCS-2::ABC, P. stutzeri DSM 10701 pBBR1MCS-2::ABC, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABC, Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABC, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABCM, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABCM, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABCM, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABCM, Pseudomonas putida 50194 pBBR1MCS-2::ABCM, P. brassicacearum DSM 13227 pBBR1MCS-2::ABCM, P. stutzeri DSM 10701 pBBR1MCS-2::ABCM, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABCM, Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABCM, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABM, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABM, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABM, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABM, Pseudomonas putida 50194 pBBR1MCS-2::ABM, P. brassicacearum DSM 13227 pBBR1MCS-2::ABM, P. stutzeri DSM 10701 pBBR1MCS-2::ABM, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABM and Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABM generated in Example 21 are cultured on LB agar kanamycin (50 μg/ml) plates. The subsequent culturing for the production of the rhamnolipids takes place as described in Example 12. The sample preparation for the following chromatographic analyses and the chromatographic analyses themselves are carried out as described in Example 4.
While the Pseudomonas strains Pseudomonas fluorescens DSM 50090, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2, Pseudomonas putida DSM 6899 pBBR1MCS-2, Pseudomonas putida DSM 50204 pBBR1MCS-2, Pseudomonas putida 50194 pBBR1MCS-2, P. brassicacearum DSM 13227 pBBR1MCS-2, P. stutzeri DSM 10701 pBBR1MCS-2, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2, Pseudomonas fulva DSM 17717 pBBR1MCS-2 produce no rhamnolipids, in the recombinant strains Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::AB, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::AB, Pseudomonas putida DSM 6899 pBBR1MCS-2::AB, Pseudomonas putida DSM 50204 pBBR1MCS-2::AB, Pseudomonas putida 50194 pBBR1MCS-2::AB, P. brassicacearum DSM 13227 pBBR1MCS-2::AB, P. stutzeri DSM 10701 pBBR1MCS-2::AB, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::AB, Pseudomonas fulva DSM 17717 pBBR1MCS-2::AB, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABM, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABM, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABM, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABM, Pseudomonas putida 50194 pBBR1MCS-2::ABM, P. brassicacearum DSM 13227 pBBR1MCS-2::ABM, P. stutzeri DSM 10701 pBBR1MCS-2::ABM, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABM and Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABM the formation of monorhamnosyl lipids and in the strains Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABC, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABC, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABC, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABC, Pseudomonas putida 50194 pBBR1MCS-2::ABC, P. brassicacearum DSM 13227 pBBR1MCS-2::ABC, P. stutzeri DSM 10701 pBBR1MCS-2::ABC, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABC, Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABC, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABCM, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABCM, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABCM, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABCM, Pseudomonas putida 50194 pBBR1MCS-2::ABCM, P. Brassicacearum DSM 13227 pBBR1MCS-2::ABCM, P. stutzeri DSM 10701 pBBR1MCS-2::ABCM, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABCM and Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABCM the formation of mono- and dirhamnosyl lipids is detectable.
Moreover, fewer monorhamnosyl lipids are formed by the recombinant Pseudomonas strains Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABM, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABM, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABM, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABM, Pseudomonas putida 50194 pBBR1MCS-2::ABM, P. brassicacearum DSM 13227 pBBR1MCS-2::ABM, P. stutzeri DSM 10701 pBBR1MCS-2::ABM, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABM Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABM and by the recombinant Pseudomonas strains Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABCM, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABCM, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABCM, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABCM, Pseudomonas putida 50194 pBBR1MCS-2::ABCM, P. brassicacearum DSM 13227 pBBR1MCS-2::ABCM, P. stutzeri DSM 10701 pBBR1MCS-2::ABCM, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABCM and Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABCM fewer mono- and dirhamnosyl lipids are formed than by the respective reference strains without the P. aeruginosa gene pa1131 Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::AB, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::AB, Pseudomonas putida DSM 6899 pBBR1MCS-2::AB, Pseudomonas putida DSM 50204 pBBR1MCS-2::AB, Pseudomonas putida 50194 pBBR1MCS-2::AB, P. brassicacearum DSM 13227 pBBR1MCS-2::AB, P. stutzeri DSM 10701 pBBR1MCS-2::AB, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::AB and Pseudomonas fulva DSM 17717 pBBR1MCS-2::AB and Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABC, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABC, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABC, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABC, Pseudomonas putida 50194 pBBR1MCS-2::ABC, P. brassicacearum DSM 13227 pBBR1MCS-2::ABC, P. stutzeri DSM 10701 pBBR1MCS-2::ABC, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2::ABC and Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABC without amplification of the pa1131 gene from Pseudomonas aeruginosa. 
23. Construction of the Vectors pBBR1MCS-2::ABPAO1-C1 and pBBR1MCS-2::ABPA7-CE264 for the Heterologous Expression of Alternative rhIA, rhIB and rhIC Genes from Pseudomonas aeruginosa PAO1, Pseudomonas aeruginosa PA7, Pseudomonas aeruginosa 1 and Burkholderia thailandensis E264 in P. putida 
For the heterologous expression of the genes rhIA, rhIB and rhIC from Pseudomonas aeruginosa PAO1 and Pseudomonas aeruginosa PA7, the plasmids pBBR1MCS-2::ABPAO1 (Seq ID No. 62) and pBBR1MCS-2::ABPA7 (Seq ID No. 63) are first constructed. For this, the synthetic operons rhIABPAO1 (Seq ID No. 64) and rhIABPA7 (Seq ID No. 65) are synthesized by the company DNA 2.0 (Menlo Park, Calif., U.S.A) and intercloned in the commercial vector pJ294 (DNA 2.0). The basis for the synthesis is the already known genomic sequence of the strains Pseudomonas aeruginosa PAO1 and Pseudomonas aeruginosa PA7. Starting from the vectors pJ294::ABPAO1 and pJ294::ABPA7, the synthetic operons are cleaved from the vectors by means of KpnI and XbaI and subsequently ligated into the expression vector pBBR1MCS-2 (Seq ID No. 49) (Kovach et al., 1995: Four new derivatives of the broad-host-range cloning vector pBBR1MCS carrying different antibiotic-resistance cassettes. Gene, 166:175-176) cleaved using KpnI and XbaI. The resulting plasmids pBBR1MCS-2::ABPAO1 (Seq ID No. 62) and pBBR1MCS-2::ABPA7 (Seq ID No. 63) are 7332 and 7354 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) takes place in the manner known to the person skilled in the art. The authenticity of the insert is checked by DNA sequence analysis.
In the second step, the plasmids pBBR1MCS-2::ABPAO1-C1 (Seq ID No. 66) and pBBR1MCS-2::ABPA7-CE264 (Seq ID No. 67) are produced. For this, the rhIC genes from Pseudomonas aeruginosa 1 (Seq ID No. 68) and Burkholderia thailandensis E264 (Seq ID No. 76) are synthesized by the company DNA 2.0 (Menlo Park, Calif., U.S.A) and intercloned in the commercial vector pJ294 (DNA 2.0). The basis for the synthesis is the already known genomic sequence of the strains Pseudomonas aeruginosa 1 and Burkholderia thailandensis E264. Starting from the vectors pJ294::C1 and pJ294::CE264, the rhIC genes are cleaved from the vectors by means of Xba and SacI and subsequently ligated into the vectors pBBR1MCS-2::ABPAO1 (Seq ID No. 62) and pBBR1MCS-2::ABPA7 (Seq ID No. 63) likewise cleaved using Xba and SacI. The resulting plasmids pBBR1MCS-2::ABPAO1-C1 (Seq ID No. 66) and pBBR1MCS-2::ABPA7-CE264 (Seq ID No. 67) are 8325 and 8335 base pairs in size. The ligation and the transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) takes place in the manner known to the person skilled in the art. The authenticity of the insert is checked by DNA sequence analysis.
The transformation of Pseudomonas putida KT2440 and GPp104 using the vectors pBBR1MCS-2, pBBR1MCS-2::ABPAO1-C1 and pBBR1MCS-2::ABPA7-CE264 takes place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of every 10 clones was isolated and analyzed. The strains obtained carrying the plasmids are named P. putida KT2440 pBBR1MCS-2, P. putida KT2440 pBBR1MCS-2::ABPAO1-C1, P. putida KT2440 pBBR1MCS-2::ABPA7-CE264, P. putida GPp104 pBBR1MCS-2, P. putida GPp104 pBBR1MCS-2::ABPAO1-C1 and P. putida GPp104 pBBR1MCS-2::ABPA7-CE264.
24. Quantification of the Rhamnolipid Production by Recombinant P. putida Strains Having Alternative rhIA, rhIB and rhIC Genes from Pseudomonas aeruginosa PAO1, Pseudomonas aeruginosa PA7, Pseudomonas aeruginosa 1 and Burkholderia thailandensis E264
The recombinant strains P. putida strains generated in Example 23 are cultured on LB agar kanamycin (50 μg/ml) plates. The subsequent culturing for the production of the rhamnolipids takes place as described in Example 12. The sample preparation for the following chromatographic analyses and the chromatographic analyses themselves are carried out as described in Example 4.
While the strains P. putida KT2440 pBBR1MCS-2 and P. putida GPp104 pBBR1MCS-2 are not able to produce mono- and dirhamnosyl lipids, the strains P. putida KT2440 pBBR1MCS-2::ABPAO1-C1, P. putida KT2440 pBBR1MCS-2::ABPA7-CE264, P. putida GPp104 pBBR1MCS-2::ABPAO1-C1 and P. putida GPp104 pBBR1MCS-2::ABPA7-CE264 form both mono- as well as dirhamnosyl lipids. It is shown that the strains are able to produce more mono- and dirhamnosyl lipids with an attenuation of the polyhydroxybutyrate formation (P. putida GPp104 pBBR1MCS-2::ABPAO1-C1 and P. putida GPp104 pBBR1MCS-2::ABPA7-CE264) than the strains without attenuation of the polyhydroxybutyrate formation (P. putida KT2440 pBBR1MCS-2::ABPAO1-C1 and P. putida KT2440 pBBR1MCS-2::ABPA7-CE264).
25. Construction of the Vectors pBBR1MCS-2::AB_rfbBDAC, pBBR1MCS-2::ABM_rfbBDAC and pBBR1MCS-2::ABMC_rfbBDAC for the Overexpression of the P. putida rfbBDAC Operon in P. putida and E. coli 
For the construction of the vectors pBBR1MCS-2::AB_rfbBDAC, pBBR1MCS-2::ABM_rfbBDAC and pBBR1MCS-2::ABMC_rfbBDAC for the overexpression of the P. putida rfbBDAC operon in P. putida and E. coli, the P. putida rfbBDAC operon was first amplified by PCR. The vector pBBR1MCS-2::rfbBDAC (Seq ID No. 45) served as matrix for a PCR. The following oligonucleotides were used:









RL_AgeI-fw:



(Seq ID No. 71)



5′-TATATATAACCGGTATTAATGCAGCTGGCACGAC-3′



 



RL_AgeI_rev:



(Seq ID No. 72)



5′-GGCCGACCGGTACTAGTGGA-3′






The PCR was carried out using the PhusionTM High-Fidelity Master Mix of New England Biolabs (Frankfurt) polymerase. It took place in the manner known to the person skilled in the art. The target sequence (lac promoter and rfbBDAC) was intercloned in the Trenzyme alligator cloning system. E. coli DH5α (New England Biolabs; Frankfurt) transformants were selected and the plasmid DNA of different candidates was isolated and sequenced. After the sequence had been checked and examined for correctness, the vector was cleaved using AgeI. The target fragment was ligated into the vectors pBBR1MCS-2::AB (Seq ID No. 38), pBBR1MCS-2::ABM (Seq ID No. 42) and pBBR1MCS-2::ABMC (Seq ID No. 51) likewise cleaved using AgeI by means of conventional ligation methods. The resulting vectors pBBR1MCS-2::AB_rfbBDAC (Seq ID No. 73), pBBR1MCS-2::ABM_rfbBDAC (Seq ID No. 74) and pBBR1MCS-2::ABMC_rfbBDAC (Seq ID No. 75) have sizes of 11960, 13289 and 14250 base pairs. The inserts of the vectors were sequenced. The carrying-out of the PCR, the checking of the successful amplification of the PCR by means of agarose gel electrophoresis, ethidium bromide staining of the DNA, determination of the PCR fragment size, purification of the PCR products and DNA concentration determination took place in the manner known to the person skilled in the art. The transformation of Pseudomonas putida KT2440 using the vectors pBBR1MCS-2::AB_rfbBDAC, pBBR1MCS-2::ABM_rfbBDAC and pBBR1MCS-2::ABMC_rfbBDAC took place as previously described (Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854). The plasmid DNA of every 10 clones was isolated and analyzed. The strains obtained carrying the plasmids are named P. putida KT2440 pBBR1MCS-2::AB_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABM_rfbBDAC and P. putida KT2440 pBBR1MCS-2::ABMC_rfbBDAC.
26. Quantification of the Rhamnolipid Production by Recombinant P. putida KT2440 pBBR1MCS-2::AB_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABM_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABMC_rfbBDAC, P. putida KT2440 pBBR1MCS-2::AB, P. putida KT2440 pBBR1MCS-2::ABM, P. putida KT2440 pBBR1MCS-2::ABC and P. putida KT2440 pBBR1MCS-2::ABMC
The recombinant strains P. putida strains generated in the Examples 2, 7 and 25 are cultured on LB agar-kanamycin (50 μg/ml) plates. The subsequent culturing for the production of the rhamnolipids takes place as described in Example 12. The sample preparation for the following chromatographic analyses and the chromatographic analyses themselves take place as described in Example 4.
It is shown that P. putida KT2440 pBBR1MCS-2::AB_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABM_rfbBDAC, P. putida KT2440 pBBR1MCS-2::AB and P. putida KT2440 pBBR1MCS-2::ABM are able to form monorhamnosyl lipids, while P. putida KT2440 pBBR1MCS-2::ABMC_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABC and P. putida KT2440 pBBR1MCS-2::ABMC are able to form mono- and dirhamnosyl lipids.
Furthermore, it is shown that P. putida KT2440 pBBR1MCS-2::ABM_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABM, KT2440 pBBR1MCS-2::ABMC_rfbBDAC and KT2440 pBBR1MCS-2::ABMC are able to form more mono- and dirhamnosyl lipids than the corresponding control strains P. putida KT2440 pBBR1MCS-2::AB_rfbBDAC, P. putida KT2440 pBBR1MCS-2::AB, KT2440 pBBR1MCS-2::ABC_rfbBDAC and KT2440 pBBR1MCS-2::ABC without amplification of the Pseudomonas aeruginosa gene pa1131.
Finally, it is shown that P. putida KT2440 pBBR1MCS-2::AB_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABM_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC, P. putida KT2440 pBBR1MCS-2::ABMC_rfbBDAC are able to form more mono- (P. putida KT2440 pBBR1MCS-2::AB_rfbBDAC and P. putida KT2440 pBBR1MCS-2::ABM_rfbBDAC) and mono- and dirhamnosyl lipids (P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC and P. putida KT2440 pBBR1MCS-2::ABMC_rfbBDAC) than the respective control strains P. putida KT2440 pBBR1MCS-2::AB, P. putida KT2440 pBBR1MCS-2::ABM, P. putida KT2440 pBBR1MCS-2::ABC, P. putida KT2440 pBBR1MCS-2::ABMC without amplification of the P. putida genes rfbBDAC.
27. Generation of Recombinant E. coli W3110 pBBR1MCS-2::AB, E. coli W3110 pBBR1MCS-2::ABM, E. coli W3110 pBBR1MCS-2::ABC, E. coli W3110 pBBR1MCS-2::ABCM, E. coli W3110 pBBR1MCS-2::AB_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC and E. coli W3110 pBBR1MCS-2::ABCM_rfbBDAC
The transformation of E. coli W3110 took place as described previously (Miller J H. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Plainview, N.Y.: Cold Spring Harbor Lab. Press; 1992) by means of electroporation. The plasmid DNA of every 10 clones was isolated and analyzed. The obtained strains carrying the plasmids were named E. coli W3110 pBBR1MCS-2::AB, E. coli W3110 pBBR1MCS-2::ABM, E. coli W3110 pBBR1MCS-2::ABC, E. coli W3110 pBBR1MCS-2::ABCM, E. coli W3110 pBBR1MCS-2::AB_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC and E. coli W3110 pBBR1MCS-2::ABCM_rfbBDAC.
28. Quantification of the Rhamnolipid Production by Recombinant E. coli W3110 pBBR1MCS-2::AB, E. coli W3110 pBBR1MCS-2::ABM, E. coli W3110 pBBR1MCS-2::ABC, E. coli W3110 pBBR1MCS-2::ABCM, E. coli W3110 pBBR1MCS-2::AB_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC and E. coli W3110 pBBR1MCS-2::ABCM_rfbBDAC
The recombinant E. coli strains generated in Example 27 are cultured on LB agar kanamycin (50 μg/ml) plates. The subsequent culturing for the production of the rhamnolipids takes place as described in Example 10. The sample preparation for the following chromatographic analyses and the chromatographic analyses themselves take place as described in Example 4.
It is shown that E. coli W3110 pBBR1MCS-2::AB, E. coli W3110 pBBR1MCS-2::ABM, E. coli W3110 pBBR1MCS-2::AB_rfbBDAC and E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC are able to form monorhamnosyl lipids, while E. coli W3110 pBBR1MCS-2::ABC, E. coli W3110 pBBR1MCS-2::ABCM, E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC and E. coli W3110 pBBR1MCS-2::ABCM_rfbBDAC are able to form mono- and dirhamnosyl lipids. Furthermore, it is shown that E. coli W3110 pBBR1MCS-2::ABM and E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC form more monorhamnosyl lipids than E. coli W3110 pBBR1MCS-2::AB and E. coli W3110 pBBR1MCS-2::AB_rfbBDAC without amplification of the Pseudomonas aeruginosa gene pa1131.
Furthermore, it is shown that E. coli W3110 pBBR1MCS-2::ABCM and E. coli W3110 pBBR1MCS-2::ABCM_rfbBDAC form more mono- and dirhamnosyl lipids than E. coli W3110 pBBR1MCS-2::ABC and E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC without amplification of the Pseudomonas aeruginosa gene pa1131. Furthermore, it is shown that E. coli W3110 pBBR1MCS-2::ABM and E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC form more monorhamnosyl lipids than E. coli W3110 pBBR1MCS-2::AB and E. coli W3110 pBBR1MCS-2::AB_rfbBDAC without amplification of the Pseudomonas aeruginosa gene pa1131.
Finally, it is shown that E. coli W3110 pBBR1MCS-2::AB_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC and E. coli W3110 pBBR1MCS-2::ABCM_rfbBDAC are able to form more mono- (E. coli W3110 pBBR1MCS-2::AB_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC) and mono- and dirhamnosyl lipids (E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC and E. coli W3110 pBBR1MCS-2::ABCM_rfbBDAC) than the respective control strains E. coli W3110 pBBR1MCS-2::AB, E. coli W3110 pBBR1MCS-2::ABM, E. coli W3110 pBBR1MCS-2::ABC and E. coli W3110 pBBR1MCS-2::ABCM without amplification of the P. putida genes rfbBDAC.

## Claims

1. A genetically modified cell, which is able to form at least one rhamnolipid of general formula (I),



wherein
m=2, 1 or 0,
n=1 or 0,
R1 and R2 are organic residues having 2 to 24 carbon atoms,
said cell having been genetically modified such that, compared to its wild-type, the cell has increased activity of at least one of the enzymes E1, E2 and E3, wherein:
the enzyme E1 has at least 95% amino acid identity to a sequence selected from SEQ ID NO: 18, 78, 80, 82, or 2;
the enzyme E2 has at least 95% amino acid identity to a sequence selected from SEQ ID NO: 20, 84, 86, 88, or 4;
the enzyme E3 has at least 95% amino acid identity to a sequence selected from SEQ ID NO: 22, 90, 92, or 6;
wherein said cell, compared to its wild-type further has increased activity of an enzyme E8, which catalyses rhamnolipid export from the cell into the surrounding medium; and
wherein E8 has at least 95% amino acid identity to SEQ ID NO: 8.

2. The genetically modified cell of claim 1, wherein said cell has increased activities of an enzyme combination selected from E1E2, E2E3 and E1E2E3.

3. The genetically modified cell of claim 2, wherein said cell has an increased activity of the enzyme combination E1E2E3 and n is =1.

4. The genetically modified cell of claim 1, wherein said cell is selected from a genus of the group consisting of Aspergillus, Corynebacterium, Brevibacterium, Bacillus, Acinetobacter, Alcaligenes, Lactobacillus, Paracoccus, Lactococcus, Candida, Pichia, Hansenula, Kluyveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Pseudomonas, Rhodospirillum, Rhodobacter, Burkholderia, Clostridium and Cupriavidus.

5. The genetically modified cell of claim 1, wherein said cell is a bacterial cell.

6. The genetically modified cell of claim 1, wherein the wild-type of said cell forms polyhydroxyalkanoates having chain lengths of C6 to C16.

7. The genetically modified cell of claim 6, wherein said cell, compared to its wild-type, has a decreased activity of at least one enzyme E9 or E10, wherein
E9 has at least 95% identity to the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 32, and
E10 has at least 95% identity to the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 36.

8. The genetically modified cell of claim 1, wherein said cell, compared to its wild-type, has increased activity of at least one enzyme selected from the group consisting of:
E4, which has at least 95% amino acid identity to SEQ ID NO: 10,
E5, which has at least 95% amino acid identity to SEQ ID NO: 12,
E6, which has at least 95% amino acid identity to SEQ ID NO: 16, and
E7, which has at least 95% amino acid identity to SEQ ID NO: 14.

9. The genetically modified cell of claim 8, wherein said cell has increased activity of each of the enzymes E4, E5, E6, and E7.

10. The genetically modified cell of claim 1, wherein said genetic modification comprises introduction into said cell of at least one vector comprising at least one nucleic acid sequence selected from:
a sequence with at least 95% identity to SEQ ID NO: 17, 77, 79, 81, or 1;
a sequence with at least 95% identity to SEQ ID NO: 19, 83, 85, 87, or 3; and
a sequence with at least 95% identity to SEQ ID NO: 21, 89, 91, or 5.

11. A method for producing rhamnolipids of general formula (I)



wherein

m=2, 1 or 0,
n=1 or 0
R1 and R2 are organic residues having 2 to 24 carbon atoms,
said method comprising:
I) contacting the genetically modified cell of claim 1 with a medium containing a carbon source; and
II) culturing the cell under conditions in which the cell forms rhamnolipids from the carbon source.