Engineered glucosyltransferases

Abstract

Disclosed herein are glucosyltransferases with modified amino acid sequences. Such engineered enzymes synthesize insoluble alpha-glucan products having decreased molecular weight. Further disclosed are reactions and methods in which engineered glucosyltransferases are used to produce insoluble alpha-glucan.

1 - 18 . (canceled) 
     
     
         19 . An isolated composition comprising insoluble alpha-glucan having at least 50% alpha-1,3 glycosidic linkages,
 wherein the insoluble alpha-glucan is a product of a non-native glucosyltransferase comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:66, wherein said amino acid sequence has at least one amino acid substitution at a position corresponding with amino acid residue Leu-282, Pro-319, Ser-322, Asn-326, Asn-327, Trp-340, Asn-342, Asp-344, Lys-347, Asn-350, Thr-354, or Gln-385 of SEQ ID NO:66.   
     
     
         20 . The isolated composition of  claim 19 , wherein:
 the amino acid substitution at the position corresponding with amino acid residue Leu-282 is with an Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr residue;   (ii) the amino acid substitution at the position corresponding with amino acid residue Pro-319 is with a Leu, Val, or Ile residue;   (iii) the amino acid substitution at the position corresponding with amino acid residue Ser-322 is with an Ala, Cys, Glu, Phe, His, Ile, Met, Asn, Arg, Thr, Val, or Tyr residue;   (iv) the amino acid substitution at the position corresponding with amino acid residue Asn-326 is with a Glu, Gln, Ile, Thr, Asp, Asn, Leu, Val, or Ser residue;   (v) the amino acid substitution at the position corresponding with amino acid residue Asn-327 is with an Asp or Glu residue;   (vi) the amino acid substitution at the position corresponding with amino acid residue Trp-340 is with a Val, Asp, Cys, Ile, Leu, Glu, or Ser residue;   (vii) the amino acid substitution at the position corresponding with amino acid residue Asn-342 is with an Ala, Asp, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Thr, Val, or Trp residue;   (viii) the amino acid substitution at the position corresponding with amino acid residue Asp-344 is with an Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr residue;   (ix) the amino acid substitution at the position corresponding with amino acid residue Lys-347 is with an Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, or Tyr residue;   (x) the amino acid substitution at the position corresponding with amino acid residue Asn-350 is with a Pro or Gly residue;   (xi) the amino acid substitution at the position corresponding with amino acid residue Thr-354 is with a Pro or Gly residue; and/or   (xii) the amino acid substitution at the position corresponding with amino acid residue Gin-385 is with an Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr residue.   
     
     
         21 . The isolated composition of  claim 19 , wherein the non-native glucosyltransferase comprises at least one amino acid substitution at a position corresponding with amino acid residue Leu-282, Ser-322, Asn-342, Asp-344, Lys-347, or Gln-385 of SEQ ID NO:66. 
     
     
         22 . The isolated composition of  claim 21 , wherein:
 (i) the amino acid substitution at the position corresponding with amino acid residue Leu-282 is with an Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr residue;   (ii) the amino acid substitution at the position corresponding with amino acid residue Ser-322 is with an Ala, Cys, Glu, Phe, His, Ile, Met, Asn, Arg, Thr, Val, or Tyr residue;   (iii) the amino acid substitution at the position corresponding with amino acid residue Asn-342 is with an Ala, Asp, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Thr, Val, or Trp residue;   (iv) the amino acid substitution at the position corresponding with amino acid residue Asp-344 is with an Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr residue;   (v) the amino acid substitution at the position corresponding with amino acid residue Lys-347 is with an Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, or Tyr residue; and/or   (vi) the amino acid substitution at the position corresponding with amino acid residue Gin-385 is with an Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr residue.   
     
     
         23 . The isolated composition of  claim 19 , wherein the non-native glucosyltransferase comprises at least one amino acid substitution at a position corresponding with amino acid residue Pro-319, Asn-326, Asn-327, Asn-350, or Thr-354 of SEQ ID NO:66. 
     
     
         24 . The isolated composition of  claim 23 , wherein:
 (i) the amino acid substitution at the position corresponding with amino acid residue Pro-319 is with a Leu, Val, or Ile residue;   (ii) the amino acid substitution at the position corresponding with amino acid residue Asn-326 is with a Glu, Gln, Ile, Thr, Asp, Asn, Leu, Val, or Ser residue;   (iii) the amino acid substitution at the position corresponding with amino acid residue Asn-327 is with an Asp or Glu residue;   (iv) the amino acid substitution at the position corresponding with amino acid residue Asn-350 is with a Pro or Gly residue; and/or   (v) the amino acid substitution at the position corresponding with amino acid residue Thr-354 is with a Pro or Gly residue.   
     
     
         25 . The isolated composition of  claim 19 , wherein the non-native glucosyltransferase comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO:66. 
     
     
         26 . The composition of  claim 19 , further comprising the non-native glucosyltransferase enzyme. 
     
     
         27 . The isolated composition of  claim 19 , wherein the insoluble alpha-glucan comprises at least about 80% alpha-1,3 glycosidic linkages. 
     
     
         28 . The isolated composition of  claim 27 , wherein the insoluble alpha-glucan comprises at least about 90% alpha-1,3 glycosidic linkages. 
     
     
         29 . The isolated composition of  claim 19 , wherein the insoluble alpha-glucan has a weight average degree of polymerization (DPw) of less than about 300. 
     
     
         30 . The isolated composition of  claim 29 , wherein the insoluble alpha-glucan has a DPw of less than about 150. 
     
     
         31 . The isolated composition of  claim 30 , wherein the insoluble alpha-glucan has a DPw of less than about 75. 
     
     
         32 . The isolated composition of  claim 19 , further comprising fructose.