# US 10,308,691 B2 · Methods for tuning carotenoid production levels and compositions in rhodosporidium and rhodotorula genera

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- Number: US 10,308,691 B2
- Document ID: US10308691B2
- Status: Granted
- Kind: B2
- Application number: 15/535,466
- Filed: 2015-12-14
- Published: 2017-12-21
- Granted: 2019-06-04
- Earliest claimed: 2014-12-15
- Assignees: Temasek Life Sciences Laboratory Limited
- Applicants: Temasek Life Sciences Laboratory Limited
- Inventors: Yanbin Liu; Chong Mei Koh; Lianghui Ji
- Web page: https://patentgrep.com/patent/US10308691B2

## CPC

- C07K 14/37: from fungi
- C12N 9/0008: {acting on the aldehyde or oxo group of donors (1.2)}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/80: for fungi
- 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)
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- 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)}

## Abstract

The present invention relates to the field of fungal biotechnology, more particularly to genetic engineering methods for the production of carotenoids in fungal hosts selected from Rhodospordium and Rhodotorula genera.

## Claims

### Claim 1

A method for tuning the production level and composition of carotenoids in a fungal host comprising:
(a) genetically manipulating a fungal host selected from the group consisting of Rhodosporidium and Rodotorula, wherein the genetic manipulation comprises deleting all or part of one or more polynucleotides selected from the group consisting of:
(i) a polynucleotide encoding a polypeptide having a sequence set forth in SEQ ID NO:12 or at least 95% identity thereto,
(ii) a polynucleotide encoding a polypeptide having a sequence set forth in SEQ ID NO:18 or at least 95% identity thereto, and
(iii) a polynucleotide encoding a polypeptide having a sequence set forth in SEQ ID NO:21 or at least 95% identity thereto,
wherein the deletion of all or part of polynucleotide (i) totally inactivates the enzyme activity of the polypeptide encoded by polynucleotide (i),
wherein the deletion of all or part of polynucleotide (ii) totally inactivates the enzyme activity of the polypeptide encoded by polynucleotide (ii), and
wherein the deletion of all or part of polynucleotide (iii) totally inactivates the enzyme activity of the polypeptide encoded by polynucleotide (iii); and
(b) growing the fungal host to produce carotenoids, wherein the carotenoids are selected from the group consisting of lycopene, beta-carotene, gamma-carotene, torulene and torularhodin or derivatives thereof,
whereby the production level or composition of the carotenoids is tuned compared to a fungal host not having the genetic manipulation.

### Claim 2 (depends on 1)

The method of claim 1, wherein the deletion is made by homologous recombination.

### Claim 3 (depends on 1)

The method of claim 1, wherein the deletion is made using an artificial nuclease selected from a Zinc finger nuclease or a Cas9-gRNA complex.

### Claim 4 (depends on 1)

The method of claim 1, wherein the genetic manipulation further comprises over-expressing one or more polynucleotides selected from the group consisting of
(iv) a polynucleotide encoding a polypeptide having a sequence set forth in SEQ ID NO:2 or at least 95% identity thereto,
(v) a polynucleotide encoding a polypeptide having a sequence set forth in SEQ ID NO:4 or at least 95% identity thereto,
(vi) a polynucleotide encoding a polypeptide having a sequence set forth in SEQ ID NO:6 or at least 95% identity thereto, and
(vii) a polynucleotide encoding a polypeptide having a sequence set forth in SEQ ID NO:15 or at least 95% identity thereto,
wherein the over-expression is compared to a fungal host not having the genetic manipulation.

### Claim 5 (depends on 4)

The method of claim 4, wherein the one or more polynucleotides are over-expressed by introducing one or more DNA molecules into the fungal host, wherein each DNA molecule comprises one or more constructs and wherein each construct comprises a heterologous promoter operatively linked to one of the polynucleotides operatively linked to a transcriptional terminator.

### Claim 6 (depends on 4)

The method of claim 4, wherein
polynucleotide (i) has the sequence set forth in SEQ ID NO:10 or 11,
polynucleotide (ii) has the sequence set forth in SEQ ID NO:16 or 17,
polynucleotide (iii) has the sequence set forth in SEQ ID NO:19 or 20,
polynucleotide (iv) has the sequence set forth in SEQ ID NO:1,
polynucleotide (v) has the sequence set forth in SEQ ID NO:3,
polynucleotide (vi) has the sequence set forth in SEQ ID NO:5,
polynucleotide (vii) has the sequence set forth in SEQ ID NO:13 or 14.

### Claim 7 (depends on 1)

The method of claim 1, wherein the genetic manipulation further comprises down-regulating one or more different polynucleotides selected from the group consisting of
(iv) a polynucleotide encoding a polypeptide having a sequence set forth in SEQ ID NO:2 or at least 95% identity thereto,
(v) a polynucleotide encoding a polypeptide having a sequence set forth in SEQ ID NO:4 or at least 95% identity thereto,
(vi) a polynucleotide encoding a polypeptide having a sequence set forth in SEQ ID NO:6 or at least 95% identity thereto, and
(vii) a polynucleotide encoding a polypeptide having a sequence set forth in SEQ ID NO:15 or at least 95% identity thereto,
wherein the down-regulation is compared to a fungal host not having the genetic manipulation.

### Claim 8 (depends on 7)

The method of claim 7, wherein the one or more polynucleotides are down-regulated by RNAi, artificial transcriptional repressor or weak promoter.

### Claim 9 (depends on 7)

The method of claim 7, wherein
polynucleotide (i) has the sequence set forth in SEQ ID NO:10 or 11,
polynucleotide (ii) has the sequence set forth in SEQ ID NO:16 or 17,
polynucleotide (iii) has the sequence set forth in SEQ ID NO:19 or 20,
polynucleotide (iv) has the sequence set forth in SEQ ID NO:1,
polynucleotide (v) has the sequence set forth in SEQ ID NO:3,
polynucleotide (vi) has the sequence set forth in SEQ ID NO:5,
polynucleotide (vii) has the sequence set forth in SEQ ID NO:13 or 14.

### Claim 10 (depends on 1)

The method of claim 1, wherein the fungal host is grown in a medium comprising at least 5% of a carbon source at a temperature from about 25° C. to about 35° C.

### Claim 11 (depends on 10)

The method of claim 10, wherein the carbon source is glucose, mannose, glycerol, sucrose, xylose or combinations thereof.

### Claim 12 (depends on 10)

The method of claim 10, wherein the medium comprises 30-100 g/L glucose, 1.5 g/L yeast extract, 0.5 g/L (NH4)2SO4, 2.05 g/L K2HPO4, 1.45 g/L KH2PO4, 0.6 g/L MgSO4, 0.3 g/L NaCl, 10 mg CaCl2, 1 mg/L FeSO4, 0.5 mg/L ZnSO4, 0.5 mg/L CuSO4, 0.5 mg/L H3BO4, 0.5 mg/L MnSO4, 0.5 mg/L NaMoO4, wherein pH of the medium is from about 5 to about 7.

### Claim 13 (depends on 10)

The method of claim 10, wherein the fungal host is grown under illumination.

### Claim 14 (depends on 13)

The method of claim 13, wherein the carbon source is glucose, mannose, glycerol, sucrose, xylose or combinations thereof.

### Claim 15 (depends on 13)

The method of claim 13, wherein the medium comprises 30-100 g/L glucose, 1.5 g/L yeast extract, 0.5 g/L (NH4)2SO4, 2.05 g/L K2HPO4, 1.45 g/L KH2PO4, 0.6 g/L MgSO4, 0.3 g/L NaCl, 10 mg CaCl2, 1 mg/L FeSO4, 0.5 mg/L ZnSO4, 0.5 mg/L CuSO4, 0.5 mg/L H3BO4, 0.5 mg/L MnSO4, 0.5 mg/L NaMoO4, wherein pH of the medium is from about 5 to about 7.

### Claim 16 (depends on 1)

The method of claim 1, wherein
polynucleotide (i) has the sequence set forth in SEQ ID NO:10 or 11,
polynucleotide (ii) has the sequence set forth in SEQ ID NO:16 or 17, and
polynucleotide (iii) has the sequence set forth in SEQ ID NO:19 or 20.