# US 11,028,373 B2 · Engineered glucosyltransferases

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- Number: US 11,028,373 B2
- Document ID: US11028373B2
- Status: Granted
- Kind: B2
- Application number: 16/381,010
- Filed: 2019-04-11
- Published: 2019-09-26
- Granted: 2021-06-08
- Earliest claimed: 2016-09-14
- Assignees: Nutrition & Biosciences USA 4, Inc.
- Applicants: Nutrition & Biosciences USA 4, Inc.
- Inventors: Yougen Li; Mark S. Payne; Jared B. Parker; Slavko Kralj; Veli Alkan; Richard R. Bott; Robert Dicosimo; Qiong Cheng; Ellen D. Semke; Susan Marie Hennessey
- Web page: https://patentgrep.com/patent/US11028373B2

## CPC

- C12N 9/1051: {Hexosyltransferases (2.4.1)}
- C08B 37/0009: {alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof}
- C12P 19/04: Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
- C12P 19/18: produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C08B: POLYSACCHARIDES; DERIVATIVES THEREOF (polysaccharides containing less than six saccharide radicals attached to each other by glycosidic linkages C07H; fermentation or enzyme-using processes C12P19/00; sugar industry C13; production of cellulose D21)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## 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.

## Claims

### Claim 1

A non-native glucosyltransferase comprising an amino acid substitution at a position corresponding with amino acid residue Phe-607 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) position, 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.

### Claim 2 (depends on 1)

The non-native glucosyltransferase of claim 1, wherein
the amino acid substitution at the position corresponding with amino acid residue Phe-607 is with a Trp, Tyr, or Asn residue.

### Claim 3 (depends on 1)

The non-native glucosyltransferase of claim 1, two or more amino acid substitutions.

### Claim 4 (depends on 3)

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 Ala-510 of SEQ ID NO:62.

### Claim 5 (depends on 3)

The non-native glucosyltransferase of claim 3, wherein at least one of the two or more amino acid substitutions is at a position corresponding with amino acid residue Leu-428, Ala-472, Leu-513, Met-529, Asn-613, Gln-616, Ser-631, Gly-633, Phe-634, Thr-635, or Phe-951 of SEQ ID NO:62.

### Claim 6 (depends on 1)

The non-native glucosyltransferase of claim 1, wherein the alpha-glucan is insoluble and comprises at least about 50% alpha-1,3 linkages.

### Claim 7 (depends on 1)

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.

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 6)

The non-native glucosyltransferase of claim 6, wherein the alpha-glucan comprises at least about 90% alpha-1,3 linkages.

### Claim 10 (depends on 8)

The non-native glucosyltransferase of claim 8, 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.

### Claim 11 (depends on 1)

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.

### Claim 12 (depends on 1)

A reaction composition comprising water, sucrose, and a non-native glucosyltransferase according to claim 1.

### Claim 13 (depends on 1)

A method of producing alpha-glucan comprising:
contacting at least water, sucrose, and a non-native glucosyltransferase enzyme according to claim 1, whereby alpha-glucan is produced.

### Claim 14 (depends on 7)

The non-native glucosyltransferase of claim 7, 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.

### Claim 15 (depends on 14)

The non-native glucosyltransferase of claim 14, 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.

### Claim 16 (depends on 15)

The non-native glucosyltransferase of claim 15, 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.

### Claim 17 (depends on 16)

The non-native glucosyltransferase of claim 16, 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.

### Claim 18 (depends on 10)

The non-native glucosyltransferase of claim 10, 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.

### Claim 19 (depends on 18)

The non-native glucosyltransferase of claim 18, 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.

### Claim 20 (depends on 19)

The non-native glucosyltransferase of claim 19, 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.

### Claim 21 (depends on 1)

The non-native glucosyltransferase of claim 1, wherein the alpha-glucan has a weight average degree of polymerization (DPw) of at least 100.

### Claim 22 (depends on 13)

The method of claim 13, further comprising isolating the alpha-glucan.