# US 8,772,575 B2 · Nucleic acids useful in the manufacture of oil

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- Number: US 8,772,575 B2
- Document ID: US8772575B2
- Status: Granted
- Kind: B2
- Application number: 13/628,039
- Filed: 2012-09-26
- Published: 2013-04-11
- Granted: 2014-07-08
- Earliest claimed: 2008-11-28
- Assignees: Solazyme, Inc.
- Applicants: Scott Franklin; Aravind Somanchi; Karen Espina; George Rudenko; Penelope Chua
- Inventors: Scott Franklin; Aravind Somanchi; Karen Espina; George Rudenko; Penelope Chua
- Web page: https://patentgrep.com/patent/US8772575B2

## CPC

- C12P 7/6427: Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
- C07C 1/2078: {by a transformation in which at least one -C(=O)-O- moiety is eliminated}
- C10L 1/02: essentially based on components consisting of carbon, hydrogen, and oxygen only
- C12N 9/00: Enzymes; Proenzymes; Compositions thereof (preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; {enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113}); Processes for preparing, activating, inhibiting, separating or purifying enzymes (preparation of malt C12C1/00)
- C12N 9/16: acting on ester bonds (3.1)
- C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)}
- C12N 15/79: Vectors or expression systems specially adapted for eukaryotic hosts
- C12P 7/20: Glycerol
- C12P 7/64: Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P 7/6409: Fatty acids
- C12P 7/6418: by hydrolysis of fatty acid esters
- C12P 7/6458: by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
- C12P 7/6463: obtained from glyceride producing microorganisms, e.g. single cell oil
- C12P 7/649: Biodiesel, i.e. fatty acid alkyl esters
- C07K 2319/01: containing a localisation/targetting motif
- C12Y 301/02014: Oleoyl-[acyl-carrier-protein] hydrolase (3.1.2.14), i.e. ACP-thioesterase
- C12Y 302/01026: Beta-fructofuranosidase (3.2.1.26), i.e. invertase
- Y02E 50/10: Biofuels, e.g. bio-diesel
- Y02P 20/52: using catalysts, e.g. selective catalysts
- Y02P 60/87: Re-use of by-products of food processing for fodder production
- Y02T 50/678: Aviation using fuels of non-fossil origin
- 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)}
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C10L: FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G OR C10K; LIQUIFIED PETROLEUM GAS; USE OF ADDITIVES TO FUELS OR FIRES; FIRE-LIGHTERS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02T: CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO TRANSPORTATION

## Abstract

Novel gene sequences from microalgae are disclosed, as well as novel gene sequences useful in the manufacture of triglyceride oils. Also disclosed are sequences and vectors that allow microalgae to be cultivated on sugar cane and sugar beets as a feedstock. In some embodiments, the vectors are useful for the purpose of performing targeted modifications to the nuclear genome of heterotrophic microalgae.

## Claims

### Claim 1

A host cell of the family Chlorellaceae having a native 23S rRNA with at least 90% sequence identity to SEQ ID NO. 15, comprising a recombinant nucleic acid codon-optimized for Prototheca and encoding a stearoyl-ACP desaturase, the recombinant nucleic acid containing the most preferred codon of Table 1 for at least 80% of the codons that encode the stearoyl-ACP desaturase of SEQ ID NO. 145 such that the codon-optimized nucleic acid is more efficiently expressed in the host cell than a non-codon-optimized nucleic acid thereby altering the fatty acid profile of the host cell.

### Claim 2 (depends on 1)

The host cell of claim 1, wherein the host is an oleaginous cell of the family Chlorellaceae.

### Claim 3 (depends on 1)

The host cell of claim 1, wherein the host cell comprises at least 50% lipid by dry cell weight.

### Claim 4 (depends on 3)

The host cell of claim 3, wherein the host cell comprises at least 60% lipid by dry cell weight.

### Claim 5 (depends on 1)

The host cell of claim 1, wherein the nucleic acid further encodes a plastid targeting peptide, the plastid targeting peptide operable to target the stearoyl-ACP desaturase to a plastid.

### Claim 6 (depends on 5)

The host cell of claim 5, wherein the plastid targeting peptide is from microalgae.

### Claim 7 (depends on 5)

The host cell of claim 5, wherein the plastid targeting peptide has the amino acid sequence of one or more of SEQ ID NOs. 127-133.

### Claim 8 (depends on 1)

The host cell of claim 1, further comprising a promoter having a nucleotide sequence of any of SEQ ID NOs: 91-102.

### Claim 9 (depends on 1)

The host cell of claim 1, wherein the cell is of the genus Prototheca.