# US 11,306,331 B2 · Processes for the production of hydroxycinnamic acids using polypeptides having tyrosine ammonia lyase activity

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- Number: US 11,306,331 B2
- Document ID: US11306331B2
- Status: Granted
- Kind: B2
- Application number: 16/938,762
- Filed: 2020-07-24
- Published: 2020-12-24
- Granted: 2022-04-19
- Earliest claimed: 2014-07-14
- Assignees: CysBio ApS
- Applicants: CysBio ApS
- Inventors: Christian Bille Jendresen; Solvej Siedler; Steen Gustav Stahlhut; Alex Toftgaard Nielsen
- Web page: https://patentgrep.com/patent/US11306331B2

## CPC

- C12P 7/42: Hydroxy-carboxylic acids
- C12N 9/88: Lyases (4.)
- C12Y 403/01023: Tyrosine ammonia-lyase (4.3.1.23)
- 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

The present invention generally relates to the field of biotechnology as it applies to the production of hydroxycinnamic acids using polypeptides having tyrosine ammonia lyase activity. More particularly, the present invention pertains to polypeptides having tyrosine ammonia lyase activity and high substrate specificity towards tyrosine, which makes them particularly suitable in the production of p-coumaric acid and other hydroxycinnamic acids. The present invention thus provides processes for the production of p-coumaric acid and other hydroxycinnamic acids employing these polypeptides as well as recombinant host cells expressing same.

## Claims

### Claim 1

A method for producing a hydroxycinnamic acid of general formula I:
comprising:
contacting a recombinant host cell which has been genetically modified to express a heterologous polypeptide with a medium comprising a fermentable carbon substrate and/or a compound of the general formula II:
to produce a hydroxycinnamic acid of general formula I;
wherein said heterologous polypeptide is selected from:
i) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or 3; or
ii) a polypeptide comprising an amino acid sequence which has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or 3, wherein the polypeptide has tyrosine ammonia lyase activity;
wherein each of R1, R3, and R4 is hydrogen (—H), and R2 is hydroxyl (—OH), or
wherein R1 is methoxyl (—OCH3) or hydroxyl (—OH), R2 is hydroxyl (—OH), and each of R3 and R4 is hydrogen (—H); and
wherein said recombinant host cell is a bacterium or yeast.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the recombinant host cell is a bacterium.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the bacterium is of the genus Bacillus, Lactococcus, Lactobacillus, Clostridium, Corynebacterium, Geobacillus, Streptococcus, Pseudomonas, Streptomyces, Escherichia, Shigella, Acinetobacter, Citrobacter, Salmonella, Klebsiella, Enterobacter, Erwinia, Kluyvera, Serratia, Cedecea, Morganella, Hafnia, Edwardsiella, Providencia, Proteus, or Yersinia.

### Claim 4 (depends on 2)

The method according to claim 2, wherein the bacterium is of the genus Escherichia, Bacillus, Lactococcus, Lactobacillus, Corynebacterium, Streptomyces or Pseudomonas.

### Claim 5 (depends on 2)

The method according to claim 2, wherein the bacterium is selected from the group consisting of Escherichia coli, Lactococcus lactis, Bacillus subtitlis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus mojavensis, Streptomyces lividans, Streptomyces griseus, Streptomyces coelicolor, Corynebacterium glutamicum, and Pseudomonas putida.

### Claim 6 (depends on 2)

The method according to claim 2, wherein the bacterium is Escherichia coli, Lactococcus lactis or Bacillus subtitlis.

### Claim 7 (depends on 2)

The method according to claim 2, wherein the bacterium is Escherichia coli.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the recombinant host cell is a yeast.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the yeast is of the genus Saccharomyces, Pichia, Schizosacharomyces, Zygosaccharomyces, Hansenula, Pachyosolen, Kluyveromyces, Debaryomyces, Yarrowia, Candida, Cryptococcus, Komagataella, Lipomyces, Rhodospiridium, Rhodotorula, or Trichosporon.

### Claim 10 (depends on 8)

The method according to claim 8, wherein the yeast is of the genus Saccharomyces or Pichia.

### Claim 11 (depends on 8)

The method according to claim 8, wherein the yeast is selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, and Pichia kudriavzevii.

### Claim 12 (depends on 8)

The method according to claim 8, wherein the yeast is Saccharomyces cerevisiae.

### Claim 13 (depends on 1)

The method according to claim 1, wherein said recombinant host cell does not express a polypeptide having phenolic acid decarboxylase (PAD) activity.

### Claim 14 (depends on 1)

The method according to claim 1, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2 or 3.

### Claim 15

A method for producing a hydroxycinnamic acid of general formula I:
comprising:
a) contacting a compound of general formula II:
with a polypeptide to produce a hydroxycinnamic acid of general formula I, wherein the polypeptide is selected from:
i) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or 3; or
ii) a polypeptide comprising an amino acid sequence which has at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or 3, wherein the polypeptide has tyrosine ammonia lyase activity; and
b) measuring the concentration of said hydroxycinnamic acid of general formula I or recovering said hydroxycinnamic acid of general formula I;
wherein each of R1, R3, and R4 is hydrogen (—H), and R2 is hydroxyl (—OH), or
wherein R1 is methoxyl (—OCH3) or hydroxyl (—OH), R2 is hydroxyl (—OH), and each of R3 and R4 is hydrogen (—H).

### Claim 16

A method for producing a hydroxycinnamic acid of general formula I:
comprising:
a) contacting a recombinant host cell which has been genetically modified to express a heterologous polypeptide with a medium comprising a fermentable carbon substrate and/or a compound of the general formula II:
to produce a hydroxycinnamic acid of general formula I;
wherein said heterologous polypeptide is selected from:
i) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or 3; or
ii) a polypeptide comprising an amino acid sequence which has at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or 3, wherein the polypeptide has tyrosine ammonia lyase activity; and
b) measuring the concentration of said hydroxycinnamic acid of general formula I or recovering said hydroxycinnamic acid of general formula I;
wherein each of R1, R3, and R4 is hydrogen (—H), and R2 is hydroxyl (—OH), or
wherein R1 is methoxyl (—OCH3) or hydroxyl (—OH), R2 is hydroxyl (—OH), and each of R3 and R4 is hydrogen (—H).

### Claim 17 (depends on 16)

The method according to claim 16, wherein the recombinant host cell is a bacterium.

### Claim 18 (depends on 17)

The method according to claim 17, wherein the bacterium is selected from the group consisting of Escherichia coli, Lactococcus lactis, Bacillus subtitlis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus mojavensis, Streptomyces lividans, Streptomyces griseus, Streptomyces coelicolor, Corynebacterium glutamicum, and Pseudomonas putida.

### Claim 19 (depends on 17)

The method according to claim 17, wherein the bacterium is Escherichia coli.

### Claim 20 (depends on 16)

The method according to claim 16, wherein the recombinant host cell is a yeast.

### Claim 21 (depends on 20)

The method according to claim 20, wherein the yeast is selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, and Pichia kudriavzevii.

### Claim 22 (depends on 1)

The method according to claim 1, wherein the polypeptide comprises an amino acid sequence which has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or 3.

### Claim 23 (depends on 1)

The method according to claim 1, wherein the polypeptide comprises an amino acid sequence which has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or 3.