Engineered glucosyltransferases
Disclosed herein are glucosyltransferases with modified amino acid sequences. Such engineered enzymes synthesize alpha-glucan products having increased molecular weight. 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 Asn-531, Arg-534, Thr-563, Gln-588, Ile-591, Lys-593, Ile-608, Ala-610, Leu-661, Arg-722, Thr-728, Met-732, Arg-741, Tyr-848, or Ile-1453 of SEQ ID NO:62, wherein the non-native glucosyltransferase synthesizes insoluble alpha-glucan comprising 1,3-linkages, and the molecular weight of said insoluble alpha-glucan is higher 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);
wherein the non-native glucosyltransferase comprises a catalytic domain that is at least 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:
(i) the amino acid substitution at the position corresponding with amino acid residue Asn-531 is with a Gly, Leu, or Met residue;
(ii) the amino acid substitution at the position corresponding with amino acid residue Arg-534 is with a Lys, Gly, Ile, Leu, or Met residue;
(iii) the amino acid substitution at the position corresponding with amino acid residue Thr-563 is with an Ala residue;
(iv) the amino acid substitution at the position corresponding with amino acid residue Gln-588 is with a Leu residue;
(v) the amino acid substitution at the position corresponding with amino acid residue Ile-591 is with a Val, Lys, or Arg residue;
(vi) the amino acid substitution at the position corresponding with amino acid residue Lys-593 is with a Met residue;
(vii) the amino acid substitution at the position corresponding with amino acid residue Ile-608 is with a Tyr residue;
(viii) the amino acid substitution at the position corresponding with amino acid residue Ala-610 is with a Cys or Thr residue;
(ix) the amino acid substitution at the position corresponding with amino acid residue Leu-661 is with a Pro residue;
(x) the amino acid substitution at the position corresponding with amino acid residue Arg-722 is with a His or Asn residue;
(xi) the amino acid substitution at the position corresponding with amino acid residue Thr-728 is with a Ser residue;
(xii) the amino acid substitution at the position corresponding with amino acid residue Met-732 is with a Leu residue;
(xiii) the amino acid substitution at the position corresponding with amino acid residue Arg-741 is with a Ser, Ala, Pro, Gln, or Thr residue;
(xiv) the amino acid substitution at the position corresponding with amino acid residue Tyr-848 is with a Glu residue; and/or
(xv) the amino acid substitution at the position corresponding with amino acid residue Ile-1453 is with a Gly or Met residue.
3. The non-native glucosyltransferase of claim 1 , comprising at least one amino acid substitution at a position corresponding with amino acid residue Gln-588, Arg-741, or Arg-722 of SEQ ID NO:62.
4. The non-native glucosyltransferase of claim 3 , comprising two or more amino acid substitutions at positions corresponding with amino acid residues Gln-588, Arg-741, or Arg-722 of SEQ ID NO:62.
5. The non-native glucosyltransferase of claim 1 , wherein the insoluble alpha-glucan comprises at least 50% alpha-1,3 linkages.
6. The non-native glucosyltransferase of claim 5 , wherein the insoluble alpha-glucan has a DP w of at least 100.
7. The non-native glucosyltransferase of claim 1 , comprising a catalytic domain that is at least 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.
8. The non-native glucosyltransferase of claim 1 , comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:4, SEQ ID NO:65, SEQ ID NO:30, SEQ ID NO:28, or SEQ ID NO:20.
9. The non-native glucosyltransferase of claim 5 , wherein the insoluble alpha-glucan comprises at least 90% alpha-1,3 linkages.
10. The non-native glucosyltransferase of claim 1 , wherein the molecular weight of said insoluble alpha-glucan is at least 10% higher than the molecular weight of insoluble alpha-glucan synthesized by said second glucosyltransferase.
11. A polynucleotide comprising a nucleotide sequence encoding a non-native glucosyltransferase according to claim 1 , wherein one or more regulatory sequences are operably linked to the nucleotide sequence.
12. A reaction composition comprising water, sucrose, and a non-native glucosyltransferase according to claim 1 .
13. A method of producing insoluble alpha-glucan comprising:
contacting at least water, sucrose, and a non-native glucosyltransferase enzyme according to 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 polynucleotide of claim 11 , wherein said one or more regulatory sequences include a promoter sequence.
16. The non-native glucosyltransferase of claim 3 , wherein:
(i) the amino acid substitution at the position corresponding with amino acid residue Gln-588 is with a Leu residue,
(ii) the amino acid substitution at the position corresponding with amino acid residue Arg-741 is with a Ser residue, and/or
(iii) the amino acid substitution at the position corresponding with amino acid residue Arg-722 is with a His residue.
17. The non-native glucosyltransferase of claim 16 , comprising two or more amino acid substitutions at positions corresponding with amino acid residues Gln-588, Arg-741, or Arg-722 of SEQ ID NO:62.
18. The non-native glucosyltransferase of claim 6 , wherein the insoluble alpha-glucan has a DP w of at least 650.
19. The non-native glucosyltransferase of claim 7 , comprising a catalytic domain that is at least 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.
20. The non-native glucosyltransferase of claim 19 , comprising a catalytic domain that is at least 95% identical to residues 55-960 of SEQ ID NO:4 or residues 54-957 of SEQ ID NO:65.
21. The non-native glucosyltransferase of claim 19 , comprising a catalytic domain that is at least 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.
22. The non-native glucosyltransferase of claim 21 , comprising a catalytic domain that is at least 97% identical to residues 55-960 of SEQ ID NO:4 or residues 54-957 of SEQ ID NO:65.
23. The non-native glucosyltransferase of claim 8 , comprising an amino acid sequence that is at least 92% identical to SEQ ID NO:4, SEQ ID NO:65, SEQ ID NO:30, SEQ ID NO:28, or SEQ ID NO:20.
24. The non-native glucosyltransferase of claim 23 , comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:4, SEQ ID NO:65, SEQ ID NO:30, SEQ ID NO:28, or SEQ ID NO:20.
25. The non-native glucosyltransferase of claim 24 , comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:4 or SEQ ID NO:65.
26. The non-native glucosyltransferase of claim 24 , comprising an amino acid sequence that is at least 97% identical to SEQ ID NO:4, SEQ ID NO:65, SEQ ID NO:30, SEQ ID NO:28, or SEQ ID NO:20.
27. The non-native glucosyltransferase of claim 26 , comprising an amino acid sequence that is at least 97% identical to SEQ ID NO:4 or SEQ ID NO:65.