Engineered glucosyltransferases

Patent metadata

Field	Value
Publication number	US-10865393-B2
Application number	US-201816127293-A
Country	US
Kind code	B2
Filing date	Sep 11, 2018
Priority date	Sep 13, 2017
Publication date	Dec 15, 2020
Grant date	Dec 15, 2020

Abstract

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.

First claim

What is claimed is:

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.

Assignees

• Dupont Ind Biosciences Usa Llc

Inventors

• Li Yougen
• Semke Ellen D
• Cheng Qiong
• Lai Jian Ping

Classifications

• C12Y204/01005

  Dextransucrase (2.4.1.5) · CPC title

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title

• C12P19/18

  produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins · CPC title

• C12Y204/01

  Hexosyltransferases (2.4.1) · CPC title

• C12P19/04

  Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds · CPC title