Production and use of phosphoethanolamine cellulose and derivatives

Abstract

Phosphoethanolamine cellulose and methods of making and using it are disclosed. In particular, the invention relates to a method of producing a phosphoethanolamine cellulose biosynthetically using a BcsG phosphoethanolamine transferase for cellulose modification. Recombinant constructs encoding BcsG are described, including constructs encoding BcsG by itself or in combination with BcsE and BcsF, which increase the extent of cellulose modification and the amount of modified cellulose produced. Production of phosphoethanolamine cellulose in cell culture and derivatization of phosphoethanolamine cellulose are also described.

1 . A cellulose-producing host cell comprising a recombinant polynucleotide encoding a BcsG phosphoethanolamine transferase operably linked to a promoter. 
     
     
         2 . The cellulose-producing host cell of  claim 1 , wherein the recombinant polynucleotide is provided by a plasmid or viral vector. 
     
     
         3 . The cellulose-producing host cell of  claim 1 , wherein the recombinant nucleic acid is integrated into the host cell genome. 
     
     
         4 . The cellulose-producing host cell of  claim 1 , wherein the host cell further comprises a recombinant polynucleotide comprising a BcsE gene or BcsF gene operably linked to a promoter. 
     
     
         5 . (canceled) 
     
     
         6 . The cellulose-producing host cell of  claim 1 , wherein the recombinant polynucleotide comprises a multicistronic vector expressing BcsG, BcsE, and BcsF. 
     
     
         7 . (canceled) 
     
     
         8 . The cellulose-producing host cell of  claim 1 , wherein the recombinant polynucleotide comprises a bcsEFG operon. 
     
     
         9 . The cellulose-producing host cell of  claim 1 , wherein the host cell is a bacterial cell, a plant cell, or an algae cell. 
     
     
         10 . The cellulose-producing host cell of  claim 1 , wherein the bacterial cell is a Gram-negative bacterium. 
     
     
         11 . The cellulose-producing host cell of  claim 10 , wherein the Gram-negative bacterium belongs to a genus selected from the group consisting of  Acetobacter, Agrobacterium, Escherichia , and  Salmonella.    
     
     
         12 . The cellulose-producing host cell of  claim 11 , wherein the Gram-negative bacterium is selected from the group consisting of  Acetobacter xylinum, Escherichia coli , and  Salmonella enterica.    
     
     
         13 . The cellulose-producing host cell of  claim 1 , wherein cellulose production is upregulated by cyclic di-GMP. 
     
     
         14 . The cellulose-producing host cell of  claim 1 , further comprising a recombinant polynucleotide comprising a promoter operably linked to a polynucleotide encoding diguanylate cyclase. 
     
     
         15 - 17 . (canceled) 
     
     
         18 . A method of producing a phosphoethanolamine cellulose, the method comprising:
 a) culturing the cellulose-producing host cell of  claim 1  under conditions suitable for expression of the BcsG phosphoethanolamine transferase, wherein the phosphoethanolamine cellulose is produced; and   b) isolating the phosphoethanolamine cellulose.   
     
     
         19 . The method of  claim 18 , wherein the temperature is in a range from about 25° C. to about 37° C. 
     
     
         20 . The method of  claim 18 , wherein said culturing is performed at a temperature below 30° C. 
     
     
         21 . The method of  claim 20 , wherein the temperature is in a range from about 26° C. to about 28° C. 
     
     
         22 . (canceled) 
     
     
         23 . The method of  claim 18 , wherein said culturing is performed in a growth media comprising one or more carbon sources selected from the group consisting of glucose, fructose, acetate, or glycerol. 
     
     
         24 . The method of  claim 18 , further comprising increasing cellulose production by contacting the cellulose-producing host cell with cyclic di-GMP. 
     
     
         25 . The method of  claim 18 , wherein cyclic di-GMP levels in the cell are increased by transfecting the cellulose-producing host cell with a recombinant polynucleotide comprising a promoter operably linked to a polynucleotide encoding diguanylate cyclase. 
     
     
         26 . The method of  claim 25 , wherein the promoter is an inducible promoter. 
     
     
         27 . The method of  claim 25 , wherein the recombinant polynucleotide is provided by a vector. 
     
     
         28 . (canceled) 
     
     
         29 . A composition comprising a phosphoethanolamine cellulose ester, wherein at least one hydroxyl group of the phosphoethanolamine cellulose is esterified. 
     
     
         30 . The composition of  claim 29 , wherein said at least one hydroxyl group is esterified with an organic acid, acid anhydride, acid chloride, or inorganic acid. 
     
     
         31 . The composition of  claim 30 , wherein the organic acid is selected from the group consisting of acetic acid, propanoic acid, and butyric acid. 
     
     
         32 . The composition of  claim 30 , wherein the inorganic acid is selected from the group consisting of nitric acid and sulfuric acid. 
     
     
         33 . A composition comprising a phosphoethanolamine cellulose ether, wherein at least one hydroxyl group of the phosphoethanolamine cellulose is etherified. 
     
     
         34 . The composition of  claim 33 , wherein the phosphoethanolamine cellulose ether is an alkyl ether, a hydroxyalkyl ether, or a carboxyalkyl ether. 
     
     
         35 . A composition comprising a phosphoethanolamine cellulose, wherein at least one amine group is modified. 
     
     
         36 . The composition of  claim 35 , wherein said at least one amine group is alkylated, acylated, or sulfonated. 
     
     
         37 . The composition of  claim 35 , wherein said at least one amine group is conjugated to an agent. 
     
     
         38 . The composition of  claim 37 , wherein the agent is a peptide, antibody, enzyme, nucleic acid, dye, ligand, or drug. 
     
     
         39 . A method of hydrolyzing a phosphoethanolamine cellulose, the method comprising contacting the phosphoethanolamine cellulose with one or more cellulases. 
     
     
         40 . The method of  claim 39 , wherein said one or more cellulases are endocellulases, exocellulases, beta-glucosidases, oxidative cellulases, cellulose phosphorylases, or a combination thereof.