Engineered glucosyltransferases
Disclosed herein are glucosyltransferases with modified amino acid sequences. Such engineered enzymes exhibit improved alpha-glucan product yields and/or lower leucrose yields, for example. Further disclosed are reactions and methods in which engineered glucosyltransferases are used to produce alpha-glucan.
1. A non-native glucosyltransferase comprising at least two amino acid substitutions at positions corresponding with amino acid residues Gln-588, Phe-607, or Arg-741 of SEQ ID NO:62,
wherein the non-native glucosyltransferase synthesizes insoluble alpha-glucan comprising 1,3-linkages, and
wherein the non-native glucosyltransferase has:
(i) an insoluble alpha-glucan yield that is higher than the insoluble alpha-glucan yield of a second glucosyltransferase that only differs from the non-native glucosyltransferase at the substitution positions, and/or
(ii) a leucrose yield that is lower than the leucrose yield of the second glucosyltransferase;
wherein the non-native glucosyltransferase comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of residues 55-960 of SEQ ID NO:4 or the amino acid sequence of residues 54-957 of SEQ ID NO:65.
2. The non-native glucosyltransferase of claim 1 , wherein the glucosyltransferase comprises amino acid substitutions at positions corresponding with amino acid residues Gln-588, Phe-607 and Arg-741 of SEQ ID NO:62.
3. The non-native glucosyltransferase of claim 1 , wherein:
(i) the amino acid substitution at the position corresponding with amino acid residue Gln-588 is with a Leu, Ala, or Val residue;
(ii) the amino acid substitution at the position corresponding with amino acid residue Phe-607 is with a Trp, Tyr, or Asn residue; and/or
(iii) the amino acid substitution at the position corresponding with amino acid residue Arg-741 is with a Ser or Thr residue.
4. The non-native glucosyltransferase of claim 1 , wherein the glucosyltransferase further comprises at least one amino acid substitution at a position corresponding with amino acid residue Ala-510 and/or Asp-948 of SEQ ID NO:62.
5. The non-native glucosyltransferase of claim 1 , wherein the glucosyltransferase further comprises at least one amino acid substitution at a position corresponding with amino acid residue Ser-631, Ser-710, Arg-722, and/or Thr-877 of SEQ ID NO:62.
6. The non-native glucosyltransferase of claim 1 , wherein the glucosyltransferase further comprises at least one amino acid substitution at a position corresponding with amino acid residue Val-1188, Met-1253, and/or Gln-957 of SEQ ID NO:62.
7. The non-native glucosyltransferase of claim 1 , wherein the insoluble alpha-glucan comprises at least 50% alpha-1,3 linkages.
8. The non-native glucosyltransferase of claim 1 , comprising an amino acid sequence that has at least 92% sequence identity to the amino acid sequence of residues 55-960 of SEQ ID NO:4 or the amino acid sequence of residues 54-957 of SEQ ID NO:65.
9. The non-native glucosyltransferase of claim 1 , comprising an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:65.
10. The non-native glucosyltransferase of claim 7 , wherein the insoluble alpha-glucan comprises at least 90% alpha-1,3 linkages.
11. The non-native glucosyltransferase of claim 1 , wherein the insoluble alpha-glucan yield is at least 10% higher than the insoluble alpha-glucan yield of the second glucosyltransferase.
12. A reaction composition comprising water, sucrose, and the non-native glucosyltransferase of claim 1 .
13. A method of producing insoluble alpha-glucan comprising:
contacting at least water, sucrose, and the non-native glucosyltransferase of claim 1 , whereby insoluble alpha-glucan is produced.
14. The method of claim 13 , wherein the method further comprises isolating the insoluble alpha-glucan.
15. The non-native glucosyltransferase of claim 4 , wherein:
(i) the amino acid substitution at the position corresponding with amino acid residue Ala-510 is with an Asp, Glu, Ile, or Val residue; and/or
(ii) the amino acid substitution at the position corresponding with amino acid residue Asp-948 is with a Gly, Val, or Ala residue.
16. The non-native glucosyltransferase of claim 5 , wherein:
(i) the amino acid substitution at the position corresponding with amino acid residue Ser-631 is with a Thr, Asp, Glu, or Arg residue;
(ii) the amino acid substitution at the position corresponding with amino acid residue Ser-710 is with a Gly, Ala, or Val residue;
(iii) the amino acid substitution at the position corresponding with amino acid residue Arg-722 is with a His or Lys residue; and/or
(iv) the amino acid substitution at the position corresponding with amino acid residue Thr-877 is with a Lys, His, or Arg residue.
17. The non-native glucosyltransferase of claim 6 , wherein:
(i) the amino acid substitution at the position corresponding with amino acid residue Val-1188 is with a Glu or Asp residue;
(ii) the amino acid substitution at the position corresponding with amino acid residue Met-1253 is with an Ile, Leu, Ala, or Val residue; and/or
(iii) the amino acid substitution at the position corresponding with amino acid residue Gln-957 is with a Pro residue.
18. The non-native glucosyltransferase of claim 8 , comprising an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of residues 55-960 of SEQ ID NO:4 or the amino acid sequence of residues 54-957 of SEQ ID NO:65.
19. The non-native glucosyltransferase of claim 18 , comprising an amino acid sequence that has at least 97% sequence identity to the amino acid sequence of residues 55-960 of SEQ ID NO:4 or the amino acid sequence of residues 54-957 of SEQ ID NO:65.
20. The non-native glucosyltransferase of claim 9 , comprising an amino acid sequence that has at least 92% sequence identity to the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:65.
21. The non-native glucosyltransferase of claim 20 , comprising an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:65.
22. The non-native glucosyltransferase of claim 20 , comprising an amino acid sequence that has at least 97% sequence identity to the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:65.
23. The non-native glucosyltransferase of claim 7 , wherein the insoluble alpha-glucan has a weight average degree of polymerization (DP w ) of at least 100.