# US 11,028,413 B2 · Microbial engineering for the production of chemical and pharmaceutical products from the isoprenoid pathway

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

- Number: US 11,028,413 B2
- Document ID: US11028413B2
- Status: Granted
- Kind: B2
- Application number: 16/809,838
- Filed: 2020-03-05
- Published: 2020-09-10
- Granted: 2021-06-08
- Earliest claimed: 2009-11-10
- Assignees: Massachusetts Institute of Technology; National University of Singapore
- Applicants: Massachusetts Institute of Technology; National University of Singapore
- Inventors: Parayil K. Ajikumar; Gregory Stephanopoulos; Heng Phon Too
- Web page: https://patentgrep.com/patent/US11028413B2

## CPC

- C12P 5/007: {containing one or more isoprene units, i.e. terpenes (carotenes C12P23/00)}
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12P 7/42: Hydroxy-carboxylic acids
- C12P 9/00: Preparation of organic compounds containing a metal or atom other than H, N, C, O, S or halogen {(phosphoglycerides, C12P7/6481)}
- C12P 15/00: Preparation of compounds containing at least three condensed carbocyclic rings {(gibbanes C12P27/00; naphthacenes C12P29/00)}
- C12P 17/02: Oxygen as only ring hetero atoms
- C12P 19/56: having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
- 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)
- C12Q 1/689: for bacteria
- C12Q 2600/158: Expression markers
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- 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
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The invention relates to the production of one or more terpenoids through microbial engineering, and relates to the manufacture of products comprising terpenoids.

## Claims

### Claim 1

A method for making a pharmaceutical product, the method comprising:
providing an Escherichia coli (E. coli) that produces isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) through an upstream methylerythritol pathway (MEP) and converts the IPP and DMAPP to amorphadiene or derivative thereof through a recombinantly expressed downstream synthesis pathway comprising farnesyl diphosphate synthase and amorphadiene synthase;
culturing the E. coli to produce the amorphadiene or derivative thereof, wherein the accumulation of indole in the culture is controlled to below 100 mg/L to thereby increase production of amorphadiene or derivative thereof; and
incorporating the amorphadiene or derivative thereof, into a pharmaceutical product.

### Claim 2 (depends on 1)

The method of claim 1, wherein accumulation of indole in the culture is controlled by balancing the upstream MEP pathway with the downstream terpenoid synthesis pathway.

### Claim 3 (depends on 1)

The method of claim 1, further comprising measuring the amount or concentration of indole continuously or intermittently.

### Claim 4 (depends on 1)

The method of claim 1, wherein accumulation of indole in the culture is maintained to below 50 mg/L.

### Claim 5 (depends on 1)

The method of claim 1, wherein accumulation of indole in the culture is maintained to below 10 mg/L.

### Claim 6 (depends on 1)

The method of claim 1, wherein the amorphadiene or derivative thereof is produced at 10 mg/L or more.

### Claim 7 (depends on 1)

The method of claim 1, wherein the amorphadiene or derivative thereof is produced at 100 mg/L or more.

### Claim 8 (depends on 1)

The method of claim 1, wherein the E. coli has additional copies of one or more of the dxs, idi, ispD, and ispF genes of the MEP pathway.

### Claim 9 (depends on 8)

The method of claim 8, wherein the E. coli has a heterologous dxs-idi-ispDF operon.

### Claim 10 (depends on 1)

The method of claim 1, wherein the recombinantly expressed downstream synthesis pathway further comprises one or more of amorpha-4,11-diene monooxygenase, and artemisinic aldehyde delta-11(13) reductase.

### Claim 11 (depends on 1)

The method of claim 1, wherein the amorphadiene derivative is artemisinin.

### Claim 12

A method for making a terpenoid, the method comprising:
providing an Escherichia coli (E. coli) that produces isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) through an upstream methylerythritol pathway (MEP) and converts the IPP and DMAPP to amorphadiene or derivative thereof through a recombinantly expressed downstream synthesis pathway comprising farnesyl diphosphate synthase and amorphadiene synthase; and
culturing the E. coli to produce the amorphadiene or derivative thereof, wherein the accumulation of indole in the culture is controlled to below 100 mg/L to thereby increase terpenoid production; and recovering the amorphadiene or derivative thereof.

### Claim 13 (depends on 12)

The method of claim 12, wherein accumulation of indole in the culture is controlled by balancing the upstream MEP pathway with the downstream terpenoid synthesis pathway.

### Claim 14 (depends on 12)

The method of claim 12, further comprising, measuring the amount or concentration of indole continuously or intermittently.

### Claim 15 (depends on 12)

The method of claim 12, wherein accumulation of indole in the culture is maintained to below 50 mg/L.

### Claim 16 (depends on 12)

The method of claim 12, wherein accumulation of indole in the culture is maintained to below 10 mg/L.

### Claim 17 (depends on 12)

The method of claim 12, wherein the amorphadiene or derivative thereof is produced at 10 mg/L or more.

### Claim 18 (depends on 12)

The method of claim 12, wherein the amorphadiene or derivative thereof is produced at 100 mg/L or more.

### Claim 19 (depends on 12)

The method of claim 12, wherein the E. coli has additional copies of one or more of the dxs, idi, ispD and ispF genes of the MEP pathway.

### Claim 20 (depends on 12)

The method of claim 12, wherein the E. coli has a heterologous dxs-idi-ispDF operon.

### Claim 21 (depends on 12)

The method of claim 12, wherein the recombinantly expressed downstream synthesis pathway further comprises one or more of amorpha-4,11-diene monooxygenase, and artemisinic aldehyde delta-11(13) reductase.

### Claim 22 (depends on 12)

The method of claim 12, wherein the amorphadiene derivative is artemisinin.