A Synthetic Biology Yeast Platform for the Biosynthesis of Plant Phenolic Diterpenes
International Conference on Plant Synthetic Biology and Bioengineering
2016
International Conference on Plant Synthetic Biology and Bioengineering
General Submissions
Plant foundry: Biopolymers, Biopharmaceuticals and Medically Effective Secondary Metabolites (Vaccines, antibodies, and metabolites)
Friday, December 16, 2016 - 1:15am to 1:40am
Using various EST databases from Rosmarinus officinalis and Salvia fruticosa we have isolated cytochrome P450 genes, hydroxyferruginol synthases (CYP76AH22-24) and C20-oxidases (CYP76AK6-8), which were tested for their activity in yeast. Co-expression of the genes (GGPPS, CPS, MS) for the biosynthesis of miltiradiene, the diterpene precursor of CA, along with the CYP76AH22-24 genes leads to the production of the pathway intermediates ferruginol and 11-hydroxyferruginol. These are natural products of rosemary, and were identified by mass spectrometry and NMR. Using modeling-based mutagenesis by comparing the CYP76AH22-24 enzymes to CYP76AH1, a ferruginol synthase from S. miltiorrhiza that cannot produce hydroxyferruginol, we identified three amino acid residues which are required and sufficient for the C11 hydroxylation of ferruginol. Lastly, co-expression of the C20-oxidases leads to the complete conversion of 11-hydroxyferruginol to CA (Scheler et al., 2016). Additional enzymes are being expressed to expand the repertoire of modifications on the phenolic diterpene skeleton, thereby constituting a platform for combinatorial biosynthesis of this class of biologically active diterpenoids.
Reference: Scheler et al., (2016), Elucidation of the biosynthesis of carnosic acid and its reconstitution in yeast, Nature Communications (in press).