Cells and process for the production of rhamnolipids

专利摘要:
The present invention relates to cells and nucleic acids, as well as their use for the production of rhamnolipids, as well as processes for the production of rhamnolipids.
公开号:BR112013002124B1
申请号:R112013002124-1
申请日:2011-07-20
公开日:2021-08-31
发明作者:Steffen Schaffer；Mirja Wessel；Anja Thiessenhusen；Nadine Stein
申请人:Evonik Operations Gmbh；
IPC主号:

专利说明:

Field of Invention
[0001] The present invention relates to cells and nucleic acids, as well as their use for the production of rhamnolipids, as well as processes for the production of rhamnolipids. State of the Art
[0002] Nowadays, surfactants are essentially produced on the basis of petrochemical raw materials. The use of surfactants based on renewable resources is a suitable alternative due to the predictable scarcity of petrochemical raw materials and the increasing demand for products that are based on natural resources or that are biologically degradable.
[0003] Rhamnolipids consist of one radical (monoramnosyl lipids) or two rhamnose radicals (diramnosyl lipids) and one or two 3-hydroxy fatty acid radicals (see Handbook of Hydrocarbon and Lipid Microbiology, 2010, pages 3037-51) . They have surfactant properties, which are necessary for use as a surfactant in the most varied applications (see Leitermann et al., 2009).
[0004] These lipids are produced today with wild type isolates of several bacteria pathogenic to humans and animals, especially representatives of the Pseudomonas and Burkholderia genera (see Handbook of Hydrocarbon and Lipid Microbiology, 2010, pages 3037-51) . The fact that these production organisms are capable of inducing disease significantly reduces customer acceptance of conventionally produced rhamnolipids. In addition, the higher safety requirements due to a higher volume of investments and possibly additional processing stages, also affect production costs.
[0005] Although with the help of these production organisms it is possible to obtain, in part, high product titles, as well as space-time or carbon yields, this presupposes the use of vegetable oils as a single substrate or co-substrate (see Handbook of Hydrocarbon and Lipid Microbiology, 2010, pages 3037-51). However, vegetable oils, compared to other carbon sources, such as, for example, glucose, sucrose or polysaccharides, such as, for example, starch, cellulose and hemicellulose, glycerin, CO, CO2 or CH4, are comparatively raw materials. expensive cousins. Furthermore, based on their surface-active character, rhamnolipids stand out for the fact that they tend to foam strongly in fermentation processes. This is especially the case when lipophilic substrates are used. This problem is markedly reduced when using water-soluble substrates such as, for example, glucose, sucrose, polysaccharides (starch, cellulose, hemicellulose) or glycerin.
[0006] Finally, the properties of the rhamnolipids produced by wild-type isolates have only a limited influence. Until now, these have been carried out exclusively through the optimization of process conduction (pH value, oxygen supply, medium composition, feeding strategies, nitrogen supply, temperature, selection of substrates and so on). However, a very targeted influence of certain product properties, such as, for example, the ratio of the different species of rhamnolipids (number of rhamnose and 3-hydroxy fatty acid radicals) or chain length and degree of saturation of the acid radicals 3 - hydroxyfat, it would be desirable, to be able to modulate the product properties relevant to the application.
[0007] Rhamnolipids, if they are used on a large scale as surfactants in household, cleaning, cosmetic, food processing, pharmaceutical, plant protection and other applications, should compete with the currently used surfactants. These are high-volume chemicals that can be produced at very low costs, with no apparent health risks to the customer, and with clearly defined and scalable product specifications. Therefore, rhamnolipids must be able to be produced at the lowest possible cost, without health risks for the customer and with the most defined properties possible.
[0008] In fact, rhamnolipids have already been produced in GRAS (generally as save) organisms based on favorable carbon sources, such as, for example, glucose or glycerin, however, in this case, it is exclusively monorhamnosyl- lipids (Ochsner et al. Appl Environ. Microbiol. 1995, 61(9):3503-3506).
[0009] Cha and collaborators at Bioresour Technol. 2008, 99(7):2192-9 describe, on the other hand, the production of monoramnosyllipids from soybean oil in P. putida through the introduction of the rhlA and rhlB genes from Pseudomonas aeruginosa.
[00010] Therefore, there is a growing need for cheaper and healthier production of mono- and diramnosyl-lipids with defined as well as modulating properties.
[00011] This modulation can be effected, for example, by a balanced availability of each of the enzymatic activities, which reduces the enrichment of monoramnosyl-lipids. But this modulation can also be effected, for example, through the use of enzymes with certain properties, for example, with respect to substrate specificity and thus, for example, of the chain length of the hydroxyfatty acids incorporated in rhamnolipids.
[00012] The aim of the present invention was based, therefore, on providing a possibility to produce rhamnolipids with safe production hosts from well-accessible carbon sources. Description of the Invention
[00013] Surprisingly it was found that the cells described below, as well as processes in which these cells are used, contribute to solving the aim of the invention.
[00014] The aim of the present invention are, therefore, cells, which are capable of forming rhamnolipids and compared to their wild type, show at least an increase in the activity of a gene product of homologues of the gene products rhlA, rhlB and rhlC. Another object of the invention is a process for the production of rhamnolipids using the cells mentioned above as biocatalyst and simple carbon sources.
[00015] An advantage of the present invention is that organisms can be used, which are non-pathogenic and are easily cultivated.
[00016] Another advantage is that the use of oils is not necessary as a sole substrate or co-substrate.
[00017] One more advantage is that with the aid of the invention, rhamnolipids can be produced with defined properties, as well as modulable.
[00018] One more advantage of the present invention is that diramnosyl-lipids can be produced.
[00019] Another advantage is that rhamnolipids can be produced with higher space-time and carbon yields than with cells without increasing these activities.
[00020] A cell contributes to the resolution of the objective mentioned above, preferably an isolated cell, which has are capable of forming at least one rhamnolipid of the General Formula (I) or its salt,
Formula (I), where
[00021] m = 2, 1 or 0, especially 1 or 2,
[00022] n = 1 or 0, especially 1,
[00023] R1 and R2 independently of one another represent an organic radical the same or different with 2 to 24, preferably 5 to 13 carbon atoms, especially an optionally branched, optionally substituted, especially substituted by hydroxy, optionally unsaturated alkyl radical , especially optionally one, two or three times unsaturated, preferably that selected from the group consisting of pentenyl, heptenyl, nonenyl, undecenyl and tridecenyl and (CH2)o-CH3 with being 1 to 23, preferably 4 to 12, characterized in that that this one has been genetically modified in such a way that, compared to its wild type, it has an increased activity of at least one of the E1, E2 and E3 enzymes, where the E1 enzyme is able to catalyze the 3-hydroxyalkanoyl reaction -ACP through 3-hydroxyalkanoyl-3-hydroxyoicoalkanoic acid-ACP to hydroxyalkanoyl-3-hydroxyoicoalkanoic acid, the enzyme E2 is a rhamnosyltransferase I and is able to catalyze the reaction that 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 reaction of dTDP-rhamnose and α-L -rhamnopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate for α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate, such enzymes E1, E2 and E3 being preferably selected from group consisting of
[00024] at least one E1 enzyme selected from
[00025] an E1a enzyme with polypeptide sequence SEQ ID NO: 2 or with a polypeptide sequence, wherein up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the reference sequence SEQ ID NO: 2, are modified by deletion, insertion, substitution or by a combination thereof and which still has at least 10%, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme with the reference sequence SEQ ID NO: 2, whereby by enzymatic activity for an E1a enzyme is meant the capacity , of reacting preferably 3-hydroxydecanoyl-ACP through 3-hydroxydecanoyl-3-hydroxydecanoic-ACP to hydroxydecanoyl-3-hydroxydecanoic acid,
[00026] an E1b enzyme with polypeptide sequence SEQ ID NO: 18 or with a polypeptide sequence, wherein up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the reference sequence SEQ ID NO: 18, are modified by deletion, insertion, substitution or by a combination thereof and which still has at least 10%, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme with the reference sequence SEQ ID NO: 18, whereby by enzymatic activity for an E1b enzyme is meant the capacity , of reacting preferably 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid-ACP to hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[00027] an E1c enzyme with polypeptide sequence SEQ ID NO: 78 or with a polypeptide sequence, wherein up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the reference sequence SEQ ID NO: 78, are modified by deletion, insertion, substitution or by a combination thereof and which still has at least 10%, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme with the reference sequence SEQ ID NO: 78, whereby by enzymatic activity for an E1c enzyme is meant the capacity , of reacting preferably 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid-ACP to hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[00028] an E1d enzyme with polypeptide sequence SEQ ID NO: 80 or with a polypeptide sequence, wherein up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the reference sequence SEQ ID NO: 80, are modified by deletion, insertion, substitution or by a combination thereof and which still has at least 10%, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme with the reference sequence SEQ ID NO: 80, whereby by enzymatic activity for an E1d enzyme is meant the capacity , of reacting preferably 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid-ACP to hydroxytetradecanoyl-3-hydroxytetradecanoic acid and
[00029] an E1e enzyme with polypeptide sequence SEQ ID NO: 82 or with a polypeptide sequence, wherein up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the reference sequence SEQ ID NO: 82, are modified by deletion, insertion, substitution or by a combination thereof and which still has at least 10%, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme with the reference sequence SEQ ID NO: 82, whereby by enzymatic activity for an E1e enzyme is meant the capacity , of reacting preferably 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid-ACP to hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[00030] at least one E2 enzyme with polypeptide sequence selected from
[00031] an E2a enzyme with polypeptide sequence SEQ ID NO: 4 or with a polypeptide sequence, wherein up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7 , 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the reference sequence SEQ ID NO: 4, are modified by deletion, insertion, substitution or by a combination thereof and which still has at least 10 %, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme with the reference sequence SEQ ID NO: 4, whereby by enzymatic activity for an E2a enzyme is meant the capacity, of preferably reacting dTDP-rhamnose and 3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,
[00032] an E2b enzyme with polypeptide sequence SEQ ID NO: 20 or with a polypeptide sequence, wherein up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7 , 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the reference sequence SEQ ID NO: 20, are modified by deletion, insertion, substitution or by a combination thereof and which still has at least 10 %, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme with the reference sequence SEQ ID NO: 20, whereby by enzymatic activity for an E2b enzyme is meant the capacity, of preferably reacting dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[00033] an E2c enzyme with polypeptide sequence SEQ ID NO: 84 or with a polypeptide sequence, wherein up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7 , 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the reference sequence SEQ ID NO: 84, are modified by deletion, insertion, substitution or by a combination thereof and which still has at least 10 %, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme with the reference sequence SEQ ID NO: 84, whereby by enzymatic activity for an E2c enzyme is meant the capacity, of preferably reacting dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,
[00034] an E2d enzyme with polypeptide sequence SEQ ID NO: 86 or with a polypeptide sequence, wherein up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7 , 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the reference sequence SEQ ID NO: 86, are modified by deletion, insertion, substitution or by a combination thereof and which still has at least 10 %, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme with the reference sequence SEQ ID NO: 86, whereby by enzymatic activity for an E2d enzyme is meant the capacity, preferably reacting dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid and
[00035] an E2e enzyme with polypeptide sequence SEQ ID NO: 88 or with a polypeptide sequence, wherein up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7 , 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the reference sequence SEQ ID NO: 88, are modified by deletion, insertion, substitution or by a combination thereof and which still has at least 10 %, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme with the reference sequence SEQ ID NO: 88, whereby by enzymatic activity for an E2e enzyme is meant the capacity, preferably reacting dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid and
[00036] at least one E3 enzyme selected from
[00037] an E3a enzyme with polypeptide sequence SEQ ID NO: 6 or with a polypeptide sequence, wherein up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7 , 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the reference sequence SEQ ID NO: 6, are modified by deletion, insertion, substitution or by a combination thereof and which still has at least 10 %, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme with the reference sequence SEQ ID NO: 6, whereby by enzymatic activity for an E3a enzyme is meant the capacity, of reacting preferably 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,
[00038] an E3b enzyme with polypeptide sequence SEQ ID NO: 22 or with a polypeptide sequence, wherein up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7 , 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the reference sequence SEQ ID NO: 22, are modified by deletion, insertion, substitution or by a combination thereof and which still has at least 10 %, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme with the reference sequence SEQ ID NO: 22, whereby by enzymatic activity for an E3b enzyme is meant the capacity, of reacting preferably 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,
[00039] an E3c enzyme with polypeptide sequence SEQ ID NO: 90 or with a polypeptide sequence, wherein up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7 , 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the reference sequence SEQ ID NO: 90, are modified by deletion, insertion, substitution or by a combination thereof and which still has at least 10 %, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme with the reference sequence SEQ ID NO: 90, whereby by enzymatic activity for an E3c enzyme is meant the capacity, of reacting preferably 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
[00040] an E3d enzyme with polypeptide sequence SEQ ID NO: 92 or with a polypeptide sequence, wherein up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7 , 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the reference sequence SEQ ID NO: 92, are modified by deletion, insertion, substitution or by a combination thereof and which still has at least 10 %, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme with the reference sequence SEQ ID NO: 92, whereby by enzymatic activity for an E3d enzyme is meant the capacity, of preferably reacting 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.
[00041] For an overview compare Figure 1.
[00042] By "wild-type" a cell is here designated a cell whose genome is present in a state as is naturally understood through evolution. The term is used both for the whole cell and for individual genes as well. Therefore, by the term "wild-type", those cells or those genes, whose gene sequences have been modified at least partially by man through recombinant processes, do not fall in particular. By the term "rhamnolipid" in the context of the present invention is meant a compound of the General Formula (I) or its salt.
[00043] It is evident, that the activities concretely indicated above for the enzymes E1a to E3b represent only a special model selection from a broader spectrum of activities of the enzymes mentioned above; the respective activity mentioned is the one for which a reliable measurement process is present in the given enzyme. Thus, it is obvious that an enzyme, which is going to react a substrate with a saturated Cyo-alkyl radical, unbranched equally - even if optionally with reduced activity - those substrates. which have a C6- or C16-alkyl radical, which optionally may also be branched or unsaturated.
[00044] The term "increased activity of an enzyme" should preferably be understood as increased intracellular activity.
[00045] The following embodiments for increasing enzyme activity in cells apply both for increasing the activity of the enzyme E1 to C3 as well as for all the enzymes mentioned below, whose activity can be optionally increased.
[00046] Fundamentally, an increase in enzyme activity can be obtained by increasing the copy number of the gene sequence or gene sequences, which encode the enzyme, using a strong promoter or a better ribosome binding point, weakening a down-regulation of gene expression, for example, through transcription regulators or enhancing up-regulation of gene expression, for example, through transcription regulators, modify gene codon utilization, increase the half-value time in different ways of the mRNA or enzyme, modify the regulation of gene expression or use a gene or allele, which encodes a corresponding enzyme with increased activity, and optionally combine these measures. Genetically modified cells according to the invention are produced, for example, by transformation, transduction, conjugation or by a combination of the same methods with a vector, which contains the desired gene, an allele of that gene or parts thereof and optionally a promoter that enables the expression of the gene. Heterologous expression is obtained, especially, through the integration of the gene or alleles into the chromosome of cells or into a vector that replicates extrachromosomally. An overview of the possibilities for increasing enzyme activity in cells in the example of pyruvate-carboxylase is provided by DE-A 100 31 999 which is hereby introduced as a reference and whose published content with respect to possibilities for increasing the Enzyme activity in cells forms a part of the publication of the present invention.
[00047] The expression of the enzymes or genes mentioned above and all below can be detected with the aid of separation with one- and two-dimensional protein gel and subsequent optical identification of the protein concentration with corresponding evaluation software on the gel. When the increase in an enzyme activity is based solely on an increase in the expression of the corresponding gene, then the quantification of the increase in enzyme activity can be determined simply by a comparison of one- or two-dimensional protein gel separations between wild type and the genetically modified cell. A common method for preparing protein gels in coryneform bacteria and for protein identification is the procedure described by Hermann et al. (Electrophoresis, 22: 1712.23(2001)). Protein concentration can be similarly analyzed by Western-Blot hybridization with 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, NY USA, 1989) and subsequent optical evaluation with corresponding software for the determination of concentration (Lohaus and Meyer (1989) Biospektrum, 5: 32-39; Lottspeich (1999) Angewandte Chemie 111: 2630-2647). The activity of DNA-binding proteins can be measured using the DNA-Band-Shift assay (also called gel retardation) (Wilson et al. (2001) Journal of Bacteriology, 183: 21512155). The effect of DNA-binding proteins on the expression of other genes can be detected by several 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, NY USA, 1989). Intracellular enzymatic activities can be determined by several methods described (Donahue et al. (2000) Journal of Bacteriology 182 (8): 22772284; Freedberg et al. (1973) Journal of Bacteriology 115 (3): 816-823). Provided that in the following embodiments no concrete methods are indicated for determining the activity of a certain enzyme, the determination of the increase in enzymatic activity and also the determination of the reduction of an enzymatic activity is preferably carried out by means of the methods described in Hermann and collaborators, Electophoresis, 22: 1712-23 (2001), Lohaus and collaborators, Biospektrum 5 32-39 (1998), Lottspeich, Angewandte Chemie 111: 2630-2647 (1999) and Wilson and collaborators, Journal of Bacteriology 183: 2151- 2155 (2001). If the increase in enzymatic activity is accomplished through endogenous gene mutation, then such mutations can be produced or undirected by classical methods, such as through UV irradiation or through chemicals that cause mutation or targeted through engineering methods genetics such as deletion(s), insertion(s) and/or exchange(s) of nucleotides. Through these mutations, modified cells are obtained. Particularly preferred enzyme mutants are, especially, also those enzymes, which are no more, or at least compared to the wild-type enzyme, poorly feedback, product or substrate inhibitory.
[00048] If the increase in enzyme activity is carried out by increasing the synthesis of an enzyme, then, for example, the copy number of the corresponding genes is increased or the promoter and regulatory region or the ribosome binding point, which is located upstream of the structural gene is mutated. The expression cassettes act in the same way, which are embedded upstream of the structural gene. Through inducible promoters it is additionally possible to increase expression at any desired time. Furthermore, the enzyme gene can also be attributed, however, so-called "enhancers" as regulatory sequences, which also cause an increased expression of the gene through an improved interaction between RNA polymerase and DNA. Through measures to extend the lifespan of mRNAs, expression is similarly improved. Furthermore, by preventing enzymatic protein degradation, enzymatic activity is also enhanced. The genes or gene structures are present, in this case, in plasmids with different copy numbers or are integrated and amplified in the chromosome. Alternatively, in addition, an overexpression of the respective genes can be obtained by modifying the composition of the medium and the conduct of the culture. Instructions for this purpose can be found in Martin et al., (Bio/Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya und Morinaga (Bio/Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in EP-A-0 472 869, in US 4,601,893, in Schwarzer und Pühler (Bio /Technology 9, 8487 (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. (Gene 134, 15-24 (1993)), in JP-A-10-229891, in Jensen und Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)) and in textbooks known from genetics and molecular biology.The measures mentioned above lead, as well as mutations, to genetically modified cells.
[00049] To increase the expression of the respective genes, for example, episomal plasmids are used. Plasmids or vectors include, in principle, all embodiments available to the person skilled in the art for this purpose. Such plasmids and vectors can be shown, for example, in the brochures of Novagen, Promega, New England Biolabs, Clontech or Gibco BRL. Other preferred plasmids and vectors can be found in: Glover, D.M. (1985) DNA cloning: a practical approach, Vol. I-III, 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.
[00050] The plasmid vector, which contains the gene to be amplified, is then converted, through conjugation or transformation, into the desired strain. The conjugation method is described, for example, in Schafer 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 und Shivnan, Bio/Technology 7: 1067-1070 (1989) and Tauch et al., FEMS Microbiology Letters 123: 343-347 (1994). After homologous recombination via a "cross-over" episode, the resulting strain contains at least two copies of the respective gene. By the formulation used in the above and below embodiments, "an increased activity of an Ex enzyme compared to its wild type" is preferably always meant an increased activity of the respective Ex enzyme by a factor of at least 2, particularly preferably of at least 10, furthermore preferably at least 100, further still more preferably at least 1,000 and most preferably at least 10,000. Furthermore, the cell according to the invention, which exhibits "an increased activity of an Ex compared to its wild type", especially comprises also a cell whose wild type does not or at least show any detectable activity of this enzyme Ex e that only after increasing enzymatic activity, for example through overexpression, shows a detectable activity of that Ex enzyme. In this context, the term "overexpression" or the formulation used in the embodiments below "increased expression" also comprises the In this case, a starting cell, for example a wild-type cell, shows no or at least no detectable expression and a detectable synthesis of the Ex enzyme is induced first by recombinant processes.
[00051] Amino acid residue modifications of a given polypeptide sequence, which do not lead to any substantial modifications of the properties and function of the given polypeptide, are known to the skilled person. So, for example, so-called conserved amino acids can be exchanged for one another; 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 by Asp; Gly by Pro; His for Asn or Gln; Ile by Leu or Val; Read by Met or Val; Lys for Arg or Gln or Glu; Met by Leu or Ile; Phe by Met or Leu or Tyr; Be by Thr; Thr for Ser; Trp by Tyr; Tyr by Trp or Phe; Val for Ile or Leu. Likewise, it is known that modifications, particularly at the N- or C-terminus of a polypeptide in the form of, for example, amino acid insertions or deletions, often do not exert any substantial influence on the function of the polypeptide.
[00052] The activity of an enzyme can be determined in which cells, which contain this activity, are broken down in a manner and manner known to the expert, for example, with the aid of a ball mill, a French press or an ultrasound disintegrator and Then, intact cells, cell pieces and auxiliary unfolding agents, such as glass beads, are separated by means of centrifugation for 10 minutes at 13,000 rpm and 4°C. With the resulting cell-free crude extract, enzymatic assays can then be performed with subsequent LC-ESI-MS detection of the products. Alternatively, the enzyme can be enriched in a manner and manner known to the skilled person by means of chromatographic methods (such as nickel-nitrilotriacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography or ion exchange chromatography) or it can also be purified to homogeneity.
[00053] The E1 enzyme activity is then determined with the samples obtained as described above, as follows: 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 E. coli FabD, 2 μg Mycobacterium tuberculosis FabH, 1 μg E. coli FabG, 0.1 M sodium phosphate buffer, pH 7.0 and 5 μg E1 enzyme in a final volume of 120 μL. ACP, β-mercaptoethanol and sodium phosphate buffer are pre-incubated at 37oC for 30 minutes to completely reduce ACP. The reaction is started by adding E1 enzyme. The reactions are stopped with 2 ml of water, which has been acidified to pH 2.0 with HCl and then extracted twice with 2 ml of chloroform/methanol (2:1 (v:v)). Phase separation is carried out by centrifugation (16,100 g, 5 min, room temperature). The lower organic phase is removed, completely evaporated in the vacuum centrifuge and the sediment is taken up in 50 µL of methanol. Undissolved components are sedimented by centrifugation (16,000 g, 5 minutes, room temperature) and the sample is analyzed by LC-ESI-MS. The identification of the products is carried out through the analysis of the respective mass traces and MS2 spectra.
[00054] The E2 enzyme activity is then determined with the samples obtained as described above, as follows: a standard assay may consist of 185 µL 10 mM Tris-HCL (pH 7.5), 10 µL 125 mM of dTDP-rhamnose and 50 μL of crude protein extract (about 1 mg of total protein) or purified protein in solution (5 μg of purified protein). The reaction is started by adding 10 μL 10 mM ethanolic solution of 3-hydroxydecanoyl-3-hydroxydecanoic acid (for E2a) or 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid (for E2b) and incubated for 1 hour at 30°C under agitation (600 rpm). Then the reaction is added to 1 ml of acetone. Undissolved components are sedimented by centrifugation (16,100 g, 5 minutes, room temperature) and the sample is analyzed using LS-ESI-MS. The identification of the products is carried out through the analysis of the respective mass traces and MS2 spectra.
[00055] The E3 enzyme activity is then determined with the samples obtained as described above, as follows: a standard assay may consist of 185 µL 10 mM Tris-HCL (pH 7.5), 10 µL 125 mM of dTDP-rhamnose and 50 μL of crude protein extract (about 1 mg of total protein) or purified protein in solution (5 μg of purified protein). The reaction is started by adding 10 μL 10 mM ethanolic solution of α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid (for E3a) or α-L-rhamnopyranosyl-33-hydroxytetradecanoyl-3-hydroxytetradecanoic acid (for E3b) and incubated for 1 hour at 30°C under agitation (600 rpm). Then the reaction is added to 1 ml of acetone. Undissolved components are sedimented by centrifugation (16,100 g, 5 minutes, room temperature) and the sample is analyzed using LS-ESI-MS. The identification of the products is carried out through the analysis of the respective mass traces and MS2 spectra.
[00056] According to the invention, cells are preferred, which show increased activities of the following enzyme combinations:
[00057] E1, E2, E3, E1E2, E1E3, E2E3 and E1E2E3,
[00058] of which the combination
[00059] E2, E2E3 and E1E2E3, especially E1E2E3
[00060] is particularly preferred.
[00061] In a preferred embodiment of the cell according to the invention, which presents an increased activity of the combination of E1E2E3 enzymes, n is preferably = 1.
[00062] The cells according to the invention can be prokaryotes or eukaryotes. In this case, it may be mammalian cells (such as human cells), plant cells or microorganisms such as yeasts, fungi or bacteria, with microorganisms being particularly preferred and bacteria and yeasts are the most preferred.
[00063] As bacteria, yeasts or fungi, especially those bacteria, yeasts or fungi, which are deposited with the Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Braunschweig, Germany, as strains of bacteria, yeasts or fungi are suitable. Suitable bacteria according to the invention belong to the genera, which are listed under
[00064] http://www.dsmz.de/species/bacteria.htm,
[00065] Suitable yeasts according to the invention belong to those groups, which are listed under
[00066] http://www.dsmz.de/species/yeasts.htm
[00067] and suitable fungi according to the invention are those, which are listed under
[00068] http://www.dsmz.de/species/fungi.htm.
[00069] Preferred cells according to the invention are those of the genus Aspergillus, Corynebacterium, Brevibacterium, Bacillus, Acinetobacter, Alcaligenes, Lactobacillus, Paracoccus, Lactococcus, Candida, Pichia, Hansenula, Kluyveromyces, Saccharomyces, Escherichia, Marrow, Zymobacterium Ralstonia, Pseudomonas, Rhodospirillum, Rhodobacter, Burkholderia, Clostridium und Cupriavidus, and Aspergillus nidulans, Aspergillus niger, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, B. cally. 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 , Cand ida rugosa, Corynebacterium glutamicum, Corynebacterium efficiens, Escherichia coli, Hansenula polymorpha, Kluveromyces lactis, Methylobacterium extorquens, Paracoccus versutus, Pseudomonas argentinensis, P. borbori, P. citronellolis, P. , P. pseudoalcaligenes, P. resinovorans, P. straminea, P. aurantiaca, P. aureofaciens, P. chlororaphis, P. fragi, P. lundensis, P. taetrolens, P. antarctica, P.nitroformans, '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 , 'FOR. helianthi', P. meliae, P. savastanoi, P. syringae, P. tomato, P. viridiflava, P. abietaniphila, P. acidophila, P. agarici, P. alcaliphila, P. alkanolytica, P. amyloderamosa, P. aspleniiphila , P. azotifigens, P. cannabina, P. coenobios, P. freezens, 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, Rho dobacter sphaeroides, Saccharomyces cerevisiae, Yarrowia lipolytica und Zymomonas mobilis,
[00070] especially Pseudomonas putida, Escherichia coli and Burkholderia thailandensis are particularly preferred.
[00071] Preferred cells according to the invention as wild type, are not capable of forming any or which detectable amounts of rhamnolipids and, furthermore, as wild type, they preferably do not show any or any detectable activity of the enzymes E1, E2 and E3 .
[00072] According to the invention it is advantageous if the cell according to the invention is a cell which, as wild type, are capable of forming polyhydroxyalkanoates with monoalkanoate chain lengths 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 citronellolis, Pseudomonas aeruginosa, Pseudomonas hydromonas resinamo and Ralstonia eutropha. In this context, the preferred cells according to the invention are genetically modified in such a way that they are able to form less polyhydroxyalkanoates compared to their wild type.
[00073] 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.
[00074] Such a cell, compared to its wild type, which is capable of forming less polyhydroxyalkanoates, is especially characterized by the fact that, compared to its wild type, it shows a reduced activity of at least one E9 or E10 enzyme,
[00075] wherein E9 represents a polyhydroxyalkanoate synthase, EC:2.3.1.-, especially with polypeptide sequence SEQ ID NO: 30 or SEQ ID NO: 32 or with a polypeptide sequence, where up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues with respect 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 which further shows at least 10%, preferably 50%, particularly preferably 80%, especially more than 90% of the activity enzyme of the enzyme with the respective reference sequence SEQ ID NO: 30 or SEQ ID NO: 32, whereby by enzymatic activity for an E9 enzyme is meant the ability to react 3-hydroxyalkanoyl-coenzyme A to poly-3-hydroxyoicoalkanoic acid , especially 3-hydroxytetradecanoyl-coenzyme A for poly-3-hydroxytetradecanoic acid and
[00076] E10 represents a 3-hydroxyalkanoyl-ACP:coenzyme A transferase, especially with polypeptide sequence SEQ ID NO: 34 or SEQ ID NO: 36 or with a polypeptide sequence in which up to 25%, preferably up to 20%, of particularly preferably up to 15%, especially up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the respective reference sequence SEQ ID NO: 34 or SEQ ID NO : 36, are modified by deletion, insertion, substitution or a combination thereof and which further exhibits at least 10%, preferably 50%, particularly preferably 80%, especially more than 90% of the enzyme activity of the enzyme with the respective reference sequence SEQ ID NO: 34 or SEQ ID NO: 36, whereby by enzymatic activity for an E10 enzyme is meant the ability to react 3-hydroxyalkanoyl-ACP to 3-hydroxyalkanoyl-coenzyme A, especially 3-hydroxyalkanoyl-ACP for 3-hydroxytetradecanoyl-coenzyme A.
[00077] For an overview compare Figure 1.
[00078] The activity of the E9 enzyme is then determined with the samples obtained described as above for the enzymes E1 to E3, where initially 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 are mixed. Then, 5 μg of E9 enzyme in 100 μL of tris/HCl, pH 7.5 is added and then the extinction enhancement is continuously recovered for 1 minute in the spectrophotometer at 412 nm (caused by the addition of 5.5' -dithiobis(2-nitrobenzoate) (DTNB) in free SH groups) over time (ΔE/min).
[00079] The activity of the enzyme E10 is then determined with the obtained samples described as above for the enzymes E1 to E3. The standard assay contains 3 mM MgCh, 40 μM hydroxydecanoyl-coenzyme A and 20 μM E. coli ACP in 50 mM Tris-HCl, pH 7.5, in a total volume of 2000 μL. The reaction is started by adding 5 μg of purified E10 enzyme in 50 μL of tris/HCl, pH 7.5 and incubated at 30oC for 1 hour. The reaction is stopped by adding 50% trichloroacetic acid and 10 mg/mL BSA (30 µL). The released coenzyme A is determined spectrometrically, in which the increase in extinction at 412 nm, caused by the addition of 5,5'-dithiobis(2-nitrobenzoate) (DTNB) in free SH groups) is recovered over time (ΔE/min ).
[00080] In the formulation used, it is meant by "reduced activity of an Ex enzyme", therefore preferably an activity reduced by at least 0.1, moreover, preferably by at least 0.01, moreover, even more preferably of at least 0.001 and most preferred of at least 0.0001. The formulation "reduced activity" also does not contain any detectable activity ("zero activity"). The reduction of the activity of a particular enzyme can be carried out, for example, through specific mutation or by other measures known to the expert to reduce the activity of a particular enzyme. Processes to reduce enzymatic activities in microorganisms are known to the expert.
[00081] Special molecular biology technologies are offered here. Instruction for the modification and reduction of protein expressions and associated enzyme activity reductions especially for Pseudomonas and Burkholderia, especially for disrupting special genes, the expert finds, for example, in Dubeau et al., 2009. BMC Microbiology 9:263; Singh & Rohm. Microbiology. 2008. 154:797-809 or Lee et al. FEMS Microbiol Lett. 2009. 297(1):38-48.
[00082] Preferred cells according to the invention are characterized by the fact that the reduction of enzymatic activity is obtained by modifying a gene comprising one of the aforementioned nucleic acid sequences, wherein the modification is selected from the group comprising, preferably consisting of insertion of exogenous DNA into 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 promoters and terminators or ribosome binding points, which flank the gene.
[00083] By exogenous DNA in this context, it is to be understood that DNA sequence, which is "exogenous" to the gene (and not to the organism), that is, also endogenous DNA sequences can act in this context as "exogenous DNA".
[00084] In this context it is especially preferable that the gene is interrupted by the insertion of a selection marker gene, therefore, the exogenous DNA is a selection marker gene, where preferably the insertion was carried out through homologous recombination at the gene site .
[00085] In a preferred embodiment of the cell according to the invention, the cell is Pseudomonas putida cells, which present a reduced synthesis of polyhydroxyalkanoate compared to its wild type. Such cells are described, for example, in Ren et al., Journal Applied Microbiology and Biotechnology 1998 Jun, 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 Microbiol. 2010. 12(1):207-21 as KT42C1 and in Ouyang et al. Macromol Biosci. 2007. 7(2):227-33 as KTOY01 and KTOY02 and represent preferred cells according to the invention.
[00086] In case the cell according to the invention is capable of forming a rhamnolipid with m=1, it is preferable that the radical determined by R1 and R2

[00087] is derived from hydroxyoctanoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydecenoic acid, 3-hydroxydecenoyl-3-hydroxyoctanoic acid, 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-hydroxydodecanoic acid, 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-hydroxy-hexadecanoyl-3-hydroxytetradecanoic acid,
[00088] 3-hydroxytetradecanoyl-3-hydroxy-hexadecanoic acid or 3-hydroxy-hexadecanoyl-3-hydroxy-hexadecanoic acid.
[00089] It is evident to the expert that a cell according to the invention is also capable of forming mixtures of several rhamnolipids of Formula General Formula (I). In this context it is stated that the cells according to the invention are capable of forming mixtures of rhamnolipids of the General Formula (I), which are characterized by the fact that in more than 80% by weight, preferably in more than 90% by weight particularly preferably more than 95% by weight of the formed rhamnolipids, n is 1 and the radical determined by R1 and R2 is less than 10% by weight, preferably less than 5% by weight, particularly preferably less than 2% by weight of the formed rhamnolipids is derived from 3-hydroxydecanoyl-3-hydroxyoctanoic acid or 3-hydroxyoctanoyl-3-hydroxydecanoic acid, the % by weight indicated referring to the sum of all formed rhamnolipids of the General Formula ( I).
[00090] It is advantageous, if the cell according to the invention was additionally genetically modified in relation to E1 to E3 in such a way that, compared to its wild type, it presents an increased activity, as specified below, of at least minus one of the enzymes selected from the group consisting of
[00091] at least one E4 enzyme, a dTTP:α-glucose-1-phosphate thymidylyltransferase, EC 2.7.7.24, especially one with a polypeptide sequence SEQ ID NO: 10 or with a polypeptide sequence, where up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues relative to the reference sequence SEQ ID NO: 10 are modified by deletion, insertion, substitution or a combination thereof and which further exhibits at least 10%, preferably 50%, particularly preferably 80%, especially more than 90% of the enzyme activity of the enzyme with the sequence of reference SEQ ID NO: 10, whereby enzymatic activity for an E4 enzyme is understood as the ability to react α-D-glucose-1-phosphate and dTTP to dTDP-glucose,
[00092] at least one E5 enzyme, a dTTP-glucose-4,6-hydrolase, EC 4.2.1.46, especially one with a polypeptide sequence SEQ ID NO: 12 or with a polypeptide sequence, wherein up to 25% preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the reference sequence SEQ ID NO: 12 are modified by deletion, insertion, substitution or a combination thereof and which further exhibits at least 10%, preferably 50%, particularly preferably 80%, especially more than 90% of the enzyme activity of the enzyme with the reference sequence SEQ ID NO: 12, whereby by enzymatic activity for an E5 enzyme is meant the ability to react dTDP-glucose to dTDP-4-dehydro-6-deoxy-D-glucose,
[00093] at least one E6 enzyme, a dTDP-4-dehydrorhamnose-3,5-epimerase, EC 5.1.3.13, especially one with a polypeptide sequence SEQ ID NO: 14 or with a polypeptide sequence, where up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues relative to the reference sequence SEQ ID NO: 14 are modified by deletion, insertion, substitution or a combination thereof and which further shows at least 10%, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme with the reference sequence SEQ ID NO: 14, whereby by enzymatic activity for an E6 enzyme is meant the ability to react dTDP-4-dehydro-6-deoxy-D-glucose to dTDP-4-dehydro- 6--deoxy-L-mannose and
[00094] at least one E7 enzyme, a dTDP-4-dehydrorhamnose reductase, EC 1.1.1.133, especially one with a polypeptide sequence SEQ ID NO: 16 or with a polypeptide sequence, wherein up to 25% preferably up to 20%, particularly preferably up to 15%, especially up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the reference sequence SEQ ID NO: 16 are modified by deletion, insertion, substitution or a combination thereof and which further exhibits at least 10%, preferably 50%, particularly preferably 80%, especially more than 90% of the enzyme activity of the enzyme with the reference sequence SEQ ID NO: 16, whereby by enzymatic activity for an E7 enzyme is meant the ability to react dTDP-4-dehydro-6-deoxy-L-mannose to dTDP-6-deoxy-L-mannose.
[00095] The activity of the E4 enzyme is determined with the samples obtained as described above for the enzymes E1 to E3, where α-D-glucose-1-phosphate (1.3 mM) is incubated with dTTP (5 mM) and 5 mg of purified E4 enzyme in 50 μL of sodium phosphate buffer, pH 8.5 and after incubation for 5, 10 and 20 minutes at 30oC, the reaction is stopped by adding 20 μL of chloroform. The mixture is then vortexed and centrifuged for 5 minutes at 16,000 g at room temperature. The aqueous phase is transferred to a new reaction vessel and the organic phase is again extracted with 80 µL of water. The two aqueous phases are combined and analyzed by means of HPLC. In that case, a Phenosphere-ODS2 column (250 x 4.6 mm; Phenomenex, Torrance, USA) or a Spheresorb-ODS2 column (250 x 4.6 mm; Waters, Milford, USA) is used. The analytes are eluted with a flow rate of 1 mL min-1 with 0.5 M KH2PO4 (eluent A) for 15 minutes, followed by a linear gradient of up to 80% eluent A and 20% methanol during a period of 14 minutes with a flow rate of 0.7 ml min-1. Analytes, which elute from the ODS2 columns, are then injected into a Phenosphere-SAX ion exchange column (250 x 4.6 mm; Phenomenex, Torrance, USA) and the analytes are eluted at a flow rate of 1 mL min-1 and with a linear gradient of ammonium formate (2 to 600 mM over 25 minutes). The quantification of dTDP-glucose is then carried out through its UV absorption with a diode array detector (DAD). The maximum absorption of thymidine is found at 267 nm. Calibration is performed using authentic nucleotide sugar (Sigma-Aldrich, Munich, USA).
[00096] The activity of the E5 enzyme is then determined with the samples obtained as described above for the enzymes E1 through E3, in which dTDP-α-D-glucose (1.3 mM) is incubated with 5 μg of enzyme E5 purified in 50 μL of sodium phosphate buffer, pH 8.5 and after incubation for 5, 10 and 20 minutes at 30oC, the reaction is stopped by adding 20 μL of chloroform. The mixture is then vortexed and centrifuged for 5 minutes at 16,000 g at room temperature. The aqueous phase is transferred to a new reaction vessel and the organic phase is again extracted with 80 µL of water. The two aqueous phases are combined and analyzed by means of HPLC. In that case, a Phenosphere-ODS2 column (250 x 4.6 mm; Phenomenex, Torrance, USA) or a Spheresorb-ODS2 column (250 x 4.6 mm; Waters, Milford, USA) is used. The analytes are eluted with a flow rate of 1 mL min-1 with 0.5 M KH2PO4 (eluent A) for 15 minutes, followed by a linear gradient of up to 80% eluent A and 20% methanol during a period of 14 minutes with a flow rate of 0.7 ml min-1. The analytes, which elute from the ODS2 columns, are then injected into a Phenosphere-SAX ion exchange column (250 x 4.6 mm; Phenomenex, Torrance, USA) and the analytes are eluted at a flow rate of 1 mL min-1 and with a linear gradient of ammonium formate (2 to 600 mM over 25 minutes). The quantification of dTDP-glucose and dTDP-4-dehydro-6-deoxy-D-glucose is then carried out through their UV absorption with a diode array detector (DAD). The maximum absorption of thymidine is found at 267 nm. Calibration is performed using authentic nucleotide sugar (Sigma-Aldrich, Munich, USA).
[00097] The activity of the E6 enzyme is then determined with the samples obtained as described above for the enzymes E1 to E3, in which initially dTDP-α-D-glucose (1.3 mM) is incubated with 5 μg of purified E5 enzyme in 50 μL of sodium phosphate buffer, pH 8.5, for 10 minutes at 30oC. Then, 0.5 μg of purified E6 enzyme is added and after incubation for 5, 10 and 20 minutes at 30oC, the reaction is stopped by adding 20 μL of chloroform. The mixture is then vortexed and centrifuged for 5 minutes at 16,000 g at room temperature. The aqueous phase is transferred to a new reaction vessel and the organic phase is again extracted with 80 µL of water. The two aqueous phases are combined and analyzed by means of HPLC. In that case, a Phenosphere-ODS2 column (250 x 4.6 mm; Phenomenex, Torrance, USA) or a Spheresorb-ODS2 column (250 x 4.6 mm; Waters, Milford, USA) is used. The analytes are eluted with a flow rate of 1 mL min-1 with 0.5 M KH2PO4 (eluent A) for 15 minutes, followed by a linear gradient of up to 80% eluent A and 20% methanol during a period of 14 minutes with a flow rate of 0.7 ml min-1. The analytes, which elute from the ODS2 columns, are then injected into a Phenosphere-SAX ion exchange column (250 x 4.6 mm; Phenomenex, Torrance, USA) and the analytes are eluted at a flow rate of 1 mL min-1 and with a linear gradient of ammonium formate (2 to 600 mM over 25 minutes). The quantification of dTDP-glucose, dTDP-4-dehydro-6-deoxy-D-glucose and dTDP-6-deoxy-L-mannose is then carried out through their UV absorption with a diode array detector ( DAD). The maximum absorption of thymidine is found at 267 nm. Calibration is performed using authentic nucleotide sugar (Sigma-Aldrich, Munich, USA).
[00098] The activity of the E7 enzyme is then determined with the samples obtained as described for the enzymes E1 to E3, in which initially dTDP-α-D-glucose (1.3 mM) is incubated with 5 μg of enzyme E5 purified in 50 μL of sodium phosphate buffer, pH 8.5, for 10 minutes at 30oC. Then, 5 μg of purified E6 enzyme and 0.5 μg of purified E7 enzyme as well as NADPH (10 mM) are added and after incubation for 5, 10 and 20 minutes at 30oC, the reaction is stopped by addition 20 µL of chloroform. The mixture is then vortexed and centrifuged for 5 minutes at 16,000 g at room temperature. The aqueous phase is transferred to a new reaction vessel and the organic phase is again extracted with 80 µL of water. The two aqueous phases are combined and analyzed by means of HPLC. In that case, a Phenosphere-ODS2 column (250 x 4.6 mm; Phenomenex, Torrance, USA) or a Spheresorb-ODS2 column (250 x 4.6 mm; Waters, Milford, USA) is used. The analytes are eluted with a flow rate of 1 mL min-1 with 0.5 M KH2PO4 (eluent A) for 15 minutes, followed by a linear gradient of up to 80% eluent A and 20% methanol during a period of 14 minutes with a flow rate of 0.7 ml min-1. The analytes, which elute from the ODS2 columns, are then injected into a Phenosphere-SAX ion exchange column (250 x 4.6 mm; Phenomenex, Torrance, USA) and the analytes are eluted at a flow rate of 1 mL min-1 and with a linear gradient of ammonium formate (2 to 600 mM over 25 minutes). 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 is performed , then through its UV absorption with a diode array detector (DAD). The maximum absorption of thymidine is found at 267 nm. Calibration is performed using authentic nucleotide sugar (Sigma-Aldrich, Munich, USA).
[00099] According to the invention, cells are preferred, which show increased activities of the following enzyme combinations:
[000100] E4E5, E4E6, E4E7, E5E6, E5E7, E6E7, E4E5E6, E4E5E7, E5E6E7, E4E6E7, E4E5E6E7,
[000101] of which the combination
[000102] E4E5E6E7
[000103] is particularly preferred.
[000104] According to the invention it may be advantageous, if the cell according to the invention in the fatty acid biosynthesis was genetically modified in such a way that the enzymatic reactions, which lead to the reaction of acyl-ACP and malonyl-coenzyme A for 3-ketoacyl-ACP, they are reinforced. Additionally or alternatively, it may be advantageous according to the invention, if the cell according to the invention in fatty acid biosynthesis has been genetically modified in such a way that the enzymatic reactions, which lead to the reaction of (R)-3-hydroxyalkanoyl -ACP to trans-2-enoyl-ACP and/or the reaction of trans-2-enoyl-ACP to acyl-ACP are weakened.
[000105] Likewise it may be advantageous, if the cell according to the invention in the e-oxidation of fatty acids has been genetically modified in such a way, that the enzymatic reactions, which lead to the reaction of acyl-coenzyme A to trans-2 -enoyl-coenzyme A and/or the reaction of trans-2-enoyl-coenzyme A to (S)-3-hydroxyalkanoyl-coenzyme A, are reinforced. Additionally or alternatively, it may be advantageous according to the invention, if the cell according to the invention in the β-fatty acid oxidation has been genetically modified in such a way that the enzymatic reactions, which lead to the reaction of (S)-3 - hydroxyalkanoyl-coenzyme A to 3-ketoacyl-coenzyme A and/or the reaction of 3-ketoacyl-coenzyme A to acyl-coenzyme A and acetyl-coenzyme A are weakened.
[000106] For an overview compare Figure 1.
[000107] Since the cells according to the invention can be used advantageously for the production of rhamnolipids and since these lipids are then optionally purified, it is advantageous if the cells according to the invention show an activity of at least one E8 enzyme relative to its wild type, which catalyzes the export of a rhamnolipid of General Formula (I) from the cell to the environment.
[000108] In this context E8 proteins selected from the group consisting of
[000109] an E8 enzyme with polypeptide sequence SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 or with a polypeptide sequence, wherein up to 25%, preferably up to 30% , particularly preferably up to 25%, especially up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues with respect to the respective reference sequence SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 are modified by deletion, insertion, substitution or a combination thereof and which further shows at least 50%, preferably 65%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme with the respective reference sequence SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28, whereby by enzymatic activity for an E8 enzyme is meant the ability, to export a rhamnolipid of General Formula (I) from the cell to the environment.
[000110] Another preferred embodiment of the cells according to the invention is characterized in that it presents at least one of the nucleic acids or vectors according to the invention mentioned below.
[000111] Cells according to the invention can be used advantageously for the production of rhamnolipids.
[000112] Thus, another object of the invention is the use of the cells according to the invention for the production of compounds of Formula General Formula (I).
[000113] Another objective of the present invention is a process for the production of rhamnolipids of the General Formula (I), in which
[000114] m = 2, 1 or 0, especially 1 or 0,
[000115] n = 1 or 2, especially 1,
[000116] R1 and R2 = independently of each other, an organic radical the same or different with 2 to 24, preferably 5 to 13 carbon atoms, especially an alkyl radical especially optionally branched, optionally substituted, especially substituted by hydroxy, optionally unsaturated, especially optionally one, two or three times unsaturated, preferably that selected from the group consisting of pentyl, heptenyl, nonenyl, undecenyl and tridecenyl and (CH2)o-CH3 with being 1 to 23, preferably 4 to 12, comprising the stages of process
[000117] contacting the cells according to the invention with a medium containing a carbon source
[000118] cultivate the cells under conditions that allow the cell to form rhamnolipid from the carbon source and
[000119] optionally isolating the formed rhamnolipids.
[000120] The genetically modified cells according to the invention can be contacted continuously or discontinuously in the batch process (batch cultivation) or in the fed-batch process or in the repetitive fed-batch process (repeated -fed-batch) in order to produce the products mentioned above with the nutrient medium and thus be cultivated. A semi-continuous process as described in GB-A-1009370 is also conceivable. A summary of known cultivation methods are described in Chmiel's manual ("Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik" (Gustav Fischer Verlag, Stuttgart, 1991)) or in the Storhas manual ("Bioreaktoren und periphere Einrichtungen", Vieweg Verlag, Braunschweig /Wiesbaden, 1994).
[000121] The culture medium to be used must adequately satisfy the claims of the respective strains. Descriptions of culture media from various yeast strains are contained, for example, in "Nonconventional yeast in biotechnology" (editor Klaus Wolf, Springer-Verlag, Berlin, 1996).
[000122] As a carbon source, carbohydrates can be used, such as, for example, glucose, sucrose, arabinose, xylose, lactose, fructose, maltose, molasses, starch, cellulose and hemicellulose, vegetable and animal oils and greases, such as, for example, soybean oil, thistle oil, peanut oil, hemp oil, jatropha oil, coconut nut fat, pumpkin seed oil, linseed oil, corn oil, poppy oil, evening primrose oil , olive oil, palm kernel oil, palm oil, rapeseed oil, sesame oil, sunflower oil, grape seed oil, walnut oil, wheat bud oil and coconut fat, 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 esters , as well as mixtures of fatty acids, mono, di- and triglycerides with the acids s already mentioned fatty substances, alcohols such as, for example, glycerin, ethanol and methanol, hydrocarbons such as methanol, carbon-containing gases and gas mixtures such as CO, CO2, synthesis and 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. Particularly preferred is the use of carbohydrates, especially monosaccharides, oligosaccharides or polysaccharides, as a carbon source, as described in US 601,494 and US 6,136,576, as well as hydrocarbons, especially alkanes, alkenes and alkynes, as well as acids monocarboxylic derivatives of these and of the mono-, di- and triglycerides derived from these monocarboxylic acids, as well as of glycerin and acetate. The mono-, di- and triglycerides, containing the esterification products of glycine with caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, arachidonic acid, behenic acid, oleic acid, acid linolenic acid, linolenic acid and/or gamma-linolenic acid are very particularly preferred.
[000123] A great advantage of the present invention is that the cells according to the invention are able to form rhamnolipids from the simplest carbon sources, such as, for example, glucose, sucrose or glycerin, so that the supply is not necessary. of long-chain carbon sources in the medium during the process according to the invention. Thus, in case of deficient availability, it is advantageous that the medium in stage I) of the process according to the invention does not contain any or any detectable amounts of carboxylic acids with a chain length greater than six carbon atoms or esters or glycerides that can be derived from those.
[000124] As a source of nitrogen, organic nitrogen compounds such as peptones, yeast extract, meat extract, malt extract, maize, soy flour and urea or inorganic compounds such as ammonium sulfate, chloride ammonium, ammonium phosphate, ammonium carbonate and ammonium nitrate, ammonia, ammonium hydroxide or ammonia water. Nitrogen sources can be used individually or as a mixture.
[000125] As a source of phosphorus phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or their salts containing sodium can be used. The culture medium must also contain metal salts such as, for example, magnesium sulphate or iron sulphate, which are necessary for growth. Finally, essential growth substances such as amino acids and vitamins can be used in addition to the substances mentioned above. In addition, suitable precursors can be added to the culture medium. The raw materials mentioned can be added to the crop in the form of a single preparation or fed during cultivation in an appropriate manner. To control the pH of the culture, basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid are suitably used. To control foam development, defoamers such as, for example, polyglycolic fatty acid esters can be used. To maintain plasmid stability, selectively acting substances such as, for example, antibiotics can be added to the medium. To maintain aerobic conditions, oxygen or oxygenated gas mixtures, such as, for example, air, are introduced into the crop.
[000126] The culture temperature is normally at more than 20oC, preferably at more than 25oC, it can also matter at more than 40oC, and advantageously a cultivation temperature of 95oC is not exceeded, particularly preferably 90oC and more preferably 80°C.
[000127] In stage III) of the process according to the invention, the rhamnolipids formed by the cells can be optionally isolated from the cells and/or the nutrient medium, and for the isolation all methods known to the expert are included for the isolation of low molecular weight substances of complex compositions, such as, for example, filtration, extraction, adsorption (chromatography) or crystallization. In addition, the product phase contains biomass radicals and various impurities, such as oils, fatty acids and other components of the nutrient medium. The separation of impurities is preferably carried out in a solvent-free process. Thus, for example, the product phase can be diluted with water, to facilitate the adjustment of the pH value. Then, the aqueous and product phases can be homogenized, whereby the rhamnolipids are converted to a water-soluble form by lowering or increasing the pH value by means of acids or lyes. Potentially, solubilization of the rhamnolipids in the aqueous phase can be supported by incubation at higher temperatures, for example, at 60 to 90°C and constant mixing. By subsequently raising or lowering the pH value by means of lyes or acids, the rhamnolipids can then be converted again into a water-insoluble form, so that they can be easily separated from the aqueous phase. Then, the product phase can be washed further one or more times with water to remove water-soluble impurities.
[000128] Oil remains, for example, can be separated by extraction using suitable solvents, advantageously by means of organic solvents. As the solvent, powder is an alkane such as, for example, n-hexane.
[000129] The separation of the product from the aqueous phase can be carried out alternatively to the solvent-free process described above, with a suitable solvent, for example, with an ester, such as, for example, with ethyl acetate or butyl acetate. The mentioned extraction stages can be carried out in the desired order. Here, solvents are preferably used, especially organic solvents. The solvent used is n-pentanol. To remove the solvent, for example, a distillation is carried out. Thereafter, the lyophilized product, for example by means of chromatographic methods, can be further purified. At this point, precipitation by suitable solvents, extraction by suitable solvents, complexation, for example, by means of cyclodextrins or cyclodextrin derivatives, crystallization, purification or isolation by means of chromatographic methods or the conversion of rhamnolipids into slightly separable derivatives.
[000130] The rhamnolipids that can be produced with the process according to the invention are also object of the present invention, especially also the mixtures of rhamnolipids described above, which can be produced with the process according to the invention.
[000131] The rhamnolipids and mixtures can be produced with the process according to the invention, can be used advantageously in cleaning agents, in cosmetic or pharmaceutical formulations as well as in plant protection formulations.
[000132] Thus, another objective of the present invention is the use of the rhamnolipids obtained with the process according to the invention, for the preparation of cosmetic, dermatological or pharmaceutical formulations, plant protection formulations, as well as treatment products and cleaning and surfactant concentrates.
[000133] By the term "treatment products" is understood here a formulation, which serves the purpose of containing an article in its original form, to attenuate or avoid the effects of external factors (for example, time, light, temperature, pressure, contamination, chemical reaction with other reactive compounds that come into contact with the article), such as, for example, aging, contamination, material fatigue, bleaching or even improving the desired positive properties of the article. For the last point, it is mentioned, for example, a better shine of the hair or a greater elasticity of the considered article.
[000134] By "plant protection formulations" are understood those formulations, which according to the type of their preparation, are evidently used for the protection of plants; this is especially the case, then, if at least one compound from the classes of herbicides, fungicides, insecticides, acaricides, nematicides, bird-eating protective substances, plant nutritive substances, and soil structure improvers is contained in the formulation. Rhamnolipids produced with the process according to the invention are preferably used according to the invention, and treatment and cleaning products for domestic use, industry, especially for hard surfaces, leather or textiles.
[000135] A contribution to the solution of the task is provided by an isolated nucleic acid, which presents in each case a sequence selected from the three groups [A1 to G1], A2 to G2] and [A3 to G3], in which
[000136] the group [A1 to G1] consists of the following sequences:
[000137] A1a) a sequence according to SEQ ID NO: 1, wherein that sequence encodes a protein, which is capable of
[000138] react 3-hydroxydecanoyl-ACP through 3-hydroxydecanoyl-3-hydroxydecanoyl-ACP to 3-hydroxydecanoyl-3-hydroxydecanoic acid,
[000139] B1a) an intron-free sequence, which is derived from a sequence according to A1a) and which encodes the same protein or peptide as the sequence according to SEQ ID NO:1,
[000140] C1a) a sequence, which encodes a protein or peptide, which comprises the amino acid sequence according to SEQ ID NO: 2 and which is preferably capable of
[000141] react 3-hydroxydecanoyl-ACP through 3-hydroxydecanoyl-3-hydroxydecanoyl-ACP to 3-hydroxydecanoyl-3-hydroxydecanoic acid,
[000142] D1a) a sequence, which is identical with a sequence according to one of the groups A1a) to C1a), particularly preferably according to the A1a group), by at least 70%, particularly preferably in at least 90%, moreover preferably at least 95% and most preferably at least 99%, which sequence preferably encodes a protein or peptide, which is capable of
[000143] react 3-hydroxydecanoyl-ACP through 3-hydroxydecanoyl-3-hydroxydecanoyl-ACP to 3-hydroxydecanoyl-3-hydroxydecanoic acid,
[000144] E1a) a sequence, which hybridizes with the opposite strand of a sequence according to one of the groups A1a) to D1a), particularly preferably according to the A1a group) or would hybridize considering the degeneration of the genetic code, being that such sequence preferably encodes a protein or peptide, which is capable of
[000145] react 3-hydroxydecanoyl-ACP through 3-hydroxydecanoyl-3-hydroxydecanoyl-ACP to 3-hydroxydecanoyl-3-hydroxydecanoic acid,
[000146] F1a) a derivative, obtained through substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, furthermore preferably of at least 5 bases and most preferably of at least less than 10 bases, however, preferably not more than 100 bases, particularly preferably not more than 50 bases and most preferably not more than 25 bases, of a sequence according to one of the groups A1a) to E1a ), particularly preferably according to the A1a group), which derivative preferably encodes a protein or peptide, which is capable of
[000147] react 3-hydroxydecanoyl-ACP through 3-hydroxydecanoyl-3-hydroxydecanoyl-ACP to 3-hydroxydecanoyl-3-hydroxydecanoic acid,
[000148] G1a) a sequence complementary to a sequence according to one of the groups A1a) to F1a), particularly preferably according to the group A1a),
[000149] A1b) a sequence according to SEQ ID NO: 17, which sequence encodes a protein, which is capable of
[000150] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000151] B1b) an intron-free sequence, which is derived from a sequence according to A1b) and which encodes the same protein or peptide as the sequence according to SEQ ID NO: 17,
[000152] C1b) a sequence, which encodes a protein or peptide, which comprises the amino acid sequence according to SEQ ID NO: 18 and which is preferably capable of
[000153] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000154] D1b) a sequence, which is identical with a sequence according to one of groups A1b) to C1b), particularly preferably according to group A1b), by at least 70%, particularly preferably by at least minus 90%, furthermore preferably by at least 95% and most preferably by at least 99%, which sequence preferably encodes a protein or peptide, which is capable of
[000155] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000156] E1b) a sequence, which hybridizes with the opposite strand of a sequence according to one of the groups A1b) to D1b), particularly preferably according to the A1b group) or would hybridize considering the degeneration of the genetic code, being that such sequence preferably encodes a protein or peptide, which is capable of
[000157] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000158] F1b) a derivative, obtained through 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 at least 10 bases, however, preferably not more than 100 bases, particularly preferably not more than 50 bases and most preferably not more than 25 bases, of a sequence according to one of the groups A1b) to E1b ), particularly preferably according to the A1b group), which derivative preferably encodes a protein or peptide, which is capable of
[000159] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000160] G1b) a sequence complementary to a sequence according to one of the groups A1b) to F1b), particularly preferably according to the group A1b) and
[000161] A1c) a sequence according to SEQ ID NO: 77, which sequence encodes a protein, which is capable of
[000162] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000163] B1c) an intron-free sequence, which is derived from a sequence according to A1c) and which encodes the same protein or peptide as the sequence according to SEQ ID NO: 77,
[000164] C1c) a sequence, which encodes a protein or peptide, which comprises the amino acid sequence according to SEQ ID NO: 78 and which is preferably capable of
[000165] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000166] D1c) a sequence, which is identical with a sequence according to one of groups A1c) to C1c), particularly preferably according to group A1c), by at least 70%, particularly preferably by at least minus 90%, furthermore preferably by at least 95% and most preferably by at least 99%, which sequence preferably encodes a protein or peptide, which is capable of
[000167] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000168] E1c) a sequence, which hybridizes with the opposite strand of a sequence according to one of the groups A1c) to D1c), particularly preferably according to the A1c group) or would hybridize considering the degeneration of the genetic code, being that such sequence preferably encodes a protein or peptide, which is capable of
[000169] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000170] F1c) a derivative, obtained through 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 less than 10 bases, however, preferably not more than 100 bases, particularly preferably not more than 50 bases and most preferably not more than 25 bases, of a sequence according to one of the groups A1c) to E1c ), particularly preferably according to the A1c group), which derivative preferably encodes a protein or peptide, which is capable of
[000171] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000172] G1c) a sequence complementary to a sequence according to one of the groups A1c) to F1c), particularly preferably according to the group A1c) and
[000173] A1d) a sequence according to SEQ ID NO: 79, which sequence encodes a protein, which is capable of
[000174] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000175] B1d) an intron-free sequence, which is derived from a sequence according to A1d) and which encodes the same protein or peptide as the sequence according to SEQ ID NO: 79,
[000176] C1d) a sequence, which encodes a protein or peptide, which comprises the amino acid sequence according to SEQ ID NO: 80 and which is preferably capable of
[000177] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000178] D1d) a sequence, which is identical with a sequence according to one of groups A1d) to C1d), particularly preferably according to group A1d), by at least 70%, particularly preferably by at least minus 90%, furthermore preferably by at least 95% and most preferably by at least 99%, which sequence preferably encodes a protein or peptide, which is capable of
[000179] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000180] E1d) a sequence, which hybridizes with the opposite strand of a sequence according to one of the groups A1d) to D1d), particularly preferably according to the A1d group) or would hybridize considering the degeneration of the genetic code, being that such sequence preferably encodes a protein or peptide, which is capable of
[000181] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000182] F1d) a derivative, obtained through substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, further preferably of at least 5 bases and most preferably of at least at least 10 bases, however, preferably not more than 100 bases, particularly preferably not more than 50 bases and most preferably not more than 25 bases, of a sequence according to one of the groups A1d) to E1d ), particularly preferably according to the A1d group), which derivative preferably encodes a protein or peptide, which is capable of
[000183] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000184] G1d) a sequence complementary to a sequence according to one of the groups A1d) to F1d), particularly preferably according to the group A1d) and
[000185] A1e) a sequence according to SEQ ID NO: 81, which sequence encodes a protein, which is capable of
[000186] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000187] B1e) an intron-free sequence, which is derived from a sequence according to A1e) and which encodes the same protein or peptide as the sequence according to SEQ ID NO: 81,
[000188] C1e) a sequence, which encodes a protein or peptide, which comprises the amino acid sequence according to SEQ ID NO: 82 and which is preferably capable of
[000189] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000190] D1e) a sequence, which is identical with a sequence according to one of the groups A1e) to C1e), particularly preferably according to the A1e group), by at least 70%, particularly preferably by at least minus 90%, furthermore preferably by at least 95% and most preferably by at least 99%, which sequence preferably encodes a protein or peptide, which is capable of
[000191] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000192] E1e) a sequence, which hybridizes with the opposite strand of a sequence according to one of the groups A1e) to D1e), particularly preferably according to the A1e group) or would hybridize considering the degeneration of the genetic code, being that such sequence preferably encodes a protein or peptide, which is capable of
[000193] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000194] F1e) a derivative, obtained through substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, further preferably of at least 5 bases and most preferably of at least less than 10 bases, however, preferably not more than 100 bases, particularly preferably not more than 50 bases and most preferably not more than 25 bases, of a sequence according to one of the groups A1e) to E1e ), particularly preferably according to the A1e group), which derivative preferably encodes a protein or peptide, which is capable of
[000195] react 3-hydroxytetradecanoyl-ACP through 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000196] G1e) a sequence complementary to a sequence according to one of the groups A1e) to F1e), particularly preferably according to the group A1e) and
[000197] the group {A2 to G2] consists of the following sequences:
[000198] A2a) a sequence according to SEQ ID NO: 3, which sequence encodes a protein, which is capable of
[000199] react dTDP-rhamnose and 3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,
[000200] B2a) an intron-free sequence, which is derived from a sequence according to A2a) and which encodes the same protein or peptide as the sequence according to SEQ ID NO: 3,
[000201] C2a) a sequence, which encodes a protein or peptide, which comprises the amino acid sequence according to SEQ ID NO: 4 and which is preferably capable of
[000202] react dTDP-rhamnose and 3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,
[000203] D2a) a sequence, which is identical with a sequence according to one of the groups A2a) to C2a), particularly preferably according to the A2a group), by at least 80%, particularly preferably by at least minus 90%, furthermore preferably by at least 95% and most preferably by at least 99%, which sequence preferably encodes a protein or peptide, which is capable of
[000204] react dTDP-rhamnose and 3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,
[000205] E2a) a sequence, which hybridizes with the opposite strand of a sequence according to one of the groups A2a) to D2a), particularly preferably according to the A2a group) or would hybridize considering the degeneration of the genetic code, being that such sequence preferably encodes a protein or peptide, which is capable of
[000206] react dTDP-rhamnose and 3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,
[000207] F2a) a derivative, obtained through 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 at least 10 bases, however, preferably not more than 100 bases, particularly preferably not more than 50 bases and most preferably not more than 25 bases, of a sequence according to one of the groups A2a) to E2a ), particularly preferably according to the A2a group), which derivative preferably encodes a protein or peptide, which is capable of
[000208] react dTDP-rhamnose and 3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,
[000209] G2a) a sequence complementary to a sequence according to one of the groups A2a) to F2a), particularly preferably according to the group A2a),
[000210] A2b) a sequence according to SEQ ID NO: 19, which sequence encodes a protein, which is capable of
[000211] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxyteradecanoic acid,
[000212] B2b) an intron-free sequence, which is derived from a sequence according to A2b) and which encodes the same protein or peptide as the sequence according to SEQ ID NO: 19,
[000213] C2b) a sequence, which encodes a protein or peptide, which comprises the amino acid sequence according to SEQ ID NO: 20 and which is preferably capable of
[000214] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000215] D2b) a sequence, which is identical with a sequence according to one of the groups A2b) to C2b), particularly preferably according to the A2b group), by at least 70%, particularly preferably by at least minus 90%, furthermore preferably by at least 95% and most preferably by at least 99%, which sequence preferably encodes a protein or peptide, which is capable of
[000216] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000217] E2b) a sequence, which hybridizes with the opposite strand of a sequence according to one of the groups A2b) to D2b), particularly preferably according to the A2b group) or would hybridize considering the degeneration of the genetic code, being that such sequence preferably encodes a protein or peptide, which is capable of
[000218] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxydecanoic acid,
[000219] F2b) a derivative, obtained through 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 at least 10 bases, however, preferably not more than 100 bases, particularly preferably not more than 50 bases and most preferably not more than 25 bases, of a sequence according to one of the groups A2b) to E2b ), particularly preferably according to the A2b group), which derivative preferably encodes a protein or peptide, which is capable of
[000220] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000221] G2b) a sequence complementary to a sequence according to one of the groups A2b) to F2b), particularly preferably according to the group A2b),
[000222] A2c) a sequence according to SEQ ID NO: 83, which sequence encodes a protein, which is capable of
[000223] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000224] B2c) an intron-free sequence, which is derived from a sequence according to A2c) and which encodes the same protein or peptide as the sequence according to SEQ ID NO: 83,
[000225] C2c) a sequence, which encodes a protein or peptide, which comprises the amino acid sequence according to SEQ ID NO: 84 and which is preferably capable of
[000226] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000227] D2c) a sequence, which is identical with a sequence according to one of the groups A2c) to C2c), particularly preferably according to the A2c group), by at least 70%, particularly preferably by at least minus 90%, furthermore preferably by at least 95% and most preferably by at least 99%, which sequence preferably encodes a protein or peptide, which is capable of
[000228] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000229] E2c) a sequence, which hybridizes with the opposite strand of a sequence according to one of the groups A2c) to D2c), particularly preferably according to the A2c group) or would hybridize considering the degeneration of the genetic code, being that such sequence preferably encodes a protein or peptide, which is capable of
[000230] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxydecanoic acid,
[000231] F2c) a derivative, obtained through 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 less than 10 bases, however, preferably not more than 100 bases, particularly preferably not more than 50 bases and most preferably not more than 25 bases, of a sequence according to one of the groups A2c) to E2c ), particularly preferably according to the A2c group), which derivative preferably encodes a protein or peptide, which is capable of
[000232] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000233] G2c) a sequence complementary to a sequence according to one of the groups A2c) to F2c), particularly preferably according to the group A2c),
[000234] A2d) a sequence according to SEQ ID NO: 85, which sequence encodes a protein, which is capable of
[000235] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000236] B2d) an intron-free sequence, which is derived from a sequence according to A2d) and which encodes the same protein or peptide as the sequence according to SEQ ID NO: 85,
[000237] C2d) a sequence, which encodes a protein or peptide, which comprises the amino acid sequence according to SEQ ID NO: 86 and which is preferably capable of
[000238] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000239] D2d) a sequence, which is identical with a sequence according to one of the groups A2d) to C2d), particularly preferably according to the A2d group), by at least 70%, particularly preferably by at least minus 90%, furthermore preferably by at least 95% and most preferably by at least 99%, which sequence preferably encodes a protein or peptide, which is capable of
[000240] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000241] E2d) a sequence, which hybridizes with the opposite strand of a sequence according to one of the groups A2d) to D2d), particularly preferably according to the A2d group) or would hybridize considering the degeneration of the genetic code, being that such sequence preferably encodes a protein or peptide, which is capable of
[000242] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxydecanoic acid,
[000243] F2d) a derivative, obtained through 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 less than 10 bases, however, preferably not more than 100 bases, particularly preferably not more than 50 bases and most preferably not more than 25 bases, of a sequence according to one of the groups A2d) to E2d ), particularly preferably according to the A2d group), which derivative preferably encodes a protein or peptide, which is capable of
[000244] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000245] G2d) a sequence complementary to a sequence according to one of the groups A2d) to F2d), particularly preferably according to the group A2d) and
[000246] A2e) a sequence according to SEQ ID NO: 87, which sequence encodes a protein, which is capable of
[000247] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000248] B2e) an intron-free sequence, which is derived from a sequence according to A2e) and which encodes the same protein or peptide as the sequence according to SEQ ID NO: 87,
[000249] C2e) a sequence, which encodes a protein or peptide, which comprises the amino acid sequence according to SEQ ID NO: 88 and which is preferably capable of
[000250] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000251] D2e) a sequence, which is identical with a sequence according to one of the groups A2e) to C2d), particularly preferably according to the A2e group), by at least 70%, particularly preferably by at least minus 90%, furthermore preferably by at least 95% and most preferably by at least 99%, which sequence preferably encodes a protein or peptide, which is capable of
[000252] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000253] E2e) a sequence, which hybridizes with the opposite strand of a sequence according to one of the groups A2e) to D2e), particularly preferably according to the A2e group) or would hybridize considering the degeneration of the genetic code, being that such sequence preferably encodes a protein or peptide, which is capable of
[000254] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxydecanoic acid,
[000255] F2e) a derivative, obtained through substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, further preferably of at least 5 bases and most preferably of at least at least 10 bases, however, preferably not more than 100 bases, particularly preferably not more than 50 bases and most preferably not more than 25 bases, of a sequence according to one of the groups A2e) to E2e ), particularly preferably according to the A2e group), which derivative preferably encodes a protein or peptide, which is capable of
[000256] react dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
[000257] G2e) a sequence complementary to a sequence according to one of the groups A2e) to F2e), particularly preferably according to the group A2e) and
[000258] the group [A3 to G3] consists of the following sequences:
[000259] A3a) a sequence according to SEQ ID NO: 5, which sequence encodes a protein, which is capable of
[000260] react 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,
[000261] B3a) an intron-free sequence, which is derived from a sequence according to A3a) and which encodes the same protein or peptide as the sequence according to SEQ ID NO: 5,
[000262] C3a) a sequence, which encodes a protein or peptide, which comprises the amino acid sequence according to SEQ ID NO: 6 and which is preferably capable of
[000263] react 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,
[000264] D3a) a sequence, which is identical with a sequence according to one of groups A3a) to C3a), particularly preferably according to group A3a), by at least 80%, particularly preferably by at least minus 90%, furthermore preferably by at least 95% and most preferably by at least 99%, which sequence preferably encodes a protein or peptide, which is capable of
[000265] react 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,
[000266] E3a) a sequence, which hybridizes with the opposite strand of a sequence according to one of the groups A3a) to D3a), particularly preferably according to the A3a group) or would hybridize considering the degeneration of the genetic code, being that such sequence preferably encodes a protein or peptide, which is capable of
[000267] react 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,
[000268] F3a) a derivative, obtained through 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 less than 10 bases, however, preferably not more than 100 bases, particularly preferably not more than 50 bases and most preferably not more than 25 bases, of a sequence according to one of the groups A3a) to E3a ), particularly preferably according to the A3a group), which derivative preferably encodes a protein or peptide, which is capable of
[000269] react 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,
[000270] G3a) a sequence complementary to a sequence according to one of the groups A3a) to F3a), particularly preferably according to the group A3a),
[000271] A3b) a sequence according to SEQ ID NO: 21, which sequence encodes a protein, which is capable of
[000272] react 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,
[000273] B3b) an intron-free sequence, which is derived from a sequence according to A3b) and which encodes the same protein or peptide as the sequence according to SEQ ID NO: 21,
[000274] C3b) a sequence, which encodes a protein or peptide, which comprises the amino acid sequence according to SEQ ID NO: 22 and which is preferably capable of
[000275] react 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,
[000276] D3b) a sequence, which is identical with a sequence according to one of the groups A3b) to C3b), particularly preferably according to the A3b group), by at least 60%, particularly preferably by at least minus 90%, furthermore preferably by at least 95% and most preferably by at least 99%, which sequence preferably encodes a protein or peptide, which is capable of
[000277] react 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,
[000278] E3b) a sequence, which hybridizes with the opposite strand of a sequence according to one of the groups A3b) to D3b), particularly preferably according to the A3b group) or would hybridize considering the degeneration of the genetic code, being that such sequence preferably encodes a protein or peptide, which is capable of
[000279] react 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,
[000280] F3b) a derivative, obtained through 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 at least 10 bases, however, preferably not more than 100 bases, particularly preferably not more than 50 bases and most preferably not more than 25 bases, of a sequence according to one of the groups A3b) to E3b ), particularly preferably according to the A3b group), which derivative preferably encodes a protein or peptide, which is capable of
[000281] react 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
[000282] G3b) a sequence complementary to a sequence according to one of the groups A3b) to F3b), particularly preferably according to the A3b group),
[000283] A3c) a sequence according to a SEQ ID NO: 89, which sequence encodes a protein, which is capable of
[000284] react 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,
[000285] B3c) an intron-free sequence, which is derived from a sequence according to A3c) and which encodes the same protein or peptide as the sequence according to SEQ ID NO: 89,
[000286] C3c) a sequence, which encodes a protein or peptide, which comprises the amino acid sequence according to SEQ ID NO: 90 and which is preferably capable of
[000287] react 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,
[000288] D3c) a sequence, which is identical with a sequence according to one of the groups A3c) to C3c), particularly preferably according to the A3c group), by at least 60%, particularly preferably by at least minus 90%, furthermore preferably by at least 95% and most preferably by at least 99%, which sequence preferably encodes a protein or peptide, which is capable of
[000289] react 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,
[000290] E3c) a sequence, which hybridizes with the opposite strand of a sequence according to one of the groups A3c) to D3c), particularly preferably according to the A3c group) or would hybridize considering the degeneration of the genetic code, being that such sequence preferably encodes a protein or peptide, which is capable of
[000291] react 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,
[000292] F3c) a derivative, obtained through 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 less than 10 bases, however, preferably not more than 100 bases, particularly preferably not more than 50 bases and most preferably not more than 25 bases, of a sequence according to one of the groups A3c) to E3c ), particularly preferably according to the A3c group), which derivative preferably encodes a protein or peptide, which is capable of
[000293] react 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
[000294] G3c) a sequence complementary to a sequence according to one of the groups A3c) to F3c), particularly preferably according to the group A3c) and
[000295] A3d) a sequence according to a SEQ ID NO: 91, which sequence encodes a protein, which is capable of
[000296] react 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,
[000297] B3d) an intron-free sequence, which is derived from a sequence according to A3d) and which encodes the same protein or peptide as the sequence according to SEQ ID NO: 91,
[000298] C3d) a sequence, which encodes a protein or peptide, which comprises the amino acid sequence according to SEQ ID NO: 92 and which is preferably capable of
[000299] react 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,
[000300] D3d) a sequence, which is identical with a sequence according to one of the groups A3d) to C3d), particularly preferably according to the A3d group), by at least 60%, particularly preferably by at least minus 90%, furthermore preferably by at least 95% and most preferably by at least 99%, which sequence preferably encodes a protein or peptide, which is capable of
[000301] react 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,
[000302] E3d) a sequence, which hybridizes with the opposite strand of a sequence according to one of the groups A3d) to D3d), particularly preferably according to the A3d group) or would hybridize considering the degeneration of the genetic code, being that such sequence preferably encodes a protein or peptide, which is capable of
[000303] react 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,
[000304] F3d) a derivative, obtained through substitution, addition, inversion and/or deletion of at least one base, preferably of at least 2 bases, further preferably of at least 5 bases and most preferably of at least less than 10 bases, however, preferably not more than 100 bases, particularly preferably not more than 50 bases and most preferably not more than 25 bases, of a sequence according to one of the groups A3d) to E3d ), particularly preferably according to the A3d group), which derivative preferably encodes a protein or peptide, which is capable of
[000305] react 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
[000306] G3d) a sequence complementary to a sequence according to one of the groups A3d) to F3d), particularly preferably according to the group A3d).
[000307] The "nucleotide identity" or "amino acid identity" is determined here with the aid of known processes. In general, particular computer programs with algorithms considering special needs are used.
[000308] Preferred processes for the determination of identity initially produce the greatest agreement between the sequences to be compared. Computer programs for the determination of identity comprise, however, are not restricted to, the GCG program package, including
[000309] 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 other sources (BLAST Handbuch, Altschul S. et al., NCBI NLM NIH Bethesda ND 22894, Altschul S. et al., above).
[000310] The well-known Smith-Waterman algorithm can also be used for nucleotide identity determination.
[000311] Preferred parameters for determining "nucleotide identity" when using the BLASTN program (Altschul, S. et al., Journal of Molecular Biology 215 (1990), pages 403-410 are: expect threshold: 10 word size: 28 match score: 1 mismatch score: -2 gap costs: linear
[000312] The above parameters are the parameters missing in the nucleotide sequence comparison. The GAP program is equally suitable to be used with the above parameters.
[000313] Preferred parameters for determining "amino acid identity" when using the BLASTP program (Altschul, S. et al., Journal of Molecular Biology 215 (1990), pages 403-410 are: expect threshold: 10 word size: 3 matrix: BLOSUM62 gap costs: existence: 11; extension: 1 compositional adjustments: conditional compositional score matrix adjustment.
[000314] The above parameters are the parameters missing in the amino acid sequence comparison. The GAP program is equally suitable to be used with the above parameters.
[000315] A 60% identity according to the above algorithm in the context of the present invention means 60% identity. The same applies for higher identities.
[000316] The characteristic "sequence, which hybridizes with the opposite strand of a sequence or would hybridize considering the degeneration of the genetic code" points to a sequence, which hybridizes under stringent conditions with the opposite strand of a reference sequence or would hybridize considering the degeneration of the genetic code. For example, hybridizations can be carried out at 68oC in 2 x SSC or according to the protocol of the kit with the brand digoxigenin from 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 65oC with 2 x SSC; 0.1% SDS.
[000317] Derivatives of DNA isolated according to the invention, which can be obtained according to alternative F1, F2) or F3) through substitution, addition, inversion and/or deletion of one or more bases of a sequence of according to one of the groups A1) to E1, A2) to E2) and A2) to E3), belong especially those sequences, which in the protein, which they encode, lead to conservative amino acid exchanges, such as, for example, to exchange of glycine for alanine or of asparaginic acid for glutamic acid. Such neutral-function mutations are referred to as sense mutations and do not lead to any fundamental change in the activity of the polypeptide. Furthermore, it is known that changes in the N and/or C-terminus of a polypeptide do not substantially impair its function or may even stabilize it, so that as a result of this, DNA sequences, in which at the 3' end or at the 5' end of the sequence with the nucleic acids according to the invention, bases are added, are comprised by the present invention. The specialist finds data for this purpose, among others, 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.
[000318] The nucleic acid according to the invention is preferably a vector, especially an expression vector or a gene overexpression cassette. Vectors include all vectors known to the skilled person, which are conventionally used to insert DNA into a host cell. These vectors can either replicate autonomously since they have replication origins such as those of the 2μ-plasmid or ARS (autonomous replicating sequences) or integrate into chromosomes (non-replicating plasmids). By vectors is also meant linear DNA fragments, which do not have any origins of replication, such as gene insertion or gene overexpression cassettes. Gene overexpression cassettes conventionally consist of a marker, the genes to be overexpressed, as well as regulatory bands relevant to gene expression, such as promoters and terminators. Preferred vectors are selected from the group comprising plasmids and cassettes, such as E. coli yeast integrative plasmids, particularly preferred are expression vectors, gene insertion or gene overexpression cassettes, especially vectors SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 45 and SEQ ID NO: 47 described below.
[000319] 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 promoter or regulatable, which is suitable for the expression of the polypeptide encoded by such DNA sequences in the cell of a microorganism, preferably of a bacterial, yeast or fungal cell, in which 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. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea, P. aurantiaca, P. aureofaciens, P. chlororaphis, P. fragi, P. lundensis, P. taetrolens, P. antarctica, P.nitroformans, '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 , 'FOR. helianthi', P. meliae, P. savastanoi, P. syringae, P. tomato, P. viridiflava, P. abietaniphila, P. acidophila, P. agarici, P. alcaliphila, P. alkanolytica, P. amyloderamosa, P. aspleniiphila , P. azotifigens, P. cannabina, P. coenobios, P. freezens, 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, Rho dobacter sphaeroides, Saccharomyces cerevisiae, Yarrowia lipolytica, Zymomonas mobilis, especially 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 Lacl 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 promoters that can be induced are lac, lacUV5, tac, trc (in each case in the presence of the Lacl 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 in the presence of the Lacl repressor in cells according to the invention), SP6 and T7 (in the presence of the gene encoding the cognate RNA polymerase, whose expression, in turn, is regulated) . The vector according to the invention should comprise, in addition to a promoter, preferably a ribosome binding point, as well as a terminator. In that case, it is particularly preferred that the nucleic acid according to the invention is incorporated into a vector expression cassette comprising the promoter, the ribosome binding point and the terminator. In addition to the structural elements mentioned above, the vector may further comprise selection genes known to the skilled person.
[000320] All percentages (%) indicated, when not otherwise indicated, are percent by mass. In the examples listed below, the present invention is described by way of examples, without the invention, whose scope of application results from the entire descriptive report and claims, being restricted to the embodiments mentioned in the examples. Brief Description of Figures:
[000321] Figure 1: fatty acid biosynthesis, fatty acid e-oxidation and linkage of these pathways of metabolism with the biosynthesis of rhamnolipids (enzymes E1, E2 and E3) and polyhydroxyalkanoates (enzymes E9 and E10). Carbon fluxes in fatty acid biosynthesis, fatty acid e-oxidation, rhamnolipid biosynthesis and polyhydroxyalkanoate biosynthesis are shown. Consumption and formation of coenzymes, redox equivalents and nucleotides are not shown.
[000322] Figure 2: Diramnosyl-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 hours, 72 hours and 96 hours of culture in CMP medium. The analysis of the rhamnolipid concentration was carried out by means of HPLC.
[000323] Figure 3: Monoramnosyl-lipid formation (peak surface/OD 600 nm) of the recombinant strains P. putida KT2440 pBBR1MCS-2, pBBR1MCS2::AB and pBBR1MCS2::ABM as well as GPp104 pBBR1MCS-2, pBBR1MCS-2 ::AB and pBBR1MCS-2::ABM after 48 hours, 72 hours and 96 hours of cultivation in CMP medium. The analysis of the rhamnolipid concentration was carried out by means of HPLC. Examples: Construction of a pBBR1MCS-2::AB vector for the heterologous expression of the rhlA and rhlB gene of Pseudomonas aeruginosa 1707 in Pseudomonas putida
[000324] For the heterologous expression of the rh1A and rh1B genes from Pseudomonas aeruginosa DSM1707, the plasmid pBBR1MCS-2::AB (SEQ ID NO: 38) was constructed. For this, the synthetic rhlAB operon (SEQ ID NO: 37) from the company GeneArt AG (Regensburg) was synthesized and intermediately cloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of Pseudomonas aeruginosa DSM1707. Starting from vector pMA::AB, the synthetic operon was cut from the vector via Bg/II and Xbal and then ligated into the expression vector pBBR1MCS-2 (SEQ ID NO:49) cut with BamHl and Xbal (described by 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. Ligation as well as transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) was carried out in a manner and manner known to the skilled person. Insert authenticity was verified by DNA sequence analysis.
The transformation of Pseudomonas putida KT2440 and Gpp104 with the vectors pBBR1MCS-2 (SEQ ID NO: 49) and pBBR1MCS-2::AB was performed as described above (Iwasaki and collaborators Biosci. Biotech. Biochem. 1994 58( 5):851-854). Plasmid DNA from 10 clones was isolated and analyzed. The strains obtained carrying plasmids were mentioned from P. putida KT2440 pBBR1MCS-2, P. putida GPp104pBBR1MCS-2, P. putida KT2440 pBBR1MCS-2::AB or P. putida GPp104 pBBR1MCS-2::AB. 2. Construction of a pBBR1MCS-2::AB vector for the heterologous expression of the rhlA, rhlB and rhlC genes from Pseudomonas aeruginosa DSM1707 in Pseudomonas putida
[000326] For the heterologous expression of the rhlA, rhlB and rhlC genes of Pseudomonas aeruginosa DSM1707, plasmid pBBR1MCS-2::AB (SEQ ID NO: 40) was constructed. For this purpose, the synthetic operon rhlABC SEQ ID NO: 39) from the company GeneArt AG (Regensburg) was synthesized and intermediately cloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of Pseudomonas aeruginosa DSM1707. Starting from the pMA::ABC vector, the synthetic operon was cut from the vector via Bg/II and Xbal and then ligated into the expression vector pBBR1MCS-2 (SEQ ID NO: 49) cut with BamHl and Xbal (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. Ligation as well as transformation of chemically competent E. coli cells (Gibco-BRL, Karlsruhe) was carried out in a manner and manner known to the skilled person. Insert authenticity was verified by DNA sequence analysis.
Transformation of Pseudomonas putida KT2440 and Gpp104 with the pBBR1MCS-2::ABC vector was performed as described above (Iwasaki and coworkers Biosci. Biotech. Biochem. 1994.58(5):851-854). Plasmid DNA from every 10 clones was isolated and analyzed. The strains obtained carrying plasmids were mentioned from P. putida KT2440 pBBR1MCS-2::ABC or P. putida GPp104 pBBR1MCS-2::ABC. 3. Construction of a pBBR1MCS-2::ABM vector for the heterologous expression of the rhlA, rhlB and pa1131 genes of Pseudomonas aeruginosa DSM1707 in Pseudomonas putida
[000328] For the heterologous expression of the rh1A, rhlB and pa1131 genes of Pseudomonas aeruginosa DSM1707, plasmid pBBR1MCS-2::ABM (SEQ ID NO: 42) was constructed. For this purpose, the synthetic operon rhlAB-pa1131 SEQ ID NO: 41) from the company GeneArt AG (Regensburg) was synthesized and intermediately cloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of Pseudomonas aeruginosa DSM1707. Starting from the pMA::ABM vector, the synthetic operon was cut from the vector via Bg/II and Xbal and then ligated into the expression vector pBBR1MCS-2 (SEQ ID NO:49) cut with BamHl and Xbal (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) has a size of 8702 base pairs. Ligation as well as transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) was carried out in a manner and manner known to the skilled person. Insert authenticity was verified by DNA sequence analysis.
Transformation of Pseudomonas putida KT2440 and Gpp104 with the pBBR1MCS-2::ABM vector was performed as described above (Iwasaki et al. Biosci. Biotech. Biochem. 1994.58(5):851-854). Plasmid DNA from every 10 clones was isolated and analyzed. The strains obtained carrying plasmids were mentioned from P. putida KT2440 pBBR1MCS-2::ABM or P. putida GPp104 pBBR1MCS-2::ABM. 4. Quantification of rhamnolipid production by recombinant P. putida strains
[000330] 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 GPp104pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABM were grown on LB-agar-kanamycin plates (50 µg/ml).
[000331] 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 x 2 H2O, 0.2% (w/v) NaNO3, 0.04% ( w/v) of MgSO4 x H2O, 0.01% (w/v) of CaCl2 x 2 H2O and 0.2% (v/v) of a solution of trace elements. This consists of 0.2% (w/v) FeSO4 x 7 H2O, 0.15% (w/v) MnSO4 x H2O, and 0.06% (w/v) (NH4)MO7O24 x 4 H2O. The pH value of the medium was pH adjusted to 6.7 with NaOH and the medium was then sterilized by autoclave (121oC, 20 minutes). It was not necessary to adjust the pH value during cultivation.
[000332] To investigate the production of rhamnolipids in the shaker flask, a pre-culture was initially prepared. For this purpose, an inoculation loop of a strain freshly streaked on the 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 grown in LB medium, to which 50 μg/ml of kanamycin was added. The strains were cultivated at 30oC and 200 rpm overnight.
[000333] Precultures were used, to inoculate 50 ml of CMP medium in the 250 ml Erlenmeyer flask (initial OD600 of 0.1). Cultures were grown at 200 rpm and 30oC for a maximum of 120 hours. Within 24 hours, a 1 mL sample of broth was removed from the culture flask. The preparation of the sample for the next chromatographic analyzes was carried out as follows:
[000334] With a displacement pipette (Combitip), 1 mL of acetone was previously introduced into a 2 mL reaction vessel and the reaction vessel was immediately closed to minimize evaporation. This was followed by the addition of 1 ml of broth. After swirling the broth/acetone mixture, it was centrifuged for 3 minutes at 13,000 rpm and 800 μL of the supernatant was transferred to an HPLC container.
[000335] For the detection and quantification of rhamnolipids, an Evaporative Light Scattering Detektor (Sedex LT-ELSD model 85LT) was used. The measurement itself was performed using the Agilent Technologies 1200 Series (Santa Clara, California) and with the Zorbax SB-C8 Rapid Resolution column (4.6 x 150 mm, 3.5 µm, Agilent). The injection volume amounted to 5 μL and the method duration was 20 minutes. As mobile phase 0.1% TFA (trifluoroacetic acid, solution A) and methanol (solution B) were used. Column temperature imported at 40oC. The ELSD (detector temperature 60oC) and the DAD (diode array detector, 210 nm) served as detectors. The gradient used in the method was:


[000336] While P. putida KT2440 pBBR1MCS-2 and Gpp104 pBBR1MCS-2 do not produce rhamnolipids, in the recombinant strains P. putida KT2440 pBBR1MCS-2::AB, P. putida KT2440 pBBR1MCS-2::ABC, P. putida KT2440 pB -2::ABM, P. putida GPp104 pBBR1MCS-2::AB, P. putida GPp104 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABM it was possible to verify the formation of several species of rhamnolipids (Figure 2 and 3).
[000337] Through the introduction of pBBR1MCS-2::AB or pBBR1MCS-2::ABM in P. putida, monorhamnosyl-lipids could be generated (Figure 3). Since there was no reference material for monorhamnosyl-lipids, product identification was carried out by analyzing the corresponding mass traces and MS2 spectra in LC-MS.
[000338] If additionally rhlC (pBBR1MCS-2::ABC) was introduced into the strains, then mono- and diramnosyl-lipids were produced (Figure 2).
[000339] Direct comparison of rhamnolipid formation by P. putida pBBR1MCS-2::AB or P. putida pBBR1MCS-2::ABM shows that the co-expression of P. aeruginosa p3111 with P. aeruginosa rhlAB leads to an improvement in P. aeruginosa rhlAB rhamnolipid biosynthesis (Figure 3). While the P. putida KT2440 pBBR1MCS-2::AB and P. putida GPp104 pBBR1MCS-2::AB strains had produced about 39 (P. putida KT2440 pBBR1MCS-2::AB) or 23 peak areas of rhamnolipids/OD 600 nm (P. putida GPp104 pBBR1MCS-2::AB) after 120 hours, P. putida KT2440 pBBR1MCS-2::ABM and P. putida GPp104 pBBR1MCS-2::ABM strains formed about 50 (P. putida KT2440 pBBR1MCS-2::ABM) or 62 peak areas of rhamnolipids/OD 600 nm (P. putida GPp104 pBBR1MCS-2::ABM) after 120 hours.
[000340] If the synthesis of monoramnosyl-lipid of the P. putida KT2440 pBBR1MCS-2::ABM and P. putida GPp104 pBBR1MCS-2::ABM strains was compared, then in the PHA-negative mutant P. putida GPp104 pBBR1MCS-2: :ABM 62 peak areas/OD 600 nm could be detected (120 hours of culture) and with P. putida KT2440 pBBR1MCS-2::ABM 50 areas/OD 600 nm of monorhamnosyl lipids (Figure 3).
[000341] A comparative analysis of the formation of diramnosyl-lipids (mg/L/OD 600 nm) in P. putida strains KT2440 and GPp104 also showed stronger formation of diramnosyl-lipids in the PHA-negative background culture of P. putida Gpp104. P. putida GPp104 pBBR1MCS-2::ABC formed on average 113 mg/L/OD 600 nm of diramnosyl lipids (95 hours), whereas with P. putida KT2440 pBBR1MCS-2::ABC only 55 mg were detected /L/OD 600 nm of diramnosyl-lipids after 96 hours (Figure 2).
[000342] Thus, it was possible to show that the use of a background culture weakened with respect to PHA synthesis, leads to an improvement in the biosynthesis of rhamnolipids. 5. Construction of a pBBR1MCS-2::ABMC vector for the heterologous expression of the rhlaA, rhlB, pa1131 and rhlC genes from Pseudomonas aeruginosa DSM1707 in Pseudomonas putida
[000343] For the heterologous expression of the rhlaA, rhlB, pa1131 and rhlC genes of Pseudomonas aeruginosa DSM1707, plasmid pBBR1MCS-2::ABMC (SEQ ID NO:51) was constructed. For this purpose, the synthetic operon rhlAB-pa1131-rhlC (SEQ ID NO: 50) from the company GeneArt AG (Regensburg) was synthesized and intermediately cloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of Pseudomonas aeruginosa DSM1707. Starting from the pMA::ABMC vector, the synthetic operon was cut from the vector via Bg/II and Xbal and then ligated into the pBBR1MCS-2 expression vector (SEQ ID NO: 49 cut with BamHI and Xbal (Kovach and Collaborators, 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) has a size of 9663 base pairs. The binding as well as the transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) was carried out in a manner and manner known to the expert. of DNA sequence.
Transformation of Pseudomonas putida KT2440 and Gpp104 with the vector pBBR1MCS-2::ABMC was performed as described above (Iwasaki and coworkers Biosci. Biotech. Biochem. 1994. 58(5):851-854). Plasmid DNA from every 10 clones was isolated and analyzed. The strains obtained carrying plasmids were mentioned from P. putida KT2440 pBBR1MCS-2::ABMC or P. putida GPp104 pBBR1MCS-2::ABMC. 6. Qualitative comparison of rhamnolipid production using recombinant P. putida strains and P. aeruginosa strains
The recombinant strains P. putida GPp104 pBBR1MCS-2 and P. putida GPp104 pBBR1MCS-2::ABMC as well as P. aeruginosa DSM 19880 were grown on LB-kanamycin agar plates (50 µg/ml; P. putida ) or LB-agar (P. aeruginosa).
[000346] For the production of the rhamnolipids the medium designated below as CMP medium was used. This consisted of 2% (w/v) glucose, 0.007% (w/v) KH2PO4, 0.11% Na2HPO4 x 2 H2O, 0.2% (w/v) NaNO3, 0.04% ( w/v) of MgSO4 x H2O, 0.01% (w/v) of CaCl2 x 2 H2O and 0.2% (v/v) of a solution of trace elements. This consists of 0.2% (w/v) FeSO4 x 7 H2O, 0.15% (w/v) MnSO4 x H2O, and 0.06% (w/v) (NH4)MO7O24 x 4 H2O. The medium had the pH value adjusted to 6.7 with NaOH and the medium was then sterilized by means of an autoclave (121oC, 20 minutes). It was not necessary to adjust the pH value during cultivation.
[000347] To investigate the production of rhamnolipids in the shaker flask, a pre-culture was initially prepared. For this purpose, an inoculation loop of a strain freshly streaked on the 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 cultivated in LB medium, to which 50 μg/ml of kanamycin was added. P. aeruginosa was grown on LB medium. The strains were cultivated at 30oC and 200 rpm overnight.
[000348] Precultures were used, to inoculate 50 ml of CMP medium in the 250 ml Erlenmeyer flask (initial OD600 of 0.1). Cultures were grown at 200 rpm and 30oC for a maximum of 120 hours. Within 24 hours, a 1 mL sample of broth was removed from the culture flask. The preparation of the sample for the next chromatographic analyzes was carried out as follows:
[000349] With a displacement pipette (Combitip), 1 mL of acetone was previously introduced into a 2 mL reaction vessel and the reaction vessel was immediately closed to minimize evaporation. This was followed by the addition of 1 ml of broth. After swirling the broth/acetone mixture, it was centrifuged for 3 minutes at 13,000 rpm and 800 μL of the supernatant was transferred to an HPLC container.
[000350] For the identification of the resulting products, 5 μL of were pipetted in an UPLC Accela installation (Thermo Scientific, Dreieich). The substances to be investigated were analyzed with a semi UPLC column "Pursuit XRs ULTRA (C8, 2.8 μm, 2.1 x 100 mm) Varian, Darmstadt). Separation was carried out within 25 minutes by means of a gradient consisting of in mobile phase A1 (H2O, 0.1% (v/v) TFA) and in mobile phase B1 (methanol, 0.1% (v/v) TFA) with a flow rate of 0.3 ml/ min at 40oC. The time course of the gradient was as follows: time [minutes] mobile phase A1 [%] mobile phase B1 [%]

[000351] Detection was carried out by means of the DAD detector in the wavelength range 200 - 600 nm, as well as mass selectively with a LTQ-FT high resolution FT-ICR mass spectrometer (Thermo Scientific, Dreieich ) in the m/z scan range of 100 - 1000. Ionization was carried out by means of ESI (electrospray ionization). Exact masses and empirical chemical formulas were determined with the aid of the FT-ICR mass analyzer, with a resolution of R = 10000 and a measurement accuracy of < 2 ppm. The identification of the products was carried out by analyzing the corresponding mass traces and MS2 spectra. In order to compare the strains, the peak areas of the corresponding substances were compared. As shown in Figure 4, the P. putida strain GPp104 pBBR1MCS-2 did not form any rhamnolipids. P. putida GPp104 pBBR1MCS-2::ABMC as well as P. aeruginosa DSM 19880 formed rhamnolipids, the ratio of di- and monorhamnosyl lipids formed in P. putida GPp104 pBBR1MCS-2::ABMC being approximately 4:1 , in P. aeruginosa DSM 19880, of approximately 2:1. Furthermore, the strain P. putida GPp104 pBBR1MCS-2::ABMC, unlike P. aeruginosa DSM 19880, did not form any or only very few rhamnolipids with a radical determined by R1 and R2 derived from 3-hydroxyoctanoyl-3-acid hydroxydecanoic or 3-hydroxydecanoyl-3-hydroxyoctanoic acid. 7. Construction of a pBBR1MCS-2::rfbBDAC and pBBR1MCS-2::ABCrfbBDAC vector for heterologous expression in Pseudomonas putida
[000352] At Trenzyme GmbH (Konstanz), the rfbBDAC rfbBDAC biosynthesis operon was amplified starting from the chromosomal DNA of Pseudomonas putida KT2440. For this, the following initiators were used:
[000353] RL1: 5'- TATATATAGAATTCGCGTCATCTGTCTACGACAACAC -3' (SEQ ID NO: 48)
[000354] RL2: 5'- TATATATAGAATTCGGCTGCGCTACCGCAGCCCTTC -3' (SEQ ID NO: 43)
[000355] The PCR product obtained was intermediately cloned in Trenzyme's Alligator Cloning system and transformed into E. coli DH5 α (New England Biolabs; Frankfurt). Vectors from several candidates were analyzed and sequenced. After successful and flawless sequencing, the vector was cut using EcoRI and the rfbBDAC target fragment was isolated. For another intermediate cloning, 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 cut from it. way and way. The cut target fragment (rfbBDAC) and the cut vector (pBBR1MCS-2) were joined by conventional ligation. The resulting pBBR1MCS-2::rfbBDAC (SEQ ID NO: 45) vector was similarly transformed into E. coli DH5α (New England Biolabs, Frankfurt). Some candidate transformants were screened with respect to efficient plasmid uptake.
[000356] The vector pBBR1MCS-2::rfbBDAC served as a template for a PCR. The following oligonucleotides were used:
[000357] RL_XbaI-fw: 5'- TATATATATCTAGAATTAATGCAGCTGGCACGAC -3' (SEQ ID NO: 44)
[000358] RL_Xba_rev: 5'-GGCCGCTCTAGAACTAGTGGA -3' (SEQ ID NO: 46)
[000359] PCR was performed with PhusionTM High-Fidelity Master Mix polymerase from New England Biolabs (Frankfurt). It was carried out in a manner and manner known to the expert. The target sequence (Iac promoter and rfbBDAC) was intermediately cloned in the Trenzyme Alligator Cloning system. E. coli DH5α New England Biolabs transformants (Frankfurt) were selected and plasmid DNA from several candidates was isolated and sequenced. After the sequence was verified and searched for accuracy, the vector was cut with Xbal. The target fragment was ligated with XbaI cut pBBR1MCS-2::ABC (see above) by standard ligation methods. The target vector pBBR1MCS-2::ABC_rfbBDAC (SEQ ID NO: 47) obtained had a size of 12249 base pairs. Insertion of the vector was sequenced. Performing the PCR, verifying successful PCR amplification using agarose gel electrophoresis, labeling the DNA with ethidium bromide, determining the size of the PCR fragment, purifying the PCR products and determining the DNA concentration was carried out in a manner and manner known to the expert.
[000360] Transformation of Pseudomonas putida KT2440 and Gpp104 with the vector pBBR1MCS-2::ABC_rfbBDAC was performed as described above (Iwasaki and collaborators Biosci. Biotech. Biochem. 1994. 58(5):851-854). Plasmid DNA from each 10 clones was isolated and analyzed. The strains obtained carrying plasmids are mentioned from P. putida KT2440 pBBR1MCS-2::ABC_rfbBDAC and P. putida GPp104 pBBR1MCS-2::ABC_rfbBDAC. 8. Quantification of rhamnolipid production by recombinant P. putida strains with and without overexpression of the rfbBDAC operon
[000361] 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 grown on LB-kanamycin agar plates (50 μg/ml).
[000362] For the production of 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 x 2 H2O, 0.2% (w/v) NaNO3, 0.04% ( w/v) of MgSO4 x H2O, 0.01% (w/v) of CaCl2 x 2 H2O and 0.2% (v/v) of a solution of trace elements. This consists of 0.2% (w/v) FeSO4 x 7 H2O, 0.15% (w/v) MnSO4 x H2O and 0.06% (w/v) (NH4)MO7O24 x 4 H2O. The medium has the pH value adjusted to 6.7 with NaOH and the medium is then sterilized by means of an autoclave (121oC, 20 minutes). It is not necessary to adjust the pH value during cultivation.
[000363] To investigate the production of rhamnolipids in the shaker flask, a pre-culture was initially prepared. For this purpose, an inoculation loop of a strain freshly streaked on the 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 grown in LB medium, to which 50 μg/ml of kanamycin was added. Cultivation of P. putida strains was carried out at 30oC and 200 rpm overnight.
[000364] Precultures are used, to inoculate 50 ml of CMP medium in the 250 ml Erlenmeyer flask (initial OD600 of 0.1). Cultures are grown at 200 rpm and 30oC for a maximum of 120 hours. Within 24 hours, a 1 mL sample of broth was removed from the culture flask. The preparation of the sample for the next chromatographic analyzes is carried out as follows:
[000365] With a displacement pipette (Combitip), 1 mL of acetone was previously introduced into a 2 mL reaction vessel and the reaction vessel was immediately closed to minimize evaporation. This was followed by the addition of 1 ml of broth. After swirling the broth/acetone mixture, it was centrifuged for 3 minutes at 13,000 rpm and 800 μL of the supernatant was transferred to an HPLC container.
[000366] For the detection and quantification of rhamnolipids, an Evaporative Light Scattering detector (Sedex LT-ELSD model 85LT) is used. The measurement itself is performed using the Agilent Technologies 1200 Series (Santa Clara, California) and with the Zorbax SB-C8 Rapid Resolution column (4.6 x 150 mm, 3.5 µm, Agilent). The injection volume is 5 μL and the method duration is 20 minutes. As mobile phase 0.1% TFA (trifluoroacetic acid, solution A) and methanol (solution B) are used. Column temperature matters at 40oC. The ELSD (detector temperature 60oC) and the DAD (diode array detector, 210 nm) serve as detectors. The gradient used in the method is: t [minutes] % by volume, of solution B flow [ml/minutes]

[000367] While P. putida KT2440 pBBR1MCS-2 and Gpp104 pBBR1MCS-2 do not produce any rhamnolipids, in the recombinant strains of 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 can be verified.
[000368] 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 compared to P. putida GPp104-2 pBBR1 ::ABC, an increased formation of di- and monorhamnosyl-lipids. This clearly indicates the positive influence of increased rfbBDAC expression on the formation of mono- and diramnosyl-lipids.
[000369] Comparing mono- and diramnosyl-lipid biosynthesis of P. putida KT2440 strains pBBR1MCS-2::ABC_rfbBDAC and P. putida GPp104 pBBR1MCS-2::ABC_rfbBDAC, then in the negative mutant PHA P. putida GPB1MCS-2 p :ABC_rfbBDAC an increased synthesis of mono- and diramnosyl-lipids is detected.
[000370] As already described above, when using an inactivated background culture in PHA synthesis, the biosynthesis of rhamnolipids increases. 9. Generation of recombinant E. coli W3110 pBBR1MCS-2:: ABC and E. coli W3110 pBBR1MCS-2::ABCrfbBDAC
[000371] Transformation of E. coli W3110 was performed as described above (Miller JH. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Plainview, NY: Cold Spring Harbor Lab. Press; 1992) by means of electroporation. Plasmid DNA from each 10 clones was isolated and analyzed. The strains obtained carrying the plasmids were mentioned from E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC and E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC. 10. Quantification of rhamnolipid production by recombinant E. coli strains with and without overexpression of the rfbBDAC operon
[000372] Recombinant strains E. coli W3110 pBBR1MCS-2; E. coli W3110 pBBR1MCS-2::ABC and E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC are grown on LB-kanamycin agar plates (50 µg/ml).
[000373] For the production of 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 x 2 H2O, 0.2% (w/v) NaNO3, 0.04% ( w/v) of MgSO4 x H2O, 0.01% (w/v) of CaCl2 x 2 H2O and 0.2% (v/v) of a solution of trace elements. This consists of 0.2% (w/v) FeSO4 x 7 H2O, 0.15% (w/v) MnSO4 x H2O and 0.06% (w/v) (NH4)MO7O24 x 4 H2O. The medium has the pH value adjusted to 6.7 with NaOH and the medium is then sterilized by means of an autoclave (121oC, 20 minutes). It is not necessary to adjust the pH value during cultivation.
[000374] To investigate the production of rhamnolipids in the shaker flask, a pre-culture was initially prepared. For this purpose, an inoculation loop of a strain freshly streaked on the LB agar plate was used and 10 mL of LB medium was inoculated into a 100 mL Erlenmeyer flask. All recombinant E coli strains are grown in LB medium, to which 50 μg/ml of kanamycin has been added. The cultivation of E. coli strains was carried out at 37oC and 200 rpm overnight. Precultures were used to inoculate 50 ml of CMP medium in the 250 ml Erlenmeyer flask (initial OD600 of 0.1). Cultures are grown at 200 rpm and 30oC for a maximum of 120 hours. Within 24 hours, a 1 mL sample of broth was removed from the culture flask. The preparation of the sample for the next chromatographic analyzes was carried out as follows:
[000375] With a displacement pipette (Combitip), 1 mL of acetone was previously introduced into a 2 mL reaction vessel and the reaction vessel was immediately closed to minimize evaporation. This was followed by the addition of 1 ml of broth. After swirling the broth/acetone mixture, it was centrifuged for 3 minutes at 13,000 rpm and 800 μL of the supernatant was transferred to an HPLC container.
[000376] For the detection and quantification of rhamnolipids, an Evaporative Light Scattering Detektor (Sedex LT-ELSD model 85LT) was used. The measurement itself was performed using the Agilent Technologies 1200 Series (Santa Clara, California) and with the Zorbax SB-C8 Rapid Resolution column (4.6 x 150 mm, 3.5 µm, Agilent). The injection volume is 5 μL and the method duration is 20 minutes. As mobile phase 0.1% TFA (trifluoroacetic acid, solution A) and methanol (solution B) were used. Column temperature matters at 40oC. The ELSD (detector temperature 60oC) and the DAD (diode array detector, 210 nm) serve as detectors. The gradient used in the method is: t [min] % by volume, of solution B flow [mL/min]

[000377] While E. coli W3110 pBBR1MCS-2 does not produce any rhamnolipids, in the recombinant strains of E. coli pBBR1MCS-2::ABC and E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC the formation of mono- and diramnosyl lipids, with E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC forming significantly more mono- and diramnosyl lipids than E. coli W3110 pBBR1MCS-2::ABC. This shows that the heterologous expression of rhlABC from Pseudomonas aeruginosa DSM1707 leads to the formation of mono- and diramnosyl-lipids in E. coli. This also shows the positive influence of increased rfbBDAC expression on the formation of mono- and diramnosyl-lipids. 11. Construction of a vector pBBR1MCS-2::ABC-BTHII1077-II1080II1081 for the heterologous expression of the rhlA, rhlB and rhlC genes from Pseudomonas aeruginosa DSM1707, as well as the BTHII1077, BTII1080 and BTII1081 genes from Burkholderia thailandensis E264 in putty
[000378] For the heterologous expression of the genes rhlA, rhlB and rhlC of Pseudomonas aeruginosa DSM1707, as well as of the genes BTH_II1077, BT_II1080 and BT_II1081 of Burkholderia thailandensis E264 in Pseudomonas putida, the plasmid is built TH77-IIMCS-2:pB101:10 -II1081 (SEQ ID NO:69). For this, the synthetic operon BTH_II1077, BT_II1080 and BT_II1081 (SEQ ID NO: 70) from the DNA 2.0 company (Menlo Park, CA, USA) is synthesized and intermediately cloned in the commercial vector pJ294 (DNA 2.0; Menlo Park, California) . The basis for the synthesis is the genomic sequence of the B. thailandensis E264 strain. Starting from vector pJ294-BTH II1074-II1080-II1081, the synthetic operon is cut from this vector via Xbal and then ligated into vector pBBR1MCS-2::ABC (SEQ ID NO: 40) equally cut with Xbal. The target vector pBBR1MCS-2::ABC-BHT_II1077-II1080-II1081 (SEQ ID NO:40) obtained has a size of 13768 base pairs. The insertion of the vector is sequenced. Performing the PCR, verifying successful PCR amplification by means of agarose gel electrophoresis, labeling the DNA with ethidium bromide, determining the size of the PCR fragment, purifying the PCR products and determining the DNA concentration is performed in a manner and manner known to the expert.
Transformation of Pseudomonas putida KT2440 and Gpp104 with the vector pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 (SEQ ID NO:69) is performed as described above (Iwasaki and collaborators Biosci. Biotech. Biochem. 1994 58(5):851-854). Plasmid DNA from every 10 clones is isolated and analyzed. The strains obtained, carrying the plasmids, are mentioned from P. putida KT2440 pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 or P. putida GPp104 pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081. 12. Quantification of rhamnolipid production by recombinant P. putida strains with and without BTHII1077, BTII1080 and BT II1081 genes from B. thailandensis E264
[000380] The recombinant P. putida strains generated in examples 1, 2 and 11 - P. putida strains 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-BTH10_II1077-II1077 -II1081 P. putida GPp104 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABC-BTH_II1077-II1080-II1081 are grown on LB-kanamycin agar plates (50 µg/ml).
[000381] For the production of rhamnolipids, the medium designated below as M9 medium was 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) v) of NH4Cl, 0.049% (w/v) of MgSO4 x 7 H2O and 0.1% (v/v) of a solution of trace elements. This consists of 1.78% (w/v) FeSO4 x 7 H2O, 0.191% (w/v) MnSO4 x 7 H2O, 3.65% (w/v) HCl, 0.187% (w/v) of ZnSO4 x 7 H2O, 0.084% (v/v) of Na-EDTA x 2 H2O, 0.03% (v/v) of H3BO3, 0.025% (w/v) of Na2MoO4 and 0.47% (w/ v) of CaCl2 x 2 H2O. The medium has the pH value adjusted to 7.4 with NH4O and the medium is then sterilized by means of an autoclave (121oC, 20 minutes). It is not necessary to adjust the pH value during cultivation.
[000382] To investigate the production of rhamnolipids in the shaker flask, a pre-culture was initially prepared. For this purpose, an inoculation loop of a strain freshly streaked on the LB agar plate was used and 10 mL of LB medium was inoculated into a 100 mL Erlenmeyer flask. All recombinant P. putida strains are grown in LB medium, to which 50 μg/ml of kanamycin has been added. Cultivation of P. putida strains was carried out at 37oC and 200 rpm overnight.
[000383] Precultures were used, to inoculate 50 mL of M9 medium (+ 50 μg/mL kanamycin) in the 250 mL Erlenmeyer flask (initial OD600 of 0.1). Cultures are grown at 200 rpm and 30oC. Within 24 hours, a 1 mL sample of broth was removed from the culture flask. The preparation of the sample for the next chromatographic analyzes and the chromatographic analysis itself are carried out as described in example 4.
[000384] 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 substantially more monoramnosyl-lipids than P. putida KT2440 pBBR1MCS-2::AB and P. putida GPp104 pBBR1MCS-2::AB strains. This proves that the increase of BTH_II1077-II1080-II1081 of B. thailandensis E264 increases the formation of monoramnosyl-lipids in P. putida strains with the rhlAB genes of Pseudomonas aeruginosa DSM1707.
[000385] Furthermore, it is demonstrated, 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 substantially more mono- and diramnosyl lipids than the P. putida KT2440 pBBR1MCS-2::ABC and P. putida GPp104 pBBR1MCS-2::ABC strains. This proves that the increase of BTH_II1077-II1080-II1081 of B. thailandensis E264 increases the formation of mono- and diramnosyl-lipids in P. putida strains with the rhlABC genes of Pseudomonas aeruginosa DSM1707.
[000386] Finally, it is shown that the weakening of the formation of polyhydroxybutyrate in the background culture of P. putida GPp104 in relation to the P. putida strain KT2440 leads to an increase in the formation of rhamnolipids, whereas the P. putida strains 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-BTH10_II1077-II1077 -II1081 are capable of forming substantially less mono- (or mono- and diramnosyl-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 pBBR1MCS-2::ABCM vector for the heterologous expression of the rhlA, rhlB, pa1131 and rhlC genes from Pseudomonas aeruginosa DSM1707 in Pseudomonas putida
[000387] For the heterologous expression of the rhlA, rhlB, pa1131 and rhlC genes of Pseudomonas aeruginosa DSM1707, plasmid pBBR1MCS-2::ABCM (SEQ ID NO:58) was constructed. For this, the pa1131 gene (SEQ ID NO: 59), starting from the genomic DNA of the Pseudomonas aeruginosa strain (PAO1 (DSM 1707) was amplified with the oligonucleotides
[000388] MFS2.0_xbaI_fw: 5'- AGGAAATCTAGATGAGAGGCCGGCAAGGATAC-3' (SEQ ID NO: 60)
[000389] MFS2.0_XbaI_rev:5'- CCAGGTTCTAGACGCCAGGATTGAACAGTACC-3' (SEQ ID NO: 61).
[000390] The amplification of the PCR product (1483 base pairs) was carried out with the polymerase PhusionTM High-Fidelity Master Mix from New England Biolabs (Frankfurt). The PCR product was cut with Xbal and the vector pBBR1MCS-2::ABC (SEQ ID NO: 40) also cut with Xbal, was ligated by means of the Fast Link Ligation kit (Epicentre Technologies; Madison, WI, USA). The target vector pBBR1MCS-2::ABCM (SEQ ID NO: 58) obtained has a size of 9892 base pairs. Insertion of the vector was sequenced. Chromosomal DNA was isolated using the DNeasy Blood and Tissue kit (Qiagen; Hilden) according to the manufacturer's instructions. Performing the PCR, verifying successful amplification using agarose gel electrophoresis, labeling the DNA with ethidium bromide, determining the size of the PCR fragment, purifying the PCR products and determining the DNA concentration was carried out in a manner and manner known to the expert.
Transformation of Pseudomonas putida KT2440 and Gpp104 with the pBBR1MCS-2::ABCM vector was performed as described above (Iwasaki and coworkers Biosci. Biotech. Biochem. 1994. 58(5):851-854). Plasmid DNA from each 10 clones was isolated and analyzed. The strains obtained, carrying the plasmids, were mentioned from P. putida KT2440 pBBR1MCS-2::ABCM or P. putida GPp104 pBBR1MCS-2::ABCM. 14. Quantification of rhamnolipid production by recombinant P. putida strains with and without overexpression of the pa1131 gene of Pseudomonas aeruginosa DSC 1707
The recombinant strains generated in examples 2 and 13 - P. putida KT2440 pBBR1MCS-2::ABC, P. putida KT2440 pBBR1MCS-2::ABCM, P. putida KT2440 pBBR1MCS-2::ABC and P. putida strains Gpp104 pBBR1MCS-2::ABCM were grown on LB-kanamycin agar plates (50 µg/ml). The subsequent cultivation for the production of the rhamnolipids was carried out as described in example 12.
[000393] The preparation of samples for the subsequent chromatographic analyzes and the chromatographic analyzes themselves were carried out as described in example 4. The results are illustrated in the following table.
[000394] Illustration of di- and monorhamnosyl-lipids through P. putida strains with and without P. aeruginosa pa1131 gene overexpression after 48 hour incubation


[000395] The results show, that the overexpression of the P. aeruginosa pa1131 gene in the two background cultures (KT2440: wild type or Gpp104 with formation of inactivated polyhydroxybutyrate) leads to an increased formation of di- and monorhamnosyl-lipids. The results further show that the impairment of polyhydroxybutyrate formation in Gpp104 generally leads to an increased formation of rhamnosyl lipids. 15. Construction of a pEC-X99A::AB vector for the heterologous expression of the rh1A and rhlB gene of Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum
[000396] For the heterologous expression of the rh1A and rh1B genes of Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum, the plasmid pEC-XT99A::AB (SEQ ID NO:52) is constructed. For this, the synthetic rhlAB operon (SEQ ID NO: 37) from the company GeneArt AG (Regensburg) was synthesized and intermediately cloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of Pseudomonas aeruginosa DSM1707. Starting from vector pMA::AB, the synthetic operon was cut from the vector via Bg/II and Xbal and then ligated into the expression vector pEC-XT99A (US patent 7118904) cut with BamHl and Xbal. The resulting plasmid pEC-XT99A::AB (SEQ ID NO:52) is 9793 base pairs in size. Ligation as well as transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) was carried out in a manner and manner known to the skilled person. Insert authenticity was verified by DNA sequence analysis.
[000397] Transformation of C. glutamicum ATCC13032 with the pEC-XT99A::AB vector is performed as described above (Liebl et al., FEMS Microbiol. Lettl 53:299-303 (1989)). Selection of transformants is performed on LBHIS agar plates (18.5 g/L boullion brain-heart infusion, 0.5 M sorbitol, 5 g/L bacto-tripton, 2.5 g/L bacto -yeast extract, 5 g/L of NaCl and 18 g/L of bacto-agar, supplemented with 5 mg/L of tetracycline). Plates were incubated for two days at 33oC. The strain obtained, containing plasmids, is mentioned from C. glutamicum pEC-XT99A::AB. 16. Construction of a pEC-XT99A::ABC vector for the heterologous expression of the rhlA, rhlB and rhlC genes from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum
[000398] For the heterologous expression of the rhlA, rhlB and rhlC genes of Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum, the plasmid pEC-XT99A::ABC is constructed (SEQ ID NO:53). For this purpose, the synthetic rhlABC operon (SEQ ID NO: 39) from the company GeneArt AG (Regensburg) was synthesized and intermediately cloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of Pseudomonas aeruginosa DSM1707. Starting from the pMA::ABC vector, the synthetic operon is cut from the vector via Bg/II and Xbal and then ligated into the pEC-XT99A expression vector cut with BamHl and Xbal (US patent 7118904). The resulting plasmid pEC-XT99A::ABC (SEQ ID NO:53) is 10780 base pairs in size. Ligation as well as transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) is carried out in a manner and manner known to the skilled person. The authenticity of the insert is verified using DNA sequence analysis.
Transformation of C. glutamicum ATCC 13032 with the pEC-XT99A::ABC vector is performed as described above (Liebl et al., FEMS Microbiol. Lett. 53:299-303 (1989)). Selection of transformants is performed on LBHIS agar plates (18.5 g/L boullion brain-heart infusion, 0.5 M sorbitol, 5 g/L bacto-tripton, 2.5 g/L bacto -yeast extract, 5 g/L of NaCl and 18 g/L of bacto-agar, supplemented with 5 mg/L of tetracycline). Plates were incubated for two days at 33oC. The plasmid-containing strain obtained is mentioned from C. glutamicum pEC-XT99A::ABC. 17. Construction of a pEC-XT99A::ABM vector for the heterologous expression of the rhlA, rhlB and pa1131 genes of Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum
[000400] For the heterologous expression of the genes rhlA, rhlB and pa1131 of Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum, the plasmid pEC-XT99A::ABM is constructed (SEQ ID NO:54). For this, the synthetic rhlABM operon (SEQ ID NO: 41) from the company GeneArt AG (Regensburg) was synthesized and intermediately cloned in the commercial vector pMA (GeneArt AG). The basis for the synthesis was the already known genomic sequence of Pseudomonas aeruginosa DSM1707. Starting from the pMA::ABM vector, the synthetic operon is cut from the vector via Bg/II and Xbal and then ligated into the pEC-XT99A expression vector cut with BamHl and Xbal (US patent 7118904). The resulting plasmid pEC-XT99A::ABM (SEQ ID NO: 54) is 11073 base pairs in size. Ligation as well as transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) is carried out in a manner and manner known to the skilled person. The authenticity of the insert is verified using DNA sequence analysis.
[000401] Transformation of C. glutamicum ATCC 13032 with the pEC-XT99A::ABM vector is performed as described above (Liebl et al., FEMS Microbiol. Lett. 53:299-303 (1989)). Selection of transformants is performed on LBHIS agar plates (18.5 g/L boullion brain-heart infusion, 0.5 M sorbitol, 5 g/L bacto-tripton, 2.5 g/L bacto -yeast extract, 5 g/L of NaCl and 18 g/L of bacto-agar, supplemented with 5 mg/L of tetracycline). Plates were incubated for two days at 33oC. The strain obtained, containing plasmids, is mentioned from C. glutamicum pEC-XT99A::ABM. 18. Construction of a pEC-XT99A::ABMC vector for the heterologous expression of the rhlA, rhlB, pa1131 and rhlC genes from Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum
[000402] For the heterologous expression of the genes rhlA, rhlB, pa1131 and rhlC of Pseudomonas aeruginosa DSM1707 in Corynebacterium glutamicum, the plasmid pEC-XT99A::ABCM is constructed (SEQ ID NO: 55). For this, the pa1131 gene (SEQ ID NO: 59) is amplified starting from genomic DNAs of the strain Pseudomonas aeruginosa PAO1 (DSM 1707) with the oligonucleotides
[000403] MFS2.0_xbaI_fw: 5'- AGGAAATCTAGATGAGAGGCCGGCAAGGATAC-3' (SEQ ID NO: 60)
[000404] MFS2.0_XbaI_rev:5'- CCAGGTTCTAGACGCCAGGATTGAACAGTACC-3' (SEQ ID NO: 61).
[000405] The amplification of the PCR product (1483 base pairs) was carried out with the polymerase PhusionTM High-Fidelity Master Mix from New England Biolabs (Frankfurt). The PCR product was cut with Xbal and ligated into vector pBBR1MCS-2::ABC (SEQ ID NO: 40) also cut with Xbal, by means of the Fast Link Ligation ligation kit (Epicentre Technologies; Madison, WI, USA). The pEC-XT99A::ABCM (SEQ ID NO: 55) target vector obtained has a size of 12263 base pairs. Insertion of the vector was sequenced. Chromosomal DNA was isolated using the DNeasy Blood and Tissue kit (Qiagen; Hilden) according to the manufacturer's instructions. Performing the PCR, verifying successful PCR amplification using agarose gel electrophoresis, labeling the DNA with ethidium bromide, determining the size of the PCR fragment, purifying the PCR products and determining the concentration of DNA was performed in a manner and manner known to the expert.
[000406] Transformation of C. glutamicum ATCC 13032 with the pEC-XT99A::ABCM vector is performed as described above (Liebl et al., FEMS Microbiol. Lett. 53:299-303 (1989)). Selection of transformants is performed on LBHIS agar plates (18.5 g/L boullion brain-heart infusion, 0.5 M sorbitol, 5 g/L bacto-tripton, 2.5 g/L bacto -yeast extract, 5 g/L of NaCl and 18 g/L of bacto-agar, supplemented with 5 mg/L of tetracycline). Plates were incubated for two days at 33oC. The strain obtained, containing plasmids, is mentioned from C. glutamicum pEC-XT99A::ABCM. 19. Construction of a pVWEX1::rfbBDAC vector for heterologous expression in C. glutamicum
[000407] For the heterologous expression of the P. putida rfbBDAC gene under control of the lac promoter in C. glutamicum, the vector pVWEX1::rfbBDAC (SEQ ID NO: 57) was constructed. For this, the vector pBBR1MCS-2::rfbBDAC (SEQ ID NO: 45) is digested with Xbal and the rfbBDAC gene from P. putida KT2440 and the fragment containing the lac promoter (3840 base pairs) are ligated into the vector pVWEX1 ( SEQ ID NO: 56) digested with Xbal. The resulting plasmid pVWEX1::rfbBDAC (SEQ ID NO: 57) is 12311 base pairs in size. Ligation as well as transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) is carried out in a manner and manner known to the skilled person. The authenticity of the insert is verified using DNA sequence analysis.
[000408] 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 with the vector pVWEX1: :rfbBDAC is performed as described above (Liebl et al., FEMS Microbiol. Lett. 53:299-303 (1989)). Selection of transformants is performed on LBHIS agar plates (18.5 g/L boullion brain-heart infusion, 0.5 M sorbitol, 5 g/L bacto-tripton, 2.5 g/L bacto -yeast extract, 5 g/L of NaCl and 18 g/L of bacto-agar, supplemented with 5 mg/L of kanamycin). Plates were incubated for two days at 33oC. The plasmid-containing strains obtained are mentioned from C. glutamicum pEC-XT99A pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::AB pVWEX1::rfbBDAC, C. glutamicum pEC-XT99A::ABM pVWEX1:,CrfBB glutamicum pEC-XT99A::ABC pVWEX1::rfbBDAC and C. glutamicum pEC-XT99A::ABCM pVWEX1::rfbBDAC. 20. Quantification of rhamnolipid production by recombinant C. glutamicum strains
[000409] The recombinant C. glutamicum strains generated in examples 15 to 19 - strains 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. pVWEX1::rfbBDAC, are grown on mg/L tetracycline or 5 mg/L tetracycline and 25 plates mg/L of kanamycin. To investigate the production of rhamnolipids in the shaker flask, pre-cultures are initially prepared. For this, an inoculation loop of a strain freshly streaked on the LBHIS agar plate is used and 10 mL of LBHIS medium (18.5 g/L of boullion brain-heart infusion, 0.5 M sorbitol) is inoculated. , 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) in a 100 mL Erlenmeyer flask. The strains are cultivated at 33oC and 200 rpm overnight. The next morning, 50 mL of CGXII medium (with 5 mg/L tetracycline or 5 mg/L tetracycline and 25 mg/L kanamycin) in a 500 mL Erlenmeyer flask with baffles are inoculated with 1 mL of the preculture (initial OD600 0.1). CGXII medium:
[000410] 20 g/L of (NH4)2SO4 (Merck)
[000411] 5 g/L of urea (Merck)
[000412] 1 g/L of KH2PO4 (Merck)
[000413] 0.25 g/L of MgSO4 . 7 H2O (Merck)
[000414] 10 mg/L of CaCl2 (Merck)
[000415] 42 g/L of MOPS (Roth)
[000416] 0.2 mg/L of biotin (Merck)
[000417] 1 ml/L of saline solution traces
[000418] adjust pH 7 with NaOH
[000419] after autoclaving, add 1 ml/L of protocatechuic acid (30 g/L dissolved in diluted NaOH, sterilized) and 40 g/L of glucose (Merck). Traces of saline solution:
[000420] 10 g/L of FeSO4 • 7 H2O (Merck)
[000421] 10 g/L of MnSO4 • H2O (Merck)
[000422] 1 g/L of ZnSO4 • 7 H2O (Merck)
[000423] 0.2 g/L of CuSO4 • 5 H2O (Merck)
[000424] 20 mg/L of NiCh • 6 H2O (Merck)
[000425] acidify to dissolve with HCl to pH 1.
[000426] Cultures are grown at 200 rpm and 33oC up to an optical density (600 nm) of 0.4 - 0.6. At this optical density, cultures are induced by adding IPTG (isopropyl-β-thiogalactopyranoside; 1 mM final concentration). The following expression is also carried out at 33oC and 200 rpm for 72 hours. Within 24 hours, a 1 mL sample of broth is taken from the culture flask. Sample preparation for the following chromatographic analyzes and the chromatographic analyzes themselves are carried out as described in example 4.
[000427] While C. glutamicum pEC-XT99A does not produce any 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 rhamnolipid formation can be detected. Based on reference materials, it is demonstrated that C. glutamicum pEC-XT99A::AB and C. glutamicum pEC-XT99A::ABC, C. glutamicum pEC-XT99A::ABM and C. glutamicum pec-XT99A:: ABCM are able to form diramnosyl-lipids and monoramnosyl-lipids. Furthermore, it is demonstrated that C. glutamicum pEC-XT99A::ABM and C. glutamicum pec-XT99A::ABCM are able to form more monorhamnosyl-lipids or diramnosyl-lipids and monorhamnosyl-lipids than the respective reference strains , C. glutamicum pEC-XT99A::AB and C. glutamicum pEC-XT99A::ABC without augmentation of the Pseudomonas aeruginosa pa1131 gene.
[000428] Furthermore, it is demonstrated 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 were substantially more mono- (C. glutamicum pEC-XT99A::AB pVWEX1::rfbBDAC and C. glutamicum pEC-XT99A::ABM pVWEX1::: rfbBDAC) or mono- and diramnosyl lipids (C. glutamicum pEC-XT99A::ABC pVWEX1::rfbBDAC and C. glutamicum pEC-XT99A::ABCM pVWEX1::rfbBDAC) than C. glutamicum pEC-XT99A:: strains ABM, C. glutamicum pEC-XT99A::ABC and C. glutamicum pEC-XT99A::ABCM without increase of P. putida rfbBDA genes. 21. Construction of Pseudomonas strains carrying plasmids pBBR1MCS-2, pBBR1MCS-2::AB, pBBR1MCS-2::ABC, pBBR1MCS-2::ABM and pBBR1MCS-2::ABCM
[000429] Plasmids pBBR1MCS-2, pBBR1MCS-2::AB, pBBR1MCS-2::ABC, pBBR1MCS-2::ABM and pBBR1MCS-2::ABCM are introduced by means of electroporation into 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 as described. above (Iwasaki and coworkers Biosci. Biotech. Biochem. 1994, 58(5):851-854). The selection of transformants is performed on nutrient agar plates (5 g/L peptone; 3 g/L meat extract; 15 g/L agar; pH 7; supplemented with 50 mg/L kanamycin). Plates are incubated for two days at 30oC, respectively, 28oC. The strains obtained, carrying the plasmids, are mentioned Pseudomonas fluorescens DSM 50090 pBBR1MCS-2, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2, Pseudomonas putida DSM 6899 pBBR1MCS-2, Pseudomonas putida DSM 50204 pBBR1B1MCS-2, Pseudomonas putida 50204 pBBR1MCS-2, Pseudomonas putida 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 DSMBS1 pBBR58, Pseudomonas fluorescens DSM 50090 pBBR58 pBBR58 - 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 99, Pseudomonas -2::ABC, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABC, Pseudomonas putida DSM 50 204 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 pB :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::BBR1MCS 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 4S166 pB :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, putida 2:ABMMCS pBBR1MCS 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 rhamnolipid production by recombinant Pseudomonas strains
Recombinant Pseudomonas strains generated in example 21 - Pseudomonas fluorescens DSM 50090, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2, Pseudomonas putida DSM 6899 pBBR1MCS-2, Pseudomonas putida DSM 50204 pBBR1Bmonas-2, Pseudomonas fluorescens-2, Pseudomonas DSM 50204 pBBR1Bmonas-2, 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 DSMBS1 pBBR58, Pseudomonas fluorescens DSM 50090 pBBR58 pBBR58 -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 pseudomonas59, Pseudomonas -2::ABC, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABC, Pseudomonas putida D SM 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 ::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 pBUD1MCS, putida 2:AB1CMS 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 ::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 pBUD1MCS, putida-2:BR1MCS 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 stut zeri DSM 4166 pBBR1MCS-2::ABM and Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABM are grown on LB-kanamycin agar plates (50 µg/ml). The subsequent cultivation for the production of rhamnolipids is carried out as described in example 12. The preparation of samples for the following chromatographic analyzes and the chromatographic analyzes themselves are carried out as described in example 4.
While the strains of Pseudomonas Pseudomonas fluorescens DSM 50090, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2, Pseudomonas putida DSM 6899 pBBR1MCS-2, Pseudomonas putida DSM 50204 pBBR1MCS-2, Pseudomonas putida pBBR1MCS-2, Pseudomonas putida DSM-1MCS-2, Pseudomonas putida PMC1MCS-2, Pseudomonas putida pMC1MCS-2, Pseudomonas putida pMC132 pMCS. -2, P. stutzeri DSM 10701 pBBR1MCS-2, Pseudomonas stutzeri DSM 4166 pBBR1MCS-2, Pseudomonas fulva DSM 17717 pBBR1MCS-2 do not produce any rhamnolipids, in the recombinant strains Pseudomonas fluorescens DSM 50090 pBBR1MCS-2 pBBR1MCS-2 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 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 4166 pBBR1MCS-2::ABM and Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABM the formation of mono- and diramnosyl-lipids can be verified, as well as in 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.brasicacearum DSM 13227 pBBR127 pBBR1 , 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:ABC, Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABC, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2: pBBR1MCS-2::ABCM, Pseudomonas putida DSM 6899 pBBR1MCS-2::ABCM, Pseudomonas putida DSM 50204 pBBR1MCS-2::ABCM, Pseudomonas putida 50194 pBBR1MCS-2::AB CM, 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: formation: of mono- and diramnosyl-lipids.
[000432] Furthermore, through the recombinant Pseudomonas strains, Pseudomonas fluorescens DSM 50090 pBBR1MCS-2::ABM, Pseudomonas fluorescens DSM 9958 pBBR1MCS-2::ABM, Pseudomonas putida DSM 6899 pBBR1MCS-2::204 putida DSMmonas 50 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 DSM1:4166 pBBR ABM and Pseudomonas fulva DSM 17717 pBBR1MCS-2::ABM, are formed less mono- and diramnosyl-lipids than through the respective reference strains without the pa1131 gene of P. aeruginosa 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 1 7717 pBBR1MCS-2::AB or 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 p 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 17717 pBBR1MCS-2::ABC, without augmentation of the Pseudomonas aeruginosa pa1131 gene. 23. Construction of the vectors pBBR1MCS-2::ABPAO1-C1 and pBBR1MCS-2::ABPA7-CE264 for the heterologous expression of alternative rhlA, rhlB and rhlC genes of Pseudomonas aeruginosa PAO1, Pseudomonas aeruginosa PA7, Pseudomonas thailandia 264 1264 in P. putida
[000433] For the heterologous expression of the alternative rhlA, rhlB and rhlC genes of Pseudomonas aeruginosa PAO1 or Pseudomonas aeruginosa PA7, the plasmids pBBR1MCS-2::ABPAO1 (SEQ ID NO: 62) and pBBR1PA7MCS-2::: (SEQ ID NO:63). For this, the synthetic operons rhlABPAO1 (SEQ ID NO: 64) and rhlABPA77 (SEQ ID NO: 65) from the company DNA 2.0 (Menlo Park, CA, USA) are synthesized and intermediately cloned in the commercial vector pJ294 (DNA 2, 0). The basis for the synthesis is the already known genomic sequence of the Pseudomonas aeruginosa PAO1 and Pseudomonas aeruginosa PA7 strains. Starting from vectors pH294::ABPAO1 and pJ294::ABPA7, the synthetic operons are cut from the vectors by means of Kpnl and Xbal and then ligated into the expression vector pBBR1MCS-2 cut with Kpnl and Xbal (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). The resulting plasmids pBBR1MCS-2::ABPAO1 (SEQ ID NO: 62) and pBBR1MCS-2::ABPA7 (SEQ ID NO: 63) are 7332 or 7354 base pairs in size. Ligation as well as transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) is carried out in a manner and manner known to the skilled person. The authenticity of the insert is verified by means of DNA sequence analysis.
[000434] In the second stage, plasmids pBBR1MCS-2::ABPAO1-C1 (SEQ ID NO: 66) and pBBR1MCS-2::ABPA7-CE264 (SEQ ID NO: 67) are produced. For this purpose, the rhlC genes from Pseudomonas aeruginosa 1 (SEQ ID NO: 68) and Burkholderia thailandensis E264 (SEQ ID NO: 76) from the company DNA 2.0 (Menlo Park, CA, USA) are synthesized and intermediately cloned in the commercial vector pH294 (DNA 2.0). The basis for the synthesis is the already known genomic sequence of the Pseudomonas aeruginosa 1 and Burkholderia thailandensis E264 strains. Starting from vectors pJ294::C1 and pJ294::CE264, the rhlC genes are cut from the vectors via Xba and Sacl and then ligated with vectors pBBR1MCS-2::ABPAO1 (SEQ ID NO: 62) or pBBR1MCS -2::ABPA7 (SEQ ID NO:63) equally cut with Xba and Sacl. Plasmids pBBR1MCS-2::ABPAO1-C1 (SEQ ID NO: 66) and pBBR1MCS-2::ABPA7-CE264 (SEQ ID NO: 67) have sizes of 8325 or 8335 base pairs. Ligation as well as transformation of chemically competent E. coli DH5α cells (Gibco-BRL, Karlsruhe) is carried out in a manner and manner known to the skilled person. The authenticity of the insert is verified using DNA sequence analysis.
Transformation of Pseudomonas putida KT2440 and Gpp104 with the vectors pBBR1MCS-2, pBBR1MCS-2::ABPAO1-C1 and pBBR1MCS-2::ABPA7-CE264 is carried out as described above (Iwasaki and collaborators Biosci. Biotech. Biochem. 1994. 58(5):851-854). Plasmid DNAs from every 10 clones are isolated and analyzed. The strains obtained carrying the plasmids are mentioned from 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 rhamnolipid production by recombinant P. putida strains with rhlA, rhlB and rhlC genes from Pseudomonas aeruginosa PAO1, Pseudomonas aeruginosa PA7, Pseudomonas aeruginosa 1 and aeruginosa PAO1, Pseudomonas aeruginosa 1, Pseudomonas aeruginosa PAO1
[000436] The recombinant P. putida strains generated in example 23 are grown on LB-kanamycin agar plates (50 µg/ml). The subsequent cultivation for the production of rhamnolipids is carried out as described in example 12. The preparation of the samples for the following chromatographic analyzes and the chromatographic analyzes themselves were carried out as described in example 4.
While the P. putida KT2440 pBBR1MCS-2 and P. putida GPp104 pBBR1MCS-2 strains are not capable of producing mono- and diramnosyl-lipids, the P. putida KT2440 pBBR1MCS-2::ABPAO1-C1 strains, 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 diramnosyl-lipids. It is demonstrated that strains with impaired polyhydroxybutyrate formation (P. putida GPp104 pBBR1MCS-2::ABPAO1-C1 and P. putida GPp104 pBBR1MCS-2::ABPA7-CE264) are able to produce more mono- and diramnosyl lipids, than strains without impairment of polyhydroxybutyrate formation (P. putida KT2440 pBBR1MCS-2::ABPAO1-C1 and P. putida KT2440 pBBR1MCS-2::ABPA7-CE264). 25. Construction of pBBR1MCS-2::ABrfbBDAC, pBBRIMCS-2::ABMrfbBDAC and pBBR1MCS-2::ABMCrfbBDAC vectors for the overexpression of P. putida rfbBDAC-operon in P. putida and E. coli'
[000438] For the construction of vectors pBBR1MCS-2::AB_rfbBDAC, pBBR1MCS-2::ABM_rfbBDAC and pBBR1MCS-2::ABMC_rfbBDAC for the overexpression of P. putida rfbBDAC-operon in P. putida and E. coli, P. putida rfbBDAC-operon was initially amplified by means of PCR. The vector pBBR1MCS-2::rfbBDAC (SEQ ID NO: 45) served as a template for a PCR. The following oligonucleotides were used:
[000439] RL_AgeI-fw: 5'- TATATATAACCGGTATTAATGCAGCTGGCACGAC -3' (SEQ ID NO: 71)
[000440] RL_AgeI_rev: 5'-GGCCGACCGGTACTAGTGGA -3' (SEQ ID NO: 72)
[000441] PCR was performed with PhusionTM High-Fidelity Master Mix polymerase from New England Biolabs (Frankfurt). It was carried out in a manner and manner known to the expert. The target sequence (Iac promoter and rfbBDAC) was intermediately cloned in the Trenzyme Alligator Cloning system. E. coli DH5α transformants (New England Biolabs; Frankfurt) were selected and plasmid DNAs from various candidates were isolated and sequenced. After the sequence was verified and searched for accuracy, the vector was cut with Agel. The target fragment was ligated into the equally cut pBBR1MCS-2::AB (SEQ ID NO: 38), pBBR1MCS-2::ABM (SEQ ID NO: 42) and pBBR1MCS-2::ABMC (SEQ ID NO: 51) vectors with Agel by conventional binding methods. The resulting 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) vectors have sizes of 11960, 13289 or 14250 base pairs. The insertions of the vectors were sequenced. Performing PCRs, verifying successful PCR amplification using agarose gel electrophoresis, labeling DNA with ethidium bromide, determining the size of PCR fragments, purifying PCR products and determining concentration of DNA was performed in a manner and manner known to the expert.
The transformation of Pseudomonas putida KT2440 with the vectors pBBR1MCS-2::AB_rfbBDAC, pBBR1MCS-2::ABM_rfbBDAC and pBBR1MCS-2::ABMC_rfbBDAC was carried out as described above (Iwasaki et al. Biosci. Biochem. 1994. Biotech. 58(5):851-854). Plasmid DNA from 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 rhamnolipid production by P. putida KT2440 pBBR1MCS-2::ABrfbBDAC, P. putida KT2440 pBBRIMCS-2::ABMrfbBDAC, P. putida KT2440 pBBR1MCS-2::ABCrfbBDAC, P. putida KT2440 pBBRIMCS-2::ABMrfbBDAC :ABMCrfbBDAC, 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 recombinant
[000443] The recombinant P. putida strains generated in examples 2, 7 and 25, are grown on LB-kanamycin agar plates (50 µg/ml). The subsequent cultivation for the production of rhamnolipids is carried out as described in example 12. The preparation of samples for the following chromatographic analyzes and the chromatographic analyzes themselves are carried out as described in example 4.
[000444] 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 ::ABMC are able to form mono- and diramnosyl-lipids.
[000445] Furthermore, it is demonstrated, 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 capable to form more mono- and diramnosyl 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 pBBR ::ABC without augmentation of Pseudomonas aeruginosa pa1131 gene.
[000446] 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 KTMC40 ::ABMC_rfbBDAC are able to form more mono- (P. putida KT2440 pBBR1MCS-2::AB_rfbBDAC and P. putida KT2440 pBBR1MCS-2::ABM_rfbBDAC) or mono- and diramnosyl-lipids (P. putida KT2440 pBBR1MCS-2::BBR1MCS-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 augmentation of P. putida rfbBDAC gene. 27. Generation of 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::ABrfbBDAC, E. coli W3110 pBBR1MCS-2::ABMrfbBDAC, E. coli W3110 pBBRIMCS-2::ABCrfbBDAC and E. coli W3110 pBBR1MCS-2::ABCM recombinant rfbBDAC
[000447] Transformation of E. coli W3110 was performed as described above (Miller JH. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Plainview, NY: Cold Spring Harbor Lab. Press; 1992) by means of electroporation. Plasmid DNA from each 10 clones was isolated and analyzed. The strains obtained carrying the plasmids were mentioned 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_r. 28. Quantification of rhamnolipid production by 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 pBBRIMCS-2::ABMrfbBDAC, E. coli W3110 pBBR1MCS-2::ABCrfbBDAC and E. coli W3110 pBBR1MCS-2::ABCMrfbB recombinants
[000448] The recombinant E. coli strains generated in example 27 are grown on LB-kanamycin agar plates (50 µg/ml). The subsequent cultivation for the production of the rhamnolipids is carried out as described in example 10. The preparation of samples for the following chromatographic analyzes and the chromatographic analyzes themselves are carried out as described in example 4.
[000449] It is demonstrated 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 diramnosyl-lipids. Furthermore, it is shown that E. coli W3110 pBBR1MCS-2::ABM and E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC form more monoramnosyllipids than E. coli W3110 pBBR1MCS-2::AB and E. coli W3110 pBBR1MCS-2::AB_rfbBDAC without augmentation of Pseudomonas aeruginosa pa1131 gene.
[000450] Furthermore, it is shown that E. coli W3110 pBBR1MCS-2::ABCM and E. coli W3110 pBBR1MCS-2::ABCM_rfbBDAC form more mono- and diramnosyl-lipids than E. coli W3110 pBBR1MCS-2: :ABC and E. coli W3110 pBBR1MCS-2::ABC_rfbBDAC without augmentation of Pseudomonas aeruginosa pa1131 gene. Furthermore, it is shown that E. coli W3110 pBBR1MCS-2::ABM and E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC form more monoramnosyl-lipids than E. coli W3110 pBBR1MCS-2::AB and E. coli W3110 pBBR1MCS-2::AB_rfbBDAC without augmentation of Pseudomonas aeruginosa pa1131 gene. Finally, it is demonstrated, 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_f are able to form more mono- ( E. coli W3110 pBBR1MCS-2::AB_rfbBDAC, E. coli W3110 pBBR1MCS-2::ABM_rfbBDAC) or mono- and diramnosyl-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 augmentation of the P. putida rfbBDAC gene.

权利要求:
Claims (9)
[0001]
1. Cell, selected from microorganisms, characterized by the fact that it is capable of forming a rhamnolipid of General Formula (I),
[0002]
2. Cell according to claim 1, characterized in that it also has an increased activity of the E3 enzyme, and the E3 enzyme is a rhamnosyltransferase II and is capable of catalyzing the conversion of dTDP-rhamnose and α-L-rhamnopyranosyl -3-hydroxyalkanoyl-3-hydroxyalkanoate for α-L-rhamnopyranosyl-(1-2)-α-L-rhamnopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate; and wherein the E3 enzyme is selected from: an E3a enzyme having a polypeptide sequence of SEQ ID NO: 6, an E3b enzyme having a polypeptide sequence of SEQ ID NO: 22, an E3c enzyme having the polypeptide sequence of SEQ ID NO: 90, and an E3d enzyme having a polypeptide sequence of SEQ ID NO: 92.
[0003]
3. Cell according to claim 1 or 2, characterized in that it has an increased activity of the combination of E1E2E3 enzymes.
[0004]
4. Cell, according to claim 3, characterized in that, with respect to Formula (I), n = 1.
[0005]
5. Cell according to any one of claims 1 to 4, characterized in that it is selected from a genus from 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.
[0006]
6. Cell according to any one of claims 1 to 5, characterized in that it has a reduced activity of an E9 or E10 enzyme compared to its wild type, where E9 has the polypeptide sequence of SEQ ID NO: 30 or SEQ ID NO: 32 and where E10 has a polypeptide sequence of SEQ ID NO: 34 or SEQ ID NO: 36.
[0007]
7. Cell according to any one of claims 1 to 6, characterized in that, compared to its wild type, it has an increased activity of an E8 enzyme, which catalyzes the export of a rhamnolipid of the General Formula (I) from the cell to the surrounding medium, preferably with polypeptide sequence SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28.
[0008]
8. Cell according to any one of claims 1 to 7, characterized in that it contains a nucleic acid that has the following sequences A1, A2 and A3: the group [A1] consists of the following sequences: (A1a) a sequence according to SEQ ID NO: 1, wherein that sequence encodes a protein, which is capable of converting 3-hydroxydecanoyl-ACP to 3-hydroxydecanoyl-3-hydroxydecanoic acid via 3-hydroxydecanoyl-3-hydroxydecanoyl-ACP, ( A1b) a sequence according to SEQ ID NO: 17, which sequence encodes a protein, which is capable of converting 3-hydroxytetradecanoyl-ACP to 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid via 3-hydroxytetradecanoyl-3-hydroxytetradecanoyl -ACP, the group [A2] consists of the following sequences: (A2a) a sequence according to SEQ ID NO: 3, which sequence encodes a protein, which is capable of converting dTDP-rhamnose and 3-hydroxydecanoyl-acid 3-hydroxydecanoic to α-L-rhamnopyranosyl-3-hydroxy acid oxidecanoyl-3-hydroxydecanoic acid, (A2b) a sequence according to SEQ ID NO: 19, which sequence encodes a protein, which is capable of converting dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α- L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxyteradecanoic, and the group [A3] consists of the following sequences: (A3a) a sequence according to SEQ ID NO: 5, which sequence encodes a protein, which is capable of 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, (A3b) a sequence according to SEQ ID NO: 21, which sequence encodes a protein, which is capable of converting dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to α-L-rhamnopyranosyl acid -(1-2)-α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, or a vector, especially a vector of expression or a gene overexpression cassette, comprising a nucleic acid sequence selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 47 and from acids nucleic selected from the three aforementioned groups [A1], [A2] and [A3].
[0009]
9. Process for the production of rhamnolipids of the General Formula (I), characterized in that it comprises the process steps: (I) contacting a cell, as defined in any one of claims 1 to 7, with a medium containing a carbon source, (II) culturing the cells under conditions that allow the cell to form rhamnolipid from the carbon source, and (III) optionally isolating the formed rhamnolipids.

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CN110248952A|2017-02-06|2019-09-17|罗格斯技术有限责任公司|The decoloration of rhamnolipid composition is concentrated|

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US20200214959A1|2017-08-24|2020-07-09|Evonik Operations Gmbh|Rhamnolipid derivatives as emulsifiers and dispersing aids|

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CN108060111B|2017-10-27|2021-06-04|中国科学院微生物研究所|Pseudomonas aeruginosa for increasing rhamnolipid yield and construction method thereof|

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WO2021190993A1|2020-03-24|2021-09-30|Evonik Operations Gmbh|Compositions containing rhamnolipide, alkyl polyglycoside and acyl lactylate|

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WO2022017844A1|2020-07-22|2022-01-27|Evonik Operations Gmbh|New rhamnolipid oligo-esters|

法律状态:
2016-10-04| B25A| Requested transfer of rights approved|Owner name: EVONIK DEGUSSA GMBH (DE) |

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-06-04| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-12-24| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-08-25| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-12-15| B25D| Requested change of name of applicant approved|Owner name: EVONIK OPERATIONS GMBH (DE) |

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2021-02-17| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-06-08| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-06-15| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-08-31| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 20/07/2011, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |

优先权:

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申请号 | 申请日 | 专利标题

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DE102010032484A|DE102010032484A1|2010-07-28|2010-07-28|Cells and methods for producing rhamnolipids|

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DE102010032484.1|2010-07-28|

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PCT/EP2011/062441|WO2012013554A1|2010-07-28|2011-07-20|Cells and method for producing rhamnolipids|BR122020023808-9A| BR122020023808B1|2010-07-28|2011-07-20|BACTERIAL CELL THAT IS CAPABLE OF FORMING AT LEAST ONE GENERAL FORMULARAMNOLIPIDS, PROCESS FOR THE PRODUCTION OF GENERAL FORMULARAMNOLIPIDS, USE OF RAMNOLIPIDS AND VECTOR|