# US 10,246,694 B2 · Phosphoketolases for improved production of acetyl coenzyme A-derived metabolites, isoprene, isoprenoid precursors, and isoprenoids

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- Number: US 10,246,694 B2
- Document ID: US10246694B2
- Status: Granted
- Kind: B2
- Application number: 14/783,391
- Filed: 2014-04-10
- Published: 2016-03-10
- Granted: 2019-04-02
- Earliest claimed: 2013-04-10
- Assignees: Danisco US Inc.; The Goodyear Tire & Rubber Company
- Applicants: Danisco US Inc.; The Goodyear Tire & Rubber Company
- Inventors: Zachary Q. Beck; Jeffrey W. Munos; Derek H. Wells; Jian Yao
- Web page: https://patentgrep.com/patent/US10246694B2

## CPC

- C12N 9/88: Lyases (4.)
- C12P 5/007: {containing one or more isoprene units, i.e. terpenes (carotenes C12P23/00)}
- C12P 5/026: {Unsaturated compounds, i.e. alkenes, alkynes or allenes}
- C12P 19/32: having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
- C12Q 1/527: involving lyase
- C12Y 401/02009: Phosphoketolase (4.1.2.9)
- 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)}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Y: ENZYMES

## Abstract

This present invention relates to cultured recombinant cells comprising a heterologous phosphoketolase (PKL) polypeptide that are capable of increased production of acetyl coenzyme A-derived metabolites, as well as methods for producing and using the same. In some embodiments, the recombinant cells further comprise one or more mevalonate (MVA) pathway polypeptides for the production of isoprenoid precursors, isoprene and isoprenoids.

## Claims

### Claim 1

A recombinant cell capable of increased carbon flux through the phosphoketolase pathway, wherein the recombinant cell comprises: (i) a heterologous nucleic acid sequence encoding a polypeptide having phosphoketolase activity, wherein the polypeptide comprises at least 65% sequence identity to SEQ ID NO:8 or SEQ ID NO:11 and (ii) one or more nucleic acids encoding one or more polypeptides of the complete mevalonate (MVA) pathway, wherein the recombinant cell is a recombinant Escherichia coli cell, and wherein
(1) said recombinant cell comprising said polypeptide having phosphoketolase activity of (i) has a Performance Index value of greater than 1.0 in one or more of the following parameters: (a) cell growth on glucose, (b) cell growth on xylose, (c) production of intracellular acetyl-phosphate or (d) cell growth on glucose-6-phosphate, wherein the Performance Index value is calculated as activity per unit relative to a corresponding cell expressing a phosphoketolase from E. gallinarum or
(2) said polypeptide having phosphoketolase activity of (i) has a Performance Index value of greater than 1.0 in one or more of the following parameters: (e) protein solubility, (f) protein expression, or (g) fructose-6-phosphate (F6P) Specific Activity, wherein the Performance Index value is calculated as activity per unit relative to a phosphoketolase from E. gallinarum.

### Claim 2 (depends on 1)

The recombinant cell of claim 1, wherein the polypeptide comprises at least 65% sequence identity to SEQ ID NO:8 and wherein the polypeptide comprises at least 90% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31.

### Claim 3 (depends on 1)

The recombinant cell of claim 1, wherein the polypeptide comprises at least 65% sequence identity to SEQ ID NO:11 and wherein the polypeptide comprises at least 90% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47.

### Claim 4 (depends on 1)

The recombinant cell of claim 1, wherein culturing of the recombinant cell in a suitable media increases one or more of an intracellular amount of erythrose 4-phosphate, an intracellular amount of glyceraldehyde 3-phosphate, or intracellular amount phosphate.

### Claim 5 (depends on 1)

The recombinant cell of claim 1, wherein the polypeptide having phosphoketolase activity is capable of synthesizing glyceraldehyde 3-phosphate and acetyl phosphate from xylulose 5-phosphate.

### Claim 6 (depends on 1)

The recombinant cell of claim 1, wherein the polypeptide having phosphoketolase activity is capable of synthesizing erythrose 4-phosphate and acetyl phosphate from fructose 6-phosphate.

### Claim 7 (depends on 1)

The recombinant cell of claim 1, wherein the one or more polypeptides of the complete MVA pathway is selected from (a) acetyl-CoA acetyltransferase; (b) 3-hydroxy-3-methylglutaryl-CoA synthase; (c) 3-hydroxy-3-methylglutaryl-CoA reductase; (d) mevalonate kinase; (e) phosphomevalonate kinase; and (f) diphosphomevalonate decarboxylase.

### Claim 8 (depends on 1)

The recombinant cell of claim 1, further comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide, wherein culturing of the recombinant cell in a suitable media provides for the production of isoprene with a Performance Index value of greater than 1.0 in one or more of the following parameters: (h) isoprene yield or (i) isoprene specific productivity.

### Claim 9 (depends on 8)

The recombinant cell of claim 8, wherein the isoprene synthase polypeptide is a plant isoprene synthase polypeptide.

### Claim 10 (depends on 9)

The recombinant cell of claim 9, wherein the plant isoprene synthase polypeptide is a polypeptide from Pueraria or Populus or a hybrid, Populus alba×Populus tremula.

### Claim 11 (depends on 9)

The recombinant cell of claim 9, wherein the plant isoprene synthase polypeptide is a polypeptide from Pueraria montana, Pueraria lobata, Populus tremuloides, Populus alba, Populus nigra, or Populus trichocarpa.

### Claim 12 (depends on 8)

The recombinant cell of claim 8, wherein the recombinant cell further comprises one or more nucleic acids encoding one or more 1-deoxy-D-xylulose 5-phosphate (DXP) pathway polypeptides.

### Claim 13 (depends on 1)

The recombinant cell of claim 1, further comprising a heterologous nucleic acid encoding a polyprenyl pyrophosphate synthase polypeptide, wherein culturing of the recombinant cell in a suitable media provides for the production of isoprenoids.

### Claim 14 (depends on 1)

The recombinant cell of claim 1, wherein culturing of the recombinant cell in a suitable media provides for the production of an acetyl CoA-derived metabolite.

### Claim 15 (depends on 13)

The recombinant cell of claim 13, wherein the isoprenoid is selected from the group consisting of monoterpenes, diterpenes, triterpenes, tetraterpenes, sequiterpene, and polyterpene.

### Claim 16 (depends on 13)

The recombinant cell of claim 13, wherein the isoprenoid is selected from the group consisting of abietadiene, amorphadiene, carene, α-farnesene, 13-farnesene, farnesol, geraniol, geranylgeraniol, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, 13-pinene, sabinene, γ-terpinene, terpindene and valencene.

### Claim 17 (depends on 14)

The recombinant cell of claim 14, wherein the acetyl CoA-derived metabolite is selected from the group consisting of polyketides, polyhydroxybutyrate, fatty alcohols, amino acids, and fatty acids.

### Claim 18 (depends on 14)

The recombinant cell of claim 14, wherein the acetyl CoA-derived metabolite is selected from the group consisting of acetone, isopropanol, isobutene, and propene.

### Claim 19 (depends on 8)

A method of producing isoprene comprising: (a) culturing the recombinant cell of claim 8 under conditions suitable for producing isoprene and (b) producing isoprene.

### Claim 20 (depends on 13)

A method of producing an isoprenoid comprising: (a) culturing the recombinant cell of claim 13 under conditions suitable for producing an isoprenoid and (b) producing an isoprenoid.