Engineered glucosyltransferases
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. 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,
wherein the non-native glucosyltransferase uses sucrose to synthesize insoluble alpha-glucan comprising at least 50% alpha-1,3-glycosidic linkages, and the molecular weight of said insoluble alpha-glucan is at least 10% lower than the molecular weight of insoluble alpha-glucan synthesized by a second glucosyltransferase that only differs from the non-native glucosyltransferase at the substitution position(s).
2. The non-native glucosyltransferase of claim 1 , comprising 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.
3. The non-native glucosyltransferase of claim 1 , 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 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, Gln, 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 Gln-385 is with an Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr residue.
4. The non-native glucosyltransferase of claim 1 , comprising 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.
5. The non-native glucosyltransferase of claim 1 , wherein the insoluble alpha-glucan comprises at least about 80% alpha-1,3 glycosidic linkages.
6. The non-native glucosyltransferase of claim 5 , wherein the insoluble alpha-glucan comprises at least about 90% alpha-1,3 glycosidic linkages.
7. The non-native glucosyltransferase of claim 1 , wherein the insoluble alpha-glucan has a weight average degree of polymerization (DPw) of less than about 300.
8. The non-native glucosyltransferase of claim 7 , wherein the insoluble alpha-glucan has a DPw of less than about 150.
9. The non-native glucosyltransferase of claim 8 , wherein the insoluble alpha-glucan has a DPw of less than about 75.
10. The non-native glucosyltransferase of claim 1 , wherein the molecular weight of said insoluble alpha-glucan is at least 25% lower than the molecular weight of insoluble alpha-glucan synthesized by said second glucosyltransferase.
11. A reaction composition comprising water, sucrose, and a non-native glucosyltransferase according to claim 1 .
12. A method of producing insoluble alpha-glucan comprising 1,3-glycosidic linkages, the method comprising:
contacting at least water, sucrose, and a non-native glucosyltransferase enzyme according to claim 1 , whereby insoluble alpha-glucan comprising 1,3-glycosidic linkages is produced.
13. The method of claim 12 , further comprising a step of isolating the insoluble alpha-glucan.
14. The method of claim 12 , wherein the insoluble alpha-glucan comprises at least about 80% alpha-1,3 glycosidic linkages.
15. The method of claim 12 , wherein the insoluble alpha-glucan has a weight average degree of polymerization (DPw) of less than about 300.
16. The method of claim 12 , wherein the non-native glucosyltransferase comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO:66.
17. The non-native glucosyltransferase of claim 2 , 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, Gln, Arg, Ser, Thr, Val, Trp, or Tyr residue; and/or
(vi) the amino acid substitution at the position corresponding with amino acid residue Gln-385 is with an Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr residue.
18. The non-native glucosyltransferase of claim 4 , 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.
19. The non-native glucosyltransferase of claim 1 , wherein the non-native glucosyltransferase comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO:66.