Glycolipopeptide biosurfactants

Abstract

Surfactants based on a newly discovered class of compounds include a hydrophobic lipid oligomer covalently linked to a peptide or peptide-like chain and a carbohydrate moiety, and a serine-leucinol dipeptide linked to the lipid oligomer. Such surfactants can be used to create an oil-in-water or water-in-oil emulsion by mixing together a polar component; a non-polar component; and the surfactant. Biosurfactants of the newly discovered class can be made by isolating and culturing a microorganism which produces the biosurfactant, and then isolating the biosurfactant from the culture. A microorganism can be engineered to produce biosurfactant of this newly discovered class by expressing a set of heterologous genes involved in the biosynthesis of the biosurfactant in the microorganism.

1 . A purified biosurfactant comprising a hydrophobic lipid component comprising a carboxyl end and a hydroxyl end, wherein the lipid component is covalently linked to (i) a peptide or peptide-like chain at the carboxyl end of the lipid component and (ii) a carbohydrate moiety at the hydroxyl end of the lipid component via a glycosidic linkage. 
     
     
         2 . The purified biosurfactant according to  claim 1 , wherein the peptide chain comprises in the range of between 2 and 10 amino acids. 
     
     
         3 . The purified biosurfactant according to  claim 1 , wherein the lipid component comprises in the range of between 1 and 6 alkanoic acid moieties 
     
     
         4 . The purified biosurfactant according to  claim 1 , wherein the lipid component comprises an acyle chain, and wherein the length of each said acyl chain is in the range of between C 4  to C 20 . 
     
     
         5 . The purified biosurfactant according to  claim 1 , wherein the carbohydrate moiety may be selected from saccharides including glucose, fructose, galactose, mannose, ribose, or deoxy saccharide variants including deoxyribose, fucose, or rhamnose. 
     
     
         6 . The purified biosurfactant according to  claim 1 , wherein the peptide or peptide-like chain comprises a serine-leucinol dipeptide. 
     
     
         7 . The purified biosurfactant according to  claim 1 , wherein the lipid component comprises three 3-hydroxyalkanoic acid moieties. 
     
     
         8 . The purified biosurfactant of  claim 4 , wherein the length of each acyl chain of the lipid component is C 10 . 
     
     
         9 . The purified biosurfactant according to  claim 1 , wherein the carbohydrate moiety comprises a rhamnose moiety attached to the lipid component via a glycosidic linkage. 
     
     
         10 . The purified biosurfactant according to  claim 9 , wherein the carbohydrate moiety comprises two rhamnose moieties. 
     
     
         11 . The purified biosurfactant according to  claim 1 , wherein the lipid component comprises three β-hydroxyalkanoic acid moieties, the length of each acyl chain of the lipid component is C 10 , and the carbohydrate moiety comprises a rhamnose moiety attached to the lipid component via a glycosidic linkage. 
     
     
         12 . A purified biosurfactant comprising a peptide or peptide-like portion covalently bound to a lipid portion, wherein the biosurfactant comprises the structure: 
       
         
           
           
               
               
           
         
       
       wherein R 1a  is selected from the group consisting of H, OH, OCH 3 , SH, S(CH 3 ), NH 2 , NH(CH 3 ), N(CH 3 ) 2 , and a peptide or peptide-like structure having the structure: 
       
         
           
           
               
               
           
         
       
       wherein R 1b , R 1c , and R 1d , are selected from the group consisting of H, OH, OCH 3 , SH, S(CH 3 ), NH 2 , NH(CH 3 ), and N(CH 3 ) 2 ; 
       R 2a , R 2b , R 2c , and R 2d  are each independently an amino acid side chain; 
       X 1a , X 1b , X 1c , and X 1d  are each independently selected from the group consisting of one oxygen atom and two hydrogen atoms; 
       X 2a , X 2b , X 2c , and X 2d  are each independently selected from the group consisting of NH, N(CH 3 ), and O; 
       R 3a  is selected from the group consisting of a carbohydrate portion, and a lipid selected from the group consisting of a monomer having the structure: 
       
         
           
           
               
               
           
         
       
       and an oligomer selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
       wherein X 3a , X 3b , X 3c , and X 3d  are each independently selected from the group consisting of NH, N(CH 3 ), and O; 
       R 3a , R 3b , R 3c , and R 3d  comprises a carbohydrate portion comprising a monomer selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
       wherein R 5a , R 6a , R 7a , and R 8a  are each independently selected from the group consisting of a hydrogen atom, methyl, acetyl, and a carbohydrate; 
       and R 4a , R 4b , R 4c , and R 4d  are each independently selected from the group consisting of a hydrogen atom, methyl, and a C 2  to C 19  saturated or unsaturated linear, branched-chain, cyclic, or aromatic hydrocarbon groups. 
     
     
         13 . The purified biosurfactant of  claim 12 , wherein at least one of R 6a , R 7a , and R 8a  comprises a carbohydrate comprising a monomer selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
       wherein R 5b , R 6b , R 7b , and R 8b  are each independently selected from the group consisting of a hydrogen atom, methyl, acetyl, and a carbohydrate. 
     
     
         14 . The purified biosurfactant of  claim 12 , wherein the peptide or peptide-like portion comprises at least one proline or proline-like monomer having the structure: 
       
         
           
           
               
               
           
         
       
       wherein X 4  is selected from the group consisting of one oxygen atom and two hydrogen atoms. 
     
     
         15 . The purified biosurfactant of  claim 14 , wherein the peptide or peptide-like portion comprises a single proline or proline-like monomer or a terminal proline or proline-like monomer having the structure: 
       
         
           
           
               
               
           
         
       
       wherein R 9  is selected from the group consisting of H, OH, OCH 3 , SH, S(CH 3 ), NH 2 , NH(CH 3 ), and N(CH 3 ) 2 ; and X 4  is selected from the group consisting of one oxygen atom and two hydrogen atoms. 
     
     
         16 . A purified biosurfactant, wherein the biosurfactant has the structure: 
       
         
           
           
               
               
           
         
       
       wherein R 5a , R 6a , R 7a , R 10 , and R 11  are each independently selected from the group consisting of a hydrogen atom and acetyl; and n 1 , n 2 , and n 3  are integers each independently selected from 1 to 7. 
     
     
         17 . The purified biosurfactant of  claim 16 , wherein the biosurfactant has the structure: 
       
         
           
           
               
               
           
         
       
       wherein R 5a , R 5b , R 6b , R 7a , R 7b , R 10 , and R 11  are each independently selected from the group consisting of a hydrogen atom and acetyl; and n 1 , n 2 , and n 3  are integers each independently selected from 1 to 7. 
     
     
         18 . The purified biosurfactant of  claim 16 , wherein the biosurfactant has the structure: 
       
         
           
           
               
               
           
         
       
     
     
         19 . The purified biosurfactant of  claim 17 , wherein the biosurfactant has the structure: 
       
         
           
           
               
               
           
         
       
     
     
         20 . The purified biosurfactant of  claim 17 , wherein the biosurfactant has the structure: 
       
         
           
           
               
               
           
         
       
     
     
         21 . A method of making the biosurfactant of  claim 1 , the method comprising the steps of:
 (a) isolating a microorganism which comprises the biosurfactant;   (b) placing the microorganism in a culture under conditions that promote the synthesis of the biosurfactant; and   (c) isolating the biosurfactant from the culture.   
     
     
         22 . The method according to  claim 21 , wherein the microorganism belongs to the genus and species  Variovorax paradoxus  and is strain RKNM-096 as deposited at the NRRL under accession number B-67038. 
     
     
         23 . An organism consisting of  Variovorax paradoxus , strain B-67038, Agricultural Research Service Culture Collection accession number B-67038. 
     
     
         24 . An emulsified oil-in-water or water-in-oil composition comprising a polar component, a non-polar component, and the biosurfactant as claimed in  claim 1 . 
     
     
         25 . An isolated microorganism engineered to produce the biosurfactant of  claim 1 , wherein a set of heterologous genes exhibiting at least 70% similarity to SEQ IDs 3, 5, 7, 9, 11 and 13 have been introduced into the microorganism. 
     
     
         26 . A method of modifying natural glyclolipopeptide surfactants by adding additional rhamnose moieties using recombinantly expressed RIpE [SEQ IDs 11, 12, 23 and 24].