Using retrosynthesis in synthetic biology and metabolic engineering
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Transcript of Using retrosynthesis in synthetic biology and metabolic engineering
Jean-LoupFaulon20161
Jean-LoupFaulon
jfaulon.com [email protected]
UsingRetroSynthesisinSynthe/cBiologyandMetabolic
Engineering
and Synthetic Biologyi n s t i t u t e o f S y s t e m s
iSSB
mSSB iGEM
Jean-LoupFaulon20162
CHASSIS
TARGET
EFFECTOR
CHASSIS
TARGET(signal)
Metabolicengineering Biosensorengineering
Findingmetabolicpathwaysbetweentwopoolsofcompounds
Jean-LoupFaulon20163
CHASSIS
TARGET
Findingpathwaysfromchassistotarget
Forward-synthesis
Findingpathwaysbetweentwopoolsofcompounds
SINK
SOURCE
Retro-synthesis
SOURCE
SINK
metabolicreac8on
Reversedmetabolicreac8on
Jean-LoupFaulon20164
Findingpathwaysbetweentwopoolsofcompounds:state-of-the-art
BNICE SimPheny/GEM-Path RetroPath
Jean-LoupFaulon20165
Findingpathwaysbetweentwopoolsofcompounds:theworkflow
Jean-LoupFaulon20166
UsingtheworkflowtofindretrosyntheMcpathwaystoproduce3-methylphenolinE.coli
SINK
SOURCE-SOURCE:trimethylphenol-SINK:E.colimetabolites-RULES:reversedMetaNetXreacEons
Jean-LoupFaulon20167
SINK
SOURCE
37productsgeneratedbyFIRE FILTER15productstobecomenewsubstratesfornextiteraEon
1SOURCEsubstrate
UsingtheworkflowtofindretrosyntheMcpathwaystoproduce3-methylphenolinE.coli
Jean-LoupFaulon20168
SINK
SOURCE
Iter2:15substrates1281productsgeneratedbyFIRE214newsubstratesIter3:214substrates25016productsgeneratedbyFIRE2789newsubstrates
UsingtheworkflowtofindretrosyntheMcpathwaystoproduce3-methylphenolinE.coli
Jean-LoupFaulon20169
SINK
SOURCE
FinalretrosyntheEcnetwork:• 1compoundinSOURCE• 163compoundsinSINK• 26312compoundstotal• 4soluEonpathways
UsingtheworkflowtofindretrosyntheMcpathwaystoproduce3-methylphenolinE.coli
L-tyrosineC3-methyltransferase(Streptomyceslavendulae)
Jean-LoupFaulon201610
L-Glutamate Oxaloacetate 2-Oxaloglutarate L-Aspartate
Transaminase(2.6.1)
BNICERULE SIMPHENYRULE
[H][#7]([H])-[#6](-[#6]-[#6]-[#6](-[#8])=O)-[#6](-[#8])=O.[#6,#1]-[#6](-[#6,#1])=O>>[#8]-[#6](=O)-[#6]-[#6]-[#6](=O)-[#6](-[#8])=O.[H][#7]([H])-[#6](-[#6,#1])-[#6,#1]
[#6,#1]-[#6](-[#6,#1])=O>>[H][#7]([H])-[#6](-[#6,#1])-[#6,#1]
Findingpathwaysbetweentwopoolsofcompounds:allthatmaOersisthesetofreacMonrules
• 86mulE-substratesrulesmanuallycuratedatthethirdEClevelclass(HenryCS,etal.BiotechnolBioeng.2010;106(3):462-73)
• Rulescover30%ofallknowmetabolicreacEons(havinganECnumber)
• 50rulesmanuallycreated,allmono-substrate(Yim,H.etal.NatChemBiol,20117(7):p.445-5)
• Rulescover20%ofallknowmetabolicreacEons(havinganECnumber)
Jean-LoupFaulon201611
-1(4−10)-1(15−17)+1(4−17)+1(15−10)
2.6.1
L-Glutamate Oxaloacetate 2-Oxaloglutarate L-Aspartate
RetroPathrulecomputaMonworkflow
[#6:1]-[#6:2]-[#6:4](-[#7:10])-[#6:5](-[#8:6])=[O:7]>>[#6:1]-[#6:2]-[#6:4](=O)-[#6:5](-[#8:6])=[O:7].[#7:10]-[#6](-[#6]-[#6](-[#8])=O)-[#6](-[#8])=O
Substrate+Oxaloacetate=Product+L-Aspartate
D=4
[#6:4]-[#7:10]>>[#6:4]=O.[#7:10]-[#6](-[#6]-[#6](-[#8])=O)-[#6](-[#8])=O
D=0Substrate+Oxaloacetate=Product+L-Aspartate
• From12000(forD=∞)to6000(forD=0)rules• Rulescover100%ofallknowmetabolicreacEons(havinganECnumber)
Jean-LoupFaulon201612
SOURCE:E.colimodeliJO1366(752compounds)
RULES:BNICE,SimPheny,RetroPathD=8onMetaNetXE.colireac8ons
Numberofproductsgenerated
inModel inBRENDA TotalSimPheny 376/752 2874 9782BNICE 546/752 3526 150228RetroPathD=8 633/752 6298 14178
SOURCE
SINK
HowwellcanthedifferentrulesystemsbuildanExtendedMetabolicSpaceforE.coli?
• Carbonell,etal.ACSSynth.Biol2014+addeddistribuEonsforSimPhenyandBNICE
SINK:BRENDA
SimPheny
Jean-LoupFaulon201613
?
RetroPathapplicaMonforflavanones
SINK:Flavanones
RULES:FromD=∞toD=4onMetaNetXreac8ons
SOURCE
SINK
SOURCE:E.colimodeliJO1366
Jean-LoupFaulon201614
AllowingpromiscuousreacEonrules(D=4)
SpecificreacEonrulesonly(D=∞)
UsingdiametertocontrolreacMonspecificityanddesignnovelmetabolicpathways
11pathwaysenumeratedfollowingCarbonelletal.BMCSysBio2012
1pathway
Jean-LoupFaulon201615
SearchingforenzymesequencescatalyzingreacMons
Tensorproduct(MachinelearningSVMandGP)
Enzymesequence?
www.jfaulon.com/category/tools/
(µM)
PredicMngKMvalues
• Protein-protein:MarEn,Roe,FaulonBioinforma/cs2005• Drug-target:Oprea,Trospha,Faulon,Rintoul,Nat.Chem.Bio2007• Enzyme-reacEon:Faulonetal.Bioinforma/cs2008
Melloretal.ACSSynth.Bio.2016
SOURCE
SINK
Jean-LoupFaulon201616
EvaluaMngreacMonthermodynamicfeasibilityandproducttoxicity
Kanamycin
Screen-168compounds
Q2=0.71
StructureToxicityRelaMonship
Model
MeasureE.colibacterialgrowth(IC50)toxicity?
• PlansonAGetal.Biotechnology&Bioengineering2011
RejectreacEonsthermodynamicallyunfavorable(Jankowskietal.BiophysJ,2008)andscorefavorablereacEonsthoughproducttoxicitySOURCE
SINK
www.jfaulon.com/category/tools/
Jean-LoupFaulon201617
DESIGN:Rankingpathways
• Benchmark:rankingthepathwaysforaminoacidbiosynthesisinE.coli
Allna8vepathwaysfoundinthetop10pathwaysreturnedbytherankingfunc8on
11.1510.9710.4610.4410.0810.039.939.579.527.757.74
scoregenesscorereacMonsscorepathways
Scoreandselectbestpathway(s)
λpathλgene
λtox
S
• CarbonellP,etal.BMCSystemsBiology2011• CarbonellP,etal.ACSSynthBiol.2014• CarbonellPetal.NucleicAcidRes.2014
GPandSVMtensorproduct
ToxicityTheoreEcalflux
xtms.issb.genopole.fr
Jean-LoupFaulon201618
Emzyme6.2.1.12(4CL)
L:Streptomycesmari8mus
M:Arabidopsisthaliana
H:Streptomycescoelocolor
Emzyme2.3.1.74(CHS)
L:Bacillussub8lis
H:Arabidopsisthaliana
Emzyme5.5.1.6(CHI)
L:Arabidopsisthaliana
H:Arabidopsisthaliana
EnzymeproducMvityvs.predictedscore
L-Phenylalanine
Cinnamoyl-CoA
Pinocembrin-chalcone
Pinocembrin
5.5.1.6
Trans-Cinnamate
CoA
ATP
Malonyl-CoA
2.3.1.74
6.2.1.12
4.3.1.25
BUILDANDTEST:ToppathwayconstrucMonandvalidaMonoftherankingfuncMon
[Pinocembrin-H]-
• FeherTetal.BiotechJ.2014
L/M/H:Low/Medium/HighpredictedacMvity
Jean-LoupFaulon201619
LEARNwithfluxanalysis
E.colimodel:FeistAetal.MolSystBiol2007+heterologouspathway2038reacEons(includingtransport)1043metabolites
@24haserinducEon:Growthrate:0.4847h-1Trans-cinnamate:49.0mg/LPinocembrin:<1.0mg/L
Fumarate
cinnamicacid-dihydrodiol
TCACycle
Trans-2,3-dihydroxycinnamate
Growth
2-Hydroxy-6-oxonona-trienedioate
1.14.12.19
1.3.1.87
1.13.11.16
3.7.1.-
L-Phenylalanine
Cinnamoyl-CoA
Pinocembrin-chalcone
Pinocembrin
5.5.1.6
Trans-Cinnamate
CoA
ATP
2.3.1.74
6.2.1.12
4.3.1.25
Malonyl-ACP
FaOyAcids
1.1695
2.3.1.39
Malonyl-CoA
6.4.1.2
Acetyl-CoA
9.24e-70.4847
0.0026
0.2909
0.2935
1.1695
9.24e-7
Weneedtoboostmalonyl-CoA
FluxesinmmolgDW-1h-1
Jean-LoupFaulon201620FluxesinmmolgDW-1h-1
4.
Coxiella
1.
Rhizobium 3.
E. coli
2.
Geobacter
Fumarate
cinnamicacid-dihydrodiol
TCACycle
Trans-2,3-dihydroxycinnamate
Growth
2-Hydroxy-6-oxonona-trienedioate
1.14.12.19
1.3.1.87
1.13.11.16
3.7.1.-
L-Phenylalanine
Cinnamoyl-CoA
Pinocembrin-chalcone
Pinocembrin
5.5.1.6
Trans-Cinnamate
CoA
ATP
2.3.1.74
6.2.1.12
4.3.1.25
Malonyl-ACP
FaOyAcids
1.1695
2.3.1.39
Malonyl-CoA
6.4.1.2
Acetyl-CoA
9.24e-70.4847
0.0026
0.2909
0.2935
1.1695
9.24e-7
LEARNàBacktoDESIGN
• FeherTetal.BiotechJ.2014
• Melloretal.ACSSynth.Bio.2016
Jean-LoupFaulon201621
Fumarate
cinnamicacid-dihydrodiol
TCACycle
Trans-2,3-dihydroxycinnamate
Growth
2-Hydroxy-6-oxonona-trienedioate
1.14.12.19
1.3.1.87
1.13.11.16
3.7.1.-
L-Phenylalanine
Cinnamoyl-CoA
Pinocembrin-chalcone
Pinocembrin
5.5.1.6
Trans-Cinnamate
CoA
ATP
2.3.1.74
6.2.1.12
4.3.1.25
Malonyl-ACP
FaOyAcids
1.1695
FluxesinmmolgDW-1h-1
2.3.1.39
Malonyl-CoA
0.3412
6.4.1.2
Acetyl-CoA Malonate
6.2.1.-
0.00120.06139.24e-70.4847
0.0050
0.6932
0.0026
0.2909
0.69950.2935
0.33761.1695
0.00129.24e-7
@24haserinducEon:Growthrate:0.0613h-1Trans-cinnamate:50.44mg/LPinocembrin:20.92mg/L
• FernandezAetal.J.Biotechnology2014
@48haserinducEon:Pinocembrin:27mg/L
1.
Rhizobium
Pathway ranking:
3.
E. coli
4.
Coxiella
2.
Geobacter
LEARNàBacktoDESIGN
Jean-LoupFaulon201622
Biosensorengineering
• DelepineBetal.NAR2016
SOURCEDrugBankHMDB(biomarkers)Tox21
SINKRegulonDBRegTransBaseRegPreciseBioNemo
RULESD=∞onRhea,MetaCycandBRENDAreac8ons
EFFECTOR
CHASSIS
TARGET(signal)
www.sensipath.micalis.fr
Jean-LoupFaulon201623
Usingmetabolicengineeringtoengineeringbiosensors
• LibisV,etal.ACSSynthBio2016
Jean-LoupFaulon201624
10 100 10000
5
10
15
20
Concentration (µM)
Rela
tive
fluor
esce
nce
(A.U
.)
1 10 100 10001
2
3
4
5
6
7
8
Concentration (µM)
Rela
tive
fluor
esce
nce
(A.U
.)
10 100 10000
20
40
60
Concentration (µM)
Rela
tive
fluor
esce
nce
(A.U
.)
0 50 100
1.0
1.2
1.4
1.6
1.8
Concentration (µM)
Rela
tive
fluor
esce
nce
(A.U
.)
10 100 10000
10
20
10 100 10000
10
20
0 50 100
1.0
1.9
2.8
Without metabolic module Without metabolic module
Without metabolic moduleWithout metabolic module1 10 100 1000
0
4
8
12
E.colibiosensorsinE.colimanymoleculesofinterest
• LibisV,etal.ACSSynthBio2016
Jean-LoupFaulon201625
Usingbiosensorsformetabolicengineering
TyrR
FapR
FdeR
Pathwayscanbedynamicallyregulatedandvariantscanbescreenedviafluorescentbiosensor
• FeherTetal.Fron/erinBioengineering&Biotechnology,2015
>100mg/L
2.5mg/L
Jean-LoupFaulon201626
LACTOSE GALACTOSE+GLUCOSE
ALLOLACTOSELacI LacZYA
+
TheundergroundacEvityof𝛽-GalactosidaseispresentonlywhenLacIispresentThisnetworkmoEfisconservedbyevoluEon(RWheatleyetal.,2013JBiolChem)
WhensyntheMcbiologybringsnewsystemsbiologyquesMons
𝛽-Gal
host Sensed compound intermediates regulator
E. coli lactose allolactose LacI E. coli nitroglycerin nitrite NarL
Thauera aromatica
toluene benzylsuccinate TutBC
Mycobacterium tuberculosis
cholesterol
cholest-4-en-3-one; 3-oxocholest-4-en-26-oyl-CoA
KstR
Mycobacterium smegmatis
cholesterol
cholest-4-en-3-one ; 3-oxo-4-cholestenoic acid
KstR
Paracoccus sp. L-gluconate L-5-ketogluconate ; D-idonate
LgnR
Azoarcus sp. 3-methylbenzoate 3-methylbenzoyl-CoA MbdR Sphingobium sp.
ferulate feruloyl-CoA FerR
Rhodopseudomonas palustris
p-Coumarate p-Coumaroyl-CoA CouR
Comamonas testosteroni
benzoic acid benzoyl-CoA GenR
Thermus thermophilus
Phenylacetic acid phenylacetyl-CoA PaaR
• Libis,Delepine,FaulonCurrentOpinionMicrobiology,2016
Jean-LoupFaulon201627
Acknowledgements
ThomasDuigou,IGR,MicalisINRA (reacMonruleworkflow)DhaferLhabib,Post-doc,MicalisINRA (retrosynthesisalgorithm)BaudoinDelepine,PhDstudent,MicalisINRA (biosensordesign)MathildeKoch,PhDstudent,MicalisINRA (biosensordesign&engineering)PabloCarbonell,SeniorResearcher,U.Manchester (metabolicpathwayscoring)JoeMellor,Post-doc,U.Manchester (MachineLearning)VincentLibis,PhDstudent,MicalisINRA (biosensorengineering)HeykelTrabelsi,Post-doc,MicalisINRA (metabolicengineering)AmirPandi,PhDstudent,MicalisINRA (biosensorengineering)CecileJacry,Tech.iSSB,Evry (metabolicengineering)IoanaPopescu,MCF,iSSB,Evry (metabolicengineering)CyrillePauthenier,Abolis,Evry (metabolicengineering)