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Laboratoire d’Ingénierie des Protéines Membranaires
CGM-CNRS Gif-sur-Yvette, France.
Denis POMPON
Reconstruction in yeast of human
steroid metabolic pathway as a tool
for drug discovery and biosynthesis
Glycolyis
sterols
Simple carbon sources
cholesterol
Membranes
Bile acids
Steroid hormones
Vitamin D
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
CORTISOL
Steroid hormones
STEROLS
CORTISOL
Steroid hormones
Adrenal gland
Cholesterol ester
Cholesterol
CholesterolCholesterol
Multi- organtransport &metabolism
Multiplesubcellularbiosynthesislocation
High organizedtissue dependent spatial organization
Highly branched multi-step biosynthetic pathway with complexregulation
Simple unicellulareukaryote microorganism
Redesigninganimal steroid hormones biosynthesis for yeast
TOOLS
Humanization of yeast sterol biosynthesis
MODELS
Make Yeast Human
Redesigninganimal 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.
O
C O
CH3
OHP450c17
O
C O
CH2OH
OH
11-Deoxycortisol
P450c21
17a-HydroxyProgesterone
HO
C O
CH3
O
C O
CH3
Pregnenolone
Progesterone
3-HSDH
EndoplasmicReticulum
Mitochondria
P450sccADX, ADR
HO
Cholesterol
HO
C O
CH3
Pregnenolone
ADX, ADR
C O
CH2OH
OHHO
O
Cortisol
O
C O
CH2OH
OH
11-Deoxycortisol
P450c11O
C O
CH3
OHP450c17
O
C O
CH3
OHP450c17
O
C O
CH3
OH
O
C O
CH3
OHP450c17
O
C O
CH2OH
OH
11-Deoxycortisol
P450c21
17a-HydroxyProgesterone
O
C O
CH2OH
OH
11-Deoxycortisol
P450c21
17a-HydroxyProgesterone
O
C O
CH2OH
OH
O
C O
CH2OH
OH
11-Deoxycortisol
P450c21
17a-HydroxyProgesterone
HO
C O
CH3
O
C O
CH3
Pregnenolone
Progesterone
3-HSDHHO
C O
CH3
O
C O
CH3
Pregnenolone
Progesterone
3-HSDHHO
C O
CH3
O
C O
CH3
Pregnenolone
Progesterone
3-HSDHHO
C O
CH3
HO
C O
CH3
O
C O
CH3
O
C O
CH3
Pregnenolone
Progesterone
3-HSDH
EndoplasmicReticulum
EndoplasmicReticulum
EndoplasmicReticulum
Mitochondria
P450sccADX, ADR
HO
Cholesterol
HO
C O
CH3
Pregnenolone
P450sccADX, ADR
HO
Cholesterol
HO
C O
CH3
Pregnenolone
HOHO
Cholesterol
HO
C O
CH3
HO
C O
CH3
Pregnenolone
ADX, ADR
C O
CH2OH
OHHO
O
Cortisol
O
C O
CH2OH
OH
11-Deoxycortisol
P450c11ADX, ADR
C O
CH2OH
OHHO
O
Cortisol
O
C O
CH2OH
OH
11-Deoxycortisol
P450c11
C O
CH2OH
OHHO
O
Cortisol
O
C O
CH2OH
OH
11-Deoxycortisol
P450c11
C O
CH2OH
OHHO
O
C O
CH2OH
OHHO
O
Cortisol
O
C O
CH2OH
OH
O
C O
CH2OH
OH
11-Deoxycortisol
P450c11
No cholesterol in yeast
Cortisol biosynthesis in animalsRequired enzymes absent
Required electron transport chains absent
Dealing with subcellular transport of heterologous enzymes
Dealing with subcellular traffic of lipophilic intermediates
Campesterol(plant sterol)
Substitute for cholesterol with a yeast metabolism derived sterol
OH
Ergosterol (yeast)
112
34
56
7
8
9
11
10
CH3
CH3
CH3
CH3
CH3
CH3
1918
21
12
14
13
15
1617
20
22
2324
2526
27
28
Cholesterol (animals)
CH3
CH3
CH3
OH
CH3
CH3
Gene critical for differences
Sterol7 reductase
Sterol 24(25) reductase
Only present in animals
Only present in yeast
S-adenosyl sterol methylene transferase (ERG6 p)22-desaturase (ERG5 p)
Sterol 24(28) reductase
ERGOSTEROLErgosterol
Yeast specific
Campesterol
CH3
CH3
CH3
OH
CH3
CH3
CH3
Engineered yeast
Simple carbon sources
Common toyeast and animals
Lanosterol
STE
RO
LS
Squalene
CH3
CH3
CH3 CH3
CH3
CH3CH3
CH3
CH3
CH3
CH3
CH3 CH3
CH3
CH3CH3
CH3
CH3
Zymosterol
Cholesterol
CH3
CH3
CH3
OH
CH3
CH3
Animal specific
H
Pregnenolone
Hydrocortisone
Steroid biosynthesis
CH3
CH3
OCH3
O
HOHO
HO
ergosterol
HO
ZymosterolSAM-transferase
8-7 isomerase
5-desaturase, cyt. b522-desaturase
disruption
HO
Ergosta 5-ene ol(campesterol)
HOHO
A. thaliana7- reductase
NADPH
P450 SCC
HO
Pregnenolone
ADX
ADRNADPH
NADP +
HO
Campesterol
O
Dealing with intracellular targeting and compartmentalization
sterols
Plasma membrane Endoplasmic reticulum
Mitochondria
sterols progesterone 11-deoxycortisol
Exogenoussterols
Lipid droplet
neosynthesis
storage
ADR(FAD)
e-
NADPH
NADP+
11-deoxycortisol cortisolP450 C11 + ADX +ADR
Two similar reactions occur on internal mitochondrial membrane in animal and involve the same electron transfer chain …..
-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
P450 SCC
CholesterolPregnenolone
Cholesterol side chain cleavage reaction
Active inputPassive output
P450 SCC + ADX + ADRcholesterol pregnenolone
Cholesterol, in contrast to oxysteroids, is insoluble and needs complex and function critical transport machinery to enter mitochondria in animals.
Yeast reconstruction of the campesterol side chain cleavage
SCCsterol ADX ADR
human Transporter Mito (IM) Mito (LM) Mito (IM)
Electron transport chain
yeast Plasma membrane
Plasma membrane
Cytosolendoplasmic reticulum
Met-mature P450SCC
Gpa1pPlasma
membrane
Dpm1pendoplasmic
reticulum
Porinmitochondria
Subcellularmarkers
Doublelabelling
Confocale immunofluorescence analysis of Met-mat SCClocalization in yeastImmuno-localization
SCC ADX ADRNADPH
e-e-
campesterol
pregnenolone
11-deoxycortisol
Pregnenolone acetate production by the strain CA10/pCD63
0
200
400
600
800
- 50 100 150 200 250culture time (h)
BatchFed-batch
Stationary phase
OD
at
600n
m
Pre
gnen
olon
e ac
etat
e (m
g/l)
erg5
A. thaliana 7 –sterol reductase
bovine adrenodoxin reductase
m-P450 SCCM-ADX
Yeast reconstruction of the 11-hydroxylase activity
P450 C11 ADX ADR
human Mito (I.M.) Mito (matrix) Mito (I.M.)
Electron transport chain
yeast COX fusionMito (I.M.)
COX fusionMito (matrix)
P450 C11 does not fold outside of mitochondrial context
EndogenousARH1pMito (I.M.)
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.
?ADRADR does not fold inside of themitochondrial context
HO
C O
CH3Pregnenolone
17Progesterone
O
C O
CH3Progesterone
3-HSDH
O
C O
CH3
OH
P450c17-hydroxy
O
C O
CH2OH
OH
11-Deoxycortisol
P450c21
CPRNADPH
NADP+
electrons
The microsomal part of the cortisol biosynthesis
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 differentfrom intermediates involved in parasitic activities making sometime identification of corresponding interfering genes very tricky.
Parasitic aldo-ketoreductase activity from enzymes of central carbon cycle
acting on steroid
progesterone
11-deoxy cortisol
3-HSD
CYP21A1
CYP17A1
CYP11B1, ADX, Arh1p
HYDROCORTISONE
progesterone
3-HSD
CYP21A1
CYP17A1
CYP11B1, ADX, Arh1p
HYDROCORTISONE
17-OH progesterone
Unexpected retro-inhibition of sterol synthesis by pregnenolone upon disruption
of parasitic Atf2p acetylase activity.
ergosta 5-enol
P450 SCC
3-HSD
progesterone
pregnenolone
NADPH
ER & plasma membrane
Reticulum
ergosta 5- enol
P450 SCC
3-HSD
progesterone
pregnenolone
NADPH
ER & plasma membrane
Reticulum
Inhibition
Pregnenoloneacetate
ATF2p Pregnenoloneacetate
ATF2p17, 20-dihydroxypregn-4-
ene-3-one
Gcy1pYpr1p
11,17, 20- tri-hydroxypregn-4-ene-3-one
CYP11B1, ADX, Arh1p
Yeast Gcy1p and Ypr1p are aldo-keto-reductases
of central carbon metabolism
17, 20-dihydroxypregn-4-ene-3-one
Gcy1pYpr1p
11,17, 20- tri-hydroxypregn-4-ene-3-one
CYP11B1, ADX, Arh1p
Yeast Gcy1p and Ypr1p are aldo-keto-reductases
of central carbon metabolism
Pregnenolone
Progesterone
21-OH P 17-OH P
17, 21-OH P
3-HSD
cortisol
P450 C17
MC
P450 C11
Pregnenolone
Progesterone
21-OH P 17-OH P
17, 21-OH P
3-HSD
P450 C21
MC
P450 C17
P450 C21
Gene dosage effectsGene dosage effect on a branched pathway dramatically affect the output of the biosynthesis
In the absence of regulation tight tuning is requested to accumulate
correct end-product0
5
10
15
20
25
30
35
40
CB DA
17, 2
1 O
H
21-O
Hpro
ges
tero
ne
17-O
H
0
5
10
15
20
25
30
35
40
CCBB DDAA
17, 2
1 O
H
21-O
Hpro
ges
tero
ne
17-O
H
Global pathway engineering of yeast for cortisol production
native parts engineered parts
Yeast culturesupernatant
Yeast ARH1
Overexpression by promoter exchanges
Aldo-ketoreductase (gcy1)Yeast
Yeast
O-acetyl transferase (atf2)Yeast
Aldo-ketoreductase (ypr1)
Yeast 22-desaturaseGene inactivation
Mature ADR
Mature P450 SCC
Bovine&
human
Mature ADX
COX VIp-P450 C11
3-HSD
Plasmids
A. thaliana 7-sterol reductase
Bovine P450 C17
Bovine P450 C21
Yeast &bovine COX VIp-ADX
Genomic integrations of transgenes
Yeast ARH1
Overexpression by promoter exchanges
Yeast ARH1
Overexpression by promoter exchanges
Aldo-ketoreductase (gcy1)Yeast
Yeast
O-acetyl transferase (atf2)Yeast
Aldo-ketoreductase (ypr1)
Yeast 22-desaturaseGene inactivation
Aldo-ketoreductase (gcy1)Yeast
Yeast
O-acetyl transferase (atf2)Yeast
Aldo-ketoreductase (ypr1)
Yeast 22-desaturaseGene inactivation
Mature ADR
Mature P450 SCC
Bovine&
human
Mature ADX
COX VIp-P450 C11
3-HSD
Plasmids
Mature ADR
Mature P450 SCC
Bovine&
human
Mature ADX
COX VIp-P450 C11
3-HSD
Plasmids
A. thaliana 7-sterol reductase
Bovine P450 C17
Bovine P450 C21
Yeast &bovine COX VIp-ADX
Genomic integrations of transgenesA. thaliana 7-sterol reductase
Bovine P450 C17
Bovine P450 C21
Yeast &bovine COX VIp-ADX
Genomic integrations of transgenes
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
mevalonate
HMG-CoA reductase
HMG-CoA
HMG-CoA synthase
acetoacetyl-CoA
acetoacetyl-CoA thiolaseacetyl-CoA
mevalonatephosphate
mevalonatekinase
mevalonatediphosphate
mevalonatephosphate kinase
isopentenyl diphosphate dimethylallyl diphosphate
geranyl phosphate
farnesyl phosphate
squalene
MVPP decarboxylase
FPP synthase
squalenesynthase
IPP isomerase
mevalonate
HMG-CoA reductase
HMG-CoA
HMG-CoA synthase
acetoacetyl-CoA
acetoacetyl-CoA thiolaseacetyl-CoA
mevalonatephosphate
mevalonatekinase
mevalonatediphosphate
mevalonatephosphate kinase
isopentenyl diphosphate dimethylallyl diphosphate
geranyl phosphate
farnesyl phosphate
squalene
MVPP decarboxylase
FPP synthase
squalenesynthase
IPP isomerase
CH3
CH3
CH3 CH3
CH3
CH3CH3
CH3
CH3
squalene
CH3
CH3
CH3 CH3
CH3
CH3CH3
CH3
CH3
O
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
Squalene epoxide
Squaleneepoxidase
+ CPR
Lanosterolsynthase
Lanosterol14-demethylase
+CPR + b5
Sterol 14-reductase4,4’-demethylation
(multistep, 3 genes)
Zymosterol(cholesta 8, 24(25) diene 3-ol)
Antifungal targets
CH3
CH3
CH3 CH3
CH3
CH3CH3
CH3
CH3
CH3
CH3
CH3 CH3
CH3
CH3CH3
CH3
CH3
squalene
CH3
CH3
CH3 CH3
CH3
CH3CH3
CH3
CH3
O
CH3
CH3
CH3 CH3
CH3
CH3CH3
CH3
CH3
O
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
Squalene epoxide
Squaleneepoxidase
+ CPR
Lanosterolsynthase
Lanosterol14-demethylase
+CPR + b5
Sterol 14-reductase4,4’-demethylation
(multistep, 3 genes)
Zymosterol(cholesta 8, 24(25) diene 3-ol)
Antifungal targets
Sterol biosynthesis in animal and yeast
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
CH4
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
CH2
CH3
CH3
CH3
OH
CH3
CH3
CH2
CH3
CH3
CH3
OH
CH3
CH3
CH2
CH3
CH3
CH3
OH
CH3
CH3
CH2
CH3
CH3
CH3
OH
CH3
CH3
CH2
CH3
CH3
CH3
OH
CH3
CH3
CH2
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
CH3
CH3
CH3
CH3
OH
CH3
CH3
CH3
Cholesterol (animal) Ergosterol (levures)
ERG28,7 isomerase
ERG35- desaturase
7- reductase
24(25)- reductase
- reductase
22-desaturase
zymosterol Erg6p
cholesterolcholesterol ergosterol
OH
Ergosterol
112
34
56
7
8
9
11
10
CH3
CH3
CH3
CH3
CH3
CH3
1918
21
12
14
13
15
1617
20
22
2324
2526
27
28
Cholesterol
CH3
CH3
CH3CH3
CH3
OH
Disruptions of yeast genes
22-sterol desaturase
S-sterol transferase-sterol reductase
Additions of human gene
7-sterol reductase
24(25)-sterol reductase
Humanisation of yeast genes
HMG CoA reductase
Lanosterol demethylase
Sterol 8,7 isomerase
Sterol 5-desaturase
Cytochrome b5
P450-reductase
Statin drugs
Antifungal drugs
Sigma receptor analog
Redox environment
YeastHuman
HMG-CoA reductase HMG-CoA reductase
Action
Lanosterol demethylase Lanosterol demethylase
7-8 sterol isomerase
SAM-sterol transferaseabsent
7-8 sterol isomerase
5- sterol desaturase
7-sterol reductase absent
absent
5- sterol desaturase
22- sterol desaturase
24(25)-sterol reductase absent
absent
substitution
substitution
substitution
deletion
substitution
introduction
substitution
introduction
deletion24(28)-sterol reductase
Cholesterol Ergosterol Cholesterol
Sterol biosynthesis is a multi dimensional network
Enzymes have preferential but wide substrate specificities
allowing multiple alternate paths
Storage
Sterol 1 Sterol 2 Sterol 3 Sterol 4 Sterol 5 Final sterol
Biosynthesis
Storage
Sterol 1 Sterol 2 Sterol 3 Sterol 4 Sterol 5 Final sterol
Biosynthesis
Storage-retrieval-transport mechanisms
are tightly coupled to biosynthesis
Pathological state or interaction with xenobiotics can reprogram sterolmetabolic network leading to physiological dysfunctions
lan
os
tero
l
8-
7 st
ero
l is
om
eras
e
5-
ster
ol
des
atu
rase
7-
ster
ol
red
uct
ase
24
(25)
- r
edu
ctas
e
24
(28)
- r
edu
ctas
e
SA
Ms
tero
l
-met
hyl
-tr
ansf
eras
e
22
- d
esat
ura
se
s
tero
l d
emet
hyl
ase
ster
ol
red
uct
ase
’
ste
rol
dem
eth
ylas
e
4 5 6 7 8 9 101 2 3Ahuman C5
(cholesterol)
Yeast () IE5,7,22
(ergosterol)
GP 8E 5,22
E5
FC8,24, C5,7,24
C5,7,22,244
H E 5,22 E5P 7
D4 P 8
C8,24 C5,7,22,24C5,7,24 C5,7,22
E4 P 7
C5,24c8,24 c5,7
C5,7,24 C5,7,22,24
B4,10 P 7,8
C5(cholesterol)
C4 P 7,8
C5 C5,22C5,24
C5,22;24 C5.7,22,24
C8,24
E 5,7
E5,7,22
WT yeast
C8,24
C5
Human
cholesterol ergosterol
C8,24
C5, 24
C5, 22,24
Expression of7- and 24-reductase
C5,7,24
C8,22,24 ?
C5
C5, 22
C5,7
C5 ,7,22,24
C5,7,22
Deletion ofSAM-sterol transferase
yeast
Non-physiological pathway accumulate sterol intermediates
Time series of output
Retention time
coun
t
Isotope shift propagation analysis
12C
13C
Analysis of pathologies or drug perturbations induced reprogramming of sterol metabolic network fluxes
Sterol type
cholesterol
Static view (sterol content)
Sterol type
cholesterol
Static view (sterol content)
Sterol type
cholesterol
Sterol type
cholesterol
Static view (sterol content)
Dynamic view (synthesis rate)
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 efficientartificial 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.
Laboratory of Membrane Protein EngineeringCentre de Génétique Moléculaire, CNRS, Gif-sur-Yvette (France)
ESTBB, University of Bordeaux (France)Transgene SA (Strasbourg, France)Sanofi-Aventis (Vitry, France)
Steroid biosynthesis in yeast
Contact : Dr. Denis POMPON mail: [email protected]
Thank to ……..
Humanized yeast
Muriel Merkamm, Gilles TruanPhilippe Urban
Steven Kelly, Diane KellyJosie Parker
STEROLTALK FP6 European program
