title: Production of the forskolin precursor 11β-hydroxy-manoyl oxide in yeast using surrogate enzymatic activities.
abstract: BACKGROUND
  Several plant diterpenes have important biological properties. Among them, forskolin is a complex labdane-type diterpene whose biological activity stems from its ability to activate adenylyl cyclase and to elevate intracellular cAMP levels. As such, it is used in the control of blood pressure, in the protection from congestive heart failure, and in weight-loss supplements. Chemical synthesis of forskolin is challenging, and production of forskolin in engineered microbes could provide a sustainable source. To this end, we set out to establish a platform for the production of forskolin and related epoxy-labdanes in yeast.
  
  
  RESULTS
  Since the forskolin biosynthetic pathway has only been partially elucidated, and enzymes involved in terpene biosynthesis frequently exhibit relaxed substrate specificity, we explored the possibility of reconstructing missing steps of this pathway employing surrogate enzymes. Using CYP76AH24, a Salvia pomifera cytochrome P450 responsible for the oxidation of C-12 and C-11 of the abietane skeleton en route to carnosic acid, we were able to produce the forskolin precursor 11β-hydroxy-manoyl oxide in yeast. To improve 11β-hydroxy-manoyl oxide production, we undertook a chassis engineering effort involving the combination of three heterozygous yeast gene deletions (mct1/MCT1, whi2/WHI2, gdh1/GDH1) and obtained a 9.5-fold increase in 11β-hydroxy-manoyl oxide titers, reaching 21.2 mg L(-1).
  
  
  CONCLUSIONS
  In this study, we identify a surrogate enzyme for the specific and efficient hydroxylation of manoyl oxide at position C-11β and establish a platform that will facilitate the synthesis of a broad range of tricyclic (8,13)-epoxy-labdanes in yeast. This platform forms a basis for the heterologous production of forskolin and will facilitate the elucidation of subsequent steps of forskolin biosynthesis. In addition, this study highlights the usefulness of using surrogate enzymes for the production of intermediates of complex biosynthetic pathways. The combination of heterozygous deletions and the improved yeast strain reported here will provide a useful tool for the production of numerous other isoprenoids.
authors.affiliation: Department of Biochemistry, School of Medicine, University of Crete, P.O. Box 2208, 71003, Heraklion, Greece. igneacodruta@yahoo.com.
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, 15771, Athens, Greece. eioannou@pharm.uoa.gr.
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, 15771, Athens, Greece. pgeorgantea@pharm.uoa.gr.
authors.forename: Panagiota
authors.initials: P
authors.lastname: Georgantea
authors.affiliation: Institute of Applied Biosciences - Centre for Research and Technology Hellas (INAB-CERTH), P.O. Box 60361, 57001, Thermi, Thessaloniki, Greece. ftrikka@gmail.com.
authors.forename: Fotini A
authors.initials: FA
authors.lastname: Trikka
authors.affiliation: Department of Biochemistry, School of Medicine, University of Crete, P.O. Box 2208, 71003, Heraklion, Greece. ath.anastasia@gmail.com.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Athanasakoglou
authors.affiliation: Mediterranean Agronomic Institute of Chania, P.O. Box 85, 73100, Chania, Greece. sofia@maich.gr.
authors.forename: Sofia
authors.initials: S
authors.lastname: Loupassaki
authors.affiliation: Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis Zografou, 15771, Athens, Greece. roussis@pharm.uoa.gr.
authors.forename: Vassilios
authors.initials: V
authors.lastname: Roussis
authors.affiliation: Institute of Applied Biosciences - Centre for Research and Technology Hellas (INAB-CERTH), P.O. Box 60361, 57001, Thermi, Thessaloniki, Greece. antoniosmakris@yahoo.gr.
authors.forename: Antonios M
authors.initials: AM
authors.lastname: Makris
authors.affiliation: Department of Biochemistry, School of Medicine, University of Crete, P.O. Box 2208, 71003, Heraklion, Greece. s.kampranis@med.uoc.gr.
authors.forename: Sotirios C
authors.initials: SC
authors.lastname: Kampranis
mesh_terms: D045784:Abietanes / Q000096:biosynthesis / Q000737:chemistry; D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D053898:Biosynthetic Pathways; D005576:Colforsin / Q000737:chemistry / Q000378:metabolism*; D004224:Diterpenes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008798:Mevalonic Acid / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D013729:Terpenes / Q000737:chemistry / Q000378:metabolism
chemical_list: D045784:Abietanes; D004224:Diterpenes; D013729:Terpenes; C475535:manoyl oxide; D005576:Colforsin; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase; C018381:salvin; D008798:Mevalonic Acid