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US-12618051-B2 - Engineered alpha-1,3 branching enzymes

US12618051B2US 12618051 B2US12618051 B2US 12618051B2US-12618051-B2

Abstract

Disclosed herein are glucosyltransferases with modified amino acid sequences. Such engineered enzymes have modified alpha-1,3-branching activity. Further disclosed are reactions and methods in which engineered glucosyltransferases can be used to add one or more alpha-1,3 branches to a suitable acceptor such as glucan.

Inventors

  • Yougen Li
  • Ellen D. Semke

Assignees

  • Nutrition & Biosciences USA 4, Inc.

Dates

Publication Date
20260505
Application Date
20200708

Claims (18)

  1. 1 . A non-native glucosyltransferase comprising at least one amino acid substitution at a position corresponding with amino acid residue Ser-734, Ile-735, Ile-737, Ile-740, Asp-744, His-771, Val-772, Ser-773, Val-775, Ser-778, Ala-779, Asp-780, Ile-845, Val-846, Asp-852, Ile-853, Asp-855, Ser-858, Asn-859, Ala-1232, Tyr-1234, or Asn-1237 of SEQ ID NO:4, wherein the non-native glucosyltransferase is capable of forming at least one alpha-1,3 branch from an acceptor molecule, wherein said at least one alpha-1,3 branch consists of a single glucose unit, wherein the non-native glucosyltransferase comprises an amino acid sequence that shares at least 80% sequence identity with residues 477-1322 of SEQ ID NO:2, and wherein said amino acid sequence does not share 100% sequence identity with residues 477-1322 of SEQ ID NO:2.
  2. 2 . The non-native glucosyltransferase of claim 1 , wherein: the amino acid substitution at the position corresponding with amino acid residue Ser-734 is with a Cys, Asp, Gly, His, Lys, Leu, Met, Asn, Thr, or Val residue; the amino acid substitution at the position corresponding with amino acid residue Ile-735 is with an Ala, Leu, or Val residue; the amino acid substitution at the position corresponding with amino acid residue Ile-737 is with an Ala, Cys, Asp, Gly, His, Leu, Met, Asn, Ser, Val, Trp, or Tyr residue; the amino acid substitution at the position corresponding with amino acid residue Ile-740 is with an Ala, Leu, or Val residue; the amino acid substitution at the position corresponding with amino acid residue Asp-744 is with an Ala, Cys, Glu, Phe, Gly, His, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr residue; the amino acid substitution at the position corresponding with amino acid residue His-771 is with an Ala residue; the amino acid substitution at the position corresponding with amino acid residue Val-772 is with an Ala or Leu residue; the amino acid substitution at the position corresponding with amino acid residue Ser-773 is with an Ala or Asn residue; the amino acid substitution at the position corresponding with amino acid residue Val-775 is with an Ala residue; the amino acid substitution at the position corresponding with amino acid residue Ser-778 is with a Trp residue; the amino acid substitution at the position corresponding with amino acid residue Ala-779 is with an Asp, Gly, or Ser residue; the amino acid substitution at the position corresponding with amino acid residue Asp-780 is with an Ala, Gln, or Tyr residue; the amino acid substitution at the position corresponding with amino acid residue Ile-845 is with an Ala or Phe residue; the amino acid substitution at the position corresponding with amino acid residue Val-846 is with an Ala, Ile, or Thr residue; the amino acid substitution at the position corresponding with amino acid residue Asp-852 is with an Ala, Glu, Leu, or Asn residue; the amino acid substitution at the position corresponding with amino acid residue Ile-853 is with a Val residue; the amino acid substitution at the position corresponding with amino acid residue Asp-855 is with an Ala, Gly, or Ser residue; the amino acid substitution at the position corresponding with amino acid residue Ser-858 is with an Ala, Gly, Gln, or Arg residue; the amino acid substitution at the position corresponding with amino acid residue Asn-859 is with an Ala, Asp, Glu, Lys, Ser, or Thr residue; the amino acid substitution at the position corresponding with amino acid residue Ala-1232 is with a Cys, Asp, Glu, Phe, Gly, His, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, or Tyr residue; the amino acid substitution at the position corresponding with amino acid residue Tyr-1234 is with a Cys, Glu, His, Leu, Met, Thr, Val, or Trp residue; and/or the amino acid substitution at the position corresponding with amino acid residue Asn-1237 is with an Asp or Gly residue.
  3. 3 . The non-native glucosyltransferase of claim 1 , comprising two or more amino acid substitutions, wherein at least one of the substitutions is at a position corresponding with amino acid residue Ser-734, Ile-735, Ile-737, Ile-740, Asp-744, His-771, Val-772, Ser-773, Val-775, Ser-778, Ala-779, Asp-780, Ile-845, Val-846, Asp-852, Ile-853, Asp-855, Ser-858, Asn-859, Ala-1232, Tyr-1234, or Asn-1237 of SEQ ID NO:4.
  4. 4 . The non-native glucosyltransferase of claim 3 , wherein at least one of the substitutions is at a position corresponding with amino acid residue Ser-734, Ile-735, Ser-778, Asp-780, Ile-845, Asp-852, Ile-853, Asp-855, Ala-1232, or Tyr-1234 of SEQ ID NO:4.
  5. 5 . The non-native glucosyltransferase of claim 1 , wherein the acceptor molecule comprises glucan.
  6. 6 . The non-native glucosyltransferase of claim 5 , wherein the glucan is soluble glucan.
  7. 7 . The non-native glucosyltransferase of claim 5 , wherein the glucan comprises alpha-glucan.
  8. 8 . The non-native glucosyltransferase of claim 7 , wherein the alpha-glucan comprises dextran.
  9. 9 . The non-native glucosyltransferase of claim 1 , wherein the amino acid sequence of the non-native glucosyltransferase only differs from SEQ ID NO:2 at the substitution position(s), wherein the alpha-1,3 branching activity of the non-native glucosyltransferase is at least 50% of the alpha-1,3 branching activity of SEQ ID NO:2.
  10. 10 . The non-native glucosyltransferase of claim 1 , wherein the non-native glucosyltransferase comprises an amino acid sequence that shares at least 90% sequence identity with residues 477-1322 of SEQ ID NO:2.
  11. 11 . The non-native glucosyltransferase of claim 1 , wherein the non-native glucosyltransferase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2.
  12. 12 . A polynucleotide comprising a nucleotide sequence encoding a non-native glucosyltransferase according to claim 1 .
  13. 13 . A reaction composition comprising water, sucrose, an acceptor molecule, and a non-native glucosyltransferase according to claim 1 .
  14. 14 . The reaction composition of claim 13 , wherein the acceptor molecule comprises dextran.
  15. 15 . A method of producing a glucan composition that comprises at least one alpha-1,3 branch, the method comprising: (a) contacting at least water, sucrose, a glucan substrate, and a non-native glucosyltransferase according to claim 1 , whereby a glucan composition comprising at least one alpha-1,3 branch is produced; and (b) optionally isolating the glucan composition produced in step (a).
  16. 16 . The polynucleotide of claim 12 , wherein one or more regulatory sequences are operably linked to the nucleotide sequence.
  17. 17 . The polynucleotide of claim 16 , wherein said one or more regulatory sequences include a promoter sequence.
  18. 18 . The non-native glucosyltransferase of claim 4 , wherein: the amino acid substitution at the position corresponding with amino acid residue Ser-734 is with a Cys residue; the amino acid substitution at the position corresponding with amino acid residue Ile-735 is with a Val residue; the amino acid substitution at the position corresponding with amino acid residue Ser-778 is with a Trp residue; the amino acid substitution at the position corresponding with amino acid residue Asp-780 is with a Tyr residue; the amino acid substitution at the position corresponding with amino acid residue Ile-845 is with a Phe residue; the amino acid substitution at the position corresponding with amino acid residue Asp-852 is with a Glu residue; the amino acid substitution at the position corresponding with amino acid residue Ile-853 is with a Val residue; the amino acid substitution at the position corresponding with amino acid residue Asp-855 is with a Gly residue; the amino acid substitution at the position corresponding with amino acid residue Ala-1232 is with a Gly, Met, Ser, or Val residue; and/or the amino acid substitution at the position corresponding with amino acid residue Tyr-1234 is with a Trp residue.

Description

This application is a National Stage application of International Application No. PCT/US2020/041093 (filed Jul. 8, 2020), which claims the benefit of U.S. Provisional Application No. 62/871,796 (filed Jul. 9, 2019), both of which prior applications are incorporated herein by reference in their entirety. FIELD The present disclosure is in the field of enzyme catalysis. For example, the disclosure pertains to alpha-1,3 branching enzymes with modified amino acid sequences. Such modified enzymes can be used to synthesize glucan products with at least one alpha-1,3 branch, for example. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 20200702_CL6645PCT_SequenceListing.txt created on Jul. 2, 2020, and having a size of about 91000 bytes and is filed concurrently with the specification. The sequence listing contained in this ASCII-formatted document is part of the specification and is herein incorporated by reference in its entirety. BACKGROUND The enzymatic addition of alpha-1,3 branches to acceptor molecules has been reported. Early biochemical studies of Leuconostoc mesenteroides and Streptococcus mutans suggested that alpha-1,3-branching enzymes exist and play a role, for example, in branching of exopolysaccharides (e.g., Vote and Robyt, 1983, Carb. Res. 119:141-156; Remaud et al., 1992, J. Carb. Chem. 11:359-378; Walker, 1980, Carb. Res. 82:404-410). More recently, Vuillemin et al. (2016, J. Biol. Chem. 14:7687-7702) and Remaud-Simeon et al. (U.S. Patent Appl. Publ. No. 2016/0136199) directly identified alpha-1,3-branching enzymes from Leuconostoc citreum and Leuconostoc fallax and use thereof to introduce alpha-1,3 branches to dextran. These enzymes were characterized as members of the GH70 family of glucosyltransferases (sucrases), which include enzymes with other activities such as alpha-1,2-branching or alpha-glucan polymerization (Vuillemin et al., ibid.). While advances have been made in understanding and using alpha-1,3-branching enzymes, less attention appears to have been drawn to modulating the activity of these enzymes. Such modulated enzymes could prove to be a valuable tool for providing glucan products with defined structure for specific applications. Addressing this technological gap, disclosed herein are alpha-1,3-branching enzymes with modified amino acid sequences that have altered alpha-1,3-branching activity. SUMMARY In one embodiment, the present disclosure concerns a composition comprising a non-native glucosyltransferase comprising at least one amino acid substitution at a position corresponding with amino acid residue Ser-734, Ile-735, Ile-737, Ile-740, Asp-744, His-771, Val-772, Ser-773, Val-775, Ser-778, Ala-779, Asp-780, Ile-845, Val-846, Asp-852, Ile-853, Asp-855, Ser-858, Asn-859, Ala-1232, Tyr-1234, or Asn-1237 of SEQ ID NO:4, wherein the non-native glucosyltransferase is capable of forming at least one alpha-1,3 branch from an acceptor molecule. In another embodiment, the present disclosure concerns a polynucleotide comprising a nucleotide sequence encoding a non-native glucosyltransferase as disclosed herein, optionally wherein one or more regulatory sequences are operably linked to the nucleotide sequence, and preferably wherein the one or more regulatory sequences include a promoter sequence. In another embodiment, the present disclosure concerns a reaction composition comprising water, sucrose, an acceptor molecule, and a non-native glucosyltransferase as disclosed herein. In another embodiment, the present disclosure concerns a method of producing a glucan composition that comprises at least one alpha-1,3 branch, the method comprising: (a) contacting at least water, sucrose, a glucan substrate, and a non-native glucosyltransferase enzyme according to claim 1, whereby a glucan composition comprising at least one alpha-1,3 branch is produced; and (b) optionally isolating the glucan composition produced in step (a). In another embodiment, the present disclosure concerns a method of preparing a polynucleotide sequence encoding a non-native glucosyltransferase, the method comprising: (a) identifying a polynucleotide sequence encoding a parent glucosyltransferase that (i) comprises an amino acid sequence that is at least about 40% identical to SEQ ID NO:3 or positions 477-1322 of SEQ ID NO:2, and (ii) is capable of forming at least one alpha-1,3 branch from an acceptor molecule; and (b) modifying the polynucleotide sequence identified in step (a) to substitute at least one amino acid of the parent glucosyltransferase at a position corresponding with amino acid residue Ser-734, Ile-735, Ile-737, Ile-740, Asp-744, His-771, Val-772, Ser-773, Val-775, Ser-778, Ala-779, Asp-780, Ile-845, Val-846, Asp-852, Ile-853, Asp-855, Ser-858, Asn-859, Ala-1232, Tyr-1234, or Asn-1237 of SEQ ID NO:4, thereby providing a polynucleotide sequence encoding a n