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Reconstruction in yeast of human steroid metabolic pathway as a tool for drug discovery and biosynthesis Denis POMPON Laboratoire d’Ingénierie des Protéines Membranaires CGM-CNRS Gif-sur-Yvette, France. Glycolyis Simple carbon sources Sterol biosynthesis is a major target for drugs (cholesterol lowering & antifungal drug) Defect in sterol synthesis or in transport is found in several genetic diseases sterols STEROLS cholesterol CORTISOL Membranes Steroid hormones Bile acids Steroid hormones Vitamin D Adrenal gland Cholesterol ester Cholesterol High organized tissue dependent spatial organization Multiple subcellular biosynthesis location Simple unicellular eukaryote microorganism Highly branched multi-step biosynthetic pathway with complex regulation Multi- organ transport & metabolism Make Yeast Human TOOLS MODELS Redesigning animal steroid hormones biosynthesis for yeast Humanization of yeast sterol biosynthesis Redesigning animal steroid hormones biosynthesis for yeast Self-sufficient biosynthesis from simple carbon source Mimetic but not necessarily a copy of natural process Target a single end-product instead of the natural hormones spectra Optimized for productivity and not to be a model Biosynthesis involved large number of membrane bound enzymes needing eukaryote environment. No cholesterol in yeast Cortisol biosynthesis in animals Required enzymes absent Cholesterol Required electron transport chains absent CH 3 C O HO Pregnenolone CH 3 C O P450scc ADX, ADR Pregnenolone HO HO 3-HSDH Mitochondria Dealing with subcellular transport of heterologous enzymes CH OH 2 C O OH Cortisol HO O P450c11 Dealing with subcellular traffic of lipophilic intermediates O Endoplasmic Reticulum CH OH 2 C O OH Progesterone CH 3 C O OH P450c21 O 17a-Hydroxy P450c17 CH OH 2 C O OH Progesterone ADX, ADR O CH 3 C O 11-Deoxycortisol O 11-Deoxycortisol Substitute for cholesterol with a yeast metabolism derived sterol Campesterol (plant sterol) S-adenosyl sterol methylene transferase (ERG6 p) D22-desaturase (ERG5 p) 28 CH3 Sterol 24(28) reductase 21 22 CH3 18 19 CH3 11 CH3 12 24 20 23 17 13 CH3 26 25 CH3 27 16 9 2 HO 1 3 10 8 5 4 14 15 Sterol 24(25) reductase 7 6 Sterol D7 reductase CH3 Ergosterol (yeast) CH3 CH3 CH3 CH3 Gene critical for differences Only present in yeast Only present in animals HO Cholesterol (animals) H3C CH3 ERGOSTEROL CH3 CH3 Simple carbon sources CH3 CH3 Ergosterol CH3 Common to yeast and animals STEROLS H3C Squalene CH3 Lanosterol Zymosterol Yeast specific Campesterol Cholesterol H 3C CH 3 CH 3 H C 3 CH3 H3C CH3 H3C CH3 O CH3 CH3 CH3 CH3 CH3 HO HO HO Pregnenolone Animal specific Steroid biosynthesis Hydrocortisone Engineered yeast Campesterol Zymosterol SAM-transferase D8-7 isomerase D22-desaturase disruption D5-desaturase, cyt. b5 NADPH HO HO HO ergosterol NADPH A. thaliana D7- reductase HO NADP + P450 SCC ADR HO ADX O HO HO HO HO Pregnenolone Ergosta 5-ene ol (campesterol) Dealing with intracellular targeting and compartmentalization Plasma membrane Endoplasmic reticulum sterols Exogenous sterols neosynthesis sterols progesterone storage Lipid droplet Mitochondria 11-deoxy cortisol Two similar reactions occur on internal mitochondrial membrane in animal and involve the same electron transfer chain ….. P450 SCC + ADX + ADR cholesterol pregnenolone P450 C11 + ADX +ADR 11-deoxycortisol cortisol Cholesterol side chain cleavage reaction NADPH ADR e- (FAD) NADP+ P450 SCC Passive output Active input Pregnenolone Cholesterol Cholesterol, in contrast to oxysteroids, is insoluble and needs complex and function critical transport machinery to enter mitochondria in animals. -Intra-mitochondrial transport of cholesterol (campesterol) cannot be rebuilt in yeast -Signal sequence engineering allowed mitochondrial import in yeast of the 4 required components of the animal system but AdRed enzyme does not fold properly in yeast mitochondria Yeast reconstruction of the campesterol side chain cleavage sterol SCC human Transporter Mito (IM) yeast Plasma membrane Plasma membrane Electron transport chain ADX ADR campesterol SCC Confocale immunofluorescence analysis of Met-mat SCC localization in yeast Immuno-localization Subcellular markers Met-mature P450SCC Mito (LM) Mito (IM) Cytosol endoplasmic reticulum NADPH e- ADX e- ADR Double labelling Gpa1p Plasma membrane Dpm1p endoplasmic reticulum Porin mitochondria pregnenolone 11-deoxycortisol Pregnenolone acetate production by the strain CA10/pCD63 erg5 bovine adrenodoxin reductase m-P450 SCC M-ADX 800 Fedbatch Stationary phase OD at 600nm A. thaliana D7 –sterol reductase Pregnenolone acetate (mg/l) Batch 600 400 200 0 - 50 100 150 200 culture time (h) 250 Yeast reconstruction of the 11-hydroxylase activity Electron transport chain P450 C11 ADX ADR human Mito (I.M.) Mito (matrix) yeast COX fusion Mito (I.M.) COX fusion Mito (matrix) P450 C11 does not fold outside of mitochondrial context ADR does not fold inside of the mitochondrial context Yeast ARH1p has an unrelated essential function in yeast in iron transport but shows a significant sequence similarity with human ADR. Experiment demonstrated that ARH1p can nicely substitute for human ADR in the mammalian electron transfer chain. ? ADR Mito (I.M.) EndogenousARH1p Mito (I.M.) The microsomal part of the cortisol biosynthesis Pregnenolone CH 3 C O 3-HSDH HO 17a-hydroxy Progesterone Progesterone CH 3 C O OH CH 3 C O O electrons P450c17 NADPH CPR NADP+ P450c21 CH OH 2 C O OH O 11-Deoxycortisol O Side reactions and end-point control. Side reactions are linked to endogenous enzyme activities acting on intermediates or final product of the artificial metabolic pathways Natural substrates of these interacting enzymes can be very different from intermediates involved in parasitic activities making sometime identification of corresponding interfering genes very tricky. Unexpected retro-inhibition of sterol synthesis by pregnenolone upon disruption of parasitic Atf2p acetylase activity. Parasitic aldo-ketoreductase activity from enzymes of central carbon cycle acting on steroid ER & plasma membrane Inhibition 3-HSD progesterone progesterone ergosta --enol 5 enol CYP17A1 17-OH progesterone NADPH P450 SCC CYP21A1 pregnenolone Yeast Gcy1p and Ypr1p are aldo-keto-reductases of central carbon metabolism ATF2p Pregnenolone acetate 11-deoxy cortisol Reticulum CYP11B1, ADX, Arh1p 3 -HSD Gcy1p Ypr1p 17a, 20-dihydroxypregn -4ene-3-one CYP11B1, ADX , Arh1p 11,17a, 20- tri-hydroxy pregn-4-ene-3-one progesterone HYDROCORTISONE Gene dosage effects Pregnenolone P450 C21 21-OH P P450 C11 35 P450 C17 30 17 17-OH -OH P P 20 P450 C17 P450 C21 25 Gene dosage effect on a branched pathway dramatically affect the output of the biosynthesis 17, 21 OH Progesterone progesterone 40 17-OH 21-OH 3-HSD In the absence of regulation tight tuning is requested to accumulate correct end-product 15 10 5 MC 17, 21-OH P 0 A cortisol B C D Global pathway engineering of yeast for cortisol production Genomic integrations of transgenes D7-sterol reductase A. thaliana Bovine P450 C17 Bovine P450 C21 Yeast &bovine COX VIp-ADX Plasmids native parts engineered parts Mature P450 SCC Mature ADX 3-HSD Mature ADR COX VIp-P450 C11 Bovine & human Overexpression by promoter exchanges ARH1 Yeast Gene inactivation Yeast Yeast Yeast Yeast D22-desaturase Aldo-ketoreductase (gcy1) Aldo-ketoreductase (ypr1) O-acetyl transferase (atf2) Yeast culture supernatant Humanization of yeast sterol biosynthesis Mimic as closely as possible human behavior. “In vivo” metabolome model aware of compartmentalization Basic and applied tool for drug design. Approach the complexity real complexity. No optimization for specific end-product Conservative approach taking advantage of a model eukaryote organism acetyl-C oA acetoacetyl-C oA thiolase acetoacetyl-C oA H3C CH3 CH3 H MG -C oA synthase CH3 H3C H3C CH3 CH3 CH3 CH3 CH3 CH3 H MG -C oA CH3 CH3 CH3 CH3 CH3 CH3 CH3 H MG -C oA reductase H3C mevalonate squalene mevalonate kinase CH3 Squalene epoxidase + CPR CH3 O H3C Lanosterol synthase CH3 HO CH3 CH3 Squalene epoxide mevalonate phosphate MV P P decarboxylase isopentenyl diphosphate mevalonate phosphate kinase mevalonate diphosphate I P P isomerase dimethylallyl diphosphate Antifungal targets Zymosterol (cholesta 8, 24(25) diene 3-ol) H3C H3C H3C CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 geranyl phosphate HO HO CH3 CH3 HO farnesyl phosphate CH3 CH3 Sterol 14reductase 4,4’-demethylation (multistep, 3 genes) squalene synthase CH3 CH3 CH3 CH3 F P P synthase Lanosterol 14-demethylase +CPR + b5 squalene zymosterol H2C H3C CH3 H3C Erg6p CH3 CH3 CH3 CH3 CH3 CH3 CH3 8,7 isomerase HO H3C HO ERG2 H2C H3 C CH3 CH3 CH3 CH3 CH3 CH3 CH3 Sterol biosynthesis in animal and yeast CH3 D5- desaturase HO H3C H2C ERG3 HO H3C CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 D7- reductase HO H3C CH4 - reductase H3C HO H3C CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 HO 24(25)- reductase H3C 22-desaturase HO H3C H3C CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 HO HO Cholesterol (animal) cholesterol cholesterol Ergosterol (levures) ergosterol 28 CH3 Disruptions of yeast genes SAM-sterol transferase -sterol reductase 21 Ergosterol CH3 18 19 CH3 22 11 CH3 12 24 20 23 17 13 16 CH3 26 25 CH3 27 D22-sterol desaturase 9 2 HO 1 3 10 8 5 4 14 15 7 6 Additions of human gene D24(25)-sterol reductase D7-sterol reductase Humanisation of yeast genes HMG CoA reductase Statin drugs Lanosterol demethylase Antifungal drugs CH3 CH3 CH3 Sterol 8,7 isomerase Sigma receptor analog CH3 CH3 Sterol 5-desaturase Cytochrome b5 P450-reductase Cholesterol Redox environment HO Human HMG-CoA reductase HMG-CoA reductase Action substitution Lanosterol demethylase Lanosterol demethylase substitution absent Yeast SAM-sterol transferase deletion D7-8 sterol isomerase D7-8 sterol isomerase substitution D5- sterol desaturase D5- sterol desaturase substitution absent introduction D22- sterol desaturase substitution absent introduction D7-sterol reductase absent D24(25)-sterol reductase absent Cholesterol D24(28)-sterol reductase Ergosterol deletion Cholesterol Sterol biosynthesis is a multi dimensional network Enzymes have preferential but wide substrate specificities allowing multiple alternate paths Storage-retrieval-transport mechanisms are tightly coupled to biosynthesis Storage Sterol 1 Sterol 2 Sterol 3 Sterol 4 Sterol 5 Final sterol Biosynthesis Pathological state or interaction with xenobiotics can reprogram sterol metabolic network leading to physiological dysfunctions human 5 6 7 8 9 D22desaturase D24(28)reductase D24(25)reductase SAM sterol -methyltransferase 4 D7-sterol reductase ,’-sterol demethylase 3 D5-sterol desaturase D1-sterol reductase 2 D8-7 sterol isomerase 1-sterol demethylase 1 10 C5 A (cholesterol) YD4,10 P 7,8 C5 C5,22 C Y D4 P8 D lanosterol Y D4 P 7,8 YD4 P7 C5 (cholesterol) B C5,24 C5,22;24 C5.7,22,24 C8,24 C5,7,22,24 C5,7,24 C5,7,22 C5,24 E c8,24 c5,7 C5,7,24 C5,7,22,24 C8,24, C5,7,24 YD4 F YP 8 G YP 7 H E 5,22 Yeast (Y) I E5,7,22 (ergosterol) C5,7,22,24 E 5,22 E5 E5 Non-physiological pathway accumulate sterol intermediates Human C8,24 WT yeast C8,24 yeast Expression of D7- and D24-reductase Deletion of SAM-sterol transferase C8,24 C8,22,24 ? E 5,7 C5, 24 C5 cholesterol C5,7 C5,7,24 C5 ,7,22,24 C5,7,22 C5 E5,7,22 ergosterol C5, 22,24 C5, 22 Analysis of pathologies or drug perturbations induced reprogramming of sterol metabolic network fluxes Isotope shift propagation analysis Time series of output Static view Static(sterol view (sterol content) content) 12C count cholesterol 13C Sterol type Dynamic view (synthesis rate) cholesterol Sterol type Retention time Some concluding remarks Metabolic network reconstruction in yeast constitutes a powerful tool both for drug development and production. Nature selected biosynthetic strategies are not unique and efficient artificial alternate self-sufficient routes can be build for biotechnological purposes. Natural organ, cellular and subcellular compartmentalization is not a request for steroid hormone synthesis Critical yeast functions controlling membrane integrity can be massively engineered keeping a viable host physiology. Enzyme functions frequently exhibit surprising plasticity which can be both source of side reactions and advantage when reconstructing heterologous metabolic pathways. Thank to …….. Steroid biosynthesis in yeast Humanized yeast Muriel Merkamm, Gilles Truan Philippe Urban Steven Kelly, Diane Kelly Josie Parker STEROLTALK ESTBB, University of Bordeaux (France) Transgene SA (Strasbourg, France) Sanofi-Aventis (Vitry, France) Laboratory of Membrane Protein Engineering Centre de Génétique Moléculaire, CNRS, Gif-sur-Yvette (France) Contact : Dr. Denis POMPON mail: [email protected] FP6 European program
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