# US 9,518,276 B2 · Genetically modified fungi and their use in lipid production

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- Number: US 9,518,276 B2
- Document ID: US9518276B2
- Status: Granted
- Kind: B2
- Application number: 13/806,281
- Filed: 2011-06-21
- Published: 2013-04-25
- Granted: 2016-12-13
- Earliest claimed: 2010-06-24
- Assignees: Teknologian tutkimuskeskus VTT Oy
- Applicants: Kari Koivuranta; Laura Ruohonen; Merja Penttilä
- Inventors: Kari Koivuranta; Laura Ruohonen; Merja Penttilä
- Web page: https://patentgrep.com/patent/US9518276B2

## CPC

- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 9/0008: {acting on the aldehyde or oxo group of donors (1.2)}
- C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)}
- C12N 9/88: Lyases (4.)
- C12N 9/93: {Ligases (6)}
- C12N 15/80: for fungi
- C12P 7/6463: obtained from glyceride producing microorganisms, e.g. single cell oil
- C12P 7/649: Biodiesel, i.e. fatty acid alkyl esters
- C12Y 102/0101: Acetaldehyde dehydrogenase (acetylating) (1.2.1.10)
- C12Y 203/0102: Diacylglycerol O-acyltransferase (2.3.1.20)
- C12Y 401/01001: Pyruvate decarboxylase (4.1.1.1)
- C12Y 602/01001: Acetate-CoA ligase (6.2.1.1)
- Y02E 50/10: Biofuels, e.g. bio-diesel
- Y02P 20/52: using catalysts, e.g. selective catalysts
- 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)}
- 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

## Abstract

The invention refers to fungal cells, and especially to oleaginous fungal cells that have been genetically modified to produce enzymes of the pyruvate dehydrogenase bypass route to enhance their lipid production. Especially the cells are modified to over-express genes encoding pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) and/or acetyl-CoA synthetase (ACS), optionally together with a gene encoding diacylglycerol acyltransferase (DAT), or to express genes encoding PDC together with ALD and/or ACS. Methods of producing lipids, biofuels and lubricants using the modified fungi are also disclosed as well as expression cassettes useful therein. A new enzyme having phospholipid: diacylglycerol acyltransferase (PDAT) activity and a polynucleotide encoding it are also disclosed, which are useful in the lipid production. A recombinant Cryptococcus cell and its construction is described.

## Claims

### Claim 1

A genetically modified oleaginous fungal cell comprising:
a) a nucleic acid with enhanced expression encoding a pyruvate decarboxylase (PDC), and
b) at least one nucleic acid with enhanced expression encoding an enzyme selected from the group consisting of acetaldehyde dehydrogenase (ALD), acetyl-CoA synthetase (ACS) and diacylglycerol acyltransferase (DAT),
wherein said genetically modified oleaginous fungal cell has enhanced lipid production in an aerobic process compared to a genetically unmodified oleaginous fungal cell.

### Claim 2 (depends on 1)

The genetically modified oleaginous fungal cell of claim 1, wherein the encoded PDC is a PDC of a Crabtree-positive organism.

### Claim 3 (depends on 2)

The genetically modified oleaginous fungal cell of claim 2, wherein the encoded PDC is a PDC of Saccharomyces cerevisiae, preferably PDC 1 of S. cerevisiae.

### Claim 4 (depends on 1)

The genetically modified oleaginous fungal cell of claim 1, wherein the nucleic acid encoding ACS is a gene that is not under glucose repression or its gene product is not subject to post-translational regulation.

### Claim 5 (depends on 4)

The genetically modified oleaginous fungal cell of claim 4, wherein the encoded ACS is a fungal ACS, preferably Saccharomyces cerevisiae ACS2.

### Claim 6 (depends on 1)

The genetically modified oleaginous fungal cell of claim 1, which is a yeast cell selected from the genera Cryptococcus, Candida, Galactomyces, Hansenula, Lipomyces, Rhodosporidium, Rhodotorula, Trichosporon and Yarrowia, preferably from the group consisting of Candida sp., Cryptococcus curvatus, Cryptococcus albidus, Galactomyces geotrichum, Hansenula ciferri, Lipomyces lipofer, Lipomyces ssp., Lipomyces starkeyi, Lipomyces tetrasporus, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulans and Yarrowia lipolytica, or a filamentous fungal cell selected from the genera Aspergillus, Cunninghamella, Fusarium, Glomus, Humicola, Mortierella, Mucor, Penicillium, Pythium and Rhizopus, preferably from the group consisting of Aspergillus nidulans, Aspergillus oryzae, Aspergillus terreus, Aspergillus niger, Cuninghamella japonica, Fusarium oxysporum, Glomus caledonius, Humicola lanuginose, Mortierella isabellina, Mortierella pusilla, Mortierella vinacea, Mucor circinelloides, Mucor plumbeus, Mucor ramanniana, Penicillium lilacinum, Penicillium spinulosum, Pythium ultimum and Rhizopus oryzae.

### Claim 7 (depends on 6)

The genetically modified oleaginous fungal cell of claim 6, which is from the genera Cryptococcus or Mucor, preferably it is a cell of Cryptococcus curvatus or Mucor circinelloides.

### Claim 8 (depends on 1)

A method of preparing an oleaginous fungal cell of claim 1, said method comprising transforming a fungal cell with
a) a nucleic acid with enhanced expression encoding a pyruvate decarboxylase (PDC) enzyme, and
b) at least one nucleic acid with enhanced expression encoding an enzyme selected from the group consisting of acetaldehyde dehydrogenase (ALD), acetyl-CoA synthetase (ACS) and diacylglycerol acyltransferase (DAT).

### Claim 9 (depends on 1)

Use of a genetically modified fungal cell of claim 1 for producing lipids, precursors of functional fatty acids, functional fatty acids, biofuels, biodiesel, renewable diesel or lubricants.

### Claim 10 (depends on 3)

The genetically modified oleaginous fungal cell of claim 3, where in the PDC1 is from S. cerevisiae which encodes the amino acid sequence SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97.

### Claim 11 (depends on 5)

The genetically modified oleaginous fungal cell of claim 5, where in the ACS2 is from S. cerevisiae which encodes the amino acid sequence SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 92.

### Claim 12 (depends on 1)

The genetically modified oleaginous fungal cell of claim 1, wherein the at least one nucleic acid with enhanced expression encoding an enzyme is selected from the group consisting of acetyl-CoA synthetase (ACS) and diacylglycerol acyltransferase (DAT).

### Claim 13 (depends on 1)

The genetically modified oleaginous fungal cell of claim 1, wherein said genetically modified oleaginous fungal cell has a 33% to 83% higher triacylglycerol yield on biomass compared to the genetically unmodified oleaginous fungal cell.