# US 10,870,843 B2 · Engineered phenylalanine ammonia lyase polypeptides

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- Number: US 10,870,843 B2
- Document ID: US10870843B2
- Status: Granted
- Kind: B2
- Application number: 16/601,993
- Filed: 2019-10-15
- Published: 2020-01-30
- Granted: 2020-12-22
- Earliest claimed: 2013-04-18
- Assignees: Codexis, Inc.
- Applicants: Codexis, Inc.
- Inventors: Gjalt W. Huisman; Nicholas J. Agard; Benjamin Mijts; Jonathan Vroom; Xiyun Zhang
- Web page: https://patentgrep.com/patent/US10870843B2

## CPC

- C12N 9/88: Lyases (4.)
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12Y 403/01005: Phenylalanine ammonia-lyase (4.3.1.5) (C12Y403/01023 - C12Y403/01025 takes precedence)
- C12Y 403/01024: Phenylalanine ammonia-lyase (4.3.1.24)
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Y: ENZYMES
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present invention provides engineered phenylalanine ammonia-lyase (PAL) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered phenylalanine ammonia-lyase (PAL) polypeptides.

## Claims

### Claim 1

An engineered polynucleotide encoding a polypeptide comprising an amino acid sequence with at least 95% sequence identity to reference sequence SEQ ID NO:24, wherein said polypeptide comprises a mutation at position 509, wherein said positions are numbered with reference to SEQ ID NO: 24.

### Claim 2 (depends on 1)

The engineered polynucleotide of claim 1, wherein said encoded engineered polypeptide further comprises at least one amino acid residue differences as compared to SEQ ID NO:24, wherein the amino acid residue differences are at one or more amino acid positions selected from 20, 24, 27, 39, 43, 45, 47, 54, 58, 59, 62, 70, 73, 80, 82, 91, 94, 98, 104, 105, 110, 112, 115, 117, 118, 119, 121, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 134, 135, 139, 140, 141, 142, 143, 144, 145, 146, 147, 149, 150, 151, 153, 154, 156, 157, 158, 159, 172, 174, 175, 176, 177, 178, 180, 187, 191, 195, 199, 205, 206, 210, 212, 213, 214, 232, 240, 243, 245, 247, 248, 250, 256, 257, 266, 270, 275, 278, 279, 285, 286, 289, 290, 292, 304, 305, 307, 308, 309, 319, 321, 326, 331, 332, 334, 349, 355, 364, 365, 369, 370, 371, 372, 374, 375, 377, 378, 379, 381, 382, 383, 384, 385, 387, 389, 394, 396, 399, 400, 403, 404, 407, 417, 418, 425, 431, 432, 433, 434, 435, 436, 437, 438, 439, 443, 446, 447, 453, 456, 459, 460, 461, 463, 471, 472, 473, 474, 475, 476, 477, 478, 479, 482, 483, 503, 507, 521, 522, 524, 525, 528, 538, 546, 547, 551, 558, 560, 564, 565, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:24.

### Claim 3 (depends on 2)

The engineered polynucleotide of claim 2, wherein said encoded polypeptide further comprises at least one amino acid residue difference as compared to SEQ ID NO:24, selected from 112C, 134Q, 158H, 180A, 195E, 240R/W, 243I/L, 245L, 256G, 257W/A, 270K, 304H, 307Q/M, 308Q, 326F, 349M, 353A/N, 364Q, 394V, 399N, 400K, 404A, 443H, 453G, 459F, 460G, 463N, 474Q, 521S, 522Y/F/N, 528L, 546R, and 564 G/L/M, wherein said positions are numbered with reference to SEQ ID NO:24.

### Claim 4 (depends on 1)

The engineered polynucleotide of claim 1, wherein said encoded engineered polypeptide exhibits at least one improved property selected from enhanced catalytic activity, reduced sensitivity to proteolysis, and increased tolerance to acidic pH, as compared to the reference sequence SEQ ID NO:24.

### Claim 5 (depends on 4)

The engineered polynucleotide of claim 4, wherein said encoded polypeptide exhibits at least one improved property selected from reduced sensitivity to proteolysis, and increased tolerance to acidic pH.

### Claim 6 (depends on 5)

The engineered polynucleotide of claim 5, wherein said encoded polypeptide is resistant to proteolysis by at least one digestive tract enzyme, wherein said engineered polypeptide is resistant to proteolysis by chymotrypsin, trypsin, carboxypeptidases, and/or elastases.

### Claim 7 (depends on 1)

The engineered polynucleotide of claim 1, wherein said encoded polypeptide is deimmunized.

### Claim 8 (depends on 1)

The engineered polynucleotide of claim 1, wherein said encoded polypeptide has phenylalanine ammonia lyase activity.

### Claim 9 (depends on 1)

The engineered polynucleotide of claim 1, wherein said polynucleotide is purified.

### Claim 10 (depends on 1)

The engineered polynucleotide of claim 1, wherein said polynucleotide is operably linked to at least one control sequence.

### Claim 11 (depends on 1)

The engineered polynucleotide of claim 1, wherein said polynucleotide is codon-optimized.

### Claim 12 (depends on 10)

An expression vector comprising at least one polynucleotide of claim 10.

### Claim 13 (depends on 11)

An expression vector comprising at least one polynucleotide of claim 11.

### Claim 14 (depends on 12)

The expression vector of claim 12, wherein said control sequence is a promoter.

### Claim 15 (depends on 11)

The expression vector of claim 11, further comprising at least one control sequence, wherein said control sequence is a promoter.

### Claim 16 (depends on 12)

A host cell transformed with at least one expression vector of claim 12.

### Claim 17 (depends on 13)

A host cell transformed with at least one expression vector of claim 13.

### Claim 18 (depends on 14)

A host cell transformed with at least one expression vector of claim 14.

### Claim 19 (depends on 15)

A host cell transformed with at least one expression vector of claim 15.

### Claim 20 (depends on 12)

A method of producing an engineered polypeptide in a host cell comprising culturing a host cell comprising at least one expression vector of claim 12, under suitable culture conditions, such that at least one engineered polypeptide is produced.

### Claim 21 (depends on 20)

The method of claim 20, further comprising recovering at least one engineered polypeptide from the culture and/or host cells.

### Claim 22 (depends on 21)

The method of claim 21, further comprising the step of purifying said at least one engineered polypeptide.

### Claim 23 (depends on 1)

A composition comprising at least one engineered polynucleotide of claim 1.