# US 10,865,397 B2 · Recombinant polynucleotide sequence for producing astaxanthin and uses thereof

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- Number: US 10,865,397 B2
- Document ID: US10865397B2
- Status: Granted
- Kind: B2
- Application number: 16/200,269
- Filed: 2018-11-26
- Published: 2019-05-16
- Granted: 2020-12-15
- Earliest claimed: 2014-05-16
- Assignees: Academia Sinica
- Applicants: Academia Sinica
- Inventors: Jui-Jen Chang; Caroline Thia; Hao-Yeh Lin; Yu-Ju Lin; Chieh-Chen Huang; Wen-Hsiung Li
- Web page: https://patentgrep.com/patent/US10865397B2

## CPC

- C12N 9/14: Hydrolases (3)
- C12N 15/815: {for yeasts other than Saccharomyces}
- C12P 23/00: Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes (containing heterorings C12P17/00)
- 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

Disclosed herein are recombinant polynucleotide sequences, vectors, host cells and methods for producing astaxanthin. The recombinant polynucleotide sequence is designed to provide a higher level of astaxanthin precursors via a shorter metabolic pathway, and thereby attains higher level of end products (e.g., astaxanthin) with desired stereoisomeric form and/or esterified form.

## Claims

### Claim 1

A recombinant polynucleotide sequence for producing astaxanthin, or a precursor or a derivative thereof, in a host cell, comprising,
a first gene cassette comprising a first promoter, and a first nucleic acid sequence operatively linked to the first promoter and encodes a geranylgeranyl pyrophosphate synthase;
a second gene cassette comprising a second promoter, and a second nucleic acid sequence operatively linked to the second promoter and encodes a 3-hydroxy-3-methylglutaryl-coenzyme A reductase;
a third gene cassette comprising a third promoter, and a third nucleic acid sequence operatively linked to the third promoter and encodes a phytoene desaturase; and
a fourth gene cassette comprising a fourth promoter, and a fourth nucleic acid sequence operatively linked to the fourth promoter and encodes a bi-functional enzyme that possesses the respective functions of a phytoene synthase and a lycopene cyclase; and
the 3′-end of each gene cassette of the recombinant polynucleotide sequence is homologous to the 5′-end of the next gene cassette downstream thereto,
wherein the first, second, third, and fourth nucleic acid sequences respectively comprise the sequences of SEQ ID NOs: 1, 2, 3, and 4,
wherein the host cell is Kluveromyces marxianus, and
wherein the derivative of astaxanthin is an astaxanthin monoester or an astaxanthin diester.

### Claim 2 (depends on 1)

The recombinant polynucleotide sequence of claim 1, wherein
the astaxanthin is 3S, 3'S-astaxanthin or 3R, 3′R-astaxanthin; and
the precursor of astaxanthin is geranylgeranyl-pyrophosphate, phenicoxanthin, lycopene, echinenone, canthaxanthin, phytoene, zeaxanthin, β-cryptoxanthin, or β-carotene.

### Claim 3 (depends on 1)

The recombinant polynucleotide sequence of claim 1, further comprising,
a fifth gene cassette comprising a fifth promoter, and a fifth nucleic acid sequence operatively linked to the fifth promoter and encodes a β-carotene hydroxylase; and
a sixth gene cassette comprising a sixth promoter and a sixth nucleic acid sequence operatively linked to the sixth promoter and encodes a β-carotene ketolase.

### Claim 4 (depends on 3)

The recombinant polynucleotide sequence of claim 3, wherein the fifth nucleic acid sequence comprises any of the sequence of SEQ ID NO: 5, 6, or 7, and the sixth nucleic acid sequence comprises the sequence of SEQ ID NO: 8.

### Claim 5 (depends on 4)

The recombinant polynucleotide sequence of claim 4, wherein the first to sixth promoters are different from one another.

### Claim 6 (depends on 1)

The recombinant polynucleotide sequence of claim 1, further comprising,
a seventh gene cassette comprising a seventh promoter, and a seventh nucleic acid sequence operatively linked to the seventh promoter and encodes a P450 reductase; and
an eighth gene cassette comprising an eighth promoter, and an eighth nucleic acid sequence operatively linked to the eighth promoter and encodes a β-carotene oxygenase.

### Claim 7 (depends on 6)

The recombinant polynucleotide sequence of claim 6, wherein the seventh nucleic acid sequence comprises the sequence of SEQ ID NO: 9, and the eighth nucleic acid sequence comprises the sequence of SEQ ID NO: 10.

### Claim 8 (depends on 7)

The recombinant polynucleotide sequence of claim 7, wherein the first, second, third, fourth, seventh, and eighth promoters are different from one another.

### Claim 9 (depends on 1)

The recombinant polynucleotide sequence of claim 1, further comprising a marker gene cassette, which comprises a marker promoter and a marker gene operatively linked to the marker promoter.