Harnessing yeast subcellular compartments for the production of plant terpenoids

1. Harnessing yeast subcellular compartments for the production of plant terpenoids • ——翟芳

2. Terpenoids • Synthesized by all organisms • A large and diverse class of biological compounds • 分子式为异戊二烯单位倍数的烃类及其含氧衍生物 • 生物功能：激素信号、电子运输、蛋白质修饰、生物膜结构和功能完整性 • 抗生素、植物抗毒素、色素、芳香化合物 • 商业用途：粘合剂、着色剂、香水、调料、药物等

3. Terpenoidsbiosynthetic pathway • MEP途径：大多数细菌 • MVA途径：古生菌和大多数真核生物

4. 酵母中的MVA途径

5. Enzymes that use IPP, DMAPP, and FDP isoprene units in eukaryotes are compartment-alizedwithin the cell. In S. cerevisiae, the MVA pathway provides all terpenoid-backbone precursors for molecules destined to both cytoplasmic and intracellular compartments. • Given that ubiquinone, heme A, and geranylgeranyldiphosphate are biosynthesized in the mitochondria, we reasoned that mitochondrial FDP might be harnessed for the production of selected terpenoids.

6. Construction of yeast expression vectors • Integrating plasmid pδ-UB • The promoter and 5’-UTRs of the copper-inducible promoter CUP1 were amplified by PCR from W303-1A yeast genomic DNA. • The 3’-UTR and terminator of CYC1 were PCR-amplified from yeast genomic DNA using primers that introduced multiple cloning sites. pδ-tHMG pδE-AtFDPS pδE-CsTPS1 pRS303N-CsTPS1 pδE-ADS pδE

7. Construction of yeast expression vectors

8. S. cerevisiae strains

9. Add copper chelator BCS

10. Results

11. Results

12. Discussion • The use of microbial platforms for terpene production has significant advantages: 1 the availability of data on metabolic networks 2 the ease of reconstructing and redesigning co- mpletepathways 3 the ability to scale up the fermentation process

13. Discussion • The use of a heterologous arabidopsis or human FDPS allowed enhanced production of valencene in S. cerevisiae. • In yeast, FDPS is under complex regulation; for instance, tRNA levels appear to regulate Erg20p levels. It is therefore plausible that the use of a heterologous FDPS circumvents this control mechanism.

14. Discussion • The higher efficiency of the BDXe strain might be due to its being a diploid, and not a labora-tory strain that accumulated mutations affec-ting MVA and terpenoid pathways.

15. Discussion • Valencene production levels were low compared to the levels of amorphadiene and to those repor-ted for other plant sesquiterpenes produced in yeast. • Indeed, the activity of valencene synthase is low relative to that of other terpene synthases.

16. Discussion • The enhanced levels of sesquiterpenes produced following mitochondrial targeting of terpene synthases, as compared to cytosolic terpenesynthases, suggest that the FDP pool is higher in the yeast mitochondria, FDP is more accessible for sesquiterpenebiosynthesis, or the terpene synthases are more active in the yeast mitochondria.

17. Discussion • Overall, it can be suggested that in yeast as well as in plants, the mitochondria possess strong potential as a factory for sesquiterpeneproduction. • The possibility of jointly harnessing different intracellular compartments, e.g. mitochondria and cytosol, for the production of terpenesof interest opens new and intriguing possibilities for the metabolic engineering of pathways leading to valuable natural compounds.