A Synthetic Biology Yeast Platform for the Biosynthesis of Plant Phenolic Diterpenes

The phenolic diterpenes carnosic acid (CA) and carnosol (CO) are of special interest due to their use as food additive and various biological activities. They are present in species of the Lamiaceae family such as rosemary (Rosmarinus officinalis) and various species of sage (e.g. Salvia fruticosa). Several studies over the past years report on the anti-oxidative, anti-cancer, anti-microbial and anti-inflammatory activities of PDs. Furthermore, the biosynthesis of the related tanshinone diterpenoids from Salvia miltiorrhiza, which also have high antioxidant, antibiotic and anti-inflammatory activities, is presumed to proceed via CA. Thus, elucidation of the biosynthesis of CA represents a key step in understanding the genesis of phenolic diterpenes and will provide the basis for metabolic engineering of this class of compounds. To facilitate the co-expression of multiple genes in the yeast Saccharomyces cerevisiae, a Golden Gate based modular cloning system was established with a set of parts including promoters and terminators.

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).