Biological processes for the production of aryl sulfates

Abstract

The present invention generally relates to the field of biotechnology as it applies to the production of aryl sulfates using polypeptides or recombinant cells comprising said polypeptides. More particularly, the present invention pertains to polypeptides having aryl sulfotransferase activity, recombinant host cells expressing same and processes for the production of aryl sulfates employing these polypeptides or recombinant host cells.

1 . A process for the production of a sulfated phenolic compound comprising:
 (i′) contacting a medium comprising a phenolic compound with a first recombinant host cell, wherein the first recombinant host cell comprises a heterologous polypeptide having an aryl sulfotransferase activity; or   (i″) contacting a medium comprising a fermentable carbon substrate with a first recombinant host cell; wherein the first recombinant host cell comprises a heterologous polypeptide having an aryl sulfotransferase activity; or   (i′″) contacting a medium comprising a precursor of a phenolic compound with a first recombinant host cell, wherein the first recombinant host cell comprises a heterologous polypeptide having an aryl sulfotransferase activity.   
     
     
         2 - 38 . (canceled) 
     
     
         39 . The process according to  claim 1 , further comprising:
 (ii) culturing the first recombinant host cell under suitable conditions for the production of the corresponding sulfated phenolic compound; and   (iii) optionally, recovering said sulfated phenolic compound.   
     
     
         40 . The process according to  claim 1 , wherein the heterologous polypeptide having an aryl sulfotransferase activity is a sulfotransferase 1A1 enzyme. 
     
     
         41 . The process according to  claim 1 , wherein the heterologous polypeptide having an aryl sulfotransferase activity is selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13;   b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, wherein the polypeptide has aryl sulfotransferase activity; and   c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has aryl sulfotransferase activity.   
     
     
         42 . The process according to  claim 1 , wherein the heterologous polypeptide is selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1;   b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the polypeptide has aryl sulfotransferase activity; and   c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has aryl sulfotransferase activity.   
     
     
         43 . The process according to  claim 1 , wherein the first recombinant host cell has been further modified to have an increased expression of an ATP sulfurylase protein as compared to an identical host cell that does not carry said modification. 
     
     
         44 . The process according to  claim 43 , wherein the ATP sulfurylase is encoded by the genes cysD and cysN. 
     
     
         45 . The process according to  claim 1 , wherein said first recombinant host cell has been further modified to have an increased expression of an APS kinase protein as compared to an identical host cell that does not carry said modification. 
     
     
         46 . The process according to  claim 45 , wherein the said APS kinase is encoded by the gene cysC. 
     
     
         47 . The process according to  claim 1 , wherein said first recombinant host cell has been further modified to have an increased expression of a PAP phosphatase protein as compared to an identical host cell that does not carry said modification. 
     
     
         48 . The process according to  claim 47 , wherein said PAP phosphatase is encoded by the gene cycQ. 
     
     
         49 . The process according to  claim 1 , wherein said first recombinant host cell further comprises a heterologous polypeptide having a tyrosine ammonia lyase activity. 
     
     
         50 . The process according to  claim 1 , wherein in step (i′), (i″) or (i′″) the medium is further contacted with a second recombinant host cell comprising a heterologous polypeptide having a tyrosine ammonia lyase activity. 
     
     
         51 . The process according to  claim 49 , wherein the heterologous polypeptide having a tyrosine ammonia lyase activity is selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23;   b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, wherein the polypeptide has tyrosine ammonia lyase activity; and   c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has tyrosine ammonia lyase activity.   
     
     
         52 . The process according to  claim 49 , wherein the heterologous polypeptide having a tyrosine ammonia lyase activity is selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 14;   b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14, wherein the polypeptide has tyrosine ammonia lyase activity; and   c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 14, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has tyrosine ammonia lyase activity.   
     
     
         53 . The process according to  claim 1 , wherein the phenolic compound is represented by the general formula (I): 
       
         
           
           
               
               
           
         
         wherein at least one of R 1 , R 2 , R 3 , R 4 , or R 5  is an hydroxyl group (—OH); 
         wherein R 1 , R 2 , R 3 , R 4 , R 5  and R 6  are independently selected from the group consisting of: halide, hydrogen, hydroxyl (—OH), —OR 7 , —OCOR 7 , —NR 7 R 8 , —COR 7 , —COOR 7 , —SR 7 , —OSO 3 R 7 , —OCSR 7 , —POR 7 R 8 , alkyl, alkenyl, alkynyl, aryl, and heteroaryl, wherein R 7 , and R 8  are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, and heteroaryl; 
         wherein R 1 , R 2 , R 3 , R 4 , R 5  and R 6 , are optionally linked with a bridge member Y n  thereby forming one or more rings, Y n  being a bond or a C 1-12  alkyl or an aryl, a carbocyclic, a heterocyclic or a heteroaromatic structure having 1-3 rings, 3-8 ring members in each and 0 to 4 heteroatoms, or a heteroalkyl comprising 1 to 12 heteroatoms selected from the group consisting of N, O, S, S(O) 1-2  and carbonyl, and wherein n is an integer between 1 and 12. 
       
     
     
         54 . A process according to  claim 1 , wherein the phenolic compound is represented by the general formula (II): 
       
         
           
           
               
               
           
         
         wherein at least one of R 1 , R 2 , R 3 , R 4 , or R 5  is an hydroxyl group (—OH); 
         wherein R 1 , R 2 , R 3 , R 4 , R 5  and R 6  are independently selected from the group consisting of halide, hydrogen, hydroxyl (—OH), —OR 7 , —OCOR 7 , —NR 7 R 8 , —COR 7 , —COOR 7 , —SR 7 , —OSO 3 R 7 , —OCSR 7 , —POR 7 R 8 , alkyl, alkenyl, alkynyl, aryl, and heteroaryl, wherein R 7 , and R 8  are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, and heteroaryl; 
         wherein R 1 , R 2 , R 3 , R 4 , R 5  and R 6 , are optionally linked with a bridge member Y n , thereby forming one or more rings, Y n  being a bond or a C 1-12  alkyl or an aryl, a carbocyclic, a heterocyclic or a heteroaromatic structure having 1-3 rings, 3-8 ring members in each and 0 to 4 heteroatoms, or a heteroalkyl comprising 1 to 12 heteroatoms selected from the group consisting of N, O, S, S(O) 1-2  and carbonyl, and wherein n is an integer between 1 and 12. 
       
     
     
         55 . The process according to  claim 1 , wherein the precursor of a phenolic compound in step (i′″) is a compound of the general Formula (p-I): 
       
         
           
           
               
               
           
         
         wherein at least one of R 1 , R 2 , R 3 , R 4 , or R 5  is an hydroxyl group (—OH); 
         wherein R 1 , R 2 , R 3 , R 4 , R 5  and R 6  are independently selected from the group consisting of halide, hydrogen, hydroxyl (—OH), —OR 7 , —OCOR 7 , —NR 7 R 8 , —COR 7 , —COOR 7 , —SR 7 , —OSO 3 R 7 , —OCSR 7 , —POR 7 R 8 , alkyl, alkenyl, alkynyl, aryl, and heteroaryl; wherein R 7 , and R 8  are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, and heteroaryl; 
         wherein R 1 , R 2 , R 3 , R 4 , R 5  and R 6 , are optionally linked with a bridge member Y n , thereby forming one or more rings, Y n  being a bond or a C 1-12  alkyl or an aryl, a carbocyclic, a heterocyclic or a heteroaromatic structure having 1-3 rings, 3-8 ring members in each and 0 to 4 heteroatoms, or a heteroalkyl comprising 1 to 12 heteroatoms selected from the group consisting of N, O, S, S(O) 1-2  and carbonyl, and wherein n is an integer between 1 and 12. 
       
     
     
         56 . The process according to  claim 53 , wherein R 6  is —COOR 7  and R 7  is hydrogen. 
     
     
         57 . The process according to  claim 53 , wherein R 3  is hydroxyl (—OH). 
     
     
         58 . The process according to  claim 53 , wherein each of R 1 , R 2 , R 4  and R 5  is hydrogen, R 3  is hydroxyl (—OH) and R 6  is —COOH. 
     
     
         59 . A recombinant host cell comprising a first heterologous polypeptide having aryl sulfotransferase activity, such as a polypeptide selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13;   b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, wherein the polypeptide has aryl sulfotransferase activity; and   c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has aryl sulfotransferase activity.   
     
     
         60 . The recombinant host cell according to  claim 59 , wherein the heterologous polypeptide is selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1;   b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the polypeptide has aryl sulfotransferase activity; and   c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has aryl sulfotransferase activity.   
     
     
         61 . The recombinant host cell according to  claim 59 , wherein the recombinant host cell has further been modified to have an increased protein expression of an ATP sulfurylase compared to an identical host cell that does not carry said modification. 
     
     
         62 . The recombinant host cell according to  claim 61 , wherein said ATP sulfurylase is encoded by the genes cysD and cysN. 
     
     
         63 . The recombinant host cell according to  claim 59 , wherein the recombinant host cell has further been modified to have an increased expression of an APS kinase protein as compared to an identical host cell that does not carry said modification. 
     
     
         64 . The recombinant host cell according to  claim 63 , wherein said APS kinase is encoded by the gene cysC. 
     
     
         65 . The recombinant host cell according to  claim 59 , wherein the recombinant host cell has further been modified to have an increased expression of a PAP phosphatase protein as compared to an identical host cell that does not carry said modification. 
     
     
         66 . The recombinant host cell according to  claim 65 , wherein said PAP phosphatase is encoded by the gene cycQ. 
     
     
         67 . The recombinant host cell according to  claim 59 , further comprising a second heterologous polypeptide having tyrosine ammonia lyase activity, wherein said polypeptide is selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23;   b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, wherein the polypeptide has tyrosine ammonia lyase activity; and   c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has tyrosine ammonia lyase activity.   
     
     
         68 . The recombinant host cell according to  claim 59 , further comprising a second heterologous polypeptide having tyrosine ammonia lyase activity, wherein the heterologous polypeptide is selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 14;   b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14, wherein the polypeptide has tyrosine ammonia lyase activity; and   c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 14, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has tyrosine ammonia lyase activity.   
     
     
         69 . The recombinant host cell according to  claim 59 , which is employed as first recombinant host cell in the process according to  claim 1 . 
     
     
         70 . Process for the production of a sulfated phenolic compound, such as zosteric acid, the method comprises sulfating a phenolic compound, such as p-coumaric acid, using a polypeptide having aryl sulfotransferase activity, such as a polypeptide selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13;   b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, wherein the polypeptide has aryl sulfotransferase activity; and   c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has aryl sulfotransferase activity.   
     
     
         71 . The process according to  claim 70 , wherein the polypeptide is selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1;   b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the polypeptide has aryl sulfotransferase activity; and   c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has aryl sulfotransferase activity.   
     
     
         72 . A composition comprising:
 a first recombinant host cell comprising a heterologous polypeptide having arylsulfotransferase activity, such as a polypeptide selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13; 
 b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, wherein the polypeptide has aryl sulfotransferase activity; and 
 c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has aryl sulfotransferase activity; and 
   a second recombinant host cell comprising a heterologous polypeptide having tyrosine ammonia lyase activity, such as a polypeptide selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23; 
 b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, wherein the polypeptide has tyrosine ammonia lyase activity; and 
 c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has tyrosine ammonia lyase activity. 
   
     
     
         73 . The composition according to  claim 72 , comprising:
 a first recombinant host cell comprising a heterologous polypeptide having arylsulfotransferase activity selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1; 
 b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the polypeptide has aryl sulfotransferase activity; and 
 c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has aryl sulfotransferase activity; and 
   a second recombinant host cell comprising a heterologous polypeptide having tyrosine ammonia lyase activity selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 14; 
 b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14, wherein the polypeptide has tyrosine ammonia lyase activity; and 
   c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 14, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has tyrosine ammonia lyase activity.   
     
     
         74 . A composition comprising:
 a first polypeptide having aryl sulfotransferase activity, such as a polypeptide selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13; 
 b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, wherein the polypeptide has aryl sulfotransferase activity; and 
 c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has aryl sulfotransferase activity; and 
   a second polypeptide having tyrosine ammonia lyase activity, such as a polypeptide selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23; 
 b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, wherein the polypeptide has tyrosine ammonia lyase activity; and 
 c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 
   14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has tyrosine ammonia lyase activity.   
     
     
         75 . The composition according to  claim 74 , comprising:
 a first polypeptide having aryl sulfotransferase activity selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1; 
 b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the polypeptide has aryl sulfotransferase activity; and 
 c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has aryl sulfotransferase activity; and 
   a second polypeptide having tyrosine ammonia lyase activity selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 14; 
 b) a polypeptide comprising an amino acid sequence which has at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14, wherein the polypeptide has tyrosine ammonia lyase activity; and 
 c) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 14, wherein 1 to 50 amino acid residues are substituted, deleted and/or inserted, wherein the polypeptide has tyrosine ammonia lyase activity.