Question2

toc =Text of the Question:= Jay Keasling at the University of California-Berkeley has come up with a means of biosynthetically generating the anti-malarial compound artemisinin. To do this, Jay had to go aorund the normal means by which //E. coli// produces isoprenoids. Diagram the alternative pathway that Jay used. Cite appropriate literature sources.

=Normal Process by which E. Coli produces isoprenoids:= Two Isoprenoid biosynthetic pathways exist that synthesize two key precursors of artemisinin: isopentenyl pyrophosphate (IPP), and its isomer dimethylallyl pyrophosphate (DMAPP). Eukaryotes other than plants use only the mevalonate-dependent (MEV) isoprenoid pathway to convert acetyl-CoA to IPP. Plants use both the MEW and the mevalonate-independent (deoxyxylulose 5-phosphate-DXP) pathway. Prokaryotes use the DXP pathway to produce IPP and DMAPP separately through a branch point. The use of the DXP pathway in E. Coli has proven to be ineffective in producing isoprenoids. However, genetic introduction of MEV pathway genes into the E. Coli DXP pathway along with a synthetic amorphadiene synthase gene has resulted in a biosynthetically useful method for production of artemisinin.



=Alternative Process:=

There are two basic pathways by which artemisinin is produced: the mevalonate (MEV) pathway in eukaryotes and the deoxyxylulose 5-phosphate (DXP) pathway in prokaryotes. Dr Keasling engineered a pathway using four genes from MEV, namely MevT, pMevB, pMBI, and pMBIS, encoding the mevalonate-dependent pathway and inserted them into E. coli. These genes were functional and expressed IPP and DMAPP, which are the essential precursors to FPP. The pMevT plasmid was transformed into pMBI or pMBIS, which encoded the biosynthetic pathway of isoprenoids.

The second figure shows the overview of the MEV and DXP pathways. The four MEV genes that are inserted into the DXP pathway are MevT, pMevT, pMBI, and pMBIS. These lead to the production of IPP and DMAPP which yields FPP.

The fourth figure shows the conversion of FPP to Amorphadiene through amorphadiene synthesase. Through cytochrome P450 oxiodreductase, Amorphadiene is converted to Artemisinin in a series of reactions, with Artemisinic Acid as an intermediate.





=Appropriate Literature Choices:=

2.1. [|Martin, V. J. J., D. J. Pitera, et al. (2003). "Engineering a mevalonate pathway in Escherichia coli for production of terpenoids." Nat Biotech 21(7): 796-802.] 2.2. [|Ro, D.-K., E. M. Paradise, et al. (2006). "Production of the antimalarial drug precursor artemisinic acid in engineered yeast." Nature 440(7086): 940-943.] 2.3. [|Lee, S. Y., H. U. Kim, et al. (2009). "Metabolic engineering of microorganisms: general strategies and drug production." Drug Discovery Today 14(1-2): 78-88.] 2.4. http://www.lbl.gov/Science-Articles/Archive/assets/images/2004/Mar-24/Engineering_terpenoids.pdf