Engineered glucosyltransferases
View Patent ↗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 one amino acid substitution at a position corresponding with amino acid residue Ala-510 of SEQ ID NO:62
wherein the non-native glucosyltransferase synthesizes alpha-glucan comprising 1,3-linkages, and
wherein the non-native glucosyltransferase has:
(i) an alpha-glucan yield that is higher than the alpha-glucan yield of a second glucosyltransferase that only differs from the non-native glucosyltransferase at the substitution position(s), and/or
(ii) a leucrose yield that is lower than the leucrose yield of the second glucosyltransferase;
wherein the non-native glucosyltransferase comprises a catalytic domain that is at least about 90% identical to residues 55-960 of SEQ ID NO:4, residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20.
2. The non-native glucosyltransferase of claim 1 , wherein the amino acid substitution at the position corresponding with amino acid residue Ala-510 is with a Glu, Ile, Val, or Asp residue.
3. The non-native glucosyltransferase of claim 1 , comprising two or more amino acid substitutions.
4. The non-native glucosyltransferase of claim 3 , wherein one of the two or more amino acid substitutions is at a position corresponding with amino acid residue Phe-607 of SEQ ID NO:62.
5. The non-native glucosyltransferase of claim 1 , wherein the alpha-glucan is insoluble and comprises at least about 50% alpha-1,3 linkages, and optionally wherein the alpha-glucan has a weight average degree of polymerization (DP w ) of at least 100.
6. The non-native glucosyltransferase of claim 1 , comprising a catalytic domain that is at least about 92% identical to residues 55-960 of SEQ ID NO:4, residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20.
7. The non-native glucosyltransferase of claim 1 , comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO:4, SEQ ID NO:65, SEQ ID NO:30, SEQ ID NO:28, or SEQ ID NO:20.
8. The non-native glucosyltransferase of claim 5 , wherein the non-native glucosyltransferase synthesizes insoluble alpha-1,3-glucan having at least about 90% alpha-1,3-linkages.
9. The non-native glucosyltransferase of claim 7 , comprising an amino acid sequence that is at least about 92% identical to SEQ ID NO:4, SEQ ID NO:65, SEQ ID NO:30, SEQ ID NO:28, or SEQ ID NO:20.
10. The non-native glucosyltransferase of claim 1 , wherein the alpha-glucan yield is at least about 10% higher than the alpha-glucan yield of the second glucosyltransferase.
11. A reaction composition comprising water, sucrose, and a non-native glucosyltransferase according to claim 1 .
12. A method of producing alpha-glucan comprising:
(a) contacting at least water, sucrose, and a non-native glucosyltransferase enzyme according to claim 1 , whereby alpha-glucan is produced; and
(b) optionally, isolating the alpha-glucan produced in step (a).
13. The non-native glucosyltransferase of claim 6 , comprising a catalytic domain that is at least about 93% identical to residues 55-960 of SEQ ID NO:4, residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20.
14. The non-native glucosyltransferase of claim 13 , comprising a catalytic domain that is at least about 95% identical to residues 55-960 of SEQ ID NO:4, residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20.
15. The non-native glucosyltransferase of claim 14 , comprising a catalytic domain that is at least about 96% identical to residues 55-960 of SEQ ID NO:4, residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20.
16. The non-native glucosyltransferase of claim 15 , comprising a catalytic domain that is at least about 97% identical to residues 55-960 of SEQ ID NO:4, residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20.
17. The non-native glucosyltransferase of claim 9 , comprising an amino acid sequence that is at least about 93% identical to SEQ ID NO:4, SEQ ID NO:65, SEQ ID NO:30, SEQ ID NO:28, or SEQ ID NO:20.
18. The non-native glucosyltransferase of claim 17 , comprising an amino acid sequence that is at least about 95% identical to SEQ ID NO:4, SEQ ID NO:65, SEQ ID NO:30, SEQ ID NO:28, or SEQ ID NO:20.
19. The non-native glucosyltransferase of claim 18 , comprising an amino acid sequence that is at least about 96% identical to SEQ ID NO:4, SEQ ID NO:65, SEQ ID NO:30, SEQ ID NO:28, or SEQ ID NO:20.
20. The non-native glucosyltransferase of claim 19 , comprising an amino acid sequence that is at least about 97% identical to SEQ ID NO:4, SEQ ID NO:65, SEQ ID NO:30, SEQ ID NO:28, or SEQ ID NO:20.