Rhamnolipid-producing cell having reduced glucose dehydrogenase activity

Abstract

Cells which make rhamnolipids and are genetically modified such that they have a decreased activity, compared to the wild type thereof, are provided. A glucose dehydrogenase and a method for producing rhamnolipids using the cells according to the invention are also provided.

1 : A cell able to make at least one rhamnolipid, wherein the cell has been genetically modified such that it, compared to the wild type thereof, has a decreased activity of at least one enzyme E 1 , which catalyses the conversion of D-glucose and quinone to D-glucono-1,5-lactone and quinol. 
     
     
         2 : The cell according to  claim 1 , wherein the cell is able to make, as the at least one rhamnolipid, a compound of the general formula (I), 
       
         
           
           
               
               
           
         
         where 
         m=1, 
         n=1 or 0, and 
         the radical determined via R 1  and R 2  is 
       
       
         
           
           
               
               
           
         
         derived from 3-hydroxyoctanoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydecenoic acid, 3-hydroxydecenoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydodecanoic acid, 3-hydroxydodecanoyl-3-hydroxyoctanoic acid, 3-hydroxyoctanoyl-3-hydroxydodecenoic acid, 3-hydroxydodecenoyl-3-hydroxyoctanoic acid, 3-hydroxydecanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxydecenoic acid, 3-hydroxydecenoyl-3-hydroxydecanoic acid, 3-hydroxydecenoyl-3-hydroxydecenoic acid, 3-hydroxydecanoyl-3-hydroxydodecanoic acid, 3-hydroxydodecanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxydodecenoic acid, 3-hydroxydecanoyl-3-hydroxytetradecenoic acid, 3-hydroxytetradecanoyl-3-hydroxydecenoic acid, 3-hydroxydodecenoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxytetradecanoic acid, 3-hydroxytetradecanoyl-3-hydroxydecanoic acid, 3-hydroxydecanoyl-3-hydroxytetradecenoic acid, 3-hydroxytetradecenoyl-3-hydroxydecanoic acid, 3-hydroxydodecanoyl-3-hydroxydodecanoic acid, 3-hydroxydodecenoyl-3-hydroxydodecanoic acid, 3-hydroxydodecanoyl-3-hydroxydodecenoic acid, 3-hydroxydodecanoyl-3-hydroxytetradecanoic acid, 3-hydroxytetradecanoyl-3-hydroxydodecanoic acid, 3-hydroxytetradecanoyl-3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoyl-3-hydroxytetradecanoic acid, 3-hydroxytetradecanoyl-3-hydroxyhexadecanoic acid or 3-hydroxyhexadecanoyl-3-hydroxyhexadecanoic acid. 
       
     
     
         3 : The cell according to  claim 1 , wherein E 1  is a glucose 1-dehydrogenase of EC 1.1.5.2. 
     
     
         4 : The cell according to  claim 1 , wherein the cell is selected from the group consisting of  Burkholderia  sp.,  Burkholderia thailandensis, Pseudomonas  sp.,  Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas oleovorans, Pseudomonas stutzeri, Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas citronellolis, Pseudomonas resinovorans, Comamonas testosteroni, Aeromonas hydrophila, Cupriavidus necator, Alcaligenes latus  and  Ralstonia eutropha.    
     
     
         5 : The cell according to  claim 1 , wherein the cell has been genetically modified such that it, compared to the wild type thereof, has an increased activity of at least one of the enzymes selected from the group consisting of E 2 , E 3  and E 4 ,
 the enzyme E 2  being able to catalyse the conversion of 3-hydroxyalkanoyl-ACP via 3-hydroxyalkanoyl-3-hydroxyalkanoic acid-ACP to hydroxyalkanoyl-3-hydroxyalkanoic acid,   the enzyme E 3  being a rhamnosyltransferase I and being able to catalyse the conversion of dTDP-rhamnose and 3-hydroxyalkanoyl-3-hydroxyalkanoate to α-L-rhamnopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate, and   the enzyme E 4  being a rhamnosyltransferase II and being able to catalyse 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.   
     
     
         6 : The cell according to  claim 5 , wherein E 2 , E 3  and E 4  are encoded by an rhlA gene, an rhlB gene and an rhlC gene, respectively, or are enzymes having a polypeptide sequence in which up to 25%, of the amino acid residues are modified with respect to the enzymes encoded by an rhl gene 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 of the enzymes encoded by an rhl gene. 
     
     
         7 : The cell according to  claim 5 , wherein the cell has increased activities of the following enzyme combinations selected from E 3 , E 3 E 4 , and E 2 E 3 E 4 . 
     
     
         8 : The cell according to  claim 1 , wherein the cell, compared to the wild type thereof, has an increased activity of at least one of the enzymes selected from the group consisting of
 at least one enzyme E 5 , a dTTP:α-D-glucose-1-phosphate thymidylyltransferase of EC 2.7.7.24,   at least one enzyme E 6 , a dTDP-glucose 4,6-hydrolyase of EC 4.2.1.46,   at least one enzyme E 7 , a dTDP-4-dehydrorhamnose 3,5-epimerase of EC 5.1.3.13, and   at least one enzyme E 8 , a dTDP-4-dehydrorhamnose reductase of EC 1.1.1.133.   
     
     
         9 : The cell according to  claim 1 , wherein the cell, compared to the wild type thereof, has an increased activity of at least one enzyme E 13 , which catalyses the export of a rhamnolipid from the cell into the surrounding medium, selected from the group consisting of enzymes having polypeptide sequence AAG04520.1, AJY02996.1, ZP_05590661.1, YP_439278.1, YP_440069.1, ZP_04969301.1, ZP_04520234.1, YP_335528.1, YP_001075859.1, YP_001061817.1, ZP_02487499.1, YP_337251.1, ZP_04897712.1, ZP_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, EGD05166.1, ZP_02458677.1, ZP_02465793.1, YP_001578240.1, ZP_04944344.1, YP_771932.1, ZP_02889166.1, YP_002232614.1, ZP_03574808.1, 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 having a polypeptide sequence in which up to 25% of the amino acid residues are modified with respect to the particular 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 having the particular aforementioned accession number. 
     
     
         10 : A method for producing a rhamnolipid, comprising:
 I) contacting a cell according to  claim 1 , combining measures as appropriate, with a medium containing a carbon source;   II) culturing the cell under conditions allowing the cell to make the rhamnolipid from the carbon source; and   III) optionally, isolating the rhamnolipid made.   
     
     
         11 : A method, comprising:
 adding the rhamnolipid obtained using the method according to  claim 10  to a mixture,   wherein the mixture is a cosmetic formulation, dermatological formulation, pharmaceutical formulation, crop-protection formulation, care product, cleaning agent, or surfactant concentrate.