Microbial engineering for the production of chemical and pharmaceutical products from the isoprenoid pathway

Patent metadata

Field	Value
Publication number	US-9796980-B2
Application number	US-201615152736-A
Country	US
Kind code	B2
Filing date	May 12, 2016
Priority date	Nov 10, 2009
Publication date	Oct 24, 2017
Grant date	Oct 24, 2017

Abstract

The invention relates to recombinant expression of a taxadiene synthase enzyme and a geranylgeranyl diphosphate synthase (GGPPS) enzyme in cells and the production of terpenoids.

First claim

What is claimed is:

1. An E. coli cell producing a monoterpenoid, diterpenoid, or sesquiterpenoid compound, the E. coli cell comprising:
an upstream MEP pathway module that comprises a duplicate copy of dxs, idi, ispD and ispF genes integrated into the chromosome, so as to increase metabolic flux through the MEP pathway; and
a downstream terpenoid synthesis pathway module comprising a heterologous GPPS, GGPPS, or FPPS enzyme, and heterologous terpenoid synthase enzyme producing a monoterpenoid, diterpenoid, or sesquiterpenoid compound;
the expression or activity of the downstream and upstream pathway modules being balanced such that indole production is less than 100 mg/L in culture.

2. The cell of claim 1 , wherein a duplicate copy of the dxs, idi, ispD and ispF genes is a dsx-idi-ispDF operon.

3. The cell of claim 1 , wherein the heterologous GPPS, GGPPS or FPPS enzyme, and the heterologous terpenoid synthase enzyme are expressed in an operon.

4. The cell of claim 1 , wherein an upstream MEP pathway module and/or a downstream terpenoid synthesis pathway module are expressed from a promoter selected from Trc, T5, and T7.

5. The cell of claim 1 , wherein the compound is a monoterpenoid.

6. The cell of claim 1 , wherein the compound is a diterpenoid.

7. The cell of claim 1 , wherein the compound is a sesquiterpenoid.

8. The cell of claim 1 , wherein the compound is taxadiene.

9. The cell of claim 1 , wherein the downstream terpenoid synthesis pathway comprises one or more cytochrome P450 enzymes.

10. The cell of claim 9 , wherein at least one cytochrome P450 enzyme is a chimeric P450 enzyme fused to a cytochrome P450 reductase.

11. The cell of claim 9 , wherein the cytochrome P450 enzyme comprises the N-terminal peptide MALLLAVF (SEQ ID NO:51).

12. The cell of claim 1 , wherein the cell comprises a GGPPS enzyme derived from a plant.

13. The cell of claim 1 , wherein the compound is citronellol, cubebol, nootkatone, cineol, limonene, eleutherobin, sarcodictyin, pseudopterosin, ginkgolide, stevioside, rebaudioside A, sclareol, labdenediol, levopimaradiene, sandracopimaradiene, or isopemaradiene.

14. The cell of claim 1 , wherein the compound is geraniol, farnesol, geranylgeraniol, linalool, limonene, pinene, or cineol.

Assignees

• Massachusetts Inst Technology
• Nat Univ Singapore

Inventors

• Ajikumar Parayil K
• Stephanopoulos Gregory
• Too Heng Phon

Classifications

• C12P17/02 Primary

  Oxygen as only ring hetero atoms · CPC title

• C12N15/70 Primary

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12P15/00

  Preparation of compounds containing at least three condensed carbocyclic rings {(gibbanes C12P27/00; naphthacenes C12P29/00)} · CPC title