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Identification and validation of candidate genes to enzyme subcellular localization in plants and yeast cells
Identification and validation of candidate genes to enzyme subcellular localization in plants and yeast cells
Natural compounds extracted from microorganisms or plants constitute an inexhaustible source of valuable molecules whose supply may be limited by biological sources. The latest advances in synthetic biology, combined with the increasing availability of extensive transcriptome and genomics data, now provide new alternatives to the production of these molecules by transferring their entire biosynthetic pathways to heterologous production platforms, such as yeasts or bacteria. Although the production of high-titer strains is still a difficult research field, it is necessary to clarify biosynthetic pathways and describe their complex subcellular structures for the effective development of such bioengineering methods. In the framework of plant and yeast examples, we describe effective ways to rationalize the study of some feature pathways, including the basis of computational applications for identifying candidate genes in transcriptome data, and the verification of their functions by improved methods. The possible resistance to Agrobacterium tumefaciens-mediated viral vectors can be obtained by direct DNA transfer mediated gene silencing. In order to identify potential changes in biosynthetic flux due to enzyme dislocations in the recombinant pathway, we also describe in detail the protocol for characterizing subcellular localization of proteins in plant cells by bio-mediated TR expression fluorescent protein fusion. A similar fluorescence program was used to locate and transfer the transferase in yeasts. Although originally developed for Catharanthus roseus in Madagascar, these methods can be applied to other plants or organisms to establish synthetic biology platforms.
