Team:Tec-Monterrey EKAM/Terpenoids

Implementation on a Platform for Terpenoid Production
The modular design strategy obviously poses a greater advantage to systems aimed at manufacturing related products, such as families of biomolecules, related to each other by common metabolic pathways of production. One such family of biomolecules is terpenoids, also known as isoprenoids, which are a kind of hydrocarbon made up of five-carbon isoprene units, arranged and modified in such a way that all of them share common physical and chemical characteristics and yet also exhibit a wide range of biological properties. Well-known cases are artemisinin, the drug with the most effective mechanism against malaria-causing P. falciparum, and taxol, a mitotic inhibitor used in chemotherapeutic cancer treatments. Both of them are produced in plants (a fern-like shrub and the bark of a tree, respectively), but their yields are usually low and the demand for their medical use is significantly high. Other examples include lycopene, the precursor in tomatoes for beta-carotene, and limonene, which is present in most cosmetic products distributed nowadays. The biological pathway through which terpenoids are synthesized in eukaryotes is the mevalonate pathway, which includes a series of molecular transformations from Acetyl-CoA to isopentenyl-5-pyrophosphate and dimethylallyl-pyrophosphate. These isomeric molecules are the common precursors for all terpenoid molecules. A bioengineering approach for terpenoid production involves using the mevalonate pathway and the appropriate synthases in order to end up with the desired molecule. The yeast P. pastoris already uses the mevalonate pathway, but it has been reported that the HMG-CoA reductase involved in its rate-limiting step is heavily regulated. This can be bypassed by a reported N-terminus truncation that renders it still functional but increases the total yield of the process. Applying the aforementioned modular biofactory design strategy to this model, it is possible to implement a biological platform for the synthesis of terpenoids. Using the production of lycopene as a proof of concept, the separate genes required include the truncated HMG-CoA reductase, GGPP synthase (CrtE), phytoene synthase (CrtB), and phytoene desaturase (CrtI), each with a transcription promoter, RBS and transcription terminator. Additionally, another module may be implemented to further optimize the platform for terpenoid production. P. pastoris utilizes a great part of the molecules produced in the mevalonate pathway to feed its sterol pathway, starting with the production of squalene. Silencing the squalene synthase gene, ERG9, would enable the use of precursors for terpenoid production. Although P. pastoris has no known RNAi mechanism, it has been shown that introducing genes for Dicer and Argonaute from Saccharomyces castellii successfully provides S. cerevisiae with the silencing mechanism. Introducing the AGO1 and DCR1 genes, together with an antisense sequence for the ERG9 gene, a separate module for the platform is generated.