title: Reconstructing the chemical diversity of labdane-type diterpene biosynthesis in yeast.
abstract: Terpenes are a large class of natural products, many of which are used in cosmetics, pharmaceuticals, or biofuels. However, terpene's industrial application is frequently hindered by limited availability of natural sources or low yields of chemical synthesis. In this report, we developed a modular platform based on standardized and exchangeable parts to reproduce and potentially expand the diversity of terpene structures in Saccharomyces cerevisiae. By combining different module-specific parts, we exploited the substrate promiscuity of class I diterpene synthases to produce an array of labdane-type scaffolds. These were subsequently modified by a scaffold decoration module consisting of a mutant library of a promiscuous cytochrome P450 to afford a range of hydroxylated diterpenes. Further P450 protein engineering yielded dedicated and efficient catalysts for specific products. Terpenes produced include precursors of pharmacologically important compounds, molecules that are difficult to obtain from natural sources, or new natural products. The approach described here provides a platform on which additional gene mining, combinatorial biosynthesis, and protein engineering efforts can be integrated to sustainably explore the terpene chemical space.
authors.affiliation: Department of Biochemistry, School of Medicine, University of Crete, P.O. Box 2208, Heraklion 71003, Greece; Mediterranean Agronomic Institute of Chania, P.O. Box 85, Chania 73100, Greece.
authors.forename: Codruta
authors.initials: C
authors.lastname: Ignea
authors.affiliation: Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens 15771, Greece.
authors.forename: Efstathia
authors.initials: E
authors.lastname: Ioannou
authors.affiliation: Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens 15771, Greece.
authors.forename: Panagiota
authors.initials: P
authors.lastname: Georgantea
authors.affiliation: Mediterranean Agronomic Institute of Chania, P.O. Box 85, Chania 73100, Greece.
authors.forename: Sofia
authors.initials: S
authors.lastname: Loupassaki
authors.affiliation: Institute of Applied Biosciences-Centre for Research and Technology Hellas (INAB-CERTH), P.O. Box 60361, Thermi, 57001 Thessaloniki, Greece.
authors.forename: Fotini A
authors.initials: FA
authors.lastname: Trikka
authors.affiliation: Institute of Applied Biosciences-Centre for Research and Technology Hellas (INAB-CERTH), P.O. Box 60361, Thermi, 57001 Thessaloniki, Greece; Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece.
authors.forename: Angelos K
authors.initials: AK
authors.lastname: Kanellis
authors.affiliation: Institute of Applied Biosciences-Centre for Research and Technology Hellas (INAB-CERTH), P.O. Box 60361, Thermi, 57001 Thessaloniki, Greece.
authors.forename: Antonios M
authors.initials: AM
authors.lastname: Makris
authors.affiliation: Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens 15771, Greece.
authors.forename: Vassilios
authors.initials: V
authors.lastname: Roussis
authors.affiliation: Department of Biochemistry, School of Medicine, University of Crete, P.O. Box 2208, Heraklion 71003, Greece; Mediterranean Agronomic Institute of Chania, P.O. Box 85, Chania 73100, Greece. Electronic address: s.kampranis@med.uoc.gr.
authors.forename: Sotirios C
authors.initials: SC
authors.lastname: Kampranis
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004224:Diterpenes / Q000378:metabolism*; D006900:Hydroxylation / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004224:Diterpenes; D029701:Saccharomyces cerevisiae Proteins; D003577:Cytochrome P-450 Enzyme System
keywords: Combinatorial biosynthesis; Cytochrome P450; Isoprenoids; Metabolic engineering; Protein engineering