# US 9,752,162 B2 · Production of mevalonate, isoprene, and isoprenoids using genes encoding polypeptides having thiolase, HMG-CoA synthase and HMG-CoA reductase enzymatic activities

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- Number: US 9,752,162 B2
- Document ID: US9752162B2
- Status: Granted
- Kind: B2
- Application number: 14/514,962
- Filed: 2014-10-15
- Published: 2015-02-05
- Granted: 2017-09-05
- Earliest claimed: 2011-04-29
- Assignees: Danisco US Inc.; The Goodyear Tire & Rubber Company
- Applicants: Danisco US Inc.; The Goodyear Tire & Rubber Company
- Inventors: Zachary Q. Beck; Michael C. Miller; Caroline M. Peres; Yuliya A. Primak; Jeff P. Pucci; Derek H. Wells
- Web page: https://patentgrep.com/patent/US9752162B2

## 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 9/1025: {Acyltransferases (2.3)}
- C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)}
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N 15/81: for yeasts
- C12P 5/002: {cyclic (compounds containing at least three condensed carbocyclic rings C12P15/00)}
- C12P 7/02: containing a hydroxy group
- C12P 7/04: acyclic
- C12P 7/42: Hydroxy-carboxylic acids
- C12Y 101/01034: Hydroxymethylglutaryl-CoA reductase (NADPH) (1.1.1.34)
- C12Y 203/01016: Acetyl-CoA C-acyltransferase (2.3.1.16)
- C12Y 203/0301: Hydroxymethylglutaryl-CoA synthase (2.3.3.10)
- Y02E 50/30: Fuel from waste, e.g. synthetic alcohol or diesel
- 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
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The invention features compositions and methods for the increased production of mevalonate, isoprene, isoprenoid precursor molecules, and/or isoprenoids in microorganisms via the heterologous expression of the mvaE and mvaS genes from the organisms Listeria grayi DSM 20601, Enterococcus faecium, Enterococcus gallinarum EG2, and Enterococcus casseliflavus.

## Claims

### Claim 1

Recombinant cells with increased production of isoprene, the cells comprising one or more heterologous nucleic acids comprising a mevalonate E (mvaE) gene and a mevalonate S (mvaS) gene selected from the organisms Listeria grayi (L. grayi), Enterococcus faecium (E. faecium), Enterococcus gallinarum (E. gallinarum), and Enterococcus casseliflavus (E. casseliflavus), wherein the mvaE gene and the mvaS gene encode polypeptides having thiolase, HMG-CoA synthase, and HMG-CoA reductase catalytic activities, and wherein the cells further comprise:
i. one or more nucleic acids encoding polypeptides of the lower MVA pathway; and
ii. a heterologous nucleic acid encoding an isoprene synthase polypeptide,
wherein the cells produce increased amounts of isoprene compared to isoprene-producing cells that do not comprise the mvaE gene and the mvaS gene.

### Claim 2 (depends on 1)

The cells of claim 1, wherein the mvaE gene and mvaS gene are from different organisms.

### Claim 3 (depends on 1)

The cells of claim 1, wherein:
a) the mvaE gene is from L. grayi and the mvaS gene is from E. faecium;
b) the mvaE gene is from L. grayi and the mvaS gene is from E. gallinarum;
c) the mvaE gene is from L. grayi and the mvaS gene is from E. casseliflavus;
d) the mvaE gene is from E. faecium and the mvaS gene is from L. grayi;
e) the mvaE gene is from E. faecium and the mvaS gene is from E. gallinarum;
f) the mvaE gene is from E. faecium and the mvaS gene is from E. casseliflavus;
g) the mvaE gene is from E. gallinarum and the mvaS gene is from L. grayi;
h) the mvaE gene is from E. gallinarum and the mvaS gene is from E. faecium;
i) the mvaE gene is from E. gallinarum and the mvaS gene is from E. casseliflavus;
j) the mvaE gene is from E. casseliflavus and the mvaS gene is from L. grayi;
k) the mvaE gene is from E. casseliflavus and the mvaS gene is from E. faecium; or
l) the mvaE gene is from E. casseliflavus and the mvaS gene is from E. gallinarum.

### Claim 4 (depends on 1)

The cells of claim 1, wherein the cells produce increased amounts of isoprene compared to isoprene-producing cells that (A) contain a mvaE gene from Enterococcus faecalis and (B) do not contain the mvaE gene and the mvaS gene from L. grayi, E. faecium, E. gallinarum, or E. casseliflavus.

### Claim 5 (depends on 1)

The cells of claim 1, wherein the nucleic acids encoding polypeptides of the lower MVA pathway comprise enzymes selected from: (a) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate; (b) an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate; and (c) an enzyme that converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate.

### Claim 6 (depends on 5)

The cells of claim 5, wherein the enzyme that phosphorylates mevalonate to mevalonate 5-phosphate is selected from the group consisting of M. mazeimevalonate kinase, M. burtonii mevalonate kinase polypeptide, Lactobacillus mevalonate kinase polypeptide, Lactobacillus sakei mevalonate kinase polypeptide, yeast mevalonate kinase polypeptide, Saccharomyces cerevisiae mevalonate kinase polypeptide, Streptococcus mevalonate kinase polypeptide, Streptococcus pneumoniae mevalonate kinase polypeptide, Streptomyces mevalonate kinase polypeptide, and Streptomyces CL190 mevalonate kinase polypeptide.

### Claim 7 (depends on 1)

The cells of claim 1, wherein the isoprene synthase polypeptide is a plant isoprene synthase polypeptide or a variant thereof.

### Claim 8 (depends on 7)

The cells of claim 7, wherein the isoprene synthase polypeptide is a polypeptide from Pueraria or Populus or a hybrid, Populus alba x Populus tremula, or a variant thereof.

### Claim 9 (depends on 7)

The cells of claim 7, wherein the plant isoprene synthase polypeptide is a Populus alba isoprene synthase polypeptide.

### Claim 10 (depends on 1)

The cells of claim 1, further comprising one or more nucleic acids encoding an isopentenyl-diphosphate delta-isomerase (IDI) polypeptide.

### Claim 11 (depends on 1)

The cells of claim 1, wherein any one or more nucleic acids is placed under an inducible promoter or a constitutive promoter.

### Claim 12 (depends on 1)

The cells of claim 1, wherein any one or more nucleic acids is cloned into a multicopy plasmid.

### Claim 13 (depends on 1)

The cells of claim 1, wherein any one or more nucleic acids is integrated into a chromosome of the cells.

### Claim 14 (depends on 1)

The cells of claim 1, wherein the cells are gram-positive bacterial cells, gram-negative bacterial cells, Escherichia cells, Pantoea cells, fungal cells, filamentous fungal cells, Trichoderma cells, Aspergillus cells, or yeast cells.

### Claim 15 (depends on 14)

The cells of claim 14, wherein the cells are selected from the group consisting of E. coli, P. citrea, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp., and P. alcaligenes cells.

### Claim 16 (depends on 15)

The cells of claim 15, wherein the cells are E. coli cells.

### Claim 17 (depends on 14)

The cells of claim 14, wherein the cells are yeast cells.

### Claim 18 (depends on 17)

The cells of claim 17, wherein the yeast cells are selected from the group consisting of Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., and Candida sp. cells.

### Claim 19 (depends on 18)

The cells of claim 18, wherein the yeast cells are Saccharomyces cerevisiae cells.

### Claim 20 (depends on 1)

A method of producing isoprene, comprising: (a) culturing the host cells of claim 1 under suitable culture conditions for the production of isoprene; and (b) producing the isoprene.

### Claim 21 (depends on 20)

The method of claim 20, further comprising (c) recovering the isoprene.