# US 2019/0169662 A1 · Variants of acetylserotonin o-methyltransferase and uses thereof

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- Number: US 2019/0169662 A1
- Document ID: US20190169662A1
- Status: Published application
- Kind: A1
- Application number: 16/302,124
- Filed: 2017-05-24
- Published: 2019-06-06
- Earliest claimed: 2016-05-24
- Assignees: Danmarks Tekniske Universitet
- Applicants: Danmarks Tekniske Universitet
- Inventors: Hao LUO
- Web page: https://patentgrep.com/patent/US20190169662A1

## CPC

- C12P 17/10: Nitrogen as only ring hetero atom
- C12N 9/1007: {Methyltransferases (general) (2.1.1.)}
- C12Y 201/01004: Acetylserotonin O-methyltransferase (2.1.1.4)
- 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)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

Described herein are variants of acetylserotonin O-methyltransferase (ASMT) as well as vectors and recombinant microbial host cells expressing such ASMT variant and their use in producing melatonin and related compounds. Preferred ASMT variants provide for a higher turnover of N-acetylserotonin into melatonin.

## Claims

### Claim 1

A variant of Homo sapiens acetylserotonin O-methyltransferase (hsASMT), the variant having at least 92% sequence identity to hsASMT (SEQ ID NO:1) and comprising one or more mutations in the segment corresponding to residues A258 to T272 in Homo sapiens ASMT (SEQ ID NO:1), providing for an increased catalytic activity in converting N-acetylserotonin to melatonin as compared to hsASMT.

### Claim 2 (depends on 1)

The variant ASMT of claim 1, having at least 50% increased catalytic activity in converting N-acetylserotonin to melatonin as compared to hsASMT.

### Claim 3 (depends on 1)

The variant ASMT of claim 1, having at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to Homo sapiens ASMT (SEQ ID NO:1).

### Claim 4 (depends on 1)

The variant ASMT of claim 1, comprising a mutation in a residue selected from the group consisting of A258, G260, and T272.

### Claim 5 (depends on 4)

The variant ASMT of claim 4, wherein the mutation is an amino acid substitution selected from the group consisting of
(a) A258E;
(b) G260D, G260N, G260L, and G260I; and
(c) T272A and T272G.

### Claim 6

A variant of hsASMT, the variant comprising SEQ ID NO:1, or a catalytically active fragment thereof, having a A258E, G260D or T272A mutation.

### Claim 7 (depends on 1)

A nucleic acid sequence encoding the variant ASMT of claim 1.

### Claim 8 (depends on 7)

A vector comprising the nucleic acid sequence of claim 7, optionally operably linked to one or more expression control sequences.

### Claim 9 (depends on 1)

A recombinant host cell comprising the variant ASMT of claim 1.

### Claim 10 (depends on 9)

The recombinant host cell of claim 9, which is a mammalian cell, a bacterial cell, a yeast cell, a filamentous fungal cell or an algal cell.

### Claim 11 (depends on 10)

The recombinant host cell of claim 10, which is an Escherichia cell.

### Claim 12 (depends on 9)

The recombinant host cell of claim 9, comprising a native or heterologous biosynthetic pathway for producing N-acetylserotonin and a native or heterologous pathway for recycling S-adenosyl-L-homocysteine (SAH) into S-adenosyl-L-methionine (SAM).

### Claim 13 (depends on 12)

The recombinant host cell of claim 12, comprising a deletion or downregulation of a gene encoding a cyclopropane fatty acyl phospholipid synthase.

### Claim 14

A recombinant host cell derived from an E. coli cell, comprising an ASMT, a native or heterologous biosynthetic pathway for producing N-acetylserotonin, a native or heterologous pathway for recycling SAH into SAM, and a deletion or downregulation of the cfa gene.

### Claim 15 (depends on 12)

A method of producing melatonin, comprising culturing the recombinant host cell of claim 12 in a medium comprising a carbon source, and, optionally, isolating melatonin.

### Claim 16 (depends on 14)

A method of producing melatonin, comprising culturing the recombinant host cell of claim 14 in a medium comprising a carbon source, and, optionally, isolating melatonin.

### Claim 17 (depends on 6)

A recombinant host cell comprising the variant ASMT of claim 6.

### Claim 18 (depends on 17)

The recombinant host cell of claim 17, comprising a native or heterologous biosynthetic pathway for producing N-acetylserotonin and a native or heterologous pathway for recycling SAH into SAM.

### Claim 19 (depends on 18)

The recombinant host cell of claim 18, comprising a deletion or downregulation of a gene encoding a cyclopropane fatty acyl phospholipid synthase.

### Claim 20 (depends on 18)

A method of producing melatonin, comprising culturing the recombinant host cell of claim 18 in a medium comprising a carbon source, and, optionally, isolating melatonin.