# US 2014/0154765 A1 · Production of acetyl-coenzyme a derived isoprenoids

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2014/0154765 A1
- Document ID: US20140154765A1
- Status: Published application
- Kind: A1
- Application number: 14/062,798
- Filed: 2013-10-24
- Published: 2014-06-05
- Earliest claimed: 2011-11-09
- Assignees: Amyris, Inc.
- Applicants: Amyris, Inc.
- Inventors: Timothy Stevens GARDNER; Kristy Michelle Hawkins; Adam Leon Meadows; Annie Ening Tsong; Yoseph Tsegaye
- Web page: https://patentgrep.com/patent/US20140154765A1

## CPC

- C12P 5/007: {containing one or more isoprene units, i.e. terpenes (carotenes C12P23/00)}
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 9/0008: {acting on the aldehyde or oxo group of donors (1.2)}
- C12N 9/1288: {Transferases for other substituted phosphate groups (2.7.8)}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 15/81: for yeasts
- C12P 7/42: Hydroxy-carboxylic acids
- 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)
- C12Y 101/01034: Hydroxymethylglutaryl-CoA reductase (NADPH) (1.1.1.34)
- C12Y 102/0101: Acetaldehyde dehydrogenase (acetylating) (1.2.1.10)
- C12Y 207/08: Transferases for other substituted phosphate groups (2.7.8)
- 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)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

Provided herein are compositions and methods for the heterologous production of acetyl-CoA-derived isoprenoids in a host cell. In some embodiments, the host cell is genetically modified to comprise a heterologous nucleotide sequence encoding an acetaldehyde dehydrogenase, acetylating (ADA, E.C. 1.2.1.10) and an MEV pathway comprising an NADH-using HMG-CoA reductase. In some embodiments, the host cell is genetically modified to comprise a heterologous nucleotide sequence encoding an ADA and an MEV pathway comprising an acetoacetyl-CoA synthase. In some embodiments, the genetically modified host cell further comprises one or more heterologous nucleotide sequences encoding a phosphoketolase and a phosphotransacetylase. In some embodiments, the genetically modified host cell further comprises a functional disruption of the native PDH-bypass. The compositions and methods described herein provide an energy-efficient yet redox balanced route for the heterologous production of acetyl-CoA-derived isoprenoids.

## Claims

### Claim at position 1

1-55. (canceled)

### Claim 56

A genetically modified yeast cell capable of producing an isoprenoid, the cell comprising:
(a) a heterologous nucleic acid encoding an acylating acetylaldehyde dehydrogenase (ADA; EC 1.2.1.10); and optionally one or more heterologous nucleic acids encoding one or more enzymes of a mevalonate (MEV) pathway for making isopentenyl pyrophosphate;
(b) a heterologous nucleic acid encoding a phosphoketolase (PK; EC 4.1.2.9, EC 4.1.2.22); and
(c) a heterologous nucleic acid encoding a phosphotransacetylase (PTA; EC 2.3.1.8).

### Claim 57 (depends on 56)

The genetically modified yeast cell of claim 56, further comprising a functional disruption of one or more enzymes of the native pyruvate dehydrogenase (PDH)-bypass selected from the group consisting of acetyl-CoA synthetase 1 (ACS1), acetyl-CoA synthetase 2 (ACS2), and aldehyde dehydrogenase 6 (ALD6).

### Claim 58 (depends on 56)

The genetically modified yeast cell of claim 56, further comprising a heterologous nucleic acid encoding an enzyme that condenses acetyl-CoA with malonyl-CoA to form acetoacetyl-CoA.

### Claim 59 (depends on 58)

The genetically modified yeast cell of claim 58, wherein the enzyme that condenses acetyl-CoA with malonyl-CoA to form acetoacetyl-CoA is an acetyl-CoA:malonyl-CoA acyltransferase.

### Claim 60 (depends on 56)

The genetically modified yeast cell of claim 56, wherein the one or more enzymes of the MEV pathway comprise an NADH-using enzyme that converts 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to mevalonate.

### Claim 61 (depends on 60)

The genetically modified yeast cell of claim 60, wherein the one or more enzymes of the MEV pathway comprise an NADH-using HMG-CoA reductase (HMGr).

### Claim 62 (depends on 57)

The genetically modified host cell of claim 57, wherein ACS1 is functionally disrupted.

### Claim 63 (depends on 57)

The genetically modified host cell of claim 57, wherein ACS2 is functionally disrupted.

### Claim 64 (depends on 57)

The genetically modified host cell of claim 57, wherein ALD6 is functionally disrupted.

### Claim 65 (depends on 57)

The genetically modified host cell of claim 57, wherein ACS1 and ACS2 are functionally disrupted.

### Claim 66 (depends on 57)

The genetically modified yeast cell of claim 57, wherein ACS1, ACS2 and ALD6 are functionally disrupted.

### Claim 67 (depends on 56)

The genetically modified yeast cell of claim 56, wherein the one or more enzymes of the MEV pathway comprise an enzyme that condenses two molecules of acetyl-CoA to form acetoacetyl-CoA.

### Claim 68 (depends on 56)

The genetically modified yeast cell of claim 56, wherein the one or more enzymes of the MEV pathway comprise an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA.

### Claim 69 (depends on 56)

The genetically modified yeast cell of claim 56, wherein the one or more enzymes of the MEV pathway comprise an NAPDH-using enzyme that converts HMG-CoA to mevalonate.

### Claim 70 (depends on 56)

The genetically modified yeast cell of claim 56, wherein the one or more enzymes of the MEV pathway comprise an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate.

### Claim 71 (depends on 56)

The genetically modified yeast cell of claim 56, wherein the one or more enzymes of the MEV pathway comprise an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate.

### Claim 72 (depends on 56)

The genetically modified yeast cell of claim 56, wherein the one or more enzymes of the MEV pathway comprise an enzyme that converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate.

### Claim 73 (depends on 56)

The genetically modified yeast cell of claim 56, wherein the one or more enzymes of the MEV pathway are selected from HMG-CoA synthase, mevalonate kinase, phosphomevalonate kinase and mevalonate pyrophosphate decarboxylase.

### Claim 74 (depends on 56)

The genetically modified yeast cell of claim 56, wherein the cell comprises a plurality of heterologous nucleic acids encoding all of the enzymes of the MEV pathway.

### Claim 75 (depends on 56)

The genetically modified yeast cell of claim 56, wherein the one or more heterologous nucleic acids encoding one or more enzymes of the MEV pathway are under control of a single transcriptional regulator.

### Claim 76 (depends on 56)

The genetically modified yeast cell of claim 56, further comprising a heterologous nucleic acid encoding an enzyme that can convert isopentenyl pyrophosphate (IPP) into dimethylallyl pyrophosphate (DMAPP).

### Claim 77 (depends on 56)

The genetically modified yeast cell of claim 56, further comprising a heterologous nucleic acid encoding an enzyme that can condense IPP and/or DMAPP molecules to form a polyprenyl compound.

### Claim 78 (depends on 56)

The genetically modified yeast cell of claim 56, further comprising a heterologous nucleic acid encoding an enzyme that can modify IPP or a polyprenyl to form an isoprenoid compound.

### Claim 79 (depends on 78)

The genetically modified yeast cell of claim 78, wherein the enzyme that can modify IPP or a polyprenyl to form an isoprenoid compound is selected from the group consisting of carene synthase, geraniol synthase, linalool synthase, limonene synthase, myrcene synthase, ocimene synthase, α-pinene synthase, β-pinene synthase, γ-terpinene synthase, terpinolene synthase, amorphadiene synthase, α-farnesene synthase, β-farnesene synthase, farnesol synthase, nerolidol synthase, patchouliol synthase, nootkatone synthase, and abietadiene synthase.

### Claim 80 (depends on 78)

The genetically modified yeast cell of claim 78, wherein the isoprenoid is selected from the group consisting of a hemiterpene, monoterpene, diterpene, triterpene, tetraterpene, sesquiterpene, and polyterpene.

### Claim 81 (depends on 78)

The genetically modified yeast cell of claim 78, wherein the isoprenoid is a C5-C20 isoprenoid.

### Claim 82 (depends on 78)

The genetically modified yeast cell of claim 78, wherein the isoprenoid is selected from the group consisting of abietadiene, amorphadiene, carene, α-farnesene, β-farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, β-pinene, sabinene, γ-terpinene, terpinolene, and valencene.

### Claim 83 (depends on 56)

The genetically modified yeast cell of claim 56, wherein the yeast is Saccharomyces cerevisiae.

### Claim 84 (depends on 56)

A method for producing an isoprenoid comprising:
(a) culturing a population of the genetically modified yeast cell of claim 56 in a medium with a carbon source under conditions suitable for making said isoprenoid compound; and
(b) recovering said isoprenoid compound from the medium.

### Claim 85

A genetically modified yeast cell capable of producing an isoprenoid, the cell comprising:
(a) one or more heterologous nucleic acids encoding one or more enzymes of a mevalonate (MEV) pathway for making isopentenyl pyrophosphate; and
(b) a heterologous nucleic acid encoding a phosphoketolase (PK; EC 4.1.2.9, EC 4.1.2.22).

### Claim 86 (depends on 85)

The genetically modified host cell of claim 85, further comprising a heterologous nucleic acid encoding a phosphotransacetylase (PTA; EC 2.3.1.8).

### Claim 87 (depends on 85)

The genetically modified yeast cell of claim 85, further comprising a heterologous nucleic acid encoding an enzyme that condenses acetyl-CoA with malonyl-CoA to form acetoacetyl-CoA.

### Claim 88 (depends on 85)

The genetically modified yeast cell of claim 85, wherein the one or more enzymes of the MEV pathway comprise an enzyme that condenses two molecules of acetyl-CoA to form acetoacetyl-CoA.

### Claim 89 (depends on 85)

The genetically modified yeast cell of claim 85, wherein the one or more enzymes of the MEV pathway comprise an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA.

### Claim 90 (depends on 85)

The genetically modified yeast cell of claim 85, wherein the one or more enzymes of the MEV pathway comprise an enzyme that converts HMG-CoA to mevalonate.

### Claim 91 (depends on 85)

The genetically modified yeast cell of claim 85, wherein the one or more enzymes of the MEV pathway comprise an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate.

### Claim 92 (depends on 85)

The genetically modified yeast cell of claim 85, wherein the one or more enzymes of the MEV pathway comprise an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate.

### Claim 93 (depends on 85)

The genetically modified yeast cell of claim 85, wherein the one or more enzymes of the MEV pathway comprise an enzyme that converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate.

### Claim 94 (depends on 85)

The genetically modified yeast cell of claim 85, wherein the one or more enzymes of the MEV pathway are selected from HMG-CoA synthase, mevalonate kinase, phosphomevalonate kinase and mevalonate pyrophosphate decarboxylase.

### Claim 95 (depends on 85)

The genetically modified yeast cell of claim 85, wherein the cell comprises a plurality of heterologous nucleic acids encoding all of the enzymes of the MEV pathway.

### Claim 96 (depends on 85)

The genetically modified yeast cell of claim 85, wherein the one or more heterologous nucleic acids encoding one or more enzymes of the MEV pathway are under control of a single transcriptional regulator.

### Claim 97 (depends on 85)

The genetically modified yeast cell of claim 85, further comprising a heterologous nucleic acid encoding an enzyme that can convert isopentenyl pyrophosphate (IPP) into dimethylallyl pyrophosphate (DMAPP).

### Claim 98 (depends on 85)

The genetically modified yeast cell of claim 85, further comprising a heterologous nucleic acid encoding an enzyme that can condense IPP and/or DMAPP molecules to form a polyprenyl compound.

### Claim 99 (depends on 85)

The genetically modified yeast cell of claim 85, further comprising a heterologous nucleic acid encoding an enzyme that can modify IPP or a polyprenyl to form an isoprenoid compound.

### Claim 100 (depends on 99)

The genetically modified yeast cell of claim 99, wherein the enzyme that can modify IPP or a polyprenyl to form an isoprenoid compound is selected from the group consisting of carene synthase, geraniol synthase, linalool synthase, limonene synthase, myrcene synthase, ocimene synthase, α-pinene synthase, β-pinene synthase, γ-terpinene synthase, terpinolene synthase, amorphadiene synthase, α-farnesene synthase, β-farnesene synthase, farnesol synthase, nerolidol synthase, patchouliol synthase, nootkatone synthase, and abietadiene synthase.

### Claim 101 (depends on 99)

The genetically modified yeast cell of claim 99, wherein the isoprenoid is selected from the group consisting of a hemiterpene, monoterpene, diterpene, triterpene, tetraterpene, sesquiterpene, and polyterpene.

### Claim 102 (depends on 99)

The genetically modified yeast cell of claim 99, wherein the isoprenoid is a C5-C20 isoprenoid.

### Claim 103 (depends on 99)

The genetically modified yeast cell of claim 99, wherein the isoprenoid is selected from the group consisting of abietadiene, amorphadiene, carene, α-farnesene, β-farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, β-pinene, sabinene, γ-terpinene, terpinolene, and valencene.

### Claim 104 (depends on 85)

The genetically modified yeast cell of claim 85, wherein the yeast is Saccharomyces cerevisiae.

### Claim 105 (depends on 85)

A method for producing an isoprenoid comprising:
(a) culturing a population of the genetically modified yeast cell of claim 85 in a medium with a carbon source under conditions suitable for making said isoprenoid compound; and
(b) recovering said isoprenoid compound from the medium.

### Claim 106

A genetically modified yeast cell capable of producing an isoprenoid, the cell comprising:
(a) one or more heterologous nucleic acids encoding one or more enzymes of a mevalonate (MEV) pathway for making isopentenyl pyrophosphate; and
(b) a heterologous nucleic acid encoding an enzyme that condenses acetyl-CoA with malonyl-CoA to form acetoacetyl-CoA.

### Claim 107 (depends on 106)

The genetically modified yeast cell of claim 106, wherein the enzyme that condenses acetyl-CoA with malonyl-CoA to form acetoacetyl-CoA is an acetyl-CoA:malonyl-CoA acyltransferase.

### Claim 108 (depends on 106)

The genetically modified yeast cell of claim 106, wherein the one or more enzymes of the MEV pathway comprise an enzyme that condenses two molecules of acetyl-CoA to form acetoacetyl-CoA.

### Claim 109 (depends on 106)

The genetically modified yeast cell of claim 106, wherein the one or more enzymes of the MEV pathway comprise an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA.

### Claim 110 (depends on 106)

The genetically modified yeast cell of claim 106, wherein the one or more enzymes of the MEV pathway comprise an enzyme that converts HMG-CoA to mevalonate.

### Claim 111 (depends on 106)

The genetically modified yeast cell of claim 106, wherein the one or more enzymes of the MEV pathway comprise an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate.

### Claim 112 (depends on 106)

The genetically modified yeast cell of claim 106, wherein the one or more enzymes of the MEV pathway comprise an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate.

### Claim 113 (depends on 106)

The genetically modified yeast cell of claim 106, wherein the one or more enzymes of the MEV pathway comprise an enzyme that converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate.

### Claim 114 (depends on 106)

The genetically modified yeast cell of claim 106, wherein the one or more enzymes of the MEV pathway are selected from HMG-CoA synthase, mevalonate kinase, phosphomevalonate kinase and mevalonate pyrophosphate decarboxylase.

### Claim 115 (depends on 106)

The genetically modified yeast cell of claim 106, wherein the cell comprises a plurality of heterologous nucleic acids encoding all of the enzymes of the MEV pathway.

### Claim 116 (depends on 106)

The genetically modified yeast cell of claim 106, wherein the one or more heterologous nucleic acids encoding one or more enzymes of the MEV pathway are under control of a single transcriptional regulator.

### Claim 117 (depends on 106)

The genetically modified yeast cell of claim 106, further comprising a heterologous nucleic acid encoding an enzyme that can convert isopentenyl pyrophosphate (IPP) into dimethylallyl pyrophosphate (DMAPP).

### Claim 118 (depends on 106)

The genetically modified yeast cell of claim 106, further comprising a heterologous nucleic acid encoding an enzyme that can condense IPP and/or DMAPP molecules to form a polyprenyl compound.

### Claim 119 (depends on 106)

The genetically modified yeast cell of claim 106, further comprising a heterologous nucleic acid encoding an enzyme that can modify IPP or a polyprenyl to form an isoprenoid compound.

### Claim 120 (depends on 119)

The genetically modified yeast cell of claim 119, wherein the enzyme that can modify IPP or a polyprenyl to form an isoprenoid compound is selected from the group consisting of carene synthase, geraniol synthase, linalool synthase, limonene synthase, myrcene synthase, ocimene synthase, α-pinene synthase, β-pinene synthase, γ-terpinene synthase, terpinolene synthase, amorphadiene synthase, α-farnesene synthase, β-farnesene synthase, farnesol synthase, nerolidol synthase, patchouliol synthase, nootkatone synthase, and abietadiene synthase.

### Claim 121 (depends on 119)

The genetically modified yeast cell of claim 119, wherein the isoprenoid is selected from the group consisting of a hemiterpene, monoterpene, diterpene, triterpene, tetraterpene, sesquiterpene, and polyterpene.

### Claim 122 (depends on 119)

The genetically modified yeast cell of claim 119, wherein the isoprenoid is a C5-C20 isoprenoid.

### Claim 123 (depends on 119)

The genetically modified yeast cell of claim 119, wherein the isoprenoid is selected from the group consisting of abietadiene, amorphadiene, carene, α-farnesene, β-farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, β-pinene, sabinene, γ-terpinene, terpinolene, and valencene.

### Claim 124 (depends on 106)

The genetically modified yeast cell of claim 106, wherein the yeast is Saccharomyces cerevisiae.

### Claim 125 (depends on 106)

A method for producing an isoprenoid comprising:
(a) culturing a population of the genetically modified yeast cell of claim 106 in a medium with a carbon source under conditions suitable for making said isoprenoid compound; and
(b) recovering said isoprenoid compound from the medium.