# US 11,441,130 B2 · Omega-hydroxylase-related fusion polypeptides with improved properties

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- Number: US 11,441,130 B2
- Document ID: US11441130B2
- Status: Granted
- Kind: B2
- Application number: 15/319,272
- Filed: 2015-06-16
- Published: 2017-05-25
- Granted: 2022-09-13
- Earliest claimed: 2014-06-16
- Assignees: Genomatica, Inc.
- Applicants: Genomatica, Inc.
- Inventors: Baolong Zhu; Andreas W. Schirmer; Cindy Chang
- Web page: https://patentgrep.com/patent/US11441130B2

## CPC

- C12N 9/0077: {with a reduced iron-sulfur protein as one donor (1.14.15)}
- C12P 7/6409: Fatty acids
- Y02E 50/10: Biofuels, e.g. bio-diesel
- 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)}
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The disclosure relates to omega-hydroxylase-related fusion polypeptides that result in improved omega-hydroxylated fatty acid derivative production when expressed in recombinant host cells. The disclosure further relates to microorganisms for expressing the omega-hydroxylase-related fusion polypeptides for the production of omega-hydroxylated fatty acid derivatives.

## Claims

### Claim 1

A CYP153A-reductase hybrid fusion polypeptide variant comprising at least 95% sequence identity to SEQ ID NO: 38 and having at least one mutation at an amino acid position corresponding to amino acid position 9, 10, 12, 13, 14, 28, 61, 119, 327, 413, 703, 745, 747, 749, 757, 770, 771 and 784.

### Claim 2 (depends on 1)

The CYP153A-reductase hybrid fusion polypeptide variant of claim 1, wherein the CYP153A-reductase hybrid fusion polypeptide variant is a hybrid cyp153ARedRhF fusion protein variant.

### Claim 3 (depends on 1)

The CYP153A-reductase hybrid fusion polypeptide variant of claim 1, wherein said mutation is selected from the group consisting of D9N, D9K, D10Y, Q12W, Q12R, Q12T, S13K, R14F, Q28M, Q28T, N61L, K119R, P327D, Y413R, L703G, P745R, D747N, E749L, E757A, T770G, V771F, and M784I.

### Claim 4 (depends on 3)

The CYP153A-reductase hybrid fusion polypeptide variant of claim 3, wherein expression of said CYP153A-reductase hybrid fusion polypeptide variant in a recombinant host cell results in a higher titer of an omega-hydroxylated fatty acid as compared to a titer of an omega-hydroxylated fatty acid produced by expression of a CYP153A-reductase hybrid fusion polypeptide of SEQ ID NO: 38.

### Claim 5 (depends on 3)

The CYP153A-reductase hybrid fusion polypeptide variant of claim 3, comprising any one of SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, and SEQ ID NO: 96.