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