# US 11,441,131 B2 · Heparosan synthases and use thereof for saccharide synthesis

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- Number: US 11,441,131 B2
- Document ID: US11441131B2
- Status: Granted
- Kind: B2
- Application number: 16/906,898
- Filed: 2020-06-19
- Published: 2021-02-11
- Granted: 2022-09-13
- Earliest claimed: 2019-06-21
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Xi Chen; Lan Na; Hai Yu; John B. McArthur
- Web page: https://patentgrep.com/patent/US11441131B2

## CPC

- C12N 9/1051: {Hexosyltransferases (2.4.1)}
- C12P 19/00: Preparation of compounds containing saccharide radicals (ketoaldonic acids C12P7/58)
- C12P 19/04: Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
- C12Y 204/01: Hexosyltransferases (2.4.1)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)}
- C12Y: ENZYMES

## Abstract

Heparosan synthase variants having improved expression levels, enhanced thermal stability, and/or reduced reverse glycosylation activity are provided. Methods for making oligosaccharides and polysaccharides, including heparin analogs and heparan sulfate analogs, are also described.

## Claims

### Claim 1

A Pasteurella multocida heparosan synthase 2 (PmHS2) variant consisting essentially of a polypeptide having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:1, which comprises a mutation at one or more positions corresponding to Y165, R184, L185, F187, I188, T189, D211, N237, K379, L462, T464, T466, V467, S485, G486, M487, and D489 in SEQ ID NO:1.

### Claim 2 (depends on 1)

The PmHS2 variant of claim 1, which consists of the polypeptide having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:1.

### Claim 3

A Pasteurella multocida heparosan synthase 2 (PmHS2) variant consisting essentially of a polypeptide having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:3, which comprises a mutation at one or more positions corresponding to Y176, R195, L196, F198, I199, T200, D222, N248, K390, L473, T475, T477, V478, S496, G497, M498, and D500 in SEQ ID NO:3.

### Claim 4 (depends on 3)

The PmHS2 variant of claim 3, which consists of the polypeptide having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:3.

### Claim 5 (depends on 3)

The PmHS2 variant of claim 3, which comprises one or more mutations corresponding to Y176A, R195A, L196A, F198A, I199A, T200A, D222N, N248A, K390A, L473A, T475A, T477A, V478A, S496N, S496A, G497 deletion, M498A, and D500N in SEQ ID NO:3.

### Claim 6 (depends on 5)

The PmHS2 variant of claim 5, which comprises the mutation D222N or D500N in SEQ ID NO:3.

### Claim 7 (depends on 5)

The PmHS2 variant of claim 5, which comprises the mutations D410N, D412N, and D500N in SEQ ID NO:3.

### Claim 8 (depends on 5)

The PmHS2 variant of claim 5, which comprises the mutation with G497 deletion in SEQ ID NO:3.

### Claim 9 (depends on 5)

The PmHS2 variant of claim 5, which comprises the mutations Y176A, R195A, L196A, F198A, I199A, T200A, and/or N248A in SEQ ID NO:3.

### Claim 10 (depends on 5)

The PmHS2 variant of claim 5, which comprises the mutations K390A, L473A, T475A, T477A, and/or V478A in SEQ ID NO:3.

### Claim 11 (depends on 5)

The PmHS2 variant of claim 5, which comprises the mutations S496N, S496A, and/or M498A in SEQ ID NO:3.

### Claim 12 (depends on 1)

A method of preparing an oligosaccharide, the method comprising:
forming a mixture comprising (i) PmHS2 variant of claim 1, (ii) an acceptor sugar, and (iii) a nucleotide sugar comprising a nucleotide moiety and a donor sugar moiety, and
maintaining the mixture under conditions sufficient to transfer the donor sugar moiety to the acceptor sugar.

### Claim 13 (depends on 12)

The method of claim 12, wherein the nucleotide sugar in the mixture is formed by converting a sugar starting material to the nucleotide sugar.

### Claim 14 (depends on 12)

The method of claim 12, wherein the acceptor sugar is selected from the group consisting of a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, a pentasaccharide, a hexasaccharide, a heptasaccharide, an octasaccharide, and a nonasaccharide.

### Claim 15 (depends on 3)

A method of preparing an oligosaccharide, the method comprising:
forming a mixture comprising (i) PmHS2 variant of claim 3, (ii) an acceptor sugar, and (iii) a nucleotide sugar comprising a nucleotide moiety and a donor sugar moiety, and
maintaining the mixture under conditions sufficient to transfer the donor sugar moiety to the acceptor sugar.

### Claim 16 (depends on 15)

The method of claim 15, wherein the nucleotide sugar in the mixture is formed by converting a sugar starting material to the nucleotide sugar.

### Claim 17 (depends on 15)

The method of claim 15, wherein the acceptor sugar is selected from the group consisting of a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, a pentasaccharide, a hexasaccharide, a heptasaccharide, an octasaccharide, and a nonasaccharide.