DE102012201360A1 - New cell capable of forming rhamnolipid compound, which is useful for the production of cosmetic, dermatological or pharmaceutical formulations, of plant protection formulations and of care and cleaning agents and surfactant concentrates - Google Patents

Abstract

Cell capable of forming at least one rhamnolipid compound (I), is new, where the cells are genetically modified in such a way that they are compared to their wild type and have an increased activity of at least one of the enzymes E 1, E 2 and E 3, the enzyme E is able to catalyze the conversion of 3-hydroxyacyl-[acyl-carrier-protein] to 3-ketoacyl-[acyl-carrier protein] or 3-hydroxyacyl-coenzyme A to 3-ketoacyl-coenzyme A, E 3 is able to catalyze the conversion of acyl-[acyl-carrier protein] to acyl-coenzyme A, and E 4 is able to catalyze the conversion of NADP+ to NADPH. Cell capable of forming at least one rhamnolipid compound of formula (I), is new, where the cells are genetically modified in such a way that they are compared to their wild type and have an increased activity of at least one of the enzymes E 1, E 2 and E 3, the enzyme E is able to catalyze the conversion of 3-hydroxyacyl-[acyl-carrier-protein] to 3-ketoacyl-[acyl-carrier protein] or 3-hydroxyacyl-coenzyme A to 3-ketoacyl-coenzyme A, E 3 is able to catalyze the conversion of acyl-[acyl-carrier protein] to acyl-coenzyme A, and E 4 is able to catalyze the conversion of NADP+ to NADPH. R1, R2 : 2-24 organic group (optionally branched, substituted and saturated by hydroxy) or mono-, di-, or triunsaturated alkyl group; m : 0-2, preferably 0-1; and n : 0 or 1 (preferred). An independent claim is included for preparing (I), comprising contacting the cell with a medium containing a carbon source, culturing the cell under conditions that make it possible for the cell to form rhamnolipid from the carbon source, and optionally isolating the formed rhamnolipids. [Image].

Description

Field of the invention

The invention relates to cells and nucleic acids and their use for the production of rhamnolipids as well as processes for the preparation of rhamnolipids.

State of the art

Surfactants are nowadays produced essentially based on petrochemical raw materials. The use of surfactants based on renewable raw materials is a suitable alternative due to the foreseeable shortage of petrochemical raw materials and increasing demand for products based on renewable raw materials or biodegradable.

Rhamnolipids consist of one (monorhamnosyl lipids) or two rhamnose residues (dirhamnosyl lipids) and one or two 3-hydroxy fatty acid residues (see Handbook of Hydrocarbon and Lipid Microbiology, 2010, pages 3037-51 ). They possess surface-active properties that are required for use as surfactant in a wide variety of applications (see Leitermann et al., 2009 ).

These lipids are nowadays produced with wild-type isolates of various human and animal pathogenic bacteria, in particular representatives of the genera Pseudomonas and Burkholderia (see Handbook of Hydrocarbon and Lipid Microbiology, 2010, pages 3037-51 ). The fact that these production organisms are able to cause disease significantly reduces the customer acceptance for the conventionally produced rhamnolipids. In addition, higher security requirements due to an increased investment volume and possibly additional processing steps also affect the production costs.

Although some of these production organisms can achieve high product titers, as well as space-time and carbon yields, this requires the use of vegetable oils as sole or co-substrate (see Handbook of Hydrocarbon and Lipid Microbiology, 2010, pages 3037-51 ). However, vegetable oils are relatively expensive raw materials compared to other carbon sources such as glucose, sucrose or polysaccharides such as starch, cellulose and hemicellulose, glycerin, CO, CO ₂ or CH ₄ . In addition, due to their surfactant character, rhamnolipids are characterized by the fact that they tend to foam strongly in fermentation processes. This is especially the case when lipophilic substrates are used. This problem is significantly reduced when using water-soluble substrates such as glucose, sucrose, polysaccharides (starch, cellulose, hemicellulose) or glycerol.

Finally, the properties of the produced by the wild-type isolates rhamnolipids are only partially influenced. So far, this has been done exclusively by optimizing process control (pH, oxygen supply, media composition, feeding strategies, nitrogen supply, temperature, substrate selection, etc.). However, a very targeted influencing of certain product properties, such as the ratio of the different rhamnolipid species (number of rhamnose and 3-hydroxy fatty acid residues) or chain length and degree of saturation of the 3-hydroxy fatty acid residues, would be desirable in order to be able to modulate the product properties relevant to the application.

If ramnolipids are to be used extensively as surfactants in household, cleaning, cosmetic, food processing, pharmaceutical, crop protection and other applications, they will have to compete with the surfactants used today. These are large volume chemicals that can be manufactured at very low cost, with no apparent health risks for the customer and with clearly defined and modulable product specifications. Therefore, rhamnolipids must be able to be produced at the lowest possible cost, without health risks for the customer and with as defined properties as possible.

Although rhamnolipids have already been produced in GRAS organisms (generally regarded as save) based on favorable carbon sources, such as glucose or glycerol, these are exclusively monorhamnosyl lipids ( Ochsner et al. Appl. Environ. Microbiol. 1995. 61 (9): 3503-3506 ).

Cha et al. in Bioresour Technol. 2008. 99 (7): 2192-9 describe, on the other hand, the production of monorhamnosyl lipids from soybean oil in P. putida by introducing the genes rhlA and rhlB from Pseudomonas aeruginosa.

There is therefore an increasing demand for the cost-effective and health-safe production of mono- and dirhamnosyl lipids with defined and modulatable properties.

This modulation can be carried out, for example, by a balanced provision of the individual enzyme activities, which reduces the accumulation of monorhamnosyl lipids. However, this modulation may also be accomplished, for example, by the use of enzymes having certain properties, e.g. regarding substrate specificity and thus about the chain length of the hydroxyfatty acids incorporated in rhamnolipids.

The present invention therefore an object of the invention to provide a way to produce rhamnolipids with safe production hosts from readily available carbon sources.

Description of the invention

Surprisingly, it has been found that the cells described below and methods in which these cells are used contribute to solving the problem addressed by the invention.

The present invention therefore relates to cells which are capable of forming rhamnolipids, having an increased activity of an enzyme compared to their wild type, which catalyzes the export of the rhamnolipids from the cell into the surrounding medium and an increased activity of at least one of the enzymes selected from 3 Ketoacyl-ACP reductase, acyl-ACP: coenzyme A transacylase and NADP ⁺ reductase.

Another object of the invention is a process for the preparation of rhamnolipids using the aforementioned cells as a biocatalyst.

An advantage of the present invention is that organisms can be used which are non-pathogenic and easy to cultivate.

Yet another advantage of the present invention is that a wide variety of carbon sources can be used.

Another advantage is that using oils as the sole or co-substrate is not necessary in all circumstances.

Another advantage is that it is possible with the aid of the invention to produce rhamnolipids with defined and modulatable properties.

Yet another advantage of the present invention is that dirhamnosyllipids can be prepared.

Another advantage is that rhamnolipids can be produced with higher space-time and carbon yields than with cells without enhancing these activities.

allgemeine Formel (I) wobei
m = 2, 1 oder 0, insbesondere 1 oder 0,
n = 1 oder 0, insbesondere 1,
R¹ und R² = unabhängig voneinander gleicher oder verschiedener organischer Rest mit 2 bis 24, insbesondere gegebenenfalls verzweigter, gegebenenfalls substituierter, insbesondere hydroxy-substituierter, gegebenenfalls ungesättigter, insbesondere gegebenenfalls einfach, zweifach oder dreifach ungesättigter, Alkylrest,
dadurch gekennzeichnet, dass sie derart gentechnisch verändert wurde, dass sie verglichen zu ihrem Wildtyp eine gesteigerte Aktivität mindestens eines Enzyms E₁, welches den Export eines Rhamnolipids der allgemeinen Formel (I) aus der Zelle in das umgebende Medium katalysiert aufweist,
und eine verglichen zu ihrem Wildtyp gesteigerte Aktivität mindestens eines der Enzyme ausgewählt aus E₂, E₃ und E₄ aufweist, wobei
E₂ die Umsetzung von 3-Hydroxyacyl-[Acyl-Carrier-Protein] zu 3-Ketoacyl-[Acyl-Carrier-Protein] oder 3-Hydroxyacyl-Coenzym A zu 3-Ketoacyl-Coenzym A, bevorzugt von 3-Hydroxyacyl-Coenzym A zu 3-Ketoacyl-Coenzym A zu katalysieren vermag,
E₃ die Umsetzung von Acyl-[Acyl-Carrier-Protein] zu Acyl-Coenzym A zu katalysieren vermag und
E₄ die Umsetzung von NADP+ zu NADPH zu katalysieren vermag. The present invention therefore relates to a cell, preferably an isolated, which is capable of forming at least one rhamnolipid of the general formula (I),

general formula (I) wherein
m = 2, 1 or 0, in particular 1 or 0,
n = 1 or 0, in particular 1,
R ¹ and R ² = independently of one another the same or different organic radical with 2 to 24, in particular optionally branched, optionally substituted, in particular hydroxy-substituted, optionally unsaturated, in particular optionally mono-, di- or triunsaturated, alkyl radical,
characterized in that it has been genetically engineered such that, compared to its wild type, it has an increased activity of at least one enzyme E ₁ which catalyzes the export of a rhamnolipid of the general formula (I) from the cell into the surrounding medium,
and an increased activity compared to its wild-type activity of at least one of the enzymes selected from E ₂ , E ₃ and E ₄ , wherein
E ₂ the conversion of 3-hydroxyacyl [acyl carrier protein] to 3-ketoacyl [acyl carrier protein] or 3-hydroxyacyl-coenzyme A to 3-ketoacyl-coenzyme A, preferably 3-hydroxyacyl-coenzyme A catalyzes A to 3-ketoacyl coenzyme A,
E _{3 is} able to catalyze the conversion of acyl- [acyl carrier protein] to acyl-coenzyme A and
E ₄ catalyzes the conversion of NADP + to NADPH.

By "wild-type" of a cell is meant herein a cell whose genome is in a state as naturally evolved by evolution.The term is used for both the entire cell and for individual genes therefore in particular not such cells or genes whose gene sequences have been at least partially modified by humans by means of recombinant methods.

The term "rhamnolipid" in connection with the present invention is understood to mean a compound of the general formula (I) or its salt.

The accession numbers cited in connection with the present invention correspond to the ProteinBank database entries of the NCBI dated 26.01.2012; As a rule, the version number of the entry is identified by ".Ziffer" such as ".1".

The cells of the invention may be prokaryotes or eukaryotes. These 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 being most preferred.

Particularly suitable bacteria, yeasts or fungi are those bacteria, yeasts or fungi which are deposited in the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Braunschweig, Germany, as bacterial, yeast or fungal strains. Bacteria suitable according to the invention belong to the genera under
http://www.dsmz.de/species/bacteria.htm
Yeasts which are suitable according to the invention belong to those genera which are listed under
http://www.dsmz.de/species/yeasts.htm
are listed and suitable fungi according to the invention are those under
http://www.dsmz.de/species/fungi.htm
are listed.

Cells preferred according to the invention are those of the genera Aspergillus, Corynebacterium, Brevibacterium, Bacillus, Acinetobacter, Alcaligenes, Lactobacillus, Paracoccus, Lactococcus, Candida, Pichia, Hansenula, Issatchenkia, Kluyveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Pseudomonas, Rhodospirillum , Rhodobacter, Burkholderia, Clostridium and Cupriavidus, wherein Aspergillus nidulans, Aspergillus niger, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Burkholderia andropogonis, B. brasilensis, B. caledonica, B.caribensis, B.caryophylli, B. fungorum, B. gladioli, B. glathei, B. glumae, B. graminis, B. hospita, B. kururiensis, B. phenazinium, B. phymatum, B. phytofirmans, B. plantarii, B. saccharomyces, 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, Issatchenkia orientalis, Kluveromyces lactis, Methylobacterium extorquens, Paracoccus versutus, Pseudomonas argentinensis, P. borbori, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea, P. aurantiaca, P. aureofaciens, P. chlororaphis, P. fragi, P. lundensis, P. taetrolens, P. antarctica, P. azotoformans, 'P , blakefordae ', P. brassicacearum, P. brenneri, P. cedrina, P. corrugata, P. fluorescens, P. gessardii, P. libanensis, P. mandelii, P. marginalis, P. mediterranea, P. meridiana, P. migulae , P. Mucidolens, P. orientalis, P. panacis, P. proteolytica, P. rhodesiae, P. synxantha, P. thivervalensis, P. tolaasii, P. veronii, P. denitrificans, P. pertucinogena, P. cremoricolorata, P fulva, P. monteilii, P. mosselii, P. parafulva, P. putida, P. balearica, P. stutzeri, P. amygdali, P. avellanae, P. caricapapayae, P. cichorii, P. coronafaciens, P. ficuserectae , 'P. helianthi ', P. meliae, P. savastanoi, P. syringae, P. tomato, P. viridiflava, P. abietaniphila, P. acidophila, P. agarici, P. alcaliphila, P. alkanolytica, P. amyloderamosa, P. asplenii , P. azotifigens, P. cannabina, P. coenobios, P. congelans, P. costantinii, P. cruciviae, P. delhiensis, P. excibis, P. extremorientalis, P. frederiksbergensis, P. fuscovaginae, P. gelidicola, P grimontii, P. indica, P. jessenii, P. jinjuensis, P. kilonensis, P. knackmussii, P. koreensis, P. lini, P. lutea, P. moraviensis, P. otitidis, P. pachastrellae, P. palleroniana , P. papaveris, P. peli, P. perolens, P. poae, P. pohangensis, P. psychrophila, P. psychrotolerans, P. rathonis, P. reptilivora, P. resiniphila, P. rhizosphaerae, P. rubescens, P salomonii, P. segitis, P. septica, P. simiae, P. suis, P. thermotolerans, P. aeruginosa, P. tremae, P. trivialis, P. turbinellae, P. tuticorinensis, P. umsongensis, P. vancouverensis , P. vranovensis, P. xanthomarina, Ralstonia eutropha, Rhodospirillum rubrum, Rho dobacter sphaeroides, Saccharomyces cerevisiae, Yarrowia lipolytica and Zymomonas mobilis, in particular Pseudomonas putida, Escherichia coli and Burkholderia thailandensis are particularly preferred.

In preferred cells according to the invention, E _{1 is} selected from the group consisting of enzymes E ₁ having polypeptide sequence Seq ID No. 2, Seq ID No. 4, Seq ID No. 6, Seq ID No. 8, ZP_05590661.1, YP_439278.1, YP_440069.1, ZP_04969301.1, ZP_04520234.1, YP_335528.1, YP_001075859.1, YP_001061817.1, ZP_02487499.1, YP_337251.1, ZP_04897712.1, ZP_04810190.1, YP_990322.1, ZP_02476924.1, ZP_04899735. 1, ZP_04893873.1, ZP_02365982.1, YP_001062909.1, YP_105611.1, ZP_03794061.1, ZP_03457011.1, ZP_02385401.1, ZP_02370552.1, YP_105236.1, ZP_04905097.1, YP_776387.1, YP_001811690.1, YP_004348730.1, YP_004348708.1, YP_371320.1, YP_623145.1, YP_001778810.1, YP_002234933.1, CCE52909.1, YP_002908248.1, ZP_04954557.1, ZP_04956038.1, ZP_02408950.1, ZP_02375897.1, ZP_02389908. 1, YP_439274.1, YP_001074762.1, YP_337247.1, YP_110559.1, ZP_02495927.1, YP_111360.1, YP_105608.1, ZP_02487826.1, ZP_02358947.1, YP_001078605.1, ZP_00438000.1, ZP_00440993.1, ZP_02477260.1, YP_371317.1, YP_001778807.1, ZP_02382843.1, YP_ 002234936.1, YP_623142.1, ZP_02907618.1, ZP_02891478.1, YP_776390.1, ZP_04943308.1, YP_001811693.1, ZP_02503985.1, YP_004362740.1, YP_002908245.1, YP_004348705.1, ZP_02408798.1, ZP_02417250.1, ZP_0494404.1, ZP_04944344.1, YP_771932.1, ZP_02889166.1, YP_002232614.1, ZP_03574808.1, ZP_02906105.1, YP_001806764.1, YP_ YP_001117913.1, YP_106647.1, YP_001763368.1, ZP_02479535.1, ZP_02461743.1, YP_560998.1, YP_331651.1, ZP_04893070.1, YP_003606714.1, ZP_02503995.1, ZP_06840428.1, YP_104288.1, ZP_02487849.1, ZP_02353848.1, YP_367475.1, ZP_02377399.1, ZP_02372143.1, YP_001897562.1, ZP_02361066.1, YP_440582.1, ZP_03268453.1, AET90544.1, YP_003908738.1, YP_004230049.1, ZP_02885418. 1 or ZP_02511831.1 or with a polypeptide sequence of up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 % of the amino acid residues are changed from the respective Seq ID No. 2, Seq ID No. 4, Seq ID No. 6, Seq ID No. 8 or the aforementioned Accession Number by deletion, insertion, substitution or a combination thereof and which are still at least 10 %, preferably 50%, more preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme having the respective reference sequence Seq ID No. 2, Seq ID No. 4, Seq ID No. 6, Seq ID No. 8 or has enzymatic activity for an enzyme E _{1 is} the ability to export a rhamnolipid of the general formula (I) from the cell into the surrounding medium.

It is preferred according to the invention that E _{2 is} a 3-ketoacyl-ACP reductase of EC 1.1.1.100 or a 3-ketoacyl-coenzyme A reductase of EC 1.1.1.35 or of EC 1.1.1.36. Particularly preferred E ₂ are selected from
Enzyme E _2a with polypeptide sequence Seq ID No. 10 or with a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues compared to Seq ID No. 10 are modified by deletion, insertion, substitution or a combination thereof and which is at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic Activity of the enzyme having the reference sequence Seq ID No. 10, wherein Under enzymatic activity for an enzyme E _{2a is} the ability to convert 3-hydroxydecanoyl-coenzyme A into 3-ketodecanoyl-coenzyme A and
Enzyme E _2b having polypeptide sequence EGM23089.1, YP_346919.1, YP_258374.1, YP_001187200.1 or YP_004380924.1 or having a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues are different from the aforementioned accession number by deletion, insertion, substitution or a combination thereof and which is at least 10%, preferably 50%, especially preferably 80%, in particular more than 90%, of the enzymatic activity of the enzyme having the reference sequence of the aforementioned accession number, wherein enzymatic activity for an enzyme E _{2b is} the ability to convert 3-hydroxytetradecanoyl-coenzyme A to 3-ketotetradecanoyl-coenzyme A. ,

It is preferred according to the invention that E _{3 is} an acyl-ACP: coenzyme A transacylase. Particularly preferred E ₃ are selected from the group consisting of enzymes E _3a with polypeptide sequence Seq ID No. 12, Seq ID No. 14, NP_249421.1, AAT51199.1, ZP_01364106.1, AEO76792.1, YP_792557.1, ZP_04932415. 1, YP_001350135.1, ZP_08139631.1, YP_004703658.1, YP_004379169.1, YP_609790.1, ACA03779.1, YP_001670627.1, NP_743567.1, YP_001269622.1, YP_001186851.1, Q9KJH8.1, AAK71349.1, ADR61731.1, AAU44816.1, YP_001747923.1, ACH70299.1, ZP_07774051.1, YP_002871082.1, AAK81868.1, AAQ16175.1, YP_004352505.1, YP_258557.1, ACA60824.1, AEV61413.1, YP_004475334. 1, AAA25978.1, YP_347066.1, EGH58099.1, BAB32432.1, EGH64812.1, ZP_05640568.1, EFW81598.1, EGH11618.1, YP_276116.1, ZP_07264431.1, EGH73565.1, EGH95622.1, YP_237050.1, EGH29888.1, ZP_06498781.1, ZP_06456665.1, EGH79586.1, NP_794008.1, ZP_03399268.1, ZP_05639386.1, EGH50352.1, EGH22129.1, ZP_07005523.1, ZP_06458504.1, EFW79804. 1, YP_275219.1, EGH85976.1, EGH22392.1, EGH66549.1, ZP_03398232.1, ZP_07229875.1, EGH10011.1, EGH43364.1, YP_236 199.1, ZP_07261632.1, ZP_04589662.1, EGH54896.1, EGH29417.1, ZP_06499968.1, EGH97259.1, NP_793082.1 or EGH90852.1 or with a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues relative to the respective Seq ID No. 12, SEQ ID No. 14 or the aforementioned Accession Number Deletion, insertion, substitution or a combination thereof, and which are at least 10%, preferably 50%, more preferably 80%, in particular more than 90%, of the enzymatic activity of the enzyme having the respective reference sequence Seq ID No. 12, SEQ ID NO 14, or enzymatic activity for an enzyme E _{3a is} the ability to react 3-hydroxydecanoyl [acyl carrier protein] and coenzyme A to 3-hydroxydecanoyl coenzyme A and acyl carrier protein and
Enzyme E _3b with polypeptide sequence AAK71350.1 or with a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues compared to the AAK71350.1 by deletion, insertion, substitution or a combination thereof are modified and the at least 10%, preferably 50%, more preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme the reference sequence has AAK71350.1, wherein enzymatic activity for an enzyme E _{3b is} understood as the ability to react 3-hydroxytetradecanoyl [acyl carrier protein] and coenzyme A to 3-hydroxytetradecanoyl coenzyme A and acyl carrier protein.

It is inventively preferred that E _{4 is} selected from
Enzymes E _4a transhydrogenases of EC 1.6.1.1 and EC 1.6.1.2, wherein enzyme E _{4a can be} a energy-independent (EC 1.6.1.1) or an energy-dependent (EC 1.6.1.2) transhydrogenase,
Enzyme E _4b NADP + -dependent glyceraldehyde-3-phosphate dehydrogenases of EC 1.2.1.9, EC 1.2.1.13 and EC 1.2.1.59 and
Enzyme E _4c NADP + -dependent pyruvate dehydrogenases of EC 1.2.1.51.

Particularly preferred E ₄ are selected from
Enzyme E _4a with polypeptide sequence Seq ID No. 16, Seq ID No. 18 or Seq ID No. 20 or with a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues over the respective SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20 by deletion, insertion, substitution or a combination thereof and which still has at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90%, of the enzymatic activity of the enzyme with the respective reference sequence Seq ID No. 16, SEQ ID No. 18 or SEQ ID No. 20 in which enzymatic activity for an enzyme E _{4a is} understood as the ability to convert NADP + to NADPH,
Enzyme E _4b having polypeptide sequence Seq ID No. 22 or having a polypeptide sequence of up to 25%, preferably up to 20%, more preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues compared to Seq ID No. 22 are altered by deletion, insertion, substitution or a combination thereof and which is at least 10%, preferably 50%, more preferably 80%, in particular more than 90% of the enzymatic Activity of the enzyme having the reference sequence Seq ID No. 22, wherein enzymatic activity for an enzyme E _{4a is} the ability to convert D-glyceraldehyde-3-phosphate to 1,3-bisphospho-D-glycerate, and
Enzyme E _4c with polypeptide sequence Seq ID No. 24 or Seq ID No. 26 or with a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues are different from the respective Seq ID No. 24 or SEQ ID No. 26 by deletion, insertion, substitution or a combination thereof and which is still at least 10%, preferably 50% , more preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme having the respective reference sequence SEQ ID NO: 24 or SEQ ID NO: 26, whereby enzymatic activity for an enzyme E _{4c is} understood to mean the ability to produce pyruvate and coenzyme A (CoA) to convert acetyl coenzyme A and CO ₂ .

According to the invention, cells are preferred which have increased activities of the following enzyme combinations:
E ₂ , E ₃ , E ₄ , E ₂ E ₃ , E ₂ E ₄ , E ₃ E ₄ and E ₂ E ₃ E ₄ ,
of which the combinations
E ₂ , E ₃ , E ₂ E ₃ , E ₂ E ₄ , E ₃ E ₄ and E ₂ E ₃ E ₄
are particularly preferred.

The parent strains of the cells according to the invention may be natural rhamnolipid producers, cells which already produce wild-type rhamnolipids, or cells which were only allowed to produce rhamnolipid by genetic engineering.

In both cases, preferred cells according to the invention benefit from the fact that they have an increased activity of at least one of the enzymes E ₅ , E ₆ and E ₇ compared to their wild type, wherein the enzyme E _{5 is} the reaction of 3-hydroxyalkanoyl-ACP via 3-hydroxyalkanoyl- 3-hydroxyalkanoic acid ACP to catalyze hydroxyalkanoyl-3-hydroxyalkanoic acid, the enzyme E _{6 is} a rhamnosyltransferase I and the reaction of dTDP-rhamnose and 3-hydroxyalkanoyl-3-hydroxyalkanoate to α-L-rhamnopyranosyl-3-hydroxyalkanoyl-3 -Hydroxyalkanoate and the enzyme E _{7 is} a rhamnosyltransferase II and the conversion of dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate into α-L-rhamnopyranosyl- (1-2) -α- L-rhamnopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate can catalyze, wherein for the enzymes E ₅ , E ₆ and E _{7 is} preferably:
Enzyme E ₅ is selected from the group consisting of
at least one enzyme E _5a having polypeptide sequence Seq ID No. 28, or having a polypeptide sequence of up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5 , 4, 3, 2, 1% of the amino acid residues with respect to the reference sequence Seq ID No. 28 have been altered by deletion, insertion, substitution or a combination thereof and which are still at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90%, of the enzymatic activity of the enzyme having the reference sequence Seq ID No. 28, whereby enzymatic activity for an enzyme E _{5a is} the ability to convert 3-hydroxydecanoyl-ACP via 3 Hydroxydecanoyl-3-hydroxydecanoic acid ACP to hydroxydecanoyl-3-hydroxydecanoic acid, at least one enzyme E _5b having polypeptide sequence SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, YP_439272.1 , YP_111362.1, YP_110557.1, YP_105231.1, ZP_02461688.1, ZP_02358949.1, ZP_01769192.1, ZP_04893165.1, ZP_02265387.2, ZP_02511781.1, ZP_03456835.1, ZP_03794633.1, YP_990329.1, ZP_02408727 .1, YP_002908243.1, ZP_04884056.1, YP_004348703.1, ZP_04905334.1, ZP_02376540.1, EGC99875.1, ZP_02907621.1, YP_001811696.1, ZP_02466678.1, ZP_02891475.1, YP_776393.1, YP_002234939.1 , YP_001778804.1, YP_371314.1, ZP_04943305.1, YP_623139.1, ZP_02417235.1 or ZP_04892059.1 or with a polypeptide sequence of up to 25%, preferably up to 20%, more preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues relative to the respective reference sequence Seq ID No. 30, SEQ ID NO 32, SEQ ID NO: 34, SEQ ID NO: 36 or the aforementioned accession number are altered by deletion, insertion, substitution or a combination thereof and which is at least 10%, preferably 50%, more preferably 80%, in particular more than 90 % of the enzymatic activity of the enzyme having the respective reference sequence Seq ID No. 30, Seq ID No. 32, Seq ID No. 34, Seq ID No. 36 or the aforementioned accession number, wherein, under enzymatic activity, for an enzyme E _5b, the ability reacting 3-hydroxytetradecanoyl-ACP via 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid ACP to give hydroxytetradecanoyl-3-hydroxytetradecanoic acid,
Enzyme E ₆ is selected from the group consisting of
at least one enzyme E _6a having polypeptide sequence Seq ID No. 38, YP_001347032.1, CBI71029.1, YP_002439138.1, CBI71031.1, NP_252168.1, CBI71034.1, CBI71028.1, AAA62129.1 or ZP_04929750.1 or with a polypeptide sequence of up to 25%, preferably up to 20%, more preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues relative to the respective reference sequence Seq ID No. 38 or the aforementioned accession number are modified by deletion, insertion, substitution or a combination thereof and which is at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme with the respective SEQ ID NO: 38 or the aforementioned accession number, wherein enzymatic activity for an enzyme E _{6a is} the ability to convert dTDP-rhamnose and 3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-3-hydroxydecanoyl-3 To convert hydroxydecanoic acid,
at least one enzyme E _6b having polypeptide sequence Seq ID No. 40, Seq ID No. 42, Seq ID No. 44, Seq ID No. 46, YP_440074.1, ZP_05590657.1, ZP_04520374.1, ZP_00438360.2, ZP_00438209.2 , YP_001074761.1, ZP_04811084.1, YP_110558.1, YP_111361.1, ZP_02492857.1, YP_337246.1, YP_001061811.1, YP_105607.1, ZP_02371503.1, ZP_02503962.1, ZP_03456839.1, ZP_02461690.1, ZP_03794634 .1, ZP_01769736.1, ZP_01769308.1, ZP_02358948.1, ZP_02487736.1, ZP_02408758.1, YP_002234937.1, ZP_02891477.1, YP_001778806.1, YP_623141.1, YP_838721.1, ZP_04943307.1, YP_776391.1 , YP_004348704.1, ZP_02907619.1, YP_371316.1, ZP_02389948.1, YP_001811694.1, YP_002908244.1, ZP_02511808.1, ZP_02376542.1, EGC99877.1, ZP_02451760.1 or ZP_02414414.1 or with a polypeptide sequence in the up to 25%, preferably up to 20%, more preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues relative to the respective reference sequence Seq ID No. 40, Seq ID No. 42, Seq ID No. 44, Seq ID No. 46 or above Once the accession number have been changed by deletion, insertion, substitution or a combination thereof, the at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90%, of the enzymatic activity of the enzyme having the respective reference sequence Seq ID No. 40 SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46 or the aforementioned accession number, wherein enzymatic activity for an enzyme E _{6b is} understood as meaning the ability to add dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid reacting α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, and
Enzyme E ₇ is selected from the group consisting of
at least one enzyme E _7a with polypeptide sequence Seq ID No. 48 or with a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues relative to the reference sequence Seq ID No. 48 are modified by deletion, insertion, substitution or a combination thereof and which is at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90 % of the enzymatic activity of the enzyme having the reference sequence Seq ID No. 48, whereby enzymatic activity for an enzyme E _{7a is} understood to mean the ability to form α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid dTDP-rhamnose and Reacting L-rhamnopyranosyl- (1-2) -α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,
at least one enzyme E _7b with polypeptide sequence Seq ID No. 50, Seq ID No. 52, Seq ID No. 54 YP_440071.1, ZP_02375899.1, ZP_02466676.1, YP_001075863.1, ZP_02408796.1, YP_335530.1, ZP_01769176. 1, YP_105609.1, ZP_01770867.1, ZP_04520873.1, YP_110560.1, YP_001024014.1, ZP_03450125.1, YP_001061813.1, YP_111359.1, ZP_00440994.2, ZP_03456926.1, ZP_02358946.1, ZP_ ZP_02461478.1, ZP_02503929.1, ZP_02511832.1, YP_004348706.1, ZP_04898742.1, YP_002908246.1, ZP_02382844.1, EGD05167.1, YP_001778808.1, YP_001811692.1, YP_002234935.1, YP_371318.1, YP_623143.1, YP_776389.1, ZP_02891479.1, ZP_02907617.1, ZP_02417424.1 or ZP_04898743.1 or with a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10 , 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues over the respective reference sequence SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or the aforementioned accession number by deletion, insertion , Substitution or a combination thereof, and which is at least 10%, preferably 50%, more preferably 80%, in particular more than 90%, of the enzymatic activity of the enzyme having the reference sequence Seq ID No. 50, SEQ ID NO. 52, Seq ID No. 54 or the aforementioned accession number, whereby enzymatic activity for an enzyme E _{7b is} understood to mean the ability dTDP -Rhamnose and α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to give α-L-rhamnopyranosyl- (1-2) -α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid.

It is apparent that the above specific activities for the enzymes E _5a to E _{7b represent} only a specific exemplary selection of a broader spectrum of activity of the aforementioned enzymes; the activity referred to is the one for which a reliable measurement method for a given enzyme is present. Thus, it is apparent that an enzyme which is a substrate having an unbranched, saturated C ₁₀ alkyl radical also - albeit optionally with reduced activity - will react those substrates which have a C ₆ - or C ₁₆ alkyl radical, which may also be may be branched or unsaturated.

Cells preferred according to the invention are able to form no or no detectable amounts of rhamnolipids as wild-type and moreover preferably have no or no detectable activity of the enzymes E ₅ , E ₆ and E ₇ as wild-type.

According to the invention, cells are preferred which have increased activities of the following enzyme combinations:
E ₅ , E ₆ , E ₇ , E ₅ E ₆ , E ₅ E ₇ , E ₆ E ₇ and E ₅ E ₆ E ₇ ,
of which the combination
E ₆ , E ₆ E ₇ and E ₅ E ₆ E ₇ , in particular E ₅ E ₆ E ₇
is particularly preferred.

In a preferred embodiment of the cell according to the invention, which has an increased activity of the enzyme combination E ₅ E ₆ E ₇ , n is preferably = 1.

It is furthermore advantageous for the rhamnolipid yield if the cell according to the invention, in addition to or as an alternative to the modified activities of the enzymes E ₅ , E ₆ and / or E _{7, has been} genetically engineered in such a way that it is enhanced compared to its wild type, as specified below in each case Activity of at least one of the enzymes selected from the group consisting of
at least one enzyme E ₈ , a dTTP: α-D-glucose-1-phosphate thymidylyltransferase, EC 2.7.7.24, in particular one with polypeptide sequence Seq ID No. 56 or with a polypeptide sequence in which up to 25%, preferably up to 20% , more preferably up to 15%, especially up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues over the reference sequence SEQ ID NO: 56 by deletion, insertion, substitution or a combination thereof are modified and still at least 10%, preferably 50%, more preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme having the reference sequence Seq ID No. 56, wherein understood by enzymatic activity for an enzyme E ₈ the ability is to convert α-D-glucose-1-phosphate and dTTP to dTDP-glucose, at least one enzyme E ₉ , a dTTP-glucose-4,6-hydrolyase, EC 4.2.1.46, in particular one with polypeptide sequence Seq ID No. 58 or with a polypeptide sequence of up to 25%, preferably up to 20%, especially be preferably up to 15%, in particular 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: 58 are altered by deletion, insertion, substitution or a combination thereof and which still has at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90%, of the enzymatic activity of the enzyme having the reference sequence Seq ID No. 58, whereby enzymatic activity for an enzyme E _{9 is} understood as the ability convert dTDP-glucose to dTDP-4-dehydro-6-deoxy-D-glucose,
at least one enzyme E ₁₀ , a dTDP-4-dehydrorhamnose-3,5-epimerase, EC 5.1.3.13, in particular one with polypeptide sequence Seq ID No. 60 or with a polypeptide sequence in which up to 25%, preferably up to 20%, more preferably, up to 15%, especially up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues over the reference sequence SEQ ID NO: 60 altered by deletion, insertion, substitution or a combination thereof and which still has at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90%, of the enzymatic activity of the enzyme having the reference sequence Seq ID No. 60, whereby enzymatic activity for an enzyme E _{10 is} understood as the ability , dTDP-4-dehydro-6-deoxy-D- Reacting glucose to dTDP-4-dehydro-6-deoxy-L-mannose and at least one enzyme E ₁₁ , a dTDP-4-dehydrorhamnose reductase, EC 1.1.1.133, in particular one with polypeptide sequence Seq ID No. 62 or with a polypeptide sequence in which up to 25%, preferably up to 20%, more preferably up to 15%, in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues compared to the reference sequence Seq ID no 62 is modified by deletion, insertion, substitution or a combination thereof and which still has at least 10%, preferably 50%, particularly preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme having the reference sequence Seq ID No. 62, wherein enzymatic activity for an enzyme E _{11 is} understood to include the ability to react dTDP-4-dehydro-6-deoxy-L-mannose to form dTDP-6-deoxy-L-mannose.

According to the invention, cells are preferred which have increased activities of the following enzyme combinations with regard to the enzymes E ₈ to E ₁₁ :
E ₈ E ₉ , E ₈ E ₁₀ , E ₈ E ₁₁ , E ₉ E ₁₀ , E ₉ E ₁₁ , E ₁₀ E ₁₁ , E ₈ E ₉ E ₁₀ , E ₈ E ₉ E ₁₁ , E ₉ E ₁₀ E ₁₁ , E ₈ E ₁₀ E ₁₁ , E ₈ E ₉ E ₁₀ E ₁₁ ,
of which the combination
E ₈ E ₉ E ₁₀ E ₁₁
is particularly preferred.

According to the invention, cells are preferred which have increased activities of the following enzyme combinations with regard to the enzymes E ₂ to E ₁₁ :
E ₂ E ₅ E ₆ , E ₃ E ₅ E ₆ , E ₄ E ₅ E ₆ , E ₂ E ₅ E ₆ E ₇ , E ₃ E ₅ E ₆ E ₇ , E ₄ E ₅ E ₆ E ₇ , E ₂ E ₅ E ₆ E ₈ E ₉ E ₁₀ E ₁₁ , E ₃ E ₅ E ₆ E ₈ E ₉ E ₁₀ E ₁₁ , E ₄ E ₅ E ₆ E ₈ E ₉ E ₁₀ E ₁₁ , E ₂ E ₅ E ₆ E ₇ E ₈ E ₉ E ₁₀ E ₁₁ , E ₃ E ₅ E ₆ E ₇ E ₈ E ₉ E ₁₀ E ₁₁ , E ₄ E ₅ E ₆ E ₇ E ₈ E ₉ E ₁₀ E ₁₁ , E ₃ E ₄ E ₅ E ₆ E ₈ E ₉ E ₁₀ E ₁₁ , E ₂ E ₄ E ₅ E ₆ E ₈ E ₉ E ₁₀ E ₁₁ , E ₂ E ₃ E ₄ E ₅ E ₆ E ₈ E ₉ E ₁₀ E _11, E ₃ E ₄ E ₅ E ₆ E ₇ E ₈ E ₉ E ₁₀ E ₁₁ , E ₂ E ₄ E ₅ E ₆ E ₇ E ₈ E ₉ E ₁₀ E ₁₁ and E ₂ E ₃ E ₄ E ₅ E ₆ E ₇ E ₈ E ₉ E ₁₀ E ₁₁ ,
of which the combinations
E ₂ E ₅ E ₆ E ₈ E ₉ E ₁₀ E ₁₁ , E ₃ E ₅ E ₆ E ₈ E ₉ E ₁₀ E ₁₁ , E ₄ E ₅ E ₆ E ₈ E ₉ E ₁₀ E ₁₁ , E ₂ E ₅ E ₆ E ₇ E ₈ E ₉ E ₁₀ E ₁₁ , E ₃ E ₅ E ₆ E ₇ E ₈ E ₉ E ₁₀ E ₁₁ , E ₄ E ₅ E ₆ E ₇ E ₈ E ₉ E ₁₀ E ₁₁ , E ₃ E ₄ E ₅ E ₆ E ₈ E ₉ E ₁₀ E ₁₁ , E ₂ E ₄ E ₅ E ₆ E ₈ E ₉ E ₁₀ E ₁₁ , E ₂ E ₃ E ₄ E ₅ E ₆ E ₈ E ₉ E ₁₀ E _11, E ₃ E ₄ E ₅ E ₆ E ₇ E ₈ E ₉ E ₁₀ E ₁₁ , E ₂ E ₄ E ₅ E ₆ E ₇ E ₈ E ₉ E ₁₀ E ₁₁ and E ₂ E ₃ E ₄ E ₅ E ₆ E ₇ E ₈ E ₉ E ₁₀ E ₁₁
are particularly preferred.

It is furthermore advantageous according to the invention if the cell according to the invention is a cell which is able to form wild-type polyhydroxyalkanoates with chain lengths of the monoalkanoate of C ₆ to C ₁₆ . Such cells include Burkholderia sp., Burkholderia thailandensis, Pseudomonas sp., Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas oleovorans, Pseudomonas stutzeri, Pseudomonas fluorescens, Pseudomonas citronellolis, Pseudomonas resinovorans, Comamonas testosteroni, Aeromonas hydrophila, Cupriavidus necator, Alcaligenes latus and Ralstonia eutropha , In this context, preferred cells of the invention are engineered such that they are capable of forming less polyhydroxyalkanoates compared to their wild-type.

Such cells are for example in Ren et al., Journal of 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 described as KTOY01 and KTOY02.

Such a high-net-worth cell to form such a polyhydroxyalkanoate compared to its wild type is characterized in particular by having a reduced activity of at least one enzyme E ₁₂ compared to its wild-type,
where E _{12 is} a polyhydroxyalkanoate synthase, EC: 2.3.1.-, in particular with polypeptide sequence Seq ID No. 64 or SEQ ID No. 66 or with a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to in particular up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the amino acid residues relative to the respective reference sequence SEQ ID NO: 64 or SEQ ID NO: 66 by deletion, insertion, substitution or a combination thereof is altered and which still has at least 10%, preferably 50%, more preferably 80%, in particular more than 90% of the enzymatic activity of the enzyme having the respective Seq ID No. 64 or SEQ ID No. 66, wherein enzymatic activity for an enzyme E _{12 is} understood to be the ability to convert 3-hydroxyalkanoyl coenzyme A to poly-3-hydroxyalkanoic acid, in particular 3-hydroxydecanoyl coenzyme A to poly-3-hydroxydecanoic acid.

Furthermore, in a further preferred embodiment of the cells according to the invention, the recombinant cell may additionally be genetically engineered in such a way that it has a reduced carbon flux from the carbon source available for its growth and as an energy source compared to its wild type Rhamnolipids of the general formula (I) are having.

This embodiment comprises recombinant cells according to the invention, which are additionally characterized compared to their wild type in that they produce less C ₁ , C ₂ , C ₃ and / or C _{4 by-} products from the carbon source available for their growth and as an energy source ,

The required genetic modification can be done by manipulation of the recombinant cell, whereby the metabolism of the C ₁ , C ₂ and / or C _{3 by-} products, such as CO ₂ , formate, acetate, butyrate, propionate, D or L-lactate, ethanol, butanol, propanol, isopropanol, 2,3-butanediol, acetoin, methylglyoxal or acetone is produced, reduced or interrupted.

For this purpose, in the cells of this embodiment preferred according to the invention, for example, at least one activity is reduced by at least one of the following enzymes as compared to the wild type of the cell:
E ₁₃ , which catalyzes the reaction of acetone and NAD (P) H ⁺ to isopropanol and NAD (P) ⁺ ;
E ₁₄ , which catalyzes the conversion of pyruvate and CoA to formate and acetyl-CoA
E ₁₅ , which activates an enzyme E ₁₄ ;
E ₁₆ , which catalyzes the reaction of pyruvate, H ₂ O and ferricytochrome b ₁ to acetate, CO ₂ and ferrocytochrome b ₁ ;
E which catalyzes the conversion of S-methylmalonyl-CoA to propanoyl-CoA and CO _{2 17;}
E ₁₉ which catalyzes the conversion of acetyl phosphate and ADP / P _i to acetate and ATP / PP _i ;
E ₂₀ , which catalyzes the conversion of acetyl CoA and P _i (orthophosphate) to acetyl phosphate and CoA;
E ₂₁ , which catalyzes the reaction of pyruvate and NAD (P) H ⁺ to D-lactate and NAD (P) ⁺ ;
E ₂₂ , which catalyzes the conversion of acetyl-CoA to acetaldehyde;
E ₂₃ , which catalyzes the conversion of acetaldehyde to ethanol;
Which catalyzes the conversion of methylglyoxal to Glyceronphosphat and orthophosphate E _24;
E ₂₅ , an enzyme or enzyme complex that catalyzes the conversion of formate to CO ₂ ;
E ₃₆ , which catalyzes the reaction of propionyl phosphate and ADP to propionate and ATP;
E ₃₇ , which catalyzes the reaction of propionyl CoA and P _i to propionyl phosphate and CoA;
E _38, which catalyzes the conversion of pyruvate to acetaldehyde and CO _2;
E ₃₉ , which catalyzes the reaction of D-xylulose-5-phosphate and phosphate to acetyl phosphate and D-glyceraldehyde-3-phosphates and H ₂ O;
E ₄₀ , which catalyzes the reaction of (S) -methylmalonyl-CoA and pyruvate to propionyl-CoA and oxaloacetate;
E ₄₁ , which catalyzes the conversion of (S) -methylmalonyl-CoA to succinyl-CoA;
E ₄₂ , which catalyzes the conversion of 2 pyruvate to 2-acetolactate and CO ₂ ;
E ₄₃ , which catalyzes the conversion of 2-acetolactate to acetoin and CO ₂ ;
E ₄₄ , which catalyzes the reaction of acetoin and NAD (P) H ⁺ to butane-2,3-diol and NAD (P) ⁺ ;
E ₄₅ , which catalyzes the reaction of butyraldehyde and NAD (P) H ⁺ to butanol and NAD (P) ⁺ ;
Which catalyzes the conversion of butyryl-CoA and NAD (P) H ⁺ to butyraldehyde and NAD (P) ⁺ E _46;
E ₄₇ , which catalyzes the reaction of acetoacetate and H ⁺ to acetone and CO ₂ ;
E ₄₈ , which catalyzes the reaction of acetone and NAD (P) H ⁺ to propanol and NAD (P) ⁺ ;
E ₄₉ , which catalyzes the conversion of pyruvate and NAD (P) H ⁺ to L-lactate and NAD (P) ⁺ .

In preferred embodiments of the cells according to the invention,
E ₁₃ , an isopropanol dehydrogenase (EC 1.1.1.80);
E _14, a formate C-acetyltransferase (EC 2.3.1.54);
E _15, a formate C-acetyltransferase activating enzyme;
E ₁₆ a pyruvate: ferricytochrome b ₁ oxidoreductase (EC 1.2.2.2);
E _{17 is} a methylmalonyl-CoA decarboxylase (EC 4.1.1.41);
E ₁₉ an ATP / PP _i : acetate phosphotransferase (EC 2.7.2.1 or EC 2.7.2.1) or a propionate / acetate kinase (EC 2.7.2.15);
E ₂₀ an acetyl-CoA: phosphate acetyltransferase (EC 2.3.1.8);
E _{21 is} a D-lactate dehydrogenase (EC 1.1.1.28);
E ₂₂ is an acetaldehyde dehydrogenase (CoA acetylierend) (EC 1.2.1.10);
E ₂₃ an NAD-dependent alcohol dehydrogenase (EC 1.1.1.1);
E _{24 is} a glycerone phosphate phospholyase (EC 4.2.3.3);
E ₂₅ , a formate dehydrogenase (EC 1.2.1.2);
E _{26 is} a propionate kinase (EC 2.7.2.15);
E _{27 is} a phosphate propionyltransferase (EC 2.3.1.8);
E _{28 is} a pyruvate decarboxylase (EC 4.1.1.1);
E _{29 is} a phosphoketolase (EC 4.1.2.9);
E _{30 is} a methylmalonyl-CoA-carboxytransferase (EC 2.1.3.1);
E _{31 is} a methylmalonyl-CoA mutase (EC 5.4.99.2);
E ₃₂ an acetolactate synthase (EC 2.2.1.6);
E ₃₃ an acetolactate decarboxylase (EC 4.1.1.5);
E ₃₄ is a butanediol dehydrogenase (EC 1.1.1.4 or EC 1.1.1.76);
E _{35 is} a butanol dehydrogenase (EC 1.1.1.1 or EC 1.1.1.2);
E _{36 is} a butyraldehyde dehydrogenase (EC 1.2.1.3, EC 1.2.1.4 or EC 1.2.1.5);
E ₃₇ an acetoacetate decarboxylase (EC 4.1.1.4);
E _{38 is} a propanol dehydrogenase (EC 1.1.1.1 or EC 1.1.1.2);
E ₃₉ an L-lactate dehydrogenase (EC 1.1.1.27).

In a preferred embodiment, the cells according to the invention have a reduced activity of at least 2, more preferably at least 3, even more preferably at least 5, of the said enzymes compared with their wild-type.

In a particularly preferred embodiment of the invention, the activity of at least one of the enzymes E ₁₉ -E _{22 is} reduced. Such a reduction in enzyme activity may be beneficial as it eliminates the energy-consuming import and activation of glucose, thereby providing the cell with more energy to import and activate sucrose and to fix carbon dioxide.

According to particularly preferred embodiments of the invention, the cell has a reduced activity of at least one enzyme E ₁₃ -E ₃₉ in comparison to its wild type, wherein:
E ₁₃ is encoded by a sadH gene;
E ₁₄ is encoded by a tdcE gene;
E ₁₅ is encoded by a pflA or pflB gene;
E ₁₆ is encoded by a poxB gene;
E ₁₇ is encoded by a ygfG gene;
E ₁₉ is encoded by an ackA or tdcD gene;
E ₂₀ is encoded by a pta gene;
E ₂₁ is encoded by an ldhA gene;
E ₂₂ is encoded by an adhE gene;
E ₂₃ is encoded by an adhE gene;
E ₂₄ is encoded by a mgsA gene;
E ₂₅ is encoded by a fdnG, fdnH, fdnI, fdnF, fdnG, fdoI or fdnH gene;
E ₂₆ is encoded by a tdcD gene
E ₂₇ is encoded by a pta gene;
E ₂₈ is encoded by a pdc gene;
E ₂₉ is encoded by an xpk1 or xpk2 gene
E ₃₀ is encoded by a methylmalonyl-CoA carboxytransferase gene;
E ₃₁ is encoded by a sbm or a mcmA and a mcmB gene;
E ₃₂ is encoded by a gene as S, ilvB, ilvM, ilvN, ilvG, ilvI or ilvH;
E ₃₃ is encoded by a alsD gene;
E ₃₄ is encoded by a butB gene;
E ₃₅ is encoded by an adhE gene;
E ₃₆ is encoded by an adhE, bdhA or bdhB gene;
E ₃₇ is encoded by an adc gene;
E ₃₈ is encoded by an adh gene;
E ₃₉ is encoded by an ldhL gene.

The nucleotide sequences of the abovementioned genes, their corresponding protein sequence and other genes for the enzymes E ₁₃ -E ₈₀ can also be taken from the KEGG, NCBI, UniProt or EMBL database.

Furthermore, in a further, preferred embodiment of the cell according to the invention, it may additionally be genetically engineered in such a way that it has an increased carbon flux from the carbon source available for its growth and as an energy source to acetyl coenzyme A in comparison to its wild type ,

The requisite genetic modification can be done by manipulation of the recombinant cell, which enhances the metabolism that catalyzes the conversion of simple carbon sources to acetyl coenzyme A.

In this embodiment of the invention, the activity of at least one of the following enzymes is preferably enhanced in comparison to the wild-type:
E ₄₀ , which catalyzes the conversion of glucose and ATP to glucose 6-phosphate and ADP;
E ₄₁ , which catalyzes the conversion of glucose-6-phosphate to fructose-6-phosphate;
E ₄₂ , which catalyzes the conversion of fructose 6-phosphate and ATP into fructose 1,6-bisphosphate and ADP;
E ₄₃ , which catalyzes the reaction of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate;
E ₄₄ , which catalyzes the reaction of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate;
E ₄₅ , which catalyzes the reaction of glyceraldehyde 3-phosphate, P _i and NAD ⁺ to 1,3-bisphosphoglycerate and NADH;
_E46 , which catalyzes the conversion of 1,3-bisphosphoglycerate and ADP to 3-phosphoglycerate and ATP;
E ₄₇ , which catalyzes the conversion of 3-phosphoglycerate to 2-phosphoglycerate;
E ₄₈ which catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate and H ₂ O;
E ₄₉ , which catalyzes the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP;
E ₅₀ , which catalyzes the conversion of pyruvate, coenzyme A and NAD ⁺ to acetyl coenzyme A and NADH;
E ₅₁ , which catalyzes the conversion of glucose-6-phosphate and NAD (P) ⁺ to 6-phosphoglucono-δ-lactone and NAD (P) H;
E ₅₂ , which catalyzes the conversion of 6-phosphoglucono-δ-lactone and H ₂ O to 6-phosphogluconate;
E ₅₃ , which catalyzes the reaction of 6-phosphogluconate and NAD (P) ⁺ to ribulose-5-phosphate and NAD (P) H;
E ₅₄ , which catalyzes the conversion of ribulose-5-phosphate to ribose-5-phosphate;
E ₅₅ , which catalyzes the conversion of ribulose-5-phosphate to xylulose-5-phosphate;
E ₅₆ , which catalyzes the reaction of xylulose-5-phosphate and ribose-5-phosphate to glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate;
E ₅₇ , which catalyzes the reaction of erythrose 4-phosphate and fructose 6-phosphate to glyceraldehyde 3-phosphate and sedoheptulose 7-phosphate;
E ₅₈ , which catalyzes the uptake of glycerol;
E ₅₉ , which catalyzes the conversion of glycerol and ATP to glycerol-3-phosphate and ADP;
E ₆₀ , which catalyzes the reaction of glycerol-3-phosphate and NAD (P) ⁺ to dihydroxyacetone phosphate and NAD (P) H;
E ₆₁ , which catalyzes the reaction of glycerol-3-phosphate and NAD (P) ⁺ to glyceraldehyde-3-phosphate and NAD (P) H;
E ₆₂ , which catalyzes the reaction of glycerol and NAD (P) ⁺ to dihydroxyacetone and NAD (P) H;
E ₆₃ , which catalyzes the reaction of dihydroxyacetone and ATP to dihydroxyacetone phosphate and ADP;
E ₆₄ , E ₆₅ , E ₆₆ and E ₆₇ , which are components of a glucose-specific phosphoenolpyruvate phosphotransferase system and, as such, catalyze the import of glucose and in sum the conversion of phosphoenolpyruvate and glucose to pyruvate and glucose-6-phosphate;
_E68 , which catalyzes the reaction of pyruvate NAD (P) H ⁺ and CO ₂ to malate and NAD (P) ⁺ ;
E ₆₉ which binds cAMP;
_E70 , which catalyzes the conversion of glucose and an oxidized electron acceptor to gluconate and a reduced electron acceptor;
E ₇₁ , which catalyzes the reaction of gluconate and an oxidized electron acceptor into 2-ketogluconate and a reduced electron acceptor;
E ₇₂ , which catalyzes the conversion of gluconate and ATP to 6-phosphogluconate and ADP;
E ₇₃ , which catalyzes the conversion of 2-ketogluconate-6-phosphate and NAD (P) H to 6-phosphogluconate and NAD (P) ⁺ ;
E ₇₄ , which catalyzes the conversion of 2-ketogluconate and ATP to 2-ketogluconate-6-phosphate and ADP;
E ₇₅ , which catalyzes the conversion of 6-phosphogluconate to 2-keto-3-deoxy-6-phosphogluconate and H ₂ O;
E ₇₆ , which catalyzes the reaction of 2-keto-3-deoxy-6-phosphogluconate to glyceraldehyde-3-phosphate and pyruvate;
E ₇₇ , which catalyzes the transport of glucose through the cytoplasmic membrane into the cell;
E ₇₈ , which catalyzes the transport of gluconate through the cytoplasmic membrane into the cell;
E ₇₉ , which catalyzes the transport of 2-ketogluconate through the cytoplasmic membrane into the cell;
E ₈₀ , which catalyzes the transport of glucose through the outer membrane into the cell;
In preferred embodiments of the cells according to the invention,
E _{40 is} a hexokinase (EC 2.7.1.1);
E _{41 is} a glucose phosphate isomerase (EC 5.3.1.9);
E ₄₂ a phosphofructokinase (EC 2.7.1.11);
E _{43 is} a fructose-1,6-bisphosphate aldolase (EC 4.2.1.13);
E _{44 is} a triose phosphate isomerase (EC 5.3.1.1);
E _{45 is} a glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9, EC 1.2.1.12, EC 1.2.1.13 or EC 1.2.1.59);
E ₄₆ is a phosphoglycerate kinase (EC 2.7.2.3 or EC 2.7.2.10);
E ₄₇ a phosphoglycerate mutase (EC 5.4.2.1);
E ₄₈ is an enolase (EC 4.2.1.11);
E ₄₉ a pyruvate kinase (EC 2.7.1.40);
E ₅₀ a pyruvate dehydrogenase (EC 1.2.1.51 or EC 1.2.4.1);
E _{51 is} a glucose-6-phosphate dehydrogenase (EC 1.1.1.49);
E _{52 is} a 6-phosphogluconolactonase (EC 3.1.1.31);
E _{53 is} a 6-phosphogluconate dehydrogenase (EC 1.1.1.44);
E ₅₄ a ribose 5-phosphate isomerase (EC 5.3.1.6);
E _{55 is} a ribulose-5-phosphate-4-epimerase (EC 5.1.3.4);
E ₅₆ a transketolase (EC 2.2.1.1);
E ₅₇ is a transaldolase (EC 2.2.1.2);
E _{58 is} a glycerol facilitator;
E ₅₉ a glycerol kinase (EC 2.7.1.30);
E _{60 is} a glycerol-3-phosphate dehydrogenase (EC 1.1.1.8 or EC 1.1.1.94);
E _{61 is} a glycerol-3-phosphate-1-dehydrogenase (EC 1.1.1.177);
E _{62 is} a glycerol-2-dehydrogenase (EC 1.1.1.156);
E _{63 is} a dihydroxyacetone kinase (EC 2.7.1.29);
E ₆₄ and E ₆₅ a PEP-dependent phosphotransferase system Enzyme II (EC 2.7.1.69);
E _{66 is} a PEP-dependent phosphotransferase system Enzyme I (EC 2.7.3.9);
E ₆₇ a phosphohistidine protein (HPr) -hexose phosphotransferase component of the PEP-dependent phosphotransferase system;
_E68 a malate dehydrogenase ((S) -malate: NAD (P) ⁺ oxidoreductase) (EC 1.1.1.38, EC 1.1.1.39 or EC 1.1.1.40);
E _{69 is} a cAMP receptor protein
E _{70 is} a glucose dehydrogenase;
E ₇₁ a gluconate oxidase (EC 1.1.99.3);
E ₇₂ a gluconate kinase (EC 2.7.1.12);
E _{73 is} a 2-ketogluconate-6-phosphate dehydrogenase (EC 1.1.1.25);
E ₇₄ a ketogluconate kinase (EC 2.7.1.13);
E ₇₅ a 6-phosphogluconate dehydratase (EC 4.2.1.12);
E ₇₆ 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.2.1.14);
E ₇₇ a glucose-specific ABC transporter;
E ₇₈ a gluconate importer;
E ₇₉ a 2-ketogluconate importer;
E _{80 is} a glucose-specific porin;

In one embodiment, the cells according to the invention have an increased activity compared to their wild type of at least one, preferably at least 2, more preferably at least 3, most preferably at least 5 of said enzymes.

It may be advantageous under the condition of the use of sucrose as the carbon source, if the cell of the invention has a genetically engineered enzyme activity of enzymes E ₈₁ to E ₉₀ , as described for the enzymes, as described in the WO2011154503 as E ₁ to E _{10 are} disclosed. In the WO2011154503 Preferred enzyme activity combinations described with respect to these ten enzymes are also preferred in the present invention in the embodiment of the cell according to the invention described in this section; Preferred enzymes E ₈₁ to E ₉₀ in connection with the present invention are also those described in the WO2011154503 are mentioned as preferred enzymes E ₁ to E ₁₀ . The disclosure of the WO2011154503 with respect to these enzymes is hereby introduced as a disclosure of the present application.

Accordingly, by the term "decreased activity of an enzyme E _x " as used herein, it is preferred to have a factor of at least 0.5, more preferably at least 0.1, above more preferably of at least 0.01, more preferably still more preferably at least 0.001, and most preferably at least 0.0001 decreased activity. The phrase "decreased activity" also does not include any detectable activity ("zero activity"). The reduction of the activity of a specific enzyme can be carried out, for example, by targeted mutation or by other measures known to those skilled in the art for reducing the activity of a particular enzyme. Methods for reducing enzymatic activities in microorganisms are known to those skilled in the art.

In particular molecular biological techniques are available here. Instructions for modifying and reducing protein expressions and associated enzyme activity reduction especially for Pseudomonas and Burkholderia, in particular for interrupting special genes, the skilled worker 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 ,

Preferred cells according to the invention are characterized in that the reduction of the enzymatic activity is achieved by modification of a gene comprising one of the nucleic acid sequences coding for the polypeptide sequences around Seq ID No. 64 or SEQ ID No. 66, in particular Seq ID No. 63 or SEQ ID NO: 65, wherein the modification is selected from the group comprising, preferably consisting of, insertion of foreign 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 sites flanking the gene.

By foreign DNA in this context is meant any DNA sequence that is "foreign" to the gene (and not to the organism), i. Endogenous DNA sequences can also function as "foreign DNA" in this context.

In this connection, it is particularly preferred that the gene is disrupted by insertion of a selection marker gene, thus the foreign DNA is a selection marker gene, preferably insertion being by homologous recombination into the gene locus.

In a preferred embodiment of the cell according to the invention, the cell is Pseudomonas putida cells which have a reduced polyhydroxyalkanoate synthesis compared to their wild type. Such cells are for example in Ren et al., Journal of 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 described as KTOY01 and KTOY02 and represent preferred cells according to the invention.

• i) Acetyl-Coenzym A und Carboxybiotin-Carboxyl-Carrier-Protein zu Malonyl-Coenzym A und Biotin-Carboxyl-Carrier-Protein (katalysiert durch Acetyl-Coenzym A-Carboxyltransferasen der EC 6.4.1.2),
• ii) Coenzym A-[4'-Phosphopantethein] und Apo-[Acyl-Carrier-Protein] zu Adenosin-3',5'-Bisphosphat und Holo-[Acyl-Carrier-Protein] (katalysiert durch Holo-[Acyl-Carrier-Protein]-Synthasen der EC 2.7.8.7),
• iii) Malonyl-Coenzym A und Holo-[Acyl-Carrier-Protein] zu Malonyl-[Acyl-Carrier-Protein] (katalysiert durch Malonyl-Coenzym A:[Acyl-Carrier-Protein]-Transacylasen der EC 2.3.1.39),
• iv) Acyl-[Acyl-Carrier-Protein] und Malonyl-[Acyl-Carrier-Protein] zu 3-Ketoacyl-[Acyl-Carrier-Protein], CO₂ und Acyl-Carrier-Protein, (katalysiert durch 3-Ketoacyl-[Acyl-Carrier-Protein]-Synthetasen der EC 2.3.1.31),
• v) 3-Ketoacyl-[Acyl-Carrier-Protein], NADPH⁺ und H⁺ zu 3-Hydroxyacyl-[Acyl-Carrier-Protein] und NADP⁺ (katalysiert durch 3-Ketoacyl-[Acyl-Carrier-Protein]-Reduktasen der EC 1.1.1.100),
• vi) 3-Hydroxyacyl-[Acyl-Carrier-Protein] zu 2-Enoyl-[Acyl-Carrier-Protein] (katalysiert durch 3- Hydroxyacyl-[Acyl-Carrier-Protein]-Dehydratasen der EC 4.2.1.60),
• vii) Biotin-Carboxyl-Carrier-Protein, ATP und CO₂ zu Carboxybiotin-Carboxyl-Carrier-Protein, ADP und P_i (katalysiert durch Biotin-Carboxylasen der EC 6.3.4.14),
• viii) Biotin, ATP und Apo-[Acetyl-Coenzym A:CO₂-Ligase (ADP-bildend)] zu [Acetyl-Coenzym A:CO₂-Ligase (ADP-bildend)], AMP und PP_i (katalysiert durch Biotin-[Acetyl-Coenzym A-Carboxylase]-Ligasen der EC 6.3.4.15)

führen, im Vergleich zum Wildtyp der Zelle verstärkt werden. It may be advantageous according to the invention if the cell according to the invention has been genetically engineered in fatty acid biosynthesis such that the enzymatic reactions necessary for the conversion of

• i) Acetyl Coenzyme A and Carboxybiotin Carboxyl Carrier Protein to Malonyl Coenzyme A and Biotin Carboxyl Carrier Protein (Catalysed by Acetyl Coenzyme A Carboxyl Transferases of EC 6.4.1.2),
• ii) coenzyme A- [4'-phosphopantetheine] and apo [acyl carrier protein] to adenosine 3 ', 5'-bisphosphate and holo [acyl carrier protein] (catalyzed by holo- [acyl carrier Protein] synthases of EC 2.7.8.7),
• iii) malonyl coenzyme A and holo [acyl carrier protein] to malonyl [acyl carrier protein] (catalyzed by malonyl coenzyme A: [acyl carrier protein] transacylases of EC 2.3.1.39),
• iv) acyl [acyl carrier protein] and malonyl [acyl carrier protein] to 3-ketoacyl [acyl carrier protein], CO ₂ and acyl carrier protein (catalyzed by 3-ketoacyl) [Acyl carrier protein] synthetases of EC 2.3.1.31),
• v) 3-ketoacyl [acyl carrier protein], NADPH ⁺ and H ⁺ to 3-hydroxyacyl [acyl carrier protein] and NADP ⁺ (catalyzed by 3-ketoacyl [acyl carrier protein] reductases EC 1.1.1.100),
• vi) 3-hydroxyacyl [acyl carrier protein] to 2-enoyl [acyl carrier protein] (catalyzed by 3-hydroxyacyl [acyl carrier protein] dehydratases of EC 4.2.1.60),
• vii) biotin-carboxyl carrier protein, ATP and CO ₂ to carboxybiotin-carboxyl carrier protein, ADP and P _i (catalyzed by biotin-carboxylases of EC 6.3.4.14),
• viii) biotin, ATP and apo [acetyl coenzyme A: CO ₂ ligase (ADP-forming)] to [acetyl-coenzyme A: CO ₂ ligase (ADP-forming)], AMP and PP _i (catalyzed by biotin - [acetyl-coenzyme A carboxylase] ligases of EC 6.3.4.15)

lead to be amplified compared to the wild type of the cell.

• i) ein Acyl-Carrier-Protein und/oder
• ii) ein Biotin-Carboxyl-Carrier-Protein

im Vergleich zum Wildtyp der Zelle verstärkt wurden. It may also be advantageous if the cell according to the invention has been genetically engineered in fatty acid biosynthesis such that

• i) an acyl carrier protein and / or
• ii) a biotin-carboxyl carrier protein

were amplified compared to the wild type of the cell.

It may also be advantageous if the cell according to the invention has been genetically engineered in fatty acid biosynthesis such that expression of the genes encoding the enzymes acetyl-coenzyme A carboxylases of EC 6.4.1.2, holo [acyl carrier protein ] Synthases of EC 2.7.8.7, malonyl coenzyme A: [acyl carrier protein] transacylases of EC 2.3.1.39, 3-ketoacyl [acyl carrier protein] synthetases of EC 2.3.1.31, 3- Ketoacyl [acyl carrier protein] reductases of EC 1.1.1.100, 3-hydroxyacyl [acyl carrier protein] dehydratases of EC 4.2.1.60, biotin carboxylases of EC 6.3.4.14 and / or biotin [ Acetyl-coenzyme A carboxylase] ligases of EC 6.3.4.15 encode repressing transcriptional regulator compared to the wild type of the cell is attenuated.

• i) Acyl-Coenzym A und NADP⁺ zu 2-Enoyl-Coenzym A, NADPH⁺ und H⁺ (katalysiert durch Acetyl-Coenzym A:C-Acetyltransferasen der EC 2.3.1.16),
• ii) 2-Enoyl-Coenzym A zu 3-Hydroxyalkanoyl-Coenzym A (katalysiert durch 2-Enoyl-Coenzym A-Hydratasen der EC 4.2.1.17),
• iii) 3-Hydroxyacyl-Coenzym A zu 3-Ketoacyl-Coenzym A (katalysiert durch 3-Hydroxyacyl-Coenzym A-Dehydrogenasen der EC 1.1.1.35) und/oder
• iv) 3-Ketoacyl-Coenzym A zu Acyl-Coenzym A und Acetyl-Coenzym A (katalysiert durch Acetyl-Coenzym A:C-Acetyltransferasen der EC 2.3.1.16)

führen, im Vergleich zum Wildtyp der Zelle verstärkt werden. It may also be advantageous if the cell according to the invention has been genetically engineered in the β-oxidation of fatty acids in such a way that the enzymatic reactions necessary for the conversion of

• i) acyl coenzyme A and NADP ⁺ to 2-enoyl coenzyme A, NADPH ⁺ and H ⁺ (catalyzed by acetyl coenzyme A: C-acetyltransferases of EC 2.3.1.16),
• ii) 2-enoyl coenzyme A to 3-hydroxyalkanoyl coenzyme A (catalyzed by 2-enoyl-coenzyme A hydratases of EC 4.2.1.17),
• iii) 3-hydroxyacyl-coenzyme A to 3-ketoacyl-coenzyme A (catalyzed by 3-hydroxyacyl-coenzyme A dehydrogenases of EC 1.1.1.35) and / or
• iv) 3-ketoacyl coenzyme A to acyl coenzyme A and acetyl coenzyme A (catalyzed by acetyl coenzyme A: C-acetyltransferases of EC 2.3.1.16)

lead to be amplified compared to the wild type of the cell.

The term "enhanced activity of an enzyme" in the context of the present invention is preferably to be understood as increased intracellular activity.

In principle, an increase in enzymatic activity can be achieved by increasing the copy number of the gene sequence or of the gene sequences which code for the enzyme, using a strong promoter or an improved ribosome binding site, attenuating a negative regulation of gene expression, for example by transcriptional regulators, or enhances positive regulation of gene expression by, for example, transcriptional regulators, modifies the codon usage of the gene, variously increases the half-life of the mRNA or enzyme, modifies regulation of expression of the gene, or utilizes a gene or allele that is of interest corresponding enzyme encoded with an increased activity and optionally combines these measures. Genetically modified cells according to the invention are produced for example by transformation, transduction, conjugation or a combination of these methods with a vector which contains the desired gene, an allele of this gene or parts thereof and optionally a promoter which enables the expression of the gene. In particular, heterologous expression is achieved by integration of the gene or alleles into the chromosome of the cell or an extrachromosomally replicating vector.

An overview of the possibilities for increasing the enzyme activity in cells using the example of pyruvate carboxylase gives DE-A-100 31 999 , which is hereby incorporated by reference, and the disclosure of which relates to the potential for increasing enzyme activity in cells forms part of the disclosure of the present invention.

The expression of the above and all of the enzymes or genes mentioned below can be detected in the gel with the aid of 1- and 2-dimensional protein gel separation and subsequent optical identification of the protein concentration with appropriate evaluation software. If the increase in enzyme activity is based solely on an increase in the expression of the corresponding gene, the quantification of the increase in enzyme activity can be easily determined by comparing the 1- or 2-dimensional protein separations between wild type and genetically engineered cell. A common method for preparing the protein gels in coryneform bacteria and for identifying the proteins is that of Hermann et al. (Electrophoresis, 22: 1712.23 (2001) ) procedure described. Protein concentration can also be assessed by Western blot hybridization with one to be detected Protein specific antibody ( 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 appropriate software for concentration determination ( Lohaus and Meyer (1989) Biospektrum, 5: 32-39 ; Lottspeich (1999) Angewandte Chemie 111: 2630-2647 ) to be analyzed. The activity of DNA-binding proteins can be measured by DNA band-shift assays (also called gel retardation) ( Wilson et al. (2001) Journal of Bacteriology, 183: 2151-2155 ).

The effect of DNA-binding proteins on the expression of other genes can be demonstrated by various well-described methods of the reporter gene assay ( Sambrook et al., Molecular Cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989 ). The intracellular enzymatic activities can be determined by various methods described ( Donahue et al. (2000) Journal of Bacteriology 182 (19): 5624-5627 ; Ray et al. (2000) Journal of Bacteriology 182 (8): 2277-2284 ; Freedberg et al. (1973) Journal of Bacteriology 115 (3): 816-823 ). Unless specific methods for the determination of the activity of a particular enzyme are specified in the following, the determination of the increase in the enzyme activity and also the determination of the reduction of an enzyme activity is preferably carried out by means of in Hermann et al., Electrophoresis, 22: 1712-23 (2001) . Lohaus et al., Biospektrum 5 32-39 (1998) . Lottspeich, Angewandte Chemie 111: 2630-2647 (1999) and Wilson et al., Journal of Bacteriology 183: 2151-2155 (2001) described methods. If the increase in enzyme activity is accomplished by mutation of the endogenous gene, such mutations can be generated either undirected by classical methods, such as by UV irradiation or by mutagenic chemicals, or specifically by genetic engineering methods such as deletion (s), insertion (s) and / or nucleotide exchange (s). These mutations result in altered cells. Particularly preferred mutants of enzymes are, in particular, also those enzymes which are no longer, or at least in comparison with the wild-type enzyme, reduced in the form of feedback, product or substrate inhibition.

If the increase in enzyme activity is accomplished by increasing the synthesis of an enzyme, for example, one increases the copy number of the corresponding genes or mutates the promoter and regulatory region or ribosome binding site located upstream of the structural gene. In the same way, expression cassettes act, which are installed upstream of the structural gene. Inducible promoters also make it possible to increase expression at any time. Furthermore, the enzyme gene can be assigned as regulatory sequences but also so-called "enhancers" which also cause an increased gene expression via an improved interaction between RNA polymerase and DNA. Measures to extend the lifetime of mRNA also improve expression. Furthermore, by preventing degradation of the enzyme protein, enzyme activity is also enhanced. The genes or gene constructs are either present in plasmids with different copy numbers or are integrated and amplified in the chromosome. Alternatively, overexpression of the genes in question can be achieved by changing the composition of the medium and culture. Instructions for this, the expert finds among others Martin et al. (Bio / Technology 5, 137-146 (1987)) , at Guerrero et al. (Gene 138, 35-41 (1994)) . Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)) , at Eikmanns et al. (Gene 102, 93-98 (1991)) , in EP-A-0 472 869 , in US 4,601,893 , at Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991) , at Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) , at LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)) , in WO-A-96/15246 , at Malumbres et al. (Gene 134, 15-24 (1993)) , in JP-A-10-229891 , at Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)) and in known textbooks of genetics and molecular biology. The measures described above as well as the mutations lead to genetically modified cells.

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

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

The expression "an increased activity of an enzyme E _{x, which} is increased in comparison with its wild type, is preferably always a factor greater than or equal to at least 2, particularly preferably at least 10, more preferably at least 100, moreover even more preferably at least 1,000 and most preferably at least 10,000 increased activity of the respective enzyme E _x . Furthermore, the cell according to the invention comprises "an activity of an enzyme E _x increased compared to its wild type, in particular also a cell whose wild type has no or at least no detectable activity of this enzyme E _x and which only after increasing the enzyme activity, for example by overexpression , a detectable activity of this enzyme E _x shows. In this context, the term "overexpression" or the expression "increase in expression" used in the following also encompasses the case that a starting cell, for example a wild-type cell, has no or at least no detectable expression and only by recombinant methods a detectable Synthesis of the enzyme E _{x is} induced.

Alterations of amino acid residues of a given polypeptide sequence which do not result in significant changes in the properties and function of the given polypeptide are known to those skilled in the art. Thus, for example, so-called conserved amino acids can be exchanged for each other; Examples of such suitable amino acid substitutions are: Ala versus Ser; Arg against Lys; Asn versus Gln or His; Asp against Glu; Cys versus Ser; Gln against Asn; Glu vs Asp; Gly vs Pro; His against Asn or Gln; Ile vs Leu or Val; Leu vs Met or Val; Lys versus Arg or Gln or Glu; Mead against Leu or Ile; Phe versus Met or Leu or Tyr; Ser against Thr; Thr against Ser; Trp against Tyr; Tyr against Trp or Phe; Val against Ile or Leu. It is also known that changes, especially at the N- or C-terminus of a polypeptide in the form of, for example, amino acid insertions or deletions, often have no significant effect on the function of the polypeptide.

The "amino acid identity" in connection with the enzymes used in the context of the invention is determined by means of known methods. In general, special computer programs are used with algorithms taking into account special requirements.

Preferred methods for determining identity initially produce the greatest match between the sequences to be compared. Computer identity determination programs include, but are not limited to, the GCG program package, including
CAP ( Deveroy, J. et al., Nucleic Acid Research 12 (1984), page 387, Genetics Computer Group University of Wisconsin, Medicine (Wi) , and BLASTP, BLASTN and FASTA ( Altschul, S. et al., Journal of Molecular Biology 215 (1990), pages 403-410 , The BLAST program can be obtained from the National Center For Biotechnology Information (NCBI) and from other sources ( BLAST Handbook, Altschul S. et al., NCBI NLM NIH Bethesda ND 22894 ; Altschul S. et al. , above).

The well known Smith-Waterman algorithm can also be used to determine identities.

Preferred parameters for determining the "amino acid identity" are when using the BLASTP program ( Altschul, S. et al., Journal of Molecular Biology 215 (1990), pages 403-410 : Expect Threshold: 10 Word size: 3 Matrix: BLOSUM62 Gap costs: Existence: 11; Extension: 1 Compositional adjustments: Conditional compositional score matrix adjustment

The above parameters are the default parameters in the amino acid sequence comparison. The GAP program is also suitable for use with the above parameters. An identity of 60% according to the above algorithm means 60% identity in the context of the present invention. The same applies to higher identities.

The activity of an enzyme can be determined by digesting cells containing this activity in a manner known to those skilled in the art, for example by means of a ball mill, a French press or an ultrasonic disintegrator and then intact cells, cell debris and digestion aids How to separate glass beads by centrifugation at 11,000 x g and 4 ° C for 10 minutes. Enzyme assays with subsequent LC-ESI-MS detection of the products can then be carried out with the resulting cell-free crude extract. Alternatively, the enzyme may be enriched or purified to homogeneity in a manner known to those skilled in the art by chromatographic techniques (such as nickel nitrilotriacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography or ion exchange chromatography).

It is trivial, and for the sake of completeness, that in order to determine an increased or decreased activity compared to the wild type of a cell, a wild-type reference culture is used which has been exposed to the same conditions as that of the sample to be determined.

The activity of the enzyme E ₁ can then be determined with the crude cell-free extracts obtained as described above by determining the amount of enzyme E ₁ formed. It is assumed that more enzyme E ₁ per biomass unit is able to export more rhamnolipid of the general formula (I) from the cell into the surrounding medium. Such quantification can be carried out by immunological detection by means of antibodies specific for enzyme E ₁ (see Curien, TB, Scofield, R. H (Eds.). Protein Blotting and Detection: Methods and Protocols. Methods in Molecular Biology, Vol. 536. 1st ed., Humana Press. NY USA, 2009 ) or by mass spectrometric methods (see Schmidt, A., Kellermann, J. & Lottspeich, F. A novel strategy for quantitative proteomics using isotope-coded protein labels. Proteomics 5, 4-15 (2005) ).

Alternatively, the activity of the enzyme E _{1 can} also be determined by monitoring uptake assays with radiolabeled rhamnolipids and inside-out vesicles prepared from the cells of the invention. The general procedure is for example in Nies DH. The cobalt, zinc, and cadmium efflux system CzcABC from Alcaligenes eutrophus functions as a cation-proton antiporter in Escherichia coli. J Bacteriol. 1995. 177 (10): 2707-12 or Lewinson O, Adler J, Poelarend's GJ, Mazurkiewicz P, Driessen AJ, Bibi E. The Escherichia coli multidrug transporter MdfA catalyzes both electrogenic and electroneutral transport reactions. Proc Natl Acad Sci USA 2003 Feb 18; 100 (4): 1667- 72 ,

The activity of the enzyme E ₂ is then carried out with the cell-free crude extracts or purified enzyme E ₂ obtained as described above in reaction mixtures consisting of 30 μM acetoacetyl-coenzyme A, 0.125 mM NADP ⁺ , up to 10 mg crude extract or 10 μg purified enzyme E ₂ in 400 μl of 50 mM Tris-HCl (pH 8.0). The reactions are started by adding the substrate acetoacetyl-coenzyme A and then incubated at room temperature for 2 min. The course of the reaction is monitored by following the decrease in absorbance at 340 nm, which is based on NADPH consumption due to the reduction of acetoacetyl-coenzyme A. The decrease in absorbance at 340 nm per minute in the initial linear region of the curve is used to determine the specific activity of 3-ketoacyl-coenzyme A reductase in the crude extract according to the following equation:

The activity of the enzyme E ₃ is then determined using the cell-free crude extracts or purified enzyme E _{3 obtained} in the above-described manner in 50 μl Tris / HCl, pH 7.5. The standard assay contains 3 mM MgCl ₂ , 40 μM hydroxydecanoyl coenzyme A and 20 μM E. coli ACP in 50 mM Tris-HCl, pH 7.5, in a total volume of 200 μl. The reaction is started by adding 5 μg of purified enzyme E ₃ in 50 μl Tris / HCl, pH 7.5 and incubated for 1 h at 30 ° C. The reaction is stopped by addition of 50% (w / v) trichloroacetic acid and 10 mg / ml BSA (30 μl). Released coenzyme A is determined spectrophotometrically by recording the increase in absorbance at 412 nm caused by addition of 5,5'-dithiobis (2-nitrobenzoate) (DTNB) to free SH groups over time.

The activity of the enzyme E _4a is then determined in the case of the energy-independent transhydrogenase using the cell-free crude extracts obtained as described above or purified enzyme E _4a in 50 mM Tris-HCl (pH 7.0).

The activity of the energy-independent transhydrogenase is determined by monitoring the decrease in the concentration of thionicotinamide adenine dinucleotide phosphate (tNADP ⁺ ) at 400 nm. The assay was carried out in a reaction mixture containing 0.1 mM NADH and 0.1 mM tNADP ⁺ (Sigma Chemical Co.) in 50 mM Tris-HCl (pH 7.0) at 30 ° C. The molar extinction coefficient of tNADPH at 400 nm was assumed to be 11,300 L · mol ^-1 · cm ^-1 . One unit of energy-independent transhydrogenase was defined as the activity that leads to the reduction of 1 μM tNADP ⁺ per minute under these conditions.

The activity of the energy-dependent transhydrogenase is determined in the membrane protein fraction. For this, the cells are harvested in the mid to late logarithmic phase of growth, washed once with 50 mM Tris-sulfate buffer, pH 7.8, with 10 mM MgCl ₂ (TM buffer), resuspended in TM buffer (1 g wet cell mass) per 10 ml of buffer) and digested in a French press (Aminco) at 20,000 psi. The suspension is then centrifuged at 17,000 xg for 10 min to remove whole cells and larger cell debris. The membrane fraction is recovered by centrifuging the supernatant at 120,000 x g for 2 hours. The pellet is resuspended in TM buffer with 1% bovine serum albumin so that a concentration of 10-15 mg membrane protein per ml is achieved.

The activity of the energy-dependent transhydrogenase is then determined as follows: 1 ml of TM buffer (0.1% bovine serum albumin, 0.1 mM dithiothreitol, 0.7 M sucrose) is mixed with 50 μl of the membrane protein fraction and placed in a cuvette at 37 ° C Preincubated for 5 min. The cuvette is then transferred to a spectrophotometer and the absorbance increase at 340 nm is determined after addition of 5 μl of ethanol, 50 μl of yeast alcohol dehydrogenase (4 mg / ml, Calbiochem) and 25 μl of 3 mM NAD. After 1 min, 50 μl of 16.3 mM NADP are added and the reduction of NADP is measured ("O ₂ -dependent transhydrogenase"). Once the oxygen in the cuvette is used up, the rate of NADP reduction abruptly decreases to a lower level ("energy independent transhydrogenase"). Once this reduction rate has stabilized, 10 μl of 60 mM ATP are added and the new reduction rate of NADP determined ("ATP-dependent transhydrogenase"). The activity of the two energy-dependent transhydrogenases is then corrected for the contribution of the energy-independent transhydrogenase.

The activity of the enzyme E _4b is then determined with those obtained as described above cell-free crude extracts or purified enzyme E _4c pyrophosphate / arsenate buffer (0.015 M sodium pyrophosphate buffer, pH 8.5 with 0.03 M sodium arsenate). For this purpose, the following solutions are combined in a cuvette:
2.6 ml of 0.015 M sodium pyrophosphate buffer, pH 8.5 with 0.03 M sodium arsenate
0.1 ml of 7.5 mM NADP (analytical grade)
0.1 ml of 0.1 M dithiothreitol (DTT)
0.1 ml cell-free crude extract (brought to a concentration of 10-30 μg crude extract / ml pyrophosphate / arsenate buffer immediately before use)

Then the cuvette is equilibrated in a spectrophotometer at 25 ° C for 3-5 minutes and the blank noted. Then, 0.1 ml of a 0.015 M DL-glyceraldehyde-3-phosphate solution is added, and the increase in absorbance at 340 nm is recorded for 3-5 minutes. The increase in absorbance at 340 nm per minute in the initial linear region of the curve is used to determine the specific activity of glyceraldehyde-3-phosphate dehydrogenase in the crude extract according to the following equation:

The activity of the enzyme E _4c is then determined using the cell-free crude extracts or purified enzyme E _{4c obtained} in potassium phosphate buffer 0.05 M potassium phosphate buffer, pH 7.8, as described above. The reactions included 2.5 mM NADP, 0.2 mM thiamine pyrophosphate, 0.1 mM coenzyme A, 0.3 mM dithiothreitol, 5 mM pyruvate, 1 mM magnesium chloride and 1 mg bovine serum albumin / ml in 0.05 M potassium phosphate buffer, pH 7.8. The reaction is started by addition of pyruvate and the reaction followed by spectrophotometric measurement of the formation of NADH at 25 ° C for several minutes. The increase in absorbance at 340 nm per minute in the initial linear region of the curve is used to determine the specific activity of pyruvate dehydrogenase in the crude extract according to the following equation:

The activity of the enzyme E ₅ is then determined using the samples obtained 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 E _5a ) or dodecanoyl coenzyme A (for E _5b ), 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 of enzyme E ₅ in a final volume of 120 μL. ACP, β-mercaptoethanol and sodium phosphate buffer are preincubated at 37 ° C for 30 min to completely reduce ACP. The reaction is started by addition of enzyme E ₅ . 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)). It is phase separated by centrifugation (16,100 g, 5 min, RT). The lower organic phase is removed, completely evaporated in the vacuum centrifuge and the sediment taken up in 50 .mu.l of methanol. Undissolved constituents are sedimented by centrifugation (16,100 g, 5 min, RT) and the sample is analyzed by means of LC-ESI-MS. The identification of the products is done by analyzing the corresponding mass traces and the MS ² spectra.

The activity of the enzyme E ₆ is then determined using the samples obtained as follows: a standard assay can be prepared from 185 μl 10 mM Tris-HCl (pH 7.5), 10 μl 125 mM dTDP rhamnose and 50 μl crude protein extract (ca. 1 mg total protein) or purified protein in solution (5 μg purified protein). The reaction is started by adding 10 μl of 10 mM ethanolic solution of 3-hydroxydecanoyl-3-hydroxydecanoic acid (for E _6a ) or 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid (for E _6b ) and shaking at 30 ° C. for 1 h ( 600 rpm). Subsequently, the reaction is mixed with 1 ml of acetone. Undissolved constituents are sedimented by centrifugation (16,100 g, 5 min, RT) and the sample is analyzed by means of LC-ESI-MS. The identification of the products is done by analyzing the corresponding mass traces and the MS ² spectra.

The activity of the enzyme E ₇ is then determined using the samples obtained as follows: a standard assay can be prepared from 185 μl 10 mM Tris-HCl (pH 7.5), 10 μl 125 mM dTDP rhamnose and 50 μl crude protein extract (ca. 1 mg total protein) or purified protein in solution (5 μg purified protein). The reaction is carried out by the addition of 10 μl of 10 mM ethanolic solution of α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid (for E _7a ) or α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid (for E _7b ) and incubated for 1 h at 30 ° C with shaking (600 rpm). Subsequently, the reaction is mixed with 1 ml of acetone. Undissolved constituents are sedimented by centrifugation (16,100 g, 5 min, RT) and the sample is analyzed by means of LC-ESI-MS. The identification of the products is done by analyzing the corresponding mass traces and the MS ² spectra.

The activity of the enzyme E ₈ is determined with the samples obtained as described above by reacting α-D-glucose-1-phosphate (1.3 mM) with dTTP (5 mM) and 5 μg of purified enzyme E ₈ in 50 μl of sodium phosphate. Buffer, pH 8.5, incubated and after 5, 10 and 20 min incubation at 30 ° C, the reaction is stopped by adding 20 ul chloroform. The mixture is then vortexed and centrifuged for 5 min at 16,000 g and room temperature. The aqueous phase is transferred to a new reaction vessel and the organic phase extracted again with 80 .mu.l of water. Both aqueous phases are combined and analyzed by HPLC. 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 elution of the analytes is carried out at a flow rate of 1 ml min ^-1 with 0.5 M KH ₂ PO ₄ (eluent A) for 15 min, followed by a linear gradient up to 80% Eluent A and 20% methanol over a period of 14 min at a flow rate of 0.7 ml min ^-1 . Analytes eluting 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 at a flow rate of 1 ml min ^-1 and a linear Ammonium formate gradient (2 to 600 mM over 25 min) eluted. The quantification of dTDP-glucose then takes place via its UV absorption with a photodiode array detector (DAD). The absorption maximum of thymidine is 267 nm. The calibration is carried out by means of authentic nucleotide sugar (Sigma-Aldrich, Munich, USA).

The activity of the enzyme E ₉ is then determined with the samples obtained as described above, by reacting dTDP-α-D-glucose (1.3 mM) with 5 μg of purified enzyme E ₉ in 50 μl of sodium phosphate buffer, pH 8.5, incubated and after 5, 10 and 20 min incubation at 30 ° C, the reaction is stopped by adding 20 ul chloroform. The mixture is then vortexed and centrifuged for 5 min at 16,000 g and room temperature. The aqueous phase is transferred to a new reaction vessel and the organic phase extracted again with 80 .mu.l of water. Both aqueous phases are combined and analyzed by HPLC. 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 elution of the analytes is carried out at a flow rate of 1 ml min ^-1 with 0.5 M KH ₂ PO ₄ (eluent A) for 15 min, followed by a linear gradient up to 80% Eluent A and 20% methanol over a period of 14 min at a flow rate of 0.7 ml min ^-1 . Analytes eluting 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 at a flow rate of 1 ml min ^-1 and a linear Ammonium formate gradient (2 to 600 mM over 25 min) eluted. The quantification of dTDP-glucose and dTDP-4-dehydro-6-deoxy-D-glucose then takes place via their UV absorption with a photodiode array detector (DAD). The absorption maximum of thymidine is 267 nm. The calibration is carried out by means of authentic nucleotide sugar (Sigma-Aldrich, Munich, USA).

The activity of the enzyme E ₁₀ is then determined using the samples obtained as described above, by first dTDP-α-D-glucose (1.3 mM) with 5 μg of purified enzyme E ₉ in 50 μl of sodium phosphate buffer, pH 8.5 , incubated for 10 min at 30 ° C. Subsequently, 0.5 μg of purified enzyme E _{10 are} added, and after 5, 10 and 20 min of incubation at 30 ° C, the reaction is stopped by addition of 20 μl of chloroform. The mixture is then vortexed and centrifuged for 5 min at 16,000 g and room temperature. The aqueous phase is transferred to a new reaction vessel and the organic phase extracted again with 80 .mu.l of water. Both aqueous phases are combined and analyzed by HPLC. 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 elution of the analytes is carried out at a flow rate of 1 ml min ^-1 with 0.5 M KH ₂ PO ₄ (eluent A) for 15 min, followed by a linear gradient up to 80% Eluent A and 20% methanol over a period of 14 min at a flow rate of 0.7 ml min ^-1 . Analytes eluting 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 at a flow rate of 1 ml min ^-1 and a linear Ammonium formate gradient (2 to 600 mM over 25 min) eluted. The quantification of dTDP-glucose, dTDP-4-dehydro-6-deoxy-D-glucose and dTDP-6-deoxy-L-mannose then takes place via their UV absorption with a photodiode array detector (DAD). The absorption maximum of thymidine is 267 nm. The calibration is carried out by means of authentic nucleotide sugar (Sigma-Aldrich, Munich, USA).

The activity of the enzyme E ₁₁ is then determined with the samples obtained as described by first dTDP-α-D-glucose (1.3 mM) with 5 μg of purified enzyme E ₉ in 50 μl of sodium phosphate buffer, pH 8.5, be incubated for 10 min at 30 ° C. Subsequently, 5 μg of purified enzyme E ₁₀ and 0.5 μg of purified enzyme E ₁₁ and NADPH (10 mM) are added, and after 5, 10 and 20 min incubation at 30 ° C, the reaction is stopped by addition of 20 μl chloroform. The mixture is then vortexed and centrifuged for 5 min at 16,000 g and room temperature. The aqueous phase is transferred to a new reaction vessel and the organic phase extracted again with 80 .mu.l of water. Both aqueous phases are combined and analyzed by HPLC. 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 elution of the analytes is carried out at a flow rate of 1 ml min ^-1 with 0.5 M KH ₂ PO ₄ (eluent A) for 15 min, followed by a linear gradient up to 80% Eluent A and 20% methanol over a period of 14 min at a flow rate of 0.7 ml min ^-1 . Analytes eluting 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 at a flow rate of 1 ml min ^-1 and a linear Ammonium formate gradient (2 to 600 mM over 25 min) eluted. The quantification of dTDP-glucose, dTDP-4-dehydro-6-deoxy-D-glucose, dTDP-6-deoxy-L-mannose and dTDP-4-dehydro-6-deoxy-L-mannose then takes place via their UV Absorption with a photodiode array detector (DAD). The absorption maximum of thymidine is 267 nm. The calibration is carried out by means of authentic nucleotide sugar (Sigma-Aldrich, Munich, USA).

ableitet von 3-Hydroxyoctanoyl-3-Hydroxyoctansäure, 3-Hydroxyoctanoyl-3-Hydroxydekansäure, 3-Hydroxydekanoyl-3-Hydroxyoctansäure, 3-Hydroxyoctanoyl-3-Hydroxydecensäure, 3-Hydroxydecenoyl-3-Hydroxyoctansäure, 3-Hydroxyoctanoyl-3-Hydroxydodekansäure, 3-Hydroxydodekanoyl-3-Hydroxyoctansäure, 3-Hydroxyoctanoyl-3-Hydroxydodecensäure, 3-Hydroxydodecenoyl-3-Hydroxyoctansäure, 3-Hydroxydekanoyl-3-Hydroxydekansäure, 3-Hydroxydekanoyl-3-Hydroxydecensäure, 3-Hydroxydecenoyl-3-Hydroxydekansäure, 3-Hydroxydecenoyl-3-Hydroxydecensäure, 3-Hydroxydekanoyl-3-Hydroxydodekansäure, 3-Hydroxydodekanoyl-3-Hydroxydekansäure, 3-Hydroxydekanoyl-3-Hydroxydodecensäure, 3-Hydroxydekanoyl-3-Hydroxytetradecensäure, 3-Hydroxytetradekanoyl-3-Hydroxydecensäure, 3-Hydroxydodecenoyl-3-Hydroxydekansäure, 3-Hydroxydekanoyl-3-Hydroxytetradekansäure, 3-Hydroxytetradekanoyl-3-Hydroxydekansäure, 3-Hydroxydekanoyl-3-Hydroxytetradecensäure, 3-Hydroxytetradecenoyl-3-Hydroxydekansäure, 3-Hydroxydodekanoyl-3-Hydroxydodekansäure, 3-Hydroxydodecenoyl-3-Hydroxydodekansäure, 3-Hydroxydodekanoyl-3-Hydroxydodecensäure, 3-Hydroxydodekanoyl-3-Hydroxytetradekansäure, 3-Hydroxytetradekanoyl-3-Hydroxydodekansäure, 3-Hydroxytetradekanoyl-3-Hydroxytetradekansäure, 3-Hydroxyhexadekanoyl-3-Hydroxytetradekansäure, 3-Hydroxytetradekanoyl-3-Hydroxyhexadekansäure oder 3-Hydroxyhexadekanoyl-3-Hydroxyhexadekansäure. In the event that the cell according to the invention is able to form a rhamnolipid with m = 1, it is preferred that the residue determined via R ¹ and R ²

Derived from 3-hydroxyoctanoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydecenoic acid, 3-hydroxydecenoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydodecanoic acid, 3-hydroxydodecanoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydodecenoic acid, 3-hydroxydodecenoyl-3-hydroxyoctanoic acid, 3-hydroxydecanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxydecenoic acid, 3-hydroxydecenoyl-3-hydroxydecanoic acid, 3- Hydroxydecenoyl-3-hydroxydecenoic acid, 3-hydroxydecanoyl-3-hydroxydodecanoic acid, 3-hydroxydodecanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxydodecenoic acid, 3-hydroxydecanoyl-3-hydroxytetradecenoic acid, 3-hydroxytetradecanoyl-3-hydroxydecenoic acid, 3-hydroxydodecenoyl 3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxytetradecanoic acid, 3-hydroxytetradecanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxytetradecenoic acid, 3-hydroxytetradecenoyl-3-hydroxydecanoic acid, 3-hydroxydodecanoyl-3 Hydroxydodecanoic acid, 3-hydroxydodecenoyl-3-hydroxydodecanoic acid, 3-hydroxydodecanoyl-3-hydroxydodecenoic acid, 3-hydroxydodecanoyl-3-hydroxytetradecanoic acid, 3-hydroxytetradecanoyl-3-hydroxydodecanoic acid, 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, 3-hydroxyhexadecananoyl-3-hydroxytetradecanoic acid , 3-hydroxytetradecanoyl-3-hydroxyhexadecanoic acid or 3-hydroxyhexadecananoyl-3-hydroxyhexadecanoic acid.

It is obvious to the person skilled in the art that a cell according to the invention can also form mixtures of different rhamnolipids of the general formula (I).

In this context, it is preferred that the cells according to the invention are able to form mixtures of rhamnolipids of the general formula (I), which are characterized in that in more than 80% by weight, preferably more than 90% by weight, particularly preferred more than 95% by weight of the rhamnolipids formed is n = 1 and the radical determined via R ¹ and R ^{2 is} less than 10% by weight, preferably less than 5% by weight, particularly preferably less than 2% by weight. deriving% of the formed rhamnolipids from 3-hydroxydecanoyl-3-hydroxyoctanoic acid or 3-hydroxyoctanoyl-3-hydroxydecanoic acid,
wherein the stated wt .-% based on the sum of all formed rhamnolipids of the general formula (I).

Cells according to the invention can be advantageously used for the production of rhamnolipids.

Thus, another object of the invention is the use of cells according to the invention for the preparation of compounds of general formula (I).

• I) in Kontakt Bringen der erfindungsgemäßen Zelle mit einem Medium beinhaltend eine
• Kohlenstoffquelle
• II) Kultivieren der Zelle unter Bedingungen, die es der Zelle ermöglichen, aus der
• Kohlenstoffquelle Rhamnolipid zu bilden und
• III) gegebenenfalls Isolierung der gebildeten Rhamnolipide.

A further subject of the present invention is a process for the preparation of rhamnolipids of the general formula (I)
in which
m = 2, 1 or 0, in particular 1 or 0,
n = 1 or 0, in particular 1,
R ¹ and R ² = independently of one another the same or different organic radical having 2 to 24, preferably 5 to 13 carbon atoms, in particular optionally branched, optionally substituted, in particular hydroxy-substituted, optionally unsaturated, in particular optionally mono-, di- or triunsaturated, alkyl radical, preferably those selected from the group consisting of pentenyl, heptenyl, nonenyl, undecenyl and tridekenyl and (CH ₂ ) _o -CH ₃ with o = 1 to 23, preferably 4 to 12, comprising the process steps

• I) contacting the cell of the invention with a medium containing one
• Carbon source
• II) culturing the cell under conditions that allow the cell to emerge from the
• To form carbon source rhamnolipid and
• III) optionally isolation of the formed rhamnolipids.

The genetically modified cells according to the invention can be used continuously or discontinuously in the batch process (batch culturing) or in the fed-batch process (feed process) or repeated-fed-batch process (repetitive feed process) for the purpose of producing the abovementioned products with the nutrient medium be brought into contact and thus cultivated. Also conceivable is a semi-continuous process, as described in the GB-A-1009370 is described. A summary of known cultivation methods are in the textbook of Chmiel ("Bioprocessing Technology 1. Introduction to Bioprocess Engineering" (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook of Storhas ("Bioreactors and Peripheral Facilities", Vieweg Verlag, Braunschweig / Wiesbaden, 1994) described.

The culture medium to be used must suitably satisfy the requirements of the respective strains. Descriptions of culture media of various yeast strains are described, for example, in "Nonconventional yeast in biotechnology" (US Pat. Ed. Klaus Wolf, Springer-Verlag Berlin, 1996 ) contain.

As a carbon source carbohydrates such. As glucose, sucrose, arabinose, xylose, lactose, fructose, maltose, molasses, starch, cellulose and hemicellulose, vegetable and animal oils and fats such. As soybean oil, thistle oil, peanut oil, hemp oil, jatropha oil, coconut oil, 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, grapeseed oil, walnut oil, wheat germ oil and coconut oil, fatty acids such. As caprylic, capric, lauric, myristic, palmitic, palmitolenic, stearic, arachidonic, behenic, oleic, linoleic, linolenic, gamma-linolenic and their methyl or ethyl esters and fatty acid mixtures, mono-, di- and triglycerides with just mentioned fatty acids , Alcohols such. As glycerol, ethanol and methanol, hydrocarbons such as methane, carbonaceous gases and gas mixtures such as CO, CO ₂ , synthesis or flue gas, amino acids such as L-glutamate or L-valine or organic acids such. As acetic acid can be used. These substances can be used individually or as a mixture. Particular preference is given to the use of carbohydrates, in particular of monosaccharides, oligosaccharides or polysaccharides, as carbon source as described in US Pat US 6,01,494 and US 6,136,576 and hydrocarbons, in particular alkanes, alkenes and alkynes and the monocarboxylic acids derived therefrom and the mono-, di- and triglycerides derived from these monocarboxylic acids, as well as glycerol and acetate. Very particular preference is given to mono-, di- and triglycerides containing the esterification products of glycerol with caprylic, capric, lauric, myristic, palmitic, palmitolenic, stearic, arachidonic, behenic, oleic, linoleic, linolenic and / or gamma-linolenic acid.

A major advantage of the present invention is that the cells of the invention are able to form rhamnolipids from simplest carbon sources such as glucose, sucrose or glycerol, so that it is not necessary to provide longer-chain C sources in the medium during the process according to the invention. Thus, in the absence of availability, it is advantageous that the medium in step I) of the process of the invention contains no or no detectable amounts of carboxylic acids having a chain length greater than six carbon atoms or esters or glycerides derivable therefrom.

As the nitrogen source, organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, ammonia, ammonium hydroxide or ammonia water can be used. The nitrogen sources can be used singly or as a mixture.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source. The culture medium must continue to contain salts of metals such. As magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth factors such as amino acids and vitamins can be used in addition to the above-mentioned substances. In addition, suitable precursors can be added to the culture medium. The said feedstocks may be added to the culture in the form of a one-time batch or fed in a suitable manner during the cultivation. For pH control 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 the Foaming can anti-foaming agents such. B. fatty acid polyglycol esters are used. To maintain the stability of plasmids, the medium suitable selective substances such. B. antibiotics are added. In order to maintain aerobic conditions, oxygen or oxygen-containing gas mixtures such. For example, air is introduced into the culture.

The temperature of the culture is usually more than 20 ° C, preferably more than 25 ° C, it may also be more than 40 ° C, advantageously a cultivation temperature of 95 ° C, more preferably 90 ° C and most preferably 80 ° C is not exceeded.

In step III) of the method according to the invention, the rhamnolipids formed by the cells can optionally be isolated from the cells and / or the nutrient medium, all methods known to those skilled in the art for isolating low molecular weight substances from complex compositions being suitable for isolation, for example filtration, extraction , Adsorption (chromatography) or crystallization.

In addition, the product phase contains residues of biomass and various impurities, such as oils, fatty acids and other nutrient media components. The separation of the impurities is preferably carried out in a solvent-free process. For example, the product phase may be diluted with water to facilitate adjustment of the pH. The product and aqueous phases can then be homogenized by converting the rhamnolipids into a water-soluble form by lowering or raising the pH by means of acids or alkalis. Potentially, solubilization of the rhamnolipids in the aqueous phase can be achieved by incubation at higher temperatures, e.g. at 60 to 90 ° C, and constant mixing are supported. By subsequently raising or lowering the pH through lyes or acids, the rhamnolipids can then be reconverted to a water-insoluble form so that they can be easily separated from the aqueous phase. The product phase can then be washed one or more times with water to remove water-soluble impurities.

Oil residues can be separated, for example by extraction by means of suitable solvents advantageously by means of organic solvents. The preferred solvent is an alkane, e.g. n-hexane.

Separation of the product from the aqueous phase may alternatively be carried out with the solvent-free process described above with a suitable solvent, e.g. an ester such as ethyl acetate or butyl acetate. The said extraction steps can be carried out in any order.

In this case, preference is given to using solvents, in particular organic solvents. The solvent used is n-pentanol. To remove the solvent, for example, a distillation. Subsequently, the lyophilized product, for example by means of chromatographic methods, be further purified. By way of example, the precipitation by means of suitable solvents, the extraction by means of suitable solvents, the complexation, for example by means of cyclodextrins or cyclodextrin derivatives, crystallization, purification or isolation by means of chromatographic methods or the conversion of the rhamnolipids into readily separable derivatives may be mentioned at this point.

The rhamnolipids which can be prepared by the process according to the invention are likewise the subject matter of the present invention, in particular also the rhamnolipid mixtures which can be prepared above and which can be prepared by the process according to the invention.

The rhamnolipids and mixtures which can be prepared by the process according to the invention can advantageously be used in cleaning compositions, in cosmetic or pharmaceutical formulations and in crop protection formulations.

Thus, a further object of the present invention is the use of the rhamnolipids obtained by the process according to the invention for the production of cosmetic, dermatological or pharmaceutical formulations, crop protection formulations and of care and cleaning agents and surfactant concentrates.

The term "care products" is understood here to mean a formulation which fulfills the purpose of obtaining an object in its original form, the effects of external influences (eg time, light, temperature, pressure, pollution, chemical reaction with others, with the object in contact passing reactive compounds) such as aging, soiling, material fatigue, fading, reducing or avoiding or even improving desired positive properties of the article. For the last point is about an improved hair shine or greater elasticity of the object considered called.

By "crop protection formulations" are meant those formulations which are obviously used for plant protection by the nature of their preparation; this is particularly the case if the formulation contains at least one compound from the classes of herbicides, fungicides, insecticides, acaricides, nematicides, bird repellents, plant nutrients and soil conditioners. According to the invention, rhamnolipids prepared by the process according to the invention are preferably used in household and industrial care and cleaning preparations, in particular for hard surfaces, leather or textiles.

In the examples given below, the present invention is described by way of example, without the invention, the scope of application of which is apparent from the entire description and the claims, to be limited to the embodiments mentioned in the examples.

The following figures are part of the examples:

: Comparison of dirhamnosyl lipid concentration in mg / L per optical density (600 nm). The measured values were determined after 24 h production phase of recombinant P. putida strains.

Examples:

Construction of a vector pBBR1MCS-2 :: ABCM-rhlG for the heterologous expression of Pseudomonas aeruginosa DSM1707 genes rhlA, rhlB, rhlC, pa1131 and rhlG in Pseudomonas putida

amplifiziert. Die Amplifikation des PCR-Produktes (1076 Basenpaare) wurde mit der Phusion^TM High-Fidelity Master Mix Polymerase von New England Biolabs (Frankfurt) durchgeführt. Das PCR-Produkt wurde mit AgeI und SacI geschnitten und in den ebenfalls mit AgeI und SacI geschnittenen Vektor pBBR1MCS-2::ABCM (Seq ID Nr. 70) mittels Fast Link Ligation Kit (Epicentre Technologies; Madison, WI, USA) ligiert. Der erhaltene Zielvektor pBBR1MCS-2::ABCM-rhlG (Seq ID Nr. 67) hat eine Größe von 10262 Basenpaaren. Das Insert des Vektors wurde sequenziert. Die chromosomale DNA wurde mittels DNeasy Blood and Tissue Kit (Qiagen; Hilden) nach Herstellerangaben isoliert. Die Durchführung der PCR, die Überprüfung der erfolgreichen Amplifikation der PCR mittels Agarosegel-Elektrophorese, Ethidiumbromidfärbung der DNA, Bestimmung der PCR-Fragmentgröße, Reinigung der PCR-Produkte und DNA-Konzentrationsbestimmung erfolgte in dem Fachmann bekannter Art und Weise. Die Transformation von Pseudomonas putida GPp104 und Pseudomonas putida KT2440 mit dem Vektor pBBR1MCS-2::ABCM-rhlG erfolgte wie vorbeschrieben ( Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5): 851–854 ). Die Plasmid-DNA von je 10 Klonen wurde isoliert und analysiert. Die erhaltenen, das Plasmid tragenden Stämme wurden P. putida GPp104 pBBR1MCS-2::ABCM-rhlG bzw. P. putida KT2440 pBBR1MCS-2::ABCM-rhlG genannt. For the heterologous expression of Pseudomonas aeruginosa DSM1707 genes rhlA, rhlB, rhlC, pa1131 and rhlG, the plasmid pBBR1MCS-2 :: ABCM-rhlG (SEQ ID NO: 67) was constructed. For this purpose, the gene rhlG (Seq ID 9) starting from genomic DNA of the strain Pseudomonas aeruginosa PAO1 (DSM 1707) with the oligonucleotides

amplified. Amplification of the PCR product (1076 base pairs) was performed with the Phusion ^™ High-Fidelity Master Mix Polymerase from New England Biolabs (Frankfurt). The PCR product was cut with AgeI and SacI and ligated into the AgeI and SacI-cut vector pBBR1MCS-2 :: ABCM (SEQ ID NO: 70) using the Fast Link Ligation Kit (Epicenter Technologies, Madison, WI, USA). The resulting target pBBR1MCS-2 :: ABCM-rhlG (SEQ ID NO: 67) has a size of 10262 base pairs. The insert of the vector was sequenced. Chromosomal DNA was isolated by DNeasy Blood and Tissue Kit (Qiagen; Hilden) according to the manufacturer's instructions. The performance of the PCR, the verification of the successful amplification of the PCR by means of agarose gel electrophoresis, ethidium bromide staining of the DNA, determination of the PCR fragment size, purification of the PCR products and DNA concentration determination were carried out in a manner known to the person skilled in the art. The transformation of Pseudomonas putida GPp104 and Pseudomonas putida KT2440 with the vector pBBR1MCS-2 :: ABCM-rhlG was carried out as described above ( Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58 (5): 851-854 ). The plasmid DNA of 10 clones each was isolated and analyzed. The resulting plasmid-carrying strains were named P. putida GPp104 pBBR1MCS-2 :: ABCM-rhlG and P. putida KT2440 pBBR1MCS-2 :: ABCM-rhlG, respectively.

Construction of a vector pBBR1MCS-2 :: ABCM-phaG for the heterologous expression of Pseudomonas aeruginosa DSM1707 genes rhlA, rhlB, rhlC, pa1131 and phag in Pseudomonas putida

amplifiziert. Die Amplifikation des PCR-Produktes (975 Basenpaare) wurde mit der Phusion^TM High-Fidelity Master Mix Polymerase von New England Biolabs (Frankfurt) durchgeführt. Das PCR-Produkt wurde mit AgeI und SacI geschnitten und in den ebenfalls mit AgeI und SacI geschnittenen Vektor pBBR1MCS-2::ABCM (Seq ID 70) mittels Fast Link Ligation Kit (Epicentre Technologies; Madison, WI, USA) ligiert. Der erhaltene Zielvektor pBBR1MCS-2::ABCM-phaG (Seq ID Nr. 71) hat eine Größe von 10385 Basenpaaren. Das Insert des Vektors wurde sequenziert. Die chromosomale DNA wurde mittels DNeasy Blood and Tissue Kit (Qiagen; Hilden) nach Herstellerangaben isoliert. Die Durchführung der PCR, die Überprüfung der erfolgreichen Amplifikation der PCR mittels Agarosegel-Elektrophorese, Ethidiumbromidfärbung der DNA, Bestimmung der PCR-Fragmentgröße, Reinigung der PCR-Produkte und DNA-Konzentrationsbestimmung erfolgte in dem Fachmann bekannter Art und Weise. Die Transformation von Pseudomonas putida GPp104 und Pseudomonas putida KT2440 mit dem Vektor pBBR1MCS-2::ABCM-phaG erfolgte wie vorbeschrieben ( Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5): 851–854 ). Die Plasmid-DNA von je 10 Klonen wurde isoliert und analysiert. Die erhaltenen, das Plasmid tragenden Stämme wurden P. putida GPp104 pBBR1MCS-2::ABCM-phaG bzw. P. putida KT2440 pBBR1MCS-2::ABCM-phaG genannt. For the heterologous expression of the Pseudomonas aeruginosa DSM1707 genes rhlA, rhlB, rhlC, pa1131 and phaG, the plasmid pBBR1MCS-2 :: ABCM-phaG (Seq ID No. 71) was constructed. For this purpose, the gene phaG (Seq ID 13) starting from genomic DNA of the strain Pseudomonas aeruginosa PAO1 (DSM 1707) with the oligonucleotides

amplified. Amplification of the PCR product (975 base pairs) was performed with the Phusion ^™ High-Fidelity Master Mix Polymerase from New England Biolabs (Frankfurt). The PCR product was cut with AgeI and SacI and ligated into the AgeI and SacI-cut vector pBBR1MCS-2 :: ABCM (Seq ID 70) using Fast Link Ligation Kit (Epicenter Technologies, Madison, WI, USA). The resulting target pBBR1MCS-2 :: ABCM-phaG (SEQ ID NO: 71) has a size of 10385 base pairs. The insert of the vector was sequenced. Chromosomal DNA was isolated by DNeasy Blood and Tissue Kit (Qiagen; Hilden) according to the manufacturer's instructions. The performance of the PCR, the verification of the successful amplification of the PCR by means of agarose gel electrophoresis, ethidium bromide staining of the DNA, determination of the PCR fragment size, purification of the PCR products and DNA concentration determination were carried out in a manner known to the person skilled in the art. The transformation of Pseudomonas putida GPp104 and Pseudomonas putida KT2440 with the vector pBBR1MCS-2 :: ABCM-phaG was carried out as described above ( Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58 (5): 851-854 ). The plasmid DNA of 10 clones each was isolated and analyzed. The resulting plasmid-carrying strains were named P. putida GPp104 pBBR1MCS-2 :: ABCM-phaG and P. putida KT2440 pBBR1MCS-2 :: ABCM-phaG, respectively.

Construction of a vector pBBR1MCS-2 :: ABCM-udhA for the heterologous expression of Pseudomonas aeruginosa DSM1707 genes rhlA, rhlB, rhlC, pa1131 and the Escherichia coli gene udhA in Pseudomonas putida

amplifiziert. Die Amplifikation des PCR-Produktes (1482 Basenpaare) wurde mit der Phusion^TM High-Fidelity Master Mix Polymerase von New England Biolabs (Frankfurt) durchgeführt. Das PCR-Produkt wurde mit SacI geschnitten und in den ebenfalls mit SacI geschnittenen Vektor pBBR1MCS-2::ABCM (Seq ID 70) mittels Fast Link Ligation Kit (Epicentre Technologies; Madison, WI, USA) ligiert. Der erhaltene Zielvektor pBBR1MCS-2::ABCM-udhA (Seq ID Nr. 74) hat eine Größe von 11356 Basenpaaren. Das Insert des Vektors wurde sequenziert. Die chromosomale DNA wurde mittels DNeasy Blood and Tissue Kit (Qiagen; Hilden) nach Herstellerangaben isoliert. Die Durchführung der PCR, die Überprüfung der erfolgreichen Amplifikation der PCR mittels Agarosegel-Elektrophorese, Ethidiumbromidfärbung der DNA, Bestimmung der PCR-Fragmentgröße, Reinigung der PCR-Produkte und DNA-Konzentrationsbestimmung erfolgte in dem Fachmann bekannter Art und Weise. Die Transformation von Pseudomonas putida GPp104 und Pseudomonas putida KT2440 mit dem Vektor pBBR1MCS-2::ABCM-udhA erfolgte wie vorbeschrieben ( Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5): 851–854 ). Die Plasmid-DNA von je 10 Klonen wurde isoliert und analysiert. Die erhaltenen, das Plasmid tragenden Stämme wurden P. putida GPp104 pBBR1MCS-2::ABCM-udhA bzw. P. putida KT2440 pBBR1MCS-2::ABCM-udhA genannt. For the heterologous expression of the Pseudomonas aeruginosa DSM1707 genes rhlA, rhlB, rhlC, pa1131 and the Escherichia coli gene udhA, the plasmid pBBR1MCS-2 :: ABCM-udhA (SEQ ID NO: 74) was constructed. For this, the gene udhA (Seq ID 19) was prepared starting from genomic DNA of the strain E. coli W3110 with the oligonucleotides

amplified. Amplification of the PCR product (1482 base pairs) was performed with the Phusion ^™ High-Fidelity Master Mix Polymerase from New England Biolabs (Frankfurt). The PCR product was cut with SacI and ligated into the SacI-cut vector pBBR1MCS-2 :: ABCM (SEQ ID 70) using Fast Link Ligation Kit (Epicenter Technologies, Madison, WI, USA). The resulting target pBBR1MCS-2 :: ABCM-udhA (SEQ ID NO: 74) has a size of 11356 base pairs. The insert of the vector was sequenced. Chromosomal DNA was isolated by DNeasy Blood and Tissue Kit (Qiagen; Hilden) according to the manufacturer's instructions. The performance of the PCR, the verification of the successful amplification of the PCR by means of agarose gel electrophoresis, ethidium bromide staining of the DNA, determination of the PCR fragment size, purification of the PCR products and DNA concentration determination were carried out in a manner known to the person skilled in the art. The transformation of Pseudomonas putida GPp104 and Pseudomonas putida KT2440 with the vector pBBR1MCS-2 :: ABCM-udhA was carried out as described above ( Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58 (5): 851-854 ). The plasmid DNA of 10 clones each was isolated and analyzed. The resulting plasmid-carrying strains were named P. putida GPp104 pBBR1MCS-2 :: ABCM-udhA and P. putida KT2440 pBBR1MCS-2 :: ABCM-udhA, respectively.

Construction of a vector pBBR1MCS-2 :: ABCM-pntAB for the heterologous expression of Pseudomonas aeruginosa DSM1707 genes rhlA, rhlB, rhlC, pa1131 and the Escherichia coli genes pntAB in Pseudomonas putida

amplifiziert. Die Amplifikation des PCR-Produktes (2993 Basenpaare) wurde mit der Phusion^TM High-Fidelity Master Mix Polymerase von New England Biolabs (Frankfurt) durchgeführt. Das PCR-Produkt wurde mit SacI geschnitten und in den ebenfalls mit SacI geschnittenen Vektor pBBR1MCS-2::ABCM (Seq ID 70) mittels Fast Link Ligation Kit (Epicentre Technologies; Madison, WI, USA) ligiert. Der erhaltene Zielvektor pBBR1MCS-2::ABCM-pntAB (Seq ID Nr. 77) hat eine Größe von 12867 Basenpaaren. Das Insert des Vektors wurde sequenziert. Die chromosomale DNA wurde mittels DNeasy Blood and Tissue Kit (Qiagen; Hilden) nach Herstellerangaben isoliert. Die Durchführung der PCR, die Überprüfung der erfolgreichen Amplifikation der PCR mittels Agarosegel-Elektrophorese, Ethidiumbromidfärbung der DNA, Bestimmung der PCR-Fragmentgröße, Reinigung der PCR-Produkte und DNA-Konzentrationsbestimmung erfolgte in dem Fachmann bekannter Art und Weise. Die Transformation von Pseudomonas putida GPp104 und Pseudomonas putida KT2440 mit dem Vektor pBBR1MCS-2::ABCM-pntAB erfolgte wie vorbeschrieben ( Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5): 851–854 ). Die Plasmid-DNA von je 10 Klonen wurde isoliert und analysiert. Die erhaltenen, das Plasmid tragenden Stämme wurden P. putida GPp104 pBBR1MCS-2::ABCM-pntAB und P. putida KT2440 pBBR1MCS-2::ABCM-pntAB genannt. For the heterologous expression of the Pseudomonas aeruginosa DSM1707 genes rhlA, rhlB, rhlC, pa1131 and the Escherichia coli gene pntAB, the plasmid pBBR1MCS-2 :: ABCM-pntAB (SEQ ID NO: 77) was constructed. For this purpose, the genes pntA (Seq ID 15) and pntB (Seq ID 17) starting from genomic DNA of the strain E. coli W3110 with the oligonucleotides

amplified. Amplification of the PCR product (2993 base pairs) was performed with the Phusion ^™ High-Fidelity Master Mix Polymerase from New England Biolabs (Frankfurt). The PCR product was cut with SacI and ligated into the SacI-cut vector pBBR1MCS-2 :: ABCM (SEQ ID 70) using Fast Link Ligation Kit (Epicenter Technologies, Madison, WI, USA). The resulting target pBBR1MCS-2 :: ABCM-pntAB (SEQ ID NO: 77) has a size of 12867 base pairs. The insert of the vector was sequenced. Chromosomal DNA was isolated by DNeasy Blood and Tissue Kit (Qiagen; Hilden) according to the manufacturer's instructions. The performance of the PCR, the verification of the successful amplification of the PCR by means of agarose gel electrophoresis, ethidium bromide staining of the DNA, determination of the PCR fragment size, purification of the PCR products and DNA concentration determination were carried out in a manner known to the person skilled in the art. The transformation of Pseudomonas putida GPp104 and Pseudomonas putida KT2440 with the vector pBBR1MCS-2 :: ABCM-pntAB was carried out as described above ( Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58 (5): 851-854 ). The plasmid DNA of 10 clones each was isolated and analyzed. The resulting plasmid-carrying strains were named P. putida GPp104 pBBR1MCS-2 :: ABCM-pntAB and P. putida KT2440 pBBR1MCS-2 :: ABCM-pntAB.

Construction of a vector pBBR1MCS-2 :: ABCM-gapC for the heterologous expression of Pseudomonas aeruginosa DSM1707 genes rhlA, rhlB, rhlC, pa1131 and the Clostridium acetobutylicum gene gapC in Pseudomonas putida

amplifiziert. Die Amplifikation des PCR-Produktes (1119 Basenpaare) wurde mit der Phusion^TM High-Fidelity Master Mix Polymerase von New England Biolabs (Frankfurt) durchgeführt. Das PCR-Produkt wurde mit SacI und AgeI geschnitten und in den ebenfalls mit SacI und AgeI geschnittenen Vektor pBBR1MCS-2::ABCM (Seq ID 70) mittels Fast Link Ligation Kit (Epicentre Technologies; Madison, WI, USA) ligiert. Der erhaltene Zielvektor pBBR1MCS-2::ABCM-gapC (Seq ID Nr. 80) hat eine Größe von 10528 Basenpaaren. Das Insert des Vektors wurde sequenziert. Die chromosomale DNA wurde mittels DNeasy Blood and Tissue Kit (Qiagen; Hilden) nach Herstellerangaben isoliert. Die Durchführung der PCR, die Überprüfung der erfolgreichen Amplifikation der PCR mittels Agarosegel-Elektrophorese, Ethidiumbromidfärbung der DNA, Bestimmung der PCR-Fragmentgröße, Reinigung der PCR-Produkte und DNA-Konzentrationsbestimmung erfolgte in dem Fachmann bekannter Art und Weise. Die Transformation von Pseudomonas putida GPp104 und Pseudomonas putida KT2440 mit dem Vektor pBBR1MCS-2::ABCM-gapC erfolgte wie vorbeschrieben ( Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58(5): 851–854 ). Die Plasmid-DNA von je 10 Klonen wurde isoliert und analysiert. Die erhaltenen, das Plasmid tragenden Stämme wurden P. putida GPp104 pBBR1MCS-2::ABCM-gapC bzw. P. putida KT2440 pBBR1MCS-2::ABCM-gapC genannt. For the heterologous expression of the Pseudomonas aeruginosa DSM1707 genes rhlA, rhlB, rhlC, pa1131 and the Clostridium acetobutylicum gene gapC, the plasmid pBBR1MCS-2 :: ABCM-gapC (SEQ ID NO: 80) was constructed. For this purpose, the gene gapC (Seq ID 21), starting from genomic DNA of the strain Clostridium acetobutylicum with the oligonucleotides

amplified. Amplification of the PCR product (1119 base pairs) was performed with the Phusion ^™ High-Fidelity Master Mix Polymerase from New England Biolabs (Frankfurt). The PCR product was cut with SacI and AgeI and ligated into the SacI and AgeI vector pBBR1MCS-2 :: ABCM (Seq ID 70) using the Fast Link Ligation Kit (Epicenter Technologies, Madison, WI, USA). The resulting target pBBR1MCS-2 :: ABCM-gapC (SEQ ID NO: 80) has a size of 10528 base pairs. The insert of the vector was sequenced. Chromosomal DNA was isolated by DNeasy Blood and Tissue Kit (Qiagen; Hilden) according to the manufacturer's instructions. The performance of the PCR, the verification of the successful amplification of the PCR by means of agarose gel electrophoresis, ethidium bromide staining of the DNA, determination of the PCR fragment size, purification of the PCR products and DNA concentration determination were carried out in a manner known to the person skilled in the art. The transformation of Pseudomonas putida GPp104 and Pseudomonas putida KT2440 with the vector pBBR1MCS-2 :: ABCM-gapC was carried out as described above ( Iwasaki et al. Biosci. Biotech. Biochem. 1994. 58 (5): 851-854 ). The plasmid DNA of 10 clones each was isolated and analyzed. The resulting strains carrying the plasmid were named P. putida GPp104 pBBR1MCS-2 :: ABCM-gapC and P. putida KT2440 pBBR1MCS-2 :: ABCM-gapC, respectively.

Quantification of rhamnolipid production by recombinant, P. rhlA, rhlB and rhlC expressing P. putida strains with and without overexpression of the pa1131 gene and phaG gene and the pa1131 gene and rhlG gene and the pa1131 gene and udhA, respectively Gene or pa1131 gene and pntAB gene or pa1131 gene and gapC gene

The recombinant strains P.putida KT2440 pBBR1MCS-2, P.putida KT2440 pBBR1MCS-2 :: ABC, P.putida KT2440 pBBR1MCS-2 :: ABCM-rhlG, P.putida KT2440 pBBR1MCS-2 :: ABCM-phaG, P. putida KT2440 pBBR1MCS-2 :: ABCM-gapC, P.putida KT2440 pBBR1MCS-2 :: ABCM-pntAB, P.putida KT2440 pBBR1MCS-2 :: ABCM-udhA, P.putida GPp104 pBBR1MCS-2, P.putida GPp104 pBBR1MCS -2 :: ABC, P.putida GPp104 pBBR1MCS-2 :: ABCM-rhlG, P.putida GPp104 pBBR1MCS-2 :: ABCM-phaG, P.putida GPp104 pBBR1MCS-2 :: ABCM-gapC, P.putida GPp104 pBBR1MCS -2 :: ABCM-pntAB and P. putida GPp104 pBBR1MCS-2 :: ABCM-udhA were cultured on LB agar kanamycin (50 μg / ml) plates.

For the production of rhamnolipids, the medium referred to below as M9 medium was used. This medium consists of 2% (w / v) glucose, 0.3% (w / v) KH ₂ PO ₄ , 0.679% Na ₂ HPO ₄ , 0.05% (w / v) NaCl, 0.2% ( w / v) NH ₄ Cl, 0.049% (w / v) MgSO ₄ .7H ₂ O and 0.1% (v / v) of a trace element solution. These consists of 1.78% (w / v) FeSO ₄ .7H ₂ O, 0.191% (w / v) MnCl ₂ .7H ₂ O, 3.65% (w / v) HCl, 0.187% (w / v). v) ZnSO ₄ .7H ₂ O, 0.084% (v / v) Na-EDTA x 2H ₂ O, 0.03% (v / v) H ₃ BO ₃ , 0.025% (w / v) Na ₂ MoO ₄ × 2 H ₂ O and 0.47% (w / v) CaCl ₂ × 2 H ₂ O. The pH of the medium was adjusted to 7.4 with NH ₄ OH and following the medium by means of autoclave (121 ° C , 20 min) sterilized. It was not necessary to adjust the pH during the cultivation.

To investigate the production of rhamnolipid in a shake flask, a preculture was first prepared. For this purpose, a colony of a strain freshly streaked out on an LB agar plate was used and 10 ml of LB medium were inoculated in a 100 ml Erlenmeyer flask. All recombinant P. putida strains were cultured in LB medium to which 50 μg / ml kanamycin was added. Cultivation of the P. putida strains was carried out at 30 ° C. and 200 rpm overnight.

The precultures were used to inoculate 50 ml of M9 medium (+50 μg / ml kanamycin) in the 250 ml Erlenmeyer flask (start OD ₆₀₀ 0.1). The cultures were cultured at 200 rpm and 30 ° C. After 24 hours, a sample of 1 ml of culture broth was removed from the culture flask. The sample preparation for the subsequent chromatographic analyzes was carried out as follows:
With a displacement pipette (Combitip) 1 ml of acetone was placed in a 2 ml reaction vessel and the reaction vessel to minimize evaporation immediately closed. This was followed by the addition of 1 ml of culture broth. After vortexing the culture broth / acetone mixture, this was spun down for 3 min at 13,000 rpm and 800 μl of the supernatant were transferred to an HPLC vessel.

An Evaporative Light Scattering Detector (Sedex LT-ELSD Model 85LT) was used to detect and quantify rhamnolipids. The actual measurement was performed using Agilent Technologies 1200 Series (Santa Clara, California) and the Zorbax SB-C8 Rapid Resolution column (4.6 x 150 mm, 3.5 μm, Agilent). The injection volume was 5 μl and the running time of the method was 20 min. As the mobile phase, aqueous 0.1% TFA (trifluoroacetic acid, solution A) and methanol (solution B) were used. The column temperature was 40 ° C. The detectors used were the ELSD (detector temperature 60 ° C.) and the DAD (diode array, 210 nm). The gradient used in the method was: t [min] Solution B vol .-% Flow [ml / min] 0.00 70% 1.00 15.00 100% 1.00 15,01 70% 1.00 20.00 70% 1.00

The results are in shown. While P. putida KT2440 pBBR1MCS-2 and P. putida GPp104 pBBR1MCS-2 did not produce rhamnolipids, in the recombinant strains P. putida KT2440 pBBR1MCS-2 :: ABC, P. putida KT2440 pBBR1MCS-2 :: ABCM-rhlG, P putida KT2440 pBBR1MCS-2 :: ABCM-phaG, P.putida KT2440 pBBR1MCS-2 :: ABCM-gapC, P.putida KT2440 pBBR1MCS-2 :: ABCM-pntAB, P.putida KT2440 pBBR1MCS-2 :: ABCM-udhA , P.putida GPp104 pBBR1MCS-2 :: ABC, P.putida GPp104 pBBR1MCS-2 :: ABCM-rhlG, P.putida GPp104 pBBR1MCS-2 :: ABCM-phaG, P.putida GPp104 pBBR1MCS-2 :: ABCM-gapC , P. putida GPp104 pBBR1MCS- 2 :: ABCM-pntAB and P. putida GPp104 pBBR1MCS-2 :: ABCM-udhA detectable formation of rhamnolipids. The results show that for the expression of the P. aeruginosa genes rhlA, rhlB and rhlC, additional expression of the P. aeruginosa genes pa1131 and rhlG, as well as pa1131 and phaG in both parent backgrounds of P. putida (KT2440: wild-type or GPp104 with inactivated Polyhydroxybutyrate formation) led to increased formation of rhamnolipids. It can also be stated that the likewise additional expression (to the P. aeruginosa genes rhlA, rhlB and rhlC) of the P. aeruginosa pa1131 gene together with the udhA gene from E. coli and the pa1131 gene from P. aeruginosa together with the pntAB genes from E. coli and the pa1131 gene from P. aeruginosa together with the gapC gene from Clostridium acetobutylicum in both parent backgrounds of P. putida (KT2440: wild type or GPp104 with inactivated polyhydroxybutyrate formation) increased rhamnolipid formation.

The results further show that the attenuation of polyhydroxybutyrate formation in GPp104 (P. putida KT2440 pBBR1MCS-2 :: ABCM-rhlG, P.putida KT2440 pBBR1MCS-2 :: ABCM-phaG, P.putida KT2440 pBBR1MCS-2 :: ABCM-gapC, P. putida KT2440 pBBR1MCS-2 :: ABCM-pntAB and P. putida KT2440 pBBR1MCS-2 :: ABCM-udhA to P. putida GPp104 pBBR1MCS-2 :: ABCM-rhlG, P. putida GPp104 pBBR1MCS-2 :: ABCM-phaG, P.putida GPp104 pBBR1MCS-2 :: ABCM-gapC, P.putida GPp104 pBBR1MCS-2 :: ABCM-pntAB and P.putida GPp104 pBBR1MCS-2 :: ABCM-udhA) for increased formation of Rhamnolipids resulted.
Generation of recombinant E. coli W3110 pBBR1MCS-2, E. coli W3110 pBBR1MCS-2 :: ABC, E. coli W3110 pBBR1MCS-2 :: ABCM-rhlG, E. coli W3110 pBBR1MCS-2 :: ABCM-phaG, E. coli W3110 pBBR1MCS-2 :: ABCM-udhA, E. coli W3110 pBBR1MCS-2 :: ABCM-pntAB and E. coli W3110 pBBR1MCS-2 :: ABCM-gapC

Transformation of E. coli W3110 is performed as previously described (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 electroporation. The plasmid DNA of 10 clones each is isolated and analyzed. The resulting plasmid-carrying strains are E. coli W3110 pBBR1MCS-2, E. coli W3110 pBBR1MCS-2 :: ABC, E. coli W3110 pBBR1MCS-2 :: ABCM-rhlG, E. coli W3110 pBBR1MCS-2 :: ABCM -phaG, E. coli W3110 pBBR1MCS-2 :: ABCM-udhA, E. coli W3110 pBBR1MCS-2 :: ABCM-pntAB and E. coli W3110 pBBR1MCS-2 :: ABCM-gapC.

Quantification of rhamnolipid production by recombinant E. coli W3110 strains

The recombinant strains E. coli W3110 pBBR1MCS-2, E. coli W3110 pBBR1MCS-2 :: ABC, E. coli W3110 pBBR1MCS-2 :: ABCM-rhlG, E. coli W3110 pBBR1MCS-2 :: ABCM-phaG, E. coli W3110 pBBR1MCS-2 :: ABCM-udhA, E. coli W3110 pBBR1MCS-2 :: ABCM-pntAB and E. coli W3110 pBBR1MCS-2 :: ABCM-gapC are grown on LB agar kanamycin (50 μg / ml) - Cultivated plates.

For the production of rhamnolipids, the medium referred to below as M9 medium is used. This medium consists of 2% (w / v) glucose, 0.3% (w / v) KH ₂ PO ₄ , 0.679% Na ₂ HPO ₄ , 0.05% (w / v) NaCl, 0.2% ( w / v) NH ₄ Cl, 0.049% (w / v) MgSO ₄ .7H ₂ O and 0.1% (v / v) of a trace element solution. This consists of 1.78% (w / v) FeSO ₄ .7H ₂ O, 0.191% (w / v) MnCl ₂ .7H ₂ O, 3.65% (w / v) HCl, 0.187% (w / v) ZnSO ₄ .7H ₂ O, 0.084% (v / v) Na-EDTA x 2H ₂ O, 0.03% (v / v) H ₃ BO ₃ , 0.025% (w / v) Na ₂ MoO ₄ × 2 H ₂ O and 0.47% (w / v) CaCl ₂ × 2 H ₂ O. The pH of the medium is adjusted to 7.4 with NH ₄ OH and following the medium by means of autoclave (121 ° C, 20 min).

To study the production of rhamnolipids in a shake flask, a preculture is first prepared. For this purpose, a colony of a strain freshly streaked out on an LB agar plate is used and 10 ml of LB medium are inoculated in a 100 ml Erlenmeyer flask. All recombinant E. coli W3110 strains are cultured in LB medium to which 50 μg / ml kanamycin is added. The cultivation of the E. coli W3110 strains takes place at 37 ° C. and 200 rpm overnight.

The precultures are used to inoculate 50 ml of M9 medium (+50 μg / ml kanamycin) in the 250 ml Erlenmeyer flask (start OD ₆₀₀ 0.1). The cultures are cultured at 200 rpm and 37 ° C to an optical density (600 nm) of 0.4-0.6. At this optical density, cultures are induced by the addition of IPTG (isopropyl-β-D-thiogalactopyranoside, 1 mM final concentration). The subsequent expression is also carried out at 37 ° C and 200 rpm for max. 72 h. At intervals of 24 h, a sample of 1 ml of culture broth is removed from the culture flask. The sample preparation for the subsequent chromatographic analyzes is carried out as follows:
With a displacement pipette (Combitip) 1 ml of acetone are placed in a 2 ml reaction vessel and the reaction vessel to minimize evaporation immediately closed. This is followed by the addition of 1 ml of culture broth. After vortexing the culture broth / acetone mixture, this is centrifuged off for 3 min at 13,000 rpm and 800 μl of the supernatant are transferred to an HPLC vessel.

An Evaporative Light Scattering Detector (Sedex LT-ELSD Model 85LT) is used to detect and quantify rhamnolipids. The actual measurement is performed using Agilent Technologies 1200 Series (Santa Clara, California) and the Zorbax SB-C8 Rapid Resolution column (4.6 × 150 mm, 3.5 μm, Agilent). The injection volume is 5 μl and the run time of the method is 20 min. The mobile phase used is aqueous 0.1% TFA (trifluoroacetic acid, solution A) and methanol (solution B). The column temperature is 40 ° C. The detectors used are the ELSD (detector temperature 60 ° C) and the DAD (diode array, 210 nm). The gradient used in the method is: t [min] Solution B vol .-% Flow [ml / min] 0.00 70% 1.00 15.00 100% 1.00 15,01 70% 1.00 20.00 70% 1.00

While E. coli W3110 pBBR1MCS-2 does not produce rhamnolipids, in the recombinant strains E. coli W3110 pBBR1MCS-2 :: ABC, E. coli W3110 pBBR1MCS-2 :: ABCM-rhlG, E. coli W3110 pBBR1MCS-2 :: ABCM-phaG, E. coli W3110 pBBR1MCS-2 :: ABCM-gapC, E. coli W3110 pBBR1MCS-2 :: ABCM-pntAB and E. coli W3110 pBBR1MCS-2 :: ABCM-udhA detectable formation of rhamnolipids. It can be seen that the expression of the P. aeruginosa genes rhlA, rhlB and rhlC, additional expression of the P. aeruginosa genes pa1131 and rhlG, as well as pa1131 and phaG in E. coli W3110 leads to increased formation of rhamnolipids. It is further noted that the additional expression (to the P. aeruginosa genes rhlA, rhlB and rhlC) of the P. aeruginosa pa1131 gene together with the udhA gene from E. coli and the pa1131 gene from P. aeruginosa are also related with the pntAB genes from E. coli, as well as the pa1131 gene from P. aeruginosa together with the gapC gene from Clostridium acetobutylicum in E. coli W3110 leads to an increased rhamnolipid formation.

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Claims (15)

Cell which is able to form at least one rhamnolipid of the general formula (I),

general formula (I) where m = 2, 1 or 0, in particular 1 or 0, n = 1 or 0, in particular 1, R ¹ and R ² = independently identical or different organic radical with 2 to 24, in particular optionally branched, optionally substituted, in particular hydroxy-substituted, optionally unsaturated, in particular optionally mono-, di- or triunsaturated, alkyl radical, characterized in that it has been genetically engineered so that compared to its wild type an increased activity of at least one enzyme E ₁ , which is the export of a rhamnolipid of the general formula (I) from the cell catalyzed in the surrounding medium, and an activity increased compared to its wild type at least one of the enzymes selected from E ₂ , E ₃ and E ₄ , wherein E _{2 is} the reaction of 3 Hydroxyacyl [acyl carrier protein] to 3-ketoacyl [acyl carrier protein] or 3-hydroxyacyl coenzyme A to 3-ketoacyl coenzyme A can catalyze E ₃ to catalyze the conversion of acyl [acyl carrier protein] to acyl-coenzyme A and E _{4 is} able to catalyze the conversion of NADP + to NADPH. A cell according to claim 1, characterized in that E _{1 is} selected from the group consisting of enzymes E ₁ having polypeptide sequence Seq ID No. 2, Seq ID No. 4, Seq ID No. 6, Seq ID No. 8, ZP_05590661.1 , YP_439278.1, YP_440069.1, ZP_04969301.1, ZP_04520234.1, YP_335528.1, YP_001075859.1, YP_001061817.1, ZP_02487499.1, YP_337251.1, ZP_04897712.1, ZP_04810190.1, YP_990322.1, ZP_02476924 .1, ZP_04899735.1, ZP_04893873.1, ZP_02365982.1, YP_001062909.1, YP_105611.1, ZP_03794061.1, ZP_03457011.1, ZP_02385401.1, ZP_02370552.1, YP_105236.1, ZP_04905097.1, YP_776387.1 , YP_001811690.1, YP_004348730.1, YP_004348708.1, YP_371320.1, YP_623145.1, YP_001778810.1, YP_002234933.1, CCE52909.1, YP_002908248.1, ZP_04954557.1, ZP_04956038.1, ZP_02408950.1, ZP_02375897 .1, ZP_02389908.1, YP_439274.1, YP_001074762.1, YP_337247.1, YP_110559.1, ZP_02495927.1, YP_111360.1, YP_105608.1, ZP_02487826.1, ZP_02358947.1, YP_001078605.1, ZP_00438000.1 , ZP_00440993.1, ZP_02477260.1, YP_371317.1, YP_001778807.1, ZP_0 ZP_02891478.1, YP_776390.1, ZP_04943308.1, YP_001811693.1, ZP_02503985.1, YP_004362740.1, YP_002908245.1, YP_004348705.1, ZP_02408798.1, ZP_02417250.1, EGD05166.1, ZP_02458677.1, ZP_02465793.1, YP_001578240.1, ZP_04944344.1, YP_ ZP_02874806.1, ZP_02906105.1, YP_001806764.1, YP_619912.1, YP_001117913.1, YP_106647.1, YP_001763368.1, ZP_02479535.1, ZP_02461743.1, YP_560998.1, YP_331651.1, ZP_04893070.1, YP_003606714.1, ZP_02503995.1, ZP_06840428.1, YP_104288.1, ZP_02487849.1, ZP_02353848.1, YP_367475.1, ZP_02377399.1, ZP_02372143.1, YP_001897562.1, ZP_02361066. 1, YP_440582.1, ZP_03268453.1, AET90544.1, YP_003908738.1, YP_004230049.1, ZP_02885418.1 or ZP_02511831.1 or with a polypeptide sequence in which up to 25% of the amino acid residues are opposite to the respective Seq ID No. 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or the aforesaid accession number are altered by deletion, insertion, substitution or a combination thereof and which still have at least 10% of the enzymatic activity of the enzyme with the respective reference sequence Seq ID No. 2, Seq ID No. 4, Seq ID No. 6, Seq ID No. 8 or the aforementioned Accession Number, wherein under enzymatic activity for e in enzyme E ₁ the ability is understood to export a rhamnolipid of general formula (I) from the cell into the surrounding medium. Cell according to claim 1 or 2, characterized in that E _{2 is} selected from the group consisting of 3-ketoacyl-ACP reductase of EC 1.1.1.100 or 3-ketoacyl-coenzyme A reductase of EC 1.1.1.35 or EC 1.1. 1.36, in particular from enzymes E _2a having polypeptide sequence Seq ID No. 10 or with a polypeptide sequence in which up to 25% of the amino acid residues have been changed by Seq ID No. 10 by deletion, insertion, substitution or a combination thereof and which is still at least 10 % of the enzymatic activity of the enzyme having the reference sequence Seq ID No. 10, wherein enzymatic activity for an enzyme E _{2a is} the ability to convert 3-hydroxydecanoyl-coenzyme A to 3-ketodecanoyl coenzyme A and enzymes E _2b having polypeptide sequence EGM23089 .1, YP_346919.1, YP_258374.1, YP_001187200.1 or YP_004380924.1 or with a polypeptide sequence in which up to 25% of the amino acid residues are opposite to the aforementioned accession number by deletion, insertion, sub institution or a combination thereof and which still has at least 10% of the enzymatic activity of the enzyme with the reference sequence of the aforementioned accession number, whereby enzymatic activity for an enzyme E _{2b is} understood as the ability to convert 3-hydroxytetradecanoyl-coenzyme A into 3-ketotetradecanoyl Coenzyme A to implement. Cell according to at least one of the preceding claims, characterized in that E _{3 is} selected from the group consisting of acyl-ACP: coenzyme A transacylases, in particular from enzymes E _3a with polypeptide sequence SEQ ID NO: 12, SEQ ID NO: 14, NP_249421 .1, AAT51199.1, ZP_01364106.1, AEO76792.1, YP_792557.1, ZP_04932415.1, YP_001350135.1, ZP_08139631.1, YP_004703658.1, YP_004379169.1, YP_609790.1, ACA03779.1, YP_001670627.1 , NP_743567.1, YP_001269622.1, YP_001186851.1, Q9KJH8.1, AAK71349.1, ADR61731.1, AAU44816.1, YP_001747923.1, ACH70299.1, ZP_07774051.1, YP_002871082.1, AAK81868.1, AAQ16175 .1, YP_004352505.1, YP_258557.1, ACA60824.1, AEV61413.1, YP_004475334.1, AAA25978.1, YP_347066.1, EGH58099.1, BAB32432.1, EGH64812.1, ZP_05640568.1, EFW81598.1 , EGH11618.1, YP_276116.1, ZP_07264431.1, EGH73565.1, EGH95622.1, YP_237050.1, EGH29888.1, ZP_06498781.1, ZP_06456665.1, EGH79586.1, NP_794008.1, ZP_03399268.1, ZP_05639386 .1, EGH50352.1, EGH22129.1, ZP_07005523.1, ZP_06458504.1, EFW79 804.1, YP_275219.1, EGH85976.1, EGH22392.1, EGH66549.1, ZP_03398232.1, ZP_07229875.1, EGH10011.1, EGH43364.1, YP_236199.1, ZP_07261632.1, ZP_04589662.1, EGH54896.1, EGH29417.1, ZP_06499968.1, EGH97259.1, NP_793082.1 or EGH90852.1 or with a polypeptide sequence in which up to 25% of the amino acid residues are opposite to the respective SEQ ID NO: 12, SEQ ID NO: 14 or the aforementioned Accession Number by Deletion, insertion, substitution or a combination thereof and which still has at least 10% of the enzymatic activity of the enzyme with the respective reference sequence SEQ ID NO: 12, SEQ ID NO: 14 or the aforementioned accession number, wherein under enzymatic activity for an enzyme E _{3 is} the ability to convert 3-hydroxydecanoyl [acyl carrier protein] and coenzyme A into 3-hydroxydecanoyl-coenzyme A and acyl carrier protein, and enzymes E _3b with polypeptide sequence AAK71350.1 or with a polypeptide sequence at the up to 25% of the amino acid residues over the AAK71350.1 are modified by deletion, insertion, substitution or a combination thereof and which still has at least 10% of the enzymatic activity of the enzyme with the reference sequence AAK71350.1, whereby enzymatic activity for an enzyme E _{3b is} understood to mean the ability to Hydroxytetradecanoyl [acyl carrier protein] and coenzyme A to 3-hydroxytetradecanoyl coenzyme A and acyl carrier protein implement. Cell according to at least one of the preceding claims, characterized in that E _{4 is} selected from the group consisting of enzymes E _4a transhydrogenases of EC 1.6.1.1 and EC 1.6.1.2, in particular from enzymes E _4a with polypeptide sequence Seq ID No. 16, Seq ID No. 18 or Seq ID No. 20 or with a polypeptide sequence in which up to 25% of the amino acid residues are opposite to the respective Seq ID No. 16, Seq ID No. 18 or SEQ ID NO: 20 are altered by deletion, insertion, substitution, or a combination thereof, and the remaining at least 10% of the enzymatic activity of the enzyme having the respective reference sequence Seq ID NO. 16, SEQ ID NO. 18 or SEQ ID No. 20 where enzymatic activity for an enzyme E _{4a is} the ability to convert NADP + to NADPH, enzymes E _4b NADP + -dependent glyceraldehyde-3-phosphate dehydrogenases of EC 1.2.1.9, EC 1.2.1.13 and EC 1.2.1.59, in particular from enzymes E _4b with polypeptide sequence Seq ID No. 22 or with a polypeptide sequence in which up to 25% of the amino acid residues have been changed by Seq ID No. 22 by deletion, insertion, substitution or a combination thereof and which is still at least 10 % of the enzymatic activity of the enzyme having the reference sequence Seq ID No. 22, wherein enzymatic activity for an enzyme E _{4a is} the ability to convert D-glyceraldehyde-3-phosphate to 1,3-bisphospho-D-glycerate, and Enzyme E _4c NADP + -dependent pyruvate dehydrogenases of EC 1.2.1.51, in particular from enzymes E _4c with polypeptide sequence Seq ID No. 24 or SEQ ID No. 26 or with a polypeptide sequence in which up to 25% of the amino acid residues opposite to the respective Seq ID No. 24 or SEQ ID NO: 26 by deletion, insertion, substitution, or a combination thereof, and which still has at least 10% of the enzymatic activity of the enzyme having the respective reference sequence Seq ID No. 24 or SEQ ID No. 26, wherein enzymatic activity for an enzyme E _{4c is} understood to be the ability to convert pyruvate and coenzyme A (CoA) to acetyl-coenzyme A and CO ₂ . Cell according to at least one of the preceding claims, characterized in that it has an increased activity of at least one of the enzymes E ₅ , E ₆ and E ₇ compared to their wild type, wherein the enzyme E _{5 is} the reaction of 3-hydroxyalkanoyl-ACP over 3 Hydroxyalkanoyl-3-hydroxyalkanoic acid ACP to catalyze hydroxyalkanoyl-3-hydroxyalkanoic acid, the enzyme E _{6 is} a rhamnosyltransferase I and the conversion of dTDP-rhamnose and 3-hydroxyalkanoyl-3-hydroxyalkanoate to α-L-rhamnopyranosyl-3-hydroxyalkanoyl Catalyzes 3-hydroxyalkanoate and the enzyme E _{7 is} a rhamnosyltransferase II and the conversion of dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate to α-L-rhamnopyranosyl- (1-2) - α-L-rhamnopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate is able to catalyze. Cell according to any one of the preceding claims, characterized in that E _{5 is} selected from the group consisting of at least one enzyme E _5a with polypeptide sequence Seq ID No. 28 or with a polypeptide sequence in which up to 25% of the amino acid residues are opposite to the reference sequence Seq ID No. 28 are modified by deletion, insertion, substitution or a combination thereof and which still has at least 10% of the enzymatic activity of the enzyme with the reference sequence Seq ID No. 28, whereby enzymatic activity for an enzyme E _{5a is} understood as the ability Reacting 3-hydroxydecanoyl-ACP via 3-hydroxydecanoyl-3-hydroxydecanoic acid ACP to hydroxydecanoyl-3-hydroxydecanoic acid, and at least one enzyme E _5b having polypeptide sequence SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, Seq ID No. 36, YP_439272.1, YP_111362.1, YP_110557.1, YP_105231.1, ZP_02461688.1, ZP_02358949.1, ZP_01769192.1, ZP_04893165.1, ZP_02265387.2, ZP_02511781.1, ZP_03456835.1, ZP_03794 633.1, YP_990329.1, ZP_02408727.1, YP_002908243.1, ZP_04884056.1, YP_004348703.1, ZP_04905334.1, ZP_02376540.1, EGC99875.1, ZP_02907621.1, YP_001811696.1, ZP_02466678.1, ZP_02891475.1, YP_776393.1, YP_002234939.1, YP_001778804.1, YP_371314.1, ZP_04943305.1, YP_623139.1, ZP_02417235.1 or ZP_04892059.1 or with a polypeptide sequence in which up to 25% of the amino acid residues are opposite to the respective reference sequence Seq. ID No. 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36 or the aforementioned accession number are altered by deletion, insertion, substitution or a combination thereof and which still have at least 10% of the enzymatic activity of the enzyme with the respective SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36 or the aforementioned accession number, wherein enzymatic activity for an enzyme E _{5b is} understood as the ability to convert 3-hydroxytetradecanoyl-ACP 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid ACP to hydroxytetradecanoyl-3 Implement hydroxytetradecanoic acid. Cell according to at least one of the preceding claims, characterized in that E _{6 is} selected from the group consisting of at least one enzyme E _6a with polypeptide sequence Seq ID No. 38, YP_001347032.1, CBI71029.1, YP_002439138.1, CBI71031.1, NP_252168.1, CBI71034.1, CBI71028.1, AAA62129.1 or ZP_04929750.1 or with a polypeptide sequence in which up to 25% of the amino acid residues relative to the respective Reference Sequence Seq ID No. 38 or the aforementioned accession number are modified by deletion, insertion, substitution or a combination thereof and which still has at least 10% of the enzymatic activity of the enzyme with the respective reference sequence Seq ID No. 38 or Accession number enzymatic activity for an enzyme E _{6a is} the ability to convert dTDP-rhamnose and 3-hydroxydecanoyl-3-hydroxydecanoic acid to α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid, and at least one enzyme E _6b with polypeptide sequence Seq 40, Seq ID No. 42, Seq ID No. 44, Seq ID No. 46, YP_440074.1, ZP_05590657.1, ZP_04520374.1, ZP_00438360.2, ZP_00438209.2, YP_001074761.1, ZP_04811084.1, YP_110558 .1, YP_111361.1, ZP_02492857.1, YP_337246.1, YP_001061811.1, YP_105607.1, ZP_02371503.1, ZP_02503962.1, ZP_03456839.1, ZP_02461690.1, ZP_03794634.1, ZP_01769736.1, ZP_01769308.1 , ZP_02358948.1, ZP_02487736.1, ZP_02408758.1, YP_002234937.1, ZP_02891 477.1, YP_001778806.1, YP_623141.1, YP_838721.1, ZP_04943307.1, YP_776391.1, YP_004348704.1, ZP_02907619.1, YP_371316.1, ZP_02389948.1, YP_001811694.1, YP_002908244.1, ZP_02511808.1, ZP_02376542.1, EGC99877.1, ZP_02451760.1 or ZP_02414414.1 or with a polypeptide sequence in which up to 25% of the amino acid residues are opposite to the respective reference sequence SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, Seq ID No. 46 or the aforementioned accession number are altered by deletion, insertion, substitution or a combination thereof and which still contain at least 10% of the enzymatic activity of the enzyme with the respective reference sequence Seq ID No. 40, SEQ ID No. 42, SEQ ID NO 44, SEQ ID NO: 46 or the aforementioned accession number, enzymatic activity for an enzyme E _{6b being} understood as the ability to convert dTDP-rhamnose and 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid into α-L-rhamnopyranosyl-3-hydroxytetradecanoyl- Implement 3-hydroxytetradecanoic acid. Cell according to at least one of the preceding claims, characterized in that E _{7 is} selected from the group consisting of at least one enzyme E _7a with polypeptide sequence Seq ID No. 48 or with a polypeptide sequence in which up to 25% of the amino acid residues compared to the reference sequence Seq ID No. 48 are altered by deletion, insertion, substitution or a combination thereof and which still has at least 10% of the enzymatic activity of the enzyme with the reference sequence Seq ID No. 48, whereby enzymatic activity for an enzyme E _{7a is} understood as the ability reacting dTDP-rhamnose and α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid to form α-L-rhamnopyranosyl- (1-2) -α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid, and at least one enzyme E _7b with polypeptide sequence SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 YP_440071.1, ZP_02375899.1, ZP_02466676.1, YP_001075863.1, ZP_02408796.1, YP_335530.1, ZP_01769176.1, YP_105609. 1, ZP_017708 67.1, ZP_04520873.1, YP_110560.1, YP_001024014.1, ZP_03450125.1, YP_001061813.1, YP_111359.1, ZP_00440994.2, ZP_03456926.1, ZP_02358946.1, ZP_00438001.2, ZP_02461478.1, ZP_02503929.1, ZP_02511832.1, YP_004348706.1, ZP_04898742.1, YP_002908246.1, ZP_02382844.1, EGD05167.1, YP_001778808.1, YP_001811692.1, YP_002234935.1, YP_371318.1, YP_623143.1, YP_776389.1, ZP_02891479. 1, ZP_02907617.1, ZP_02417424.1 or ZP_04898743.1 or with a polypeptide sequence in which up to 25% of the amino acid residues are opposite to the respective reference sequence Seq ID No. 50, SEQ ID No. 52, SEQ ID No. 54 or the aforementioned accession number are modified by deletion, insertion, substitution or a combination thereof and which still has at least 10% of the enzymatic activity of the enzyme having the reference sequence Seq ID No. 50, Seq ID No. 52, Seq ID No. 54 or the aforementioned Accession Number, wherein is understood by enzymatic activity for an enzyme E _7b the ability dTDP-rhamnose and α-L-rhamnopyran osyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid to give α-L-rhamnopyranosyl- (1-2) -α-L-rhamnopyranosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid. Cell according to at least one of the preceding claims, characterized in that it is selected from a genus selected 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. Cell according to at least one of the preceding claims, characterized in that, compared to its wild-type, it has an increased activity of at least one of the enzymes selected from the group consisting of at least one enzyme E ₈ , a dTTP: α-D-glucose-1-phosphate thymidylyltransferase, EC 2.7.7.24, especially with polypeptide sequence SEQ ID NO: 56 or with a polypeptide sequence opposite to up to 25% of the amino acid residues SEQ ID NO: 56 are altered by deletion, insertion, substitution or a combination thereof and which still has at least 10% of the enzymatic activity of the enzyme having the reference sequence Seq ID No. 56, wherein under enzymatic activity for an enzyme E ₈ the Ability to convert α-D-glucose-1-phosphate and dTTP to dTDP-glucose, at least one enzyme E ₉ , a dTTP-glucose-4,6-hydrolyase, EC 4.2.1.46, in particular with polypeptide sequence Seq. 58 or with a polypeptide sequence in which up to 25% of the amino acid residues have been altered by reference to the reference sequence SEQ ID NO: 58 by deletion, insertion, substitution or a combination thereof and which still contains at least 10% of the enzymatic activity of the enzyme with the reference sequence Seq. ID 58, wherein enzymatic activity for an enzyme E _{9 is} the ability to convert dTDP-glucose to dTDP-4-dehydro-6-deoxy-D-glucose, at least one enzyme E ₁₀ , a dTDP 4-dehydrorhamnose-3,5-epimerase, EC 5.1.3.13, in particular with polypeptide sequence SEQ ID NO: 60 or with a polypeptide sequence in which up to 25% of the amino acid residues are opposite to the reference sequence SEQ ID NO: 60 by deletion, insertion, substitution or a combination thereof, and which still has at least 10% of the enzymatic activity of the enzyme having the reference sequence Seq ID No. 60, whereby enzymatic activity for an enzyme E _{10 is} understood to be the ability dTDP-4-dehydro-6-deoxy D-glucose to convert dTDP-4-dehydro-6-deoxy-L-mannose and at least one enzyme E ₁₁ , a dTDP-4-Dehydrorhamnose reductase, EC 1.1.1.133, in particular with polypeptide sequence Seq ID No. 62 or with a polypeptide sequence in which up to 25% of the amino acid residues are changed from the reference sequence SEQ ID NO: 62 by deletion, insertion, substitution or a combination thereof and which still has at least 10% of the enzymatic activity of the enzyme with the reference sequences z Seq ID No. 62, wherein enzymatic activity for an enzyme E _{11 is} understood to include the ability to react dTDP-4-dehydro-6-deoxy-L-mannose to form dTDP-6-deoxy-L-mannose. Cell according to at least one of the preceding claims, characterized in that it is capable of forming as wild-type polyhydroxyalkanoates with chain lengths of C ₆ to C ₁₆ , especially those genetically engineered such that it is capable of producing less polyhydroxyalkanoates compared to their wild type. Cell according to claim 12, characterized in that the cell is selected from the group consisting of P. putida GPp121, P.putida GPp122, P.putida GPp123, P.putida GPp124 and P.putida GPp104, P.putida KT42C1, P. putida KTOY01 or P. putida KTOY02. A further subject of the present invention is a process for the preparation of rhamnolipids of the general formula (I) comprising the method steps I) contacting a cell according to any one of claims 1 to 11 with a medium comprising a carbon source II) culturing the cell under conditions that allow the cell to form rhamnolipid from the carbon source and III) optionally isolation of the formed rhamnolipids. Use of the rhamnolipids obtained by the process according to claim 12 for the preparation of cosmetic, dermatological or pharmaceutical formulations, of crop protection formulations and of care and cleaning agents and surfactant concentrates.

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