Alpha-glucose-1-phosphate synthesis from sucrose and glucan synthesis using glucan phosphorylases

Patent metadata

Field	Value
Publication number	US-2021163902-A1
Application number	US-201917261937-A
Country	US
Kind code	A1
Filing date	Jul 18, 2019
Priority date	Jul 23, 2018
Publication date	Jun 3, 2021
Grant date	—

Abstract

Reactions are disclosed herein comprising water, alpha-glucose-1-phosphate (alpha-G1P), an acceptor molecule, and an alpha-1,4-glucan phosphorylase. Novel alpha-1,4-glucan phosphorylase enzymes are also disclosed. Additional disclosures herein regard sucrose phosphorylase enzymes and methods of use thereof to produce alpha-G1P.

First claim

What is claimed is:

1 . A method for producing alpha-1,4-glucan, said method comprising:
(a) contacting at least water, alpha-glucose-1-phosphate (alpha-G1P), an acceptor molecule, and an alpha-1,4-glucan phosphorylase comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:32, SEQ ID NO:30, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, or SEQ ID NO:52, wherein alpha-1,4-glucan is produced; and (b) optionally, isolating the alpha-1,4-glucan produced in step (a).

2 . The method of claim 1 , wherein the acceptor molecule comprises a monosaccharide, disaccharide, or oligosaccharide.

3 . The method of claim 1 , wherein the acceptor molecule comprises alpha-1,4 glycosidic linkages and has a degree of polymerization (DP) of 2 to 35.

4 . The method of claim 1 , wherein the acceptor molecule comprises a polysaccharide.

5 . The method of claim 1 , wherein the alpha-1,4-glucan has at least about 90% alpha-1,4 glycosidic linkages.

6 . The method of claim 5 , wherein the alpha-1,4-glucan has at least about 99% alpha-1,4 glycosidic linkages.

7 . The method of claim 1 , wherein the degree of polymerization (DP) of the alpha-1,4-glucan is at least 3.

8 . The method of claim 1 , wherein said alpha-G1P is provided in step (a) by providing a second reaction, wherein the products of the second reaction comprise alpha-G1P.

9 . The method of claim 8 , wherein the second reaction is provided in the same vessel in which step (a) is performed, and wherein the second reaction is performed before and/or continuously with step (a).

10 . The method of claim 8 , wherein the second reaction produces alpha-G1P by contacting (i) water, (ii) inorganic phosphate, (iii) a glucose-comprising disaccharide, oligosaccharide, or polysaccharide, and (iv) a phosphorylase that phosphorolyzes the disaccharide, oligosaccharide, or polysaccharide.

11 . The method of claim 10 , wherein the second reaction comprises:
(I) water, inorganic phosphate, sucrose, and a sucrose phosphorylase; (II) water, inorganic phosphate, starch, and a starch phosphorylase; or (III) water, inorganic phosphate, cellodextrin, and a cellodextrin phosphorylase.

12 . The method of claim 10 , wherein the second reaction comprises water, inorganic phosphate, sucrose, and a sucrose phosphorylase comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:8, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24.

13 . A reaction composition comprising at least water, alpha-glucose-1-phosphate (alpha-G1P), an acceptor molecule, and an alpha-1,4-glucan phosphorylase comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:32, SEQ ID NO:30, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, or SEQ ID NO:52, wherein the alpha-1,4-glucan phosphorylase synthesizes alpha-1,4-glucan.

14 . A composition comprising an isolated alpha-1,4-glucan phosphorylase that comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, or SEQ ID NO:52.

15 . A composition comprising a polynucleotide that comprises a nucleotide sequence encoding an isolated alpha-1,4-glucan phosphorylase according to claim 14 , optionally wherein one or more regulatory sequences are operably linked to the nucleotide sequence, and preferably wherein said one or more regulatory sequences include a promoter sequence.

16 . A method for producing alpha-glucose-1-phosphate (alpha-G1P), said method comprising:
(a) contacting at least water, inorganic phosphate, sucrose, and a sucrose phosphorylase comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:20, or SEQ ID NO:22, wherein alpha-G1P is produced; and (b) optionally, isolating the alpha-G1P produced in step (a).

17 . A reaction composition comprising at least water, inorganic phosphate, sucrose, and a sucrose phosphorylase comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:20, or SEQ ID NO:22, wherein the sucrose phosphorylase produces alpha-glucose-1-phosphate (alpha-G1P).

18 . A composition comprising an isolated sucrose phosphorylase that comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:22.

19 . A composition comprising a polynucleotide that comprises a nucleotide sequence encoding an isolated sucrose phosphorylase according to claim 18 , optionally wherein one or more regulatory sequences are operably linked to the nucleotide sequence, and preferably wherein said one or more regulatory sequences include a promoter sequence.

Assignees

• Danisco Us Inc

Inventors

• Kralj Slavko
• Yu Zheyong
• Zhang Zhenghong

Classifications

• C12P19/02

  Monosaccharides (2-ketogulonic acid C12P7/60) · CPC title

• C12Y204/01001

  Phosphorylase (2.4.1.1) · CPC title

• C12P19/04 Primary

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

• C12Y204/01007

  Sucrose phosphorylase (2.4.1.7) · CPC title

• C12N9/1051 Primary

  Hexosyltransferases (2.4.1) · CPC title