Laboratoire d’Ingénierie des Protéines Membranaires CGM-CNRS Gif-sur-Yvette, France.

1. 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.

2. Simple carbon sources Glycolyis sterols cholesterol Steroid hormones Membranes Bile acids Vitamin D Steroid hormones Steroid hormones Sterol and steroid hormone biosynthesis pathway in animals 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

3. Adrenal gland

4. Cholesterol ester Cholesterol

5. 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

6. TOOLS MODELS Redesigning animal steroid hormones biosynthesis for yeast Humanization of yeast sterol biosynthesis Make Yeast Human

7. 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 Biosynthesis involved large number of membrane bound enzymes needing eukaryote environment. Optimized for productivity and not to be a model

8. No cholesterol in yeast Cortisol biosynthesis in animals Required enzymes absent Required electron transport chains absent Dealing with subcellular transport of heterologous enzymes Dealing with subcellular traffic of lipophilic intermediates

9. Substitute for cholesterol with a yeast metabolism derived sterol Campesterol (plant sterol)

10. S-adenosyl sterol methylene transferase (ERG6 p) D22-desaturase (ERG5 p) Sterol 24(28) reductase Sterol 24(25) reductase Sterol D7 reductase Only present in yeast Only present in animals 28 CH3 21 22 CH3 CH3 26 24 18 20 25 CH3 23 19 CH3 11 12 17 CH3 27 13 16 9 2 1 1 14 15 10 8 5 7 3 4 6 CH3 Ergosterol (yeast) CH3 CH3 CH3 CH3 Gene critical for differences Cholesterol (animals)

11. ERGOSTEROL H H C C C C H H 3 3 3 3 C C H H 3 3 C C H H Simple carbon sources 3 3 C C H H C C H H 3 3 3 3 C C H H 3 3 Squalene C C H H 3 3 H H C C 3 3 Lanosterol Common to yeast and animals STEROLS Yeast specific Zymosterol Campesterol Cholesterol H C H C C H 3 3 3 H C C H C H 3 3 3 C H 3 C H H C 3 3 O C H C H 3 3 C H 3 C H 3 C H 3 H O H O H O Pregnenolone Steroid biosynthesis Engineered yeast Animal specific Hydrocortisone

12. Campesterol HO NADPH + P450 SCC NADP NADPH ADR A. thaliana D7- reductase ADX O HO HO HO Pregnenolone HO Ergosta 5-ene ol (campesterol) Zymosterol SAM-transferase D8-7 isomerase D22-desaturase disruption D5-desaturase, cyt. b5 ergosterol HO HO HO HO

13. Dealing with intracellular targeting and compartmentalization Plasma membrane Endoplasmic reticulum sterols Exogenous sterols neosynthesis 11-deoxy cortisol sterols progesterone storage Mitochondria Lipid droplet

14. Two similar reactions occur on internal mitochondrial membrane in animal and involve the same electron transfer chain ….. P450 SCC + ADX + ADR P450 C11 + ADX +ADR 11-deoxycortisol cortisol cholesterol pregnenolone 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

15. Yeast reconstruction of the campesterol side chain cleavage endoplasmic reticulum yeast Plasma membrane Plasma membrane Cytosol NADPH campesterol ADR ADX SCC e- e- Immuno-localization pregnenolone 11-deoxycortisol Electron transport chain sterol SCC ADX ADR human Transporter Mito (IM) Mito (LM) Mito (IM)

16. OD at 600nm Pregnenolone acetate production by the strain CA10/pCD63 erg5 Fed- batch Stationaryphase Batch 800 A. thaliana D7 –sterol reductase 600 Pregnenolone acetate (mg/l) bovine adrenodoxin reductase 400 200 0 m-P450 SCC - 50 100 150 200 250 culture time (h) M-ADX

17. Yeast reconstruction of the 11b-hydroxylase activity EndogenousARH1p Mito (I.M.) yeast COX fusion Mito (I.M.) COX fusion Mito (matrix) P450 C11 does not fold outside of 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. Electron transport chain P450 C11 ADX ADR human Mito (I.M.) Mito (matrix) Mito (I.M.) ADR ADR does not fold inside of the mitochondrial context

18. The microsomal part of the cortisol biosynthesis C H 3 Progesterone C O C H 3 C O 3b-HSDH H O O electrons C H P450c17 3 -hydroxy NADPH C O O H CPR C H O H NADP+ 2 P450c21 C O O O H 11-Deoxycortisol O Pregnenolone 17 a Progesterone

19. 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.

20. Parasitic aldo-ketoreductase activity from enzymes of central carbon cycle acting on steroid Inhibition b b 3 3 - - HSD HSD Yeast Yeast Gcy1p Gcy1p and and Ypr1p are Ypr1p are progesterone progesterone aldo aldo - - keto keto - - reductases reductases CYP17A1 CYP17A1 of of central central carbon carbon metabolism metabolism 17-OH progesterone CYP21A1 CYP21A1 Gcy1p Gcy1p ATF2p ATF2p Pregnenolone Pregnenolone Ypr1p Ypr1p 11-deoxy cortisol a a 17 17 , 20 , 20 - - dihydroxypregn dihydroxypregn - - 4 4 - - acetate acetate ene ene - - 3 3 - - one one CYP11B1 CYP11B1 , , ADX ADX , , CYP11B1, ADX CYP11B1, ADX , , Arh1p Arh1p Arh1p Arh1p b b a a 11 11 ,17 ,17 , 20 , 20 - - tri tri - - hydroxy hydroxy pregn pregn - - 4 4 - - ene ene - - 3 3 - - one one HYDROCORTISONE HYDROCORTISONE Gene dosage effects Pregnenolone Pregnenolone Gene dosage effect on a branched pathway dramatically affect the output of the biosynthesis b b 3 3 - - HSD HSD 40 40 Progesterone Progesterone 35 35 progesterone progesterone P450 C21 P450 C17 30 30 17, 21 OH 17, 21 OH OH OH OH OH - - - - 25 25 17 17 21 21 In the absence of regulation tight tuning is requested to accumulate correct end-product 17 OH P 21 21 - - OH P OH P 20 20 17 - - OH P 15 15 P450 C11 P450 C17 P450 C21 10 10 5 5 MC MC 17, 21 17, 21 - - OH P OH P 0 0 A A A C C C D D D B B B cortisol Unexpected retro-inhibition of sterol synthesis by pregnenolone upon disruption of parasitic Atf2p acetylase activity. ER & plasma membrane ER & plasma membrane enol ergosta 5 ergosta 5 - - enol NADPH NADPH P450 SCC P450 SCC pregnenolone pregnenolone Reticulum Reticulum b b 3 3 - - HSD HSD progesterone progesterone

21. Global pathway engineering of yeast for cortisol production native parts engineered parts Yeast culture supernatant

22. 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

23. cholesterol ergosterol cholesterol Sterol biosynthesis in animal and yeast

24. 28 Disruptions of yeast genes SAM-sterol transferase 24(28)-sterol reductase D22-sterol desaturase Additions of human gene D24(25)-sterol reductase D7-sterol reductase Humanisation of yeast genes Statin drugs HMG CoA reductase Lanosterol demethylase Antifungal drugs Sterol 8,7 isomerase Sigma receptor analog Sterol 5-desaturase Cytochrome b5 Redox environment P450-reductase CH3 21 Ergosterol 22 CH3 CH3 26 24 18 20 25 CH3 23 19 CH3 11 12 17 CH3 27 13 16 9 2 1 1 14 15 10 8 5 7 3 4 6 CH3 CH3 CH3 CH3 CH3 Cholesterol H O

25. substitution substitution deletion substitution substitution introduction substitution introduction deletion Human Yeast Action HMG-CoA reductase HMG-CoA reductase Lanosterol demethylase Lanosterol demethylase absent SAM-sterol transferase D7-8 sterol isomerase D7-8 sterol isomerase D5- sterol desaturase D5- sterol desaturase absent D7-sterol reductase absent D22- sterol desaturase absent D24(25)-sterol reductase absent D24(28)-sterol reductase Ergosterol Cholesterol Cholesterol

26. Storage-retrieval-transport mechanisms are tightly coupled to biosynthesis Sterol biosynthesis is a multi dimensional network Enzymes have preferential but wide substrate specificities allowing multiple alternate paths Pathological state or interaction with xenobiotics can reprogram sterol metabolic network leading to physiological dysfunctions

27. SAMsterol 28-methyl- transferase 4,4’-sterol demethylase D 22- desaturase 14-sterol demethylase D 5-sterol desaturase D 8-7 sterol isomerase D14-sterol reductase D 24(28)- reductase D 7-sterol reductase D 24(25)- reductase 1 2 3 4 5 6 7 8 9 10 A C5 (cholesterol) human C5 (cholesterol) • D 4,10 P 7,8 B C5 C5,22 C5,24 C5,22;24 C5.7,22,24 C • D 4 P 7,8 C8,24 C5,7,22,24 C5,7,24 C5,7,22 D • D 4 P 8 C5,24 c8,24 c5,7 C5,7,24 C5,7,22,24 E • D 4 P 7 F C8,24, C5,7,24 C5,7,22,24 Y D 4 E 5,22 E5 G Y P 8 H E 5,22 E5 Y P 7 E5,7,22 (ergosterol) I Yeast (Y) lanosterol

28. yeast Expression of D7- and D24-reductase Deletion ofSAM-sterol transferase C8,24 C8,22,24 ? C5,7,22 C5,7 C5,7,24 C5, 24 C5 C5 ,7,22,24 C5, 22,24 C5, 22 Non-physiological pathway accumulate sterol intermediates WT yeast Human C8,24 C8,24 E 5,7 C5 E5,7,22 cholesterol ergosterol

29. Analysis of pathologies or drug perturbations induced reprogramming of sterol metabolic network fluxes Time series of output Static view (sterol content) 13C count 12C Dynamic view (synthesis rate) Retention time Isotope shift propagation analysis

30. 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.

31. Thank to …….. Steroid biosynthesis in yeast Humanized yeast Muriel Merkamm, Gilles Truan Philippe Urban Steven Kelly, Diane Kelly Josie Parker FP6 European program 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: pompon@cgm.cnrs-gif.fr